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2018-04-03T05:48:59.092Z

1970-01-01T00:00:00.000Z

24749334

s2

Inactivation of vesicular stomatitis virus by disinfectants. Twenty-four chemical disinfectants considered to be viricidal were tested. Ten disinfectants were not viricidal for vesicular stomatitis virus within 10 min at 20 C when an LD(50) titer of 10(8.5) virus units per 0.1 ml were to be inactivated. Quantitative inactivation experiments were done with acid, alkaline, and a substituted phenolic disinfectant to determine the kinetics of the virus inactivation. Substituted phenolic disinfectants, halogens, and cresylic and hydrochloric acids were viricidal. Basic compounds such as lye and sodium metasilicate were not viricidal. MATERIALS AND METHODS The techniques used to obtain these data were the same as described previously (8) with the following exceptions. Disinfectant tests. Virus-disinfectant contact times were 5 and 10 min. The 30-min interval was eliminated since 10 min has been established as a maximal practical exposure time for effective viricides in animal disease control work. Speed of inactivation. Experiments were completed with seven time intervals. These were 0.5, 1, 2, 3, 4, 7.5, and 10 min. Additional time intervals of 30 and 120 min and 24 hr were used for NaOH. The disinfectant was diluted at the end of each time interval by the addition of 9 ml of phosphate-buffered saline (PBS) to the disinfectant-virus mixture, and, where acid or base was used, sterile 0.1 N NaOH or 0.1 N HCI was added to neutralize the pH of the PBS. Quantitative determinations of virus titer were measured by using serial 10-fold dilutions of virusdisinfectant mixture in embryonating chicken eggs for each time interval. The titer of virus was estimated by the LD5o method of Reed and Muench (7). Disinfectants. The 24 disinfectants are listed in Table 1. RESULTS Of 24 disinfectants, 10 were not viricidal with the previously described procedure. Alkaline chemicals tested were not viricidal for VSV, e.g., VSV survived for 24 hr in 10% NaOH (pH 12.2). Virulent virus was demonstrated after treatment with 10% KOH (pH 13.3), 10% Na2CO3 (pH 11.1), and 5% Na2SiO3 (pH 12.1) for 10 min. The quatemary ammonium compound, disinfectant G, was not viricidal at concentrations of 0.1 to 5.0%. In addition, sulfuric acid, acetic acid, isopropanol, ethyl alcohol, and Formalin were not viricidal at concentrations tested. The results are listed in Table 2 with the range of concentrations. Identical results were obtained for at least four replications of each disinfectant. The 14 other disinfectants were viricidal, but in some cases at concentrations greater than suggested by the manufacturer. The minimum viricidal concentration and the range of concentrations tested are listed in Table 3. Consistent results were not obtained with disinfectant E. The manufacturer's lot 1 was viricidal, whereas a second lot was not viricidal at the same concentration. The speed of inactivation was determined with NaOH, acetic acid, and disinfectant L. The death curve with NaOH and acetic acid was not linear, being initially rapid, followed by a decrease in rate. Virulent virus was not detectable after 30 min with 5% acetic acid, whereas, with 10% NaOH, it was still virulent after 24 hr. Disinfectant L at 2% concentration was rapidly viricidal and virulent virus was not detectable after 2 min. The virus survived 0.5% Table 1. Table 1. b Results variable depending on lot tested. propanol were not viricidal at a rate rapid enough to be useful against VSV. Alkaline chemicals have been employed as viricides in vesicular disease outbreaks in the United States because of their application for the inactivation of foot-and-mouth disease (FMD) virus. FMD virus was much more susceptible to change in pH as a means of virus destruction than VSV (4). VSV was resistant to destruction by alkaline chemicals in the present study. None of the alkaline chemicals, NaOH, KOH, Na2CO3, or Na2SiO3, was effective against VSV. However, in the United States, when a vesicular condition is found, it must be assumed to be FMD and chemicals used must be recognized as effective against FMD virus until the agent is proved to be VSV. Whereas acids have also been used to control VSV, it was determined that acetic acid was of only marginal value, since variable results were obtained with a 5 % concentration and lower concentrations were not viricidal. The results obtained with mineral acids depended on the particular acid, possibly owing to the different degrees of ionization (pK). The pK values of the mineral acids were hydrochloric acid, 0.784; sulfuric acid, 0.510; and acetic acid, 0.004. The most consistent viricides for VSV were the phenolic types when a sufficient concentration was used, but the effective concentration was higher than the manufacturer's recommendations in some cases. Organic iodine (U) and sodium hypochlorite were both active viricides. The virus was inactivated in 10 min even with the presence of the chorioallantoic membrane and allantoic fluid in the virus mixture. However, halogens are more susceptible to inactivation by organic material than other disinfectants. The rate of inactivation was determined for three different types of disinfectants. Disinfectant L (2%) was rapidly viricidal by an apparent first-order reaction, but, when it was diluted to 0.5%, a diphasic inactivation curve was evident. The diphasic curve was also evident for acetic acid and NaOH, even though the pH of the NaOH remained stable at pH 12.2 over the time of exposure. It appeared that the survivor curve was diphasic when the concentration of disinfectant was below that which was rapidly viricidal. Disinfectants at viricidal concentrations produced logarithmic virus survival curves or curves with a slight change in slope. The survival of virulent virus particles may have been due to the size of the aggregate and degree of aggregation. When a sufficiently high concentration of phenolic and halogen type of disinfectants was present, the additional time required to inactivate the virus was minimal. Based on laboratory evidence, substituted phenolics, halogens, or cresylic acids are recommended for use when vesicular stomatitis virus is to be destroyed on an infected premise. These chemicals are in the proper concentration under clean conditions and can be used with greater personal safety than acids and bases.

v3-fos

2017-07-06T04:33:04.858Z

1970-10-01T00:00:00.000Z

237232758

s2

Microbiological Examination of Cocoa Powder Total aerobic plate counts of 36 cocoa powders ranged from 100 to 27,000 per g, with 86% having counts of less than 9,300 per g. Bacillus and Micrococcus were the only genera identified. Limited information available on the bacteriology of cocoa powders used for the manufacture of chocolate-flavored milk indicates that the microflora is mainly aerobic sporeforming bacilli and micrococci (2). The microflora of the cocoa powders apparently results from contamination of the cocoa beans after fermentation (4). This note confirms the earlier reports on the bacteria isolated from cocoa powders. Thirty-six cocoa powders were obtained from 10 different manufacturers and were analyzed for coliforms and total aerobic plate counts by using Violet Red Bile Agar and Standard Plate Count Agar, respectively. In general, plating procedures outlined in Standard Methods for the Examination of Dairy Products were used (1). Isolates were obtained randomly from the total aerobic count plates by the method of Harrison (3). The isolates were cultured routinely in Brain Heart Infusion broth and agar slants. The tests carried out to aid in the identification of each isolate are presented in Table 1. A total of 37 features were recorded, coded, and transferred to IBM data cards. The computer used was the IBM 360-50 with a program of a simple ratio determination prepared to determine the similarity between a pair of cultures (6). Characteristics of each species from the genera Micrococcus and Bacillus were taken from Bergey's Manual and placed on IBM cards. Similarity values were then determined between these known species, and the unknown isolates having the greater similarity of characteristics to those of the known species were classified as being the same species. The total aerobic plate counts ranged from 100 to 27,000 per g and can be divided into three distinct groups as shown in Table 2. The results show that 31 samples (86.1 %) had counts of less than 9,300 per g. Of these, 12 (39%) had counts of less than 1,500 per g. The Growth at 7 C 21 C 32 C 40 C SOC counts of over 15,000 per g included two over 22,000 per g and three between 15,000 and 17,000 per g. No coliforms were detected in any samples. Study of the isolates indicated that Bacillus and Micrococcus were the only genera comprising the microflora of the samples. All samples contained bacilli but only nine contained micrococci. Cocoa powders may be a source of contamination of the finished product, but the numbers added are generally small. Normal storage conditions after processing would be sufficient to prevent growth of these organisms as far as spoilage is concerned. A total of 519 isolates were obtained from 36 cocoa powder samples. Of the isolates from the cocoa powder samples plated and incubated at 32 C, only 10 were of the genus Micrococcus and the rest were of the genus Bacillus. Computer analysis determined that there were 4 Micrococcus species and 20 Bacillus species represented in the 519 isolates. The identified species and the num- In conclusion, total numbers of aerobic bacteria added to chocolate-flavored milk by cocoa powders would be insignificant, cocoa powders are not generally a source of coliform contamination, the genera found in cocoa powders plated at 32 C are Micrococcus and Bacillus as determined by numerical taxonomy, and bacilli are

v3-fos

2020-01-02T21:54:39.337Z

1970-02-01T00:00:00.000Z

237233701

s2

Herbicide Transformation A strain of Fusarium solani isolated from soil by enrichment techniques used propanil (3′, 4′-dichloropropionanilide) as a sole source of organic carbon and energy for growth in pure culture. The primary product of the transformation of propanil by F. solani was isolated and identified as 3,4-dichloroaniline (DCA). This compound accumulated in the medium to a level (80 μg/ml) which stopped further herbicide utilization. Herbicide utilization by F. solani was influenced by various environmental and nutritional factors. It was more sensitive to acid than alkaline pH. Added glucose and yeast extract increased the rate of propanil decomposition, and the reduced aeration retarded growth of the fungus and herbicide utilization. The growth of F. solani on propionate was inhibited by added DCA. to be a significant agricultural chemical, perhaps rivaling even the auxin-derived herbicides such as 2,4-D (2,4-dichlorophenoxyacetic acid). Information regarding its fate in nature, including the identification of organisms able to transform it, will be of prime importance in its ultimate evaluation. Earlier work from this laboratory (2) gave evidence for the oxidative alteration of this compound by microorganisms in soil. It was of interest, then, to attempt the isolation in pure culture of organisms responsible for its transformation in soil. MATERIALS AND METHODS Culture. The propanil-degrading fungus, Fusarium solani (Martius) Saccardo (Collin Booth, Commonwealth Mycological Institute, Surrey, England, personal communication) was an isolate obtained from Nixon Sandy loam soil by conventional enrichment techniques (Lanzilotta, Ph.D. Thesis, Rutgers University, 1968). The isolate was selected specifically for its ability to grow in mineral salts medium with propanil as sole source of organic carbon. The fungus was maintained by transfer on Czapek-Dox agar slants. Tests showed that continued subculturing on media free of propanil did not result in loss of ability to degrade the herbicide. Media. The propanil-mineral salts medium consisted of propanil, 0.16 g; NH4Cl, 0.2 g; MgSO4. 7H20, 0.1 g; KH2PO4, 1.19 g; K2HPO4, 0.216 g; NaCl, 0.1 g; CaCl2-2H20, 0.01 g; Fe2(SO4)3, 0.0005 g; and 1,000 ml of distilled water. The final medium (pH of 6.0) could be sterilized by autoclaving (121 C) without significant hydrolysis of the herbicide. In autoradiochromatographic studies of propanil metabolites, the medium was modified to contain 0.10 g of propanil per liter. An absolute ethyl alcohol solution containing 4.6 mg of ring-labeled 14C-propanil per ml (specific activity, 2.18 mc/g) was prepared, and 0.1 ml (1.0 j,c) of this solution was added to each 50 ml of medium before autoclaving. It was predetermined that the ratio of unlabeled to "4C. labeled propanil used here was suitable for autoradiochromatographic identification of the herbicide and its metabolites. In a study of the effect of 3,4-dichloroaniline (DCA) on the growth of F. solani, a medium was used which lacked NaCl, but was otherwise identical in salt composition to that described above; it contained sodium propionate at a level of 0.4 g/liter at pH 7.4. In experiments designed to examine the effect of pH on fungal degradation of propanil in the basal salts medium, the ratio but not the molar concentration of monoand dibasic phosphate salts was varied to obtain the desired initial pH. The effects of glucose (0.05%), yeast extract (0.01%), Hoagland's trace element solution (1.0 ml/liter), and reduced aeration on the decomposition of propanil by F. solani were determined in a series of separate simple tests. In each case the medium was at pH 7.5. The glucose and trace elements were added to the propanil-mineral medium before autoclaving. The yeast extract was filter-sterilized and added aseptically after autoclaving. All flasks were incubated on a rotary shaker, except for one series which remained static to reduce aeration. Solvent extractions. Primary aromatic amines were recovered from culture media as follows. The solution was brought to approximately pH 13 with NaOH and extracted twice with a half-volume of chloroform. The chloroform extracts were combined and partitioned twice with a half-volume of 1.0 N HCl. The acidic aqueous extracts were combined, brought to pH 13 by the addition of solid NaOH, and then extracted twice with a half-volume of chloroform. The chloroform extract was dried over anhydrous Na2CO3 and then concentrated in a flash evaporator (Buchler Instruments, Fort Lee, N.J.). A portion of solid residue was recrystallized twice from ligroin and stored. To obtain acidic, neutral, and basic metabolites for analysis by autoradiochromatography, the following extraction procedure was employed. The medium was extracted with two half-volumes of chloroform. It was then adjusted to pH 1.0 with concentrated HCl and reextracted with chloroform as before. The acidified medium was then adjusted to pH 13.0 with solid NaOH, and the extraction procedure repeated once again. The three chloroform extracts were combined, dried over anhydrous Na2CO3, and concentrated to dryness in a flash-evaporator. The residue was dissolved by using 10 ml of acetone for each 50 ml of medium originally extracted, and the acetone solutions were used for autoradiochromatography. The foregoing extractions were also performed on uninoculated medium, and the products were used as controls in all analytical work. Thin-layer chromatography. Thin-layer chromatography was performed by using silica gel strips cut from Eastman thin-layer chromatogram sheets with and without fluorescent indicator (Distillation Products Industries, Division of Eastman Kodak, Rochester, N.Y.). Benzene and acetone (95:5) and benzene alone were used to develop the chromatograms. When the chromatogram sheet was impregnated with a fluorescent indicator, the spots were located by their ability to quench fluorescence under ultraviolet radiation. Primary aromatic amines, such as DCA, were also detected as bright yellow spots immediately after spraying with Ehrlich's reagent. Extremely small quantities (<0.1 ,ug) of DCA were detected by spraying a solution of N-(1-naphthyl) ethylene diamine dihydrochloride in absolute ethyl alcohol on chromatograms previously treated with 1.0% NaNO2 in 1.0 N HCI. This process produces diazonium salts from primary aromatic amines which then couple with the ethylene derivative to form bright blue to purple spots. In autoradiochromatography, silica gel strips were spotted with 50 ,Aliters of the final acetone solutions resulting from the chloroform extracts of culture media described above. They were subsequently developed with the solvent systems indicated above and then placed in contact with no-screen medical X-ray film (Eastman Kodak Co., Rochester, N.Y.). After exposures of 2 or 7 days, the films were developed and printed. Cultural conditions. In most cases, media were dis-pensed in 100-ml portions in 250-ml Erlenmeyer flasks and autoclaved at 121 C for 15 min. Media were inoculated uniformly with a suspension of spores harvested from Czapek-Dox agar slants. The flasks were incubated on a rotary shaker at 150 rev/min at 28 C. Analytical procedures. To estimate the relatively small amount of fungal growth that occurred in cultures in which propanil served as sole substrate, it was necessary to resort to a semimicro-colorimetric procedure. In this assay, mycelium was harvested by centrifugation, washed, and oxidized by dichromate in sulfuric acid. Reduction of the yellow dichromate ion to the blue trivalent species was a function of organic matter concentration. The actual amount of organic matter or mycelium present was estimated as sucrose equivalents by reference to a standard curve developed with the aid of a Klett-Summerson colorimeter equipped with a red filter (no. 66). The procedure used to prepare the standard curve and to perform the analyses was essentially that of Lu et al. (9) and is described in detail elsewhere (Lanzilotta, 1968). In studies of the effect of DCA on fungal development with propionate as substrate, growth was estimated as dry weight of mycelium collected on tared membranes (Millipore Filter Corp., Bedford, Mass.). DCA was determined colorimetrically by the use of the Bratton-Marshall reaction (5) essentially according to Pease (11). Infrared spectroscopy of authentic DCA (melting point 72 C, recrystallized from ligroin) and unknown metabolic products isolated in crystalline form were prepared as micropellets containing 30 mg of KBr to 300 ltg of sample. Spectra were obtained with a Perkin-Elmer model 21 spectrograph. RESULTS Studies of the transformation of propanil in soil (2) had indicated that DCA was a metabolite. Subsequent studies (4) confirmed this supposition and further showed that at least part of the DCA formed in soil was subsequently converted to form 3,3' , 4, 4'-tetrachloroazobenzene (TCAB). It was of interest then to ascertain whether DCA, TCAB, or other metabolites accumulated in a mineral medium when propanil served as sole substrate for growth of the herbicide-utilizing F. solani. Initially, propanil-mineral salts medium was extracted 10 days after inoculation with the test fungus for recovery of primary aromatic amines. When examined by thin-layer chromatography, a material was recovered which was found to contain only one aromatic amine, and that compound had RF values identical to those of DCA. In addition, a sample of the same material recrystallized twice from ligroin had the same melting point of authentic DCA (72 C). 4 on thin-layer chromatography. Both react with the spray reagents, develop identical spots, and melt at 72 C. Therefore, additional evidence was required for conclusive identification. Since the infrared spectrum of 4-chloroaniline is readily distinguishable from that of DCA (Standard Infrared Spectra, Samuel P. Sadtler and Sons, Inc., Philadelphia, Pa.) and since the infrared spectrum of the unknown compound matched identically to that of an authentic sample of DCA (Fig. 1), it was concluded that the lone aniline detected on thin-layer chromatographs was, in fact, DCA. As mentioned, the solvent extraction procedure used initially was designed to recover primary aromatic amines and excluded any metabolic product not sufficiently basic to be extracted from chloroform with 1.0 N HCI, e.g., TCAB. Therefore, to obtain a more complete profile of the products of propanil metabolism in mineral medium, a second extraction procedure was applied that recovered acidic, neutral, and basic compounds of ring-labeled '4C-propanil. The autoradiochromatogram shown in Fig. 2 was developed ascendingly with benzene and acetone (95:5) and that illustrated in Fig. 3 was developed with benzene. The chromatograms were left in contact with X-ray film for 7 days, and the film was then developed and printed. The right side of both figures represents extracts of uninoculated media. Clearly, Fig. 2 and 3 show that the controls contained only one major radioactive component, propanil, and that the remaining materials were attributed to radioactive impurities reported by the manufacturer and amounted to 1.4%7. The absence of major radioactive compounds other than propanil in the uninoculated control media is evidence that the herbicide was stable to both autoclaving and the chemical fractionation procedure. No residual propanil was detected by chromatography of extracts of inoculated media (left sides of Fig. 2 and 3). The major radioactive metabolite had the RF value of DCA in both solvent systems and responded to spray reagents as would DCA. In addition, several minor radioactive components were detected in extracts of the inoculated media in both solvent systems. Although the identity of these substances was not established, none corresponded to 3,3',4,4'tetrachloroazobenzene in RF value, and none was a primary aromatic amine. Actually, there is some doubt that they were fungal metabolites of propanil since their appearance coincided with the formation of a reddish-brown substance in the medium which was subsequently linked with a photochemical alteration of DCA. In fact, when DCA was added to medium that was sterilized but not inoculated and stored in the light, the reddish-brown material appeared after 2 to 3 days. When this medium was extracted and chromatographed, the material was resolved into three to five components, depending on the time of incubation before extraction, the type and intensity of light source, and the solvent system used. The RF values of some of these compounds matched those of the radioactive unknowns. Furthermore, like the unknowns, none of the photochemically produced substances were primary aromatic amines. Growth of F. solani on propanil as the only carbon source was limited by other than the supply of herbicide. To examine this phenomenon more fully, an experiment was designed to cor- relate growth with propanil degradation. In this study, the decomposition of propanil was conveniently followed by analysis of DCA released in the medium during fungal development (Fig. 4). It can be seen that DCA production reached a maximum in 7 days after inoculation or 5 days after the onset of propanil degradation, and, at that time, 70% of the propanil present initially in the medium had been decomposed. The course of DCA formation was essentially sigmoidal. Growth paralleled propanil decomposition and was maximum after 6 days. The decrease in mycelial mass noted from the sixth through the eighth day of incubation suggested cell leakage or lysis induced by the accumulation of a toxic level of DCA. Filaments harvested on days 8, 9 and 10 were highly vacuolated and frequently devoid of cytoplasm when examined under the microscope. It appeared that at a concentration of approximately 4 mg/50 ml, DCA interferes with metabolic activity and with the osmotic integrity of cells. When 70% (5.6 mg) of the added herbicide was transformed, sufficient DCA had accumulated in the medium to inhibit further transformation. The slight decrease in DCA concentration recorded between days 7 and 8 coincided with the formation of a reddish-brown material in the medium and therefore was attributed, at least in part, to the photochemical reactions. To test the supposition that DCA inhibits fungal metabolism, an experiment was performed to measure the effect of various concentrations of DCA on the growth of F. solani in propionate. (Propionate and DCA are produced in equimolar amounts by the hydrolysis of propanil, and it is the only substrate available to the fungus as it develops on the herbicide.) The amount of propionate supplied was approximately 10 times greater than that which the fungus obtains by complete hydrolysis of the amount of propanil (8 mg/ml) usually supplied in the medium. Nevertheless, as can be seen from the data listed in Table 1, DCA is toxic. Inhibition of growth of F. solani on propionate increased with increasing DCA concentration. The rate but not the extent of growth was reduced by 2 mg of DCA. Toxicity was more marked at 3 mg, and, at 4 mg, DCA reduced growth in 4 days by 90%. Five milligrams of DCA/50 ml of medium was completely inhibitory, and there appears to be support for the suggestion that accumulation of DCA to toxic levels inhibits the continued hydrolysis of propanil by F. solani. Results of a study of the influence of pH on the decomposition of propanil showed that the onset of decomposition was more rapid at increased pH (4.0 to 7.5). After 7 days, however. there was no significant difference in the extent of DCA formation in media of pH 5.0 to 7.5. No optimum was discernable, but there was no growth at pH 4.0. The effects of various nutritional factors and aeration on the utilization of propanil by F. solani are summarized in Table 2. No treatment decreased the 3-day lag that consistently preceded herbicide utilization by the fungus. Both glucose and yeast extract increased the subsequent rate of propanil decomposition. Only glucose increased the extent of decomposition which reached a maximum of 81% in 5 days, DISCUSSION The results of this study showed that F. solani hydrolyzed propanil and formed DCA and propionic acid. The propionate served as a source of carbon and energy for growth of the fungus, whereas DCA accumulated as the major metabolic product. No evidence could be obtained to indicate that the fungus could further transform the DCA, although such reactions as dehalogenation (8) and conversion to azobenzenes (3,4) of chloroanilines are reported to occur in soil. In addition, no phenolic products were evident which may result from the ring hydroxylation that often precedes ring rupture in the microbial dissimilation of chloroaryls (6,7,10). In contrast, the relative recalcitrance of chloroanilines is supported in work by Alexander and Lustigman (1) who demonstrated that aniline disappeared from a dilute soil suspension in 4 days, but o-, m-, and p-chloroanilines persisted for more than 64 days. The apparently nonmetabolic, photochemical products formed from DCA in mineral medium may be complex polymers of DCA in which the primary amino groups are linked covalently and, therefore, are unable to react with reagents used to detect and identify anilines. The ability of DCA to accumulate in the mineral medium and inhibit further metabolic activity of the fungus is not understood. Possibly, DCA may repress further synthesis of the acylamidase that hydrolyzes propanil or otherwise inhibits its action. This inhibition may cause suppression of growth even when the need for propanil hydrolysis is obviated by supplying the products of hydrolysis directly to the medium. This is reasonable in light of the fact that the VOL. 19, 1970 305 acylamidase involved most likely functions in the usual metabolic activity of the fungus, acting on some "normal" substrate other than propanil. In other words, bypassing the need to hydrolyze propanil for growth may not bypass the need for the acylamidase involved in that hydrolysis. Subsequent kinetic studies (Lanzilotta and Pramer, Appl. Microbiol., submitted for publication) have showed, however, that the acylamidase of F. solani does not exhibit product inhibition, at least when acetanilide is used as substrate. The increased rate of decomposition of propanil in mineral media supplemented with glucose and yeast extract was apparently a reflection of an increased cell population at the onset of propanil utilization rather than to increased activity. The inhibition of propanil decomposition by the addition of Hoagland's trace element solution may have been the result of using too high a level of one or more specific metals or to a synergistic effect resulting from a combination of metals that are not toxic individually. ACKNOWLEDGMENTS This investigation, published as part of the Journal Series of the New Jersey Agricultural Experiment Station, was supported by Public Health Service grant ES-16 from the Division of Environmental Health Sciences.

v3-fos

2020-12-10T09:04:16.744Z

1970-04-01T00:00:00.000Z

237234161

s2

Mycotoxicity of Aspergillus ochraceus to Chicks Five isolates of Aspergillus ochraceus, obtained from peanuts, were grown separately on sterile, moist corn for 14 days and fed to 1-day-old Babcock B-300 cockerels to evaluate their toxic effects. Two isolates were highly toxic, causing death of all birds during the 1st week of the experiment. Two isolates were moderately toxic, causing severe growth suppression with some deaths occurring throughout the 3-week test period. One isolate had no apparent effect. When the two most toxic isolates (diets) were diluted, survival time increased but severe growth suppression was evident. Postmortem examinations revealed a few small hemorrhages in the proventriculi of birds which died between the 2nd and 5th days. Emaciation, dehydration, and dry, firm gizzard linings were observed throughout the experiment. Extensive hepatic injury consisting of either fatty changes or necrotic foci was the principal microscopic finding. Suppression of bone marrow activity and depletion of lymphoid elements in the spleen and bursa of Fabricius were also found. The severity of the histopathological changes was directly related to the concentration of ochratoxin A in the diets. Five isolates of Aspergillus ochraceus, obtained from peanuts, were grown separately on sterile, moist corn for 14 days and fed to 1-day-old Babcock B-300 cockerels to evaluate their toxic effects. Two isolates were highly toxic, causing death of all birds during the 1st week of the experiment. Two isolates were moderately toxic, causing severe growth suppression with some deaths occurring throughout the 3-week test period. One isolate had no apparent effect. When the two most toxic isolates (diets) were diluted, survival time increased but severe growth suppression was evident. Postmortem examinations revealed a few small hemorrhages in the proventriculi of birds which died between the 2nd and 5th days. Emaciation, dehydration, and dry, firm gizzard linings were observed throughout the experiment. Extensive hepatic injury consisting of either fatty changes or necrotic foci was the principal microscopic finding. Suppression of bone marrow activity and depletion of lymphoid elements in the spleen and bursa of Fabricius were also found. The severity of the histopathological changes was directly related to the concentration of ochratoxin A in the diets. Toxic isolates of Aspergillus ochraceus Wilh. were obtained from cereal and legume products in South Africa by Scott (9) in 1965 and have since been found in foodstuffs by other investigators (4,6,14). The toxic principle was 7-carboxy-5-chloro-8-hydroxy-3 ,4-dihydro-3 R-methylisocoumarin, linked by an amide bond of the 7-carboxy-group to L-f3-phenylalanine, and was named ochratoxin A (12,13). A single 100-,ug oral dose of ochratoxin A to ducklings and rats resulted in acute hepatic injury (11) which varied from mild fatty degeneration to necrosis. Tubular necrosis of the kidney and enteritis have also been reported (8). Although A. ochraceus is widely distributed in nature (5) and is a common fungus in stored feeds and foodstuffs, the natural occurrence of ochratoxin A was not reported until 1969 (10). Two additional species of Aspergillus, A. sulphurous (Fres.) Thom and Church and A. mellus Yukawa (7), and a Penicillium species (14) are reported to produce ochratoxin A. A. ochraceus isolates of undetermined toxigenicity have been obtained from peanut kernels (1). Since toxic studies were not carried out, investigations were initiated on the mycotoxicity of A. ochraceus isolates obtained from domestic peanuts. The results of experiments in chicks are reported here. Some of the results have been published (2). MATERIALS AND METHODS Fungi. Ten isolates of A. ochraceus were initially screened for toxigenicity to 1-day-old chicks as previously described (3). Eight of these isolates, designated by "P" numbers, were obtained from R. T. Hanlin (University of Georgia, Athens). They were isolated from peanuts grown in several states. One isolate (CPES-0414) was obtained from peanuts grown locally in Southern Georgia. A reference isolate (NRRL-3174), known to produce ochratoxin A, was obtained from C. W. Hesseltine (Northern Regional Research Laboratory, Peoria, Ill.). Initially, all isolates were highly toxic and resulted in the death of all birds within a 2-week test period. After subculturing, several of these isolates lost their toxigenicity. Subsequently, five isolates were selected for further study; two with high toxicity (P-657 and NRRL-3174), two with low toxicity (P-94 and CPES-0414), and one with no apparent toxicity (P-3602). Cultures. Each isolate was grown singly in 2,800-ml Fernbach flasks containing 500 g of moist, autoclaved, cracked corn at room temperature (27 C). The flasks were shaken daily to reduce mycelial matting of the corn. After incubation for 2 weeks, each culture was dried at 50 C for 15 hr, ground, and mixed singly with a 36% protein supplement (6:4, w/w) to form a diet. Chicks. Each experimental group consisted of ten 1-day-old Babcock B-300 cockerels. A diet containing a single isolate of A. ochraceus was provided ad libitum for each group. The control group received 594 sterile corn similarily treated. Water was provided ad libitum. Additional birds were added during the experiments to evaluate the effects of dilution of the diet and age of the chicks on the mortality rates caused by two isolates (NRRL-3174 and P-657). Observations. Body weights of surviving chicks were recorded and averaged for each group at 1, 7, 14, and 21 days of age. Birds which died were examined for gross lesions as soon as possible after death. Chicks that survived the test period were killed and examined for gross lesions. All abnormalities were recorded. Tissues were collected in 10% neutral buffered Formalin, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically. The tissues examined included brain (three levels), spinal cord, eyes, dorsal root ganglia, peripheral nerve, adrenal glands, trachea, lungs, heart, aorta, spleen, bursa of Fabricius, thymus, bone marrow, crop, esophagus, gizzard, small intestine, large intestine, cloaca, pancreas, liver, kidney, gall bladder, testes, skeletal muscle, femur, and vertebrae. RESULTS Ochratoxin assays. The ochratoxin A content of undiluted NRRL-3174-infested corn was 7,925 parts per billion (ppb), whereas that of P-657 was 1,050 ppb as determined by thin-layer chromatography. No detectable ochratoxin A was found in either the CPES-0414-, P-94-, or P-3602infested corn. These analyses were made by N. D. Davis (Auburn University, Auburn, Ala.). Mortalities. The mortalities of 1-day-old chicks are presented in Table 1. Diets containing NRRL-3174-, P-657-, and P-94-infested corn caused 100%, mortality; however, those birds on P-94infested corn died at a much slower rate. CPES-0414-infested corn caused two deaths during the experiment, whereas none occurred in chicks fed the P-3602-infested corn. aDiets were prepared by mixing infested corn (6:4, w/w) with a 36% protein supplement. The use of older birds and dilutions of the NRRL-3174and P-657-infested corn slightly increased the survival times (Table 2). Growth. Birds fed corn infested with P-94 and CPES-0414 had severe growth suppression, whereas those fed corn infested with P-3602 had normal growth rates (Table 1). Gross findings. The gross postmortem findings are summarized in Table 3. The principal findings in birds fed NRRL-3174and P-657-infested corn were dehydration, emaciation, dry firm mucosal linings in the gizzards, and a few small hemorrhages in the mucosa of the proventriculi. One bird on the NRRL-3174-infested corn had an ulcerated gizzard. Emaciation was the only gross finding in the chicks fed P-94-infested corn. The chicks fed corn infested with CPES-0414 were emaciated and had enlarged, pale-red hearts and enlarged, pale-gray proventriculi. Four of these birds also had ascites, hydropericardium, and edema of the air sacs, mesentery, and pancreas. The small intestine appeared dilated. Histopathological findings. The microscopic findings are summarized in Table 4. Hepatic lesions varying from mild scattered foci of fatty changes to severe diffuse foci of necrosis occurred in 19 of 33 birds fed NRRL-3174-infested corn, 8 of 12 birds fed P-657, and 2 of 7 birds fed P-94. Only fatty changes were seen in the livers of 2 of 9 birds fed CPES-0414-infested corn. Suppression of hematopoietic activity in the bone marrow and depletion of lymphoid elements from the spleen and bursa of Fabricius were seen in chicks on all four isolates, but occurred most consistently in chicks fed NRRL-3174and P-657-infested corn. Incidental findings consisted of mild mycotic pneumonia (seven birds), bile duct proliferation in livers (four birds), focal suppurative inflammation of the gizzard (three birds), and focal fibrinosuppurative inflammation of the crop (four birds) and esophagus (one bird). DISCUSSION Corn infested with two isolates (NRRL-3174 and P-657) was highly toxic when fed to 1-day-2 old chicks. The mortality rate during the first week was 100%, which correlated with the high concentration of ochratoxin A. Dilution of these 3 toxic diets and use of older birds resulted in slightly increased survival times ( and 3rd weeks. Birds in these groups which survived the 1st week had severe growth suppression. Chicks fed corn infested with P-3602 and control chicks fed the noninfested corn diet had normal body weight gains and no mortalities. Emaciation, dehydration, and dry, firm mucosal linings in the gizzards were observed in a majority of the birds fed corn infested with NRRL-3174 and P-657. Emaciation was also apparent in the P-94 and CPES-0414 groups. These findings are probably secondary effects of the toxins since similar effects have been produced in normal chicks after water and feed were withheld for 42 hr. The hepatic fatty changes and necrotic foci found in the NRRL-3174 and P-657 groups were related to the presence of ochratoxin A. Seven chicks which were fed the highest concentration (60%) of isolate NRRL-3174 died before the 3rd day and had no liver lesions. These birds probably died before morphological changes developed in the liver. Liver lesions were a consistent finding in the chicks fed the diluted diets and in the older chicks. Chicks fed corn infested with P-657 also had consistent liver lesions with fatty changes, which represent mild hepatic injury, occurring more frequently than necrotic foci. These findings correlated closely with the ochratoxin A concentration. The two chicks affected in the P-94 group suggested that ochratoxin A was present in this corn at a level not detected by thin-layer chromatography, or some other undefined factor was involved. Suppression of bone marrow activity and depletion of lymphoid elements from the spleen and bursa of Fabricius were also frequently observed in the NRRL-3174 and P-657 groups. These changes are nonspecific and can be related to emaciation and growth suppression. Bile duct proliferation, which is considered a characteristic of aflatoxin injury, occurred in the liver of four birds. This lesion appeared to have a random distribution in the groups and was considered as an incidental finding. None of the control chicks was affected. One isolate, CPES-0414, produced effects which were entirely different from those observed with the other toxigenic isolates of A. ochraceus. The enlarged proventriculi and hearts and edematous tissues were observed only in this group of chicks. Additional experiments will be required to determine the cause of these effects which appear unrelated to ochratoxin A.

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Performance, diversity analysis and character association of black pepper (Piper nigrum L.) accessions in the high altitude of Idukki district, Kerala The experiment was conducted to evaluate black pepper accessions for growth parameters, yield attributing characters and yield. Out of the ten accessions tested, Karimunda recorded the highest fresh (1.61 kg) and dry (508.7 g) yield of berries plant-1. Fresh weight showed significant positive genotypic correlation to dry weight and while negative correlated to 100 berry volume, 100 berry weight and number of berries spike-1. Hence, selection based on number of berries spike-1, 100 berry volume and 100 berry weight may not lead to the high yielding black pepper variety. The results showed that Karimunda is the most suitable black pepper variety for high altitude areas of Idukki district. Introduction Black pepper, Piper nigrum L., referred as the 'King of spices' is the most important spice in the world. Indian pepper fetches a premium price in major international markets due to its preference and intrinsic quality (Thomas 2010). In India, Kerala is famous for black pepper cultivation and export. Idukki and Wayanad are the major pepper growing districts which together contribute 90% of the Kerala production. There exists considerable variation for yield among black pepper cultivars (Ravindran & Babu 1994). Idukki has maximum genetic diversity of black pepper. Most of the pepper farmers in the Idukki district are cultivating a minimum of five cultivars. Every traditional pepper-growing tract has its own popular cultivar. Selection of cultivars for prevailing agroclimatic conditions is important for higher productivity. The diverse climatic and soil conditions in a state like Kerala necessitates the identification and popularization of location specific varieties for the enhancement of productivity of pepper (Prasannakumari et al. 2001). The performance of black pepper varieties varies significantly in plains and high ranges owing to the difference in environmental conditions of these two regions. For evolving high yielding varieties of any crop, knowledge on the relationship between yield and yield attributing characters as well as vegetative characters is essential. Therefore, the present study was initiated with the objective of 18 Preethy et al. evaluating the performance, character association and variability studies of the released varieties as well as promising selections of black pepper. Materials and methods The experiment was conducted as a part of All India Coordinated Research Project on Spices at Cardamom Research Station, Kerala Agricultural University, Pampadumpara, Idukki during 2010-2017 (7 years). Vines of ten high yielding accessions of black pepper were collected from Panniyur Research Station (HB 20052, PRS 88), Horticultural Research and Extension Station, Sirsi (Acc no. 53, Acc. no. 106), Horticultural Research Station, Yercaud (Acc no. 33, Acc. No. 57), IISR, Kozhikode (C-1090, HP 39) and planted along with checks (Panniyur 1 and Karimunda) in a randomized block design with three replications at a spacing of 2 × 2 m. Agronomical practices were followed as per the package of practices of Kerala Agricultural University. Observations were recorded on various yield components for three cropping seasons. The pooled data for three years were subjected to statistical analysis. The simple phenotypic correlation coefficient was estimated using the formula suggested by Al-jibouri et al. (1958). Performance of different cultivars All the black pepper accessions under test, differed significantly for vegetative characters like vine column height, leaf length, leaf width and internodal length (Tables 1). Vine column height recorded highest for Acc 57 which is on par with CL 1090. Acc 33 showed highest leaf length and leaf width followed by Panniyur 1. Karimunda recorded lowest leaf length and leaf width. All the yield and yield attributing characters showed significant difference among the accessions both at 1% and 5% level (Table.3). This indicates the presence of wide variability in accessions for these characters. Karimunda recorded highest fresh weight and dry weight of berries which is statistically on par with Panniyur 1 and HB 20052. Most of the yield attributing characters was high in Panniyur 1 except volume of 100 berries. Even though Karimunda was the best performed accession for the last three years, the yield attributing characters were not that much comparable with high yielding accessions except number of berries spike -1 . However, according to Deka et al. (2016), Karimunda recorded higher number of laterals with more spread and higher number of spikes compared to the other varieties. Ibrahim et al. (1985;1987) have reported that spike yield and spike number in black pepper as important traits contributing for yield forwhich straight selection can be practiced for improvement. It may be the reason for high yielding nature of Karimunda. Variance components, heritability and genetic advance The estimates of genotypic variance (GV) ranged between 0.03-2958.02 and phenotypic variance (PV) 0. 18-21930.38. The phenotypic and genotypic variances were very far to each other and this shows the high influence of environment on morphological traits. Phenotypic coefficients of variation (PCV) ranged between 1.39-68.90; and genotypic coefficients of variation (GCV) between 1.01-25.30 (Table 3). The PCV values were relatively higher than the GCV values for all the parameters indicating environmental influence on the expression of the traits [11]. However, GCV values were near to PCV values for characters like 100 berry volume, vine column height, leaf width, internodal length. Such differences indicate very low environment variance for the phenotypic expression of these characters. Heritability (h 2 ) values ranged between 13.48% for dry weight to 95.63% for vine column height. The genetic advance as a percentage of the mean (GAM) also ranged from 1.51 for 100 berry volume to 40.00 for spike length. The combined estimation of heritability and genetic advance gives the response of a character to selection. Characters like spike length, vine column height, leaf width and internodal length was showed high heritability and high genetic advance. This indicates the suitability of these characters for selection due to additive gene action. The influence of environment to the characters governed by additive gene action is very less. Character association The character association studies done for both penotypic and genotypic correlation. Significant positive phenotypic correlation existed between dry weight and fresh weight of berries (0.92). The association between all other characters was not significant. The genotypic correlation studies revealed that, fresh weight is positively and significantly correlated only with dry weight of berries however it is significantly and negatively correlated with 100 berry weight, 100 berry Diversity analysis of black pepper volume and number of berries spike -1 . However, this is not corroborates with the findings of Sainamole et al. (2002) in which number of berries spike -1 is positively correlated to fresh yield. Therefore selection programme based on of dry weight of berries would be better than selection based on size of berries and number of berries spike -1 in black pepper. Dry weight of berries showed a negative significant genotypic correlation with 100 berry weight and 100 berry volume. Length of spike genetically correlated with vine column height which is significant and negative in direction. Same result had been reported by Sainamole et al. (2002). However, Sujatha & Namboothiri (1995) reported positive and significant influence on yield with the spike length. Leaf length showed a significant positive genotypic correlation with leaf width and internodal length. Productivity of black pepper depends on elevation, soil fertility, cultural practices, temperature, rainfall, age of the crop and climatic conditions during flowering, fruit set and development (Sivaraman et al. 1999 Introduction Small cardamom (Elettaria cardamomum Maton) is a commercial Zingiberaceous spice originated and evolved in the biodiversity rich forest ecosystem of Western Ghats, India. Besides its centre of origin, cardamom is widely cultivated in Sri Lanka, Guatemala, Papua New Guinea and Tanzania (Ravindran 2002). The diverse species has morphologically evolved into Malabar, Mysore and Vazhukka genotypes distinguished primarily based on plant stature, leaf, panicle and capsule characters. A myriad of biotic and abiotic factors acts as major 23 impediments to cardamom cultivation consequently leading to decline in gross cropped area and productivity. Among the foliar diseases, leaf blight incited by Colletotrichum spp. is noticed in all cardamom growing areas, inflicting damage invariably to all the genotypes. Divergent species belonging to the hemi-biotrophic ascomycetous pathogen, Colletotrichum incites leaf blight (Chethana et al. 2016). Though the disease is prevalent during the entire cropping season, it generally exacerbates and assumes epiphytotic proportions during post-monsoon period, probably favoured by an escalation in atmospheric temperature (Thomas & Bhai 2002). The anamorphic phase (conidia) plays a pivotal role in the epidemiology of leaf blight facilitating dissemination and pathogenesis evidently favoured by conducive environmental factors. Delineating vulnerable crop stages with respect to weather factors and disease pattern helps to formulate and implement plant protection strategies economically and efficiently. Correlating the disease progression with weather variables adequately supports decision making process besides providing an indirect indication on pathogen dynamics. The apparent infection rate (r) in conjunction with area under disease progress curve (AUDPC) values could be effectively employed as epidemiological parameters to define vertical or horizontal nature of disease resistance (Van der Plank 1963;Nagarajan 1983). Adopting plant protection measures based on apparent infection rate helps targeting the initial inocula thereby checking disease spread. Definitive information on epidemiology of cardamom leaf blight and the role of r and AUDPC parameters in disease progression as well as attempts towards deciphering the nature of resistance based on epidemiological parameters is scanty in cardamom-Colletotrichum host-pathosystem. Hence, the present investigation was formulated to delineate the relationship between disease progression vis-à-vis weather variables and an attempt was made to interpret the resistant nature of Malabar, Mysore and Vazhukka genotypes based on epidemiological parameters. Computing r and AUDPC values The apparent infection rate (r) is a parameter used to analyze the momentum of epidemic development. The apparent infection rate was calculated for each genotype based on the formula suggested by Van der Plank (1963). where, r is the apparent infection rate, x 1 is the disease index at initial time (t 1 ), x 2 is the disease index at subsequent observations (t 2 ). The apparent infection rates were further employed to identify the highest and lowest infection periods with respect to each genotypes. The AUDPC value was computed using the formula suggested by Wilcoxson et al. (1975). where, y i is the disease incidence at i th day of evaluation (initial observation), y i + 1 is the observation after the successive defined period, t i+1 -t i is the period between two observations and n is the number of successive evaluations. Results and discussion The percent disease index (PDI) observed in different cardamom genotypes along with weather variables recorded during October, 2015 to September, 2016 are presented in Table 1. Among the genotypes, Malabar comprised of 41 accessions and Mysore and Vazhukka comprised of 29 and 52 accessions, respectively. The period of observation spanned reproductive as well as vegetative phases of developmental stage and post-monsoon as well as monsoon periods as far as the weather pattern is concerned. Leaf blight incidence in cardamom genotypes and variation in weather variables In (Table 1). Crop architecture and microclimate appears to be the deciding factors which favour incidence and proliferation of various diseases in the spice ecosystem. Nevertheless, the dynamic weather variables, often manifested in the form of erratic rainfall and temperature fluctuations over a period of time have unforeseen effects which may be even pose a threat to the production system in which these spice crops are grown either as principal or component crops. Murugan et al. (2017) observed that, climate change as evidenced through rise in ambient temperature and erratic rainfall immensely contributed to disease outbreaks in cardamom, demanding nonjudicious application of fungicides. Patterns in disease progression and correlation with weather variables The disease exhibited a positive correlation with respect to T max and T min in all the genotypes, however, significant correlation was observed only in Malabar and Mysore with respect to T max . Whereas, rainfall and rainy days had negative correlation with the disease in all genotypes however found to be non-significant (Table 2). In Malabar, the disease progression followed a high-low-high pattern. The disease exhibited increasing trends during November, December, January, March, April, May, August (2002) illustrated that, interaction of components conferring genotypic resistance with environmental factors is required to modify the effects of host diversity with respect to disease progression. Categorization of accessions based on resistance towards leaf blight The reaction exhibited by the genotypes and their categorization based on resistance/ susceptibility levels towards leaf blight are presented in Table 3 Trends in r and AUDPC values The rate of disease spread as expressed in terms of r-value exhibited an erratic pattern irrespective of the genotypes. However, in general, the occurrence of anti-epidemics (where the r value attained a negative phase) was found to be uniform in all the genotypes during June-July. The average r-value was the highest (0.000429) for Malabar and least in Mysore (0.000124), whereas in Vazhukka it was 0.000186 (Table 4). The take-off level (an increase in r-value after attaining the anti-epidemic phase) was generally observed during July-August which coincided with an average T max of 24. Non-judicious application of synthetic molecules might lead to several far-reaching consequences as it contributes immensely to the evolution of novel races of pathogens with fungicide resistance and pesticide residues in the produce. Besides deciphering the speed at which the disease spreads in a population, the r-value could be effectively employed to identify the stages at which the plant protection measures can be adopted, primarily targeting the initial inoculum (X o ). In the present study it is observed that, in general, the r-value exhibited the trend of anti-epidemic phase during May-June and a take-off phase during July-August. Hence, undertaking plant protection measures with recommended fungicides before the commencement of monsoon (May-June) and during July-September targeting the low inoculum levels would have significant adverse effect on the disease progression. From the perspectives of epidemiology and host plant resistance it is concluded that, an increase in temperature during post-monsoon and precipitation have significant implications on disease progression. Introduction India is the second largest producer of onion next to China. Maharashtra state is the leading producing state in India. Onion is cultivated in three distinct seasons namely Kharif, late Kharif and Rabi. The soil moisture affect the quality of bulb and yield which is greatly influenced by the irrigation system. Onion is a shallow rooted crop needs light but frequent irrigation either by flood, sprinkler, or drip. The productivity of onion in India is 17.33 ton ha -1 which is low compared to world average. Managing the amount of applied irrigation water is critical to achieve optimum yield and quality. Most of the onion grown in India is under surface irrigation, which is relatively inexpensive, but inefficient in the amount of water use. Irrigation through drip is a new technique to increase agricultural production and to enhancing the efficiency of water use (Kusçu et al. 2009;Shock 2013;Enciso et al. 2015). Drip irrigation lends itself to automation, more so than either surface or sprinkler irrigation. Keeping this in mind, an experiment was planned on onion to study the feasibility of onion cultivation under drip irrigation. system fertigation was done with drip tank in 7 splits with 7 days intervals per day 2-3 hr. First irrigation was operated immediately after transplanting and light irrigation was done three days after transplanting for better and uniform initial establishment of crop. Materials and methods Recommended crop production and protection practices were followed as and when required to get good healthy crop. Following critical precautions were taken while conducting the experiment viz. irrigation interval followed uniformly; the operating pressure of drip system was 1.0 -1.5 kg cm -2 . In both the systems, irrigation was stopped at 15-20 days before crop harvesting. The bulbs were harvested at full maturity stage. After proper curing and neck cutting, the observations on yield and yield contributing characters and marketable bulb yield, percent of A (>6.5 cm), B (4.5-6.5 cm) and C (< 4.5 cm) grade bulbs on weight basis separately recorded and quantity of water applied was also measured. Results and discussion The results revealed that all growth and yield parameters of onion are significantly influenced by irrigation methods. The results further revealed that drip irrigation provided lower bolting (0.56%) and doubles (2.28%) as compared with surface system and it was observed that during Kharif season bolting did not record in any treatments because the average minimum temperature is above 17ºC is not favourable to initiate bolting, however doubles were recorded (Table 2). Nashik comes under shadow of a southwest monsoon; hence the erratic summer monsoon experienced by this region sets in last part of June and extends till the 2 nd week of October. The mean average rainfall varied from 548.0 mm to 862.0 mm during 2013 to 2015. Rainfall was coincided with bulb initiation and bulb development phases, due to heavy rain fall during Kharif season poor bulb development was observed. This is the reason and other climatic reasons during Kharif season 36.32% yield decreased as compared with Rabi, however by adopting drip irrigation system in Kharif season considerable yield was increased over surface irrigation method because drip irrigation raised bed system up to some extent remove excess water and avoid water logging due to slow and steady runoff water as compared with surface flat bed system where crop was affected. Over all during Kharif season in drip irrigation, the gross yield and marketable yield increased 14.27% and 18.30%, respectively. It is evidenced from the results, properly designed and managed drip irrigation raised bed has many advantages over surface irrigation including: elimination of surface runoff, high uniformity of water distribution, high water usage efficiency, flexibility in fertilization, prevention of weed growth and plant disease during rainy season. The results further revealed that highest 'A' grade (>6.0 cm) bulb (63.07%), 'B' grade (4.0-6.0 cm) bulb (24.82%) and lower 'C' grade (<4.0 cm) bulb (12.10%) were recorded in drip irrigation system during Rabi season and in Kharif also higher 'A' grade bulb (28.97%), 'B' grade bulb (36.28%) and lower 'C' grade bulb (34.76%) recorded in drip irrigation system (Table 3). Drip irrigation ensures optimum growth, better bulbing and early maturity of crops by assuring optimum soil moisture, water, air and nutrients throughout the crop growing period resulting uniform bulb obtained is directly correlated to the highest bulb size and productivity, whereas in surface irrigation yield decreased due to deep percolation and water is lost beyond the active absorption zone of The benefits of drip irrigation system over surface irrigation are illustrated in Table 4, that applied water in drip system is very lower in two seasons as compare with surface irrigation system. The 60.29 ha cm -1 and 55.65 ha cm -1 quantity of water was applied in drip system during Rabi and Kharif, respectively where as in flood system 85.35 ha cm -1 and 76.35 ha cm -1 , respectively. Thus, the drip system could save 29.36% and 27.12% water during Rabi and Kharif, respectively. The primary reasons attributed for the water savings include irrigation of a smaller portion of the soil volume, decreased surface evaporation, reduced irrigation runoff from the drip field and controlled deep percolation losses below the crop root zone, which enables higher water use efficiency in drip irrigation raised bed system, which was 562.79 kg ha -1 mm for Rabi and 418.69 kg ha -1 mm for Kharif (Fig. 2). The results were similar in line with results of Halim & Ener (2001) and Nagaz et al. (2012). Based on water consumption of crop calculated that during Rabi season 1000 lit of water is utilized for production of 5.62 kg and 3.49 kg of onion in drip and surface irrigation, respectively. Whereas in Kharif season 1000 litre of water by drip produces 4.18 kg, while in surface it is 2.67 kg, therefore drip irrigation system well suited for shallow rooted onion. The Cost: Benefits ratio also high in drip (1:2.69) while in surface irrigation it is 1: 1.68. The Cost: Benefits ratio in drip suggests that despite higher initial cost of the drip system, the drip irrigation is more profitable than the surface irrigation. Based on the obtained results of Rabi and Kharif seasons of the effect of drip irrigation on yield, yield components and morphological characteristics of onion, as well as water use efficiency and water saving it concluded that drip irrigation is highly significant effect on all studied parameters. To achieve a high production potential of onion, adopting drip irrigation should be maintained during the both Rabi and Kharif seasons. Basil is considered as a species with substantial nutritional and fertilization needs. It responds extremely well to nitrogen fertilization. The yield of basil increases when the dose of , but intensive farming that need high input of mineral fertilizers is considered as serious damage that may affect soil health, sustained production and balanced environment (Anwar et al. 2007). Taking into consideration of the deleterious effect of continuous applying of mineral fertilizer on the soil structure, organic farming could consider as a suitable replacement of inorganic fertilizer for improving microbial population, and soil organic matter (Shahram & Ordookhani 2011). Reza et al. (2015) claimed that substituting chemical fertilizers by organic manures and biofertilizers, could consider as a good farming system improve the ecosystem and soil health as a step for achieving sustainability in agriculture. Besides this, for medicinal and aromatic plants (MAPs), the real value is given to the quality rather than quantity, so that organic farming is considered as a suitable approach that enhances the performance of these crops. However, complete replacement of inorganic fertilizers by the organic manures is not advisable owing to their very low nutrient concentration and in turn requirement in huge quantities which may not be possible due to scarcity of such materials. In this endeavor, a blend of organic and inorganic fertilizers is important for improving the yield, maintaining soil health and keeping favorable ecological conditions on long-term basis. Amran (2013) and El-khyat (2013) revealed that application of organic manure along with half dose of chemical fertilizer had a positive impact on the oil yield of Pelargonium graveolens and Rosmarinus officinalis. The quality of the essential oil in basil is determined by the percentage of its volatile molecules which is affected by soil and climate conditions. Combined application of organic manure and inorganic fertilizer is considering the best tool that can be used to improve the yield and quality of these plants. Despite the importance of the basil crop, information on different aspects of growth, development, influence of organic manure and inorganic fertilizers on herbage and oil yield is very meager. In this context, this study was aimed to find out the "Influence of FYM, inorganic fertilizer and bio-fertilizers on herbage, oil yield, essential oil content and oil quality of sweet basil. Material and methods Field experiments were conducted in the experimental field at ICAR-Indian Institute of Horticultural Research (ICAR-IIHR), Bangalore during the kharif season of 2015 and 2016. The experimental station is located at an altitude of 890 m above mean sea level and 13 o 58" N latitude and 77 0 29" E longitudes. The nine treatments of experiment contain T 1 (FYM (10 t ha -1 ) + 100% recommended N through FYM), T 2 (FYM (10 t ha -1 ) + 100% recommended N through FYM + bio-fertilizer), T 3 (FYM (10 t ha -1 ) + 75% recommended N through FYM), T 4 (FYM (10 t ha -1 ) + 75% recommended N through FYM + biofertilizer), T 5 (FYM (10 t ha -1 ) + 50% recommended N through FYM), T 6 (FYM (10 t ha -1 ) + 50% recommended N through FYM + biofertilizer), T 7 (recommended FYM (10 t ha -1 only), T 8 (recommended NPK (160:80:80 kg ha -1 ) only, and T 9 (recommended FYM 10 t ha -1 ) + recommended NPK (160:80:80 kg ha -1 ). Treatments were replicated thrice in a randomized complete blocks design. Physical and chemical properties of the initial experimental soil are presented in (Table 1). The nutrients were supplied in the form of straight fertilizers like urea (160 kg N ha -1 ), single super phosphate (80 kg P 2 O 5 ha -1 ) and muriate of potash (80 kg K 2 O ha -1 ). Fifty per cent of nitrogen and full dose of phosphate and potash were applied as basal dose and the remaining fifty per cent of N was applied after 45 days of transplanting in T 8 and T 9 treatments. For biofertilizers, Arka Microbial Consortium (AMC) developed by ICAR-IIHR was used in the experiment and it contains N fixing, P and Zn solubilizing and plant growth promoting microbes in a single carrier. After 15 days of transplanting, recommended dose of AMC @5 kg ha -1 was applied at 2 cm deep to individual plants and immediately covered with soil. Similar method of application was also followed for ratoon crop after harvest of main crop in T 2, T 4, and T 6 treatments. Quantities of added fertilizers are given in (Table 2). 40 Each experimental plot size was 4.8 m long and 4 m wide with spacing of 40 cm between the plants and 60 cm between the rows. There was a space of 0.5 meter between the plots and 0.5 meter between replications. Basil variety Cim-Saumya (CIMAP) was sown in two nursery beds of 6.0 m in length with 1 m in width and 10 cm height. Forty days old (40) healthy and uniformly rooted seedlings of sweet basil were transplanted to the field. Weeding was done manually and drip irrigation was given daily for half an hour in the early stages and subsequently irrigation was given depending on the soil moisture condition. Fresh weight from each plot was converted to per hectare and it was expressed in tones (t). In order to determine the essential oil content (%), a sample of 100 g of basil fresh herb from the each plot were collected and mixed with 500 ml distilled water and then were subjected to hydrodistillation for 3 h using a Clevenger-type apparatus (Darzi et al. 2012). The quality of basil oil samples was analyzed by gas Al-mansour et al. FYM=Farm Yard Manure; Rec.=Recommended and *BF=Bio-fertilizer chromatography (Varian 3800 series) using VH-5 column for GC and VH-5 MS column for GCMS 30 m x 0.2 mm with 0.2 mm film thickness, oven temperature programmed at 60 0 C for 5 min then 210 0 C hold for 1 min then 240 0 C hold it for 1 min and helium gas as a carrier at 1 ml min -1 . Injector and detector temperature were 270 0 C and 240 0 C, respectively. Methyl chavicol and Linalool constituents of the oil were identified based on their retention time by comparing with the peak retentions times of those authentic standards obtained from Sigma, Aldrich, Bengaluru and run under identical conditions, then it were estimated in respect to total components and expressed as percentage. The data recorded from the experiment were analyzed using SAS 9.3 version of the statistical package (SAS Institute Inc 2011). Analysis of variance (ANOVA) was performed using SAS PROC ANOVA procedure. Means were separated using Fisher 's protected least significant difference (LSD) test at a probability level of p<0.01. Fresh herbage yield Fresh herbage yield of basil differed significantly due to application of different levels of N through FYM along with and without biofertilizers and inorganic fertilizer in the main crop and ratoon during two years of the experiment. It is evident from the Table 3 that the application of NPK (160:80:80 kg ha -1 ) + FYM (10 t ha -1 ) i.e., T 9 recorded significantly the highest herbage yield in the main crop (39.96 t ha -1 ) and ratoon (19.37 t ha -1 ). The lowest fresh herbage yield per hectare was obtained in T 7 applied with FYM (10 t ha -1 ) alone (22.92 t ha -1 ) and in ratoon (10.76 t ha -1 ). Similar trend was also reflected in total herbage yield of basil. Application of NPK (160:80:80 kg ha -1 ) + FYM (10 t ha -1 ) i.e., T 9 recorded significantly the highest total herbage yield (59.3 t ha -1 ) while, the lowest value (33.7 t ha -1 ) was recorded in T 7 applied with FYM alone. Nutrients through chemical fertilizer is expected to be more available that reflect on its uptake by plants leading to enhance the growth and yield. On the other hand, combined application of organic manure along with inorganic fertilizer regulated the supply of nutrients which in turn increased the yield (Merestala 1996). Similar findings were also reported by Mohamad et al. (2014) in Ocimum basilicum. Essential oil content Essential oil content plays a key factor in selecting the "adequate" combination of fertilizers in sweet basil cultivation. Application of different levels of FYM, bio-fertilizers and inorganic fertilizer on essential oil content (%) in the main crop and ratoon showed a significant difference (Table 3). Application of recommended NPK (160:80:80 kg ha -1 ) + FYM (10 t ha -1 ) recorded maximum essential oil content ( 0.48 and 0.45 %) whereas the lowest essential oil content was recorded with recommended dose of FYM alone in T 7 (0.31 and 0.17%) in the main crop and ratoon, respectively. The content of soil nutrient enhanced with application of organic manure, that had positive effect on the growth parameters, herbage and oil yield (Khalid et al. 2006). Oil yield Oil production is the most important parameter in basil farming. The results of different levels of N through FYM, bio-fertilizers and inorganic fertilizer on oil yield of main crop and ratoon recorded during the two years of the field experiment showed a significant increase in oil production which can be attributed to the increase of nitrogen doses either through organic or inorganic form (Table 3). In the present study, oil yield per hectare increased with the increase of FYM doses, but with application of NPK (160:80:80 kg ha -1 ) + FYM (10 t ha -1 ) recorded maximum oil yield in the main crop (199.7 kg ha -1 ) and in ratoon (107.58 kg ha -1 ). The lowest oil yield per hectare was obtained in T 7 applied with FYM (10 t ha -1 ) alone in the main crop (73.8 kg ha -1 ) and in ratoon (26.94 kg ha -1 ). Highest total oil yield was observed in T 9 applied with NPK (160:80:80 kg ha -1 ) + FYM (10 t ha -1 ) and the lowest total oil yield was recorded in T 7 applied with FYM alone. Integrated nutrient management improve the chemical, physical and biological soil proprieties that reflect positively on plant growth and oil yield (Patra et al. 2000). These results are similar to the observation of Zeinab (2005), Dadkh (2012) and El-naggar et al. (2015). Oil quality The most important volatile molecules identified in basil essential oil are methyl chavicol as phenolic compound and linalool as monoterpene (Mondello et al. 2002) andSajjadi (2006). The quality standard of the essential oil in basil is determined by polyphenols concentration (Toor 2006). Comparing the integration peaks, there was a significant difference in methyl chavicol percentage within the treatments. The results in ( The outcome of the present investigation revealed that the maximum fresh herbage yield, essential oil content, oil yield and its best quality was obtained with application of recommended FYM (10 t ha -1 ) + recommended NPK (160:80:80 kg ha -1 ) for both main as well as in ratoon basil crop. Addition of biofertilizers also increased the content of principle ingredient in basil essential oil (methyl chavicol and linalool). Hence, the incorporation of full dose of recommended FYM along with 50% of recommended N through inorganic fertilizer as basal and the remaining fifty per cent as top dressing at 45 days after transplanting may be recommended for basil crop to realize higher herbage and oil yield and better oil quality in sweet basil. Materials and methods Four exclusive WG districts viz., Kodagu from Karnataka, The Nilgiris from Tamil Nadu, Wayanad and Idukki from Kerala were selected for the study. These are high altitude districts have dense forest cover and enjoy higher rainfall. The altitude of Wayanad district is 700 to 1,200 masl lies in the latitude between 11°27' and 15°58' N and the longitude 75°47' and 70°2 7' E. Idukki's mean altitude is 1,200 m but many peaks rise above 2000masl and the district lies between 9°15' and 10°2' N and 76°37' to 77°25' E. The Nilgiris altitude varies between 300 m and 2,789 masl situated between 11°08' to 11°37' N and 76°27' to 77°4' E longitude. Kodagu has the lowest elevation range between 900 to 1,750 masl located between 11°56' to 12°56' N latitude and 75°22' to 76°11' E longitude. The rainfall data of these districts for hundred years (1901 to 2000) were purchased from IMD, Pune and used for rainfall analysis. A non-parametric Mann-Kendall test was used for the detection of rainfall trends (Mann, 1945;Kendall, 1975 (1968a and b). According to this test, the null hypothesis H0 assumes that there is no trend (the data is independent and randomly ordered) and this is tested against the alternative hypothesis H1, which assumes that there is a trend at P d"0.05. Results and discussion Major plantation and spice crops like tea, coffee; black pepper and cardamom are produced in higher elevation of the WG districts where their relative spread and yield are high compared to lower elevation areas or plains. These crops used to grow under rainfed condition as the rainfall receipt was high and distribution also was good. The yield of these crops now often fluctuates mainly due to higher inter-annual variability in rainfall. Results of study conducted to find out the rainfall trend of potential plantation and spice producing WG districts viz., Idukki and Wayanad in Kerala, The Nilgiris in Tamil Nadu and Kodagu (Coorg) in Karnataka are presented and discussed here. The Nilgiris Mean annual rainfall was 1839.7mm ( Rainfall analysis of western ghats (20%) like tea, coffee and the remaining areas are used for vegetables. They also stressed that many places experience sever water scarcity and moisture stress during summer months due to frequent dry spells even during monsoon periods that adversely affects the productivity of plantations and vegetable crops in this region. They also strongly advocated strengthening water harvesting structures to capture excess rainfall received between 20 th and 45 th weeks and utilize for farming. Kodagu Mean annual rainfall of this district was 2715.7mm (Table 2) with 17.0% C.V. Monthly rainfall pattern signaled that receipt was less during winter and summer months and July is considered the rainiest month (876.6mm) with a coefficient of variation of 34.8%. The rainfall amounts from southwest and northeast monsoons as well as summer rainfall were 2143.1mm, 310.5mm, 252.4 mm that respectively accounts 78.9%, 11.4%, 9.3% of annual rainfall. The rainfall receipt between May and October was more than 100 mm per month. Sufficient soil moisture would be available for crop production during these months. Mann-Kendall Statistics (S) and Sen's slope analysis have indicated that there was a positive trend in annual rainfall. Although few months showed negative trend in monthly rainfall, the 'p' values proved that as such there was no significant trend in monthly rainfall except for August where 'p' value was less than 0.05%. Raju et al. (2013) have classified the Kodagu climate as per-humid to humid type. Increasing rainfall trend was also reported by Mallappa et al. (2015) for Kodagu district. The district is popular for cardamom and coffee + black pepper cropping system and contributes substantially for national production. Ankegowda et al. (2010) have identified the length of growing period for this region between 21 st and 43 rd week (21 st June to 28 th November) with more dependable rainfall with lesser C.V. and suggested crop production operations to be carried out during this period for various crops. Idukki Idukki's mean annual rainfall was 2979.4mm (Table 3) with very high percentage of C.V. of (25.8%). Highest rainfall among months was received by July (673.7 mm) with 45.7% C.V. The monthly rainfall between May and October was above 100 mm and contribution to annual rainfall was maximum (22.61%) for July followed by June 18.92% and August (14.77%). The rainfall from southwest and northeast monsoons as well as summer rainfall were 1943.9mm, 548.1mm, 360.9mm that accounted 65.2%, 18.4%, 12.1% of annual rainfall respectively. Mann-Kendall Statistics (S) and Sen's slope results have indicated that there was a negative trend in monthly and annual rainfall except August (Table 3) and the trend was significant for months of January, March, October and December with 'p' less than 0.05%. Raju et al. (2013) have classified the Idukki's climate as Per-humid to humid type. Krishnakumar and Rao (2006) have analysed the long term rainfall data (1871-2000) of Kerala and found that the failure of northeast and premonsoon rainfall lead to droughts in 65% of the years during summer that has affected the most plantation crops and they attributed this to man-made interventions into the ecological balance particularly during the last 50 years. Krishnakumar & Rao (2008) have argued that the monthly rainfall over Kerala for the period 1871 to 1994 showed an increasing tendency during October and November but a decreasing trend was shown by December. Similar trend was more evident since 1961 onwards. Archana et al. (2014) have noticed a decreasing monthly and annual rainfall trend in Kerala which they attribute to anthropogenic green house gas (GHG) emissions due to increased fossil fuel use, land-use change due to urbanisation and deforestation, proliferation in transportation associated atmospheric pollutants. Murugan et al. (2000;2009; have reported the changes in rainfall trends of this region and discussed possible negative impacts on crops and plants as well as ecosystem hydrology because cardamom, coffee, tea and native forest plants are highly sensitive to precipitation changes. -2004), and only 28.2 percent of the years only fell under early or late monsoon years. Further, it was also noticed that if the monsoon was early that season would receive deficit rainfall, and no trend was seen when the monsoon was late. They also reported that there was decline of 6.8% in annual rainfall over the period which was more evident in monsoon rainfall since the last sixty years. Similar negative trend for the district was reported by Sushant et al. (2015). In another study by Kumar & Srinath (2011) showed that weakening of the early phase of the southwest monsoon precipitation; increasing polarisation of daily rainfall and more frequent heavy rainfall days that hastened maturation of a variety of crops. Gaetaniello et al. (2014) have observed that the variability in rainfall was more influential than the air temperature fluctuation in this district. During the study period (1951 to 2008), the monsoon rainfall decreased, while maximum and minimum daily temperature increased and farmers perceived Rainfall analysis of exclusive WG districts indicated that there was a temporal and spatial variation in rainfall receipt among these districts and high mean annual rainfall was received in Wayanad (3381.0 mm) followed by Idukki (2979.4 mm), Kodagu (2715.7 mm) and Nilgiris (1839.7). Decreasing trend in mean annual rainfall was noticed in Idukki, Wayanad and The Nilgiris, whereas in Kodagu the annual rainfall was stable (Fig. 1). The trend was significant for The Nilgiris and Wayanad. Similar trend was also observed for southwest monsoon rainfall. The decline in annual rainfall and the southwest monsoon was noticed for The Nilgiris and Wayanad. July was the rainiest month in all these districts. The negative trend was significant for January, May, June, July and August for the The Nilgiris, whereas in Kodagu no significant trend for mean monthly rainfall was observed except for August. At Idukki, significant negative changes were noticed for January, March, October and December months rainfall; whereas in Wayanad, January, March, April and July months rainfall showed significant negative trend. These negative trends in these important plantation and spices producing districts of the WG would affect the agricultural economy and hydrological systems. Rainfall analysis of western ghats The availability of quality planting material is one of the major issues that black pepper growers face in India. The conventional propagation methods have several limitations due to low success rate, poor rooting, spread of soil borne pathogens and poor survival rate of transplanted rooted cuttings (Rini et al. 2016). In order to overcome these problems, a technological intervention is needed to boost both the production and supply of quality planting materials. In the context of promoting organic cultivation, the use of biofertilizers for quality seedlings/cuttings production secures much importance because they are eco-friendly, low cost, capable of improving crop yields and quality sustainably. Biofertilizers improve growth rate of plants and soil health as they act as plant strengthners, phytostimulators, plant health improvers, and have the potential to fix nitrogen (Babalola 2014). The fertility of the soil is also restored by biofertilizers so that plants were better protected from getting any diseases (Amna 2010 T 4 0.2% Humic acid @100 ml plant -1 T 5 0.5% Fish extract @100 ml plant -1 T 6 PSB @5 g plant -1 + Azospirillum @5 g plant -1 + 0.2% Humic acid @100 ml plant -1 + 0.5% Fish extract @100 ml plant -1 T 7 PGPR Mix I @5 g plant -1 + 0.2% Humic acid @100 ml plant -1 + 0.5% Fish extract @100 ml plant -1 T 8 Untreated control Local Karimunda cuttings were planted in polythene bags size of 20 × 15 cm filled with potting mixture composed of garden soil, sand, and farm yard manure in 1:1:1 proportion. The potting mixture had an initial nutrient status of organic carbon (1.98%), N (0.704 mg/100 g) available P (4.01 mg/100 g) and K (43 mg/100 g) with a pH of 5.3. The treatments were superimposed and evaluated in vivo under greenhouse condition. Commercial formulations of phosphorus solubilizers (5x10 7 cfu), Azospirillum (5x10 7 cfu) and PGPR Mix I (Consortium of Azospirillum lipoferum, Azotobacter chroococcum, Bacillus megaterium and Bacillus sporothermodurans each with 5 × 10 7 cfu) were obtained from the Department of Agricultural Microbiology, College of Agriculture, Vellayani. The humic acid containing 12% potassium humate was used in this study. Fish extract was prepared as per the standard procedure. Equal quantity of fish and jaggery was taken, sliced, mixed and kept in closed container with periodical stirring up to 30 days, after that the solution was strained through a muslin cloth and stored. The experiment was conducted using Completely Randomized Design (CRD) with four replications. Five plants were kept in each replication.In addition to this fiveplants/ treatment were also maintained for destructive sampling to study the root characters. Talc based formulation of PSB, Azospirillum, PGPR Mix I, humic acid and fish extract individually and in combination were applied to the root zone of one month old black pepper cuttings grown in polythene bags. The treatments were given twice at fortnightly interval (when the plants were 30 and 45 days old). Observations on the height, number of leaves, leaf area and root characters were recorded 60 days after second treatment application. The experimental soil (potting media) was analyzed for physicochemical properties like pH, organic carbon and available N, P and K content. The leaf chlorophyll content was estimated as per the protocol of Sadasivam & Manickam (1992). The data collected from the experimentwere processed statistically with appropriate statistical tool for the interpretation of the results. There was no significant difference with respect to morphological characters before treatment application. Butgood improvement invegetative characterswas noted after the application of biofertilizers and organic supplements.Black pepper rooted cuttings responded well to combined inoculation of biofertilizers and organic supplements compared to individual inoculation and control ( Table 1). Application of PSB along with Azospirillum, humic acid and fish extract (T 6 ) resulted increased plant height (98.66 cm), numbers of leaves (12.16), number of roots (13.50), root length (28 cm) and highest leaf area (63.18 cm 2 ) 60 days after second treatment application compared to all the other treatments. Similar increaseof growth was recorded in black pepper for combined inoculation of biofertilizers such as Azospirillum, Phosphobacteria and VAM (Kandiannan et al. 2000;Bopaiah & Khader 1989). In addition to this, fish emulsions have been reported to increase the nitrogen accessibility (Weinert et al. 2014). According to Chen & Aviad (1990) the application of humic substances increase root length and produce more secondary roots. As a result, the plants were capable of absorbing more available nutrients from the soil which in turn resulted better establishment and subsequent growth and development. Thus, the combined inoculation of PSB and Azospirillum along with humic acid and fish extractperformed best through improving the morphological characters of the cuttings compared to their individual application. Total chlorophyll content was maximum in plants treated with PSB along with Azospirillum, humic acid and fish extract (0.64 mg/100 g) followed by the combined application of PGPR Mix I along with Azospirillum, humic acid and fish extract (0.60 mg/100 g). The least quantity of chlorophyll was observed in control plants (0.42 mg/100 g). This can be attributed to the increased uptake of nutrients leading to enhanced chlorophyll content. Pereira et al. (2015) reported that maize plants when inoculated with Azospirillum under different dosages of nitrogen enhanced the chlorophyll content. Our best treatment also includes Azospirillum as one component. This contributes to the increased chlorophyll content of the treated plants. Nutrient status of the potting mixture 60 days after second treatment application (Table 2) showed that, readily available nutrient content of potting mixture was improved through the application of biofertilizers and organic supplements compared to control. Among the treatments, PSB along with Azospirillum humic acid and fish extracttreated soil showed higher levels of organic carbon (3.17%), available phosphorus (17.83 mg/100 g) and potassium (84.66 mg/100 g) whereas, available nitrogen was highest for combined application of PGPR Mix I along with Azospirillum, humic acid and fish extract (1.05 mg/100 g). Increased availability of nutrients in the potting mixture was attributed through combined application of biofertilizers and organics. Bio inoculants have had influence in increasing the organic carbon content of turmeric (Sumathi et al. 2011). Biofertilizers like Azospirillum and PSB were highly beneficial to plants through augmentation of nitrogen and phosphorus content in soil, thus making these two essential nutrients available to the plant and also produce phytohormones like auxins (Singh et al. 2011;Rocheli et al. 2015). In our experiment also treatments consisting of PSB along with Azospirillum, humic acid and fish extract (T 6 ) resulted in high available organic carbon, P & K compared to control which is responsible for increased growth parameters observed. The increase in P availability could be attributed to the application of PSB which produces organic acids that act as a chelating agent and thereby, releases P into the soil solution and making it more available resulted in improved root growth. These findings are in agreement with the findings of Naik & Hari babu (2007) and Sharma et al. (2009) in guava. It is evident that, the combined application of PSB (5 g) along with Azospirillum (5 g), 0.2% humic acid (100 mL plant -1 ) and 0.5% fish extract (100 mL plant -1 ) followed by PGPR Mix I (5 g) along with 0.2% humic acid (100 mL plant -1 ) and 0.5% fish extract (100 mL plant -1 ) produced healthy black pepper rooted cuttings with good morphological characters than those of sole application of any of these biofertilizers and organics. Therefore, this technology could be effectively advocated to produce black pepper root cuttings with lusty growth in nurseries. 60 The demand for healthy planting material of high yielding black pepper varieties is on the increase and the country needs large quantity of quality planting material to meet the increasing demand. The present study was aimed to establish a model nursery to produce disease free healthy planting material of improved varieties of black pepper by creating awareness among farmers with good agricultural practices and by adopting nonchemical bio-intensive management strategies. et al. 2016) in different combinations were used as growth promoters as well as bioagents for incorporating into the nursery mixture. The experiment was designed in a two factor CRD with four improved varieties of IISR and five treatments. The five treatments were T1-T. harzianum + P. chlamydosporia, T2-Streptomyces strains 2+9, T3-Streptomyces strains 5+9, T4-Metalaxyl-Mz+ Carbsosulfan and T5-control without any amendments. The individual treatments were incorporated with the solarized nursery mixture separately and filled in polythene bags (15 cm × 10 cm) @250 g bag -1 . T. harzianum and P. chlamydosporia were made in liquid form with water and added @100 mL (cfu 10 9 mL -1 ) each to 100 kg potting mixture. Streptomyces spp. grown as broth culture in Nutrient broth and 100 mL (cfu 10 10 mL -1 ) mixed with 1 kg vermicompost and grown for 5 days (cfu10 8 mL -1 ) ) and applied @1 kg 100 kg -1 potting mixture. The treatment imposed poly bags were arranged inside the nursery and planted with single node virus free (indexed) rooted cuttings as mentioned above and were allowed to grow by serpentine method. When the number of rooted nodes in the serpentine reached around 10, the rooted middle cuttings were cut and separated leaving three plants at the tip and nucleus plant at the end and were kept for establishment to a 3-4 leaf stage in the same nursery. Five plants each were taken from each treatment and observed for biometric growth parameters. The biometric observations were recorded on height of the plant, fresh and dry weight of the plant, number of roots, root length and root biomass. The soil was analysed for the presence of targeted pathogens like Phytophthora capsici and nematodes (Radopholus similis and Meloidogyne incognita), pH and dehydrogenase activity (DHA). The data were analyzed by using PROC ANOVA procedure of SAS 9.3. Least square means statements were used for mean separation. After nine months of growth by serpentine method, the variety IISR Malabar Excel and IISR Thevam produced the maximum number of plants in T1 (T. harzianum + P. chlamydosporia (59 nos. and 51 nos. respectively) followed by T2 (Act 2+9) in case of Malabar Excel (45nos) and T3 (Act 5+9) (45 nos) in case of Thevam, from a single node cutting. An average of 6-7 plants/month/cutting was produced from these varieties with the treatment T1, while it was only 4 plants/month/ cutting in control. In case of IISR Girimunda, the performance was almost the same with all the three bioagent combinations when compared to Metalaxyl-Mz + Carbsosulfan and control. However, IISR Shakti showed comparatively lesser performance with bioagent combinations. The results of the study clearly showed the response of varieties to bioagents. In all cases, the number of plants produced with Metalaxyl-Mz + Carbsosulfan (T4) was comparatively lesser when compared to control ( Table 1). The root system was also healthy in all treatments except for control where the root was not profusely grown. No disease of any kind was observed in any of the plants. Though there is no difference between varieties in fresh weight of the plant, the dry weight ( Fig. 1) is significantly superior in treatment with Streptomyces strains (Act 2+9) and is at par with T. harzianum and P. chlamydosporia. Not much difference was observed in the number of leaves between treatments but, there is difference in the height of the plants (Fig. 2) where bioconsortia showed increased height when compared to Metalaxyl-Mz. + Carbsosulfan and control. Difference was observed in number of roots, root length, and root fresh and dry biomass (Tables 2 to 5). Since the nursery mixture was solarised and irrigation was limited to once in two days, there was no incidence of soil borne infections caused by Phytophthora capsici, Sclerotium rolfsii or nematodes (the common diseases otherwise observed in nurseries), in any of the treatments including control ( Table 6). The pH of the soil in untreated plants ranged from 4.55-5.66. It is interesting to note that the pH of the soil is raised to neutral in treatments with T. harzianum + P. chlamydosporia where it ranged from 6.86-7.63. In the case of Streptomyces combinations the pH ranged from 4.85 to 6.85 (Table 6) and the dehydrogenase activity which reflects the total oxidative activity of soil microflora, (Liang et al. 2014) was unaffected by the incorporation of bioagents like Trichoderma, Pochonia or Streptomyces sp. (Table 6). So without the addition of external nutrients, the micro flora enriched solarized mixture supported the growth of plants as well as prevented the incidence of infection caused by nematodes, Phytophthora or any other soil borne pathogens of black pepper. This may be due to the increased microbial activity either through the production of IAA or other growth promoting traits including siderophore production which is observed in the case such reports are available for the combined use of T. harzianum and P. chlamydosporia for the production of rooted plants of black pepper, except for the individual use of T. harzianum against foot rot and P. chlamydosporia against slow decline diseases. The present study recommend soil solarisation along with the use of Trichoderma + Pochonia combination or combination of Streptomyces strains Act 2+9 for the production of healthy rooted planting material. The effect of solarized potting mixture on growth of black pepper rooted cuttings was reported earlier (Thankamani et al. 2007). Since both Phytophthora and nematodes are serious threats of black pepper, the treatment combinations are made in such a way that the combination contain one antagonist against Phytophthora and another antagonist against nematode. Similar work was reports for the use of consortia in planting material production. Consortia of P. fluorescens + T. harzianum + Paecilomyces lilacinus were used for the production of tomato seedlings for combating nematode infection (Mukhtar 2013) and P. fluorescens + T. harzianum for the production of nematode free papaya seedlings (Rae 2007). The promotive effects of Pseudomonas and Trichoderma were quite significant in growth promotion in tomato during nursery and crop growth stages (Kumar et al. 2007 Bio-control agents for black pepper cuttings production Here we attempt to study the floral diversity of monoecious nutmeg. The study on sex differentiation and variability in monoecious nutmeg was carried out at ICAR-Indian Institute of Spices Research (IISR), Kozhikode during June to September 2015. Total of 53 trees of 18 year old were selected randomly from the germplasm maintained at ICAR IISR and were studied for sex of tree from December 2014 onwards. The sex of flowers were recorded in all the trees at monthly interval. Out of the 53 trees of the selected population 6 trees are found to be monoecious. These 6 monoecious trees are used in the present study. Within the 6 monoecious trees observations on inter and intra floral variability in monoecious for anther length, number of anthers, filament length, ovary length and flower opening type were recorded. Total of 100 flowers / month were collected from each tree and observations were recorded for variability during peak flowering season June -September, 2015. Pollen viability test was done using Acetocarmine staining method. Pollen viability estimated using glyceroacetocarmine (Marks 1954). The pollen viability percentage determined as the ratio of the number of viable pollen to the total pollen number. ANOVA for the floral attributes were done using SAS 9.3 software. Based on the observations made on the phenological measurements inter and intra flower variability in monoecious nutmeg flowers are prominent. Three types of flowers are recorded in monoecious trees namely, pistillate, staminate and hermaphrodite flowers. The structure of hermaphrodite in nutmeg is reported for the first time. The flowers are borne on the leaf axil, flowering habit of the three types of flowers are seen in cymes as well as solitary in the same tree. All the types of flowers were light creamy yellow colour in monoecious tree, with thick gamosepalous perianth which bursts as trilobed or bilobed (less frequent) lobes during anthesis as reported earlier (Armstrong & Drummond 1986). However, addition to this tetralobed and pentalobed are observed rarely in monoecious trees (Fig. 1). Tetralobed lobes are reported in M. fatua, this type of lobe opening helps in more access for insects (pollinators) to enter the flower (Sharma & Armstrong 2013). The androecium of staminate flowers consisted of adnate 7-13 anthers, with average of 9.30 anthers and 4.09 and 3.18 mm of anther length and filament length respectively, androecium with 8-10 anthers were reported earlier by Armstrong & Drummond (1986) in staminate flowers. The gynoecium consists of single ovary with a mean length of 5.28 mm with bifid stigma in the pistillate flowers (Tables 1 & 2). Intra flower variability is evident in case of hermaphrodite flowers. In hermaphrodite flower the androecium ranges with 1-4 anthers, in the form of fused filament or free filament or both; some anthers are fused with the gynoecium (Fig. 2); the length of the anther varies from 3- 3). Analysis of variance of trees for flower types and floral attributes like flower type, number of anthers, length of anthers, length of filaments and length of ovary are found to be highly significant (Table 1). High coefficient of variation is recorded for hermaphrodite (183.84%) and pistillate (171.71%) flowers ( Table 2). The pollen viability of the hermaphrodite flowers are found less as compared to the staminate flowers. The percentage of pollen viability is 79.74 and 94.77 in hermaphrodite and male flower respectively (Table 3). In the population studied, the average segregation of staminate, pistillate and hermaphrodite flower in the collected samples is 87.72, 19.22 and 5.77, respectively with approximate ratio of 17: 4:1. The occurrence of hermaphrodite flower in monoecious tree ranged from 0-9%. In 90% of the flowers studied the stamen length is found shorter than the stigma thereby excluding the chance of selfing. Moreover the pollen viability is also found less in these flowers. Variability in flowering phenology among individuals has direct impact on their fitness (Mauricio et al. 2013). However, the influence of environment factors on sex expression and floral variability need to be studied in detail in nutmeg. Cinnamon (Cinnamomum verum L.) or dalchini is one of the ancient tree spices grown in India. Though both bark as well as leaves are known to possess aromatic components mainly cinnamaldehyde and eugenol, the bark of this species is valued as a spice. It has been used in the form of dried bark, bark powder, oil and oleoresins. Cultivation of this spice in India is mainly confined to States of Andaman and Nicobar Islands, Kerala, Karnataka, North Eastern India and parts of Tamil Nadu and Maharashtra. Combined estimate of cinnamon and tejpat suggests that in India, it's being grown on 2,770 ha area with about 5,050 t production (Indian Horticulture Database 2014). However, the production is not enough to meet the domestic demand and hence, cinnamon is being imported from other countries of the world (Indian Spices 2016). Increasing the productivity through development and adoption of improved technologies is a key factor in reducing the dependence on import. Soil and climatic conditions of the Andaman and Nicobar Islands are well suited for the cultivation of cinnamon (Parthasarathy et al. 2009) and presently it is cultivated in about 150 ha yielding 40 t annually. Quality of cinnamon is assumed to be the finest in the islands compared with other parts of the country (Singh & Sankaran 2012). Large availability of interspaces in the coconut and arecanut plantations in the Andaman and Nicobar Islands could be successfully utilized for its cultivation (Waman et al. 2016). However, existing plantations in the islands are of seedling origin. One can easily notice variations in cinnamon seedlings for leaf size, shape and colour of new flush, apart from the distinct chemotypes (Krishnamoorthy et al. 1988). Ergo, considerable variability is noticed in the yields and quality of the final produce and hence, vegetative propagation is of interest (Rema et al. 1997). The present report concerned an effort to identify the most appropriate time for carrying out air layering under island condition. The present investigation was carried out in the Division of Horticulture and Forestry of ICAR-Central Island Agricultural Research Institute, Port Blair, Andaman and Nicobar Islands during 2015 and 2016. The islands exhibit typical tropical climate with average temperature of 18-31°C and annual rainfall of 3,100 mm distributed over May to December. Further, the relative humidity ranges between 60-90% in a year, while the average lies near to 70-80%. For layering, healthy shoots of 25-30 cm length and 1.0-1.5 cm thickness were selected. Leaves and small branches near the ringing area on selected shoots were removed and two circular cuts were given to the shoots for removing a ring of bark of about an inch width. Commercial formulation of rooting hormone (Lipsa, Kolkata) was used for root induction and ringed portion was covered with soil: farmyard manure (1:1) before wrapping with polythene (20 cm × 20 cm). Experiment consisted of six treatments i.e. T 1 : layering on July 3, T 2 : layering on July 23, T 3 : layering on August 12, T 4 : layering on September 1, T 5 : layering on September 21 and T 6 : layering on October 11. Experiment was laid out in completely randomized design with 20 layers in each treatment. Various parameters were recorded at the time of separation (90 days after layering) and data was subjected to analysis of variance using Web Agri Statistical Package 2.0 (WASP 2.0, ICAR-RC for Goa, Ela, India). Air layering was performed for six times during rainy season under island condition. Generally, rooting process is facilitated by the rains (Ranaware et al. 1995) and hence, the dry periods in the islands were avoided during the experimentation. Pooled analysis of two years data revealed that percentage root induction varied between 35.0% and 87.5% amongst the treatments studied (Table 1). Maximum rooting percentage was obtained in layers done on 3 rd July (87.5%) followed by those done on 23 rd July, while it was the lowest in layering performed on 21 st September. Though rooting response varied considerably during different seasons, the mean number of primary roots per layer did not vary significantly (Table 1). Length of longest root varied significantly amongst the studied treatments (Table 1). Maximum length of primary root was observed in propagules obtained from cinnamon layered on 1 st September (8.5 cm), which remained on par with layers of 23 rd July. Similar to rooting percentage, length of root was found to be the lowest (3.6 cm) in layers of 21 st September batch. Thickness of root ranged from 2.04 to 2.74 mm; however, the differences were non-significant amongst the treatments studied. Waman & Bohra Similar to present communication, significant variations for rooting response and growth parameters have been reported from different agro-ecological regions of the country viz., West Bengal (Banerjee et al. 1982), Karnataka (Hegde et al. 1989) and Maharashtra (Ranaware et al. 1995). These reports have suggested the positive effects of rainy period on layering success; however, the raining season and pattern vary significantly in different regions and hence, location specific studies are required. As no reports are available for island conditions, present study was conducted. Cinnamon propagation technique Fennel (Foeniculum vulguare mill.) is a highly cross pollinated and very important seed spice crop exhibiting 82.2% to 91.4% natural out crossing (Ramanujam et al. 1964). The hybridization of diverse genotypes followed by selection in segregating crop will be helpful in identifying heterozygous and heterogenous progeny. Insects plays an important role in maintaining heterogeneity in fennel crop. The existing varieties were developed using mass selection. Fennel seeds are having medicinal importance as carminative, cardiotonic, stimulant, vermicide and lactagogue (Lal 2014 environments and over the years for high yield and other yield attributing and quality traits. Phenotypically stable genotypes are of great importance because environmental conditions vary from season to season. Wider adaptation to a particular environment and consistent performance of recommended genotypes is one of the main objectives in breeding programme. A differential response of fennel genotypes when grown under different environments in the Rabi season has been reported by few scientists (Lal 2014;Drazic et al. 2014). Hence information on availability of stable high yielding varieties of Fennel is lacking. Keeping this in view, the present investigation was undertaken to determine the genotype x environment (year) interaction on stability parameters and to identify the stable and responsive genotypes for yield and yield contributing characters of fennel for Chhattisgarh. The inter-and intrarow spacing was kept at 45 cm and 20 cm, respectively. The recommended package of cultural practices was followed to raise a good crop. In each plot, five competitive plants were identified randomly for recording data on days to 50% flowering, plant height (cm), primary branches plant -1 , umbels plant -1 , umblets umbel -1 and seed yield plot -1 (kg). The data recorded during three years were subjected to stability analysis according to the model proposed by Eberhart & Russel (1966) and three stability parameters mean (m), regression coefficient (bi) and the deviation from linearity (S 2 di) were estimated. (Table 2). For days to flower, umbelets /umbel and umbels plant -1 FNL 69 had recorded wider stability (above average mean, bi = 1 and S 2 di = 0). The genotype FNL 70 was stable for plant height, primary branches plant -1 , umbelets umbel -1 and umbels plant -1 but had low seed yield, indicating its adaptation to stress environments (Lal 2008) while genotype FNL 71 recorded stability for plant height and umbels plant -1 . For umbels umbel -1 FNL 72 had high stability across the environments of Raigarh. The genotypes FNL 69, FNL 70 and FNL 71 were among the top entries which had their mean umbels plant -1 greater than the average of all the genotypes with regression coefficient (bi =1) and non-significant deviation for regression (S 2 di =0). This indicated their high stability over the different environments of years (Lal 2008). The genotype FNL 68 and RF 101 had above average mean for umbels plant -1 , S 2 di = 0 but the value of bi #1, indicating their adaptation to high input conditions. For umbels plant -1 genotype RF 205 and FNL 67 had bi = 1 and S 2 di = 0, but their mean was low, indicating their adaptation to stress environments (Table 3). The genotypic differences were found to be highly significant for all the traits in each environment (year). The mean genotypic values from different year were subjected to pooled analysis. The mean sum of squares (MSS) due to genotypes (G) and environments (E) were significant for all the traits except for primary branches plant -1 when tested against MSS due to genotype x environments. It revealed the non significant differential response of the varieties to the changing environments. The results were in close conformity to the findings of Lal (2008), Verma & Solanki (2015). The MSS due to G × E when tested against pooled error, were found highly significant for all the characters. Thus stability analysis was carried out for all the traits. The variance due to G × E were divided in to G × E (Linear) and due to pooled deviation (Non-linear). The G × E (Linear) mean squares were found significant for all the traits except primary branches plant -1 indicating the presence of predictable components where as significance of pooled deviation for seed yield plot -1 , days to 50% flowering, plant height (cm), primary branches plant -1 and umbels plant -1 showed the presence of non-predictable components. These observations indicated that some reliable predictions about G × E interaction as well as its unpredictable components can be made for these traits. Hence, both components contributed significantly in determining the stability of genotypes (Lal 2008;Verma & It was observed that national check (RF 205) showed wider stability for seed yield, days to flower, primary branches plant -1 , umbels plant -1 and umbelets umbel -1 while local fennel had wider stability for seed yield (kg ha -1 ), plant height, umbels plant -1 and umbelets umbel -1 . While none of the test entries showed superior stable performance over the national and local checks during all the three years. This appears to be evidence for much greater genotype × environment interaction for the entries to be evaluated than for the pure lines. This is somewhat unexpected in view of the greater homeostasis in unfavorable environments usually found in heterozygous genotypes. This needs further study. (125), highest yield plant -1 (1052.5g), yield ha -1 (337.63q) and premier fruit quality score (9.11) with maximum net return (Rs.326407.28) and benefit: cost ratio (3.41) was also reported in same treatment. Comparatively minimum time (15 hours) required for one hectare irrigation was also reported in drip irrigation on raise bed with plastic mulch. This led to lower population of white fly plant -1 (4.53), minimum weed infestation (1.53 weed m -2 ), leaf curl (5.50%) and fruit rot (5.0%) incidence than other treatment combinations. The minimum growth, yield and profitability were reported in check basin method of irrigation without mulch (T 1 treatment). to mulch includes early production, more yield and reduced insect and disease problems (Pattanaik et al. 2003). Linear Low Density Poly Ethylene (LLDPE) plastic films have been proved as better mulch because of their puncture resistance quality, thinness and lower cost (Panda 2004). Numerous experiments have reported the benefits of LLDPE mulch in several crops, but research is limited on response of chilli production in western Rajasthan by this method. Keeping this in background, the present study was undertaken to study the effect of different irrigation methods and mulching on chilli crop and compare the result with the conventional method of growing the crop under surface irrigation without mulch. Keywords A field experiment was conducted at Agricultural Research Station, Mandor, Jodhpur (Rajasthan), India during kharif seasons of year 2016-17. The soil of experimental plot was of sandy loam texture with average pH range 8.5, having organic carbon 0.55%, available N 180 kg ha -1 , P 27.5 kg ha -1 and K 250.0 kg ha -1 during experimentation. The experiment was conducted in a completely randomized design having nine treatments comprising by different irrigation methods and mulching viz., T 1 = Check basin method, T 2 = furrow irrigation method, T 3 = Raise bed with trench method, T 4 = Flat bed with drip irrigation, T 5 = Flat bed + plastic mulch + drip irrigation, T 6 = Raise bed with drip irrigation, T 7 = Raise bed + organic mulch + drip irrigation, T 8 = Raise bed + plastic mulch + drip irrigation, T 9 = Sprinkler irrigation method. In well prepared field, transplanting of Chilli seedlings variety RCh 1 of 35-40 days old were planted in pair row method with a spacing of 45cm x 45 cm/90 cm (33,333 plant ha -1 ) during last week of June. In check basin and sprinkler system of irrigation the bed size is 2 x 2 meter and in all other methods is 1 × 4 meters. The cultural practices of the crop were followed as per the recommendations. The organic material and LLDPE silver colour film of 100-micron thickness was used for mulching around the plant. The lateral lines of 12 mm diameter LLDPE pipes were laid along with crop rows. The laterals were provided with inlet drippers of 8 litre hr -1 discharge capacity. All the observations were taken from five randomly selected plant of each replication throughout the investigation period at appropriate time by adopting standard method for growth, development, fruiting behavior and yield. Seedling survival per cent (after transplanting in main field at 15 DAT and 30 DAT) was recorded by following formula; Survival percent = [Total survival transplanted plants / Total transplanted plants] × 100 Plant height (at 45 DAT and at harvesting) was measured from soil surface upto the highest shoot tip by straightening all branches. Stem girth was measured 1 cm from the base of the stem using vernier calliper. Observation of number of branches, days taken to first flower initiation, duration of fruiting period and number of fruit plant -1 was recorded by standard counting method. Number of roots, root length was measured by destructive method of uprooting the plants and taking measurement by standard method. Length of fresh fruits measured by scale and fruit diameter using vernier calliper and expressed in centimeter. Fruit set per cent was recorded by following formula; Fruit set per cent = [Total number of fruit set plant -1 / Total number of flowers plant -1 ] × 100 Fruit weight was determined by weighing method at the time of harvesting and expressed in gram fruit -1 . The total fruit yield plant -1 and hectare -1 was calculated by weighing total marketable fruits and has been expressed in gram and quiantal respectively. Further, the net return was calculated by subtracting cost of each treatment from gross return. The gross return was calculated from yield multiplied by average market rate during the period of investigation. The benefit cost ratio was calculated by dividing net return to total cost of cultivation. Benefit-Cost ratio and net profit were carried out to determine the economic feasibility of the crop using surface and drip irrigation as suggested by Tiwari et al. (1998a). The seasonal system cost of drip irrigation system included depreciation, prevailing bank interest rate, and repair and maintenance cost of the system. The fixed cost of drip irrigation system was determined to be Rs 112,000 ha -1 . The useful life of drip system was considered to be 10 years. The system cost was evaluated by distributing the fixed cost of system over life period of drip irrigation set. For calculating depreciation, the life of the drip irrigation set and 10% junk value was considered. The interest was calculated on the average of investment of the drip irrigation set taking into consideration the value of the set in the first and last year @10% per annum. Cost of repairs and maintenance of set is @2% of initial cost of the drip irrigation set per year. The cost of cultivation includes expenses incurred in land preparation, interculture operation, fertilizer, crop protection measures, irrigation water and harvesting with labour charges. Therefore, total seasonal cost was worked as: depreciation, interest, repairs and maintenance cost of set + cost of cultivation + cost of mulch. The income from produce was calculated using prevailing average market price of capsicum @ Rs 1250 q -1 . Disease incidence (leaf curl and fruit rot) and quality of fruits was measured by visual inspection (Five member team of crop experts and plant pathologist). White fly population plant -1 and weed infestation meter -2 was calculate by simple counting method. The time required for irrigation was calculated as per actual required time of irrigation of specified area by different methods of irrigation. To test the significance of variance of data obtained from crop growth, yield and economics of variance technique for completely randomized design was done by standard procedure prescribed by Panse & Sukhatme (1985). Significance of difference among the treatments effect was tested by 'F' test and critical difference (CD) was calculated, wherever the results were significant. The results revealed that, the irrigation methods and mulching are significantly influenced growth attributes at all the growth stages ( Table 1). The maximum seedling survival per cent at 15 DAT (95.10%) and 30 DAT (91.70%) was recorded in T 8 treatment, which was significantly superior to other treatment but at par with T 6, and T 7 treatments. The maximum survival per cent of seedling in T 8 treatment might be due to more favourable moisture condition for seedling transplanting and reestablishment of roots than others. The height of plant under treatment T8 (47.10 cm at 45 DAT) and treatment T6 (62.60 cm at harvest) was found highest among all other treatments and is 67.19% and 13.40% higher than the T 1 treatment. About to number of branch plant -1 , maximum value was recorded in treatment T8 (14.93) followed by treatment T7 (12.53) and the lowest value was in treatment T1 (7.17). Maximum stem girth at harvest (2.36 cm) and highest number of roots plant -1 (138.50) were observed in T 8 treatment whereas longest root system (10.50 cm) was observed in T 3 treatment. The minimum stem girth (1.68 cm) and the number of roots plant -1 (53.57) were observed in T 1 treatment whereas shortest root system (7.97 cm) was observed in T 9 treatment. The higher available moisture status in soil favourably influences the uptake of nutrients which maintains the cell turgidity, cell elongation, photosynthesis and respiration at optimum level, leading to favourable growth and development of plant in terms of plant height, number of branches plant -1 , stem girth and number of root plant -1 in the present study. The highest increase in vegetative growth in drip irrigation with mulching might be due to the availability of soil moisture as well as favourable temperature at optimum level for plant growth development (Pattanaik et al. 2003;Paul et. al. 2013). The lowest value of vegetative growth in T1 might be because of unfavourable moisture regime (moisture stress or excess moisture) in the soil through surface irrigation and competition of weeds for nutrients (Pattanaik et al. 2003;Agrawal & Agrawal 2005). The increased growth attributes might have supplied water and nutrients in adequate proportion, which resulted in triggering the production of plant growth hormone, viz., indole acetic acid (IAA) and higher number of leaves and roots throughout the cropping period (Sankar et al. 2008). The drip irrigation in combination with mulch significantly increased the yield of chilli as compared to drip irrigation without mulch (Table 2) and surface irrigation methods. The minimum days (42.38) required for first flower initiation was reported in T 9 treatment whereas the maximum days (51.39) was required in T 5 treatment. Among various treatments, highest fruit set (38.47%), length of fresh fruit (12.56 cm), diameter of fruit (3.52 cm), duration of fruiting (71.38 days), fresh weight of fruit -1 (8.42 g), maximum number of fruits plant -1 (125), highest yield plant -1 (1052.5 g) and yield ha -1 (337.63 q) was recorded under T 8 treatment, whereas lowest yield (153.45 q ha -1 ) was recorded under T 1 treatment. This might be due to water stress during the critical growth period and fruit development stage coupled with aeration problem in first few days immediately after irrigation. Another reason to get low yield by surface irrigation without mulch might be due to less availability of nutrients for crop growth due to leaching and high weed competition between the crops (Pattanaik et al. 2003). In drip irrigation system on raise bed with plastic mulch the water is applied at a low rate for a longer period at frequent intervals near the plant root zone through lower pressure delivery system, which increases the availability of nutrients near the root zone with a reduction in leaching losses and minimum weed competition. More nutrient availability, especially near the root zone might have increased the translocation of photosynthetes to storage organ of chilli resulting in an increased weight of fruits. This result corroborated the findings of Singh (2007), Sankar et al. (2008), Paul et al. (2013) and Kumar et al. (2016). Irrigation methods and mulching also significantly influenced the gross return, net return and benefit cost ratio in chill (Table 3). Maximum net profit of Rs. 326407.28 ha -1 with B: C ratio of 3.41 was recorded in T 8 treatment followed by Rs 296192.61 ha -1 with B: C ratio of 3.11 in T 5 treatment and lowest net profit of Rs 119007.80 ha -1 with a B: C ratio of 1.63 in T 1 treatment (Table 3). It is observed that, the drip 85 irrigation with mulched treatments T 5 , T 7 and T 8 gave better net return with higher B: C ratio ha -1 than their corresponding treatments without mulching in conventional irrigation method. The highest net return (US$ 7098 ha -1 ), incremental net return (US$ 1556 ha -1 ), and incremental benefit-cost ratio (7.03) were found for 50% water application with straw mulch (Biswas et al. 2015). The results are in conformity with the findings of Singh (2007), Sankar et al. (2008) and Kumar et al. (2016). Apart from reducing water consumption, drip irrigation with mulching also helps in reducing cost of cultivation and improving productivity of crops as compared to the same crops cultivated under flood method of irrigation (Paul et al. 2013). Irrigation time significantly pretentious by different irrigation methods. The minimum time required in irrigation (14.50 hours) in T 5 treatment which closely followed by T 8 treatment. Drip irrigation method with or without mulching required less irrigation time than without mulching in conventional irrigation method. There was significant effect of LLDPE mulch over drip irrigation system alone. Drip irrigation with LLDPE mulching (T 5 & T 8 treatment) saving irrigation time (21.40 hour ha -1 and 21.35 hour ha -1 ) upto 60 per cent by reducing water losses and increased irrigation efficiency. The increase in water saving per cent in trench method (T 2 ), drip irrigation system alone (T 4 ), drip irrigation system with LLDPE mulch (T 5 & T 8 ), drip irrigation with organic mulch (T 7 ) and sprinkler system (T 9 ) over conventional surface irrigation by check basin method (T 1 ) was 38.8%, 46.6%, 60.0%, 54.4% and 41.9% respectively. The highest water use efficiency of 592 kg ha -1 mm -1 was obtained with 50 per cent water application under polyethylene mulch (Biswas et al. 2015). Drip irrigation with mulching helps to achieve yield gains of upto 100 per cent, water savings of upto 40-80 per cent, and associated fertilizer, pesticide, and labour savings over conventional irrigation systems in capsicum crop (Paul et al. 2013). Similar trend has been reported in water use efficiency for okra crop by Tiwari et al. (1998a) and for tomato crop by Singh (2007). Occurrence of chilli leaf curling and fruit rot was detected throughout the investigation period. The best performance, with a marked reduction in leaf curling and fruit rot and improve fruit quality was observed in drip irrigation with LLDPE mulching (Table 3). The minimum incidence of fruit rot (5.0%), leaf curing (5.5%) and highest quality score of fruit (9.11) was observed in T 8 treatment which was closely followed by T 7 and T 5 treatments where as maximum incidence of fruit rot (20.02%) and leaf curling (15.84%) was reported in check basin method of irrigation (T 1 treatment) whereas minimum quality score (6.50) was observed in T 9 treatment. Presence of white fly and weed was observed throughout the investigation period. The minimum population of white flies (4.53 plant -1 ) and weed infestation (1.53 weed m -2 ) was observed in T 8 treatment whereas highest incidence of white flies (17.43 plant -1 ) and weed infestation (30.03 weed m -2 ) was observed in T 1 treatment. This is due to the fact that in drip irrigation with mulching significantly reduced additional moisture level in field environment which in turn increase quality of fruits and reduce disease infestation, white fly population as well as it also trim down weed seed germination, growth and development. The increase in quality of fruits was due to the effective utilization of applied nutrients, water and significantly reduced weed growth; disease incidence and increased rate of photosynthesis, sink capacity and accumulated more amounts of dry matter and finally increased quality of fruits and yield. Conventional surface irrigation methods without mulching provide favorable environmental condition for increase insect population and development of disease as well as germinate and develop high density weed plants. The beneficial effect of drip irrigation and black LLDPE mulch in capsicum, tomato and okra was also reported earlier by Horo et al. (2003); Singh (2007) The combination of raised bed + drip irrigation system with LLDPE mulching is observed to be economical and cost effective as compared with conventional surface irrigation without mulching. Thus, the use of drip irrigation system either alone or in combination with mulching, could increase the chilli yield quality of fruits and profitability. It also reduces white fly population, disease incidence (root rot and leaf curling) and minimise with crop weed competition. Drip irrigation with mulching increase water use efficiency by significant reduction in irrigation time ha -1 . It is concluded that the drip irrigation method with LLDPE mulching is suitable for chilli production in arid and semi arid condition of western Rajasthan. Swelling factor (cc g -1 ) 11.4 9.1 Patel et al. GI-4 recorded 89.67% less shattering than that of check variety, which revealed that Gujarat Isabgol 4 was non shattering in habit as compared to Gujarat Isabgol 3 which is prone to high seed shattering. The post dipped seed weight of GI 4, was 0.115 kg threshed seeds, which was 96% higher than that of the post dipped seed yield of GI-3 (0.037 kg threshed seeds) (Table 4). Hence, Gujarat Isabgol 4 recommended for cultivation.

v3-fos

2018-04-03T04:17:20.914Z

1970-03-01T00:00:00.000Z

38340690

s2

Effect of Temperature on Radiosensitivity of Newcastle Disease Virus' Newcastle disease virus was irradiated at temperatures ranging from 2.2 to 60 C. An interaction between the thermal and ionizing energy was observed in the temperature region of 49 to 60 C. At 2.2 C, the hemagglutinin was considerably more radioresistant than the infectivity property. It is believed that radiation inactivation of Newcastle disease virus infectivity at low temperatures was due to nucleic acid degradation and at higher temperatures was due to protein denaturation. The effect of ionizing radiation on viral inactivation has been studied since as early as 1939 (5). The subject matter has been discussed in great detail by Lea (8) in his classic book, Action of Radiations on Living Cells, in which the target theory was developed. Research in this area has continued, but the main emphasis of this research has been on the use of ionizing radiation as a tool to study molecular organization of viruses, particularly in relation to biological function, or particle size, or for preparing vaccines. Little consideration has been given to radiation as a means of decontaminating virus-infected foods because of the relatively high sterilizing dose required (7) which results in undesirable organoleptic changes. Thus, any treatment that might sensitize virus to radiation warrants an investigation. It was reported that the survivors of X-irradiated T5 bacteriophage are more sensitive to heat (2). Adams and Pollard (1) similarly found that irradiation of Ti bacteriophage particles sensitized them to heat, but of more importance was the finding that a greater degree of inactivation occurred when the irradiation and heating were carried out simultaneously rather than sequentially. The purpose of the present study was to determine whether there is an effect of temperature on radiosensitivity of an animal virus, since the previous work had been confined to bacterial viruses. MATERIALS AND METHODS Test virus. The virus used was Newcastle disease virus (NDV). Allantoic fluids harvested 48 to 72 hr after infection of 10-day-old chicken embryos were pooled, centrifuged at 8,500 rev/min for 20 min, and stored at -40 C in 15-ml portions until used. Irradiation of samples. The irradiation was carried out with gamma rays from a Mark I United States Atomic Energy Commission Cobalt-60 Food Irradiator. Under the conditions of the experiment, the dose rate, as determined by ferrous-ferric dosimetry, was approximately 4,850 rad/min. Three-milliliter quantities of the infective allantoic fluid were filled into glass tubes (8 by 150 mm). For temperature control during irradiation, the tubes were placed in a stainless-steel jacketed vessel containing water tempered to the desired temperature. Constant temperature was maintained by circulating properly tempered water through the jacket. The entire apparatus was contained within an insulated steel chamber which was lowered into the gamma ray field for the prescribed time. The assay method for hemagglutinin and infectivity was reported in the preceding publication (3). RESULTS AND DISCUSSION In Fig. 1 are presented the curves for inactivation of NDV infectivity by irradiation at various temperatures ranging from 2.2 to 60 C (36 to 140 F). The logarithm of the survival fraction (V/Vo) has been plotted as a function of the irradiation dose in kilorads. It is of interest to note that for irradiation at the low temperature the inactivation curve was linear, whereas the inactivation curves for irradiation at the higher temperatures were nonlinear, consisting of a fast-inactivating component followed by a slower-inactivating component. This same pattern characterized the thermal inactivation curves for NDV infectivity (3). This result may indicate that the inactivation of NDV infectivity by simultaneous irradiation and heating was due principally to the thermal effects with the irradiation augmenting the effect of heat. The inactivation rate during DiGIOIA ET AL. irradiation at lethal temperatures is the integral sum of three different components, that is, the effect of irradiation, the effect of heat, and the effect of an interaction between irradiation and heat if one is present. On the assumption that these three effects are additive, the interaction between heat and irradiation in the inactivation of NDV infectivity was determined by subtracting the slopes of the heat inactivation curves (3) from the slopes of the radiation inactivation curves for the same temperature. The radiation inactivation rates thus corrected for heating effect were plotted as a function of irradiation temperature (Fig. 2). Since both the thermal inactivation curves and the irradiation-heating curves were two-component types, two curves are shown in Fig. 2, one representing the primary slopes (fast-inactivating component) and the other reresenting the secondary slopes (slower-inactivating component). In both cases, it was indicated that there was no effect of irradiation temperature on inactivation of NDV infectivity over the temperature region of 2.2 to 49 C, but at temperatures greater than 49 C there was a marked increase in radiosensitivity. The sharp increase in radiosensitivity that occurred in the temperature region of 40 to 50 C has also been reported for Ti bacteriophage (1) and Salmonella typhimurium (9). The following theory has been postulated for the synergism between thermal and ionizing energy. The loss of biological function of some macromolecule, such as protein, by thermal denaturation requires the rupture of at least three adjacent bonds, such as hydrogen bonds, which causes the molecule to open up and lose its biological configuration. Rupture of one of these bonds by irradiation would lessen the requirement for the number of bonds to be broken by the thermal energy (1). One may take an alternative approach to this problem and correct the irradiation-heating curves for the component effect caused by irradiation. This was done in the present study, and again the interaction effect was indicated at temperatures greater than 49 C. In 1953, Epstein (4) proposed that the radiosensitive matter of virus is the nucleic acid and this has since been confirmed. However, Wilson and Pollard (11), by using a technique of charged particle bombardment with carefully controlled penetration, deduced that the radiosensitive volume for NDV is about 20 % of the whole virus. Since the total ribonucleic acid (RNA) content of the virus (about 5%) would be expected to occupy less volume than this, they proposed that some of the radiosensitive material includes protein. In the present study, it was found that an irradiation dose of 1.25 X 106 rad at 2.2 C had no effect on the hemagglutinating property of NDV. Yet an irradiation dose of this magnitude at a temperature of 2.2 C affected a 7.5 logi0 reduction in infectivity of the virus. Thus, at low temperatures of irradiation, the hemagglutinin of NDV was more radioresistant than the infectivity property. Therefore, it can be concluded that inactivation was due principally to damage to the RNA. This finding has also been reported for other animal viruses (6,10,12). However, it is believed that loss of NDV infectivity by irradiation at lethal temperatures was mainly due to protein denaturation, although some RNA degradation may have also occurred. This supposition is predicated on the result that, when an Arrhenius plot of the radiation inactivation rate constants was made, a twocomponent curve was obtained. The break in the curve occurred at a temperature between 37. 8

v3-fos

2020-08-27T09:04:47.060Z

1970-01-01T00:00:00.000Z

228589950

s2

In Vitro Test of Antibacterial Ethanol Extract, n-Hexane Fraction and Ethyl acetate Fraction of Sungkai Leaf (Peronema cenescens) Against Salmonella typhi Methods: Study included phytochemical screening and in vitro antibacterial testing of ethanol extract, n-hexane fraction and ethyl acetate of Sungkai leaves against Salmonella typhi. Results: obtained groups of chemical compounds alkaloids, flavonoids, glycosides, anthraquinones, tannins and triterpenoids/steroids on Sungkai leaf powder. Ethanol extract of Sungkai leaves obtained resistance at a concentration of 20% by 12.7 mm, and inhibition of the ethyl acetate fraction at a concentration of 20% of 14.8 mm. Conclusion: Ethyl acetate fraction of Sungkai leaves have antibacterial properties against S. typhi which is greater than ethanol extract and hexane fraction of leaf heal. INTRODUCTION ccording to WHO, typhoid fever is a global problem. The main etiologies in Indonesia are Salmonella enterica subspecies enteric serovartyphi (S.typhi) and Salmonella enterica subspecies enteric serovar paratyphi enterica. In the past two decades, there have been reports of multidrug-resistant (MDR) strains of S. typhi. This strain is resistant to chloramphenicol, trimethoprim-sulfamethoxazole, and ampicillin. Besides strains are resistant to nalidixic acid, also showing a reduced effect of ciprofloxacin which is endemic in India 1 . Indonesian people have known and used plants to treat various infectious diseases caused by microbes. This is due to public awareness of the side effects of synthetic drugs that are greater than traditional medicines as well as the government's efforts to find new drugs to prevent resistance to infectious diseases caused by microbes 2 . Sungkai (Peronemacanescens) is often referred to as teak sabrang, belonging to the Verbenaceae family. Some people in South Sumatra and Lampung use sungkai leaves as antiplasmodial and fever medicine. In the treatment of the Lembak tribe, steeping sungkai leaves are used to reduce heat, malaria and maintain health 3 . Preparation of Simplicia ofSungkai leaves were obtained from the Langkat area of North Sumatra. Identification has been carried out at Medanense Herbarium (MEDA) University of Sumatra Utara, Medan. Phytochemical Screening Screening is carried out on a simplicia powder using a screening protocol for identification of secondary metabolites for plants including examination of alkaloids, flavonoids, glycosides, anthraquinone glycosides, saponins, tannins and triterpenes/steroids 4-6 -7 . Preparation of n-hexane and ethyl acetate fractions. As much as 40 g of ethanol extract were added 40 ml of ethanol and 100 ml of distilled water were homogenized then added 50 ml of n-hexane, shaken, allowed to stand until two layers were formed, the n-hexane fraction and the water fraction. The n-hexane fraction is collected and fractionation is carried out until the n-hexane layer is clear. The fraction of water was then added 50 ml of ethyl acetate, shaken, allowed to stand until two layers formed, the ethyl acetate fraction and the water fraction. The ethyl acetate fraction was collected and fractionation was carried out until the ethyl acetate layer was clear. The fraction of n-hexane and ethyl acetate in the rotary evaporator 5 . In Vitro Antibacterial Test Pipette 0.1 ml of suspension Salmonella typhi with a concentration of 10 6 CFU / ml, was put into a sterile petri dish. Then 20 ml of Nutrient Agar (NA) medium is poured, then homogenized and allowed to stand until the media solidifies. After the solid media is then made a hole using a hole (punch hole) and then drops 0.1 ml of ethanol extract test solution with a concentration of 5%, 10%, 15% and 20%, then incubated at a temperature of 35+ 2 o C for 18-24 hours. Furthermore, the diameter of the inhibition zone is measured using a calliper. Tests carried out three times. Tests were also performed on the n-hexane fraction, sungkai leaf ethyl acetate, chloramphenicol as a positive control, and DMSO as a negative control. Phytochemical Screening for Sungkai leaf simplicia powder added Molish reagents and concentrated sulfuric acid formed a purple ring at the liquid level indicating glycosides. The addition of Mg powder concentrated hydrochloric acid and amyl alcohol, then allowed to separate gives a yellow colour indicating the presence of flavonoid compounds. The addition of Lieberman-Bourchard reagents gives a purple-red colour indicating the presence of triterpenoids/steroids on sungkai leaves. The results of phytochemical screening for simplex powder are shown in Table 1.These results are per previous studies, in which the phytochemical examination of sungkai leaves did not identify the saponin metabolites in simplicia 10 . Remarks The ethyl acetate fraction of sungkai leaves gave a yield of 14.8 mm against bacteria Salmonella typhi with inhibition zone boundaries that were considered effective according to the Indonesian Pharmacopoeia ie inhibitory diameters between 14 mm to 16 mm, and when compared with positive control using chloramphenicol the ethyl acetate inhibition zone was almost approaching the amount of inhibition zone of chloramphenicol. The results of the measurement of effective inhibition area diameter in the ethyl acetate fraction of sungkai leaves (EFSL) were obtained at a concentration of 20%. The comparison diagram of the inhibition zones of each extract and fraction can be seen in Figure 1. In previous studies showing the results of sungkai leaf extract, ethyl acetate fraction, methanol fraction had a minimum inhibitory zone and a minimum inhibitory concentration against S. aureus respectively 1024 μg/ml and 512 μg/ml, whereas against E. coli, the extract and fraction had zones inhibitors and a minimum concentration of 512 μg/ml 11 . This is due to the presence of compounds that are attracted to semi-polar solvents, such as flavonoids and tannins. The group of phytochemical compounds that are commonly associated with combating microbial resistance and having antimicrobial activity in medicinal plants are flavonoids, alkaloids, tannins, triterpenoids, essential oils, saponins, glycosides, and phenols 8 . At present, many pathogenic bacteria become resistant to various types of antibiotics that are commonly used and cause various diseases. Therefore, the search for new drugs is very important in the last decade and some plants have been used as medicine 9 . Polyphenolic compounds constitute the largest group in plants, one of which is tannins which have antibacterial activity. In general, the mechanism predicted is that the toxicity of the polyphenol compound can damage the bacterial cell membrane. The use of DMSO negative control aims to ensure that the inhibition zones that are formed are not the influence of DMSO solvents, but the kill zones that are formed purely from the active compounds contained in the ethanol extract and ethyl acetate fraction of Sungkai leaves.

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2018-04-03T04:36:20.179Z

1970-07-01T00:00:00.000Z

39585191

s2

Survey of the sensitivity of microorganisms to rubratoxin B. Of the 133 microorganisms tested, Tetrahymena pyriformis and Volvox aureus were the most sensitive to rubratoxin B, being inhibited at 25 and 50 mug/ml, respectively. tively. Virtually no information on the antimicrobial activity of rubratoxin B, a toxic secondary metabolite of Penicillium rubrum Stoll, is available. Wilson and Wilson (4) reported inhibition upon Bacillus subtilis, B. cereus, and Staphylococcus aureus by 30 mg of an acidic fraction containing this mycotoxin. Lightly seeded Chlorella pyrenoidosa-buffered agar plates inoculated with P. rubrum NRRL A-11785 produced a zone of inhibition preceding the mycelial growth (3). This paper deals with the antimicrobial activity of rubratoxin B. Rubratoxin B was produced by P. rubrum NRRL A-11785 by the method of Hayes and Wilson (2). All test strains were obtained from our culture collection or from the Southern Research Institute, Birmingham, Ala. The agar streak method with filter paper disc (Schleicher and Schuell, no. 740-E) containing 100 to 3,000 gg of rubratoxin B per disc was employed for determination of antimicrobial activity against bacteria, fungi, and algae. Control cultures with discs containing equivalent volumes of solvent (acetone or dimethyl sulfoxide) were run simultaneously. When a positive response with a bacterium was observed on agar plates, it then was tested turbidimetrically in appropriate liquid media. Inhibition of Tetrahymena pyriformis and Volvox aureus induced by rubratoxin added at time zero to liquid cultures was determined after 24, 48, and 72 hr by counting cells under low-field magnification. Rubratoxin B had no effect at 100 to 1,000 ,Ag on the algae, fungi, or gram-negative bacteria investigated. However, there was a decrease in pigment in Pseudomonas fluorescens growth adjacent to the disc containing rubratoxin (1,000 Mug). There was no difference in cell density when measured turbidimetrically in concentrations as high as 3,000 Mug of toxin per ml, but there was a marked decrease in pigment in rubratoxin- fluorescens. Four species of Bacillus including B. subtilis, two species of Micrococcus, and an S. aureus were inhibited by 1,000 Mg of rubratoxin B per disc (Table 1). Colonial characteristics of the Bacillus species were markedly changed on nutrient-agar plates (1,000 Mug). The colonial growth was thin, spreading, and mycoides-like, whereas solvent-control colonies were thicker and softer in appearance. The inhibitory effect of rubratoxin on M. lysodeikticus growth was noted in broth cultures after 18 to 24 hr. This particular organism requires 48 hr to reach the stationary growth phase. No change in growth of S. aureus was observed for 6 hr when tested against 2,000 Mg of toxin per ml in Trypticase Soy broth; however, by the 6th hr, the S. aureus growing in the presence of rubratoxin was very flocculant, and turbidity could not be determined. Growth of T. pyriformis and V. aureus was inhibited by less than 25 and 50 ,g of rubratoxin B per ml, respectively (Table 1). Some mycotoxins, such as aflatoxin, appear to be toxic to a range of biological systems including animals, plants, and microbial systems, whereas others are more restricted in action. Rubratoxin B appears to be toxic to a variety of 164 on March 18, 2020 by guest http://aem.asm.org/ Downloaded from VOL. 20, 1970 NOTES 165 higher animals (1, 4) but limited in its toxicity caused by eating moldy corn. II. Experimental production to microorganisms.

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2020-12-10T09:04:10.868Z

1970-11-01T00:00:00.000Z

237232100

s2

Heat Resistance of Xerophilic Fungi Based on Microscopical Assessment of Spore Survival An improved viable counting technique was developed to facilitate study of the heat resistance of fungal spores. Spores were heated and subsequently incubated in the same medium. After germination, hyphae and germ tubes were stained with lactofuchsin, and the germinated spores were counted with the aid of a microscope. A number of xerophilic strains were examined, mostly isolates from spoiled highmoisture prunes. Of these, ascospores of Aspergillus chevalieri, A. mangini, and Xeromyces bisporus were the most heat-resistant. A decimal reduction curve obtained for A. chevalieri was specified by a z value of 23 F and an F180 of 2.2 min. To establish a thermal process for the commercial sterilization of high-moisture prunes packed in plastic pouches, information was required on the heat resistance of a group of xerophilic fungi whose isolation and water relations were described previously (11). There are no published data on the heat resistance of these fungi, with the exception of Xeromyces bisporus Fraser (3). The difficulty of obtaining accurate viable counts of fungal spores has led to continued use of thermal death time (TDT) measurements in which only the heating time necessary to destroy all viable cells is measured. This method is much less precise than that commonly used to study bacterial heat resistance, in which the decrease in viability with time is determined quantitatively at each temperature. Our initial attempts to perform viable counts on fungal spore suspensions were of limited accuracy, as mycelia from rapidly germinating spores obscured those more slowly germinating. The maximum number of mycelia that could be counted on a standard 10-cm petri dish was less than 100, and dilution experiments indicated inaccuracies at this level of up to 20%. For these reasons, a microscopical counting method with greatly increased accuracy was developed. MATERIALS AND MEITHODS Cultures. The fungi studied are listed below. The majority were isolated from prunes (11) Two dissimilar ascospore types were isolated in strains of A. echinulatus, one producing ascospores approximately 7 gr in long axis, the other 9 ,um in long axis. Both isolates were studied and are designated types S and L, respectively, in this report. Culture media. Cultures were grown on Czapek agar as modified by Smith (14), generally containing 20% (w/w) sucrose (pH 6.5). A. carnoyi required 40% sucrose; C. xerophilum, 40% sucrose at pH 4; and X. bisporus, 55% sucrose at pH 4 for optimum growth. Conidial production by members of the A. glaucus group was enhanced by media containing 0.5% peptone. Incubation was at 25 C, conidia requiring 2 to 4 weeks and ascospores 5 to 9 weeks to ensure maturation. Inocula. Spores were dispersed by rubbing conidial chains or cleistothecia in a drop of distilled water between twocover slips. Good dispersion was achieved but with some loss of viability, presumably as a result of mechanical cell damage. Inoculum concentrations were adjusted to give an estimated viable count of 04 cells per ml, which usually corresponded to a total count of about 1.7 X 104 spores per ml. Medium. So that results could be readily related to the destruction of fungi on prunes, the heating and counting medium used was a plum extract. Fresh d'Agen plums were cooked with one-third their weight of water at 70 C for 10 min, pulped, screened, treated with a pectolytic enzyme, and ifitered. The pH of the extract was adjusted to 3.8, which was the mean pH of several samples of Australian d'Agen prunes (11). Because xerophilic species were being studied, sucrose was added to produce a medium of 200 Brix. The water activity (a,) of this medium is approximately 0.98 (10). Before use, agar (2%7, w/v) 682 was added; the medium was refiltered and pasteurized by heating at 100 C for 30 min. Heating. Heating temperatures of 50, 60, 70, 75, and 80 C were used. Melted fresh plum extract medium was equilibrated to the desired temperature, and the spore inoculum was added in the proportion of 1 part of inoculum to 9 of medium, giving an initial viable count of about 103 spores per ml. At intervals, 1-ml samples were transferred from heating tubes to 5-cm petri dishes, spread by rocking, and allowed to solidify as a thin, even layer. Heating times were calculated to the moment of plating, and temperature changes during sampling were minimized by using pipettes heated to bath temperature. For initial viable counts, samples were plated in melted media at 45 C. Incubation. To minimize changes in the water content of the medium, petri dishes were incubated in desiccators in which the a. levels were controlled by saturated solutions of appropriate salts. The incubation period was critical: experimentation showed that the maximum viable count was obtained if incubation was terminated when the largest mycelia were 300 to 500,um in diameter. Longer incubation periods resulted in confluent growth, whereas shorter periods reduced the number of germinations. Optimum incubation periods for most species were 45 hr to 4 days and increased with increase in heat treatment. With C. xerophilum and X. bisporus, optimum incubation periods were 2 and 4 weeks, respectively. Except for these two species, incubation was concluded after 2 weeks if growth had not occurred. Viable counting. Growth was determinated by adding 1 ml of lactofuchsin [0.1%o acid fuchsin in lactic acid, (2)], which killed the mycelia and stained them bright red. Excess lactofuchsin was decanted from the plate after 10 min. Counts were made over the entire plate at 40 diameters magnification, by using as guidelines 2-mm squares ruled on transparent plastic under the plate. Ungerminated spores were not visible, but hyphae were readily seen if more than 15,um in length. RESULTS The microscopical counting method proved to be an accurate but tedious technique. As many as 1,200 mycelia could be counted on a single 5-cm petri dish without confluent growth causing serious inaccuracy. Reproducibility was very high. For 52 viable counts performed in duplicate, with means between 100 and 1,200 colonies, variation from the mean averaged i 3.2%. Comparative heat resistances. The heat resistances at various temperatures of the fungi under study were compared by counting survivors after heating for 10 min. The results are shown in Tables 1 and 2, all data being based on the means of duplicate counts. Of the 16 spore types tested, ascospores of A. chevalieri, A. mangini, and X. bisporus were most heat-resistant, some spores from each remaining viable after 10 min at 80 C. Marked heat activation was PI6T AND CHRISTIAN mic plot in Fig. 1. The data were collected from several experiments, with the age of the cultures varying from 5 to 10 weeks, which may account for some of the observed scatter. Survivor curves were constructed by a least-squares technique which constrained the curves to pass through the initial point (103 viable count, zero time). By using these figures, a decimal reduction time (DRT) curve was drawn (Fig. 2). From previous work (1,. 6, 18), this has been assumed to be a straight line. As recommended by Townsend, Esty, and Baselt (15), it has been drawn through the points indicating maximum heat resistance. This line is specified by a z value of 23 F and an Fu8o of 2.2 min. DISCUSSION The microscopical method of viable counting used here has advantages of accuracy and reproducibility when compared with conventional plate counting and provides more information than does the simpler TDT technique. Heating and viable counting were carried out at the same a, to reproduce as closely as possible the condi-0' 4. 4. APPL. MICROBIOL. I r CD tions encountered by fungal spores in heattreated prunes. However, by taking smaller samples from the heating suspension and mixing with media of other a, at the time of plating, the influence of a, on growth could be dissociated readily from its effect on heat resistance. This study has been confined to xerophilic fungi of the types likely to cause spoilage in inadequately heat-processed foods of relatively low a, and pH. Within this group, the ascospores generally have greater heat resistance than the asexual spores of the same species. Of those species producing asexual spores, only C. xerophilum showed some survival after 10 min at 70 C. Among the aspergilli, the greater heat resistance of ascospores was most marked. This difference was the basis of the short steam treatments used by Warcup (16) in isolating fungi from the soil. Under appropriate conditions, isolations of Ascomycetes were increased greatly as a result of heat inactivation of most Phycomycetes and Fungi Imperfecti. Warcup (17) also found that soil samples heated at 50 to 75 C gave much higher counts of viable ascospores than unheated samples and concluded that many ascospore types are activated by sublethal heating. The results obtained with A. mangini ascospores (Table 1) show this effect. Two isolates were used, each grown under three sets of conditions, and in every case the viable counts after heating at 60 C were much greater than after heating at lower temperatures. The scatter in viable counts of A. chevalieri ascospores in the survivor curves of Fig. 1 can be attributed to at least three causes: variability in culture age, random error of sampling and counting, and the spores' "dry" nature and consequent affinity for glass and air interfaces which reduces pipetting accuracy. Survivor curves and DRT values have rarely been recorded in a manner which will permit a statistical assessment of variability (7). We attempted this by calculating the survivor curves by a least-squares fit, without constraint, giving the count at zero time the same weight as counts obtained during heating. The following DRT values and standard deviations were obtained: at 65 C, 45.9 :i 8.2 min; at 70 C, 19.8 i 4.7 min; at 75C, 5.1 1.4 min; and at 80C, 3.44 i 0.63 min. All of the values obtained by the constrained method, except that at 75 C, lay within one standard deviation of the unconstrained values, and it is clear that the differences between values obtained by the two methods would not be of great practical significance. The only previous detailed presentation of survivor curves for fungal spores was of Penicillium lapidosum Raper et Fennell (18) in which exponential plots of survival data against time of heating were not linear. This has been interpreted (12) as evidence that the exponential rate of heat destruction commonly found for bacteria does not apply to fungal spores. However, examination of the results (18) shows that this conclusion is drawn from very limited data. The curve of heat destruction of ascospores has but three points, the first two spanning a 10,000fold reduction in viability. The curve for sclerotia has a pronounced shoulder which may be explained by the finding (13) that, after several weeks of incubation, P. lapidosum sclerotia mature into cleistothecia, each containing hundreds of ascospores. Each sclerotium is, in effect, many-fold multinucleate, and a marked departure from an exponential destruction curve would be expected in the early stages of heating. Although the data reported here for A. chevalieri ascospores ( Fig. 1) do show considerable scatter and some evidence of a shoulder at two of the four temperatures, it seems reasonable to assume that the rate of heat destruction of fungal spores is exponential. From recently published data for the heat resistance of X. bisporus ascospores (3), we deduced a z value of 22 F, an F180 of 2.0 to 2.5 min, and a DRT at 80 C of between 2.7 and 3.6 min. Our data indicate a DRT at 80 C for X. bisporus ascospores of 3.3 min, in close agreement. The DRT curve obtained for A. chevalieri ascospores (Fig. 2) has an extremely high z value, 23 F (12.8 C). This z value, similar to that deduced for X. bisporus, is higher than that usually quoted for Clostridium botulinum (18 F; reference 4) and much higher than those deduced from data for P. lapidosum (18) and Byssochlamys fulva (5) of 10.3 and 9.9 F, respectively. However, A. chevalieri ascospores, with an Fj80 of 2.2 min, are much less heat-resistant than ascospores of B. fulva (5), with an F180 deduced to be 50 to 100 min, depending on heating substrate, or the sclerotia of P. Iapidoswn (18) with F180 calculated as 180 min.

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2018-04-03T04:39:06.593Z

1970-01-01T00:00:00.000Z

25000170

s2

Microbiological Aspects of Ethylene Oxide Sterilization An investigation determined the effects of environmental moisture content or water activity (Aw), exposure humidity, and sterilant concentration on the resistance of microbial spores. Decimal reduction values [expressed as D values at 54.4 C-specified concentration (milligrams per liter) of ethylene oxide] were determined from spore destruction curves of Bacillus subtilis var. niger dried on hygroscopic and nonhygroscopic surfaces. Four groups of spore preparations were preconditioned in one of four Aw environments (<0.1, 0.1, 0.5, 0.95) for 2 weeks or longer and were exposed to 500 mg of ethylene oxide per liter at 54.4 ± 3 C and 10, 50, and 95% relative humidity in a specially designed thermochemical death rate apparatus. A fifth group did not receive any preconditioning treatment and was exposed immediately after preparation, in the same apparatus at the same temperature, to ethylene oxide concentrations of 200, 400, 600, 800, and 1,200 mg/liter and relative humidities of 15, 30, 50, 60, and 90%. The resistance of the spores on both types of surfaces to ethylene oxide increased proportionately with the Aw of the conditioning environment. The study also showed that moisture in the exposure system was not as critical a variable as the ethylene oxide concentration. The spore destruction rates, irrespective of the carrier types at all concentrations and at different humidities, varied little from one another. The decimal reduction values were reduced as the ethylene oxide concentration increased, and no optimal exposure humidity concentration was observed. It has generally been accepted that moisture is essential to sterilization with ethylene oxide vapors. In fact, Kaye (3) recently reported that water must be present, not simply as an adjuvant but as a necessity, and that ethylene oxide with water is a nonspecific toxic agent for all microorganisms. The influence of moisture on gas sterilization has been the subject of numerous studies (1,2,4,5,12). These studies have considered the effect of moisture on microbial cells when exposed to ethylene oxide as well as on the sterilization of products and materials by this agent. Lea et al. (8) demonstrated the critical relation of cell moisture content to certain chemical restrictions associated with living cells. Their work suggested that the Z value (reciprocal of the decimal reduction value) will decrease as the moisture content of the cell increases. Murrell and Scott (9,10) investigated the effect of cell moisture content on the rate of microbial death at dry-heat temperatures from 70 to 120 C. They demonstrated that Bacillus spores were most resistant to dry heat when preconditioned and exposed at 20 to 40% relative humidity. Gilbert et al. (2), in studying the effects of moisture on ethylene oxide sterilization, speculated that certain cross-linkages are formed through water molecule bridges within cell proteins or between adjacent proteins which make alkylating sites more accessible to ethylene oxide. Kaye and Phillips (4) reported 20 to 40% relative humidity as optimal for the destruction of microorganisms by ethylene oxide. Kelsey (5) noted that insufficient moisture (very low humidities) prevented the bactericidal action of ethylene oxide, whereas an excess of moisture caused ethylene oxide to hydrolyze to the more ineffective compound ethylene glycol. Ernst and Shull (1) noted how barriers to diffusion of moisture could limit ethylene oxide effectiveness. They also reported that prehumidification of materials prior to exposure enhanced the penetration of water vapor through absorbent materials and thus decreased the time for sterilization. In this study, relative humidity is expressed as the ratio between the density of the actual water vapor existing and the density of saturated vapor at the existing temperature (11). Since both densities are proportional to pressure, it is more often referred to as the ratio of actual vapor pressure to the saturated pressure at the existing temperature. This definition describes relative humidity as being independent of the total pressure in the area and independent of the characteristics of the other gases in the area. Water activity (Aw) is a fundamental property of an aqueous solution and by definition is equal to the ratio of the vapor pressure of a solution to that of a solvent (13). Aw is numerically equal to the corresponding relative humidity expressed as a fraction of 100. The term "water activity" describes the status of water in a substrate in preference to relative humidity which applies more strictly to the surrounding atmosphere. The primary aim of this work was to consider how Aw and varied relative humidities affected the sporicidal nature of ethylene oxide. A second aim was to consider the relationship between ethylene oxide concentration and relative humidity. MATERIALS AND METHODS Test organism preparation. Bacillus subtilis var. niger (Ft. Detrick strain) was used for all tests. The groups of carriers inoculated with spore suspensions of this organism were prepared as previously described (7). Five groups of inoculated carriers (both types) were prepared. Four of the groups were placed in preconditioning environments before testing. The fifth group received no special preconditioning. Immediately after inoculation, the carriers of this group were transferred to sterile glassine envelopes and were exposed to the test conditions. Preconditioning. Four environments, each with a different Aw value were used to precondition the Spore-survivor curves of B. subtilis var. niger exposed to various ethylene oxide concentrations at relative humidities of 15, 30 and 50%0. Symbols: *, 200 mg/liter; 0, 400 mg/liter; 0, 600 mg/liter; A, 800 mg/liter; *, 1,000 mg/liter; A, 1,200 mg/liter. inoculated carriers. The values were <0.1, 0.1, 0.5, and 0.95 Aw, respectively. To obtain an Aw of <0.1, a desiccator containing CaCl2 was used. An atmosphere with an Aw of 0.1 was prepared by placing a saturated aqueous solution of LiClIH20 in a desic- cator. An Aw of 0.5 was prepared with a saturated aqueous solution of KNO2 in a desiccator. A saturated aqueous solution of KNO, in a desiccator was used to obtain an Aw of 0.95. Each desiccator was equipped with a relative humidity sensor (El-Tronics, Inc., Mayfield, Pa.) attached to a portable hygrometer (El-Tronics, Inc.). The desiccators were maintained at 21 3 C and conditioned for a minimum of 1 week before use. At the end of this period, the inoculated carriers (in sterile petri dishes) were placed in the preconditioning desiccators and conditioned for at least 2 weeks before use. Prior to testing, the carriers were aseptically transferred from the desiccators to sterile glassine envelopes. Exposure apparatus and procedure. The thermochemical death rate apparatus and exposure procedures previously described were used (6). Death kinetics were studied with the gas sterilant at 54.4 3 C. Ethylene oxide concentrations. Ethylene oxide concentrations were 200, 400, 500, 600, 800, 1,000, and 1,200 mg/liter. These concentrations were obtained by adjusting the pressure of the gaseous mixture within the test chamber. Exposure time. The inoculated carriers were exposed at intervals of 0 to 35 min. RESULTS AND DISCUSSION Survivor curves for B. subtilis var. niger spores inoculated on both hygroscopic and nonhygroscopic surfaces and exposed to the previously described conditions are presented in Fig. 1. 2, and 3. The thermochemical survivor curves and the decimal reduction values (Tables 1 and 2) were prepared as previously described (6). These results demonstrated the effect of three factors on the sporicidal activity of ethylene oxide during exposure of the spores in the test apparatus. These factors were preconditioned spores, various relative humidities, and various ethylene oxide concentrations. The resistance of B. subtilis var. niger spores to ethylene oxide can be influenced by the water activity of the microenvironment surrounding the spores prior to exposure. Generally, as the Aw of the preconditioning environment increased the resistance of spores on both types of carriers increased. This was demonstrated by the higher decimal reduction values (expressed as D values at 54.4C-500 mg of ethylene oxide per liter). However, the survivor patterns ( Fig. 2 and 3) as well as the decimal reduction values in Table 2, signify that moisture in the exposure system is not as critical a variable as ethylene oxide con-centration. It will be noted that at all concentrations, irrespective of the carrier types, spore destruction rates varied little at the different exposure humidities. An increase in the sporicidal effect was observed as the ethylene oxide concentration increased. The most marked effect was noted when the concentration of ethylene oxide was increased from 200 to 400 mg/liter. Considering all data at these two concentrations, irrespective of carrier materials and relative humidity, there was an average decrease in the decimal reduction values of 2.57 min. None of the other gas concentration increments was as dramatic in its effect on the decimal reduction values. Our results also provided some insight into the effect of moisture on the resistance of B. subtilis var. niger spores to ethylene oxide from two aspects-the moisture content Aw of the microenvironment of the spores and the moisture content (relative humidity) during ethylene oxide exposure. The effect of moisture on the sterilizing action of ethylene oxide seems to lie either in the moisture content of the bacterial cell at the time it contacts the sterilant or in the moisture conditions of the atmosphere (or microenvironment) surrounding the individual cells. Kelsey (5) suggested that the moisture content of the atmosphere surrounding the organism and the water content of the organism itself are more important than the overall humidity of the atmosphere surrounding the material being sterilized. Gilbert et al. (2) demonstrated that desiccated or highly dried bacterial spores which were difficult to sterilize with ethylene oxide lost their resistance when prehumidified in an atmosphere of 75 to 98% relative humidity. Kaye and Phillips (4), however, suggested that a zone of high moisture about a spore will have a diluting effect (dilution theory) on ethylene oxide and reduce its availability to the spore. This theory, as well as others, would give credence to our findings that the moisture content (Aw) of the atmosphere had more effect on the resistance of B. subtilis var. niger spores than did the overall relative humidity in the exposure chamber. It is concluded from these studies that the water content or Aw of the microenvironment of the spores influenced the ethylene oxide re-sistance more than the overall relative humidity in the sterilizing atmosphere. On the other hand, the remarkable decrease in the spore resistance noted in the presence of increased ethylene oxide concentration was strong evidence that gas concentration is an essential variable in ethylene oxide sterilization. Our study also disclosed no optimal humidity for the destruction of spores of B. subtilis var. niger when exposed under the aforementioned test conditions. The small variations observed in the decimal reduction values at various humidities were considered of little consequence in commercial gas sterilization; however, moisture can become a critical factor when packaging materials interfere with moisture and ethylene oxide diffusion or transmission.

v3-fos

2020-12-10T09:04:11.149Z

1970-09-01T00:00:00.000Z

237231545

s2

Degradation and Utilization of Hemicellulose from Intact Forages by Pure Cultures of Rumen Bacteria Several pure strains of rumen bacteria have previously been shown to degrade isolated hemicelluloses from a form insoluble in 80% acidified ethanol to a soluble form, regardless of the eventual ability of the organism to utilize the end products as energy sources. This study was undertaken to determine whether similar hemicellulose degradation or utilization, or both, occurs from intact forages. Fermentations by pure cultures were run to completion by using three maturity stages of alfalfa and two maturity stages of bromegrass as individual substrates. Organisms capable of utilizing xylan or isolated hemicelluloses could degrade and utilize intact forage hemicellulose, with the exception of two strains of Bacteroides ruminicola which were unable to degrade or utilize hemicellulose from grass hays. Intact forage hemicelluloses were extensively degraded by three cellulolytic strains that were unable to use the end products; in general, these strains degraded a considerably greater amount of hemicelluloses than the hemicellulolytic organisms. Hemicellulose degradation or utilization, or both, varied markedly with the different species and strains of bacteria, as well as with the type and maturity stage of the forage. Definite synergism was observed when a degrading nonutilizer was combined with either one of two hemicellulolytic strains on the bromegrass substrates. One hemicellulolytic strain, which could not degrade or utilize any of the intact bromegrass hemicellulose alone, almost completely utilized the end products solubilized by the nonutilizer. Similar synergism, although of lesser magnitude, was observed when alfalfa was used as a substrate. Since the hemicelluloses can constitute a considerable portion of forage carbohydrate, their use as an energy source by the ruminant animal is of special interest. Early studies, with conventional digestion trials, indicated that a definite loss of pentose occurs in the ruminant digestive tract (18)(19)(20). Since then, the in vitro fermentation of various isolated and intact forage hemicelluloses has been demonstrated with mixed cultures of rumen organisms (11, [13][14][15], as well as with several pure cultures (6,8,12,17). Dehority (6), investigating whether the xylan-utilizing species of rumen bacteria were capable of digesting isolated hemicelluloses, found that from a total of eight strains of cellulolytic bacteria only those three capable of using xylan as an energy source were able to grow on isolated hemicelluloses. The five remaining strains of cellulolytic bacteria, although unable to utilize the isolated hemicelluloses as a source of energy, degraded these materials to a form soluble in 80% acidified I Approved for publication as Journal Article no. 36-70 by the Ohio Agricultural Research and Development Center, Wooster, Ohio. ethanol. The extent of hemicellulose degradation or utilization, or both, varied between organisms and source of the hemicellulose. Degradation was defined as the conversion of ethanol-insoluble pentose to a soluble form and utilization as total pentose loss. Further studies with several additional strains isolated on a xylan medium revealed differences in the rate and extent of degradation, utilization, or both, between the isolated hemicellulose substrates and the various bacterial strains and species (8). Dehority and Scott (12) estimated hemicellulose digestion in two maturity stages each of bromegrass and alfalfa and found that digestion was appreciably decreased in the intact plant as compared with isolated hemicelluloses. In general, the extent of hemicellulose digestion varied with the maturity and type of forage, as well as with the species of bacteria. In limited synergism studies, it was observed that cellulose digestion was increased in all cases when the cellulolytic organisms were combined with Bacteroides ruminicola H8a, a noncellulolytic, hemicellulolytic organism. Kock and Kistner (17) confirmed the results of Dehority and Scott (12) on the digestibility of forage hemicelluloses. Ten strains of Butyrivibrio and two strains each of Ruminococcusflavefaciens, R. albus, and Clostridium sp., all capable of fermenting xylan, were able to solubilize the hemicellulose from ethanol-benzene-extracted low protein teff hay. Hemicellulose solubilization was based on the loss in weight of isolated holocellulose when treated with 24% KOH (w/v). In view of these data, the present study was undertaken to (i) investigate whether hemicelluloses are degraded from an intact forage in a manner similar to that observed in the isolated materials by both the utilizing and nonutilizing strains; (ii) determine the influence of plant type and maturity as well as bacterial strain differences on the extent of hemicellulose degradation; (iii) study several previously untested species and strains of rumen bacteria for their ability to degrade intact forage hemicelluloses; and (iv) investigate possible synergistic effects on hemicellulose utilization or degradation, or both, by using an intact forage substrate. One additional objective in the present study was to measure the utilization or degradation, or both, of hemicellulose from an intact forage and compare these values with those obtained from a sample of hemicellulose previously isolated from this same forage. MATERIALS AND METHODS Forage substrates consisted of boot and bloom stages of bromegrass (Bromus inermis Lincoln) and prebloom, early bloom, and late bloom stages of alfalfa (Medicago sativa Vernal). All of the forages were harvested from pure stands, artificially dried on a wagon dryer, chopped, ground through a medium screen in a large Wiley mill, and finally ground through a 40-mesh screen in a small laboratory Wiley mill. Fescue grass and isolated fescue grass hemicellulose were supplied by the late Fred Smith of the University of Minnesota. The fescue grass was chopped and ground through a 40-mesh screen in a small Wiley mill for use as a substrate. Isolated hemicellulose was prepared from the same stand of fescue grass by the procedure of Myhre and Smith (21). The anaerobic cultural techniques were similar to those described by Hungate (16), except for the modification proposed by Dehority (10) in the preparation of media. The fermentation medium contained 0.5% forage or 0.1% isolated hemicellulose, 40% clarified rumen fluid, 15% each of mineral solutions I and II of Bryant and Burkey (2), 0.0001% resazurin, 0.4% sodium carbonate, and 0.05% cysteine. The mixture was agitated with a magnetic stirrer, and 5-ml samples were pipetted anaerobically into pyrex culture tubes (16 by 150 mm), which were then closed with a size 0 rubber stopper. The individual tubes were autoclaved for 20 min at 121 C in a clamp-type rack to prevent the stoppers from blowing out. The 10 pure cultures of rumen bacteria used for this study included strains A3c and S-85 of B. succinogenes, strains Bla and B34b of R. flavefaciens, strain 7 of R. albus, strains HlOb and H17c of Butyrivibrio fibrisolvens, strains H8a and D31d of B. rwninicola (subspecies brevis and rwninicola, respectively), and strain D15d of Lachnospira multiparus. Thecharacteristics of these strains have previously been described (3)(4)(5)(6)(7)10). Strains A3c, Bla, B34b, S-85, and 7, all cellulolytic, were isolated from rumen contents with a nonselective glucose-cellobiose medium; strains H8a, H1Ob, and H17c were isolated with a xylan medium; and strains Dl5d and D31d were isolated with a pectin medium. All cultures were carried in the medium in which they were originally isolated. Inoculum cultures were grown overnight in an optically clear 0.5% cellobiose broth containing 40% clarified rumen fluid and then diluted with anaerobic dilution solution until an optical density of 0.2 had been reached. A 0.2-ml amount of this suspension was used to inoculate each 5 ml of forage media. All turbidity measurements were made in Pyrex culture tubes (16 by 150 mm) on a Bausch & Lomb Spectronic-20 colorimeter, reading at 600 nm. The fermentations were allowed to incubate for 168 hr at 39 C. The inoculum level and the fermentation time had been previously determined (8) to allow for maximum rate and completion of forage cellulose digestion. Estimation of forage hemicellulose concentration either by simple solubility methods or the more complex isolation procedures did not appear to be suitable for this study. Even though the constituent carbohydrates and per cent composition of plant hemicelluloses vary between species, approximately 80 to 90% of the hemicellulose is composed of pentoses. On this basis, it is believed that total pentose analyses would be indicative of changes in the hemicellulose fraction, and relatively simple analytical methods could be adapted for routine use. One possible source of error would be the contribution of ribose from plant and bacterial ribonucleic acid; however, this would be quite small in relation to overall pentose changes. Thus, hemicellulose concentration of the forages was estimated by hydrolysis in 1 N H2S04 and measurement of total pentose present with the orcinol reaction (1). Hexose and uronic acid interference was removed by reading at two wavelengths and treatment with an anion exchange resin, respectively (12,22). Although methods were described by Dehority (6) for estimating solubilization of total pentose in 80% ethanol with isolated hemicellulose and by Dehority and Scott (12) for the loss of total pentose when working with intact forages, procedures for measuring solubilization of pentose in 80% ethanol from the intact forage had to be developed. After a series of preliminary studies, the following procedure was adopted for routine use. The entire contents of the fermentation tube were transferred to a 40-ml glass centrifuge tube, 20 ml of a 5% acetic acid in 95% ethanol solution was added, and the contents were 363 VOL. 20,1970 mixed and allowed to stand at room temperature for 30 min. The mixture was then centrifuged 30 min at 2,000 X g, and the supernatant was decanted into a 50-ml volumetric flask. Acidified 80% ethanol solution (20 ml) was added to the residue, and the contents were mixed and recentrifuged for 20 min at 2,000 X g. The wash solution was decanted and added to the original supernatant, and the total supernatant was brought to volume with distilled water. To hydrolyze the hemicellulose in the residue, 5 ml of 1 N H2S04 was added to each tube; the tube was covered with a metal cap and autoclaved for 1 hr at 15 psi. After cooling, the hydrolysate was transferred to a 50-ml volumetric flask and brought to volume with distilled water. The insoluble material was allowed to settle, and a sample was pipetted from the upper layer for further analysis. Portions (5 ml) of both fractions were treated with 2 g (wet weight) Amberlite IRA400 anion exchange resin in the acetate form. The resin was removed by filtration and the filtrate plus washings, diluted to appropriate volumes, were than analyzed for total pentose by the orcinol method (1). Per cent transmission was read at 520 and 660 nm on an Evelyn colorimeter, which allowed correction for any color at 660 nm arising from hexose in the sample. In this study, degradation is defined as the solubilization of 80% ethanol-insoluble pentose, whereas utilization is defined as a loss in total pentose (6). Degradation and utilization values are based on duplicate fermentations in two replicates. A preliminary study involving two organisms and two different forage substrates (alfalfa and orchardgrass) was run to determine a feasible coefficient of variation for acceptance of duplicate analyses. Six replicates of each forage and organism were set up in duplicate, and coefficients of variation ranging between 5 and 10% were obtained. These were similar to the values obtained previously by Dehority (8), in a study on the digestibility of cellulose in intact forages in which a coefficient of variation of 15% was chosen as the upper limit for acceptance of the data. Thus, when a coefficient of variation larger than 15% was obtained between the four values in the present study, the fermentations were run twice again in duplicate. RESULTS Data on the utilization or degradation, or both, of hemicellulose from two maturity stages of bromegrass are presented in Table 1. It it apparent that all of the cellulolytic strains tested were capable of degrading a considerable amount of the 80% acidified ethyl alcohol-insoluble pentose to a soluble form without necessarily being able to utilize the resulting end products as energy sources. Values of 6 % or less are considered to be within the range of experimental variation and are, therefore, of doubtful significance. Those cellulolytic strains which can ferment xylan, Bla and 7, were capable of utilizing the hemicellulose, although utilization by Bla was somewhat limited. Despite this utilization, neither of these Since a number of the cellulolytic strains could degrade but not utilize hemicellulose from the intact bromegrass, it seemed desirable to determine whether the xylan-digesting strains which could not degrade the hemicellulose could utilize the material solubilized by the cellulolytic species. R. flavefaciens B34b was used as the degrading nonutilizer in combination with either B. ruminicola H8a, B. fibrisolvens H1Ob, L. multiparus Dl 5d, or all three organisms. These fermentations were run under the same conditions as described previously, by using 0.2 ml of a 0.2-optical density suspension as inoculum for each organism required (Table 1). In all cases, degradation was increased above that obtained with R. flavefaciens B34b alone. The most striking effect, however, was noted in the utilization of total pentose by the combination with B. ruminicola H8a, in which utilization increased from essentially zero for each organism alone to approximately 80 and 70%, respectively, for maturity stages I and H. A marked increase in utilization was also observed when B34b and B. fibrisolvens HlOb were combined. For both maturity stages, the increase was approximately 30%. Combination of B34b with L. multiparus D15d was unsuccessful with regard to utilizing the degraded hemicellulose, which would be in agreement with the inability of Dl5d to use pentoses or xylan as energy sources (10). When all three organisms, H8a, HiOb and Dl5d, were combined with B34b, degradation and utilization were esentially the same as obtained with the highest combination of two strains, i.e., B34b and H8a. In every case where applicable, degradation and utilization were reduced as the plant matured. In general, this reduction was in the magnitude of 15 to 20%, and the overall relationship of one species or strain to another was unchanged. A similar series of experiments was set up with three maturity stages of alfalfa, and these results are presented in Table 2. All of the cellulolytic VOL. 20, 1970 species were able to degrade the hemicellulose from intact alfalfa; however, the extent was generally lower than that observed with bromegrass. Marked reduction was obtained in the extent of utilization for strains 7 and Bla, especially in the more mature plant. Both strains of B. succinogenes degraded similar amounts of alfalfa hemicellulose, whereas a considerable difference was noted with bromegrass. In contrast to the bromgrass data, all of the hemicellulolytic and pectinolytic species degraded and utilized the alfalfa pentosans. The results with L. multiparus Dl5d were particularly interesting since this strain cannot use pentoses, xylan, or degraded bromegrass hemicellulose as energy sources. An explanation of the degradation and utilization of alfalfa hemicellulose by strain D15d is not immediately obvious. Further studies with a second strain of L. multiparus have confirmed this difference in availability of grass and alfalfa hemicellulose. Marked strain differences were observed between B. ruminicola H8a and D31d and B. fibrisolvens HlOb and H17c with the alfalfa substrate. Both H8a and HiOb utilized almost all of the hemicellulose they could degrade. On the other hand, H17c degraded considerably less hemicellulose and could utilize only a portion of the degraded material. Strain D31d degraded more hemicellulose than H8a but could not utilize even 50% of that degraded. Results of the synergism studies with the alfalfa substrates were quite similar to those obtained with bromegrass although the extent of the increases was somewhat less. In contrast, utilization of alfalfa III by the combination of B34b and H1Ob, and all alfalfa stages with B34b and Dl5d, was considerably lower than utilization by HiOb and D15d alone. Table 3 presents the results on degradation or utilization, or both, of intact fescue grass hemicellulose and the hemicellulose after isolation from the forage. In general, the overall pattern of degradation and utilization is quite similar to that observed from bromegrass. Although these values correspond rather closely to those observed with the bloom stage of the bromegrass, the maturity stage of the fescue is unknown. In agreement with the degradation values observed on the bromegrass, strain H8a was totally unable to degrade any of the intact fescue grass hemicellulose (Table 3). However, this particular strain could almost completely degrade and utilize this hemicellulose when it had been separated from the intact plant. In all cases except strain D15d, the isolated hemicellulose was degraded to a considerable extent. These data are in close agreement with those reported by Dehority (6,8) for several of these same strains on the isolated fescue grass hemicellulose substrate. DISCUSSION Dehority and Scott (12) estimated total pentose loss on the same forages with 7 of the 10 strains used in this study. The utilization values reported herein are in close agreement with their data, although a somewhat more complicated analytical procedure was used. Kock and Kistner (17) fermented ethanol-benzene-extracted intact teff hay with several strains of R. albus, R. flavefaciens, and Butyrivibrio species. Although not directly comparable because of differeing analytical methods, the present results compare favorably with their 25 to 67% range of hemicellulose solubilization. The complete inability of B. ruminicola H8a to degrade and subsequently utilize the brome hemicellulose was quite surprising, since this organism, a xylan isolate, was capable of degrading and utilizing hemicellulose isolated from several grasses (8). This observation led to a study of possible synergistic effects of combining a degrading nonutilizer, R. flavefaciens B34b, with the hemicellulolytic strains H8a or H1Ob, the pectinolytic strain D15d, or all three together. The most striking results were with the combination of strains H8a and B34b, in which utilization increased from zero for each strain alone to 80% for the organisms together. Except in the case of Dl5d, some increase in degradation and a marked increase in utilization were observed for all combinations. These data suggest that the hemicellulolytic organisms were probably fermenting the oligosaccharides and other products solubilized by strain B34b. Synergism was also studied with the alfalfa substrates; however, the magnitude of the increases observed was lower. Undoubtedly, this was because those strains that could not degrade bromegrass hemicellulose both degraded and utilized alfalfa hemicellulose to a reasonable extent. Dehority and Scott (12) previously observed a small but significant increase in the extent of cellulose digestion when they combined strain H8a, a noncellulolytic, with several cellulolytic strains. Combined with the present results, these data suggest the possibility that a physical masking effect can occur in the intact plant between the cellulose and hemicellulose components. The variation observed between bromegrass and alfalfa, particularly with strains H8a, Dl5d, and D31d, would further indicate that there are definite differences in structure (or orientation) of hemicelluloses between intact legumes and grasses. The ability of L. multiparus Dl5d to degrade and utilize alfalfa hemicellulose was quite surprising, since it was unable to ferment xylan, brome hemicellulose, or isolated fescue grass hemicellulose. The organism may be producing some type of soluble oligosaccharides which are permeable to its cell membrane. No explanation is readily available for the marked decrease in utilization when strain D15d was combined with strain B34b. The degradative end products produced by B34b would appear to differ in some way from those produced by strain D15d. Consequently, this competitive degradation would result in less useful substrate for Dl5d. For all strains and combinations of strains, hemicellulose degradation or utilization, or both, in the fescue grass appeared quite similar to that previously obtained with bromegrass. Both hemicellulolytic organisms, H8a or HlOb, were capable of increased degradation and utilization when the hemicellulose had been previously separated from other plant constituents, either chemically prior to fermentation in the case of fescue grass or by a nonutilizing cellulolytic strain, B34b, with both fescue and bromegrass. The complete inability of strain H8a to degrade any of the fescue grass hemicellulose substantiates the observation that this hemicellulolytic strain can utilize grass hemicellulose only after it has been removed from the plant in some way. Where applicable, the hemicellulose degradation patterns for each organism across the two types of forage correspond rather closely to the data obtained by Dehority and Scott (12) for cellulose digestion from these same forages. This point, the high degradation values observed for the cellulolytic isolates, and the previously reported evidence that the enzymes involved are constitutive would possibly tend to support the suggestion that hemicellulose degradation by the nonutilizing cellulose digestors is a nonspecific function closely associated with cellulase activity (9). In this case, the cellulolytic organisms could quite easily be contributing to the over all rumen fermentation of forage hemicellulose by supplying the utilizers with more soluble substrate than they alone could degrade.

v3-fos

2022-04-29T15:18:20.457Z

1970-01-01T00:00:00.000Z

248436782

s2

RESISTANCE OF Fasciola hepatica TO TRICLABENDAZOLE, CLOSANTEL, AND RAFOXANIDE IN A SHEEP FARM IN SHARAZOR DISTRICT, KURDISTAN-IRAQ Fasciolosis caused by Fasciola hepatica has long been one of the most important helminthic infections of livestock in Iraq. The control of this parasite is based on the use of anthelmintic agents, mainly triclabendazole and closantel. Sheep on a farm in Tut Aghach village, Sulaymaniyah, Iraq died from liver fluke disease in spite of previous treatment with triclabendazole. Thisstudy was conducted to investigate the effect of three commercially available anthelmintic agents – triclabendazole, closantel, and rafoxanide – against natural liver fluke infection in sheep. Fecal egg count reduction test (FECRT) was used to determine the efficacy of the drugs.Treatment with triclabendazole resulted in 73.9% reduction in fecal egg count (FEC) with the lower confidence interval of 61.0%. Closantel and rafoxanide reduced the FEC in the treated groups by 70.1% and 70.2%, respectively. This study confirms the resistance to closantel, rafoxanide, and triclabendazole of the F. hepatica from a sheep farm in Sharazor district, Sulaymaniyah, Iraq. A study of more farms from endemic areas in Iraq is required to evaluate the burden of resistance development against the common anthelmintic agents. INTRODUCTION Fasciola hepatica is a parasitic trematode found in temperate and high-altitude tropical areas, it is commonly known as liver fluke. This parasite causes liver disease (fasciolosis) in a wide range of host species. Although ruminants are most commonly affected(1), fasciolosis has been described in a range of mammals including horses(2)and camels (3). The parasite is transmitted by a snail as an intermediate host (4). The most effective and widely used anthelmintic for the treatment of fasciolosis in animals is triclabendazole (5), which is highly effective against both mature and immature stages of liver flukes (6,7).Triclabendazole was introduced in the 1980s for the therapy of liver fluke infections in livestock and it has become the drug of choice for the treatment of human fasciolosis (8).Closantel and rafoxanide are two salicylanilides which have also been used against liver flukes in livestock. However, these drugs are not as effective against immature flukes as triclabendazole (9). Fasciolosis has been previously documented from different parts of Iraq (10,11). However, no studieshave been conducted, and no evidence of resistance of F. hepatica against triclabendazole and closantel has been reported yet. Asheepfarm in Sharazor district, Sulaymaniyah city, Kurdistan region was previously treated with triclabendazole but the therapy failed to clearthe infection from the animals. Consequently, many sheep died from acute fasciolosis. Hence, this study was conducted to investigate the susceptibility of F. hepatica against the commercially available triclabendazole, rafoxanide, and closantel under field conditions. Farm and animals The study was conducted in a sheep farm in Tut Aghachvillage that is located in the northern part of Sharazor district, 43 km southeast ofSulaymaniyah city ( Fecal samples were collected from the sheep before treatment and after 14 days and were tested for the number of liver fluke eggs by Fecal Egg Count Reduction Test (FECRT) (6). Fecal Egg Count Reduction Test (FECRT) A sedimentation method was used to determine the number of fluke eggs per gram (EPG) of feces. The sample was mixed well and 10 g of feces were weighed out and mixed with water in a 500 ml beaker. The beaker was then topped up with water. Three sieves (38 µm, 150 µm, and 500 µm) were stacked with the smallest aperture at the bottom and largest at the top. The fecal water was slowly passed through the sieves followed by thorough washing with water until the water ran clear from the bottom sieve. The 500 µm sieve was removed and washing through the remaining two sieves was repeated. The 150 µm sieve was removed and the retentive on the surface of the 38 µm sieve was washed and the remaining contents were transferred into a 500 ml beaker. The beaker was topped up with water and left to stand for four minutes. The supernatant was poured off leaving approximately 100 ml of sediment and then the beaker was refilled with water and left to stand for four minutes. This process was repeated until the supernatant was clear. When clear, the supernatant was poured to 100 ml or less, if possible, without losing any sediment and the remaining content was transferred into a large square Petri dish. Four drops of methylene blue were added and the number of F. hepatica eggs counted using a dissecting microscope. The number of eggs per gram of feces(EPG)was calculated by dividing the total number of eggs by 10. Statistical analysis The Fecal egg count reduction (FECR) of the different drugs was calculated as × 10. The 95% confidence interval (CI) was calculated as { EPG day 0 -EPG day 14 EPG day 0 × 100} ± 1.96 √Y2 , where Y2 is the variance of the FECR. Fasciola hepatica was considered resistant to a drug when the average FECR by the drug was <95% and the lower 95% CI was <90% (6). Efficacies of the different drugs were compared statistically using one-way analysis of variance, followed by post hoc (Duncan). RESULTS The FECR was 100% in 11 sheep treated with triclabendazole. However, the fecal egg count increased after two weeks of treatment in two sheep (numbers 9 and 30, Table 1). The average FECR in the group of sheep treated with triclabendazole was 73.9% and the lower CI was 61.0%, which indicated that resistance has been developed by F. hepatica (Table 1, The minus signs indicate that the fecal egg count increased after treatment with the designated drug. FEC = fecal egg count. Nine of the sheep treated with rafoxanide were free from Fasciola eggs after 14 days. The FECR of rafoxanide was 70.1% with the lower CI of 57.7%, while treatment with closantel resulted in 70.2% decrease in the fecal egg count with the lower CI of 57.4%. The fecal egg count increased in one animal (number 4) following therapy with rafoxanide. The results indicated multidrug resistance of F. hepatica in the studied animals against all the tested antitrematodal agents. Comparison among the average FECR values of the three drugs revealed no statistical difference (p ≥ 0.05), meaning that none of the tested drugs proved to be better than the rest. DISCUSSION Fasciolosis is one of the most common helminthic infections inIraq (12,13). Triclabendazole is one of the most widely used drugs by farmers against liver fluke infections in Sulaymaniyah for many years. It is one of the most important antitrematodal agents to treatfasciolosis since it is the only available drug that has the ability to eliminate early immature and adult F. hepaticaat a dosage rate of 10 mg/kg (7,14). According to the best of our knowledge, noprevious studyabout the failure of this drug to eliminate fasciolosis in Closantel and rafoxanide aresalicylanilideanthelminticsused against liver flukes. They act by uncoupling the oxidative phosphorylation in the parasite (15). Development of resistance against one drug usually results in the failure of the other drug as well since they share the same mechanism of action. The efficacies of closantel and rafoxanide in this study were 70.2% and 70.1%, with a lower CI of 57.4% and 57.7%, respectively. This indicates that other members of salicylanilides such as oxyclozanide and nitroxinil would most probably be ineffective against F. hepatica in the area. Resistance development against closantel, rafoxanide, and other members of the salicylanilides has not been reported previously in Iraq. Hence, this study is considered the first report about the emergence of multidrug-resistant F.

v3-fos

2018-04-03T00:33:46.365Z

1970-07-01T00:00:00.000Z

12956407

s2

Survival of Virus in Chilled, Frozen, and Processed Oysters Samples of whole and shucked Pacific and Olympia oysters, contaminated with 104-plaque-forming units (PFU) of poliovirus Lsc-2ab per ml, were held refrigerated at two temperatures, 5 and -17.5 C. To study the survival of virus in the oysters under these conditions, samples were assayed for virus content at weekly in-tervals for as long as 12 weeks. Results indicated that poliovirus would survive in refrigerated oysters for a period varying from 30 to 90 days, depending upon -temperature. The survival rate varied from 10 to 13%. To study the extent of the hazard presented by oysters contaminated with virus, samples of whole and shucked Pacific oysters contaminated with 104 PFU of poliovirus Lsc-2ab per ml were heat processed in four ways: by stewing, frying, baking, and steaming. Results indicated that virus in oysters withstood these methods of processing. The survival rate varied from 7 to 10% and appeared dependent upon the processing method used. Heat penetration studies showed that the internal temperature in the oyster was not sufficient to inactivate all of the virus present. These results suggest that not only fresh but also refrigerated and cooked oysters can serve as vectors for the dissemination of virus disease if the shellfish are harvested from a polluted area. Research by European and East coast workers has shown that viruses can survive in the sea long enough to be taken up by shellfish (2,3,13). Similar findings have been reported recently for West coast species (8). The major site of virus accumulation in these species has proven to be the digestive tract (11,12,15). However, the extent of the hazard presented by the survival of virus, through the processes of freezing or cooking, has not been fully examined. Therefore, a study was made of virus survival in chilled and frozen oysters and in oysters processed by stewing, frying, steaming, and baking. This latter study was considered of particular value because the Pacific oyster, the principal oyster grown on the West coast, is normally cooked prior to being eaten. MATERIALS AND METHODS Oyster samples. The Pacific oysters (Crassostrea gigas) and Olympia oysters (Ostrea lurida) used in these experiments were obtained from a Shelton, Wash., oyster grower in lots of 48 to 50 oysters. The shells were cleaned of external debris and superficially disinfected by dipping in a 1%-to hypochlorite solution; they were then rinsed with tap water and dried. Oysters to be used in experiments were kept in 5-gal (ca. 19 liters) stainless-steel aquaria to which was added 3,500 ml of filtered seawater of 28%0 salinity. Water temperature was maintained at 13 C by cold-air circulation in a constant temperature bath. Aeration and water circulation were provided by means of air hoses placed in the aquaria. Virus. Attenuated poliovirus Lsc-2ab was used in all experiments. This virus is representative of enteric viruses and has been used commonly in studies dealing with the uptake of virus by shellfish. The strain was obtained from the Department of Preventive Medicine, University of Washington. The seed virus contained 3.5 X 108 virus plaque-forming units (PFU)/ ml. Stock virus was propagated on primary African green monkey (MK) kidney cell cultures. The stock virus pool, diluted with Hanks balanced salt solution (BSS) at 30 C and adjusted by plaque formation determinations to contain approximately 108 PFU/ml, was kept at -20 C until used. Tissue culture. Cell suspensions of primary MK kidney tissue (BBL) were used throughout experiments. Monoayer cultures were prepared in 3-oz prescription bottles (ca. 90 ml). Hanks BSS, to which were added 0.2% Casamino Acids and 10% calf serum, was used for cell growth, and Earle's BSS containing 2% calf serum was used for cell maintenance. One hundred units of penicillin G per milliliter, 100 jig of streptomycin sulfate per ml, and 2.5 ,ig of 58 on May 5, 2020 by guest http://aem.asm.org/ Downloaded from Fungizone per ml were incorporated in all media to prevent the growth of bacteria, molds, and yeasts. Plaque assay technique. The assay method used was essentially that of Hsuing and Melnick (9). The medium was decanted from monolayer bottles, and 0.5 ml of appropriately diluted sample was added to the bottles. All samples and all experiments were run in duplicate. Immediately after inoculation, the bottles were rotated five times to insure even distribution of the inoculum over the cell sheet. All inoculated bottles were incubated at 36 C for 1 hr and 15 min to allow for virus adsorption. After incubation, cell sheets were washed once with 4 ml of warm (30 C) Hanks BSS and then overlaid with 10 ml of growth medium containing 1.5% purified agar (Difco) and 0.0017% neutral red (BBL). This procedure was conducted in semidarkness to prevent photoinactivation of virus and cell sheets by the neutral red. After the agar solidified, the bottles were rotated flat side up and then covered and returned to the 36 C incubator. Bottles were observed for the appearance of plaques on the third to fifth days. Each plaque was marked with a Magic Marker pen, and the total number observed was written on the side of the bottle. The total count on the last day of observation was considered the final count. The sum of the counts of two bottles multiplied by the dilution factor represented the total virus content in each milliliter of inoculum. Survival of virus in chilled Olympia oysters. To determine the survival rate of virus in Olympia oysters held at storage temperatures, the following study was conducted. Forty-eight 4-year-old Olympia oysters were exposed to attenuated poliovirus I for 48 hr, dipped in a 1% hypochlorite solution to disinfect the shell surfaces, washed in distilled water, and sealed in polymylar pouches, eight per pouch. Samples were placed in a refrigerator set at 5 C and assayed for virus content at 0, 5, 10, 15, 20, 25, and 30 days. To determine the rate of virus inactivation in the various anatomical regions of the oyster, the shellfish were carefully dissected as aseptically as possible. The gills, mantle, and palps were first dissected out, then the digestive area, and the remaining body. Ten per cent (w/v) homogenates of each in nutrient broth were prepared, and the samples were clarified by low-speed centrifugation (1,200 X g for 20 min at 10 C) in a Sorvall RC 2-B refrigerated centrifuge. Serial decimal dilutions in nutrient broth were prepared, and all samples were assayed. Survival of virus in frozen oysters. To determine the ability of virus to withstand inactivation in frozen whole oysters, a long-term study was conducted. Fifty shucked Pacific oysters were inoculated directly in the gut region with an inoculum containing 104 virus PFU/ml. The inoculum consisted of poliovirus Lsc-2ab in nutrient broth. The oysters were sealed in polymylar pouches, five to a pouch, and quick frozen at 36 C in a blast freezer for 24 hr. Suspensions of poliovirus (3 ml) in nutrient broth were also quick frozen for 24 hr to serve as controls. Samples were removed from the freezer and stored frozen at -17.5 C. Samples were assayed for virus content at 0, 2, 4, 6, 8, 10, and 12 weeks, by using identical assay procedures (see above). Survival of virus in stewed, fried, baked, and steamed oysters. (i) For stewed oyster studies, 20 mediumsized shucked Pacific oysters were inoculated with 0.5 ml of an attenuated poliovirus suspension in nutrient broth containing 104 virus PFU/ml. Oyster stew, in milk, was prepared by standard cookbook methods. The inoculated oysters were placed in boiling milk in a 2.5-liter stainless steel kettle. Normal recipe procedure requires the oysters to stew for 5 min; however, cooking time was extended to 8 min. Samples of milk and oysters were removed at 0, 2, 4, and 8 min. A 5-ml amount of milk and five oysters constituted a sample. The milk was aseptically pipetted into 20-ml sterile test tubes, and the oyster samples were sealed in polymylar pouches. All samples were cooled to room temperature (23 C). Samples not assayed immediately were held at -20 C until tested. Controls consisted of inoculated oysters which were not stewed but were sealed in polymylar pouches and held at -20 C until assayed. (ii) For fried oyster studies, 20 medium-sized shucked Pacific oysters were inoculated with a poliovirus suspension in the identical manner described in the preceding experiment. The oysters were prepared for frying by standard cookbook recipes. Oysters were coated in a batter of egg, bread crumbs, and seasoning and fried in Wesson Oil at a temperature of 177 C. Frying time was extended from the 5 to 8 min called for in the recipe to 10 min. Samples of five oysters each were removed for assay at 3-, 6-, and 10-min intervals, sealed, in polymylar pouches, allowed to cool, and tested. The zero-hour and control samples consisted of inoculated but unprocessed oysters. (iii) For the baked oyster study, 20 medium-sized Pacific oysters were inoculated with poliovirus, as described in preceding experiment, and then prepared by standard cookbook methods. The oysters were rolled in a batter of egg and bread crumbs, plus seasoning, and then baked for 20 min in an oven at 121.5 C. Samples were removed for assay at 5, 15, and 20 min. Oysters used as zero-hour and control samples were inoculated and rolled in batter but not baked. (iv) For the steamed oyster study, 25 medium-sized Pacific oysters were placed in three 5-gal (ca. 19 liters) aquaria containing 3,500 ml of filtered seawater (28% salinity) at 13 C. To each aquarium was added sufficient poliovirus Lsc-2ab to yield a count of 105 virus PFU/ml of seawater. The oysters were contaminated for 48 hr and then removed from the aquaria. Shell surfaces were disinfected by dipping in a 1% hypochlorite solution and then rinsed in distilled water and dried. Oysters used in experiments were placed in metal pans and held under flowing steam for 30 min in an autoclave. Samples were removed at 0, 5, 15, 20, and 30 min, by using metal tongs. They were placed in polymylar pouches and allowed to cool to 25 C before being assayed. The control samples were contaminated oysters not subjected to steaming. Temperature determinations. Temperature determinations were made in all processing studies to correlate the rate of virus inactivation with the rise in temperature inside processed shellfish. All processing procedures were repeated in duplicate with noncontaminated oysters. Four oysters were used in each experiment. A Because of decomposition, it was impossible to dissect the oysters after more than 15 days of storage. A second study was made over a 30-day period in which virus survival in the whole oyster was measured. Results are summarized in Fig. 1. After 10 days of storage, there was less than a log reduction in the total virus present and 46% of the virus were still viable. After 30 days of storage, the oysters were badly decomposed. At this time, the total virus count was reduced by 2 logs, but 13% of the virus still remained viable even under these conditions. Survival of virus in frozen oysters. Poliovirus was found to be very stable in whole Pacific (Fig. 2). After 4 weeks of storage, the total virus count was reduced by little more than 0.5 log. By 12 weeks of storage, the original virus titer in the oysters was reduced by one log. Approximately 91% of the virus was viable after 2 weeks of storage, 40% was still viable at 6 weeks, and 10% still survived after 12 weeks of storage (Table 2). Stewed oyster studies. The inactivation of poliovirus in stewed oysters was relatively rapid. The stewing process caused the oysters to split open after 2 min of cooking time, thereby releasing some of the virus into the hot milk. This fact probably accounted for some of the virus inactivation. However, even after 8 min of stewing, 10% of the virus still survived in the oyster, and 7% was recovered from the milk ( Table 3). Fried oyster studies. The initial inactivation of poliovirus in the oyster was slow, with 61 % of the 14t 0 . (Table 4). When the exper.iment was terminated, after 10 min of frying, the virus count had been reduced by approximately 2.5 logs. This represents a 13% survival of poliovirus. Baked oyster studies. The rate of virus inactivation was initally rapid but declined as processing proceeded (Table 5). Virus titer was reduced by approximately 2.5 logs after 20 min of baking, at which time the experiment was terminated. Virus titer was rapidly reduced during the first 5 min of baking, with only 24% of the virus still surviving at this time. However, at the conclusion of the study, 13 % of the virus still survived in the baked oysters. Steamed oyster studies. The inactivation of poliovirus in steamed oysters appeared to progress in four distinct stages, two of rapid decline and two of longer, more gradual decline. The total virus count was decreased by 1 log after 15 min of steaming and by 2 logs at the end of the experiment. At this time, although the oysters had been steamed for 30 min, approximately 7% of the virus was still viable (Fig. 3). Heat penetration studies. The results of heat penetration determinations for the above studies are presented in Tables 2 through 6. Temperature rise was rapid inside oysters processed by stewing and frying, but less so in baked and steamed oysters. Routinely, exposure to a temperature of (1). In these studies, 6.5 min of heating was required for stewed oysters to reach this temperature, 5.5 min for fried oysters, 16 min for baked oysters, and 20 min for steamed oysters. Under experimental conditions, only in the case of steaming did the actual internal temperature of the processed oysters attain that of input (processing temperature). This was as expected and is due in part to the processing times used and in part to the kinetics of heat penetration. DISCUSSION Survival of virus in chilled Olympia oysters. It has been reported that enteroviruses would persist for a considerable length of time in inoculated, chilled, commercial food products. Thus, Lynt (14) observed that type 1 poliovirus and types Bi and B6 coxsackievirus would survive for at least 1 month in representative samples of commercially prepared foods stored at 10 C. The results of our studies are in agreement with the observation of Lynt and show that virus can survive for a considerable length of time in chilled oysters. After 15 days of storage at 5 C, the total poliovirus remaining in the Olympia oysters was reduced by only 60%. Significantly, three-fourths of the remaining virus was recovered from the digestive tract. After 30 days of storage, although the oysters were badly decomposed, 13%8o of the poliovirus was still viable, indicating that the breakdown products of oyster decomposition have little effect on the virus. These findings are significant since the Olympia oyster, a small cocktail oyster, is normally not held either in grocery or household refrigeration for more than 5 days. Obviously, refrigeration for this length of time will not be sufficient to inactivate even low numbers of virus. The reasons for the prolonged survival of virus in shellfish are not known. The tendency of virus to aggregate and their incorporation by ionic bonding into shellfish mucous may be means by which virus are able to survive in chilled oysters. However, further research is needed to clarify this matter. Survival of virus in frozen oysters. Lynt (14) reported that poliovirus type 1 and coxackievirus types Bi and B6 would survive for 5 months in inoculated frozen foods held at -20 C, findings subsequently confirmed by Heidelbaugh and Giron (6). Results of the present studies with Pacific oysters show that virus also survive for considerable lengths of time in frozen oysters. In our studies, the titer of poliovirus in frozen Pacific oysters was reduced by less than 10% after 4 weeks of storage at -17.5 C, and by only one log after 12 weeks of storage at this temperature. The de facto survival of virus in frozen oysters is of public health significance. Since the advent of modern freezing techniques, there is considerable interstate shipment of frozen shellfish. If the shellfish are harvested from an area contaminated with virus, or if the animals become secondarily contaminated during handling, freezing will not inactivate these pathogens. Hence, these shellfish can serve as vectors for the dissemination of virus diseases. Survival of virus in stewed, fried, baked, and steamed oysters. Viruses are known to be inactivated by heat, which causes coagulation and breakdown of the virus protein coat. However, the medium in which viruses are held has been shown to influence virus sensitivity to thermal inactivation (4,19,20). Results of processing studies show that virus in oysters survived the inactivating effects of heat. Survival of the virus ranged from 7 to 13%o in different home cooking procedures. Virus resistance to inactivation was influenced by the method of processing used. Thus, virus survival was greater in fried and baked oysters (13 %) than in steamed and stewed shellfish (770). It has been proven that most of the virus in contaminated oysters is found in the digestive tract (11,15). The heat penetration studies have shown that, at the processing times normally used to cook oysters, the internal temperature of the shellfish is not sufficient to inactivate all of the virus which may be present. These findings are of great public health significance, since it is often assumed that cooked oysters are ipso facto safe foods. We believe that the possible accumulation by, and survival of, virus in marine food products is an area in which more research should be conducted.

v3-fos

2020-12-10T09:04:11.461Z

1970-07-01T00:00:00.000Z

237230275

s2

Preservation of Mycoplasma Strains by Freezing in Liquid Nitrogen and by Lyophilization with Sucrose Procedures for maintaining Mycoplasma strains are described. Liquid nitrogen storage provided an adequate means for keeping stock cultures stable. Over 74 strains of approximately 26 species have been preserved in this way, some for as long as 9 years. Mycoplasma strains can also be recovered satisfactorily from the freeze-dried state when the procedure described includes the use of 12% (v/v) sucrose as an additive. Fifteen strains representing 12 or more species were subjected to a freeze-drying program with and without sucrose added to concentrated cell suspensions in growth medium. Cell counts indicate improved survival with sucrose. Procedures for maintaining Mycoplasma strains are described. Liquid nitrogen storage provided an adequate means for keeping stock cultures stable. Over 74 strains of approximately 26 species have been preserved in this way, some for as long as 9 years. Mycoplasma strains can also be recovered satisfactorily from the freeze-dried state when the procedure described includes the use of 12% (v/v) sucrose as an additive. Fifteen strains representing 12 or more species were subjected to a freeze-drying program with and without sucrose added to concentrated cell suspensions in growth medium. Cell counts indicate improved survival with sucrose. In 1960, the American Type Culture Collection (ATCC) initiated the development of a Mycoplasma collection to fill the need for a national repository and distribution center for these organisms of increasing importance in human and veterinary medicine. This step was made possible because it was found that mycoplasmas could be frozen and stored in liquid nitrogen refrigerators (-150 to -196 C) with little loss in viability. Such storage facilities by that time had become available at the ATCC. Furthermore, the collection was made possible by the willingness of specialists to collaborate by providing strains, guidance, and facilities for ATCC staff members to carry out certain phases of the work. Since 1960, over 74 strains of Mycoplasma representing approximately 26 species have been accessioned into the ATCC (1). Because of the expense and other impracticalities of distributing frozen cultures, freeze-drying was also investigated as a means of preserving mycoplasmas. Tully and Ruchman (8) reported recovering viable mycoplasmas from 20-year-old lyophilized cultures received from Sabin. D. G. ff. Edward (personal communication) has used lyophilization for preserving mycoplasmas since the 1950's. Morton described the methods he used for freeze-drying mycoplasmas at the American Society for Microbiology meetings in 1963. Andrews (2) mentioned the use of freeze-drying with glucose and sucrose as additives for preserving mycoplasmas. Our trials over the past 8 years have resulted in the development of the freeze-drying procedure reported here. Early attempts to freeze-dry mycoplasmas in a growth medium by using the double-vial "batch" system in use at the ATCC and detailed by Weiss (9) were relatively unsuccessful. However, we subsequently found that a single-ampoule manifold system similar to that described by Heckly et al. (5) showed promise. Furthermore, the addition to the suspending fluid of sucrose at a final concentration of 12% (v/v), previously found useful in this laboratory (4) and elsewhere (6,7) as an additive in freeze-drying of other organisms, enhanced even more the recovery of freeze-dried mycoplasmas. MATERILS AND METHODS Strains. The strains of Mycoplasma used in this study are recorded in Tables 1 and 2 and in the ATCC Catalogue of Strains (1). Cultures were incubated on a gyratory shaker rotating at 90 rev/min for 24 hr at 37 C in plastic centrifuge bottles (122 by 61.7 mm) containing broth (see below). Slow-growing species were incubated for as long as 96 hr. Media. Most cultures were grown in Heart Infusion Broth (Difco) adjusted to pH 7.5 and enriched with Harvesting procedure. Cultures were harvested by centrifugation at 13,200 X g for 20 min in a Sorvall automatic superspeed refrigerated centrifuge (RC-2). After the supernatant was decanted, the cells were resuspended in fresh, sterile broth-culture medium to produce a concentration of cells 30 times greater than that in the original culture. Purity of the culture was verified by inoculating the concentrated material on an agar gel of the recommended broth medium, on 4% (v/v) sheep blood-agar, in fluid thioglycolate medium, and by examination of a Gram-stained smear. Ampoules were cooled at a rate of 1 C/min to -40 C by means of a BF-3-2 freezer (Linde Co., Tonawanda, N.Y.) and thereafter at an uncontrolled rate to the temperature of liquid nitrogen. They were stored in a liquid nitrogen refrigerator at -150 to -196 C. Freeze-drying procedure. The concentrated culture suspension was divided into two equal portions. Fresh culture medium was added to the first portion in a ratio of 1: 1 (v/v). The second portion was mixed 1: 1 with a 24%o (w/v) sterile aqueous sucrose solution. Amounts of the respective cell suspensions (0.2 ml) PRESERVATION OF MYCOPLASMA STRAINS were dispensed into sterile cotton-plugged 1.0-ml teardrop ampoules (catalogue no. 4020, Bellco Glass Co., Inc., Vineland, N.J.). The cell suspensions were frozen by agitating the ampoules in a dry ice-ethylene glycolmonoethyl ether bath (freezing rate, approximately 100 C/min). Ampoules containing the frozen cell suspensions and still immersed in the freezing bath were then attached to a 30-port manifold connected in series by using amber latex tubing to a -79 C (dry ice-ethylene glycol-monoethyl ether) condenser and a vacuum pump as described by Heckly (5). After a vacuum of 50 Mm of Hg or less was attained (as determined with a McLeod gauge, Stokes Machine Co., Philadelphia, Pa.), the dry ice was allowed to sublime, and the bath was permitted to reach room temperature at an uncontrolled rate (during a period of approximately 3 hr). The vacuum was maintained throughout the drying (ca. 18 hr). Thereafter, the ampoules were sealed under vacuum at a point just below the cotton plug. Ampoules were stored at -70 C. Determination of viable cell counts. Prior to processing, two portions (0.1 ml) of concentrated culture were diluted 10-fold serially with appropriate growth medium. Samples from the dilution tubes were plated in triplicate. For postfreezing determinations, two ampoules of each strain were removed from frozen storage and thawed rapidly by swirling in a 37 C water bath until the contents were visibly thawed; they were then diluted and plated as above. The total viable cell counts of freeze-dried cultures were determined by rehydrating each culture in duplicate with 0.2 ml of broth, and then diluting and plating. RESULTS Of 15 strains of Mycoplasma frozen and stored in liquid nitrogen, all showed good recovery (Table 1). Some 20 strains other than those listed in Table 1 have been frozen and have remained stable for 3 years in liquid nitrogen storage. Regarding the recovery of mycoplasmas from freeze-drying, the effect of adding sucrose to the medium prior to processing is shown in Table 2. In initial trials without sucrose, recovery of M. felis was unsuccessful and that of M. hominis was poor. By using sucrose as a protective additive, the colony counts after lyophilization were appreciably improved. Because of the improvement with these strains, sucrose has been incorporated as a routine additive to all Mycoplasma strains now lyophilized by the ATCC. There was variation between the strains in their response to the addition of sucrose (Table 2). Whereas M. felis, M. hominis, and M. pneumoniae showed markedly improved recovery, M. anatis, M. bovimastitidis, and Mycoplasma species 67-166 were apparently unaffected. The rest of the strains showed moderate improvement. DISCUSSION The strains of Mycoplasma tested showed various degrees of sensitivity to freezing. For example, M. orale ATCC 23714 showed 7.9% survival, and M. arthritidis ATCC 13988 showed 95% survival. The even higher survival rates of strain numbers ATCC 25523, 25025, 25528, and 15492 can be explained at this time only by disaggregation of the cells in the freeze-thaw procedure. Of the procedures for freeze-drying mycoplasmas tried at the ATCC, the method described above not only gave the best recoveries in terms of total cell counts but also was the only one that permitted the recovery of all strains submitted to the procedure. Pending controlled experiments to determine the effect of various storage temperatures on the stability of freezedried mycoplasmas, they are being stored at -70 C in an electric ULT-659 refrigerator (Revco Manufacturing Co., Deerfield, Mich.). In general, better recovery was obtained from cultures stored in liquid nitrogen than from those freeze-dried.

v3-fos

2022-04-29T15:14:51.876Z

1970-01-01T00:00:00.000Z

248436784

s2

Study The Effect of Ketoconazole and Vitamin E on Some Arabi Ram Sperm Characteristics Experiment aims to investigate the effect of ketoconazole on certain sperm characteristic of Arabi rams .fifteen adult rams were randomly assigned in 3 groups, 5 animals in each group which treated daily as the following : control group, ket group received orally (25 mg/kg body weight of ketoconazole and ket-E group received (25 mg/kg body weight of ketoconazole+50 mg vit E / head). The study lasted 28 days. Collection of semen was done each week by artificial vagina. Sperm traits were estimated in addition to assessment the level of testosterone and some anti_oxidative enzyme and oxidative marker (MDA ). It was concluded that orally administered Ketoconazole has a negative impact on fertility of Arabi rams and vit E at 50 mg/head can prevent adverse effect of ketoconazole on sperm characteristic of Arabi rams. Introduction Reproduction is considered an important part for production process, therefore any reducing in fertility of animal can cause economic loss in animal production (1). Fertility is described as the ability of animals to create pregnancy and is kept by insemination of good-management female using good-value semen that is placed in the female reproductive tract at the suitable time. Males should have a superior reproductive performance and a good genetic profile as the males fertility and genetic efficiency appears more vital than those of the females (2). The exposure of male to some chemicals and drugs may disturb their sexual function. Some drugs can hurt spermatogonia cells that denote the male genomes, and so disturb the spermatozoa before maturation (3). The fungal diseases are rendered a serious risk for human and animal health. Fungal infections may be cause of hypersensitivity due to fungal protein, toxicity due to fungal mycotoxin. Various fungal infections are happen due to opportunist pathogen that may be endogenous(e.g,Candida infections) or are came from the environments (e,g . Cryptococcus, Aspergillus infection) (4). Ketoconazole is one of imidazole family which has a broad, spectrum antifungal activity. It comes first amongst the azole antifungal derivatives. It has established to be the most effective and most widely used antifungal azole derivatives to date [5]. As well, ketoconazole is generally used for dealing with progressive prostate cancer. However, ketoconazole has adverse effects on the male genital system in both human and animal which researcher reported that the uses of ketoconazole cause a decrease in the weight of male genital organ (6). In additionally found that reduce epididymal sperm count and serum testosterone level (7) as well as have serious adverse events effect on sperm parameters (8) subsequently on fertility capacity. Furthermore, affected a testicular structure which has a toxicity effect on the testicular tissues (9). Recent studies suggested that ketoconazole causes an increase in the level of reactive oxygen species (10 and 11). Vit. E is an additive which is termed as anti_sterility meanwhile it is essential for regular function of female genital system. (12), and (13), showed that vit E is a chief chain .Because of lipid solubility of vit. E ,it is consider the major protection lines from peroxidation of the poly-unsaturated fatty acids of phospholipid in spermatozoal membranes (14). Thus, the present study aimed to indicate and evaluate the adverse effect of ketoconazole on some semen characteristics and the protective effect of vit E on reducing these toxicity on sperm parameters of Arabi rams. Animal and semen collections The study were carried out in an animal (16). Ketoconazole administration Ketoconazole tablets 200mg/tables were bought from a local drug store. The doses were calculated according to the practice guide of dose conversion by (17). Semen Processing Collection of semen was done with an artificial vagina every week , placed in ranked test tube, located in a thermoflask at 37C°, and conveyed to the lab for valuation of semen volumes, PH , sperm counts, motility (mass and individuals), live sperm and deformities. Estimation of serum testosterone The blood collection and separation of serum was done weekly and the level of testosterone was done according to the way of (18) Acrosomal integrity of sperm The integrity of the membrane of acrosomal was determined by using eosin nigrosine stain; 20 microliters of eosin nigrosine blended with 20 microliters of semen and then was smeared on a microscopic slide for evaluation under a phase-contrast microscope (19). Estimation of Glutathiones peroxidases GPx: Level of GPx in semen was estimated with spectrophotometer using Ransel reagent from Randox-Laboratory Ltd . (Crumlens,UK ). GPx activity was indicated as unit(U) / mL semen. Estimation of activity of catalase(CAT): This way designated by (22) which used for the assessment of activity of CAT in the semen sample. Statisticals analysis: SPSS-Version 19 was used for Data analyzing. Results The result in Table 2

v3-fos

2020-12-10T09:04:12.831Z

1970-03-01T00:00:00.000Z

237231904

s2

Effect of Temperature on Radiosensitivity of Newcastle Disease Virus Newcastle disease virus was irradiated at temperatures ranging from 2.2 to 60 C. An interaction between the thermal and ionizing energy was observed in the temperature region of 49 to 60 C. At 2.2 C, the hemagglutinin was considerably more radioresistant than the infectivity property. It is believed that radiation inactivation of Newcastle disease virus infectivity at low temperatures was due to nucleic acid degradation and at higher temperatures was due to protein denaturation. The effect of ionizing radiation on viral inactivation has been studied since as early as 1939 (5). The subject matter has been discussed in great detail by Lea (8) in his classic book, Action of Radiations on Living Cells, in which the target theory was developed. Research in this area has continued, but the main emphasis of this research has been on the use of ionizing radiation as a tool to study molecular organization of viruses, particularly in relation to biological function, or particle size, or for preparing vaccines. Little consideration has been given to radiation as a means of decontaminating virus-infected foods because of the relatively high sterilizing dose required (7) which results in undesirable organoleptic changes. Thus, any treatment that might sensitize virus to radiation warrants an investigation. It was reported that the survivors of X-irradiated T5 bacteriophage are more sensitive to heat (2). Adams and Pollard (1) similarly found that irradiation of Ti bacteriophage particles sensitized them to heat, but of more importance was the finding that a greater degree of inactivation occurred when the irradiation and heating were carried out simultaneously rather than sequentially. The purpose of the present study was to determine whether there is an effect of temperature on radiosensitivity of an animal virus, since the previous work had been confined to bacterial viruses. MATERIALS AND METHODS Test virus. The virus used was Newcastle disease virus (NDV). Allantoic fluids harvested 48 to 72 hr after infection of 10-day-old chicken embryos were pooled, centrifuged at 8,500 rev/min for 20 min, and stored at -40 C in 15-ml portions until used. Irradiation of samples. The irradiation was carried out with gamma rays from a Mark I United States Atomic Energy Commission Cobalt-60 Food Irradiator. Under the conditions of the experiment, the dose rate, as determined by ferrous-ferric dosimetry, was approximately 4,850 rad/min. Three-milliliter quantities of the infective allantoic fluid were filled into glass tubes (8 by 150 mm). For temperature control during irradiation, the tubes were placed in a stainless-steel jacketed vessel containing water tempered to the desired temperature. Constant temperature was maintained by circulating properly tempered water through the jacket. The entire apparatus was contained within an insulated steel chamber which was lowered into the gamma ray field for the prescribed time. The assay method for hemagglutinin and infectivity was reported in the preceding publication (3). RESULTS AND DISCUSSION In Fig. 1 are presented the curves for inactivation of NDV infectivity by irradiation at various temperatures ranging from 2.2 to 60 C (36 to 140 F). The logarithm of the survival fraction (V/Vo) has been plotted as a function of the irradiation dose in kilorads. It is of interest to note that for irradiation at the low temperature the inactivation curve was linear, whereas the inactivation curves for irradiation at the higher temperatures were nonlinear, consisting of a fast-inactivating component followed by a slower-inactivating component. This same pattern characterized the thermal inactivation curves for NDV infectivity (3). This result may indicate that the inactivation of NDV infectivity by simultaneous irradiation and heating was due principally to the thermal effects with the irradiation augmenting the effect of heat. The inactivation rate during DiGIOIA ET AL. irradiation at lethal temperatures is the integral sum of three different components, that is, the effect of irradiation, the effect of heat, and the effect of an interaction between irradiation and heat if one is present. On the assumption that these three effects are additive, the interaction between heat and irradiation in the inactivation of NDV infectivity was determined by subtracting the slopes of the heat inactivation curves (3) from the slopes of the radiation inactivation curves for the same temperature. The radiation inactivation rates thus corrected for heating effect were plotted as a function of irradiation temperature (Fig. 2). Since both the thermal inactivation curves and the irradiation-heating curves were two-component types, two curves are shown in Fig. 2, one representing the primary slopes (fast-inactivating component) and the other reresenting the secondary slopes (slower-inactivating component). In both cases, it was indicated that there was no effect of irradiation temperature on inactivation of NDV infectivity over the temperature region of 2.2 to 49 C, but at temperatures greater than 49 C there was a marked increase in radiosensitivity. The sharp increase in radiosensitivity that occurred in the temperature region of 40 to 50 C has also been reported for Ti bacteriophage (1) and Salmonella typhimurium (9). The following theory has been postulated for the synergism between thermal and ionizing energy. The loss of biological function of some macromolecule, such as protein, by thermal denaturation requires the rupture of at least three adjacent bonds, such as hydrogen bonds, which causes the molecule to open up and lose its biological configuration. Rupture of one of these bonds by irradiation would lessen the requirement for the number of bonds to be broken by the thermal energy (1). One may take an alternative approach to this problem and correct the irradiation-heating curves for the component effect caused by irradiation. This was done in the present study, and again the interaction effect was indicated at temperatures greater than 49 C. In 1953, Epstein (4) proposed that the radiosensitive matter of virus is the nucleic acid and this has since been confirmed. However, Wilson and Pollard (11), by using a technique of charged particle bombardment with carefully controlled penetration, deduced that the radiosensitive volume for NDV is about 20 % of the whole virus. Since the total ribonucleic acid (RNA) content of the virus (about 5%) would be expected to occupy less volume than this, they proposed that some of the radiosensitive material includes protein. In the present study, it was found that an irradiation dose of 1.25 X 106 rad at 2.2 C had no effect on the hemagglutinating property of NDV. Yet an irradiation dose of this magnitude at a temperature of 2.2 C affected a 7.5 logi0 reduction in infectivity of the virus. Thus, at low temperatures of irradiation, the hemagglutinin of NDV was more radioresistant than the infectivity property. Therefore, it can be concluded that inactivation was due principally to damage to the RNA. This finding has also been reported for other animal viruses (6,10,12). However, it is believed that loss of NDV infectivity by irradiation at lethal temperatures was mainly due to protein denaturation, although some RNA degradation may have also occurred. This supposition is predicated on the result that, when an Arrhenius plot of the radiation inactivation rate constants was made, a twocomponent curve was obtained. The break in the curve occurred at a temperature between 37.8 and 43.3C. This would indicate that two different mechanisms ofinactivation were involved over the temperature range investigated.

v3-fos

2018-12-21T13:25:08.370Z

1970-01-01T00:00:00.000Z

91027667

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RESPONSE OF PEARL MILLET TO INTEGRATED USE OF ORGANICS AND FERTILIZERS In the present study, the integrated organic fertilizers were used to estimate the growth and yield of pearl millet. The treatments consisted of application of increased levels of recommended dose of fertilizer with organic manures for pearl millet. The treatments were; T1-100% RDF, T2-125% RDF, T3-75% RDF, T4-100% RDF+FYM @ 12.5 t ha-1, T5-100% RDF+Poultrymanure@5tha,T6-100 % RDF+Vermicompost @ 5 t ha-1, T7-100 % RDF+Pressmud@5tha-1, T8-125% RDF+Poultry manure @ 5 t ha-1, T9-125 % RDF+Vermicompost @ 5 t ha-1, T10-125 % RDF+Pressmud @ 5 t ha-1, T11-75% RDF+Poultry manure @ 5 t ha-1, T12-75 % RDF+Vermicompost @ 5 t ha-1 and T13-75 % RDF+Pressmud @ 5 t ha-1. The experiments were laid out in randomized block design (RBD) and replicated thrice. The result of the experiment revealed that the application of 125% recommended dose of fertilizer+vermicompost@5tha(T9), significantly increased the growth, yield, quality and nutrient uptake of pearl millet followed by, T10 (125% recommended dose of fertilizer+Pressmud @ 5 t ha-1) respectively. INTRODUCTION Pearl millet [Pennisetum glaucum) is one of the important millet crop of hot and dry areas of arid and semi-arid climatic condition [1]. It has been estimated that pearl millet embodies a tremendous productivity potential particularly in areas having extreme environmental stress condition on account of drought [2]. Pearl millet grain is more nutritious with high protein of good quality. The grain contains 11-19 per cent protein, 60-78 per cent carbohydrates and 3.0-4.6 per cent fat good amount of phosphorus and iron [3]. India is one of the main producers of pearl millet [2]. It is a dual purpose crop of arid and semiarid areas as it provides cheap food, comparatively rich in various nutrients, protein, fat, carbohydrates and minerals for poor masses and feed for poultry birds as well as green fodder for cattle [4]. Recycling of agricultural and industrial wastes and utilization in agriculture as an alternative to fertilizer is promising [5]. Integrated use of chemical fertilizers with organics has been found to be quite promising in maintaining high productivity and greater stability for crop production [6]. Vermicompost improves microbial load in soil and increases microbial availability of phosphorus and nitrogen [7]. Green revolution in India witnessed phenomenal increase in fertilizer consumption and it may not be desirable to spend huge sum of money towards the import of fertilizers [8]. More-over, the present hike in the prices of chemical fertilizers has compelled the Indian farmers to resort to imbalance nutrition for their crops and thus reduction in crop yields. At this critical juncture, there is an urgent need to optimize nutrient recycling to sustain crop production without affecting soil health and protecting the environment. Keeping in view of the above situation, the present investigation was carried out to study the integrated organic fertilizers were used to estimate the growth and yield of pearl millet. Research methodology A field experiment was conducted during 2016 at Venganur village, Perambalur district, Tamil Nadu with pearl millet cv. CO-7 to study the effect of integrated nutrient management on yield of pearl millet and soil health. The experimental soil was clay loam with a pH of 7.85, EC of 0.43 dSm -1 and CEC of 30.40 cmol (p + ) kg -1 . The available nitrogen, phosphorus, potassium and sulphur contents were 272, 14, 255 kg ha -1 and 12.5 mg kg -1 respectively. The exchangeable calcium, magnesium, potassium and sodium contents were 8.8, 7.9, 6.9 and 6.5 cmol (p + ) kg -1 respectively. The treatments consisted of application of increased levels of recommended dose of fertilizer with organic manures for pearl millet. Physico-chemical properties of initial experimental soil The initial soil was analysed for the physico-chemical properties and the results are furnished in table 1. The soil of Venganur village was found to contain 51.20, 11.10, and 36.80 per cent sand, silt and clay respectively, being to the textural class of clay loam. The bulk density, partial density, pore space, pH, electrical conductivity and cation exchange capacity of the soil were 1.25, 2.60 Mg m -3 37.5per cent, 7.85, 0.43 dSm -1 and 30.4 cmo (p + ) kg -1 respectively. The organic carbon content of soil was 0.51 g kg -1 . The available N, P and K content of soil was 271, 14 and 255 kg ha -1 respectively. The available Sulphur content was 12.5 mg kg -1 . The exchangeable calcium, magnesium, potassium and sodium content were 8.8, 7.9, 6.5 and 6.9 cmol (p + ) kg ha -1 respectively. Number of tillers hill -1 Among the different treatments tried, the maximum number of tillers per hill was noticed with application of 125% recommended fertilizer+vermicompost @ 5 t ha -1 (T9) recording 7.47 and 8.40 at 60 and 90 DAT respectively. This was followed by T10 (125% recommended fertilizers+ pressmud @ 5 t ha -1 recording 6.99 and 7.88 at 60 and 90 DAT respectively. This treatment was followed by T8, T6, T7, T5, T4, T2, and T1. The least number of tillers per hill -1 1.77 and 2.19 at 60 and 90 DAT respectively were recorded in control (T3). These results are in agreement with findings of Chellamuthu and Agrawal [10] who also reported significant improvement in growth parameters of pearl millet due to fertilizer application. Other findings [11,12] also supports these findings. Grain yield The data recorded on grain yield are presented in table 3. Among the various treatments tried, recommended 125% NPK + vermicompost @ 5 t ha -1 (T9) recorded the highest grain yield of 3,445 kg ha -1 . The treatments next in order were T10, T8, T6, T7, T5 and T4. The least grain yield of 1,140 kg ha -1 was observed under T3 (75% RDF) Stover yield The recorded data on stover yield are presented in table 3. The stover yield was significantly influenced by the application of 125% RDF+vermicompost @ 5 t ha -1 (T9), which registered the highest stover yield of 7,530 kg ha -1 . The treatments viz. , T10, T8, T6, T7, T5, T4, T12, T13, T11, T2 and T1, stood next in order of ranking. The treatment T3 75% RDF registered the lowest stover yield of 2,296 kg ha -1 The yield attributes like earhead weight, length and girth were significantly increased by the application of 125% recommended dose of fertilizers+vermicompost @ 5 t ha -1 (T9), followed by the application of 125% recommended fertilizers + pressmud @ 5 t ha -1 (T10). The increased level of fertilizer with organics significantly increased the yield components of pearl millet. Similar effect of integration of inorganic and organics sources of plant nutrients on crop production was earlier reported earlier [13][14][15]. Significantly, the higher grain yield was recorded with 120 kg N ha -1 over rest of the levels except 90 kg Nha -1 . The higher grain yield could be due to cumulative effect of improvement in yield attributes viz., number of effective tillers plant -1 , ear head length, thickness and test weight. Further, yield improvement was possible on account of better nitrogen use efficiency as was evident from the higher nitrogen uptake under this level. Stover yield was also significantly increased up to 120 kg Nha -1 . The improvement in stover yield was mainly on account of increase in growth parameters due to higher level of N 120 kg ha -1 . These data are in agreement with earlier findings [11,[16][17][18]. CONCLUSIONS AND RECOMMENDATIONS Based on the results of the present field experiment, it is concluded that, the application of 125 % recommended fertilizer+vermicompost @ 5 t ha -1 was significantly superior in performance with respect to growth, yield attributes, yield and quality attributes of pearl millet and found to be effective in improving the post-harvest soil fertility and nutrient uptake by pearl millet. It can be recommended to farmers to achieve more benefit out of giving pearl millet as a poor man crop. Mg (cmol (p + ) kg -1 ) 7.9 C Na (cmol (p + ) kg -1 ) 6.5 D K (cmol (p + ) kg -1 ) 6.9

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2018-04-03T06:23:06.960Z

1970-07-01T00:00:00.000Z

24785008

s2

Detection and Incidence of Escherichia coli on Storage Pen Surfaces of Fishing Trawlers Six methods for the detection and enumeration of Escherichia coli on the storage pen surfaces of commercial fishing trawlers and in harbor wash water were evalu- ated. E. coli was found consistently present in Boston harbor water used for washing vessel holds and was detected either in small numbers or not at all on storage pen surfaces. Violet Red Bile Agar as a primary enumeration medium was found ineffective for detection of coliforms because of the nonselective development of large numbers of other gram-negative organisms. The use of E. coli broth at 44.5 C for primary most-probable-number determinations, followed by confirmation of E. coli on Levine Eosine Methylene Blue Agar, appears to offer numerous advantages over more conventional methods of detecting E. coli for survey studies of the fishing industry, where coliform-like organisms result in many false-positive presumptives with other methods. The common practice in the New England fishing industry of using raw, untreated harbor water for washing trawler fish storage pens is of sanitary and public health significance. Such water is usually associated with sewage pollution arising locally or from large-volume municipal sewage disposal in the same locale. The industry practice of briefly hand-scrubbing pen surfaces and rinsing with untreated harbor water was previously shown (4) to achieve no detectable reduction in the bacterial load. This study is concerned with the enumeration of Escherichia coli on storage pen surfaces after washing with unchlorinated harbor water and with the examination of the various schemes available for detection and confirmation. MATERIALS AND METHODS Sampling. Alginate wool swabs were used with stainless-steel templates to obtain 1-square inch swab samples (ca. 2.54 by 2.54 cm). Immediately after all of the fish in the storage hold of a vessel were unloaded, three swab samples were obtained from different panels of the same pen and dissolved in 20 ml of 1% Calgon. Three swab samples were again obtained adjacent to the first areas sampled after the storage surfaces of the hold and pens had been hosed down and scrubbed with unchlorinated harbor water in the usual manner by the cleanup crew. Water samples were also collected from the hose used on board the vessels for washing the storage pens and from the water source itself, the harbor water adjacent to the vessels. Bacteriological tests were performed within 24 hr after samples were obtained. Bacteriological methods. Decimal dilutions of dissolved swabs and water samples were made in Nutrient Broth containing 0.5% NaCl. The detection procedures were separated into six groups, each designated with a Roman numeral (Fig. 1). Difco media were used throughout. In method I, Lauryl Tryptose (LT) Broth was used for five-tube most-probable-number (MPN) presumptive determinations for coliforms. Tubes showing gas after 48 hr of incubation at 35 C were used to inoculate tubes of Brilliant Green Lactose Bile (BGB) Broth, followed by the transfer of resulting growth onto Levine Eosine Methylene Blue (EMB) Agar and incubation at 35 C for 24 hr; the combinations of these procedures served as the confirmed test. Typical colonies of E. coli were picked and transferred onto slants of Nutrient Agar (NA), from which the indole, methyl red, Voges-Proskauer, and citrate (IMViC) tests were performed. Method II made use of five-tube MPN determinations by using tubes of BGB incubated for 48 hr at 35 C, followed by confirmation with EMB and the IMViC tests. In method III, plates of EMB Agar were inoculated directly with 0.1 ml of diluted samples which were spread with a sterile glass rod and then incubated at 35 C for 24 hr. Colonies having the typical appearance of E. coli were picked for IMViC tests. Method IV consisted first in transferring growth from all positive LT MPN tubes from method I to tubes of Escherichia coli (EC) Broth followed by incubation at 44.5 C for 48 hr. The growth from positive tubes was then streaked onto EMB Agar plates, and colonies typical of E. coli were transferred to NA slants from which the IMViC tests were performed. RESULTS Comparison of five methods for detection and enumeration of E. coli from pen surfaces and wash water. Two vessels were used for this study ( Table 1). Method IV was found to be the method of choice for enumeration of E. coli in wash water. Only methods I, II, and IV successfully recovered E. coli from pen surfaces. The use of VRB Agar as the primary medium in method V proved highly unsatisfactory for detection of E. coli in both wash water and on pen surfaces because of the development of large numbers of red coliform-like colonies, some of which exhibited zones of precipitation typical of E. coli which failed to grow in BGB Broth or EMB Agar. Considerable bifficulty was encountered with the use of EMB Agar as the primary medium in method III because of the extensive growth of organisms other than coliforms. With all five methods used, E. coli was detected in only low numbers on pen surfaces or not at all, which is most likely accounted for by the low, though consistent, incidence of E. coli in hose and harbor water ( Table 1). Incidence of coliforms and E. coli on pen surfaces and in wash water. Eleven vessels were examined between October 1967 and October 1968 by using method IV. E. coli was detected on only two vessels before washing and on three after washing. The highest number of E. coli detected was 2.6 per square inch. The samples of hose and harbor water all yielded the consistent presence of E. coli and were highest in harbor water (harbor water, 1 to 35 per ml; hose water, 0.5 to 22 per ml). Effectiveness of EC Broth as a primary enumeration medium for E. coi. The effectiveness of EC Broth incubated at 44.5 C as the primary medium for MPN determinations for E. coli was compared to the conventional method with LT Broth at 35 C. Four trawlers were examined in this study ( Table 2). E. coli was detected on two vessels before washing and on three after washing (before, 0.3 to 1 per square inch; after, 1 to 2.7 per square inch). The samples of hose and harbor water all yielded E. coli (harbor water, 5 to 35 per ml; hose water, 4 to 21 per ml). No significant loss in the final number of confirmed E. coli was encountered with the use of EC Broth at 44.5 C, which offered the advantages of (i) eliminating many false-positive presumptives otherwise obtained with LT Broth, (ii) eliminating 1 additional day of incubation, and (iii) reducing considerably the amount of media and effort involved in obtaining final confirmation and in determining the identity of organisms producing gas in positive presumptive tubes. DISCUSSION Various methods and media are presently in use for enumerating coliforms and E. coli in food products and water. To determine the MPN values of coliforms in water, the Canadian Department of Fisheries (2) recommends Lactose Broth followed by confirmation with BGB Broth; for estimating the density of E. coli on fish fillets, the suggested method is the direct inoculation of dilutions into MPN tubes of BGB Broth, followed by confirmation in EC Broth at 45 C. The American Public Health Association (1) recommends the enumeration of E. coli and coliforms in water by performing a presumptive test in LT Broth, followed by confirmation with BGB Broth or EMB Agar and completing the test with the Gram stain and observing for gas production in LT Broth. Our results indicate that the direct inoculation of diluted samples into EC Broth incubated at 45 C appears to be the method of choice for the enumeration of E. coli in fishery products and untreated harbor water used for washing vessels. VRB Agar is recommended by some workers for selectively detecting coliforms from food products of nonmarine origin. Lewis and Angelloti (5) recommend VRB Agar as a primary solid enumeration medium for detecting coliforms in foods, followed by confirmation in BGB Broth; for E. coli, they recommend MPN determinations in LT Broth, followed by confirmation in EC Broth incubated at 44.5 C. Hartman (3) observed a great variation in the percentage of confirmed coliforms when various foods were tested by use of VRB Agar as the primary detection medium. Our results indicate that VRB Agar is ineffective as a selective medium for the direct enumeration of coliforms from fishery sources and that the term coliform has no significance in the fishing industry since many gram-negative organisms gave rise to APPL. MICROBIOL. false-positive presumptive tubes of LT Broth and BGB Broth and mimicked coliforms on VRB Agar.

v3-fos

2017-10-19T03:32:48.093Z

1970-12-01T00:00:00.000Z

24759688

s2

Effect of Trace Elements on Citric Acid Fermentation by Aspergillus niger Citric acid yields of 98.7% (sugar consumption basis) were reached in shaker flasks with mutant UV-ET-71-15 of Aspergillus niger in a resin-treated sucrose medium of the following composition (g/100 ml): sucrose, 14.0; NH4NO3, 0.20; KH2PO4, 0.10; MgSO4.7H20, 0.025; and (mg/liter): FeSO4, 0.15 to 0.75; ZnSO4, 0.10; and CuSO4, 0.01. Yields of 75% were obtained in medium with resin-treated clarified syrup and 68% with ferrocyanide-treated blackstrap molasses. Optimal conditions included selection of appropriate pellets as inoculum at 3%, pH of 4.5, temperature at 30 C, agitation at 250 rev/min, and fermentation time of 8 days. The mutant tolerated high concentrations of trace elements. Among the problems associated with citric acid production by strains of Aspergillus niger under submerged growth conditions, those related to the control of the trace-element concentration play an important role. Recent contributors to this subject include Noguchi and Johnson (6), Millis et al. (5), and Clark (1). The optimal concentrations of such ions as Fe2+, Cu2+, Zn2, and Mn2+ vary so widely with the different strains under study that it is necessary to adjust the composition of the medium to avoid the inhibitory effects caused when these cations are present in toxic concentrations. Some of the methods we have previously used to solve this problem include (i) selection of tolerant strains, (ii) addition of substances to decrease the trace-element content of the fermentation media, (iii) treatment of the carbohydrate raw material to minimize inhibition effects, and (iv) development of potentially high-yielding mutants with a higher tolerance to trace elements. The present paper deals with the utilization of a high-yielding mutant of A. niger, previously obtained (10) by a combination of ultraviolet (UV) and ethyleneimine treatment, to study the effect of specific trace elements and phosphate on the citric acid fermentation of treated and untreated sugar sources. MATERIALS AND METHODS Raw materials. Refined commercial sugar from two types of sugar mills (producing sugars with relatively highor low-trace-element content), blackstrap molasses, and sugar cane liquor ("clarified syrup," International Society of Sugar Cane Technologists terminology) were studied. The blackstrap molasses was used either as received or after treatment with K4Fe(CN)6 by the method of Horitsu and Clark (3). The sucrose and clarified syrup, at a 14% concentration (sucrose basis), were passed through a glass column of Amberlite IR-120 prepared as follows. Appropriate amounts of the resin were washed in a glass column and were then washed successively with 2 N HCI, redistilled water, 1 N redistilled NH40H, and redistilled water. Strains. Aspergillus niger ATCC-233; the parent strain M-172, which was isolated from a Columbian soil (7); and two mutants, UV-6 and UV-ET-71-15, obtained from the latter (10) were transferred from the soil stock cultures through several successive potato dextrose agar slants to sucrose-salts agar slants. Mutant UV-ET-71-15 does not sporulate well in the usual media but does produce abundant spores in the following (g/100 ml): sucrose, 0.25; meat extract, 1.0; NaCl, 0.5; and agar, 1.5. After 4 to 6 days of incubation at 28 C, the spores were suspended in 10 ml of sterile distilled water to prepare the inoculum. Preparation of inoculum. The spores were harvested. Suspensions were prepared and standarized at a concentration of 107 to 2 X 107 and were then used to inoculate 50 ml of sterile seed media in 250ml Erlenmeyer flasks. These were incubated at 28 C on a rotary shaker at 250 rev/min for 17 to 24 hr. After that time, those flasks showing good formation of small, firm, spherical pellets were selected as inoculum for the fermentation media (3%, v/v). Seed media. Different seed media were tested and the following was finally selected (g/l00 ml): sucrose, 6; NH4NO3, 0.25; KH2PO4, 0.1; MgSO4.7H20, 0.025; agar, 0.2; and distilled water, 100 ml (pH 4.5). Fermentation media. Two previously reported media [A-1 and G (10)] and three basic media, as shown in Table 2, were first tested in a screening procedure by using the UV-6 mutant as a standard 888 for citric acid production in 250-ml shaker Erlenmeyer flasks at 250 rev/min with 50 ml of medium. When sucrose was not resin treated, either no trace elements or small quantities were added depending on the amounts initially present. All fermentation media were adjusted to pH 4.5 (except molasses at pH 6.8) and incubated at 30 C for 8 to 10 days. Estimation of growth and chemical determinations. Dry mycelial weight, pH, residual reducing sugars, citric acid production, and the presence of other acids were estimated by published methods (7,8,9). Citric acid yields were calculated as the anhydrous acid on the basis of sugar consumed. Oxalic and gluconic acids were detected by paper chromatography. The cations in the raw materials were determined by the methods of Noguchi and Johnson (6). The effect of resin treatment on the ion content of raw materials was estimated by means of absorption spectrophotometry. All cation values of the raw materials are expressed in ,ug/ml; those of culture media are in mg/liter. Table 1 gives the trace element content (tg/ml) of the raw materials used: commercial white sugar with relatively highor low-trace-element content, blackstrap molasses, and clarified syrup. Blackstrap molasses showed the highest content of trace elements. The concentration of the cations Fe2+, Zn2+, and Cu2+ tolerated by the UV-ET-71-15 mutant were 2.2, 3.8, and 0.5 mg/ liter, respectively, giving yields of citric acid from 3.0 to 5.0 g/100 ml in 10 days. It is apparent that this culture is highly tolerant of trace elements. RESULTS The UV-6 mutant was tested in the five different media given in Table 2. Untreated white sugar with a high content of trace metals was used in medium A-1; methanol and corn steep liquor were present in medium A-1; and small amounts of trace elements were in medium G. The highest yield was obtained in medium G. In the three other media, untreated white sugar with a low content of trace elements was added. These media differed in concentrations of NH4NO3, KH2PO4, and MgSO4. Results were inferior to those obtained with media A-1 and G, indicating that the addition of trace elements (either as such or present in corn steep liquor) resulted in better citric acid production. Similar results were obtained with mutant UV-ET-71-15. The trace elements of medium G were added to medium 3, and the citric acid yields of the parent strains (233 and M-172) and the two mutants (UV-6 and UV-ET-71-15) were determined. Data, as shown in Table 3, indicate the UV-ET-71-15 mutant to be superior with an average citric acid yield of 98 %. Instead of sugar, untreated molasses and mo- lasses treated with K4Fe(CN)6 were tested in medium 3 and in medium 3 with trace elements added. The yields obtained with mutants UV-6 and UV-ET-71-15 were higher in both media with the treated molasses; they were also higher when low concentrations of trace elements were added (Table 4). Resin treatment of sucrose, without added cations, also resulted in higher yields than the untreated sucrose in media 1 and 3 (Table 5); medium 3 was slightly superior. The effect of resin treatment on the traceelement content of sucrose and clarified syrup is shown in Table 6. Very small amounts of Fe2+ Cu2+, and Zn2+ remained after treatment, and Mn2+ was undetectable. The effect upon citric acid production of the addition of trace elements to media 1, 2, and 3 is shown ( Table 7). The tolerance of the UV-ET-71-15 mutant to high cation concentrations is again apparent (medium 2), and the best yields (62%) were once again obtained in medium 3. When the sucrose was resin treated and trace elements were added (Table 8), yields as high as 98 % in 8 days were reached in these modifications of either medium 1 or 3. Iron did not appear to be particularly critical for the UV-ET-71-15 mu- tant. In experiments with mutant UV-ET-71-15 and medium 3, neither oxalic nor gluconic acids Table 7. R = resin treatment; K = treatment with K4Fe(CN)6. bLow content of trace metals. c Values represent mean at 8 days in five tests. were detected by paper chromatography of the fermentation broths with the usual concentration of KH2PO4 (1.0 g/liter). However, when higher amounts of KH2PO4 (2.5 g/liter) were used, these other acids were present ( Table 9). The yields of citric acid were between 48.5 and 66.7% with untreated and resin-treated sucrose, respectively, at a KH2PO4 level of 0.5 g/liter. No cations were added in these cases. In other studies, blackstrap molasses and clarified syrup behaved in a similar manner as regards the response to prefermentation treatment. Several tests showed that the yields of citric acid in media with clarified syrup were better than molasses but not as good as white commercial grade cane sugar under the conditions of the experiments. The best yields obtained with these three substrates and the mutant UV-ET-71-15 are shown in Table 10. DISCUSSION Trace elements. The importance of trace elements in the submerged citric acid fermentation of raw commercial sugar substrates has been repeatedly pointed out (1,2,5,6,7,9). In the present investigation, great variability was found in the Fe2+, Zn2+, and Cu2+ content of the raw materials used. Reproducibility of the citric acid yields was not accomplished until the trace-element concentration of the medium was properly controlled. Although very small amounts of these trace elements were apparently necessary for good citric acid synthesis by the mutants under study, these cultures were capable of resisting unusually high cation concentrations (2.2, 3.8, and 0.5 mg of Fe", Zn2, and Cu2 per liter, respectively) in addition to those already present in the sugar substrate itself. The largest amounts of citric acid were, however, repeatedly obtained when only low concentrations of these three cations were added to the medium. Control of the trace element concentration has ensured reasonable reproducibility of the fermentation yields. Sugar treatment. The removal of excess trace elements by either ferrocyanide or resin treatment of sugar followed by the proper addition of trace elements was associated with good and reproducible citric acid yields. Our results confirm previous observations (3,4,6,7). Mutants. The development of mutants of A. niger allowed a higher average yield from all the crude substrates tested than the parent culture. Mutant UV-ET-71-15, obtained by a combined treatment of UV and ethyleneimine (10), was superior to the UV-6 mutant as well as to the parent strain. This culture has resisted higher concentrations of trace elements, thus resembling the mutants obtained by Gardner et al. (2) and Millis et al. (5), and has maintained its properties and characteristics after a storage period of 2 years in sterile soil. In the fermentation media selected, this mutant developed small pellets with a morphology similar to that described by Martin and Waters (4). Inoculum. The type and concentration of the inoculum was critical, with best results depending on the selection and number of pellets.

v3-fos

2019-09-17T02:47:06.051Z

2019-07-29T00:00:00.000Z

202902108

s2

Research results of local buckwheat varieties and forms of Ukrainian origin The national collection of buckwheat in Ukraine consists of 578 local samples and forms of Ukrainian origin added into the collection during 1929 to 2012. In 1994-2000 and 2014-2018, the material has been studied and described, the common characteristics of samples peculiar of a certain area of origin, feature manifestation peculiarities, material flexibility and stability of the quantitative and qualitative characteristics depending on the weather in growing years have been identified. The range of studies included sample performance indicators, architectonic values, grain characteristics, and so on. A common characteristic of local buckwheat varieties is a significant sensitivity to changes in growing conditions, which is reflected in the change of growth processes (increase in the plant height by lengthening of internodes, the number of branches of the second and higher orders, the number of leaves per plant) and adjustment of the growth duration (lengthening the duration of the growing season after the onset of more favorable conditions for growth and development). The performance indicators related to the grain characteristics are quite stable. However, the general trends characteristic of plants from a certain origin of the collection material remained unchanged. Tryhub, (2019): Local buckwheat varieties of Ukrainian origin 24 INTRODUCTION Buckwheat has not become one of the most demanded crops by the international community yet. The main reason is its limited distribution around the world and low yields. However, the conclusions made in recent years concerning the maintenance of full-fledged human life, health and outstanding buckwheat plant properties indicate a significant need in buckwheat products (Kreft, 2010, Alekseeva at al., 2005). Gradually, the buckwheat products are distributed from the major regions of its traditional consumption to the new areas, where it gains the status of the core component of a healthy diet and becomes the foundation for a healthy lifestyle. As a result of in-depth study of biochemical composition of buckwheat grant and the whole plant, involving new types of crops into the research, the scientists around the world have greatly expanded the application areas of buckwheat products from traditional to exotic. To carry out this work, the genetic material with extensive properties and characteristics, most of which is concentrated in banks of plant genetic resources, is used (Alekseeva at al., 2004). Genetic diversity preservation has long been an imperative of global importance. The work to create the banks of genetic plant resources, launched more than a hundred years ago, not only validated the feasibility of this direction development, but also confirmed an unvalued contribution to ensuring food security worldwide (Alexanian, 2003). To solve scientific problems, the collection material of various eco-geographical origin and biological status is critical. However, given the importance of addressing the issue of the plant productive potential realization in contrasting environmental conditions, the study of local varieties and forms, the research of adaptive mechanisms and biochemical plant components became of paramount importance. MATERIALS AND METHODS A part of the National Buckwheat Collection in Ukraine, which is stored in Ustymivska Experimental Station of Plant Production (Poltavs’ka region), includes over 1,600 authentic samples, of which 1.1 thousand samples are the local varieties and forms of the common buckwheat (Fagopyrum esculentum Moench.) (Tryhub at al., 2015). 578 samples in the collection have Ukrainian origin. The material included in the collection is the result of its collection by employees of the N. I. Vavilov All-Union Institute of Plant Genetic Resources (VIR, Leningrad) during the missions (Fesenko at al., 2006), by researchers of Ternopil Breeding and Research Station, the Research Institute of Agriculture and Livestock of the Western Regions of Ukraine led by Olena Alekseeva (Alekseeva, 1967), as well as employees of the National Center for Plant Genetic Resources of Ukraine, assisted by the Ustymivska Experimental Station of Plant Production (Kirjan at al. 2014) in the territory of Ukraine from 1929 to 2012. As regards the ecological and geographical origin, there are samples from 18 regions. These are the representatives of the Carpathian region, Woodlands, Forest Steppe and Steppe zones. The overwhelming majority is the samples from the North, Central and Western parts of Ukraine, and a small number comes from the Eastern and Southern regions (see Table 1 and Pic. 1). Natural-climatic zones and regions of Ukraine Number of accesses Forest Steppe 294 Kyivs’ka (part) 12 Sums’ka (part) 60 Khmel’nyts’ka 11 Ternopil’s’ka 34 Cherkas’ka 9 Poltavs’ka 70 Vinnyts’ka 58 Kharkivs’ka 40 Steppe 20 Kirovohrads’ka 8 Dnipropetrovs’ka 4 Odes’ka 8 Polissya and Carpathian region 264 Zakarpats’ka 8 Ivano-Frankivs’ka 19 L’vivs’ka 36 Rivnens’ka 11 Volyns’ka 8 Chernihivs’ka 128 Kyivs’ka (part) 8 Sums’ka (part) 24 Zhytomyrs’ka 22 Total 578 Table 1. Distribution of origin of the local sample collection by climatic zone of Ukraine Fagopyrum 36(1):23-29 (2019) 25 The research of the collection material of buckwheat varieties and forms was carried out as required by the “Complete unified classifier of the genus Buckwheat (Fagopyrum Mill.)” (Tryhub at al., 2013), “Descriptors for buckwheat (Fagopyrum spp.)” (Descriptors..., 1994) , “Guidelines for the study of collection samples of corn, sorghum and groats” (Krotov, 1968), “Analysis of the structure of buckwheat plants (Methodical recommendations)” (Bochkareva, 1994). The material study and description was carried out in several stages during 1994-2000 and 2014-2018, and the results obtained were compared to the standard variety of Ukrainka. The common characteristics of samples peculiar of a certain area of origin, feature manifestation peculiarities, material flexibility and stability of the quantitative and qualitative characteristics depending on the weather in growing years have been identified. The range of studies included the sample performance indicators (the number of seeds and buds per plant, inflorescence yield), architectonic parameters (plant height, number of branches and inflorescences, height of attachment of the inflorescences and branches, the length of the branching and grain production zone), grain characteristics (grain size, evenness and husk content), the length of the growing season and its components, and so on. RESULTS AND DISCUSSION To obtain high and stable buckwheat yields, it is required to continue creating new varieties combining high performance, friendly maturation, resistance to drought, temperatures below zero, lodging, grain shattering, pests and diseases, as well as a high grain quality. For this purpose, in selection, as noted by N.I. Vavilov, we need to use the local material subjected to prolonged exposure to natural selection and adapted to specific conditions. This material has great value, and is widely used in selection (Korynyak at al., 2017). It should be noted that in terms of soil and climatic conditions, the research region, namely the south of Poltavs’ka region (central part of Ukraine, Forest Steppe zone) is one of the most favorable for growing buckwheat. Still, it has recently been affected by the ongoing significant climate change towards higher temperatures during flowering and insufficient precipitation during initial growth, which is typical for most areas of buckwheat cultivation in Ukraine. These trends are typical for most regions of the world where the buckwheat is sown. The features of sign manifestation were identified based on a set of researches conducted in two stages. The samples were studied following a three-year cycle during 19942000 and 2014-2018. The first cycle of study (19942000) was remarkable for more favorable weather conditions with precipitation and temperature regime of the growing season close to the average long-term data. The second cycle of study (2014-2018) was characterized by a high temperature in summer and a significant lack of moisture in the spring. The data obtained in the course of study indicate a considerable variety of feature manifestations by the local plant varieties and forms. There is a considerable dependence in the levels of feature manifestation on climatic zones of the sample origin. A significant impact on the plant characteristics was exercised by the climatic factors, precipitation and temperature regime during the years of cultivation. However, a set of the studied traits showed the characteristic, genetically determined features of samples and allowed describing the quantitative and qualitative material characteristics. Given the considerable heterogeneity of the climatic zones of Ukraine in terms of soil conditions, heat and precipitation, the entire buckwheat gene pool of these areas has been further divided into smaller, but more similar regions. To characterize the samples from the western regions of Ukraine, the classification proposed of Olena Alekseeva (Alekseeva, 1999) was applied. For other areas, the author’s classification was applied. Fig. 1. Territorial distribution of origin of the local buckwheat samples (name of district and number of samples). Tryhub, (2019): Local buckwheat varieties of Ukrainian origin 26 Samples from Forest Steppe of Ukraine In terms of soil and climatic conditions, the Forest Steppe Zone was subdivided into western, central and eastern parts, each with its own characteristics, significantly differing among themselves, but with rather similar buckwheat growing conditions within each part. The western part includes the samples from Khmel’nyts’ka, Ternopil’s’ka and Vinnyts’ka regions, having more precipitation as compared to the other parts of the Forest Steppe Zone, and their more even distribution during the vegetation period, lower average daily temperatures and fertile soils. The central part of the Forest Steppe (Cherkas’ka, Poltavs’ka, Sums’ka and Kyivs’ka regions) is characterized by harsher weather conditions in terms of the temperature and humidity. Here is sufficient precipitation during the growing season, but its distribution is very uneven. The cover in most of the area is presented with fertile soil with excellent quality characteristics. The eastern part of the Forest Steppe zone includes Kharkivs’ka region and the south of Sums’ka region. This part shows the most extreme weather conditions with little precipitation and high temperatures, especially during the buckwheat flowering and graining. In general, the gene pool of this region is represented by 294 samples. The samples of this group are the most malleable genetic material grown in the area with conditions, which are the most suitable for the buckwheat cultivation. Such a genepool was the source material for most of the modern high-tech varieties, and today the scientists are searching for the forms remarkable for their performance indicators combined with resistance to abiotic environmental factors, grain quality, and so on primarily among these samples. The samples from the western part of the Forest Steppe Zone of Ukraine show an extensive diversity in terms of the key parameters of the plant organism structure, which requires their subdivision into two subgroups by the growing season duration, i.e. mid-late-ripening (78-85 days) and early-ripening (70-77 days). Mid-late-ripening samples form tall plants (1.5 m high) with a large number of internodes (9-13 pcs.), a large number of leaves and branches (9-16 pcs.). Lower interstitials are thickened, forming resistance to lodging. The samples’ yield is 180-220 g/m2, and the productivity is 1.8-2.6 g/plant. They have medium-sized grain, up to 25.6 g/1000 grain with uniformity up to 82% and husk content 22.7-23.5%. The early-ripening samples have the plants 85-110 cm high with 10 internodes and the ratio of grain formation zone vs. branching zone as 1.0-1.3. The plants form a large number of inflorescences, up to 55 pieces per plant. Samples have large dark brown grain as for the local forms; the weight of 1,000 grains is 25.826.9 g, the husk content increased to 23.8%, and a good inflorescences uniformity, i.e. 85%. The yields of these samples is within 165-200 g/m2, and the productivity is 1.6-2.2 g/plant. Plants have a significant number of leaves and the medium number of branches (5-8 pcs.), are resistant to lodging and moderately resistant to grain shattering after ripening. The central part of the Forest Steppe Zone has a more form genepool in terms of manifestation of quantitative and qualitative traits of the buckwheat plant organism. Local varieties from this part feature the medium-term ripening (70-80 days), medium and large plant height (100-150 cm), 7 to 16 internodes on the main stem and the ratio of the graining zone vs the branching zone as 1.1-1.3. The plants from this group are resistant to lodging, have many leaves and 8-14 branches (including 5-8 first-order branches) and 85 buds per plant. The grain of these samples is gray and dark brown with an average weight of 1,000 grains as 24.2-27.3 g, grain husk content as 22.1-23.5%, and 90% uniformity. The average grain yield varies between 185-236 g/m2, and the productivity – within 1.8-2.8 g/plant. The plants from the local samples of eastern part of the Forest Steppe of Ukraine are potential sources of the variety resistant to abiotic environmental factors (high temperatures and drought). Samples from this part form the medium-term ripening (75 days) plant up to 120 cm high with 5-9 internodes, 5-12 branches and 65 buds per plant. These samples are characterized by high resistance to lodging and medium resistance to the grain shattering. Grain has a dark brown color with a clear or blurry pattern in the form of dots or dashes, the weight of 1,000 grains is 23.5-26.7 g, the medium husk content (up to 23.1%) and evenness (75%). The yield of samples from this group is 168-218 g/m2 with the plant performance at 1.45-2.05 g/plant. Samples from Polissya and Carpathian region The material from this zone also features extensive diversity due to a significant difference between the weather, climate and soil conditions within the region. The samples from this area were subdivided into 3 groups: the north-western, western, northern and Carpathian. Fagopyrum 36(1):23-29 (2019) 27 The north-western part includes the samples originating from Volyns’ka and Rivnens’ka regions, the climate in which is characterized by excessive precipitation, moderate temperature conditions and poor nutritional composition of their soils. The western part (Ivano-Frankivs’ka, L’vivs’ka and Zhytomyrs’ka region) is characterized by a moderate precipitation and temperature conditions favorable for plant growth and development, with soils of varying fertility. The northern part of the Polissia zone (Chernihivs’ka and a part of Kyivs’ka and Sums’ka regions) has the most fertile soils in this region, sufficient precipitation and heat. The genepool of the Carpathian region (Zakarpats’ka and a part of Ivano-Frankivs’ka region) includes representatives of foothill and mountain areas, featuring moderate and sometimes low temperature during the growing season, often excessive precipitation and poor soils. The collection of local varieties and forms from this region includes 264 samples. The samples of the north-western part of the region are characterized by a short growing season, low (1.2 m) stem with 4-5 internodes; slight branching (3-7 branches, of which 2-3 are the first-order branches) with a ratio of the graining zone vs the branching zone as 0.8, the medium number of leaves, the sample yield at 120-180 g/ m2 per plant and the productivity as 0.8-1.0 g/plant; the medium grain particle size (up to 23.5 g/1000 grains), uniformity (80%) and husk content (up 23.5%), brown grain with a clear patter; resistant to lodging and having a medium degree of grain shattering after ripening. The samples from the western part feature a significant heterogeneity by the growing season duration; the samples vegetation varies from early-ripening (70 days) to late-ripening (90 days), and respectively, the plant height and the number of internodes ranged from 80100 cm and 5-7 pieces. to 125-130 cm and 9-12 pieces. In general, all samples were dark brown and gray, with medium and large grain size (23.7-26.8 g/1000 seeds), medium evenness (85%) and high husk content (24%); the plants have many leaves with a medium degree of grain subsidence and lodging. The samples’ yield was 175-210 g/m2, and the plant productivity – 1.6-2.2 g/plant. The samples from the northern part feature a high yield (up to 250 g/m2) and plant productivity (up to 3.0 g/plant); large grain (24.5-26.4 g/1000 grain), good evenness (up to 85%), and medium husk content (22.623.1%), brown and dark brown color. The plants of these samples are tall (1.2-1.5 m) with a large number of branches (13-18 pcs.) and buds (70 pcs.) per plant, as well as many leaves. The stem is thick and resistant to lodging. The samples from the Carpathian region (foothill areas of Zakarpats’ka and Ivano-Frankivs’ka regions) are characterized by a considerable length of the growing period (80-90 days) and tall plants (150 cm) with a significant number of branches (15 pcs.) and internodes (10-13 pcs.); the ratio of the graining zone vs the branching zone is 0.9-1.1; the plants have a large number of leaves and buds (80 pcs.); yield at 180-200 g/m2 and productivity up to 2.2 g/plant; the medium grain size (up to 25.1 g/ plant), medium husk content (23%) and evenness (85%). The plants are remarkable for a low resistance to plant lodging and grain shattering. Samples from the Carpathian mountain areas have a medium length of the growing period (75 days), the plants are lodging heavily due to thin stems and lots of leaves, the medium number of branches per plant (7-10 pcs.) and internodes per stem (6-9 pcs.); the ratio of the graining zone vs the branching zone is 0.6-0.9. The sample yield is medium, 160 g/m2 per plant, and the productivity is 0.9-1.5 g/plant, while the number of buds is 50 pcs. The grain features a medium size 21.3-24.6 g/1000, low evenness (70%) and high husk content (23.4-24.6%); gray or brown color with a distinct pattern. Samples from Steppe of Ukraine This is the least numerous group of samples available in the National Collection of Ukraine. The total gene pool of the region comprises 20 samples. However, in terms of its climatic conditions, each of the regions included in this group has the contrasting features and requires subdivision into two parts, namely the genepool of Odes’ka region, which was formed in conditions of unstable humidity, relatively poor soils and high temperature during the vegetation period, as well as samples originating from Kirovohrads’ka and Dnipropetrovs’ka regions, where the sample forming conditions are remarkable for unstable, but more moderate precipitation, more favorable temperature conditions and availability of fertile soils. The samples originating from Odes’ka region feature the early ripening (68-75 days), low height (110 cm), a small number of internodes per stem (48 pcs.), low resistance to lodging due to a thin stem, a small number of branches (5-9 pcs.) and a moderate number of leaves per plant. The graining zone vs the branching zone ratio is 1.1-1.3. The sample productivity level varies considerably, several times, depending on the weather conditions. Tryhub, (2019): Local buckwheat varieties of Ukrainian origin 28 The average yield in this group of samples ranged from 68.5 to 174 g/m2, and the plant productivity was 0.42.3 g. Samples form a quite fine grain, 21.6-23.2 g/1,000 grain, with a medium husk content – 22.8-23.4%, and low evenness (75%). The samples originating from Kirovohrads’ka and Dnipropetrovs’ka regions are more grainful (179-236 g/ m2) with the productivity of 1.9-2.6 g/plant. They also form larger (24.7-26.5 g/1,000 grain), even (85%) grain with thinner husk (up 23.1%). The samples of these plants have more leaves, branches (7-13 pcs.) and buds (80 pcs.) per plant. The plant height is medium, 125 cm, with 9-13 internodes. The plants are more resistant to the grain shattering after ripening and resistant to lodging due to the thickened lower internodes. A common characteristic of all local buckwheat varieties of Ukrainian origin is a significant sensitivity to changes in growing conditions, which is reflected in the change of the growth processes (increase in the plant height by lengthening of internodes, the number of branches of the second and higher orders, as well as the number of leaves per plant) and adjustment of the growth process duration (extension of the growing season length after the onset of more favorable conditions for growth and development). A quite stable performance is related to grain characteristics (size, husk content and uniformity, color, grain, etc.). However, the general trends, characteristic of the collection material origin, remained unchanged. Extensive variety options are important for selection, because enables finding a material with specific characteristics among varieties and forms, identifying and examining the same according to a set of indicators. While working to study and describe the sample collection, some contrasting forms in comparison with the original populations were discovered (in terms of the shape of plants and buds, flower color and size, availability and varying degrees of anthocyanin color, initial growth rate, a lack of branching, ability to counter or avoid the extreme environmental factors, etc.). This is the most valuable genepool, because it allows expanding the variety polymorphism, extending the traditional and starting the new areas of buckwheat selection or use of products obtained from its cultivation. REFERENCES 1. Alekseeva, E., 1967. Ecological groups of local buckwheat varieties of the western regions of the UkrSSR // Selection and seed-growing. Vol. 7, Ukraine, pp. 13-25. (in Russian) 2. Alekseeva, E., 1999. Breeding of Podolsk varieties of buckwheat, Chernovcy: Ruta, Ukraine, 120 p. (in Russian) 3. Alekseeva, E., Taranenko, L., Malyna, M., 2004. Genetics, selection and seedling of buckwheat, Kyiv: Vyscha shkola, Ukraine, 216 p. (in Ukrainian) 4. Alekseeva, E., Elagin, I., Taranenko, L., Bochkareva, L., Malina, M., Rarok, V., Yacishin, O., 2005. Culture of buckwheat. Vol. 1. Historical of culture, botanical and biological feature. Kamenetz Podolsky, Moshak, Ukraine, pp. 66-75. (in Russian) 5. Alexanian S.M., 2003. State and Bioresources, St. Peterburg: VIR, Russia, 180 p. (in Russian) 6. Bochkareva, L., 1994. Analysis of the structure of buckwheat plants (Methodical recommendations), Chernovcy, Ukraine, 45 p. (in Russian) 7. Descriptors for buckwheat (Fagopyrum spp.), 1994. IPGRI. 48 p. 8. Kirjan, V., Boguslavs’kyy, R., Smekalova, T., Bagmet, L., 2014. Collection of samples of the gene pool of plants for replenishment of the National genebank of plants of Ukraine (results of the expedition 20-30 August 2013) // Theoretical and applied aspects of the development of natural sciences: mater. of Intern. scient.-pract. conf. (20-21 November 2014), Poltava: PNPU, Ukraine, pp. 73-75. (in Ukraine) 9. Kreft, I., Ikeda, K., Ikeda, S., Vombergar, B., 2010. Development of functionally new food products based on buckwheat ordinary and tartary // Vesnik OrelSAU, No4 (25), Russia, pp.15-17. (in Russian) 10. Krotov, A., 1968. Guidelines for the study of collection samples of corn, sorghum and groats. Leninhrad: VIR, Russia, pp. 37-44. (in Russian) 11. Korunyak, O., Burdyha, V., Rarok, A., Rarok, V., 2017. Collection of Fagopyrum Mill world genepool: formation, studing and use of the samples. Podilian Bulletin: agriculture, engineering, economics. Kamianets-Podilskyi, Ukraine, pp. 87-93. (in Ukrainian) Fagopyrum 36(1):23-29 (2019) 29 12. Tryhub, O., Burdyga, V., 2015. Formation of the collection of the world genepool of buckwheat in Ukraine and directions its use. The manual of Ukrainian grain growers, pp. 118-123. (in Ukrainian) 13. Fesenko, N., Fesenko, N., Romanova, O., Alekseeva, E., Suvorova, G., 2006. Buckwheat. Genepool and breeding groat cultures (Theoretical basis of plant breeding) (edited by Drahavcev V.), Vol.5. St. Peterburg: VIR, Russia, 196 p. (in Russian) 14. Tryhub, O., Kharchenko, Yu., Ryabchun, V., Hryhoraschenko, L., Dokukina, K., 2013. Complete unified classifier of the genus Buckwheat (Fagopyrum Mill.), Ustymivka, Ukraine, 54 p. (in Ukrainian) IZVLEČEK Zbirko ajde Ukrajine tvori 578 lokalnih vzorcev, ki so bili nabrani in vključeni v zbirko v letih 1929 do 2012. V letih 1994-2000 in 2014-2018 so potekale raziskave in opisovanje vzorcev, zlasti običajnih lastnosti, značilnih za posamezna območja izvora, posebnosti vzorcev, variabilnost in stalnost kvantitativnih in kvalitativnih lastnosti v odvisnosti od vremena in leta pridelovanja. Raziskave so med drugim vključevale kazalce rodnosti, opis zgradbe rastlin in lastnosti zrn. Običajne lastnosti lokalnih varietet ajde so odvisne od sprememb v razmerah pridelovanja, kar se kaže v spremembah poteka rasti (večanje višine rastlin z daljšanjem internodijev, števila stranskih vej drugega in višjega reda, števila listov na rastlino). Kazalci povezani z lastnostmi zrn so precej stabilni. Vseeno pa ostajajo lastnosti rastlin glede na izvor v znatni meri nespremenjene. INTRODUCTION Buckwheat has not become one of the most demanded crops by the international community yet. The main reason is its limited distribution around the world and low yields. However, the conclusions made in recent years concerning the maintenance of full-fledged human life, health and outstanding buckwheat plant properties indicate a significant need in buckwheat products (Kreft, 2010, Alekseeva at al., 2005. Gradually, the buckwheat products are distributed from the major regions of its traditional consumption to the new areas, where it gains the status of the core component of a healthy diet and becomes the foundation for a healthy lifestyle. As a result of in-depth study of biochemical composition of buckwheat grant and the whole plant, involving new types of crops into the research, the scientists around the world have greatly expanded the application areas of buckwheat products from traditional to exotic. To carry out this work, the genetic material with extensive properties and characteristics, most of which is concentrated in banks of plant genetic resources, is used (Alekseeva at al., 2004). Genetic diversity preservation has long been an imperative of global importance. The work to create the banks of genetic plant resources, launched more than a hundred years ago, not only validated the feasibility of this direction development, but also confirmed an unvalued contribution to ensuring food security worldwide (Alexanian, 2003). To solve scientific problems, the collection material of various eco-geographical origin and biological status is critical. However, given the importance of addressing the issue of the plant productive potential realization in contrasting environmental conditions, the study of local varieties and forms, the research of adaptive mechanisms and biochemical plant components became of paramount importance. MATERIALS AND METHODS A part of the National Buckwheat Collection in Ukraine, which is stored in Ustymivska Experimental Station of Plant Production (Poltavs'ka region), includes over 1,600 authentic samples, of which 1.1 thousand samples are the local varieties and forms of the common buckwheat (Fagopyrum esculentum Moench.) (Tryhub at al., 2015). 578 samples in the collection have Ukrainian origin. The material included in the collection is the result of its collection by employees of the N. I. Vavilov All-Union Institute of Plant Genetic Resources (VIR, Leningrad) during the missions (Fesenko at al., 2006), by researchers of Ternopil Breeding and Research Station, the Research Institute of Agriculture and Livestock of the Western Regions of Ukraine led by Olena Alekseeva (Alekseeva, 1967), as well as employees of the National Center for Plant Genetic Resources of Ukraine, assisted by the Ustymivska Experimental Station of Plant Production (Kirjan at al. 2014) in the territory of Ukraine from 1929 to 2012. As regards the ecological and geographical origin, there are samples from 18 regions. These are the representatives of the Carpathian region, Woodlands, Forest Steppe and Steppe zones. The overwhelming majority is the samples from the North, Central and Western parts of Ukraine, and a small number comes from the Eastern and Southern regions (see Table 1 and Pic. 1). The research of the collection material of buckwheat varieties and forms was carried out as required by the "Complete unified classifier of the genus Buckwheat (Fagopyrum Mill.)" (Tryhub at al., 2013), "Descriptors for buckwheat (Fagopyrum spp.)" (Descriptors..., 1994) , "Guidelines for the study of collection samples of corn, sorghum and groats" (Krotov, 1968), "Analysis of the structure of buckwheat plants (Methodical recommendations)" (Bochkareva, 1994). Number of accesses The material study and description was carried out in several stages during 1994-2000 and 2014-2018, and the results obtained were compared to the standard variety of Ukrainka. The common characteristics of samples peculiar of a certain area of origin, feature manifestation peculiarities, material flexibility and stability of the quantitative and qualitative characteristics depending on the weather in growing years have been identified. The range of studies included the sample performance indicators (the number of seeds and buds per plant, inflorescence yield), architectonic parameters (plant height, number of branches and inflorescences, height of attachment of the inflorescences and branches, the length of the branching and grain production zone), grain characteristics (grain size, evenness and husk content), the length of the growing season and its components, and so on. RESULTS AND DISCUSSION To obtain high and stable buckwheat yields, it is required to continue creating new varieties combining high performance, friendly maturation, resistance to drought, temperatures below zero, lodging, grain shattering, pests and diseases, as well as a high grain quality. For this purpose, in selection, as noted by N.I. Vavilov, we need to use the local material subjected to prolonged exposure to natural selection and adapted to specific conditions. This material has great value, and is widely used in selection (Korynyak at al., 2017). It should be noted that in terms of soil and climatic conditions, the research region, namely the south of Poltavs'ka region (central part of Ukraine, Forest Steppe zone) is one of the most favorable for growing buckwheat. Still, it has recently been affected by the ongoing significant climate change towards higher temperatures during flowering and insufficient precipitation during initial growth, which is typical for most areas of buckwheat cultivation in Ukraine. These trends are typical for most regions of the world where the buckwheat is sown. The features of sign manifestation were identified based on a set of researches conducted in two stages. The samples were studied following a three-year cycle during 1994-2000 and 2014-2018. The first cycle of study (1994)(1995)(1996)(1997)(1998)(1999)(2000) was remarkable for more favorable weather conditions with precipitation and temperature regime of the growing season close to the average long-term data. The second cycle of study (2014-2018) was characterized by a high temperature in summer and a significant lack of moisture in the spring. The data obtained in the course of study indicate a considerable variety of feature manifestations by the local plant varieties and forms. There is a considerable dependence in the levels of feature manifestation on climatic zones of the sample origin. A significant impact on the plant characteristics was exercised by the climatic factors, precipitation and temperature regime during the years of cultivation. However, a set of the studied traits showed the characteristic, genetically determined features of samples and allowed describing the quantitative and qualitative material characteristics. Given the considerable heterogeneity of the climatic zones of Ukraine in terms of soil conditions, heat and precipitation, the entire buckwheat gene pool of these areas has been further divided into smaller, but more similar regions. To characterize the samples from the western regions of Ukraine, the classification proposed of Olena Alekseeva (Alekseeva, 1999) was applied. For other areas, the author's classification was applied. Samples from Forest Steppe of Ukraine In terms of soil and climatic conditions, the Forest Steppe Zone was subdivided into western, central and eastern parts, each with its own characteristics, significantly differing among themselves, but with rather similar buckwheat growing conditions within each part. The western part includes the samples from Khmel'nyts'ka, Ternopil's'ka and Vinnyts'ka regions, having more precipitation as compared to the other parts of the Forest Steppe Zone, and their more even distribution during the vegetation period, lower average daily temperatures and fertile soils. The central part of the Forest Steppe (Cherkas'ka, Poltavs'ka, Sums'ka and Kyivs'ka regions) is characterized by harsher weather conditions in terms of the temperature and humidity. Here is sufficient precipitation during the growing season, but its distribution is very uneven. The cover in most of the area is presented with fertile soil with excellent quality characteristics. The eastern part of the Forest Steppe zone includes Kharkivs'ka region and the south of Sums'ka region. This part shows the most extreme weather conditions with little precipitation and high temperatures, especially during the buckwheat flowering and graining. In general, the gene pool of this region is represented by 294 samples. The samples of this group are the most malleable genetic material grown in the area with conditions, which are the most suitable for the buckwheat cultivation. Such a genepool was the source material for most of the modern high-tech varieties, and today the scientists are searching for the forms remarkable for their performance indicators combined with resistance to abiotic environmental factors, grain quality, and so on primarily among these samples. The samples from the western part of the Forest Steppe Zone of Ukraine show an extensive diversity in terms of the key parameters of the plant organism structure, which requires their subdivision into two subgroups by the growing season duration, i.e. mid-late-ripening (78-85 days) and early-ripening (70-77 days). Mid-late-ripening samples form tall plants (1.5 m high) with a large number of internodes (9-13 pcs.), a large number of leaves and branches (9-16 pcs.). Lower interstitials are thickened, forming resistance to lodging. The samples' yield is 180-220 g/m 2 , and the productivity is 1.8-2.6 g/plant. They have medium-sized grain, up to 25.6 g/1000 grain with uniformity up to 82% and husk content 22.7-23.5%. The early-ripening samples have the plants 85-110 cm high with 10 internodes and the ratio of grain formation zone vs. branching zone as 1.0-1.3. The plants form a large number of inflorescences, up to 55 pieces per plant. Samples have large dark brown grain as for the local forms; the weight of 1,000 grains is 25.8-26.9 g, the husk content increased to 23.8%, and a good inflorescences uniformity, i.e. 85%. The yields of these samples is within 165-200 g/m 2 , and the productivity is 1.6-2.2 g/plant. Plants have a significant number of leaves and the medium number of branches (5-8 pcs.), are resistant to lodging and moderately resistant to grain shattering after ripening. The central part of the Forest Steppe Zone has a more form genepool in terms of manifestation of quantitative and qualitative traits of the buckwheat plant organism. Local varieties from this part feature the medium-term ripening (70-80 days), medium and large plant height (100-150 cm), 7 to 16 internodes on the main stem and the ratio of the graining zone vs the branching zone as 1.1-1.3. The plants from this group are resistant to lodging, have many leaves and 8-14 branches (including 5-8 first-order branches) and 85 buds per plant. The grain of these samples is gray and dark brown with an average weight of 1,000 grains as 24.2-27.3 g, grain husk content as 22.1-23.5%, and 90% uniformity. The average grain yield varies between 185-236 g/m 2 , and the productivity -within 1.8-2.8 g/plant. The plants from the local samples of eastern part of the Forest Steppe of Ukraine are potential sources of the variety resistant to abiotic environmental factors (high temperatures and drought). Samples from this part form the medium-term ripening (75 days) plant up to 120 cm high with 5-9 internodes, 5-12 branches and 65 buds per plant. These samples are characterized by high resistance to lodging and medium resistance to the grain shattering. Grain has a dark brown color with a clear or blurry pattern in the form of dots or dashes, the weight of 1,000 grains is 23.5-26.7 g, the medium husk content (up to 23.1%) and evenness (75%). The yield of samples from this group is 168-218 g/m2 with the plant performance at 1.45-2.05 g/plant. Samples from Polissya and Carpathian region The material from this zone also features extensive diversity due to a significant difference between the weather, climate and soil conditions within the region. The samples from this area were subdivided into 3 groups: the north-western, western, northern and Carpathian. The north-western part includes the samples originating from Volyns'ka and Rivnens'ka regions, the climate in which is characterized by excessive precipitation, moderate temperature conditions and poor nutritional composition of their soils. The western part (Ivano-Frankivs'ka, L'vivs'ka and Zhytomyrs'ka region) is characterized by a moderate precipitation and temperature conditions favorable for plant growth and development, with soils of varying fertility. The northern part of the Polissia zone (Chernihivs'ka and a part of Kyivs'ka and Sums'ka regions) has the most fertile soils in this region, sufficient precipitation and heat. The genepool of the Carpathian region (Zakarpats'ka and a part of Ivano-Frankivs'ka region) includes representatives of foothill and mountain areas, featuring moderate and sometimes low temperature during the growing season, often excessive precipitation and poor soils. The collection of local varieties and forms from this region includes 264 samples. The samples of the north-western part of the region are characterized by a short growing season, low (1.2 m) stem with 4-5 internodes; slight branching (3-7 branches, of which 2-3 are the first-order branches) with a ratio of the graining zone vs the branching zone as 0.8, the medium number of leaves, the sample yield at 120-180 g/ m 2 per plant and the productivity as 0.8-1.0 g/plant; the medium grain particle size (up to 23.5 g/1000 grains), uniformity (80%) and husk content (up 23.5%), brown grain with a clear patter; resistant to lodging and having a medium degree of grain shattering after ripening. The samples from the western part feature a significant heterogeneity by the growing season duration; the samples vegetation varies from early-ripening (70 days) to late-ripening (90 days), and respectively, the plant height and the number of internodes ranged from 80-100 cm and 5-7 pieces. to 125-130 cm and 9-12 pieces. In general, all samples were dark brown and gray, with medium and large grain size (23.7-26.8 g/1000 seeds), medium evenness (85%) and high husk content (24%); the plants have many leaves with a medium degree of grain subsidence and lodging. The samples' yield was 175-210 g/m 2 , and the plant productivity -1.6-2.2 g/plant. The samples from the northern part feature a high yield (up to 250 g/m 2 ) and plant productivity (up to 3.0 g/plant); large grain (24.5-26.4 g/1000 grain), good evenness (up to 85%), and medium husk content (22.6-23.1%), brown and dark brown color. The plants of these samples are tall (1.2-1.5 m) with a large number of branches (13-18 pcs.) and buds (70 pcs.) per plant, as well as many leaves. The stem is thick and resistant to lodging. The samples from the Carpathian region (foothill areas of Zakarpats'ka and Ivano-Frankivs'ka regions) are characterized by a considerable length of the growing period (80-90 days) and tall plants (150 cm) with a significant number of branches (15 pcs.) and internodes (10-13 pcs.); the ratio of the graining zone vs the branching zone is 0.9-1.1; the plants have a large number of leaves and buds (80 pcs.); yield at 180-200 g/m 2 and productivity up to 2.2 g/plant; the medium grain size (up to 25.1 g/ plant), medium husk content (23%) and evenness (85%). The plants are remarkable for a low resistance to plant lodging and grain shattering. Samples from the Carpathian mountain areas have a medium length of the growing period (75 days), the plants are lodging heavily due to thin stems and lots of leaves, the medium number of branches per plant (7-10 pcs.) and internodes per stem (6-9 pcs.); the ratio of the graining zone vs the branching zone is 0.6-0.9. The sample yield is medium, 160 g/m 2 per plant, and the productivity is 0.9-1.5 g/plant, while the number of buds is -50 pcs. The grain features a medium size 21.3-24.6 g/1000, low evenness (70%) and high husk content (23.4-24.6%); gray or brown color with a distinct pattern. Samples from Steppe of Ukraine This is the least numerous group of samples available in the National Collection of Ukraine. The total gene pool of the region comprises 20 samples. However, in terms of its climatic conditions, each of the regions included in this group has the contrasting features and requires subdivision into two parts, namely the genepool of Odes'ka region, which was formed in conditions of unstable humidity, relatively poor soils and high temperature during the vegetation period, as well as samples originating from Kirovohrads'ka and Dnipropetrovs'ka regions, where the sample forming conditions are remarkable for unstable, but more moderate precipitation, more favorable temperature conditions and availability of fertile soils. The samples originating from Odes'ka region feature the early ripening (68-75 days), low height (110 cm), a small number of internodes per stem (48 pcs.), low resistance to lodging due to a thin stem, a small number of branches (5-9 pcs.) and a moderate number of leaves per plant. The graining zone vs the branching zone ratio is 1.1-1.3. The sample productivity level varies considerably, several times, depending on the weather conditions. The average yield in this group of samples ranged from 68.5 to 174 g/m 2 , and the plant productivity was 0.4-2.3 g. Samples form a quite fine grain, 21.6-23.2 g/1,000 grain, with a medium husk content -22.8-23.4%, and low evenness (75%). The samples originating from Kirovohrads'ka and Dnipropetrovs'ka regions are more grainful (179-236 g/ m 2 ) with the productivity of 1.9-2.6 g/plant. They also form larger (24.7-26.5 g/1,000 grain), even (85%) grain with thinner husk (up 23.1%). The samples of these plants have more leaves, branches (7-13 pcs.) and buds (80 pcs.) per plant. The plant height is medium, 125 cm, with 9-13 internodes. The plants are more resistant to the grain shattering after ripening and resistant to lodging due to the thickened lower internodes. A common characteristic of all local buckwheat varieties of Ukrainian origin is a significant sensitivity to changes in growing conditions, which is reflected in the change of the growth processes (increase in the plant height by lengthening of internodes, the number of branches of the second and higher orders, as well as the number of leaves per plant) and adjustment of the growth process duration (extension of the growing season length after the onset of more favorable conditions for growth and development). A quite stable performance is related to grain characteristics (size, husk content and uniformity, color, grain, etc.). However, the general trends, characteristic of the collection material origin, remained unchanged. Extensive variety options are important for selection, because enables finding a material with specific characteristics among varieties and forms, identifying and examining the same according to a set of indicators. While working to study and describe the sample collection, some contrasting forms in comparison with the original populations were discovered (in terms of the shape of plants and buds, flower color and size, availability and varying degrees of anthocyanin color, initial growth rate, a lack of branching, ability to counter or avoid the extreme environmental factors, etc.). This is the most valuable genepool, because it allows expanding the variety polymorphism, extending the traditional and starting the new areas of buckwheat selection or use of products obtained from its cultivation.

v3-fos

2018-04-03T06:14:42.391Z

1970-09-01T00:00:00.000Z

46000408

s2

Degradation and utilization of hemicellulose from intact forages by pure cultures of rumen bacteria. Several pure strains of rumen bacteria have previously been shown to degrade isolated hemicelluloses from a form insoluble in 80% acidified ethanol to a soluble form, regardless of the eventual ability of the organism to utilize the end products as energy sources. This study was undertaken to determine whether similar hemicellulose degradation or utilization, or both, occurs from intact forages. Fermentations by pure cultures were run to completion by using three maturity stages of alfalfa and two maturity stages of bromegrass as individual substrates. Organisms capable of utilizing xylan or isolated hemicelluloses could degrade and utilize intact forage hemicellulose, with the exception of two strains of Bacteroides ruminicola which were unable to degrade or utilize hemicellulose from grass hays. Intact forage hemicelluloses were extensively degraded by three cellulolytic strains that were unable to use the end products; in general, these strains degraded a considerably greater amount of hemicelluloses than the hemicellulolytic organisms. Hemicellulose degradation or utilization, or both, varied markedly with the different species and strains of bacteria, as well as with the type and maturity stage of the forage. Definite synergism was observed when a degrading nonutilizer was combined with either one of two hemicellulolytic strains on the bromegrass substrates. One hemicellulolytic strain, which could not degrade or utilize any of the intact bromegrass hemicellulose alone, almost completely utilized the end products solubilized by the nonutilizer. Similar synergism, although of lesser magnitude, was observed when alfalfa was used as a substrate. Since the hemicelluloses can constitute a considerable portion of forage carbohydrate, their use as an energy source by the ruminant animal is of special interest. Early studies, with conventional digestion trials, indicated that a definite loss of pentose occurs in the ruminant digestive tract (18)(19)(20). Since then, the in vitro fermentation of various isolated and intact forage hemicelluloses has been demonstrated with mixed cultures of rumen organisms (11, [13][14][15], as well as with several pure cultures (6,8,12,17). Dehority (6), investigating whether the xylan-utilizing species of rumen bacteria were capable of digesting isolated hemicelluloses, found that from a total of eight strains of cellulolytic bacteria only those three capable of using xylan as an energy source were able to grow on isolated hemicelluloses. The five remaining strains of cellulolytic bacteria, although unable to utilize the isolated hemicelluloses as a source of energy, degraded these materials to a form soluble in 80% acidified I Approved for publication as Journal Article no. 36-70 by the Ohio Agricultural Research and Development Center, Wooster, Ohio. ethanol. The extent of hemicellulose degradation or utilization, or both, varied between organisms and source of the hemicellulose. Degradation was defined as the conversion of ethanol-insoluble pentose to a soluble form and utilization as total pentose loss. Further studies with several additional strains isolated on a xylan medium revealed differences in the rate and extent of degradation, utilization, or both, between the isolated hemicellulose substrates and the various bacterial strains and species (8). Dehority and Scott (12) estimated hemicellulose digestion in two maturity stages each of bromegrass and alfalfa and found that digestion was appreciably decreased in the intact plant as compared with isolated hemicelluloses. In general, the extent of hemicellulose digestion varied with the maturity and type of forage, as well as with the species of bacteria. In limited synergism studies, it was observed that cellulose digestion was increased in all cases when the cellulolytic organisms were combined with Bacteroides ruminicola H8a, a noncellulolytic, hemicellulolytic organism. Kock and Kistner (17) confirmed the results of Dehority and Scott (12) on the digestibility of forage hemicelluloses. Ten strains of Butyrivibrio and two strains each of Ruminococcusflavefaciens, R. albus, and Clostridium sp., all capable of fermenting xylan, were able to solubilize the hemicellulose from ethanol-benzene-extracted low protein teff hay. Hemicellulose solubilization was based on the loss in weight of isolated holocellulose when treated with 24% KOH (w/v). In view of these data, the present study was undertaken to (i) investigate whether hemicelluloses are degraded from an intact forage in a manner similar to that observed in the isolated materials by both the utilizing and nonutilizing strains; (ii) determine the influence of plant type and maturity as well as bacterial strain differences on the extent of hemicellulose degradation; (iii) study several previously untested species and strains of rumen bacteria for their ability to degrade intact forage hemicelluloses; and (iv) investigate possible synergistic effects on hemicellulose utilization or degradation, or both, by using an intact forage substrate. One additional objective in the present study was to measure the utilization or degradation, or both, of hemicellulose from an intact forage and compare these values with those obtained from a sample of hemicellulose previously isolated from this same forage. MATERIALS AND METHODS Forage substrates consisted of boot and bloom stages of bromegrass (Bromus inermis Lincoln) and prebloom, early bloom, and late bloom stages of alfalfa (Medicago sativa Vernal). All of the forages were harvested from pure stands, artificially dried on a wagon dryer, chopped, ground through a medium screen in a large Wiley mill, and finally ground through a 40-mesh screen in a small laboratory Wiley mill. Fescue grass and isolated fescue grass hemicellulose were supplied by the late Fred Smith of the University of Minnesota. The fescue grass was chopped and ground through a 40-mesh screen in a small Wiley mill for use as a substrate. Isolated hemicellulose was prepared from the same stand of fescue grass by the procedure of Myhre and Smith (21). The anaerobic cultural techniques were similar to those described by Hungate (16), except for the modification proposed by Dehority (10) in the preparation of media. The fermentation medium contained 0.5% forage or 0.1% isolated hemicellulose, 40% clarified rumen fluid, 15% each of mineral solutions I and II of Bryant and Burkey (2), 0.0001% resazurin, 0.4% sodium carbonate, and 0.05% cysteine. The mixture was agitated with a magnetic stirrer, and 5-ml samples were pipetted anaerobically into pyrex culture tubes (16 by 150 mm), which were then closed with a size 0 rubber stopper. The individual tubes were autoclaved for 20 min at 121 C in a clamp-type rack to prevent the stoppers from blowing out. The 10 pure cultures of rumen bacteria used for this study included strains A3c and S-85 of B. succinogenes, strains Bla and B34b of R. flavefaciens, strain 7 of R. albus, strains HlOb and H17c of Butyrivibrio fibrisolvens, strains H8a and D31d of B. rwninicola (subspecies brevis and rwninicola, respectively), and strain D15d of Lachnospira multiparus. Thecharacteristics of these strains have previously been described (3)(4)(5)(6)(7)10). Strains A3c, Bla, B34b, S-85, and 7, all cellulolytic, were isolated from rumen contents with a nonselective glucose-cellobiose medium; strains H8a, H1Ob, and H17c were isolated with a xylan medium; and strains Dl5d and D31d were isolated with a pectin medium. All cultures were carried in the medium in which they were originally isolated. Inoculum cultures were grown overnight in an optically clear 0.5% cellobiose broth containing 40% clarified rumen fluid and then diluted with anaerobic dilution solution until an optical density of 0.2 had been reached. A 0.2-ml amount of this suspension was used to inoculate each 5 ml of forage media. All turbidity measurements were made in Pyrex culture tubes (16 by 150 mm) on a Bausch & Lomb Spectronic-20 colorimeter, reading at 600 nm. The fermentations were allowed to incubate for 168 hr at 39 C. The inoculum level and the fermentation time had been previously determined (8) to allow for maximum rate and completion of forage cellulose digestion. Estimation of forage hemicellulose concentration either by simple solubility methods or the more complex isolation procedures did not appear to be suitable for this study. Even though the constituent carbohydrates and per cent composition of plant hemicelluloses vary between species, approximately 80 to 90% of the hemicellulose is composed of pen-mixed and allowed to stand at room temperature for 30 min. The mixture was then centrifuged 30 min at 2,000 X g, and the supernatant was decanted into a 50-ml volumetric flask. Acidified 80% ethanol solution (20 ml) was added to the residue, and the contents were mixed and recentrifuged for 20 min at 2,000 X g. The wash solution was decanted and added to the original supernatant, and the total supernatant was brought to volume with distilled water. To hydrolyze the hemicellulose in the residue, 5 ml of 1 N H2S04 was added to each tube; the tube was covered with a metal cap and autoclaved for 1 hr at 15 psi. After cooling, the hydrolysate was transferred to a 50-ml volumetric flask and brought to volume with distilled water. The insoluble material was allowed to settle, and a sample was pipetted from the upper layer for further analysis. Portions (5 ml) of both fractions were treated with 2 g (wet weight) Amberlite IRA400 anion exchange resin in the acetate form. The resin was removed by filtration and the filtrate plus washings, diluted to appropriate volumes, were than analyzed for total pentose by the orcinol method (1). Per cent transmission was read at 520 and 660 nm on an Evelyn colorimeter, which allowed correction for any color at 660 nm arising from hexose in the sample. In this study, degradation is defined as the solubilization of 80% ethanol-insoluble pentose, whereas utilization is defined as a loss in total pentose (6). Degradation and utilization values are based on duplicate fermentations in two replicates. A preliminary study involving two organisms and two different forage substrates (alfalfa and orchardgrass) was run to determine a feasible coefficient of variation for acceptance of duplicate analyses. Six replicates of each forage and organism were set up in duplicate, and coefficients of variation ranging between 5 and 10% were obtained. These were similar to the values obtained previously by Dehority (8), in a study on the digestibility of cellulose in intact forages in which a coefficient of variation of 15% was chosen as the upper limit for acceptance of the data. Thus, when a coefficient of variation larger than 15% was obtained between the four values in the present study, the fermentations were run twice again in duplicate. RESULTS Data on the utilization or degradation, or both, of hemicellulose from two maturity stages of bromegrass are presented in Table 1. It it apparent that all of the cellulolytic strains tested were capable of degrading a considerable amount of the 80% acidified ethyl alcohol-insoluble pentose to a soluble form without necessarily being able to utilize the resulting end products as energy sources. Values of 6 % or less are considered to be within the range of experimental variation and are, therefore, of doubtful significance. Those cellulolytic strains which can ferment xylan, Bla and 7, were capable of utilizing the hemicellulose, although utilization by Bla was somewhat limited. Despite this utilization, neither of these Since a number of the cellulolytic strains could degrade but not utilize hemicellulose from the intact bromegrass, it seemed desirable to determine whether the xylan-digesting strains which could not degrade the hemicellulose could utilize the material solubilized by the cellulolytic species. R. flavefaciens B34b was used as the degrading nonutilizer in combination with either B. ruminicola H8a, B. fibrisolvens H1Ob, L. multiparus Dl 5d, or all three organisms. These fermentations were run under the same conditions as described previously, by using 0.2 ml of a 0.2-optical density suspension as inoculum for each organism required (Table 1). In all cases, degradation was increased above that obtained with R. flavefaciens B34b alone. The most striking effect, however, was noted in the utilization of total pentose by the combination with B. ruminicola H8a, in which utilization increased from essentially zero for each organism alone to approximately 80 and 70%, respectively, for maturity stages I and H. A marked increase in utilization was also observed when B34b and B. fibrisolvens HlOb were combined. For both maturity stages, the increase was approximately 30%. Combination of B34b with L. multiparus D15d was unsuccessful with regard to utilizing the degraded hemicellulose, which would be in agreement with the inability of Dl5d to use pentoses or xylan as energy sources (10). When all three organisms, H8a, HiOb and Dl5d, were combined with B34b, degradation and utilization were esentially the same as obtained with the highest combination of two strains, i.e., B34b and H8a. In every case where applicable, degradation and utilization were reduced as the plant matured. In general, this reduction was in the magnitude of 15 to 20%, and the overall relationship of one species or strain to another was unchanged. A similar series of experiments was set up with three maturity stages of alfalfa, and these results are presented in Table 2. All of the cellulolytic VOL. 20,1970 species were able to degrade the hemicellulose from intact alfalfa; however, the extent was generally lower than that observed with bromegrass. Marked reduction was obtained in the extent of utilization for strains 7 and Bla, especially in the more mature plant. Both strains of B. succinogenes degraded similar amounts of alfalfa hemicellulose, whereas a considerable difference was noted with bromegrass. In contrast to the bromgrass data, all of the hemicellulolytic and pectinolytic species degraded and utilized the alfalfa pentosans. The results with L. multiparus Dl5d were particularly interesting since this strain cannot use pentoses, xylan, or degraded bromegrass hemicellulose as energy sources. An explanation of the degradation and utilization of alfalfa hemicellulose by strain D15d is not immediately obvious. Further studies with a second strain of L. multiparus have confirmed this difference in availability of grass and alfalfa hemicellulose. Marked strain differences were observed between B. ruminicola H8a and D31d and B. fibrisolvens HlOb and H17c with the alfalfa substrate. Both H8a and HiOb utilized almost all of the hemicellulose they could degrade. On the other hand, H17c degraded considerably less hemicellulose and could utilize only a portion of the degraded material. Strain D31d degraded more hemicellulose than H8a but could not utilize even 50% of that degraded. Results of the synergism studies with the alfalfa substrates were quite similar to those obtained with bromegrass although the extent of the increases was somewhat less. In contrast, utilization of alfalfa III by the combination of B34b and H1Ob, and all alfalfa stages with B34b and Dl5d, was considerably lower than utilization by HiOb and D15d alone. Table 3 presents the results on degradation or utilization, or both, of intact fescue grass hemicellulose and the hemicellulose after isolation from the forage. In general, the overall pattern of degradation and utilization is quite similar to that observed from bromegrass. Although these values correspond rather closely to those observed with the bloom stage of the bromegrass, the maturity stage of the fescue is unknown. In agreement with the degradation values observed on the bromegrass, strain H8a was totally unable to degrade any of the intact fescue grass hemicellulose (Table 3). However, this particular strain could almost completely degrade and utilize this hemicellulose when it had been separated from the intact plant. In all cases except strain D15d, the isolated hemicellulose was degraded to a considerable extent. These data are in close agreement with those reported by Dehority (6,8) for several of these same strains on the isolated fescue grass hemicellulose substrate. DISCUSSION Dehority and Scott (12) estimated total pentose loss on the same forages with 7 of the 10 strains used in this study. The utilization values reported herein are in close agreement with their data, although a somewhat more complicated analytical procedure was used. Kock and Kistner (17) fermented ethanol-benzene-extracted intact teff hay with several strains of R. albus, R. flavefaciens, and Butyrivibrio species. Although not directly comparable because of differeing analytical methods, the present results compare favorably with their 25 to 67% range of hemicellulose solubilization. The complete inability of B. ruminicola H8a to degrade and subsequently utilize the brome hemicellulose was quite surprising, since this organism, a xylan isolate, was capable of degrading and utilizing hemicellulose isolated from several grasses (8). This observation led to a study of possible synergistic effects of combining a degrading nonutilizer, R. flavefaciens B34b, with the hemicellulolytic strains H8a or H1Ob, the pectinolytic strain D15d, or all three together. The most striking results were with the combination of strains H8a and B34b, in which utilization increased from zero for each strain alone to 80% for the organisms together. Except in the case of Dl5d, some increase in degradation and a marked increase in utilization were observed for all combinations. These data suggest that the hemicellulolytic organisms were probably fermenting the oligosaccharides and other products solubilized by strain B34b. Synergism was also studied with the alfalfa substrates; however, the magnitude of the increases observed was lower. Undoubtedly, this was because those strains that could not degrade bromegrass hemicellulose both degraded and utilized alfalfa hemicellulose to a reasonable extent. Dehority and Scott (12) previously observed a small but significant increase in the extent of cellulose digestion when they combined strain H8a, a noncellulolytic, with several cellulolytic strains. Combined with the present results, these data suggest the possibility that a physical masking effect can occur in the intact plant between the cellulose and hemicellulose components. The variation observed between bromegrass and alfalfa, particularly with strains H8a, Dl5d, and D31d, would further indicate that there are definite differences in structure (or orientation) of hemicelluloses between intact legumes and grasses. The ability of L. multiparus Dl5d to degrade and utilize alfalfa hemicellulose was quite surprising, since it was unable to ferment xylan, brome hemicellulose, or isolated fescue grass hemicellulose. The organism may be producing some type of soluble oligosaccharides which are permeable to its cell membrane. No explanation is readily available for the marked decrease in utilization when strain D15d was combined with strain B34b. The degradative end products produced by B34b would appear to differ in some way from those produced by strain D15d. Consequently, this competitive degradation would result in less useful substrate for Dl5d. For all strains and combinations of strains, hemicellulose degradation or utilization, or both, in the fescue grass appeared quite similar to that previously obtained with bromegrass. Both hemicellulolytic organisms, H8a or HlOb, were capable of increased degradation and utilization when the hemicellulose had been previously separated from other plant constituents, either chemically prior to fermentation in the case of fescue grass or by a nonutilizing cellulolytic strain, B34b, with both fescue and bromegrass. The complete inability of strain H8a to degrade any of the fescue grass hemicellulose substantiates the observation that this hemicellulolytic strain can utilize grass hemicellulose only after it has been removed from the plant in some way. Where applicable, the hemicellulose degradation patterns for each organism across the two types of forage correspond rather closely to the data obtained by Dehority and Scott (12) for cellulose digestion from these same forages. This point, the high degradation values observed for the cellulolytic isolates, and the previously reported evidence that the enzymes involved are constitutive would possibly tend to support the suggestion that hemicellulose degradation by the nonutilizing cellulose digestors is a nonspecific function closely associated with cellulase activity (9). In this case, the cellulolytic organisms could quite easily be contributing to the over all rumen fermentation of forage hemicellulose by supplying the utilizers with more soluble substrate than they alone could degrade.

v3-fos

2020-04-30T09:08:20.987Z

1970-01-01T00:00:00.000Z

227433446

s2

Antioxidant Activity of “Kolang Kaling” Jam which is added with “Pucuk Merah” (Syzygium oleana) Fruit Juice Article History: Received: 11 February 20 Final Revision: 07 March 20 Accepted: 19 April 20 Online Publication: 20 April 20 “Kolang kaling” is the endosperm of Arenga pinnata seed that contains galactomannan so it can be made into jam. This research was aimed to study the effect of “Pucuk merah” fruit juice as a natural colorant and antioxidant sources on antioxidant activity of the jam. This research used an experimental design with different levels of the juice of “Pucuk merah” fruit (6%, 8%, 10%, and 12%). Observations were anthocyanin level (pH differential method), total phenol (by Follin-Ciocalteu method) and antioxidant activity (by DPPH method) of the jam. It was found that the addition of the concentration level of “Pucuk merah” fruit juice had a statistically significant effect on the anthocyanin and total phenol level of the jam. The more the juice was added, the higher the anthocyanin level (2.82 ± 0.13 ppm; 3.76 ± 0.29 ppm; 3.88 ± 0.11 ppm; and 4.20 ± 0.52 ppm, respectively) and the higher the total phenol level (382 ± 1.53 mg GAE/100g; 431 ± 3.06 mg GAE/100g; 540 ± 1.53 mg GAE/100g and 547 ± 6.43 mg GAE/100g, respectively). By using sensory evaluation, the best product was the addition of 12% of the juice; the antioxidant activity was 628 ppm. Research Background Jam is intermediate moisture food made from the processing of fruits, sugars or without the addition of permissible food additives [1]. The formation of gel during the processing of the fruit is highly dependent on the pectin content in the fruit pulp. In addition, the acidity and sugar added greatly determine the quality of the gel that is formed. Some fruits contain high pectin, so there is no need to add pectin to the fruit pulp in the gel formation process [2]. Pectin is a compound that includes hydrocolloids. Hydrocolloid is a water soluble polymer, capable of forming colloids and capable of thickening a solution or forming a gel from the solution. Gradually the term hydrocolloid which is short for hydrophilic colloids is replacing the term gum because it is considered the term gum is too broad meaning. Gums are molecules with high molecular weight hydrophilic as well as hydrophobic, usually colloidal and in the appropriate developer material can form gel, solution or thick suspension at very low concentrations [3] Hydrocolloid is widely used as a thickener or as a gelling agent so as to extend the shelf life of food. The thickening effect is mainly given by carboxymethyl cellulose, methyl cellulose and hydroxypropylmethyl cellulose, guar gum, locust bean gum, tara gum, konjac maanan, gum tragacanth, gum ghatti and gum Arabic. Commonly used gelling agents include modified starch, agar, carrageenans, pectins, gellan gum, alginate and methyl and hydroxypropylmethyl celluloses [4]. As in Ref. [5], the nature of gel formation varies from one type of hydrocolloid to another depending on the type. Gel may contain 99.9% water but have more characteristic properties such as solids, particularly elasticity and rigidity. In principle, the formation of a hydrocolloid gel occurs due to the formation of a net or a three-dimensional network by a primary molecule that spans the entire volume of the gel formed by trapping a certain amount of water in it. Cross-linking of polymers consisting of long-chain molecules in sufficient quantities will then form a continuous three-dimensional building so that the solvent molecules will be trapped, immobilizing solvent molecules and forming rigid and firm structures that are resistant to particular forces or pressures. Gelation is a phenomenon involving incorporation, or the occurrence of crosslinking between polymer chains. "Kolang-kaling" is an endosperm of seed of Arenga pinnata that is soft and chewy in clear white. [6] "Kolang-kaling" contains gum consisting of galactomannan. Galactomannan is a polysaccharide composed of galactose and mannose and has a soluble water property. Galactomannan is a compound that includes hydrocolloids [7]. So "kolang-kaling" could potentially be the basic ingredient of jam, so galactomannan can replace the function of pectin. However, the color of the jam has not well, so it was necessary to add fruit that has color to make the jam more interesting. Literature Review Galactomannan is a carbohydrate reserve that also serves to regulate the amount of water in seeds during germination [8]. In addition to as storage of carbohydrates, galactomannan also serves to store water to prevent the occurrence of drought in plants [9]. galactomannan is a heterogeneous polysaccharide consisting of a main chain of beta (1,4) -D manopiranose with 1 unit of alpha-D-galactopiranose branch attached to the alphaposition (1,6) [10]. the basic structures that build galactomannan are galactose and mannose [9]. The "Pucuk merah" is usually used as a hedge plant, as a protective crop on the side of the road or as an ornamental plant. This plant is one family with the guava plant that belongs to the family Myrtaceae. The fruit is small and dark red like blacberries. Anthocyanins in "Pucuk merah" are suspected was cyanidineglycoside compounds. The content of anthocyanin in "Pucuk merah" was 462.51 mg L-1 [11]. The anthocyanin content in the fruit has the potential as a natural antioxidant and natural colorant that has beneficial to health. Anthocyanins belong to a pigment called flavonoids that are generally soluble in water. The word anthocyanin comes from the Greek word "anthos" meaning flowers and "kyanos" which means dark blue [12]. Anthocyanin is a natural pigment with red, purple and blue color variations that are abundant in nature and safe to consume. It dissolves in water, and easy to use in food [13]..\ Research Objective This research aimed to study the effect of "Pucuk merah" fruit juice addition on the antioxidant activity of "Kolang kaling" jam. Materials The main ingredient used in this research was "Kolang kaling" obtained from traditional market and "pucuk merah" as natural colorant obtained from region around Andalas university campus. Experimental Design of this Research Experimental design used in this research with 4 level of juice of "Pucuk merah" fruit (6%, 8%, 10% and 12%). The Data were analysed by using analysis of Variance (ANOVA). If there was a statistically significant difference, then proceed with Duncan's New Multiple Range Test (DNMRT) at a 5% significance level. [14][15] Production of "Pucuk Merah" Fruit Juice "Pucuk merah" fruit are sorted and washed with water, then added with water (3:1) and crushed using a blender then filtered. Production of "Kolang Kaling" Pulp Refers to the method of making "kolang kaling" pulp as in Ref. [ [17], modified Mixed sample, ethanol and HCL (1 ml: 9 ml: 1 ml) and then the mixture was homogenized. After that were mixed with solution of potassium chloride buffer (pH 1; 0.4 mL) and solution of sodium acetate buffer (pH 4.5; 4 mL), and then the mixture was measured its absorbance at λ510 nm and λ700 nm used a UV-Vis spectrophotometer. Determination of Total Phenol: [18] Dissolving 10 μL of sample and 790 μL of distilled water in the reaction tube, then were added with 50 μL of Follin-Ciocalteu reagents and vortexed. After 1 minute, added with 150 μL of sodium carbonate 20% solution then vortex and place at dark room for 120 minutes. Absorbance was determined at 750 nm and the concentration of the total polyphenol was counted from the curve calibration. Results are expressed as mg/L Gallic Acid Equivalents (GAE). Sensory analysis. [19] Sensory analysis is a test that uses the human senses as a tool. This test is often used to determine agricultural and food commodities quality. The sensory analysis test used in this study is a hedonic test (preference test) by panellists. The test conducted is the acceptance test that each panellist is required to express a response about the product presented. The scale of preference was 1-5, that explanation was (1) very not like, (2) not like, (3) average, (4) like and (5) very like. Determination of Antioxidant Activity: DPPH Method (IC50) [20] One g of sample dissolved with 10 ml of methanol and homogenized, it was taken the solution (0,5; 1,0; 1,5; 2,0 ml), diluted with methanol and distilled water (1:1) until the volume of 10 ml. Took 1 ml of each and added 1 ml of DPPH, leave on for 15 minutes. The solutions were measured by a spectrophotometer with a wavelength of 517 nm. The inhibition percentage of each was counted by the equation. Note: A= Absorbance of blank B= Absorbance of sample Table 1 show that juice of "Pucuk merah" fruit contain anthocyanin as much as 21.32 ± 1.51 ppm, the total phenol as much as 832 ± 2.08 mg GAE g-1 and the pH was 4.58 ± 0.02. The pH of "Kolang kaling" was 3.45 ± 0.008. RESULT AND DISCUSSION Anthocyanins are a group of plant pigments that soluble in water that were commonly found in different types of fruits and vegetables. The pigment has a color ranging from pink, red, purple and blue that has high potential to be used as a natural colorant, replace synthetic colorant. However, anthocyanins have unstable properties on processing and storage. Several factors that influence the stability of anthocyanins are environment, pH, sugar, temperature, enzymes, light and metal ions [21]. There are 18 types of anthocyanins that have been found but only six that play an important role in food and are often found, namely pelargonidin (orange and salmon), cyanidine (magenta and crimson), delfinidine (purple, mauve and blue, peonidine (magenta), petunidine (purple) and malvidine (purple) [22]. Ref.n [23] reported that Cyanidine is found in the fruit of "Senduduk" (Melastome malabathricum) and Malvidin is found in the rind of the "Jamblang" (Syzygium cumini) fruit. Polyphenols are commonly found in fruit. Polyphenol per 100 g of fresh weight of pears, apples, grapes, berries and cherries contain up to 200-300 mg, processed products from this fruit, and still contains polyphenols in large quantities. [24]. As in [25] reported that the total phenolic content in freshly processed jams of strawberries with different maturity levels is almost the same and decreased during storage. The total phenolic of strawberry fruit, freshly processed jam, jam stored for 3 months at 4 0C and jam stored for 3 months at 20 0C were 184 mg/100g, 95 mg/100g, 97 mg/100g and 89 mg/100g respectively. Anthocyanin are a group of flavonoid is also included in the phenol group that has potential as antioxidant. From the raw material test found that the pH of "Kolang kaling" puree was 3.45 ± 0.08 whereas "Pucuk merah" fruit juice was 4.58 ± 0.02. This shows that the two main ingredients to be used are not sufficiently acidic, so in the production of jams must added with citric acid. The composition of water-soluble polysaccharide such as galactomannan, sugar and acids are essential in the formation of the gel. The value of pH of Jam The pH value of this jam ranges from 3.4 to 3.5. (Table 2). Based on the analysis of variance showed that the addition of fruit juice gave no significant effect on the pH value of jam. This is due to the addition of citric acid was in the same amount of 0.2%. Measurement of pH is one of the variables associated with durability of a product. The desired optimum pH value in the manufacture of jam ranges from 3.2 to 3.5 [2]. Table 2 shows that the pH value of this jam meets the requirements of the formation of the gel in all treatments. The pH value is often used as an indicator of food damage because the pH value is associated with the growth of spoilage microbes. Microbes can grow on food at a neutral pH. Anthocyanin of Jam Temperature and pH effect on stability of color. Increasingly heating temperature can cause loss glycosyl of the anthocyanin by hydrolysis of the glycosidic bond. The resulting compound is less stable and causes the color loss on anthocyanin of Rosella extract. [26]. Anthocyanin had positively correlated with chroma and negative correlated with the value of Hue [25]. Reducing anthocyanin can damage the color of the jam. Because of the temperature effected toward anthocyanin changes, so the fruit juice mixed with puree of "kolang kaling" at 50 0C. Although the use of these temperatures still has an effect on changes in anthocyanin content, but the decline is relatively small. The use of 50 o C could reduce anthocyanin content by 17% [26]. Ref. [11] showed that using temperature up to 700C, degradation of the color of anthocyanin less than 4%, and use 1000C of temperature cause degradation of the color of anthocyanin as much as 31%, of extract by using a mixture of water and citric acid of 3%. Table 3 show that the anthocyanin content range between 2.82 ± 0.13 to 4.20 ± 0.52 ppm. Based on the variance analysis indicated that the juice addition has significantly affect (α<0.05) on anthocyanin of the jam. The results of the analysis were shown in table 2. The analysis showed that the higher the addition of juice cause anthocyanin levels in the jam increased significantly. The lowest anthocyanin is present in jam with 6% addition of fruit juice, whereas the highest anthocyanin is in jam with the addition of 12% fruit juice. There was a statistically significant difference between the addition of 6% fruit juice with the addition of 8%, 10% and 12% fruit juice. There was no statistically significant difference between the addition of 8% fruit juice with 10% and 12% fruit juice on the anthocyanin content of jam The "Pucuk merah" contains anthocyanin of 21.32 ppm, which affects the anthocyanin content of the jam. There are several benefits of using food containing anthocyanin, such as natural dyes that can replace synthetic dyes, harmless to the body and function as an antioxidant. As in [11] reported that anthocyanin content in "Pucuk merah" was 440 to 447 mg L¬-1, it was higher than this researched. Presumably this is related to the concentration of the extract, used crushing fruit of "Pucuk merah" made from mixing water and fruit (3: 1). There are several advantages of using anthocyanin as a natural colorant in foods or beverages, that were safe and it has and also believed to play a role in biological systems, including the ability of free radical scavenging. [11]. Total Phenol of Jam The result of variance analysis show that the juice addition had significantly affect at α <0.05 on the phenol of "Kolang kaling" Jam. The results of the analysis can be seen in Table 4. The range of the total phenol was 382 ± 1.53 to 547 ± 6.43 mg GAE/100g. The more the juice addition, the higher the total of phenol in "Kolang-kaling" jam, it is influenced by the total phenol contained in the juice. Table 4 show the total phenol in "Kolang kaling" with added of 6% "Pucuk merah" fruit juice was lowest and total phenol in the jam with added of 10% "Pucuk merah" fruit juice was highest. The addition of fruit juices 10% and 12%, did not show statistically significant differences. There was a statistically difference of total phenol between the jam with the addition of 6% fruit juice and the addition of 8%, 10% and 12%, between the addition of 8% of the juice and the addition of 10% and 12% fruit juice. Phenol is sometimes called phenolic, is a chemical compound containing OH groups which is more soluble in water. As in [27] the total phenolics in Melastoma malabathricum leaves extract using water solvents, choloroform and methanol were 3344 mg GAE/100g, 92 mg GAE/100g and 3055 mg GAE/100 g, respectively. This data show that total phenolic solubility is better by using a water solvent. Based on the analysis of raw materials, fruit juice contains total phenol of 832 mg GAE /100 g, that was lower than total phenol in Melastome malabathricum leaves extract. Phenol compounds include a variety of plant-derived compounds, which have the same characteristic of an aromatic ring containing one or two hydroxyl, in which the role of several classes of phenol compounds is known, for example, lignin as the building material of cell walls, anthocyanin as the pigment of flower. The antioxidant activity of phenol compounds is formed because the ability of phenol compounds to form ions can give one electron to free radicals [13] Sensory Analysis of Jam Based on the variance analysis indicated that the juice addition had significantly different effect at α <5% on the color and the taste of "Kolang kaling" jam, but there was not significantly different on the texture of the jam ( Table 5). The more juice added to the "Kolang kaling", the higher the level of color acceptance of the panelist. The level of panellist acceptance of color ranges from 3.5 to 4.3. There was no statistically significant difference between the addition of 6% juice with 8%; 8% with 10%; and 10% with 12%. There was a statistically significant difference between the addition of juice of 6% and 10%; 6% with 12%; and 8% with 12% on the color of the jam. The color produced on the "kolang kaling" jam with "pucuk merah" fruit juice added was a purplish red with varying degrees of color. The more juice is added resulting the darker the color of jam The level of panelist's acceptance of taste ranges from 3.5 to 4.0. There was no statistically significant difference between the addition of 6% juice with 8%; and 8% with 10% and 12%. There was a statistically significant difference between the addition of juice of 6% to 10% and 12%. The highest level of acceptance of the panelists was the addition of 12% of the juice. Taste is an important parameter to panelist's acceptance, where flavour is the main parameter chosen by the consumer. The rate of addition of "Pucuk merah" fruit juice effect the sweet taste of jam produced. In addition to color and taste, texture has an important role in sensory reception. The acceptance value of textures ranges from 3.6 to 3.9. Based on statistical analysis showed that the addition of "Pucuk merah" fruit juice did not differ significantly to the texture of jam. Although there are differences in numbers, but statistically shows no significant difference. Based on the sensory test it is concluded that the panelists prefer jam with the addition of the most fruit juice, which is 12%. So the antioxidant analysis is only done on the best product by sensory product with the addition of fruit juice 12%. Activity of Antioxidant of Jam Activity of antioxidant is influenced by the content of phenol in the material. Anthocyanins are flavonoid group which is also part of the phenol compound. There was correlation between antioxidant activity (DPPH method) and total phenol content (R2 = 0.745), and antioxidant activity and flavonoid content (R2 = 0.759) on the extract of Trifolium pretense L [28]. A stronger relationship was reported by [29] the correlation between activity of antioxidant (IC50) and total phenol content in extract of Mimusops elengi was 0.9714, and the correlation between the activity of antioxidant and the total flavonoids content was 0.9993. The more the total phenol and anthocyanin contents in the material, the higher the antioxidant activity. All polysaccharides that are phosphorylated showed that a strong influence as an antioxidant and its strength is affected by the degree of substitution. The higher the degree of substitution, the stronger the antioxidant activity. Galactomannan is phosphorylated carbohydrate [30]. The raw material of this research was "kolang kaling". As in [7] reported that "kolang kaling" that made from Arenga pinnata contains galactomannan. Increasing the maturity will increase the content of galactomannan content, that were 1.27%, 2.71% and 3.72%. Galactomannan is composed of mannose and galactose with a ratio of 2: 1; 3: 1 and 5: 1 (M/G), where the increasing of mannose proportion with increasing the maturity. This study has analyzed the jam that has highest total phenol and anthocyanin content. Antioxidant activity was aqueous by determining IC50 values that are defined as the concentration of antioxidant compounds that cause the loss of 50% DPPH activity. Compounds having lower IC50 values have higher antioxidant activity. The analysis was performed on jam with the juice addition based in the best treatment (added 12%). Antioxidant activity of this jam (628 ppm) was lower than antioxidant activity of isolated pheophytin from green tea. As in [31] showed that IC50 concentration of isolated pheophytin from green tea was 573 mg/L. "Kolang kaling" with the fruit juice addition with a concentration of 12% has an IC50 value of 628 ppm. IC50 values describe the concentration of test compounds that can capture radicals by 50%. Ref. [11] reported that IC50 of "Pucuk merah" fruit extract was 0.055% (550 ppm) using a mixture of water and citric acid as solvent. It is compared with antioxidant activity in "Kolang kaling" jam added with "Pucuk merah" fruit juice, showed a very small decrease, but it was only added 12% of the juice. Maybe it is related with antioxidant activity of galactomannan in "Kolang Kaling". As in [32] has proven that water-soluble polysaccharides, which are rich in galactomannan, have antioxidant activity, with an IC50 of 330 μg mL-1. According to Ref. [33] the smaller the IC50 indicate the higher the activity of antioxidant. A compound has a very active antioxidant if the IC50 value is less than 50 ppm, said to be active if it is 50-100 ppm, is said to be less the antioxidant activity, if the IC50, 101-250 ppm, said weak if it has 250-500 ppm and said to be inactive if it is more than 500 ppm. "Pucuk merah" contain high phenol compounds; have a large contribution to the role of antioxidants, while anthocyanins are part of the total phenol. Phenol is also called phenolic compounds which are bioactive compounds that are often found in agricultural products. CONCLUSION The addition of juice of "Pucuk merah" fruit has a statistically significant effect on the anthocyanin content and total phenol of the "Kolang kaling" jam added with "Pucuk merah" fruit juice, but there is no significantly difference on the value of pH. The more the juice was added the more anthocyanin and total phenol content. Based on sensory analyses of "Kolang kaling" jam, the addition of the juice of 12% is the best product with score value of color was 4.3, the texture was 3.7, and the taste was 3.5. The addition of "Pucuk merah" fruit juice, resulting the red color of "Kolang kaling" jam, where more juice is added, the red color of the jam gets darker. "Kolang kaling" jam with the addition of the juice of 12% (based on the best product) has an IC50 value of 628 ppm. Although IC50 above 500 ppm, it showed that the jam still has antioxidant activity. In addition to "Pucuk merah", "kolang kaling" also contribute as a compound of antioxidant. Antioxidants are compounds that can delay, slow down and prevent oxidation processes, and can be used to protect food components that contain unsaturated fatty acids or have double bonds. Antioxidants can also be used to protect vitamins or pigment that also contains multiple bonds in the structure.

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Biological activities and Phytochemical analysis of Zanthoxylum armatum DC. leaves and bark extracts collected from Kumaun region, Uttarakhand, India The methanolic and chloroform extracts of leaves and bark of Zanthoxylum armatum DC. were evaluated for their phytochemical analysis and biological activities. In phytochemical analysis, fargsin was identified as major constituent in leaves methanolic and leaves chloroform extracts. t-butylamine and benzoxazole, 2-(isobutylamino) were identified as the major constituents in bark methanolic and bark chloroform extracts respectively. Both the extracts exhibited moderate antioxidant activity with IC50 values ranging from 19.42±0.07 to78.01±0.31 μg. These extracts also possess moderate anti-inflammatory activity with IB50 values ranging from 28.53 ± 0.06 to 89.80 ± 0.05 μg. Moderate anti-bacterial activity against E. coli and S. aureus has also been observed in both the extracts. The total flavonoids, orthodihydric phenols and phenolic contents were also quantified in the extracts. Based on these observations, it can be concluded that Zanthoxylum armatum DC. may be used as herbal antioxidant, food preservative, natural anti-inflammatory drug and natural bactericidal, besides generation of data base for judicious exploitation in future. INTRODUCTION for industries, medicinal plants and culinary applications. Almost all the species of genus Zanthoxylum have great ability to produce tires which could be used as encapsulates in the pharmaceutical industry, diluents and emulsifying agents [4,5,6,7,8]. Zanthoxylum armatum DC., commonly called as 'Prickly ash' or 'Timur' or 'Kababe Tejal', is a shrub or small tree which predominately grows in well drained alluvial, black soil and have a strong aroma. The plants are armed scandent or erect, 6 m tall or more, with dense foliage [9]. In India, it is found in the warmer valleys of the Himalaya from Jammu and Kashmir to Assam and Khasi (1,000 to 2,100 m), in the Eastern Ghats in Orissa and Andhra Pradesh (1,200 m) and the lesser Himalayan regions in the northeastern part of India for example, Naga Hills, Meghalaya, Mizoram, and Manipur [10,11,12,13]. Essential oils and different extracts of aqueous ethanol, dichloromethane, acetone, methanol, petroleum ether have been reported to shows many biological activities viz; larvicidal, antiviral, antifungal, keratolytic, anti-protozoan, pesticidal/insecticidal, hepatoprotective, antibacterial, antihelminthic and allelopathic [14,15]. The fruits and seeds are extensively used as tonic in fever, dyspepsia and cholera, eliminate pain, used to treat heart diseases, piles, diseases of mouth, teeth and throat disorder and the bark is used for intoxicating fishes [16]. We already have reported the chemical composition and biological activities of seeds, bark and leaves essential oil of Zanthoxylum armatum DC. collected from different altitudes of Kumaun region, Uttarakhand [17,18,19]. The present study assesses the chemical composition and biological activities of methanolic and chloroform extracts of Zanthoxylum armatum DC. leaves and bark collected from Kumaun region of Uttarakhand (India). Collection of Plant Material The plants material was collected from Aadi Kailash region (Bhimtal) (1370 m elevation), Nainital, Uttarakhand in the month of July, 2017. The plant material was properly identified by one of the author and Plant Taxonomist (DSR) and the identity was further confirmed by comparing the specimens with authentically identified specimens at the herbarium of Botanical Survey of India, Dehradun, India (BSD). The voucher specimen (GBPUH-917/28. Preparation of Extracts The bark and fresh leaves of Z. armatum were finely chopped, and shade dried and grinded. The resultant powdered material (250g) was subjected for extraction by cold percolation method in organic solvents with varying polarity. The solvents from extracts were evaporated by using rotatory vaccum evaporator and the final yield of the extracts was weighed. The yields in percentage (w/w) obtained were 2.4% leaves methanol extract, 4.8% bark methanol extract, 7.2% leaves chloroform extract and 4.4% bark chloroform extract. GC-MS Analysis GC/MS analysis of the different extracts was performed using a GC MS-QP 2010. The GC capillary column DB-5 (30 m × 0.25 mm i.d.; 0.25 µm film thickness; J&W Scientific, Agilent, Santa Clara, CA, USA) was used. Helium was used as a carrier gas with a flow rate of 1.21 mL/min, at a pressure of 73.3 kPa. The extracts were injected at temperature: 260 °C with oven temperature programme as: Initial temperature 60°C, RAMP 3°C/min upto 210°C (isotherm for 2 min), then RAMP 6°C/min upto 280°C (isotherm for 2 min), finally hold for 11 min. The compounds were identified with the help of NIST-MS, FFNSC Wiley Library, and comparing the data with literature reports and retention indices (RI) [20]. DPPH (1, 1-diphenyl -2 -picryl -hydrazyl) radical scavenging activity This activity was evaluated according to the developed protocols with slight modifications [21,22,23]. The tested extract samples (50-250 µg/mL) were taken and mixed with 5 mL of a 0.004% methanolic solution of freshly prepared DPPH. The O.D. (optical density/optical absorbance) was measured by using UV-visible spectrophotometer (Thermo Scientific EVOLUTION-201 series) at 517 nm. All the observations were recorded in triplicate with reference to the standard antioxidants catechin and BHT. Inhibition of free radical in percent (IC%) was calculated by using the equation: IC% = (A 0 -A t )/A 0 × 100 where, A 0 = absorbance value of control sample, A t = absorbance value of test sample, IC = inhibitory concentration. Percent inhibition was plotted against concentrations in graph. The standard curve was drawn using standard antioxidant (BHT and catechin) to calculate the IC 50 values for different extracts and standard. Reducing power The reducing power of extracts was evaluated by the method developed earlier and is being practiced [24]. Various amount of extracts (50-250 µg/mL) were mixed with 2.5 mL of phosphate buffer (pH= 6.6, 200 mM,) and 2.5 mL of 1% potassium ferricyanide, K 3 [FeCN 6 ]. After 20 minute incubation at 50±1ºC, 2.5 mL of trichloroacetic acid was added to the mixtures, followed by centrifugation at 650 rpm for 10 min. The supernatant (1 mL) was mixed with 5mL distilled water followed by 1 mL of 0.1% ferric chloride. The absorbance of the resultant solution was measured spectrophotometrically at 700 nm. All the readings were recorded in triplicate. Ascorbic acid was used as standard. The percent reducing power of samples was calculated using the formula: Reducing power % = (A 0 -A t )/A 0 × 100 where, A 0 = absorbance value of control sample, A t = absorbance value of test sample. Percent inhibition was plotted against concentrations in the graph. The standard curve was drawn using standard antioxidant (BHT) to evaluate the RP 50 values for standard and different extracts. Metal chelating activity The chelation of Fe+2 was evaluated using the method developed earlier [24]. 0.1 mL of 2mM FeCl2.4H2O, 0.2mL of 5mM ferrozine and 4.7 mL of methanol was added to various concentrations of test sample (50-250 µg/mL). The solutions were mixed thoroughly and incubated for 10 min. At 562 nm, the absorbance of test sample was measured in a UV spectrophotometer (Thermo Scientific EVOLUTION 201 series). All the readings were recorded in triplicate; EDTA (0.01 mM) was used as the standard. The metal-chelating activity of tested samples, expressed as percentage was calculated by using the formula: IC% = (A 0 -A t )/A 0 × 100 where, A 0 = absorbance value of control sample, A t = absorbance value of test sample, IC = inhibitory concentration. The percent of chelating ability was plotted against concentrations in graph. The standard curve was drawn using standard antioxidant (EDTA) to calculate the IC 50 values for standard and different extracts. Estimation of Phenols The phenolic assay of both the extracts of Z. armatum was calculated quantitatively by spectrophotometer in terms of total phenols, flavonoids, and orthodihydroxy phenols. The concentrations of these samples were measured with the help of working calibration curve by the relation among concentration and absorbance of the sample. Total phenolic assay The total phenols were determined by the Folin-Ciocalteu reagent (FCR) method [25]. In brief, 1 mL of the sample extract was poured into a test tube and mixed with 1 mL of 80% methanol and 8 mL of distilled water. In each sample 0.5 mL of 1 N FCR was added and mixed. After 5 min., 1 mL of saturated Na 2 CO 3 was added to the reaction mixture and allowed to stand for 60 min. The absorbance of test sample was observed at 650 nm. The standard curve was drawn using various concentrations of gallic acid and results were expressed as mg of gallic acid per gram of sample in dried weight. Estimation of flavanols Aluminum chloride colorimetric assay [26] was applied for the estimation of flavanols. 10 mg of extract were dissolved in 10 mL of 80% methanol to prepare stock solution. In a test tube, 0.1 mL of stock solution was mixed with 1.25 mL water and 0.75 mL of 5% NaNO 2. The mixture was incubated for 5 min. 0.15 mL of 10% AlCl 3 was added to the mixtures after incubation. After 6 min, 275 µL of distilled water and 0.5 mL of 1 N NaOH were added, after thoroughly mixing of the solution. At 510 nm, the intensity of pink colour was obtained. The standard curve was established using different concentrations of catechin and the concentrations were evaluated with the help of calibration curve and expressed in mg/100gm of dry material [27]. Estimation of orthodihydric phenols (OHP) 10 mg of extract was added in 10 mL of 80% methanol to prepare stock solution. 0.1 mL of the extract solution was poured in a test tube and mixed with 0.4 mL of water and 1 mL of 0.05N HCl, 1 mL of Arnow's reagent (10 g sodium nitrite and 10 g sodium molybdate made up to 100 ml with distilled water), 10 mL of water and 2 mL of 1 N NaOH. The resultant solutions were thoroughly mixed (appearance of pink colour) and at 515 nm absorbance was measured. The standard curve was prepared with the help of working standard catechol solution at different concentrations. The concentration was calculated and expressed in mg per 100gm of material [28]. In-vitro Anti-inflammatory Activity The in-vitro anti-inflammatory activity of extracts was evaluated by using inhibition of albumin denaturation technique, by the standard protocols [29,30,31]. The reaction mixture (5 mL) was comprised of 0.2 mL of egg albumin, 2.8 mL of phosphate buffer solution (pH= 6.4) and 2 mL of varying amount of extracts (25 µg/mL, 50 µg/mL, 75 µg/mL, 100 µg/mL, 125 µg/mL, 250 µg/mL and 500 µg/mL). Double distilled water was used as control. At 37±2°C, the mixtures were incubated in a BOD incubator (for 15 min and then heated for 5 min at 70°C in water bath). Subsequent to cooling, the absorbance was observed at 660nm. All the readings were observed in triplicate, diclofenac sodium was used as the standard. The percentage inhibition of protein denaturation was calculated using the formula: IB% = (A 0 -A t )/A 0 × 100 where, A 0 = absorbance value of control sample, A t = absorbance value of test sample, IB = inhibitory concentration. The extract/drug concentration for 50% inhibition (IB 50 ) was determined by plotting percentage inhibition with respect to control against treatment concentration. Antibacterial Activity The antibacterial activity was determined by using Agar well diffusion method [32,33]. It was expressed as the mean of zone of inhibition (ZOI) diameters (mm) produced by the various extracts. For screening plates were prepared by using nutrient agar. The inoculums (50µL) of different bacterial strains were spread evenly on respective plates with sterile spreader. A borer (8mm diameter) was used to cut well. 20µL of different concentrations of the extracts were poured in each well and incubated at 24 hrs at 37±2°C. The diameter of ZOI was measured and the mean was recorded. The experiment was performed in triplicate. Statistical Analysis The data were analyzed by using Analysis of Variance (ANOVA) using STPR. All the values were taken in triplicate. IC 50 was determined by linear regression analysis using MS Excel 2007. The literature search reveals no report on GC/MS analysis of chloroform and methanolic extracts of Z. armatum leaves and bark (ZNLClE, ZNLME ZNBME and ZNBClE) hence is being reported first time. The major fargsin detected in leaves has also been reported in the leaves essential oil of Z. acanthopodium from north-eastern region of India [34]. Hence, the present analysis reveals the first report on it. The study on comparative chemical composition among ZNLME, ZNBME, ZNLClE and ZNBClE has been represented in Table 1 and structures of major chemical constituents in extracts of Z. armatum has been illustrated in Figure 1. Total phenols The total phenolics content in different extracts of Zanthoxylum armatum DC. were observed in the order of: ZNLME (31.23±0.03 mg/g GAE) > ZNBME (27.58±0.05 mg/g GAE) > ZNBClE (25.02±0.04 mg/g GAE) > ZNLClE (17.74±0.02 mg/g GAE). The variation in total phenolic content in different extracts might be because of different solubility of phenolics in organic solvents with varying polarity. Among different plant extracts from different parts of plant, it has been observed that ZNLME contained maximum total phenolic content (31.23±0.03 mg/g GAE) while ZNLClE showed minimum among the extracts (17.74±0.02 mg/g GAE) ( Table 2). Total flavonoids Flavonoids are one of the most important groups of bioactive secondary metabolites in plants and are known for their health promoting propertied due to protective effects against cancer, cardiovascular disease and other diseases [35]. In the present study, the total flavonoids content in different extracts were observed in the order of: ZNLClE (77.18±0.06 mg/g CNE) > ZNBME (68.04±0.06 mg/g CNE) > ZNBClE (56.72±0.03 mg/g CNE) > ZNLME (47.63±0.14 mg/g CNE) ( Table 2). The total phenolics and flavonoids in bark ethanolic extracts from Pakistan have also been reported [36]. The results revealed the presence of total phenolics (11.66±0.33mg/g) and flavonoids (13.68±0.66mg/g) in leaves and total phenolics (16.48±1.33mg/g) and flavonoids (18.33±1.22mg/g) in bark. Our study revealed high phenolic and flavonoids content in leaves methanolic extract (31.23±0.03 mg/g GAE and 47.63±0.14 mg/g GAE respectively) and bark methanolic extract (27.58±0.05 mg/g GAE and 68.04±0.06 mg/g GAE respectively) compared to Barkatullah et al., 2017 [36] (Table 2). This discrepancy in results might be due to diverse natural habitats of sampled plants. DPPH radical scavenging activity All the extracts exhibited good radical scavenging activity as a function of their concentrations. ZNLME ( (Table 3) Kanwal et al., 2015 [37] has reported the moderate antioxidant activity of Z. armatum leaves methanolic extract by DPPH and reducing power assay. Present study exhibited good antioxidant assay in all the extracts. Among all the extracts ZNLME exhibited maximum antioxidant activity while the minimum was found in ZNLClE. The phenolics are found to be related and reported to possess the antioxidant activity [37]. In present study, a good quantitative amount of phenolics, flavonoids and orthodihydric phenols have been observed. Hence, the good antioxidant activity of extract is because of the presence of phenols. Further, it has been observed that the phenolic content in the extract exhibited positive correlation with antioxidant activity and negative with IC 50 values. The antioxidants are used to prevent the oxidative deterioration of foods and food products and can be used as food preservation for long-life of the food material. The synthetic antioxidants possess deleterious effects. Hence, in present scenario, the herbal antioxidants are in demands. Based on these facts, it can be concluded that the medicinal herb Z. armatum can be used as an herbal source of natural antioxidants and phytochemical source of nutraceuticals. In-vitro Anti-inflammatory Activity The in-vitro anti-inflammatory activity of extracts was performed by inhibition of egg albumin denaturation method as described in materials and methods section. Denaturation of proteins is well documented and is caused by inflammation process, mostly in conditions like arthritis. In protein denaturation mechanism, due to external stress, influence of chemical reactions results in distortion of protein's tertiary and secondary structure and leads to denaturation of proteins [38]. As the part of study on mechanism of anti-inflammation activity, capability of plant extracts were studied. It was observed that the extracts ZNLClE, ZNLME, ZNBClE and ZNBME inhibited the heat induced albumin denaturation in a dose dependent manner as monitored spectrophotometrically. The in-vivo anti-inflammatory activity of ethyl acetate extract from stems and roots of Z. armatum has been reported. The study revealed that the constituents like eudesmin, pinoresinol, sesamin and yangambin were responsible for in-vivo anti-inflammatory activity [39]. In present study most of the extracts exhibit moderate anti-inflammatory activity. The compounds eudesmin, sesamin and yagambin are also present in our extracts. Hence, it can be inferred that the anti-inflammatory activity is possibly due to the presence of these compounds or synergetic effect of co-occurrence of major, minor or trace constituents. Presence of polyphenolic compounds such as alkaloids, flavonoids, tannins, steroids, and phenols has also been reported to posses anti-inflammatory activity [40]. It has been reported that the total phenolics and antioxidants possess anti-inflammatory activity [41,42]. There is a positive correlation between antioxidant activity and total phenolics in present study. Hence, the constituents responsible for antioxidant activity may also be responsible for anti-inflammatory activity, i.e. there is positive co-relation between anti-inflammatory activity and phenolics/antioxidants. Antibacterial Activity The antibacterial efficiency of the ZNLClE, ZNLME, ZNBClE and ZNBME has been presented in Table 5. The extracts were tested for antibacterial activity against gram-positive and gramnegative bacteria and were found to be effective against all the tested bacterial strains as compared to the antibiotic gentamicin sulphate, taken as standard. It was observed that ZNLME showed maximum ZOI (zone of inhibition) of 17.67 mm at 500 ppm against E. coli while, ZNBME exhibits maximum ZOI of 17.67 mm at 500 ppm against S. aureus. The minimum ZOI was observed in ZNBClE (5.33 mm), at 250 ppm against E. coli while, in case of S. aureus ZNBClE showed minimum ZOI (3.67 mm) ( Table 5). The screening of antibacterial activity of different extracts of Z. armatum against E. coli has been represented in Figure 3 while the screening of antibacterial activity of different extracts of Z. armatum against S. aurens has been represented in Figure 4. [44] reported the antibacterial activity against Staphylococcus aureus, Escherichia coli, Proteus vulgaris and Pseudomonas aeruginosa in Z. armatum chloroform, methanol and acetone bark extracts collected from lesser and higher Himalaya (altitude 700-2000 m). The acetone and methanol extracts of bark were found to be more effective for S. aureus and chloroform extract for P. vulgaris. CONCLUSIONS The above results show that the Z. armatum is a good source of major compounds like eudesmin, sesamin, methyl-vanillin, linolenic acid, fargsin, γ-sitosterol, doxepin, besides other major and minor constituents. These compounds find their wide applications in perfumery, preservation, pharmacological activities and starting material for the synthesis of novel molecules. The compounds like doxepin and (Z, Z) -6, 9-cis-3, 4-epoxy-nonadecadiene has also been reported to possess anti-depressant activity and sex pheromone, respectively. The extracts have also been found to possess moderate antioxidant, anti-inflammatory and antibacterial activity. The total flavonoids, orthodihydric phenols and phenolic contents were also quantified in methanolic and chloroform extract of leaves and bark. In present scenario, food and pharmaceutical industries are in search of environmentally benign novel lead molecules from herbal origin. The present study concludes that the entire plant of Zanthoxylum armatum might be used as a good source of herbal antioxidants, food preservative, natural anti-inflammatory drug and natural anti-bacterial agent after proper clinical trials. The present study contributes for preparation of database on this species so that it can be exploited judiciously and scientifically.

v3-fos

2019-03-19T13:12:35.604Z

1970-12-01T00:00:00.000Z

83431575

s2

The selenium content of Finnish forage crops. The importance of selenium in plants was first realised in the 1930’5, when it was found that »alkali disease» and »blind staggers» ofcattle and horses in the United States of America were caused by the animals eating plants containing a high concentration of selenium (Beath el al. 1934). The biological significance of selenium was believed to be restricted to its toxicity until Schwarz and Foltz (1957) showed that Factor-3, which protects rats against liver necrosis, was a selenium compound. It was subsequently established that selenium is an essential trace element which will protect animals against a variety of diseases that were previously thought to be due primarily to vitaminE deficiency (Schwarz 1961). One of the most important diseases of farm animals, now known to be associated with a low selenium intake, is nutritional muscular degeneration (NMD) in ruminants. Research in many countries has shown that NMD in ruminants is associated with low selenium concentrations in the diet (Hartley & Grant 1961, Gardiner el al. 1962, Allaway & Hodgson 1964). This in turn has stimulated the development of accurate methods ofmeasuring small amounts of selenium in biological material (Wolf et al. 1963, Watkinson 1966, Lindberg 1968). In Finland, NMD in cattle has caused considerable economic losses (Andersson 1960, Oksanen 1965), and although the results of selenium treatment have generally been good, there have been conflicting reports on the selenium content of forage plants in Ostrobothnia, where the disease was once most common (Andersson 1960,Westermarck 1964, Oksanen 1965). In view of this, it was considered important to re-examine the selenium content ofFinnish forage plants collected from different parts of the country. In Finland, NMD in cattle has caused considerable economic losses (Andersson 1960, Oksanen 1965, and although the results of selenium treatment have generally been good, there have been conflicting reports on the selenium content of forage plants in Ostro-bothnia, where the disease was once most common (Andersson 1960,Westermarck 1964, Oksanen 1965. In view of this, it was considered important to re-examine the selenium content of Finnish forage plants collected from different parts of the country. Material and Methods During the crop years 1968 and 1969, 145 plant samples were collected from 13 different field stations belonging to the Agricultural Research Centre in Finland. The samples were ground and their dry weights determined. The selenium content ofsamples weighing 10-25 g was determined by the method of Lindberg (1968). The material was wet-ashed with a mixture of nitric and perchloric acids, the selenium extracted with toluene 3,4dithiol, and 2,3-diaminonaphthalene was used as a fluorescing reagent. The recovery of the selenium was measured by using Se 75 as a tracer, the radioactivity being measured with a Nal-crystal computer before and after handling. Results The results are set out in Table I. The mean concentration of selenium in hay and grain samples was 0.014 ppm (14 ppb) and 0.007 ppm (7 ppb), respectively. The samples with the highest concentration came from the most northerly station at Rovaniemi. There was no correlation between the selenium content in the plants and the type of soil in which the plants were grown. Discussion The low selenium values obtained in this study are similar to those reported from other countries in forage known to cause selenium deficiency diseases in animals. Oldfield et al. (1963) reported that they could provoke NMD in lambs with a hay-oat diet containing less than 0.02 ppm Se and that the disease was prevented, when the selenium concentration was raised to 0.06 ppm. Most of the field cases of selenium responsive diseases in ruminants have occurred in young lambs and calves from dams that have been kept on feeds containing less than 0.05 to 0.10 ppm Se (Allaway et al. 1967). The observed critical minimum levels of selenium required in animal diets vary from less than 0.03 to 0.3 ppm Se (Alla way et al. 1967). In preliminary investigations it was found that hay samples collected in 1965 from South-Ostrobothnia contained 0.01 to 0.02 ppm Se, while hay samples collected previously from the same area contained 0.013 to 0.081 (mean 0.032) ppm Se. A barley sample contained 0.014 and an oat sample less than 0.010 ppm Se (Oksanen 1965) These results were obtained by the use of neutron activation analysis and the accuracy was restricted by the low power of the reactors used. The fluorescence method used in the present study is suitable for determining low selenium contents in biological material (Watkinson 1966), and the use of the Se 75 tracer, to measure the loss of selenium during the analysis, increases the reliability of the method. Low selenium content in plants is not necessarily related to the soil selenium content, as selenium in the soil may occur in forms that the plant cannot utilize. For example in acid environments compounds of selenium and iron may occur, from which plants are unable to absorb selenium (Trelease & Beath 1949). Likewise, the content of sulphur in the soil affects the availability of selenium to the plant (Hurd-Karrer 1934). In Finland, especially in Ostrobothnia, there are acid »sulphate soils» rich in iron and sulphur (Kivinen 1950) and it is possible that this may account for the low selenium content of plants in this area. However, it is known that the plutonic and metamorphic rock in the whole country is especially low in selenium, about 0.06 ppm (Koljonen 1965). Consequently it appears that the low soil selenium content is the main reason for the low selenium values in forage plants in Finland. The selenium responsive diseases are not necessarily simple selenium deficiencies. Vitamin E, unsaturated fatty acids, sulphur containing amino acids and possible other factors, are involved. For example in Ostroborhnia, where NMD was a common disease prior to selenium supplementation, it was found that yearly fluctuations in incidence could be related to variations in the vitamin E content ofhay, which was dependent on harvesting conditions (Oksanen 1965, Thafvelin & Oksanen 1966. Notwithstanding this complex aetiology, NMD can be prevented and cured by selenium. From the results of the present study it would appear that the selenium content of hay and grain in Finland is so low' that selenium responsive diseases may be expected to occur anywhere in the country unless selenium supplementation is practised. Summary The total of 145 plant samples were collected from 13 field stations in Finland during the crop years 1968 and 1969 and analysed for selenium by a fluorescence method using 2,3-diaminonaphthalene as the fluorescing reagent. The loss of selenium during analysis was measured by a Se 75 tracer. The selenium content of all samples was very low. The mean value for hay samples was 0.014 ppm (range 0.002 to 0.048), and for grain sarnies 0.007 ppm (range 0.002 to 0.085). The samples with the highest selenium content came from Lapland (Rovaniemi), where the mean values for hay and grain were 0.036 ppm and 0.013 ppm respectively. There was no correlation between the plant selenium content and soil type. It appears that selenium deficiency diseases may occur anywhere in Finland unless Se supplementation is carried out.

v3-fos

2018-04-03T01:49:31.064Z

1970-06-01T00:00:00.000Z

31100912

s2

Survival and activity of frozen starter cultures for cheese manufacture. A study has been conducted on the effect of freezing and storage in liquid nitrogen on 13 strains of lactic streptococci. Cultures were frozen in droplet form and collected in mesh bags. After rapid thawing, the activity of the frozen cultures was compared with a culture of the same organism of the age usually used in cheese-making. The activities of the test and control cultures were traced simultaneously by continuous recording of the pH changes in inoculated milks. Viable counts were performed before and after freezing in liquid nitrogen and after storage in liquid nitrogen. There was no decrease in viable count or loss in activity of the cultures due to freezing and storage. Frozen cultures of some strains showed a shorter lag period after inoculation of milk than control cultures. Frozen concentrated cheese-starter cultures behaved normally in the manufacture of Cheddar cheese. At the present time, there is a widespread interest in the preservation of cheese-starter cultures by freezing in liquid nitrogen and in the use of concentrated frozen starter cultures for direct inoculation into the vat in the manufacture of cheese (1,4,9,12,13). Already such cultures are in commercial use in the United States for the setting of bulk inoculum and for direct vat inoculation for long-set cheese manufacture. The effects on the survival and activity of lactic streptococci, after freezing and thawing, of different methods, of different temperatures of freezing and storage, and of growth temperatures and pH prior to freezing have been the subjects of recent investigations (2, 5-8, 10, 11). The present paper deals with a study of the activity of lactic starters as determined by the continuous recording of pH changes in milks inoculated with frozen starter and incubated for 18 hr at 30 C. Thirteen different strains of lactic streptococci were studied after periods of storage in liquid nitrogen, the behavior of the various strains being relevant to future commercial production of frozen concentrated starter cultures. MATERIALS AND METHODS Cultivation. The medium used for the cultivation of starter cultures contained 1,000 ml of low-calcium coprecipitate whey (0.04%/o calcium) and 0.005% bromocresol purple. The pH was adjusted to 6.8, and the medium was sterilized at 121 C for 10 min in 100-ml quantities. Cultures were incubated for 6 hr at 30 C, neutralized with 0.1 N sodium hydroxide to the bromocresol purple end point, and immediately frozen. Culture HP was grown at 25 C. Culture C2 was used in cheese manufacturing experiments. Method of freezing. The neutralized culture was frozen in droplet form by discharging drops from a 10-mi pipette into a terylene mesh bag suspended in a bath of liquid nitrogen. The bag was held in place in a specially constructed holder consisting of a stainless-steel tube with a mesh bottom. When 10 ml of culture had been frozen, the bag was closed by pulling a drawstring and was transferred to a liquid nitrogen refrigerator (type LR-10, Union Carbide). To test the feasibility of freezing concentrated starter cultures in this way, cultures in the logarithmic phase of growth, produced by a continuous-culture technique, concentrated by centrifugation, and resuspended in a whey culture medium (pH 6.3) with the aid of a high-speed blendor, were frozen as a thick slurry (approximately 1012 cells/ml) in the same manner. Method of testing the effects of freezing and thawing. Viable counts were performed on all cultures immediately before freezing, immediately after freezing, and at approximately monthly intervals during the storage period. The medium used for viable counts was tryptone-yeast extract-agar (TYA) of the following composition: tryptone, 3 Skim milk was used in checking the activity of the cultures, rather than reconstituted skim milk, because the behavior of the frozen starter in different milks was of interest in the experiment. Two flasks, each containing 500-ml quantities of milk, were heated by being brought momentarily to 121 C and then cooled and inoculated with starter. One flask of milk was inoculated with the frozen 10 ml of starter and the other with 10 ml of a coprecipitate whey suspension of a 16to 18-hr milk culture. The amount of culture added to the coprecipitate whey medium was calculated so that the counts in the test and control flasks of milk would be comparable. Viable counts were made on the two flasks of milk immediately after inoculation and mixing. The two flasks were placed in a water bath at 30 C and a combined calomel-glass pH electrode was placed in each flask. The two electrodes were connected to Radiometer (model 22) pH meters which in turn were connected to a Hitachi (model QPD73) dual pen recorder. The pH change during the course of 16 to 18 hr of incubation was traced. The activities of the normal starter and the frozen starter were compared by measuring the time taken for the first indication of pH change and the time taken for the pH to drop further to 5.6 in each flask. Preliminary Cheddar cheesemaking trials. Frozen concentrated cheese starter (approximately 101" cells) was inoculated directly into 100 gal of milk in an experimental cheese vat. At the same time, another 100 gal of the same batch of milk was inoculated with 2% of the cheese-starter strain prepared in the normal way. Viable counts were made on the milk in each vat immediately after inoculation and mixing of the starter with the vat contents. Because chain disruption due to freezing and thawing has been reported (10), before counts were made, each sample of inoculated milk from the test and control vats was subjected to a blending at a standard speed with a high-speed blendor to ensure maximum disruption of chains of the streptococci to make the viable counts in both samples comparable. Counts were also performed on untreated samples of the inoculated milks for comparison. Cheddar cheese was manufactured in the two vats in the usual way, and the behavior of the normal and frozen starters during manufacture was compared. Only one strain of starter, S. lactis, C2, was tested. RESULTS Of the 13 strains of starter tested for viability before and after freezing, none showed a loss in numbers as measured by the plate-count method. None of the cultures stored for long periods, from 1 to 13 months, in liquid nitrogen showed a significant change in count during the period of storage. Microscopic examination of the culture before and after freezing showed no evidence of chain disruption due to freezing. All cultures tested showed equal or better activity than their controls, except in the case of C13 where the difference in inoculum favored the control (Table 1). Figure 1 shows a typical chart tracing the activity of a frozen starter and its control. Cheese manufacture with frozen starter proceeded normally, and the cheese was equal in quality to that made with the conventional starter, although, as expected, the higher pH in the frozen starter resulted in the renneted milk taking 10 min longer to coagulate in the test vat. Viable counts on the milk in the control and test vats after inoculation were very similar (4 X 107 ml in both vats). There was no evidence of chain disruption by concentration and freezing of the streptococcal culture. DISCUSSION The freezing of the starter culture in liquid nitrogen in droplet form presents no problems with regard to viability and activity of the culture. The better activity of the frozen culture, in terms of time taken to show the first indication of pH change in the milk, could be attributed to the fact that the starters were frozen in an active phase of growth (6 hr of incubation), whereas the control cultures were in the stationary phase of growth when transferred to the milk, as is normal with the starter inoculum prepared by conventional methods for cheese manufacture. If, in the future, cultures are grown by continuous-culture techniques, the freezing of cells in their active phase of growth could be safely achieved. A selection of more active variants may also result from continuous culture. It is possible, therefore, that a reduction in the number of cells required for vat inoculation could be made for some strains of lactic streptococci. The medium used in the present study was selected because of its possible use in the production of starter by continuous-culture technique. Its protein content was very low. In spite of this, the cells suffered no apparent damage during the processes of freezing and thawing. The fact that cultures can be stored successfully for 13 months in liquid nitrogen would suggest that they might in fact be stored indefinitely. The addition of a protective substance to the culture before freezing in liquid nitrogen is unnecessary. This has also been demonstrated by other workers (3). If the cultures are frozen or stored at higher temperatures, protective substances are necessary (7). The absence of protective substances not derived from milk constituents in a frozen starter is advantageous because such additives might not be permissible under some food regulations. Preliminary cheesemaking trials showed that, at this stage of the investigation, the concentration of cheese starters by centrifugation and their subsequent rapid freezing in liquid nitrogen followed by rapid thawing appear to present no problems in cheesemaking. This can be assumed, provided that in the continuous culture there is no selection of cells which might be lacking in enzymes essential for fast acid production or for the maturing of cheese. It is foreseen that in commercial production the concentrated suspension of a starter could be dropped at a constant rate into bags of a similar type but larger than the ones used in the present experiment. The bath of liquid nitrogen would be slowly moving to ensure that the drops do not fall in the same place consecutively to avoid clumping. Another possibility would be to discharge the concentrate on a moving Teflon belt through a bath of liquid nitrogen. The culture would be frozen as a thin film on the belt. The belt would impinge upon a scraping device which would remove the frozen concentrate as a powder into a plastic bag. Although it is possible that the frozen cultures could be stored at temperatures above that of liquid nitrogen and below -130 C without the presence of protective substances, there are advantages in the use of liquid nitrogen refrigeration. Provided the supply of liquid nitrogen is assured, there is little likelihood of breakdown of refrigeration equipment. Liquid nitrogen is not a sterile product, and its sterilization would probably be too costly for commercial use. The large numbers of quickly revived cells in the concentrated frozen starter would be expected to suppress small numbers of contaminants. In the trials carried out in the present study, although no specific tests were made, there has been no evidence of bacterial or phage contamination in the milk inoculated with starters which have been in direct contact with the liquid nitrogen. ACKNOWLEDGMENTS The author thanks E. G. Pont and G. T. Lloyd for providing their concentrated culture grown by the continuous-culture method for cheesemaking trials, G. Pettingill for very skilful technical assistance, and L. A. Hammond for the manufacture of cheese.

v3-fos

2020-12-10T09:04:16.847Z

1970-06-01T00:00:00.000Z

237231284

s2

Concentrated Cultures of Leuconostoc citrovorum Two single-strain cultures of Leuconostoc citrovorum were grown in a broth medium with automatic pH control. Culture concentrates were prepared by centrifugally harvesting the cells and resuspending them in 1/50th the original volume in 10% nonfat milk solids. The concentrates were stored in liquid nitrogen until analyzed. The maximum population attainable was approximately equal when cultures were grown at pH 6.0, 6.5, or 7.0 with sodium hydroxide or ammonium hydroxide as the neutralizer. Citrate was required in the growth medium for the cultures to be able to produce diacetyl subsequently in milk. At pH 6.0, the cells reached maximum population and ability to produce diacetyl. Organoleptic analysis by an experienced flavor panel showed a preference for cottage cheese creamed with a creaming mixture prepared with a culture concentrate rather than a normal culture. The culture concentrates maintained their viability and ability to produce diacetyl for at least 30 days when stored in liquid nitrogen. by an experienced flavor panel showed a preference for cottage cheese creamed with a creaming mixture prepared with a culture concentrate rather than a normal culture. The culture concentrates maintained their viability and ability to produce diacetyl for at least 30 days when stored in liquid nitrogen. Single-strain cultures of Leuconostoc citrovorum are employed for the enhancement and control of flavor in creamed cottage cheese (4,6). L. citrovorum is grown in successively larger volumes of milk until a sufficient volume of culture is prepared to formulate the creaming mixture. The use of a concentrated suspension of L. citrovorum for the direct inoculation of the milk to be used in the creaming mixture would eliminate the in-plant build up of cultures and reduce the time required to produce desired flavor compounds. This would also minimize many problems involving culture activity and undesirable microbial contamination. Concentrates of lactic streptococci for use in cheese manufacture have been successfully prepared and stored at subzero temperatures without loss in viability or biological activity (1,7,10). The present investigation was undertaken to study the preparation of similar concentrates of L. citrovorum. MATERIALS AND METHODS Cultures. Two cultures of L. citrovorum (strains 3 and 28) were obtained from a commercial supply laboratory. The cultures were routinely propagated in sterile litmus milk, by using a 1% inoculum and incubation at 25 C for 24 hr. The cultures were subcultured at least three time in the experimental medium prior to growing cells for preparing concentrated cultures. The cultures were stored in litmus milk at 4 C. 1 Paper number 3058, Journal Series, North Carolina State University Agricultural Experiment Station, Raleigh, N.C. Culture growth with automatic pH control. A 7.5liter fermentor equipped with autoclavable electrodes in conjunction with an automatic pH controller (New Brunswick Scientific Co., New Brunswick, N.J.) was used to grow the cultures. Four liters of broth containing 2% tryptone (Difco), 0.5% yeast extract (BBL), 1% glucose, and 0.5%o sodium citrate was autoclaved at 121 C for 15 min and aseptically transferred into the sterile fermentor. The growth medium was inoculated with 40 ml of a fresh broth culture and incubated at 25 C. The pH was controlled at the desired level with either a 20%o aqueous solution (weight per volume) of NaOH or NH40H. After the desired incubation time the cells were concentrated and stored in liquid nitrogen by using the methods described by Peebles et al. (8). Cell population. Colony counts were made with Elliker broth (Difco) plus 1.5% agar. Dilutions for plating were prepared by the technique of Peebles et al. (8), except that the initial dilution was blended for 2 min. Duplicate plates at each dilution were prepared and incubated at 32 C for 48 hr, after which all visible colonies were counted. Measurement of diacetyl production. Frozen concentrates were thawed and diluted in 10% nonfat milk solids (NFMS) to a population equivalent to a culture of the same strain grown in 10% NFMS for 18 hr at 25 C. The pH was immediately adjusted to 4.4 to 4.5 with 15% citric acid. This required 4.2 ml of the citric acid per 100 ml of milk. The level of diacetyl in the acidified cultures was determined after incubation at 25 C by the method of Pack et al. (7). The amount of sample assayed depended on the level of diacetyl but was usually 10 or 20 g. The 10-g samples were purged with nitrogen for 1 hr and the 20-g samples were purged for 1.5 hr to insure transfer of all the diacetyl to the hydroxylamine trap. A culture of the same strain 890 grown for 18 hr in 10% NFMS was acidified and assayed in a similar manner for comparison. Organoleptic analysis. Dry cottage cheese curd obtained from the North Carolina State University Creamery or a local dairy plant was creamed with a series of mixtures which were standardized to 16% fat with either 10% NFMS inoculated with a concentrated culture or a normal culture grown in 10% NFMS. The inoculated NFMS was incubated at 25 C for 6 hr at pH 4.4 to 4.5 prior to standardization and homogenization (2,400 psi). Salt (6.2%) was added to the homogenized creaming mixture before mixing with the dry curd. The creamed cottage cheese was stored at 4 C until analyzed. Members of the flavor panel were given a portion of each cheese and asked to assign a preference to each sample based on flavor and aroma. RESULTS Citrate requirement. Concentrated suspensions of L. citrovorum 3 prepared from cells grown at pH 6.0 in the broth medium without citrate did not produce diacetyl when inoculated into acidified milk (Table 1). There was no appreciable production of diacetyl until after the concentrate was subcultured three times in milk. The subcultures were prepared by using the equivalent of a 1 % inoculum and by incubating for 18 hr at 25 C. The viable populations at the time of acidification show that the lack of diacetyl production by the concentrate was not due to insufficient inoculum. A culture concentrate prepared from cells grown in the same broth containing 0.5 % sodium citrate produced a much greater amount of diacetyl in a 4-hr period than did the third subculture from the cells grown in the same broth without citrate ( Table 1). Effect of pH on growth and diacetyl production. Cultures of L. citrovorum 3 grown in the sodium citrate broth at 25 C with 20% NaOH to control the pH at 6.0, 6.5, or 7.0 attained populations in 23 hr approximately twice those obtained without pH control ( Table 2). Incubation of these cultures beyond the 23-hr period did not result in increased cell numbers. Preliminary studies have indicated that the cessation of growth was caused partly by depletion of nutrients. When the pH of the growth medium was maintained at 5.5, the population in 23 hr was about the same as that of the culture grown without pH control. The production of diacetyl by the L. citrovorum 3 concentrates decreased as the pH maintained during growth of the cells increased. Although the cells grown at pH 5.5 produced approximately 30%O more diacetyl than an equal population of cells grown at pH 6.0, only one-half the population was attained by growing the cells at pH 5.5 as at pH 6.0. Therefore, more total diacetyl could be obtained from cells grown at pH 6.0 than from cells grown at pH 5.5. Similar results were obtained when the pH of the growth medium was controlled with 20% ammonium hydroxide. Diacetyl production was also unaffected. Comparison with normal milk cultures. The results in Table 1 suggested the possibility that cells ofL. citrovorum grown in the sodium citrate broth would be more active than milk cultures with respect to diacetyl production. Results from experiments comparing diacetyl production of milk cultures to that of culture concentrates are presented in Table 3. For these studies, the culture concentrates were prepared from cells grown at pH 6.0 in sodium citrate medium with 20% NaOH as the neutralizer. For the analyses, the culture concentrates were inoculated into the acidified 10% NFMS to yield a population comparable to that of the normal milk culture. Cells from the L. citrovorum 3 culture concentrate produced considerably more diacetyl than did the cells in the normal milk culture of the same strain. The amount of diacetyl produced by the L. citrovorum 28 culture concentrate did not appear to be much greater than that formed by the normal milk culture of strain 28. However, by considering the difference in the number of cells in the samples for strain 28 at the time of acidification, the increased diacetyl production by the cells from the culture concentrate becomes more significant. These data also show L. citrovorum 28 to be considerably more active than strain 3 in production of diacetyl. Storage stability. Culture concentrates of L. citrovorum were stable to storage in liquid nitrogen (-196 C). Table 4 shows results from analyses of an L. citrovorum 3 culture concentrate after various periods of frozen storage. There was no loss of viability or diacetyl production during the 30-day storage period. Flavor analysis of creamed cottage cheese. Cottage cheese creaming mixtures were prepared using both normal milk cultures and culture concentrates of L. citrovorum 3, applied to dry cottage cheese curd, and subjected to analyses by an experienced taste panel. The results for two trials involving different lots of curd and creaming mixtures are shown in Table 5. In these experiments, the number of cells from the concentrated culture was adjusted to be equivalent to the population attained in the 18-hr milk culture. Cottage cheese made with the creaming mixture prepared with the concentrated culture showed superiority in diacetyl content, and the cheese was preferred by the taste panel. This preference was probably due to the higher level of diacetyl and, furthermore, indicated that no undesirable balance in flavor compounds resulted from the use of the concentrated culture. DISCUSSION When growing L. citrovorum cells for the preparation of culture concentrates, it is desirable to obtain the highest population possible. Although Leuconostoc species do not produce large amounts of acid, they do produce sufficient quantities to limit their growth in a broth medium. The results of this study have shown a twofold increase in the attainable population of L. citrovorum by controlling the pH at a level favorable for growth. The limitation on the population attainable may have been due to limiting nutrients or to the production of autoinhibitory metabolites other than acids. The requirement for citrate in the growth medium for subsequent production of diacetyl by L. citrovorum concurs with results reported by Harvey and Collins (3), who found the citrate permease to be inducible. Harvey and Collins (2) also suggested that the citrate-splitting enzyme, citratase, is inducible in some organisms. The increased diacetyl production by culture concentrates prepared from cells grown at lower pH levels indicates a low pH optimum for synthesis of the enzyme system of L. citrovorum required for diacetyl production. Although the cells grown at pH 5.5 were more active in producing diacetyl than those grown at pH 6.0, the latter appeared to be more advantageous for cell population. The direct inoculation of L. citrovorum culture concentrates into the acidified milk (pH 4.4 to 4.5) for preparing cottage cheese dressing does not permit growth of the culture; therefore, it is necessary to have a sufficient number of cells in the concentrate to produce the desired amount of flavor without further growth. The more active production of diacetyl by the concentrated cultures offers an added advantage of requiring fewer cells than those in a milk culture for producing a given level of diacetyl. There was a certain amount of variation in the production of diacetyl by the two strains of L. citrovorum. This indicates that some strains offer an advantage over others for the preparation of culture concentrates capable of rapid diacetyl production.

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2020-12-10T09:04:11.095Z

1970-08-01T00:00:00.000Z

237232781

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Almond Harvesting, Processing, and Microbial Flora This survey was set up on a statistical sampling plan to determine the microbial quality of almonds as they are received at the processing plant. The total aerobic bacterial count and yeast and mold count distribution were skewed by a few high counts compared with the majority of relatively low counts. Hard shell varieties of almonds had lower counts than did soft shell, and almonds with complete shells had lower counts than shelled almonds. Almonds harvested onto canvas had lower counts than those harvested by knocking onto the ground. Nuts with the least amounts of foreign material mixed into the sample had the lowest counts, as did nuts with the least amount of insect damage. Coliforms, Escherichia coli, and Streptococcus were isolated from the nuts, and their presence was correlated with soil contamination. When almonds are stored, the total plate count, the Streptococcus count, and the E. coli count after an initial drop remain nearly constant for more than 3 months. In addition to the indicator organisms, several genera of bacteria were isolated including Bacillus, Xanthomonas, Achromobacter, Pseudomonas, Micrococcus or Staphylococcus, and Brevibacterium. This survey was set up on a statistical sampling plan to determine the microbial quality of almonds as they are received at the processing plant. The total aerobic bacterial count and yeast and mold count distribution were skewed by a few high counts compared with the majority of relatively low counts. Hard shell varieties of almonds had lower counts than did soft shell, and almonds with complete shells had lower counts than shelled almonds. Almonds harvested onto canvas had lower counts than those harvested by knocking onto the ground. Nuts with the least amounts of foreign material mixed into the sample had the lowest counts, as did nuts with the least amount of insect damage. Coliforms, Escherichia coli, and Streptococcus were isolated from the nuts, and their presence was correlated with soil contamination. When almonds are stored, the total plate count, the Streptococcus count, and the E. coli count after an initial drop remain nearly constant for more than 3 months. In addition to the indicator organisms, several genera of bacteria were isolated including Bacillus, Xanthomonas, Achromobacter, Pseudomonas, Micrococcus or Staphylococcus, and Brevibacterium. Edible sweet almonds (Prunus amygdalus) have three distinct parts: the inner kernel or meat, the middle shell portion, and an outer hull. Almond varieties vary in shell texture; therefore, they are termed hard or soft shelled. The harvesting procedure starts when the almonds are partly dried on the trees. They are shaken down to collecting sheets or onto the ground and are mechanically picked up after further drying. A hulling operation then removes the outer hulls. During this operation, some nutmeats are inadvertently removed from the shell. The hulled nuts are then sent to the processing plant where the nuts are fumigated to destroy insects and eggs before shelling. After the shell has been removed, the nutmeats are processed into graded meats or almond products. This research was conducted at the request of the almond industry to determine the microbial quality of almonds as received at the processing plant. A preliminary report of this research has been published (5). There has been a continuing interest in coliforms and Escherichia coli in relation to almond contamination (7,9,13). MATERIALS AND METHODS Sampling plans. A preliminary survey showed that 10% of the almond samples were contaminated with coliforms. Therefore, our statistical sampling plan in 1966 was set up to detect coliform contamination on nutmeats in 10% of the lots with 95% confidence. A 0.25-to 0.5-lb sample (113 to 227 g) was collected from every 200th lot of nuts received at the California Al-mond Growers Exchange plant in Sacramento for a total of 172 samples. It was shown in a separate preliminary unreported study that samples so collected would represent receipts at all almond processing plants because samples from all growing areas would be included. Samples used were subsamples of those taken by automatic sampling devices used to grade each lot. The samples were carefully packaged in sterile plastic bags to prevent additional contamination. They were taken immediately upon receipt at the plant and refrigerated until transported to the laboratory where they were stored at 2 C in metal containers to prevent moisture pickup. During the 1967 season, the sampling plan was changed to include samples that contained such foreign material as soil and sticks, as well as those samples representative of harvesting methods, varieties, and growing areas. A total of 99 samples was selected. Microbial analyses. Nutmeats were removed from the sample bag or from the shell in an aseptic manner and placed in a sterile bottle. Hard-shelled varieties were cracked with a small hammer before the meat was removed with forceps. An equal weight of water was added to the approximately 10 g of nutmeats in the bottle, and the sample was shaken to wash the surface of the nutmeat. After 5 min, the sample was again shaken, and portions were removed for dilution in 0.1% peptone water (12) or inoculation of plates for microbial counts. Bacteria counts were made with plate count agar containing 100 ,ug of cycloheximide (Upjohn) per ml added to suppress mold growth. Yeast and mold counts were made with potato dextrose agar acidified to pH 3.5 with tartaric acid BAClTERIA IN ALMONDS Identification of bacteria. Routine methods were used to plate four samples of almond meats, two from almonds at the processing plant and two from pallets of bagged almonds ready for shipment after processing. From each plate, 25 colonies were picked at random and transferred to BBL Trypticase Soy Broth. The tubes were incubated at 35 C for 72 hr, and the characteristics of the colonies were noted. A loopful of broth was then streaked on BBL Trypticase Soy Agar (TSA) plates to isolate any mixed colonies (incubated 72 hr at 35 C). Colony characters were noted, and the isolated organisms were transferred to TSA slants (incubated 24 hr at 35 C). Appropriate descriptive tests were run on the isolates by use of standard methods (11). After these tests were completed, the organisms were grouped by genera (10). RESULTS Microbial counts. The total bacterial plate counts on the almonds were quite low, on the average, when compared with those of food items that are not dry processed. The highest value of bacterial counts is within the suggested limit of counts for many foods (4). During both seasons, the yeast and mold count plates contained mostly mold and very little yeast. The data shown in Table 1 illustrate the wide range and distribution of microbial counts. The median is considerably lower than the mean, indicating that the majority of counts are low, with a relatively few high-count samples that skew the data. Ninety per cent of the values found are below the value listed as the ninth decile in Table 1. To compare microbial contamination of the unprocessed nuts with that of processed nuts, a series of counts were made on processed almond meats. For 11 samples from 1966, the average count for bacteria was 580 per gram and for yeast and mold, 170 per gram. Comparable values from 1967 (the average of 12 samples) were 1,200 and 2,700 per gram, respectively. These values are somewhat lower than the values listed in Table 1 for receiving supplies, indicating a net drop in microbial count during the processing. Indicator organisms. We examined the unprocessed nuts for coliform and streptococcus content as pollution-indicator organisms in almond processing. Both types were found on the nutmeats when delivered to the processing plant. Table 2 lists the counts obtained from the samples containing these organisms. From the 1966 season, all 22 streptococcus-positive samples were isolated from soft-shell varieties that had been shelled or had split shells. Coliforms were also present in 11 Table 3 for the 1966 data indicate that coliforms were mainly present in almonds shelled before ship-ment to the processing plant and those with split shells. E. coli was present in seven (4.1 %) of the samples, a value somewhat lower than that found by Kokal and Thorpe (7). A total of 40 of the 99 samples from the 1967 sampling had coliform organisms present. Of these, only nine contained E. coli. None of the correlations calculated for numbers of coliform organisms was significant. E. coli was isolated only from nuts that had the shell removed or had split shells. Correlations of microbial counts and test variables. To give a more normal distribution to the data shown in Table 1, the microbial counts were expressed as logs from which geometric means and confidence intervals were estimated (3). The confidence intervals were based on Student's t distribution at the 95% probability level. Table 3 shows the correlations of microbial counts and some of the 1966 and 1967 test variables. Variables reported in per cent were trans- formed by arc sine square root and count data by logs. No influence of growing areas upon the microbial population was found. Variety. There were differences in microbial content among individual varieties that appear to be a reflection of the shell type or condition, rather than a difference in chemical composition among the varieties. Shell type and shell condition. Almond shells vary in texture from fragile to hard, depending on the variety. The varieties we studied were classified as hard or soft shell. Soft-shelled varieties were IXL, Jordonalo, Neplus, and Nonpareil; hard-shell varieties were Davey, Drake, Llewellyn, Mission, and Peerless. The completeness of shell is related to its hardness (Fig. 1). The influence of shell type and condition upon the microbial counts of almond meats is shown in Table 4. In general, the hard-shelled almonds had lower counts than the soft-shelled varieties. The more complete the shell, the lower FIG. 1. In-shell almonds after removal of hulls. Notice the difference in amount of shell breakage and exposure of meats between the hard-shell (left) and soft-shell (right) varieties as they appear when delivered to the processing plant. the microbial content of the meat. These data illustrate the importance of a complete shell for preventing contamination. The fact that the shells frequently crack and meats are exposed during drying on the tree, particularly with soft-shelled varieties, indicates the need to maintain relatively clean conditions during all handling steps in order to minimize contamination of the nutmeat. Method of harvest. During the 1967 season, we obtained samples that had been harvested by knocking the nuts either onto some type of ground cover (such as canvas) or onto the soil, which usually is carefully rolled and prepared in advance. Nuts without contamination, harvested by either method, were statistically compared (Table 5). Those harvested on ground cloths had significantly lower bacterial and yeast and mold counts than those picked from the ground. Contamination materials. During the 1967 season, some samples were selected from incoming shipments of almonds that contained noticeable dirt. These samples were classified and analyzed by the kind of foreign material present. The classes were: no noticeable contamination, mud balls and pieces of dried manure, mud balls only, and other foreign material (lint, ground in soil on meats, worms, rubber, rocks, etc.). The analysis shown in Table 6 illustrates the influence of the various contaminants upon the microbial counts. These data indicate that bacterial counts are related to the amount of soil mixed with the sample. The three contaminated categories (Table 6) had significantly higher bacterial counts than did samples with no foreign material. Although not shown in the table, the highest counts that we noted were obtained from samples 10,500-53,100 with soil ground into the nutmeats and not easily removed. The yeast and mold count did not follow this pattern and appeared to be unrelated to such contamination. Insect damage. The 1967 crop had an unusually large amount of insect damage, primarily due to the navel orange moth. Samples were visually graded with respect to the amount of insect damage on the nutmeat and were labeled from 1 to 5, to indicate no damage through increasingly high damage. The data (Fig. 2) show a positive relationship between the amount of insect damage and aerobic plate count (note a correlation of 0.476 from Table 3). Three samples with least insect damage had significantly lower bacterial counts than the two with the most damage. For yeast and mold content, sample categories 1 and 3 were significantly lower than 5. Types of bacteria isolated from almond meats. To determine the types of aerobic bacteria present on almond meats, a total of 25 colonies were isolated from each of two processed and two unprocessed samples. The colonies were classified as to genera by the method of Skerman (10). Most (60) of the cultures were Bacillus species, gram-positive, and catalase-positive with endospores. Numerically, the second most important group (16 isolates) was the gram-negative, catalasepositive, motile, and rod-shaped bacteria. Of these, two seemed to fit the classification of the genus Xanthomonas and two Achromobacter. Except for two nonmotile cultures, the remaining bacteria had polar flagella but could not be easily placed in a genus, although they were related to Pseudomonas as were two nonmotile cultures. The third group, containing 15 isolates, consisted of gram-positive cocci that fit into the classification of Micrococcus or Staphylococcus. The last bacterial group of seven isolates can be described as gram-positive, catalase-positive, having nonmotile rods that do not form endospores, and loosely classified in the genus Brevibacterium. Survival studies. Figure 3 shows the survival of total bacteria, E. coli, and Streptococcus species on almond meats stored in plastic bags at 2 or 24 C. The total bacterial counts were made on normal samples of almonds; for the counts of E. coli and Streptococcus species, the organisms were inoculated on almonds before storage. Figure 3 indicates that after an initial drop, the total aerobic count is nearly constant with time and shows no marked decline in 225 days at 2 C. Also, there is an initial decrease in the E. coli count which precedes a gradual leveling off. After 225 days of storage, E. coli was still present. Almonds inoculated with Streptococcus species and stored at both 2 and 24 C show curves similar in shape to those of total bacteria and E. coli survival in cold storage. The data show an initial steep drop in the count in the first several days and then a leveling off. At room temperature, the organism dies off more rapidly than at cold temperatures. Salmonella typhimurium also was inoculated on whole almond meats that were stored at 2 C. After 190 days, the organism could still be recovered, indicating a long survival time on almond meats. Thus, if the nutmeats are contaminated, the bacteria can persist for a long time. Kokal (6) reported that the tannins of walnut skins kill E. coli. In experiments with ground almond skins added to the growth medium, we were unable to demonstrate a similar influence; in fact, the unwashed skins had a stimulatory influence on E. coli, further indicating the nonantagonistic aspect of the almond to bacteria. DISCUSSION The data reported in this paper reflect the microbial population on the surface of almond meats. A portion of the bacterial contamination stems from soil and dust contact with nutmeats. This is shown by the lower counts for hard-shelled varieties which have a more complete shell and less chance for soil contamination. The effect of soil contact is also reflected in the lower counts for nuts harvested on cloths as opposed to those collected from the ground. Nuts with the lowest amount of contaminating foreign material had lower counts than those with large amounts, particularly if pieces of soil were ground into the meats. Portable catching frames or ground cloths would lessen soil and microbial contact. The increase in microbial counts with increased insect damage illustrates the need for better insect control both in the orchard and after harvest. The yeast and mold surface counts correlate with shell factors during the 1966 season but not in 1967. The correlation with rejected material is an indication of the type of spoilage included in this classification. The correlations do not explain the wide range of yeast and mold counts observed. The lack of correlations in 1967 is probably a result of the different sampling plan used that season. The indicator organisms (coliforms, E. coli, and Streptococcus) were associated with soil contamination of nutmeats, as were the total bacteria. Kokal and Thorpe (7) showed that the incidence of E. coli on almonds was associated with processing operations after harvesting, although some E. coli cells were found on nuts before harvest. The present findings indicate contamination of nutmeats before processing, so their presence on processed nuts does not necessarily indicate poor manufacturing practice. We had hoped to determine if Streptococcus or E. coli was the best indicator organism to use for measuring contamination of almonds. They both appear to be present in similar numbers and are associated with the same kind of contamination. Neither test appears to be superior, except that the Streptococcus test is easier and less time consuming. 0 The genera of bacteria isolated from the nut kernel, Bacillus, Xanthomonas, Achromobacter, Pseudomonas, Micrococcus, and Brevibacterium, would be expected to be associated with soil or plant material and to survive on the kernel. The survival of heat resistant Bacillus species on almonds could be of consequence if almonds are used as an ingredient for heat-processed food products. Survival tests for total bacterial count, streptococci, S. typhimurium, and E. coli indicate that the almond does not exert an inhibitory effect and that organisms can persist on the nuts for long periods of time. This illustrates the importance of preventing contamination of the nutmeat, particularly during normal processing when there is only a dry cleaning operation; it is not common practice to wash nutmeats. The data show that the microbial quality of the almond as it is received at the processing plant is generally good. However, the surface contamination of the nutmeat can be partially controlled VOL. 20, 1970 213 214 by lessening the degree of soil or dust contact and thereby improving the quality of the product for further processing.

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2020-12-10T09:04:12.283Z

1970-03-01T00:00:00.000Z

237232348

s2

New Antibacterial Agent Isolated from the Avocado Pear A group of eight new long-chain aliphatic compounds recently isolated from the avocado and some derivatives thereof were tested for antibacterial activity on 13 different species of bacteria and a yeast. Some of these compounds inhibited the growth of microorganisms. 1,2,4-Trihydroxy-n-hepadeca-16-en was found to be the most active, inhibiting certain gram-positive bacteria at 4 μg/ml. L. B. Jensen (U.S. Patent 2,550,254, 1951) reported that acetone extract of avocado seeds exhibited antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Aspergillus glaucus, Penicillium notatum, and Achromobacter perolens. This extract was found to be inactive against Esherichia coli, Pseudomonas fluorescens and Penicilliwn camemberti. Valeri and Gimeno (3) extracted avocado seeds with petrol ether and reported that the resulting crude wax inhibited growth of Micrococcus pyogenes and Sarcina lutea, but not growth of B. subtilis or of E. coli. The avocado extracts tested in these experiments were crude, and the compound causing the inhibition was not isolated. Recently, we succeeded in isolating from avocado seeds and fruits a family of natural compounds, all having a long aliphatic chain with one end being unsaturated and the other end highly oxygenated. The structure of these eight compounds was elucidated and a number of derivatives thereof were prepared. It was of interest to find out which of these compounds were responsible for the antibacterial activity of the avocado seed and whether any of the derivatives were also active. MATERIALS AND METHODS Compounds. The compounds reported here were isolated and prepared according to the methods described by Kashman, Neeman, and Lifshitz (1). All the compounds were further refined before the bacteriological assay by subsequent passage through silicagel columns and by recrystallization to fully free them from possible traces of impurity. The purity of each compound was ascertained by thin-layer chromatography and by nuclear magnetic resonance. P. fluorescens was grown at 22 C, B. subtilis at 30 C, and all other organisms at 37 C. Disc inhibition zone. Each compound was dissolved in CHC1, (10 mg/ml), and 5 ,liters of the solution was transferred to a sterile 6-mm diameter disc made from Whatman no. 3 filter paper, so that each disc contained 0.05 mg of the compound. To the blank discs, 5 uliters of CHC16 was added. The discs were left for about 3 hr to evaporate the solvent. A suspension containing ca. 3,000 cells per ml was prepared by washing 3-day-old slants. One-tenth milliliter of this suspension was transferred to a petri dish containing 10 ml of solid medium and spread evenly with a glass rod. Three discs, each containing 0.05 mg of the test compound, and one blank disc were put on each dish and incubated at the appropriate temperature. The diameter of the inhibition zone around the disc was measured after 48 hr. Each experiment was repeated four times. Minimal inhibitory concentration. A serial dilution assay was made to determine the minimal inhibitory concentration as described by Kavanagh (2). As the compounds do not readily dissolve in water, 20 mg of each compound was dissolved in 1 ml of absolute ethyl alcohol, and 0.05 ml (containing 1 mg of the compound) was added to the first test tube. The first test tube of the blank series contained 0.05 ml of ethyl alcohol. Growth curves. Growth curves were made as follows. Two milligrams of the compound tested was dissolved in 0.1 ml of absolute ethyl alcohol and was added to 25 ml of sterile broth in a 250-ml Erlenmeyer flask with a side arm. One milliliter of 1-day-old culture grown in slants containing ca. 107 cells was then added to the Erlenmeyer flask and shaken continuously at 37 C (30 C for B. subtilis). Plate counts were made, and the optical density of the culture was measured at predetermined intervals, by using a Spec- RESULTS AND DISCUSSION Results of the disc inhibition zone test are tabulated in Table 1. The values indicate the diameter of the inhibition zone in millimeters after 48 hr of incubation. The gram-negative organisms were slightly inhibited by the compounds, whereas the gram-positive organisms were strongly inhibited by some of the compounds, especially by 1, 2, 4-trihydroxy-n-heptadeca-16-en (compound IV). It is notable that, when the olefinic bond in this compound is fully reduced, the product (compound IX) is no longer inhibitory. Furthermore, when the hydroxyl groups on the oxidized part of compound IV are totally, or partially, acetylated, the anti- bacterial activity is greatly weakened. This can also be seen in Fig. 1, showing the effect of a concentration of 80 ,ug/ml of three compounds on S. aureus. Whereas compound IV totally inhibited the growth of the organism, the acetylenic analogue (compound III) had a limited bacteriostatic effect, if any, and the fully reduced compound IX had no effect. Similar results were obtained with B. subtilis and Saccharomyces cerevisiae. The organisms found to be affected in the first experiment performed on solid media were tested for their sensitivity to compound IV in liquid media ( Table 2). The growth of five organisms was inhibited in this experiment as it was in the first one. However, three organisms, Candida albicans, Shigella dysenteriae, and Salmonella typhi, whose growth was inhibited on solid media, gave satisfactory growth on liquid media. To investigate further the action of compound IV on these three organisms, their growth curves in the presence of the inhibitor were analyzed. The results of such an experiment with C. albicans are shown in Fig. 2. The number of the cells dropped markedly during the first 10 hr but started to increase afterward. Similar results were obtained with the other two organisms, S. dysenteriae and S. typhi. The large decrease in the number of viable cells immediately after exposure to the inhibitory compound shows that its action is bactericidal. The subsequent increase in cell count is probably due to the emergence of resistant cells. This did not occur, however, when B. cereus, B. subtilis, Saccharomyces cerevisiae and Staphylococcus aureus were tested. These microorganisms were inhibited by compound IV, which was found to be heat-resistant since its antibacterial efficiency did not decrease after sterilization.

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2019-04-02T13:07:52.179Z

1970-01-01T00:00:00.000Z

202861309

s2

Variation of rutin and quercetin contents in Tartary buckwheat germplasm This study was carried out to investigate the regional variation and effects of shape and color of seeds and sprouts on the content of rutin and quercetin in Tartary buckwheat germplasm. A total of 44 foreign Tartary buckwheat germplasms were examined and compared their rutin and quercetin contents based on the collected countries, seed shape and seed color using high-performance liquid chromatography and spectrophotometry. The results revealed that rutin and quercetin content varied at different regions. Rutin content in seed (1326.5 mg/100 g) and sprouts (5440.4 mg/ 100 g) of the accession collected from Nepal area was higher than any accession collected from other regions. In seeds, the quercetin content showed the highest value (22.5 mg/100 g) from Pakistan whereas sprouts showed the highest quercetin content (392.0 mg/100 g) from China. However, the quercetin content in sprout was 4~90 times higher than that of seeds. Taken together, the present study suggests that sprouts could be used more effectively than seeds in the case of quercetin, and strains from Nepal, Bhutan, China, and Japan have a high potential material to use seed and sprouts for buckwheat industry in making functional food and medicine. Yu et al. (2019): Rutin and quercetin in Tartary buckwheat germplasm 52 INTRODUCTION Buckwheat, which belongs to the family Polygonaceae, genus Fagopyrum, has been a popular health food in Asian and European countries for a long time (Kreft, 2003). It has been widely cultivated in Korea since it has abundant nutrients and medicinal efficacy. Buckwheat sprouts are higher in nutritional value than other crops regarding fiber, anthocyanin, and rutin contents (Shin, 2010). Unlike traditional buckwheat, Tartary buckwheat remains in the wild and is most widely grown buckwheat species in the Himalaya region (Golob et al., 2015). The main cultivation areas are Tibet or Chinese mountainous regions, such as India, Bhutan and Nepal (Kreft et al., 2003). However, they are abundant in various nutrients and high in protein (12%) and fat (3.9%) content. Also, oleic acid and linoleic acid account for 80% of total fatty acids (Park et al., 2004). Buckwheat contains many important bioactive compounds especially flavonoids that have been shown to exert excessive stress via activating antioxidants such as superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT) and glutathione reductase, (Park et al., 2000; Zhu et al., 1999). These compounds are complementary to the lack of enough activity due to environmental factors, eating habits, smoking and lifestyle habits, and by reactive oxygen species (ROS), and DNA damage (Lee et al., 2011; Park et al., 2011), organ and tissue damage (Shin et al., 1990; Graf et al., 2005). Flavonoids found in buckwheat, include rutin, c-glycosylflavones (orientin, isoorientin, vitexin, isovitexin), quercetin, and phenolic acid as chlorogenic acid (Margna et al., 1978; Watanabe et al., 2002), however, the content of rutin in Tartary buckwheat is much higher than that of common buckwheat and other crops (Wang et al., 1995; Park et al., 2005a; Park et al., 2005b). However, it has been only a few years since the cultivation of buckwheat in Korea has started, and basic ecological studies have not been systematically established yet (Park et al., 2004; Lim et al., 2009). With growing demand for food and functional foods, interest in sprouting vegetables is increasing. In the United States, Europe and Australia, sprouting vegetables account for about 30% of the vegetable stores. In Asia, interest in sprouting vegetables is increasing, mainly in Japan. Sprouting vegetables account for 10 to 20% of the market. It is estimated that the sprout vegetable market in Korea is about 2 billion won in 2005 (Lee, 2007). In recent years, buckwheat sprouts have been developed in Korea and Japan (Kim et al., 2004; Kim et al., 2007), and sprouting vegetables (Hokkai T9, Hokkai T10) were registered especially in Japan in 2007 (Suzuki, 2008). In addition, the content of quercetin, as well as rutin, was also higher in Tartary buckwheat sprouts than in common buckwheat sprouts. Thus, the consumption of buckwheat sprouts as a source of rutin is increasing (Jeon, 2012). Rutin, which is more abundant in sprouts than seeds in Tartary buckwheat, and compared than common buckwheat and has been reported to be effective against various diseases, including antioxidant activity (Lee et al., 2000), diabetes (Lee et al., 1994), antioxidative effects (Kwon et al., 1995) and the prevention of cardiovascular disease (He et al., 1995; Wojcicki et al., 1995). Previous study was conducted with buckwheat genetic resources for rutin, the comparison was made by the color of seed coat, seed type, and country of collection (Park et al., 2005). Quercetin is a flavonoid substance belonging to the flavonol family. Quercetin is mainly found in fruits and minerals, especially in onions (Formical et al., 1995). In the United States, quercetin is also known to be a representative flavonoid (Park et al., 1991), as it is known to consume 25 mg per person per day (Lamson et al., 2000). The pharmacological effects of quercetin have been extensively studied both in vivo and in vitro models and have been shown to be associated with reduced lipid peroxidation (Cavallini et al., 1978) and decreased activity of carcinogens (Edenhader et al., 1996), hypotension, (Daniel et al., 2003) and antimicrobial effects (Kimura et al., 1984). In recent years, attempts to search for natural antioxidants harmless to the human body have been studied in various ways (Halliwell et al., 1992; Lee, 2007). Recently, Korean people have much interest in using of Tartary buckwheat because of its higher rutin content and bio-active compounds than common buckwheat that has been traditionally utilized in Korea. However, there are no cultivars of Tartary buckwheat in Korea. So, it is urgent to develop the promising cultivars with high yield of seeds and herbs and good quality with high rutin concentration. MATERIALS AND METHODS 1. Materials collection and plant growth condition The experimental materials were collected from 7 countries, including China, Nepal, Bhutan, India, Japan, Fagopyrum 36(2):51-65 (2019) 53 Pakistan and Slovenia. A total of 44 kinds of Tartary buckwheat genetic resources were stored at Chungbuk National University (4°C, 30-40% RH). The genotypes used in the present study were 28 specimens from China, 5 specimens from Nepal, 3 specimens from Bhutan, 2 specimens from India, 2 specimens from Japan, 2 specimens from Pakistan, 2 specimens from Slovenia. However, the seed size of the genotypes were 6 specimens of notched, 22 specimens of round, 16 specimens of slender, and seed color were identified as 8 brown, 12 darkbrown, 15 dark-gray, and 9 gray-brown (Table 1). The above-mentioned genetic resources were cultivated in Chungbuk National University Farm in 2015 and planted on July 31, 2015, maintaining planting density at 30×10 cm. Other cultivation management was provided in accordance with the crop cultivation guideline from Rural Development Administration. A total of 44 seedlings were transferred to a seedling tray (5.1 cm × 4.7 cm), and grown for 7-days in a controlled (25°C, 14 h day/10 h night, 150 μmol.m-2.s-1 light intensity) growth chamber (GC-300 TLH, JEIO TECH). Shoot cotyledons and hypocotyls of buds which were watered twice daily and grown under conditions of 10 hours of dark conditions. Prior to analysis, the collected buds were drying using freeze-drier (FDU-1200, EYELA). After freeze-drying for 3 days, the samples were ground into fine powder with a pestle in liquid nitrogen, and stored in a cryogenic freezer (DF8517S, Ilshinbiobase, 4°C, 30-40% RH) for rutin and quercetin analysis. 2. Rutin and quercetin analysis methods 2.1. Pretreatment for rutin and quercetin analysis A portion (0.5 g) of Tartary buckwheat seeds and buds powder stored in a cryogenic freezer (DF8517S, Ilshinbiobase) was weighed into 15 ml tubes (BD, FalconTM). 10 ml of ethanol (96% Germany, MERCK) was added to each of the weighed tubes, and vortexed vigorously. The tubes were sonicated using an ultrasonic clearer (SDD300H, Seongdong) for 60 minutes at 80°C and then cooled in a refrigerated shell (IBK-1400RFD, Infobiotech) for 60 minutes at 4°C. The mixture was centrifuged at 5000 rpm for 5 min at 4oC. 2 ml of the extracted sample was filtered using a 0.45 μm PVDF membrane syringe filter (Whatman, USA) and analyzed by HPLC (Agilent 1200 series) instrument with 3 repetitions. The contents of rutin and quercetin were expressed on dry weight basis. The content of both investigated compounds was expressed in mg/100 g DW. 2.2. HPLC analysis method Methanol, acetonitrile, and water (Honeywell B & J) used in HPLC analysis were HPLC grade. For the solvent A, 0.05% TFA buffer (Sigma) was added to a total volume of 100% water. The solvent B was prepared with 60% of methanol, 40% of acetonitrile, 0.05% of TFA buffer respectively. All analyses for rutin and quercetin were performed on a HPLC system (HPLC 1200 series manufactured Origin Seed shape Seed color Notched Round Slender Total Brown Dark-brown Dark-gray Gray-brown Total China 5 14 9 28 5 7 8 8 28 Nepal N/A 4 1 5 N/A 2 3 N/A 5 Bhutan N/A N/A 3 3 2 N/A 1 N/A 3 India N/A 1 1 2 N/A 1 N/A 1 2 Japan N/A 2 N/A 2 N/A N/A 2 N/A 2 Pakistan 1 N/A 1 2 N/A 1 1 N/A 2 Slovenia N/A 1 1 2 1 1 N/A N/A 2 Total 6 22 16 44 8 12 15 9 44 *N/A=Not applicable Table 1. Country-wise number of Tartary buckwheat germplasm based on seed shape and seed color used in evaluation of rutin and quercetin contents Yu et al. (2019): Rutin and quercetin in Tartary buckwheat germplasm 54 by Agilent), equipped with a column used YMC-pack OSD-AC18 (4.6 mm ID × 250 mm, S-5 μm, YMC Co., LTD., Japan). The flow rate was 1.0 ml/min, the column temperature was 25°C, the injection volume was 10 μl, and the detection wavelength was set at 359 nm. The detailed HPLC analysis conditions are shown in Table 2. For the quantitative analysis of rutin and quercetin, the standards were made according to the protocol of Sigma, and the concentrations used in this analysis were 1, 2, 5, 10, 20, 50, 100 and 200 ppm. The pattern and retention time of rutin and quercetin observed in HPLC apparatus are shown in Fig. 1, and the equation of linear regression of rutin and quercetin y=14.993x-62.119 y=32.943x-19.09 respectively while the coefficient of determination (R2) was 0.9989 and 0.9999 in rutin and quercetin respectively. 3. Statistical processing analysis Analysis of variance was performed using SAS software (SAS Institute Inc., ver. 9.2). The significance test for the rutin and quercetin content of each country, seed shape, and seed color was performed using Duncan’s multiple range tests. Also, the correlation between rutin and quercetin content was investigated within the seeds, within the shoots and between the seeds and sprouts. RESULTS AND DISCUSSION 4. Variation of rutin and quercetin content in seed 4.1. Content, distribution and resource selection of rutin and quercetin in seed Among the 44 kinds of Tartary buckwheat genetic analyses, the average content of the two components (sum of rutin and quercetin) was 815.4 mg/100 g, and the all investigated genetic samples ranged from 308.3 to 1337.0 mg /100 g. In terms of each component analysis, the average content of rutin was 808.4 mg /100 g, the in all genetic samples ranged from 304.5 to 1326.5 mg/ 100 g. On the other hand, the average content of quercetin was 7.0 mg /100 g, and in all genetic samples ranged from 2.0 to 22.5 mg /100 g. However, the results obtained from the present study revealed that the content of rutin was higher compared to quercetin regarding all investigated samples. The distribution of rutin and quercetin contents in the tested buckwheat samples is shown in Fig. 2. The highest distribution of rutin (16 germplasms) was observed in the range of 600~800 mg/100 g followed by 12 germplasms in the range of 800~1000 mg/100 g, 7 germplasms in the range of 600 mg/100 g and 6 gerGradient Time (min) Mobile phase condition A (%): water + TFA (0.05%) B (%): MeOH (60%) + ACN (40%) + TFA (0.05%) INTRODUCTION Buckwheat, which belongs to the family Polygonaceae, genus Fagopyrum, has been a popular health food in Asian and European countries for a long time (Kreft, 2003). It has been widely cultivated in Korea since it has abundant nutrients and medicinal efficacy. Buckwheat sprouts are higher in nutritional value than other crops regarding fiber, anthocyanin, and rutin contents (Shin, 2010). Unlike traditional buckwheat, Tartary buckwheat remains in the wild and is most widely grown buckwheat species in the Himalaya region (Golob et al., 2015). The main cultivation areas are Tibet or Chinese mountainous regions, such as India, Bhutan and Nepal (Kreft et al., 2003). However, they are abundant in various nutrients and high in protein (12%) and fat (3.9%) content. Also, oleic acid and linoleic acid account for 80% of total fatty acids . Buckwheat contains many important bioactive compounds especially flavonoids that have been shown to exert excessive stress via activating antioxidants such as superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT) and glutathione reductase, (Park et al., 2000;Zhu et al., 1999). These compounds are complementary to the lack of enough activity due to environmental factors, eating habits, smoking and lifestyle habits, and by reactive oxygen species (ROS), and DNA damage Park et al., 2011), organ and tissue damage (Shin et al., 1990;Graf et al., 2005). Flavonoids found in buckwheat, include rutin, c-glycosylflavones (orientin, isoorientin, vitexin, isovitexin), quercetin, and phenolic acid as chlorogenic acid (Margna et al., 1978;Watanabe et al., 2002), however, the content of rutin in Tartary buckwheat is much higher than that of common buckwheat and other crops (Wang et al., 1995;Park et al., 2005a;Park et al., 2005b). However, it has been only a few years since the cultivation of buckwheat in Korea has started, and basic ecological studies have not been systematically established yet Lim et al., 2009). With growing demand for food and functional foods, interest in sprouting vegetables is increasing. In the United States, Europe and Australia, sprouting vegetables account for about 30% of the vegetable stores. In Asia, interest in sprouting vegetables is increasing, mainly in Japan. Sprouting vegetables account for 10 to 20% of the market. It is estimated that the sprout vegetable market in Korea is about 2 billion won in 2005 (Lee, 2007). In recent years, buckwheat sprouts have been developed in Korea and Japan (Kim et al., 2004;Kim et al., 2007), and sprouting vegetables (Hokkai T9, Hokkai T10) were registered especially in Japan in 2007 (Suzuki, 2008). In addition, the content of quercetin, as well as rutin, was also higher in Tartary buckwheat sprouts than in common buckwheat sprouts. Thus, the consumption of buckwheat sprouts as a source of rutin is increasing (Jeon, 2012). Rutin, which is more abundant in sprouts than seeds in Tartary buckwheat, and compared than common buckwheat and has been reported to be effective against various diseases, including antioxidant activity (Lee et al., 2000), diabetes (Lee et al., 1994), antioxidative effects (Kwon et al., 1995) and the prevention of cardiovascular disease (He et al., 1995;Wojcicki et al., 1995). Previous study was conducted with buckwheat genetic resources for rutin, the comparison was made by the color of seed coat, seed type, and country of collection (Park et al., 2005). Quercetin is a flavonoid substance belonging to the flavonol family. Quercetin is mainly found in fruits and minerals, especially in onions (Formical et al., 1995). In the United States, quercetin is also known to be a representative flavonoid (Park et al., 1991), as it is known to consume 25 mg per person per day (Lamson et al., 2000). The pharmacological effects of quercetin have been extensively studied both in vivo and in vitro models and have been shown to be associated with reduced lipid peroxidation (Cavallini et al., 1978) and decreased activity of carcinogens (Edenhader et al., 1996), hypotension, (Daniel et al., 2003) and antimicrobial effects (Kimura et al., 1984). In recent years, attempts to search for natural antioxidants harmless to the human body have been studied in various ways (Halliwell et al., 1992;Lee, 2007). Recently, Korean people have much interest in using of Tartary buckwheat because of its higher rutin content and bio-active compounds than common buckwheat that has been traditionally utilized in Korea. However, there are no cultivars of Tartary buckwheat in Korea. So, it is urgent to develop the promising cultivars with high yield of seeds and herbs and good quality with high rutin concentration. Materials collection and plant growth condition The experimental materials were collected from 7 countries, including China, Nepal, Bhutan, India, Japan, Pakistan and Slovenia. A total of 44 kinds of Tartary buckwheat genetic resources were stored at Chungbuk National University (4°C, 30-40% RH). The genotypes used in the present study were 28 specimens from China, 5 specimens from Nepal, 3 specimens from Bhutan, 2 specimens from India, 2 specimens from Japan, 2 specimens from Pakistan, 2 specimens from Slovenia. However, the seed size of the genotypes were 6 specimens of notched, 22 specimens of round, 16 specimens of slender, and seed color were identified as 8 brown, 12 darkbrown, 15 dark-gray, and 9 gray-brown ( Table 1). The above-mentioned genetic resources were cultivated in Chungbuk National University Farm in 2015 and planted on July 31, 2015, maintaining planting density at 30×10 cm. Other cultivation management was provided in accordance with the crop cultivation guideline from Rural Development Administration. A total of 44 seedlings were transferred to a seedling tray (5.1 cm × 4.7 cm), and grown for 7-days in a controlled (25°C, 14 h day/10 h night, 150 µmol.m -2 .s -1 light intensity) growth chamber (GC-300 TLH, JEIO TECH). Shoot cotyledons and hypocotyls of buds which were watered twice daily and grown under conditions of 10 hours of dark conditions. Prior to analysis, the collected buds were drying using freeze-drier (FDU-1200, EYELA). After freeze-drying for 3 days, the samples were ground into fine powder with a pestle in liquid nitrogen, and stored in a cryogenic freezer (DF8517S, Ilshinbiobase, 4°C, 30-40% RH) for rutin and quercetin analysis. Pretreatment for rutin and quercetin analysis A portion (0.5 g) of Tartary buckwheat seeds and buds powder stored in a cryogenic freezer (DF8517S, Ilshinbiobase) was weighed into 15 ml tubes (BD, Falcon™). 10 ml of ethanol (96% Germany, MERCK) was added to each of the weighed tubes, and vortexed vigorously. The tubes were sonicated using an ultrasonic clearer (SD-D300H, Seongdong) for 60 minutes at 80°C and then cooled in a refrigerated shell (IBK-1400RFD, Infobiotech) for 60 minutes at 4°C. The mixture was centrifuged at 5000 rpm for 5 min at 4ºC. 2 ml of the extracted sample was filtered using a 0.45 µm PVDF membrane syringe filter (Whatman, USA) and analyzed by HPLC (Agilent 1200 series) instrument with 3 repetitions. The contents of rutin and quercetin were expressed on dry weight basis. The content of both investigated compounds was expressed in mg/100 g DW. HPLC analysis method Methanol, acetonitrile, and water (Honeywell B & J) used in HPLC analysis were HPLC grade. For the solvent A, 0.05% TFA buffer (Sigma) was added to a total volume of 100% water. The solvent B was prepared with 60% of methanol, 40% of acetonitrile, 0.05% of TFA buffer respectively. All analyses for rutin and quercetin were performed on a HPLC system (HPLC 1200 series manufactured by Agilent), equipped with a column used YMC-pack OSD-AC18 (4.6 mm ID × 250 mm, S-5 µm, YMC Co., LTD., Japan). The flow rate was 1.0 ml/min, the column temperature was 25°C, the injection volume was 10 µl, and the detection wavelength was set at 359 nm. The detailed HPLC analysis conditions are shown in Table 2. For the quantitative analysis of rutin and quercetin, the standards were made according to the protocol of Sigma, and the concentrations used in this analysis were 1, 2, 5, 10, 20, 50, 100 and 200 ppm. The pattern and retention time of rutin and quercetin observed in HPLC apparatus are shown in Fig. 1, and the equation of linear regression of rutin and quercetin y=14.993x-62.119 y=32.943x-19.09 respectively while the coefficient of determination (R 2 ) was 0.9989 and 0.9999 in rutin and quercetin respectively. Statistical processing analysis Analysis of variance was performed using SAS software (SAS Institute Inc., ver. 9.2). The significance test for the rutin and quercetin content of each country, seed shape, and seed color was performed using Duncan's multiple range tests. Also, the correlation between rutin and quercetin content was investigated within the seeds, within the shoots and between the seeds and sprouts. Content, distribution and resource selection of rutin and quercetin in seed Among the 44 kinds of Tartary buckwheat genetic analyses, the average content of the two components (sum of rutin and quercetin) was 815.4 mg/100 g, and the all investigated genetic samples ranged from 308.3 to 1337.0 mg /100 g. In terms of each component analysis, the average content of rutin was 808.4 mg /100 g, the in all genetic samples ranged from 304.5 to 1326.5 mg/ 100 g. On the other hand, the average content of quercetin was 7.0 mg /100 g, and in all genetic samples ranged from 2.0 to 22.5 mg /100 g. However, the results obtained from the present study revealed that the content of rutin was higher compared to quercetin regarding all investigated samples. The distribution of rutin and quercetin contents in the tested buckwheat samples is shown in Fig. 2. The highest distribution of rutin (16 germplasms) was observed in the range of 600~800 mg/100 g followed by 12 germplasms in the range of 800~1000 mg/100 g, 7 germplasms in the range of 600 mg/100 g and 6 ger- mplasms in the range of 1000~1200 mg/100 g respectively. However, the lowest distribution (3 germplasms) of rutin was observed in the range of 1200 mg/100 g. In the case of quercetin, the highest (19 germplasms) was observed in the range of 5~10 mg / 100 g, followed by 15 germplasms with less than 5 mg/100 g, and 9 germplasms with the range of 10~15 mg/100 g. Table 3 summarizes the rutin and quercetin content, seed shape and seed color properties among the tested resources. The highest content of rutin (1326.0 mg/ 100 g) was found in CBU408 (Collected from Nepal), whereas the highest content of quercetin (22.5 mg/ 100 g) was observed in CBU456 (Collected from Pakistan). Taken together, the results obtained from the present study revealed that the quercetin exhibited the highest CV among the component tested in this study. Country-wise variation of rutin and quercetin content of seeds The total amount of the two components in seeds was measured and compared in this study. The highest total amount of the tested components (sum of rutin and quercetin) was 1002.6 mg / 100 g, followed by 854.0 mg/100 g, 838.9 mg/100 g, 813.6 mg/100 g, 802.6 mg/100 g, 715.0 mg in the order of the following manner Nepal> Bhutan> Japan> China> Pakistan> Slovenia> India. But no significant difference was observed among the germplasms (Table 4). Rutin was also found to be in the same order as the total amount of the two components. However, the total amount of rutin was in the order of 995.1 mg/100 g in Nepal, 845.2 mg / 100 g in Bhutan, 835.7 mg/100 g in Japan, 806.9 mg/100 g in China, 788.3 mg/100 g in Pakistan, 396.0 mg/100g in India. The data shown in Table 4 showed that there was no significant difference among the data examined. Quercetin showed different results compared than the rutin findings; however, the quercetin contents were 14.3 mg/100 g, 8.7 mg/100g, 7.5 mg/100 g, 7.4 mg/100 g, 6.7 mg/100 g, 3.12 mg/100 g in the order of the following manner; Pakistan> Bhutan> Nepal> India> China> Japan> Slovenia (Table 5). However, the quercetin exhibited the highest CV among the components tested in this study. Park et al. (2005) reported that rutin content was in the order of Bhutan> Slovenia> China> Pakistan> Nepal> Japan> India. Taken together, the obtained results suggest that the various concentrations of components may provide important insights regarding the development of functional components in buckwheat. Table 5 summarizes the rutin and quercetin contents in seeds regarding seed shape. The highest total amount Table 4. Statistical analysis of rutin and quercetin content in seed of Tartary buckwheat germplasm based on country of origin of the sum of these two components were 831.2 mg/ 100 g, followed by 822.8 mg/100 g, and 745.9 mg/ 100 g in the order of Slender> Round> Notched. The rutin contents were the similar as the total amount. However, the highest rutin (824.8 mg/100 g) was observed from slender shape seed followed by round shape (816.0 mg/ 100 g), and notched shape (736.8 mg/100 g). In the case of quercetin, pronounced results were observed from notched shaped seed (9.1 mg/100 g) followed by round (6.8 mg/100 g), and slender (6.4 mg/100 g) respectively in the order of Notched> Round> Slender. However, the quercetin exhibited the highest CV among the component tested in this study. A previous study reported that seed shape played a crucial role in the variation of rutin content in the Tartary buckwheat genetic resources (Park et al., 2005). However, they found that the rutin contents were in the order of Slender> Notched> Round, wherein the results showed differences compared than that of the present study. Table 6 summarizes the rutin, quercetin and total amount of these two components in grain seeds considering seed color. The total contents of the two components were measured according to their seed color. However, the highest content was exhibited from the dark-gray (865.3 mg/100 g), followed by dark-brown (806.3 mg/100 g), gray-brown (778.7 mg/100 g), and brown (776.5 mg/ 100 g) respectively. Rutin content showed the similar results as the total amount of the two components. However, the pronounced rutin content was observed from the dark-gray (858.6 mg /100 g), followed by dark-brown (799.0 mg/100 g), gray-brown (771.5 mg/ 100 g) and brown (769.7 mg/100 g). The highest content of quercetin (7.4 mg/100 g) was observed from darkbrown, followed by gray-brown (7.2 mg/100 g), graybrown and brown (6.7 mg/100 g) respectively. However, the quercetin exhibited the highest CV among the components tested in this study. Table 5. Statistical analysis of rutin and quercetin content in seed of Tartary buckwheat germplasm based on seed shape A previous study reported that seed color played an essential role in the variation of rutin and quercetin content in the Tartary buckwheat genetic resources (Park et al., 2005). Taken together, the results obtained from the present study revealed that seed color may provide insights in the variation of rutin and quercetin content in Tartary buckwheat. The variation of the content of rutin in Tartary buckwheat samples showed in the order of Dark-gray> Dark-brown (Dark)> Gray-brown> Brown. Contents, distribution and resource selection of rutin and quercetin in sprout The amount of rutin (3362.9 mg/100 g) was observed from the sprout with the total genetic resources ranges from 328.8 to 5440.4 mg/100 g whereas the average content of quercetin was 143.2 mg/100 g with the total genetic resources ranged from 53.5 to 392.0 mg/100 g. The results revealed that the content of rutin was overwhelmingly higher than that of quercetin. Considering the genetic resources between seed and sprout, rutin was increased by 0.5~10.5 times and quercetin was increased by 3.7~90.7 times. The distribution of rutin and quercetin content in the sprouts of 44 buckwheat genetic resources is shown in Fig. 3. The highest distribution of rutin (16 germplasms) was observed in the range of 3000~4000 mg/100 g fol- Table 6. Statistical analysis of rutin and quercetin content in seed of Tartary buckwheat germplasm based on seed color lowed by 8 germplasms in the range of 2000~3000 mg/ 100 g, 7 germplasms in the range of 4000~5000 mg/ 100 g and 6 germplasms in the range of 1000~1200 mg/100 g, and 3 germplasm in the range of 1000~2000 mg/100 g respectively. However, the lowest rutin frequency distribution (1 germplasm) was observed from less than 1000 mg/100 g. In the case of quercetin, the range of 100 ~ 200 mg/100 g showed the highest frequency (30 germplasms), followed by less than 100 mg/100 g (9 germplasms), and more than 300 mg/100 g (3 germplasms) respectively, while the lowest frequency (2 germplasms) was observed in the range of 200~300 mg/100 g. Table 7 summarizes the rutin quercetin contents of the sprouts regarding location, seed shape and seed color. The highest rutin amount (5440.4 mg/100 g) was obtained from CBU460 in Nepal with round shaped and dark-gray color seeds while the lowest amount (1462.4 mg/100 g) was obtained from CBU302 in China with round shaped brown color seeds. On the other hand, the highest quercetin contents (392.0 mg/100 g) was obtained from CBU302 in China with round shaped brown color seeds CBU302 and the lowest amount of quercetin (268.0 mg/100 g) was obtained from CBU460 in Nepal. Table 8 shows the rutin and quercetin amount in sprouts and their statistical analysis regarding different countries of the world. Nepal showed the highest amount of rutin (4205.0 mg/100 g), followed by Slovenia (3684.3 mg/100 g), Japan (3662.9 mg/100 g), Pakistan (3402.3 mg/100 g), China (3337.7 mg/100 g), Bhutan (3108.8 mg/1682.3 mg/100 g). In the case of quercetin content, Slovenia showed the highest amount (191.1 mg/ 100 g), followed by Nepal (165.4 mg/100 g), Japan (161.5 mg/100 g), India (145.1 mg/100 g), Bhutan (130.9 mg/100 g), Pakistan (130.9 mg/100 g) in the order of Nepal> Japan> India> China> Bhutan> Pakistan. However, no significant differences were observed among the components. Table 9 shows the rutin and quercetin contents from sprouts according to their various seed shape. The slender shaped seeds showed the highest amount of rutin (3890.2 mg/100 g), followed by round (3399.8 mg/ 100 g), and notched (2871.9 mg/100 g) in the order of Slender> Round> Notched. In the case of quercetin, the highest amount of quercetin was observed from the round shape seed (158.1 mg/100 g), whereas the lowest amount of quercetin (125.8 mg/100 g) was observed from the slender shaped seed. However, the amount of quercetin was in the order of Round> Notched> Slender. Table 10 shows the rutin and quercetin contents of the seeds in the sprouts according to seed color. The highest rutin content (3565.0 mg/100 g) was obtained from the dark-gray color seed, followed by dark-brown The correlation between rutin and quercetin in seeds is shown in Fig. 4. The obtained results demonstrated that the content of quercetin was positively correlated (r = 0.4530, p-value = 0.002) with the content of rutin. As a result, the results suggest that both rutin and quercetin selection criteria may provide crucial insights for developing varieties. Fig. 5 shows the correlation between rutin and quercetin in sprouts. The content of quercetin in the sprout was high (r = -4110, p-value = 0.0056) compared to rutin. The amount of quercetin is decreased as the amount of rutin produced when the seeds germinate, and sprouts come out as a typical quercetin glycoside (Lee et al., 2013). As a result, it would be useful to develop a variety containing high content of each component based on the criteria of rutin or quercetin using sprouts. To this end, the present study postulated that sprout would be a great choice to develop a cultivar with a high rutin and quercetin content. Fig. 6 shows the correlation between rutin content in seed and sprouts of Tartary buckwheat germplasm. The rutin content in the seeds was found to be increased with increasing the rutin content in the sprouts (R = 0.3552, p-value = 0.018). However, the content of quercetin in the seeds and the sprouts showed no correlation (R = 0.0169, p-value = 0.9134). However, the use of quercetin would be a potential choice for utilization because it increases 3.7~90.7 times in sprout compared to seeds (Fig. 7). CONCLUSION The present study was carried out to investigate the variation of rutin and quercetin contents in seeds and sprouts of 44 buckwheat genetic resources for the development of high-quality Tartary buckwheat. Rutin and quercetin content varied at different regions from which each accession was collected. Rutin content in seed and sprouts of the accession collected from Nepal area was higher than any accession collected from other regions. Accession collected from Pakistan showed lower rutin content in seeds compared to the accession collected from other regions. On the other hand, quercetin content showed the opposite results in the seeds. In the case of sprout, accession collected from Nepal showed highest rutin content compared than that of accession collected from other regions. Interestingly, the China showed the highest quercetin contents in sprouts. High rutin in seeds and sprouts of Tartary buckwheat indicates that these two components would be great materials for buckwheat industry in making functional food and medicine.

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Behavior of Salmonellae in Sliced Luncheon Meats Salmonellae inoculated onto the surface of bologna and liver cheese survived protracted storage at 5 C. Salmonellae grew when the meats were incubated at room temperature. There have been numerous reports of foodborne salmonellosis traced to delicatessens and other catering operations (2). In most of these outbreaks, poultry or poultry products have been the vehicle for the infectious agent. Heath and Kaufman (1) described an outbreak that was attributed to salads and meats purveyed by a delicatessen. Examination of the equipment on the premises disclosed that the meat-slicing machine and food grinder were contaminated and had most likely seeded the products passing through them. Equipment of this type is recognized as a problem because of contact with many different products thereby increasing the opportunities for cross-contamination to occur. Moreover, such equipment often is not adequately cleaned due to laxness by the operator or because the machinery is constructed in such a manner as to render efficient cleaning difficult. It is conceivable that a product might become contaminated with salmonellae through contact with an inadequately cleaned slicing machine. This study was undertaken to investigate the behavior of salmonellae in cooked luncheon meats held under a variety of conditions. During processing these products are cooked to an extent that precludes the survival of salmonellae. The following data are representative of what might be expected if the products were contaminated during the slicing or packaging operation. Sliced luncheon meats (bologna and liver cheese) were purchased from local retail outlets. The meat was inoculated with Salmonella typhimuriwn or S. anatum by spreading 0.1 ml of a suitable dilution of a 24-hr nutrient broth culture over the surface of the slice. The level of inoculum was chosen to give an initial load of 102 to 5 X 103 salmonellae per g of meat. All inoculated slicedmeat samples were then vacuum-packaged in I Published with the approval of the Director of the Wisconsin Experiment Station. Saran-coated mylar pouches (Oscar Mayer and Co., Madison, Wis.) and stored in the refrigerator (5 C) for 7 days before use. Viable salmonellae were enumerated by the three-tube most-probable-number method involving preenrichment at 37 C in nutrient broth for 20 to 24 hr, followed by enrichment in selenitecystine broth at 37 C for a similar period of time. One loopful of enrichment broth was streaked on Brilliant Green Sulfa agar. Somatic and flagellar agglutination tests confirmed the identity of the isolates as the salmonellae that were inoculated into the product. Figure 1 shows the behavior of S. typhimurium in luncheon meats that were vacuum-packed and held at 5 C for six weeks. There was a reduction in viable cells of 30 to 90% in the first two weeks. After this period, the population of salmonellae stabilized and there was no further decline for the remainder of the experiment. A similar survival pattern was evidenced by S. anatum. These data indicate that salmonellae will persist (although at a reduced level) throughout the shelf life of the products. Since the salmonellae survived extended holding in these products (under refrigeration), it was of interest to determine whether the organisms would grow if the products were subjected to abuse. First, several pouches of contaminated, vacuum-packed bologna and liver cheese were removed from refrigeration and left unopened at room temperature (23 C). Pouches were then opened and sampled at suitable intervals over a 48-hr period. The results are summarized in Fig. 2. With the exception of S. anatum in bologna, the salmonellae did not proliferate significantly during the initial 24-hr after removal from refrigeration. During the second 24-hr period, the number of viable S. typhimurium in bologna and liver cheese and S. anatum in bologna increased 25to 30-fold. In a second experiment, several pouches of contaminated meat were removed from the refrigerator. These pouches were opened immediately, and the contents were removed and rewrapped (without vacuum) in household Saran Wrap. The repackaged product was left at room temperature. The behavior of S. typhimurium in the rewrapped meats incubated at room temperature for 48 hr is shown in Fig. 3. Several differences in the behavior of salmonellae in vacuum-packed meats and the rewrapped meats were noted. The lag period before active growth began was about twice as long in the vacuum-packaged product as in the rewrapped product. The magnitude of the salmonella population increase under aerobic conditions was double that in the vacuum-packed product. Interestingly, a difference in the growth response of the salmonellae in the two products under aerobic conditions was observed. The bologna supported more rapid and extensive growth of both serotypes of Salmonella than did the liver cheese. The difference was not observed under conditions of anaerobic incubation. The reasons for this difference are not known, but quite possibly this is a reflection of the difference in formulation of the two products. In the final experiment, several slices of contaminated meat were left completely exposed to the room environment. Under these conditions the products browned, oiled off, and dried in a matter of a few hours. There was no multiplication by the salmonellae in this material, and, indeed, a dying off was noted as the product dried out. The data presented suggest that the reason meat products of this type are not more often associated with salmonelloses may be their relative inadequacy to serve as substrates for the proliferation of these organisms. Although the minimum number of salmonellae that are necessary to cause illness is not known, it is generally agreed that larger numbers are more likely to cause disease than smaller numbers. The evidence presented suggests that to achieve high numbers of salmonellae in products of this type, it would be necessary to inoculate the meat with very high initial loads. To do this, considerable growth of salmonellae would have to occur on the food contact surfaces of the equipment to which the product is exposed. Any degree of attention to cleaning and sanitation of this equipment should prevent a buildup of this kind.

v3-fos

2020-06-04T09:10:31.056Z

1970-01-01T00:00:00.000Z

222381166

s2

Common Insect Galls of Saskatchewan Seldom does one walk in the outdoors without coming across abnormal growths on the leaves or stems of various plants. “Pine cones” on willows or “marbles” on rose stems have cap¬ tured the curiosity of most of us. If we open one of these structures, we will find the larva of a tiny insect nestled at the centre. Such is the world of the gall insects, a select group of specialized in¬ sects that have evolved the amazing ability to regulate growth patterns of plants to their own advantages. The “pine cones” or “marbles” are actually plant structures caused by the feeding activities of the larvae of gall insects. Some, as yet unknown, substances in the larva’s saliva cause the plant cells to enlarge and multiply rapidly. The insect is thereby surrounded with thick layers of nutritive plant matter. The plant not only supplies the insect with an abun¬ dance of food, but also provides protec¬ tion from rigours of the environment and a shelter in which to pass the winter. The thick walls of the gall also give the helpless insect some protection from predators. BY J. D. SHORTHOUSE* Seldom does one walk in the outdoors without coming across abnormal growths on the leaves or stems of various plants. "Pine cones" on willows or "marbles" on rose stems have cap¬ tured the curiosity of most of us. If we open one of these structures, we will find the larva of a tiny insect nestled at the centre. Such is the world of the gall insects, a select group of specialized in¬ sects that have evolved the amazing ability to regulate growth patterns of plants to their own advantages. The "pine cones" or "marbles" are actually plant structures caused by the feeding activities of the larvae of gall insects. Some, as yet unknown, substances in the larva's saliva cause the plant cells to enlarge and multiply rapidly. The insect is thereby surrounded with thick layers of nutritive plant matter. The plant not only supplies the insect with an abun¬ dance of food, but also provides protec¬ tion from rigours of the environment and a shelter in which to pass the winter. The thick walls of the gall also give the helpless insect some protection from predators. Insect galls are better known to people than are the insects that produce them. Galls are often noticed because of their abundance, colour, or grotesque size and shape. On the other hand, the gall insects are usually small and dif¬ ficult to identify. Well over 1,500 dif¬ ferent species of gall-forming insects have been described from North America and about 100 different kinds probably occur in Saskatchewan. The gall making habit occurs in six orders of insects: beetles (Coleoptera), moths (Lepidoptera), thrips (Thysanoptera), aphids (Homoptera), flies and midges (Diptera), and sawflies and cynipid wasps (Hymenoptera). About 55% of the galls are caused by flies and midges and 35% by cynipid wasps. Little is known about the insect galls of Saskat¬ chewan and, in fact, a preliminary checklist has yet to be made. In this ar¬ ticle only the most common insect galls are discussed. One characteristic of gall insects is that they are very host specific, that is, each species of gall insect always forms galls on the same species of host plant. For some unknown reason certain plants are more attractive to gall for¬ mers than others. In Saskatchewan the plants with the most gall insects are the willows, poplars, roses and goldenrods. Large numbers of gall formers are associated with the oaks which have a restricted distribution in this province and this is one reason why Saskat¬ chewan has few kinds of galls compared to other areas of North America. Gall insects are also very specific as to the part of the plant they attack. Galls are found on the roots, stems, flowers, buds, leaves and petioles depending on the species of insect. The host plant and the part attacked usually are characteristic of the insect species and this infor¬ mation is very useful in making iden¬ tifications. Another interesting aspect of gall in¬ sects is that each species of insect produces its own kind of gall, which is remarkably constant in size and shape. There are two basic types of galls: open and closed. The open galls are con¬ sidered the more primitive. Those for¬ med by aphids (Fig. 1) are a good exam¬ ple. In the spring aphids begin feeding on the outside of the poplar leaves and as a result of their feeding, cause the leaf to fold and grow inwards to produce a pocket in which they live and feed. Young aphids are produced inside the galls and once they mature and develop wings, they escape through natural openings in the gall. Closed galls are caused by the larvae of beetles, moths, flies, sawflies and cynipid wasps. These insects lay eggs on or within healthy parts of a plant and their larvae then cause the gall to be formed. The goldenrod ball gall (Fig. 3) is one of the most common examples of a closed gall. In some parts of Saskatchewan nearly every goldenrod stem will have one or two galls. The female flies (Fig. 15) lay their eggs upon the young goldenrod stems in the spring and the gall appears a few weeks after the larvae (Fig. 14) have begun to feed on tissues of the stem. The larvae are full grown by fall and remain inside the gall throughout the winter. As soon as the snow has melted in the spring, the larvae change into pupae and about a week later the adults emerge and the cycle is repeated. More advanced insects such as the cynipid wasps form galls of greater com¬ plexity than those of aphids. Some rose galls are as large as apples while the in¬ sects that cause them are smaller than apple seeds. Insect Gall Communities Many insects besides the gall-formers often are associated with galls. Plant feeding insects are attracted to the con¬ centrations of nutritive plant cells and parasitic insects attack and feed on the larvae of the gall formers. Another group of insects, closely related to the cynipid wasps (Fig. 12), cannot form their own galls, but instead lay their eggs inside the galls of other insects (Fig. 20). During the laying process the larvae of the gall-formers are killed. When the larvae of these insects, called "inquilines" (from the Latin word for "guest"), begin feeding on the walls of the gall, they too stimulate the cells to grow and as a result each larva is en¬ closed in its own 'gall within a gall' (Fig. 21). The increased number of in¬ sects inside the modified gall attracts even more parasites. The group of insects associated with galls formed by a particular species is an excellent example of a simple insect community. Studying these insects, one is exposed to nearly all aspects of com¬ munity ecology. If you examine galls at various times throughout the summer, you will see that the numbers and kinds of occupants change as the season ad¬ vances. Ecologists call this change "suc¬ cession." When the season draws to a close and no further changes occur, the community is referred to as having reached its "climax." If one determines the feeding habits of the gall inhabitants, a food web can be construc¬ ted: for instance, when the gall formers and inquilines (guests) feed on the plant tissues and they in turn are fed upon by the parasites. Often a parasite will itself be attacked by another parasite. But in gall communities, the key character is always the gall former, for without the gall, none of the other species could exist. Studying Insect Galls As A Winter Project Most Saskatchewan students and naturalists would agree that they are restricted in the kinds of biology projects that they can attempt during the winter. Insect gall studies are an excep¬ tion, for not only can some galls be collected and studied during the winter months (those of Figs. 2, 3, 4, 7, 8 and 9), but they are actually easier to findf when the leaves have fallen and the background is snow. If at least 15 gallsl of one kind are collected in the winter and brought indoors, the insectsj thinking it is spring, will begin emerging within about 3 weeks. Galls with holes indicate that the occupants have alreadyi emerged. Glass jars make good rearing containers and when the insects crawl to the lid, they can be easily removed with a small moistened brush. A great deal can even be learned about communities! by examining these galls through the winter. To observe community suc¬ cession, arrange a series of about 2C vials (pill bottles) containing alcohol near the rearing jar. At the end of the day place all the emerged insects in a vial, use a different vial each day and mark the date on it. Comparison ol numbers and kinds of adults from the first few days of the cycle with those near the end will illustrate succession. Ii is interesting to note than in most rose gall communities the gall formers are the least common of the insects 7igs. 1 to 9 -Common insect galls found in Saskatchewan. "ig. 1. Poplar leaf gall caused by aphids of the genus Fig. 6. Willow leaf galls caused by larvae of the Pemphigus. ig. 3. Goldenrod ball gall caused by maggots of the fly Eurosta solidaginis. ig 4. Rose stem gall caused by larvae of the cynipid wasp Diplolepis multispinosus. 'ig 5. Rose leaf galls caused by larvae of the cynipid wasp Diplolepis polita. Note that the maggot has chewed a channel to the outside of the gall. This will be the escape route of the adult fly, which has inadequate mouthparts chew through the gall tissues. merging (Fig. 22) and the parasites are le most common. The succession obsered indoors will cover a shorter period lan it actually takes in nature, but the up-ihpr of emergents and the order of leir appearance will be the same. / larch, 1973 Fig. 22. Food web of the D. polita gall community. The gall-forming cynipid wasps are in the centre (a) and the gall-modifying inquiline wasps are to the lower left (b). Four species of parasitic wasps (c), whose larvae feed on the larvae of the gal 1formers and the inquilines, are shown above and to the right of the gall-formers. Note that the number of gall-formers that survive is much smaller than the number of inquilines and parasites. When studying Saskatchewan insect galls, there is also the satisfaction of breaking new ground. Little is yet known about the insects that emerge; many of the species have not even been named.

v3-fos

2018-12-11T09:19:10.315Z

1970-01-01T00:00:00.000Z

56007828

s2

Selection of solvent and extraction method for determination of antimicrobial potential of Taxus wallichiana Zucc Antimicrobial potential of different plant parts (needle, stem and bark) of Himalayan yew (Taxus wallichiana Zucc.) has been investigated with particular reference to selection of solvents and extraction methods. Two extraction methods (maceration and soxhlet), seven solvents (methanol, ethanol, acetone, chloroform, ethyl acetate, di chloro methane and petroleum ether), and 3 groups of microorganisms (bacteria, actinobacteria and fungi) were considered for detection of antimicrobial activity. While qualitative estimations were done using agar well diffusion method, quantitative analysis was based on dilution method. All the plant part showed significant activity against all 3 groups of microorganisms in qualitative bioassays; maximum being in case of needles. Among solvents, ethanolic extract of needles (maceration) showed highest antibacterial activity (15.33 ± 0.25 mm). Growth of actinobacteria was inhibited maximum (22.0±0.26 mm) by the methanolic extracts of needles (maceration). Ethyl acetate extract of needles (soxhlet) showed higher antifungal activity (8.67±0.23 mm). Antibacterial and antifungal activities were higher in maceration and soxhlet methods, respectively. The most affected group among the test microorganisms was bacteria which may be due to their prokaryotic organization. This was also supported by the low minimum inhibitory concentration (MIC) values. Di chloro methane and petroleum ether did not show any antifungal activity. The antimicrobial activity of various plant parts of T. wallichiana varied with respect to the solvent as well as the extraction method. The study will have implications in selection of the use of solvent and the extraction procedure in obtaining the antimicrobial metabolites from various plant parts of T. wallichiana. INTRODUCTION Antibiotic resistance phenomenon and development of the side effects due to consumption of microbe derived antibiotics suggested the need for alternate sources for combating the infectious diseases. In this perspective, plant based antimicrobials (derived from medicinal plants, in particular) are increasingly receiving attention for harnessing their potential in production of antimicrobial substances, as safer source of antibiotics. The Himalayan mountain ecosystem is well known for harbouring a plethora of medicinal and aromatic plants along with their domestication and cultivation for commercial purposes [1,2]. Plant extracts from a variety of medicinal plants showed potential antioxidant properties [3,4]. Now a day's, antimicrobial compounds are being utilized in various areas such as medical, pharmaceutical, textile and dairy industries, in food-based products, cosmetics, personal care products, etc. Taxus wallichiana Zucc. (English name: Himalayan Yew; Hindi name: Thuner; family: Taxaceae) is well recognized as a medicinally important evergreen tree that grows under temperate locations of Indian Himalaya. In the Indian subcontinent, the species grows in the northern hemisphere with its distribution in the hills of northern Jammu & Kashmir, Himachal Pradesh, Uttarakhand and the states in northeast namely Meghalaya, Nagaland, Arunachal Pradesh, and Manipur, at an altitude range of 1800-3300 m. The species has received considerable attention on account of its existing exploitation for the extraction of the drug (taxol) from its bark [5][6][7]. T. wallichiana is also known for its various ethanomedicinal uses [8] such as the leaf paste is used in treatment of asthma and bronchial disorders. Selection of solvent and extraction method for determination of antimicrobial potential of Taxus wallichiana Zucc. Tea, made out of the stem bark of Himalayan yew, has been popular in Himalayan tribal communities for curing cold, cough and hypertension. The species is also known as source of antioxidants [4]. However, the plant species still needs to be highlighted for its antimicrobial potential. Optimization of plant extracts for any activity, such as production of antimicrobials, is primarily important before isolation of antimicrobial compound(s). Several investigators have worked on the selection of suitable solvents for extraction along with the type of extraction method for assessing bioactive compounds including antimicrobials from different medicinal plants/parts [9][10][11][12]. Such reports are lacking in case of T. wallichiana, therefore, the focus of the present study is on the selection of solvent and extraction method in view of achieving maximum antimicrobial potential of the plant species. Therefore, the aim of the present study is to investigate the antimicrobial potential of T. wallichiana with respect to three major groups of microorganisms (bacteria, actinobacteria and fungi) considering seven solvents and two extraction methods. Study Site and Sample Collection Plant samples were collected from Jageshwar area in District Almora (29º35´-29º39´ N and 79º59´-79º53´E) of Uttarakhand, India. Herbarium of plant needle was submitted to herbarium record of G. B. Pant National Institute of Himalayan Environment and Sustainable Development, Kosi-Katarmal, Almora, Uttrakhand, India (Voucher number: GBPI 5050). The collected plant parts (needle, stem and bark) were washed, air dried and converted into fine powder for further experimental work. Extraction Needle, bark and stem were extracted through maceration and soxhlet methods, separately, using seven selected solvents (methanol, ethanol, acetone, chloroform, ethyl acetate, PET and DCM). Maceration 2 g of plant sample (needle, bark, and stem, separately) was mixed in different solvents, separately, in a ratio of 1:5 (dry powder: solvents). The mouth of conical flask was sealed with the para-film. Samples were macerated using rotary shaker (Remi) at 160 rpm for 24 h. Soxhlet 2 g of plant sample was placed in thimble and extracted with selected solvent, separately, in soxhlet extraction unit (MAC). Extraction was repeated until the sample extract became of color less. Plate based bioassays For qualitative estimation of antimicrobial potential of T. wallichiana extracts, agar plate based bioassays were performed using disc diffusion method. Bacterial and actinobacterial culture suspensions were prepared in TYE agar while fungal culture suspension was prepared in PDA. 100 µl of all the test organisms (separately) were spread uniformly on the respective agar surface (TYE agar plates for bacteria and actinobacteria, and PD agar plates for fungus) with the help of a glass spreader. Sterilized 5 mm filter paper (Whatman No. 1) discs were placed over the agar surface with the help of sterile forceps.15 µl of extract was loaded over the agar disc. The plates were then incubated at 25 ºC. The results were recorded on the basis of zone of inhibition (mm) after 24 h for bacteria and 120 h for actinobacteria and fungi. All the experiments were performed in triplicate. Quantitative Estimation/Minimum Inhibitory Concentration (MIC) MIC was determined following Clinical and Laboratory Standard Institute methodology [13]. Bacterial and actinobacterial culture suspensions were prepared in TYE while fungal cultures were prepared in PD broth. For determination of MIC, 1 ml extract was diluted using different concentration ranging from 100 to 1000 µg/ml, 1 ml test organism and 8 ml broth was taken in sterile test tube, and then incubated at 25 °C for 24 h for bacteria and 120 h for actinobacteria and fungi. Control was prepared in two sets, one containing broth medium and test organism while the other containing broth medium and extract. After 24 h, the MIC values were recorded on the basis of the lowest concentration showing absence of growth in the tubes. The test was further confirmed by plating on TYE agar and PDA medium. Statistical Analysis The data was expressed as the means ± standard errors (SE) from experiments, performed in triplicate. Statistical significance was determined using student's t-test. A p value <0.05 was considered as significant. Homogenizing grouping of all microorganisms was done separately using Duncan test in SPSS version 20. Figure 1 shows the extract yield of different plant parts of T. wallichiana (needle, stem and bark) extracted by different solvents and extraction methods. The extraction yield was recorded highest in stem in soxhlet ethanolic extract, while it was at par in case of acetone and methanol in soxhlet method. The extraction yield in bark was recorded highest in ethanolic extract in soxhlet and lowest in PET extract of maceration method. In needles, the yield was estimated higher in soxhlet methanolic and acetone extracts in comparison to the other extracts and it was recorded lowest in chloroform extract obtained by maceration method. Among all the plant parts of T. wallichiana, including the needle, stem and bark extracts following the maceration and soxhlet methods, the highest extract yield was found in needle extracts obtained by soxhlet method. Higher extract yield with polar solvents indicated towards the presence of more polar molecules in T. wallichiana. Low extract yield in ethyl acetate in comparison to other polar solvent was probably due to the poor dielectric constant. The extract weight of different part of T. wallichiana used were significantly different at p < 0.029. Qualitative plate based bioassays Needle, bark and stem extracts of T. wallichiana showed antimicrobial activity against all the three groups of microorganisms viz. bacteria, actinobacteria and fungi. Tables 1-3 and Figure 2 represent the antimicrobial activity of needle, bark and stem extracts, separately, in both the extraction methods. While all the solvents were effective in revealing the antibacterial activity in T. wallichiana extracts, PET and DCM showed selectivity in this aspect. Further, these two solvents (PET and DCM) did not show any activity against actinobacteria and fungi. Amongst bacteria, species of Bacillus were the most affected ones in terms of showing inhibition by T. wallichiana extracts, while E. coli was the least affected. On the basis of Duncan test used for homogeneous grouping of microorganisms separately, out of the two test species of Bacillus, B. subtilis showed higher inhibition in case of ethanolic extract of macerated needles and B. megaterium in case of methanolic extract of macerated needles. The inhibition of E. coli in macerated needle acetone extract, S. marcescens in macerated needle and bark methanolic extracts, P. chlororaphis in macerated needle and bark ethanolic extracts and P. palleroniana in soxhlet needle methanolic extract was at par. The actinobacterial species, N. tenirefensis and Streptomyces sp., were highly inhibited in macerated methanolic needle extract and macerated needle and bark methanolic extracts, respectively. Overall, both the extraction methods (soxhlet and maceration) were effective in revealing the antimicrobial activity. However, if compared on the basis of the groups of the microorganisms, maceration extracts were better in terms of exhibiting inhibition of bacteria and actinobacteria, while soxhlet was better in case of the species of fungi. Among the solvents, macerated ethanolic needle extract showed highest antibacterial activity (15.33 ± 0.25 mm), macerated methanolic needle extract showed highest actinobacterial (22.0±0.26 mm), and soxhlet ethyl acetate needle extract was best in exhibiting high antifungal activity (8.67±0.23 mm). Quantitative estimation The MIC was performed using five solvents i.e. chloroform, ethyl acetate, acetone, ethanol and methanol, out of which the acetone, ethanol and methanol extracts showed good results against all the group of test microorganisms. The most affected group among the three was bacteria, which is probably due to their prokaryotic organization. This was also supported by the low MIC values. Different microorganisms showed variable response towards these different extracts. The results of MIC of the needle, stem and bark extracts are presented in Tables 4-6. DISCUSSION In the present investigation, extracts of various parts of T. wallichiana have been evaluated for determination of their antimicrobial activity against three major groups of microorganisms bacteria (Gram -ve and Gram +ve), actinobacteria and fungi. The selection of seven different solvents used in this study was based on their polarity and dielectric constant which, starting from the lower dielectric constant values, can be arranged as follows: di chloro methane (1.3) < petroleum ether (4.3) < chloroform (4.3) < ethyl acetate (6) < acetone (20.7) < ethanol (24.6) < methanol (32.7). As depicted in results, extraction yield with polar solvents was higher in comparison to non-polar solvents. These variations may be due to the difference in their polarity as well as dielectric constant, which play vital role in the solubility of phytochemical compounds in respective solvents. Therefore, this result confirms the effect of solvent system on the extract yield that consequently confirms the richness of this plant species (T. wallichiana) in polar substances [14,15]. The results in the present study coincide with the earlier report of Patel et al. [16] on the antimicrobial activity of T. baccata needles which was demonstrated through the extracts made in different solvents. In the cited study, ethanol extracts of T. baccata showed highest inhibition activity against the selected test bacteria as also in the case of the present study. This preliminary investigation shows that all the polar solvent extracts, such as ethanol, methanol, acetone, ethyl acetate, were active against the tested microorganisms while the non-polar solvent extracts were not effective in inhibiting the microbial growth. These results are also supported by the previous findings of Sati et al. [11] and Nisar et al. [17]. Contrary to these results in various plant species, the extracts of Crossandra infundibuliformis prepared in PET have been reported with higher antifungal activity [18]. An earlier study carried on T. wallichiana needles growing in North-western Frontier Province, Pakistan, reported the absence of antibacterial activity in methanol fractions of the various plant parts [19]. Demonstration of the antibacterial activity in the methanolic extracts of all the plant parts (needles, stem and bark) of T. wallichiana belonging to Indian Himalayan region, in the present study, appears to be an indicative of the influence of different climatic conditions. Among the two extraction methods used, the soxhlet method resulted in higher yield in comparison to the maceration method. Interestingly, the antimicrobial activity was recorded higher in macerated extracts. This could be attributed to the higher solubility of extractable bioactive components such as phenols, flavanols, tannin and flavonoids, having antimicrobial potential. These results were also supported by the earlier findings of Felhi et al. [15,20]. The cited reports have shown that extraction in different solvents influence the extraction yield of total phenol content along with the antioxidants and antimicrobial activity. The variation in the extraction yield could also be due to the difference in polarity of the solvents used which plays a key role in increasing the solubility of phytochemical compounds [21,22]. Significant variation in MIC recorded in all the extracts demonstrated towards the variable contribution of the method of extraction and selection of solvent as well as the type of test microorganisms used. Also, variation in MIC of different plant extracts may arise from variation in their chemical constituents and their volatiles indicating the presence of one or more chemical moieties in the crude extract contributing towards the antimicrobial activity. This is proven by the varying results showing highest antifungal activity in needle ethyl acetate extract (soxhlet), and highest antibacterial activity in leaf ethanolic extract (maceration). Ethyl acetate soxhlet needle extracts are likely to contain the compounds with the capability to target eukaryotic cells, while ethanolic macerated needle extract composition is suitable to target prokaryotic cells which needs to be further explored [23,24]. On the basis of the results obtained, it is concluded that among the various plant parts, needles of T. wallichiana possess relatively higher antimicrobial activity. As needle is renewable part of the plant, its utilization for harnessing the antimicrobial potential is recommended. For further isolation of the antibacterial and antifungal compounds, maceration and soxhlet methods are also recommended, respectively. Higher antimicrobial activity achieved in the polar solvents is indicative of the presence of the antimicrobial activity in the polar compounds. On the other side, the resistance to the T. wallichiana extracts in fungi, such as Trichoderma, is indicative of the mutualistic interaction and the possibility of its use as biopesticide.

v3-fos

2021-08-13T03:56:49.123Z

1970-11-01T00:00:00.000Z

237230317

s2

Penicillic Acid Production by Blue-Eye Fungi on Various Agricultural Commodities Of 10 Penicillium species reported to cause blue-eye disease of corn, four (P. martensii, P. palitans, P. cyclopium, P. puberulum) were found capable of producing the mycotoxin penicillic acid on various agricultural commodities. Commodities with high protein contents did not support toxin synthesis. The extent of toxin production varied with the strain of mold, the commodity, and the temperature; low temperatures (1 to 10 C) favored toxin accumulation. In modern agricultural practice, high-moisture corn is frequently harvested by picker-sheller. It has been established that the higher the moisture, the greater the damage to kernels during shelling. The combination of high moisture, damaged kernels, and warm temperatures that often prevails in autumn makes such corn particularly vulnerable to molding. Although much of the corn is dried at or shortly after harvest to 12 to 14% moisture, holding periods may occur during which mold growth can be initiated. Sometimes the mold appears as a blue-green discoloration of the germ, a condition commonly referred to as blue-eye (7). Reportedly this disease of corn is caused by a wide variety of fungi, primarily species of Penicillium (7,9,10) but also including some aspergilli (8). Semeniuk and associates (10) state that the blue-eye fungi were the only ones isolated from shelled corn, stored in bins during the winter, that were capable of growth at 0.5 and 9 C. Blue-eye disease is commonly observed in corn stored during the winter in the Midwest where temperatures often hover around freezing. Kurtzman and Ciegler (Bacteriol. Proc., p. 8,1970) found that, in blue-eye-diseased corn caused by P. martensii, large quantities of penicillic acid were produced when high-moisture corn was stored at low temperatures (1 to 10 C). Penicillic acid is known to be toxic to mammals and has proved carcinogenic to rats (4). Since blue-eye disease may occur in combine-harvested corn, consideration needs to be given to the possible production of penicillic acid as representing a potential mycotoxin hazard to humans and livestock consuming this grain. The purpose of this investigation was to determine the capability of the various fungi implicated in blue-eye disease to produce penicillic acid on corn and other agricultural commodities particularly at a low temperature. MATERIALS AND METHODS Cultures. All cultures used in this investigation were obtained from the Agricultural Research Service Culture Collection at the Northern Regional Research Laboratory. They were maintained on either potatodextrose-agar or malt-yeast extract-agar slants stored at 10 C. Production of toxin. Thirty-five grams of grain or other agricultural commodity was placed in 300-ml Erlenmeyer flasks. To each flask was added 28 ml of distilled water, except for rice and cottonseed to which 14 ml of distilled water was added; the flasks were then autoclaved for 15 min at 121 C. Each flask was inoculated with 1 ml of spore suspension made by suspending spores of the various molds from 10-to 14-day-old slant cultures in 50 ml of sterile distilled water. Flasks were incubated without agitation at 1 and 20 C. All samples were prepared in duplicate, and each flask was assayed in duplicate. Extraction and assay for penicillic acid. The toxin penicillic acid was assayed fluorodensitometrically by the method of Ciegler and Kurtzman (J. Chromatogr., in press). Briefly, the method involves thin-layer chromatography ofthe unknown with known amounts of standard on silica gel (solvent, chloroform-ethyl acetate-formic acid, 60:40:1, v/v) followed by exposure of the plate to ammonia fumes. The penicillic acid-ammonia derivative is excited at 350 nm and fluoresces at 440 nm. The degree of fluorescence was determined with a Photovolt densitometer (model 530) equipped with an automatic scanning thin-layer plate stage and a recorder equipped with an integrator. A standard curve is prepared for each analysis, a linear plot being followed between 1 and 9 Ag of penicillic acid. The concentration of unknown is determined from the standard curve taking into account the dilutions involved. The penicillic acid used for standards was produced by fermentation as previously described (C. P. Kurtzman and A. Ciegler, Bacteriol. Proc., p. 8,1970). 761 For all assays, the contents of an entire flask were extracted with 250 ml of chloroform-methanol (90: 10, v/v) in a Waring Blendor for 3 min. The homogenate was filtered through anhydrous sodium sulfate, and the first 50 ml of solvent recovered was analyzed for penicillic acid. In those samples in which penicillic acid was not found, the 50 ml of solvent extract was evaporated to dryness, the residual solids were placed in solution or suspension in 3 ml of propylene glycol, and 0.1 and 0.2 ml of the glycol solution was injected into 20-g mice as a test for the potential presence of other mycotoxins. RESULTS AND DISCUSSION All Penicillium species chosen for this survey have been implicated in blue-eye disease of corn with the exception of P. granulatum NRRL 1575 (1,(8)(9)(10). This organism was included because, in a previous unpublished investigation, we determined that it also produced penicillic acid in Czapek-Dox broth. Two of the cultures, P. trzebinskii NRRL 732 and P. ochraceunm NRRL 870, were isolated from corn that had been blue-eye-damaged in the field. Two cultures of P. martensii, NRRL 3747 and 3612, were isolated from high-moisture corn that became blue-eyed after large-scale experimental storage under refrigeration. Data in Table 1 reveal that only 5 of the 16 strains tested produced penicillic acid. The production of this toxin was confirmed by thinlayer chromatography, by derivative formation with phenylhydrazine to form the hydrazone (C. P. Kurtzman and A. Ciegler, Bacteriol. Proc., p. 8,1970), and by mouse assay. In general, as we reported earlier (C. P. Kurtzman and A. Ciegler, Bacteriol. Proc., p. 8,1970), low temperature (1 C) favors the accumulation of penicillic acid although the rate of toxin formation is more rapid at a higher temperature (20 C). The exception to this was P. cyclopium NRRL 1888, which produced a greater quantity of toxin at 20 C than at 1 C. Strain specificity is also involved in the amount and type of toxin produced. P. martensii NRRL 3612 consistently produced more penicillic acid than did P. martensii NRRL 3747, but neither strain produced the tremorgenic mycotoxin (3), a capability shown by P. martensii NRRL 2034, which does not produce penicillic acid. Similar observations apply to strains of P. palitans: P. palitans NRRL 3672 produces penicillic acid but no tremorgen; the converse is true for NRRL 3468, whereas NRRL 966 produces neither toxin and appears to be nontoxigenic as determined by mouse assay. Thus, it would not be judicious to predict whether one type of mycotoxin or another might be produced on a given grain based on a mold profile. Penicillic acid production on some commodities. Drying most grains and other agricultural commodities to a low-moisture level is a generally accepted practice for safe storage of grains. However, several new procedures including refrigeration are being designed or are in use to store high-moisture corn and other cereals (5). High-moisture corn is more palatable to cattle than dry corn; it is also preferable for some industrial applications. Since blue-eye disease of corn appears to be particularly selected at low temperatures, we examined the four species found to produce penicillic acid on corn at 1 C (Table 1) for their ability to synthesize this toxin on a limited number of additional farm commodities at 20 and 1 C. Good growth and sporulation of the four species were observed on all commodities tested. In general, commodities with a high protein concentration (peanuts, soybeans, cottonseed) did not support penicillic acid synthesis by the test fungi at either temperature, whereas those substances rich in starch did when fermented at 20 C (Table 2). However, even with the starchy substrates, there is considerable variation with respect to the fungus, the grain, and the amount of toxin produced. Sorghum provided the best substrate for penicillic acid production by all four organisms at 20 and 1 C, whereas barley, oats, and wheat were poor substrates. Curiously, rice supported production by only one strain, P. cyclopium NRRL 1888. At 20 C, penicillic acid in affected commodities tends to gradually disappear, whereas at 1 C it slowly accumulates reaching levels higher than at 20 C. The reason for the loss of toxin at the higher temperature has not yet been determined. Better production of mycotoxins at low temperatures is not unusual and has been recorded for a number of fungi (2,6,11). When penicillic acid was not detected in any given flask during the survey, except for P. palitans NRRL 3468 which has been shown to produce tremorgenic toxins (3), the residue from the solvent extract was injected intraperitoneally into mice to test for the potential presence of other mycotoxins; none was found. Because of the ready growth of mold, even where toxin was not synthesized, refrigeration at 1 C of highmoisture grains does not appear to be a suitable substitute at the moment for drying grains before storage. Interfering substances. Substances that might seriously interfere with the penicillic acid assay from the various commodities used or that might be mistaken for the bright-blue fluorescentammoniated derivative of penicillic acid were not encountered, with the possible exception of soybeans. A pale-green fluorescing substance, extracted from soybeans, behaved somewhat like penicillic acid on thin-layer chromatographic plates. It did not fluoresce before treatment of the plates with ammonia, fluoresced green instead f blue, had a different excitation and emission oectrum (Fig. 1) than penicillic acid (penicillic APPL. MICROBIOL. acid: excitation, 350 nm; emission, 440 nm; unknown: excitation, 352 nm; emission, 480 nm), and had a slightly lower RF in the thin-layer chromatographic system used, 0.41 versus 0.45 (Fig. 2). Hence, the assay as developed should be suitable to detect penicillic acid in agricultural commodities.

v3-fos

2020-12-10T09:04:16.826Z

1970-07-01T00:00:00.000Z

237232852

s2

Endocarpic Microorganisms of Two Types of Windrow-Dried Peanut Fruit (Arachis hypogaea L.) The endocarpic microorganisms of peanut fruit dried in either a random windrow (plants left as they fell from the digger) or an inverted windrow (plants inverted to expose fruit to sunlight) were different from that of freshly dug fruit. Chaetomium, Penicillium, Trichoderma, Rhizoctonia, and Fusarium were the dominant fungi found associated with shells (pericarp) of freshly dug fruit. The dominant fungi of shells of windrowed fruit included Chaetomium, Rhizoctonia, Fusarium, Sclerotium, and Alternaria. Seeds of freshly dug fruit were dominated by Penicillium and Aspergillus. The only dominant species in seed of windrowed fruit was Penicillium. Microorganisms were isolated from shells and seed of freshly dug fruit at a frequency of 79% and 52%, respectively. The percentage of infestation was reduced by drying in the field. This was particularly true of the inverted windrow. The proportion of shells and seed infested with a microorganism was reduced 13% and 36%, respectively, after field drying for 5 to 7 days in random and inverted windrows. Microorganisms were isolated much more frequently from shell pieces (73%) than from seed (36%). Since the first discovery in 1960 (14) that a fungus commonly associated with peanut fruit (Arachis hypogaea L.) could produce a metabolite toxic to some animals, numerous reports on the microorganisms associated with mature fruit (5-7, 10, 12), overmature fruit (3), and damaged fruit (1,16) have appeared in the literature. As fruits mature in the soil, they become more susceptible to invasion by members of the microbial community of the surrounding soil (13). Thus, there is established an endocarpic (11) [or endogeocarpic (7)] microbial community in the fruit (5,18). Porter and Garren (18) reported that, from freshly dug fruit, microorganisms were isolated from 90% of the shells and over 63% of the seed. Other reports (2,3) indicate that fungal invasion may continue after fruits are removed from the soil. Garren (6) showed that isolation procedures, including the use of different media, temperature, and soil additives, influenced the microbial population associated with peanut fruit. In the now widespread mechanical harvesting of peanuts, plants are frequently lifted from the soil with fruits intact and windrowed in the field until the moisture content is reduced to 20 to 30%. The fruits are then combined, and drying is completed with forced air. During the period of windrow drying, changes in the microbial community associated with peanut fruit have been noted. Jackson (9,11) found that the "fungal communities from windrowed peanuts were distinctly different from communities which developed in the soil." Dickens (4) found that fielddrying was accelerated considerably in the inverted windrow and was less favorable for fungal growth than random windrows. The objectives of this study were to characterize the dominant endocarpic microorganisms of mature peanut fruit (i) at the time of digging, and (ii) after partial drying in two types of field windrows. MATERIAIS AND METHODS Peanut plants of the cultivar Virginia Bunch 46-2 were grown in a Norfolk fine, sandy loam soil at Holland, Va., in 1966Va., in , 1967Va., in , 1968Va., in , and 1969. Agronomic practices approved for Virginia-type peanuts were used. Planting dates were between 10 and 16 May. Soil fungicides and nematocides were not used. Plants were harvested during the time commercial peanuts were being harvested. Plants were mechani- and 2, 9, 21 October 1969. Plants were exposed for 5 to 7 days in a random windrow or in an inverted windrow (Fig. 1). In the random windrow, plants were left as they fell from the digger with most fruit covered with foliage and in contact with the soil. In the inverted windrow, plants were turned to expose most of the fruit to direct sunlight. After 5 to 7 days in each windrow, mature handpicked fruit was shelled, and pieces of shell (ca. 1 cm2) and seed with intact testa were surface-disinfested for 3 min in 0.5% NaOCl and plated (four per plate) on rose bengal-streptomycin-agar (15). Therefore, fungi growing onto this medium from surface-disinfested shells and seed should not have come from surface propagules, but from propagules produced by a thallus well-established therein. After incubation for 7 days at 25 C, most of the thalli that grew on to the medium from shells and seed could be identified. Approximately 1,200 shell pieces and 1,200 seed were plated in each of the 4 years of the study. At each reading, the percentage of shell pieces and seed from which at least one microorganism grew was determined. This was recorded as the proportion of shells and seed infested with some microorganism. We could identify most of the thalli of the fungi that grew from shells and seed. Thus, at each reading, we determined the isolation frequency of the dominant fungi in these shells and seed. RESULTS The percentages of shells and seed of freshly dug and windrowed peanut fruit that were infested with some microorganism during the 4 years of the study are given in Table 1. From this data, the following may be deduced. An Drying in a random windrow did not significantly reduce the number of microorganisms associated with shells. An average of 52.1 % (range 45.8 to 66.6) of freshly dug seed examined during the 4-year period yielded microorganisms. The proportion of shells and seed infested with a microorganism was reduced by an average of 31.5% by drying in a random windrow and by 60.4%o in an inverted windrow. An average of 42.2% fewer seed yielded microorganisms when dried in an inverted windrow than in a random windrow. In the whole of this study, a larger proportion of shells (73%) than seed (36%) was infested with some microorganism. The dominant shell and seed fungi (classified as a dominant if the isolation frequency from shell or seed was 6% or over) isolated during 1966, 1967, 1968, and 1969 are given in Table 2. The dominant fungi associated with shells of freshly dug peanuts included Chaetomium, Penicillium, Trichoderma, Rhizoctonia, and Fusarium. Chaetomium, Rhizoctonia, Fusarium, Sclerotium, and Alternaria dominated the shells of windrowed fruit. The dominant fungi of seed of freshly dug fruit included Penicillium and Aspergillus while that of seed from windrowed fruit was dominated only by Penicillium. More than one-fifth of the shells of freshly dug fruit yielded Chaetomium spp., mainly C. globosum Kunze ex Fr. ( Table 2). The isolation frequency of this fungus dropped slightly in the inverted windrow (15.2%) but was not changed in the random windrow (20.5%). The isolation frequency of Chaetomium from seed was much lower than that obtained from shells. In fact, only 3.9% of the seed from freshly dug fruit, 3.7% of the seed from random windrows, and 2.1 % from the inverted windrows gave rise to this fungus. Penicillium was isolated at a frequency of 17.5% from shells of freshly dug fruit (Table 2). Each windrow-type greatly reduced the isolation frequency of this fungus. Penicillium was the predominant genus in the seed of freshly dug and windrowed fruit, and approximately 28% of all seed plated from freshly dug fruit gave rise to this fungus. Its isolation frequency dropped considerably after drying in the windrow. More shells and seed yielding at least one thallus of Penicillium spp. were taken from random windrows than from inverted windrows. Many different species of this fungus were observed; however, over onehalf were P. funiculosum Thom. and P. janthinellium Biourge. A higher proportion of freshly dug fruit was infested with Trichoderma, mainly T. viride Pers. ex Fr. and other species including Trichodermalike isolates that were probably Gliocladium spp., than fruit from either windrow type ( Table 2). The type of windrow had little effect on the isolation frequency of Trichoderma. In all instances, the isolation frequencies of this fungus were higher in shells than in seed. The isolation frequency of Rhizoctonia was greater in windrowed fruit than in freshly dug fruit ( Table 2). The average isolation frequency from windrowed fruit was 6.7% compared to 3.5 % from freshly dug fruit. More shells than seed were infested with Rhizoctonia. More fruit yielding at least one thallus of Fusarium spp. were taken from windrows than were taken from freshly dug lots ( This fungus was obtained more readily from fruits taken from random windrows than from fruit taken from inverted windrows. A higher proportion of freshly dug and randomwindrowed fruit were infested with Rhizopus, mainly R. stolonifer (Ehr. ex Fr.) Vuill. and R. arrhizus A. Fischer than fruit in the inverted windrow (Table 2). This fungus was found more frequently in shells of random-windrowed fruit (5.5 %) than in shells of freshly dug fruit (4.2%). However, twice as many seed with at least one thallus of Rhizopus spp. were taken from freshly dug lots than from random windrows. The isolation frequency of Sclerotium from shells was much higher in fruits that were windrowed (8.5 %) than from freshly dug fruit (2.7 %; Table 2). More seed than shells yielded at least one thallus of Aspergillus spp. (Table 2). These were mainly A. flavus and A. niger v. Tiegh. Aspergillus spp. were isolated more readily from freshly dug fruit than from windrowed fruit. More fruit in the random than in the inverted windrow yielded at least one thallus of Aspergillus spp. Some A. flavus was isolated each year from shells and seed of freshly dug and windrowed fruit. The isolation frequencies of this species from seed of freshly dug, random, and inverted fruit were 5.2%, 3.9%, and 2.6 respectively. Shells with at least one thallus of Alternaria spp. were more numerous in windrowed fruit (8.3 %) than in freshly dug fruit (0.6%) ( Table 2). Seed were rarely infested with this fungus. In the 4 years of this study, the data (Table 1) show that without exception more shells and seed were infested in 1968 than in any other year. This may be attributed in part to the environmental conditions that prevailed during these years ( Table 3). The rainfall in October 1968, was about five times that of 1966, about four times that of 1967, and about two times that of 1969. The mean temperature for October was slightly higher during 1968 than during the other years. Also, the growing season of 1968 was prolonged because of the lateness of the first killing frost. The relation of rainfall on the windrow to changes in microbial infestation of peanut shells and seed is shown in Table 4. The differences in microbial infestation of freshly dug fruit and that of samples of the same fruit after 5 to 7 days in the windrow seemed almost inversely proportional to the amount of rain falling on the windrow. If no rain fell on the fruit while they were in the windrow, the proportion of fruit components infested with at least one microorganism decreased greatly and rapidly. For example, no rain fell on those plants dug and windrowed on 5 October 1966 and 25 October 1968, and fewer fruit parts yielding at least one microorganism were found in samples taken from these windrows than in samples taken from the windrows immediately after digging. On the other hand, much rain fell on plants dug on 2 November 1966 and 18 Octo- ber 1968, and these were the only two instances in which the proportion of fruit parts yielding at least one microorganism in samples taken from windrows was greater than that of the samples taken from the windrows immediately after digging. Frequency of shell infestation was affected more by rain than was the frequency of seed infestation. The moisture content of freshly dug fruit (determined on wet weight basis after drying for 4 hr at 130 C) during 1967 and 1968 averaged approximately 52% (Table 5). After field-drying for 5 to 7 days, fruit moisture content was reduced to 32% in the random windrow and to 22.7%, in the inverted windrow. DISCUSSION The degree of microbial infestation of shells and seed from windrowed peanut fruit was less than that of freshly dug fruit. Also, fewer shells and seed from fruit dried in inverted windrows were infested with microorganisms than were shells and seed from fruit dried in random windrows. These changes in the microbial community are to be expected, because the environment surrounding the windrowed fruit is different from that of fruit in the soil. At the time of removal from the soil, the fruit moisture averaged over 50%. However, once plants were placed in the windrow, the moisture content dropped rapidly. The drying rate of fruit was much more pronounced in the inverted windrow than in the random windrow. The average moisture content of seed dried for 6 days in the random windrow and inverted windrow in 1967 and 1968 was 31.9%zo and 22.6%, respectively. Others have reported similar findings (4,9). Fruits on plants in a windrow, especially an inverted windrow, undergo rapid dehydration which no doubt retards growth and development of the endocarpic fungi and which may account, in part, for the decrease in prevalence of the microbial populations. Another, and perhaps even greater, difference between fruit in the soil and fruit on plants in a windrow is relative humidity. The humidity level in the soil is high and remains fairly constant unless the soil moisture is depleted (19). Thus, the subterranean peanut fruits are usually surrounded by an atmosphere of very high humidity, except when surface soil is very dry. On the other hand, fruits on plants in a windrow are exposed to a wide range of relative humidities. Usually the above ground humidity is low during the day except during periods of inclement weather, and the relative humidity at night is high and often exceeds 90%. Most of the fungi found associated with peanut fruit have been called molds. Studies on molds (17) show them to be hydrotolerant, with optimum relative humidities of at least 90%. These high-optimum relative humidities for peanut fruit fungi may account in part for the reduction in the number of microorganisms present in the windrowed fruit. For example, the proportion of seed taken from windrows which yielded at least one thallus of Penicillium spp. was only 22 to 32% of that of freshly dug fruit. These factors, plus others undescribed, may act either separately or in combination to reduce the population of some of microorganisms associated with drying peanut fruit. A reduction in the isolation frequency of a fungus during windrowing may result from dehydration of the fruit and exposure to variable relative humidities. A similar reduction in the inverted windrow may be the result of more rapid dehydration and variable humidities, coupled with the effects of solar radiation. On the other hand, the isolation frequencies of some of the principal microorganisms of fruit increased while they were drying in the windrow. The isolation frequency of Fusarium and Alter-naria increased 4-fold and 16-fold, respectively, when fruits were subjected to windrow drying. Others (11) have also shown that the isolation frequency of Fusarium from peanut fruit increased when plants were windrowed. The increase in the isolation frequency of these two genera in windrowed fruit is of significance, especially since Garren et al. (8) recently reported that these fungi were capable of producing mycotoxins. McDonald and Harkness (16) showed that the isolation frequencies of most of the microorganisms associated with undug peanut fruit increased during periods of rainy weather. In our study, the isolation frequencies of most microorganisms tended to decrease more slowly in shells and seed when fruit drying in the windrows was exposed to appreciable rain than when not so exposed (Table 4). This was probably due to such factors as rehydration of the fruit, increased relative humidities, and lowered light intensities. The possibility of mycotoxin contamination of the fruit also increases under these conditions. Thus, growers must exercise extreme care in thehandling of such fruit. The isolation frequency of the well-known toxicogenic fungus A. flavus (14) was relatively low in freshly dug fruit each year of this study.

v3-fos

2018-04-03T05:23:43.587Z

1971-01-01T00:00:00.000Z

42648632

s2

Lipoprotein inhibitor of Newcastle disease virus from chicken lung. A lipoprotein inhibitor of Newcastle disease virus was obtained from chicken lung tissue by means of dilute alkaline extraction procedures. The inhibitor was further purified by ammonium sulfate fractionation, isoelectric precipitation, and density gradient centrifugation. The purified lipoprotein inhibited active Newcastle disease virus hemagglutination at a concentration of 2.0 mug/ml which represented a 30-fold purification over the original extract. Infection of chicken embryo fibroblasts by Newcastle disease virus was also inhibited by the purified lipoprotein, the degree of inhibition depending upon the inhibitor-to-virus ratio. Chemical analysis of the purified inhibitor provided a composition of 72% lipid, 26% protein, and 3% carbohydrate, although some compositional variation was observed from one preparation to another. The chloroform-soluble lipids were shown to contain 40 to 50% phospholipid and 10 to 20% cholesterol; of the fatty acids recovered from the saponified lipoprotein, 39% was palmitic, 22% oleic, and 17% stearic. Careful analyses of large quantities of the inhibitor revealed a small (0.84%) but significant content of sialic acid. Removal of sialic acid from the lipoprotein by means of digestion with neuraminidase produced a sharp diminution in inhibitory properties. A delipidized form of the inhibitor was obtained by ether extraction, and this material produced a single broad band of precipitate in gel immunodiffusion tests. A lipoprotein inhibitor of Newcastle disease virus was obtained from chicken lung tissue by means of dilute alkaline extraction procedures. The inhibitor was further purified by ammonium sulfate fractionation, isoelectric precipitation, and density gradient centrifugation. The purified lipoprotein inhibited active Newcastle disease virus hemagglutination at a concentration of 2.0 ,ug/ml which represented a 30-fold purification over the original extract. Infection of chicken embryo fibroblasts by Newcastle disease virus was also inhibited by the purified lipoprotein, the degree of inhibition depending upon the inhibitor-to-virus ratio. Chemical analysis of the purified inhibitor provided a composition of 72% lipid, 26% protein, and 3% carbohydrate, although some compositional variation was observed from one preparation to another. The chloroform-soluble lipids were shown to contain 40 to 50% phospholipid and 10 to 20% cholesterol; of the fatty acids recovered from the saponified lipoprotein, 39% was palmitic, 22% oleic, and 17% stearic. Careful analyses of large quantities of the inhibitor revealed a small (0.84%) but significant content of sialic acid. Removal of sialic acid from the lipoprotein by means of digestion with neuraminidase produced a sharp diminution in inhibitory properties. A delipidized form of the inhibitor was obtained by ether extraction, and this material produced a single broad band of precipitate in gel immunodiffusion tests. The intensive past studies on the inhibitors of myxovirus hemagglutination and infectivity have focused primarily on those inhibitors present in sera (13,17,19). Among the most active of these inhibitors are those glycoproteins classified as Francis or alpha inhibitors, the activity of which is associated with the sialic acid portion of the molecule. Removal of the sialic acid with neuraminidase causes a loss of inhibitor activity. The beta and gamma class inhibitors of serum are resistant to neuraminidase and have generally been less well characterized than those of the Francis type. In contrast to the ambitious investigations of myxovirus inhibitors on serum, tissue inhibitors, excepting those present on erythrocytes, have been largely ignored. Hirst (15) was the first to demonstrate that perfused ferret lung would first adsorb and then release influenza virus. A similar behavior of mouse, hamster, rat, guinea pig, rabbit, and chicken lung toward human and swine influenza virus, Newcastle disease virus, and mumps virus (34) has been described. It was an interesting observation that chicken lung did not adsorb Newcastle disease virus as well as the other influenza viruses and mumps virus. Investigation of the myxovirus inhibitor in mouse lung has indicated that it is easily extracted by low molarity bicarbonate solutions (4,23). No detailed chemical characterization of this solubilized inhibitor has yet been attempted; however, it was partially purified by differential centrifugation and Sephadex G-200 filtration, indicating its macromolecular nature. It was resistant to trypsin at 65 C for 30 min; however, it was sensitive to periodate and receptor-destroying enzyme, suggesting that it is a Francis-type inhibitor. Laucikova (20) has described a lipoprotein inhibitor for influenza virus which she extracted from chicken embryo chorioallantoic membranes. The chemical composition of the inhibitor varied somewhat from one preparation to another but was approximately 55% protein, 18% lipid, and 2% carbohydrate. Extraction of the lipoprotein with ether destroyed the inhibitor, indicating its dependence upon lipid for its antiviral activity. Periodate treatment, exposure to receptordestroying enzyme, or active virus also destroyed the activity of the inhibitor (21), supporting its classification as a sialic acid-dependent, Francistype inhibitor. The extraction of ox brain with a mixture of chloroform and methanol liberated an inhibitor ALLEN, BARRETT, AND CAMPBELL of influenza virus hemagglutination (31). It was described as a mucolipid consisting of as much as 20% sialic acid but no evidence was presented concerning the relation of the sialic acid to antiviral activity nor was the inhibitor described further in chemical terms. The research reported here was initiated to determine whether inhibitors of Newcastle disease virus (NDV) infection and hemmagglutination were present in chicken lungs. Since the lung is a primary tissue invaded by this virus, it was felt that the isolation of an inhibitor might be, simultaneously, an isolation of a natural receptor site for NDV. A unique lipoprotein inhibitor has been partially purified from chicken lungs and characterized in chemical terms. Its antiviral activity, as an inhibitor of viral hemagglutination and infectivity, has also been described. MATERIALS AND METHODS Unless stated to the contrary, all procedures were performed at 4 C. Purification of lipoprotein inhibitor. Approximately 25 g of minced, washed chicken lungs were homogenized in 100 ml of 0.001 M NaHCOs adjusted to pH 7.5 (26) with the aid of a Sorvall Omnimixer. After 10 min, this homogenate was diluted by the addition of 500 ml of cold bicarbonate solution. The diluted homogenate was clarified by centrifugation at 12,100 X g for 10 min. These conditions were employed for all future clarifications and for the collection of (NH4)2SO4 and isoelectric precipitates. The clarified bicarbonate extract was adjusted to 20% saturation by the addition of solid, reagent-grade (NH4)2SO4, held for 24 hr and clarified by centrifugation. The supematant was adjusted to 50% (NH4)2S04 saturation by the addition of more ammonium sulfate and again held for 24 hr before being clarified by centrifugation. The precipitate was dissolved in 0.15 M phosphate-buffered saline (pH 7.2) which was 0.01 M with respect to ethylenediaminetetraacetic acid (EDTA). This solution was dialyzed against distilled water to remove the ammonium sulfate. The dialyzed 20 to 50% ammonium sulfate fraction was adjusted to pH 5.0 by the dropwise addition of 1.0 M CH3COOH and held for 24 hr. The precipitate was collected by centrifugation and dissolved in 0.15 M phosphate-buffered saline containing 0.1 M EDTA (pH 7.5). The redissolved isoelectric precipitate was placed on top of a 20 to 50% sucrose gradient prepared at room temperature with the aid of a Beckman density gradient former. The sucrose solution was prepared in 0.15 M phosphate-buffered saline (pH 7.5) containing 0.1 M EDTA. The gradient was subjected to 90,000 X g for 90 min in the SW25 head of a Spinco model L preparative ultracentrifuge. The top band (1.130 density) was collected, diluted by the addition of an equal amount of the buffer solution, and recentrifuged on another 20 to 50% sucrose gradient. The single band was collected and dialyzed against distilled water before chemical analysis. It was dialyzed against 0.15 M phosphate-buffered saline (pH 7.2) before hemagglutination inhibition (HAl) and plaque inhibition tests. Chemical analyses. Protein determinations were performed by the method of Lowry et al. (24). Total hexose was determined by the method of Scott and Melvin (33) adapted to smaller volumes. A slight modification of the Elson and Morgan procedure (8) was used to quantitate hexosamine, and the thiobarbituric acid and diphenylamine methods of Warren (35) and Saifer and Siegel (32) were applied to the determination of sialic acid. The sum of hexose, hexosamine, and sialic acid is reported as total carbohydrate. Total lipid was measured by the gravimetric procedure of Folch et al. (11). Twenty milliliters of chloroform-methanol (2:1) at room temperature was added to 20 mg of lyophilized inhibitor which was extracted for 1 hr with magnetic stirring before being filtered upon sintered glass. The insolubles on the glass filter were extracted with an additional 20 ml of solvent. Distilled water was added to the filtrate in a biuret until the supernatant aqueous phase cleared. The lower chloroform phase was collected and dried completely under vacuum at room temperature before being weighed. Cholesterol was determined by the procedure of Zlatkis (37), and phosphorous, in the chloroform-methanol extract, was measured by Bartlett's method (3). Phosphorus was converted to phospholipid by assuming it represented 25% of the phospholipid. Fatty acids were determined as their methylated derivatives by gas chromatography (28) after saponification and petroleum ether extraction. For this analysis, 10 mg (dry weight) of lyophilized inhibitor sample was saponified with 2.5 ml of 2.5 M KOH in 85% methanol for 18 hr at 55 C. Unsaponified materials were extracted three times with 5 ml of petroleum ether, and the aqueous phase was adjusted to pH 2.0 with 5 M HCl. Fatty acids were extracted by two 5-ml applications of petroleum ether which were evaporated under vacuum and then dried over anhydrous Na2SO4. Methylation was accomplished by adding 1.0 ml of boron trifluoride-methanol reagent (25) to the dried product, followed by heat at 60 C for only 2 min. A petroleum ether extract of this mixture was washed with water before being evaporated to dryness. The methylated fatty acids were taken up in heptane and separated by a Barber-Coleman, series 5,000 gas chromatograph operated with helium gas carrier at 190 C and a 20% ethylene glycolsuccinate column on a gas pack (60 to 80 mesh). The flow rate was 80 ml/min, and the column effluent was analyzed by means of a hydrogen flame detector and paper strip recorder. Virus and inhibitor titration, tissue culturing, and plaque inhibition. NDV was used throughout this study and was perpetuated in the chorioallantoic cavity of 9or 10-day-old chick embryos, with harvesting 48 hr after inoculation. Chick embryo fibroblast (CEF) cultures were prepared from 9-or 10-day-old embryos by the method of Hoskins (16) and grown in plastic tissue culture plates (60 by 15 mm) in Eagle's growth medium (7) For the plaque inhibition test, CEF cultures with complete monolayers were rinsed twice with 5 ml of cold (4 C) Hanks balanced salt solution (BSS) (14), lacking phenol red, and placed at 4 C. The lipoprotein inhibitor was diluted twofold in Hanks BSS containing either 96 or 180 plaque-forming units (PFU) of NDV per ml. These mixtures were added to the CEF cultures and held at 4 C for 1 hr to allow virus attachment. Thereafter, the excess fluid was removed and overlay agar was added. The overlay medium was composed of Eagle's medium with 5% calf serum and 0.7% Noble special agar. After 72 hr of incubation at 39 C, neutral red agar (12% Noble special agar, 0.25% lactalbumin hydrolysate, and 0.04% neutral red in Hanks BSS) was added, and plaques were counted. Triplicate plates were prepared for each concentration of inhibitor. The hemagglutination (HA) titer of NDV was established as described by Hoskins (16). The titer of the hemagglutination inhibitor (HAI) was also determined by Hoskin's procedure (16). HAI titer is reported in terms of micrograms of inhibitor protein per milliliter which will inhibit 4 HA units of NDV at 4 C. Neuraminidase digestion. Top band inhibitor from the sucrose gradient was dissolved in 0.08 M phosphate-buffered saline (pH 6.1) at a level of 4.4 mg/ml. Neuraminidase from Clostridium perfringens type IV (Sigma Chemical Co., St. Louis) was added to a final concentration of 287 ,ug/mL. The solution contained 0.02% sodium azide to retard microbial growth. This mixture was incubated at 37 C for 45 hr with constant shaking. Appropriate controls were employed in which lipoprotein was incubated under the assay conditions without the presence ofneuraminidase. This digestion procedure is essentially that of Ada et al (1). At the end of the digestion period, the samples were dialyzed against distilled water; the dialyzates were then lyophilized and analyzed for sialic acid. The digested inhibitor and control were assayed for HAI activity after dialysis. Preparation of lipoprotein antigen, immunization, and serological testing. The lipoprotein fraction resulting from the isoelectric precipitation at pH 5.0 was dissolved in 0.01 M phosphate buffer at pH 7.0 and adjusted to 2.0 mg of protein/ml. The HAI titer of this solution was 17.5 ,g. Twenty-two milliliters of this preparation was applied to a carboxymethyl cellulose (CMC) column (70 by 1.9 cm) equilibrated with the same buffer but containing 0.02% NaNs as a preservative. The column was developed by batch elution at room temperature with 0.01 M phosphate buffers (pH 7.0 and 8.0), 0.20 M phosphate buffer (pH 9.0), and 2.0 M phosphate buffer (pH 9.5) containing 1.0 M NaCl. All buffers contained 0.02% NaN, . The column was adjusted to a flow rate of 10.0 ml/hr, and fractions were collected with the aid of a Beckman model 132 fraction collector. The first elution peak, which contained the inhibitor, was dialyzed against distilled water, lyophilized, and redissolved in 0.15 M phosphate buffer (pH 7.2). The inhibitor solution (330 pg of protein/ml) was injected intravenously into a rabbit with an irregular schedule over a period of 1 month, consuming approximately 2.0 mg of inhibitor before a capillary fluid precipitation test became positive. This antigen was also used in the immunodiffusion test. A delipidized sample of the antigen was prepared by dissolving the lyophilized CMC, peak 1 product in 0.15 M NaCl to a final concentration of 330 pug of protein/ml. Four milliliters of this solution was added dropwise, with vigorous mixing, to 50 ml of an ethanol-diethyl ether solution (1:3) at -21 C (10). After 4 hr at this temperature, the precipitate which formed was collected by centrifugation (-21 C) and washed once with approximately 25 ml of diethyl ether (-21 C). The precipitate was dissolved in 0.13 M tris(hydroxymethyl)aminomethane (Tris) buffer (pH 8.6) containing 0.06 M sodium lauryl sulfate and was then dialyzed against this buffer lacking the detergent before use in the immunodiffusion experiment. A third antigen used in the immunodiffusion test consisted of the top band sucrose gradient purified inhibitor. The gel immunodiffusion experiment was performed essentially as previously described (2). The CMC peak 1 antigen was tested at 430,ug of protein per ml and lesser concentrations. The delipidized peak 1 antigen and the undelipidized top band gradient purified antigen were tested at 430 pug of protein per ml and 1.7 mg of protein per ml, respectively. The gel plates were cut with a commercially available cutter (Consolidated Laboratories, Chicago, Ill.) which spaced the center antiserum well 7.5 mm from the peripherally located antigen reservoirs. The serological reaction was developed at room temperature, and observations were made daily until the precipitation band diffused into a broad zone. Table 1 illustrates the purification scheme used to isolate the lipoprotein inhibitor of NDV. The biological activity of all inhibitor fractions are reported as micrograms of inhibitor protein per milliliter which will inhibit 4 HA units of NDV at 4 C. The original, clarified bicarbonate homogenate of chicken lung had an HAI titer of 62.5 ,ug/ml. This value was quite constant from preparation to preparation, presumably because each lot represented a pool of several lungs. The fraction precipitating between 20 and 50% ammonium sulfate saturation improved the HAI activity by approximately fourfold. This fraction had an HAI titer of 17.5 ,ug/ml. The 0 to 20% fraction generally had a titer of 95 4g/ml. Fractions recovered in the range 50 to 75 % and greater than 75% ammonium sulfate saturation had titers in excess of 200 ,ug/ml. Ammonium sulfate fractionation removed hemoglobin from the inhibitor fraction. Isoelectric precipitation at pH 5.0 produced a material with very good HAI activity (i.e., HAI = 9 ,ug/ml). This product was poorly soluble in ordinary buffers unless stirred overnight in the cold. Sucrose banding of this material produced three bands (Fig. 1) the uppermost of which, at a density of 1.130, contained the inhibitor (HAI activity = 2.0 ,ug) in a 31-fold purification from the lung extract. The lowest band in the sucrose gradient appeared to consist of aggregated materials. RESULTS At each stage in the purification scheme, protein, carbohydrate, and lipid analyses were performed to provide insight into the chemical nature of the inhibitor. Table 2 presents a summary of these data, from which it can be seen that, as purification of the inhibitor progressed, the percentage of protein decreased and the percentage of lipid increased significantly. The purest preparation consisted of 3.6% carbohydrate, 26% protein, and 72% lipid, the sum of which account for the entire molecule. On the basis of the high lipid content, the molecule is referred to as the lipoprotein inhibitor hereafter. More definite expressions of the carbohydrate and lipid content of the lipoprotein inhibitor are presented in Table 3. Of the carbohydrate, 1 to 2% was in the form of hexose, about 1% in the form of hexosamine, and 0.82 to 0.86% in the form of sialic acid. Chloroform-soluble lipids were analyzed for cholesterol and phospholipid which accounted for 10 to 20% and 40 to 50% of these lipids, respectively. Of the fatty acids recovered (Table 4), palmitic acid was the most abundant (39.3%), followed by oleic acid (22%) ( Table 5). In the first experiment utilizing 96 PFU of virus, plaque reduction by as much as 64% was accomplished by 3.0 mg of inhibitor per ml. When the virus load was increased to 180 PFU, 3.0 mg of inhibitor per ml reduced the plaque count by only 14%, and lower concentrations were noninhibitory. Digestion of the lipoprotein inhibitor with neuraminidase for 45 hr reduced its sialic acid content from 0.84 to 0.37% and lowered its HAI activity from 2 to 20 ,ug/ml, a 10-fold increase in activity. During the digestion period 40% of the silaic acid of the inhibitor was removed. Inhibitor incubated without enzyme did not change in HAI activity nor sialic acid content. A typical elution patternof theinhibitorfraction on carboxymethyl cellulose is presented in Fig. 2. The first elution peak corresponded with the void volume of the column and had an HAI titer of 9.0 jig of protein per ml. This represented a twofold purification over the isoelectric precipitation stage. Later elution peaks were not active against NDV virus. The CMC-purified fraction was chosen as the antigen for hyperimmunization of a rabbit. Figure 3 presents the results of the Ouchterlony test after 24 hr. Precipitation was noted only when the delipidized antigen was tested against the antiserum. A rather broad band was formed. The untreated immunizing antigen failed to produce a precipitate even though it did react in the fluid capillary test used to establish antibody activity in the rabbit serum. Undelipidized sucrose gradient purified material also failed to exhibit precipitation with the antiserum. Upon observation for longer periods of time, the precipitation band broadened but could not be resolved into definite minor components. DISCUSSION The purification scheme for isolation of the lipoprotein inhibitor was devised by utilizing typical biochemical fractionation procedures and noting the improvement in HAI activity on a protein basis. Bicarbonate solutions are often used in the initial stages of cell membrane preparation (4,23,26,34). Laucikova (20,21) used bicarbonate extraction and isoelectric precipitation at pH 5.1 to separate an inhibitor of influenza virus HA from chick embryo chorioallantoic membrane extracts. Her inhibitor was composed of only 18% lipid and 55% protein and is thus quite dissimilar from the lipoprotein inhibitor described herein. Our effort to utilize diethylaminoethyl (DEAE) cellulose in the purification scheme was unsuccessful; the lipoprotein inhibitor bound so tightly to the DEAE that it could not be eluted. Philipson lipoprotein inhibitor of enteroviruses upon ionexchange chromatography. Blumenfeld (5) has reported complete retention of cell membrane sialoproteins by DEAE, so this problem is not unique to this inhibitor. On the other hand, chromatography of the active material on CMC at neutral pH demonstrated that inhibitor activity was not retained on the column but eluted with the void volume. Some noninhibitory materials were retained on the column, and this procedure was employed in the preparation of the inhibitor antigen. Because of the inability to use ion-exchange chromatography to great advantage, we attempted purification by sucrose gradient density centrifugation. Apparently, this method has not been used previously in the purification of viral inhibitors. Nevertheless, this method revealed that our isoelectrically precipitated product could be fractionated into three components, with approximately fouror fivefold improvement in purification. The lipoprotein inhibitor stratified at a density of 1.130. The precise chemical composition of the NDV lipoprotein inhibitor varied somewhat from one preparation to another (Table 3). This is a typical problem in lipoprotein chemistry (10,18,20), the causes of which are not entirely known. Nevertheless, it is obvious that the inhibitor is not chemically similar to fowl immunoglobulins (22), neither is it chemically similar to fowl interferons (9) although it will prevent cell infection like the latter. It does not, in fact, have the same chemical composition as the influenza inhibitor from chicken chorioallantoic membrane described by Laucikova (20,21). The plasma membrane from chick embryo fibroblast cultures has a composition of 25% protein, 64 to 71 % lipid, and 7 to 8% carbohydrate, very similar to that of the lipoprotein inhibitor (29). This suggests that the inhibitor is a portion of the cell membrane and adds significance to its description, for it could represent a natural receptor of the virus on chicken lung cells. The major fatty acids present in the lipoprotein inhibitor are shown in Table 4 to be palmitic, oleic, stearic, linoleic, and arachidonic. These fatty acids have been reported to be in highest concentration in the influenza virus and in-calfkidney and chick-embryo host cells (18). It has been suggested that host lipid components are incorporated into the influenza virus particles at some time during their synthesis (12). Future work will attempt to determine whether lipid incorporated into Newcastle disease virus during infection resembles the lipid obtained as lipoprotein inhibitor from host tissue. on May 5, 2020 by guest http://aem.asm.org/ Downloaded from and vaccinia viruses have all been described as lipophilic viruses (27) based on their ability to adsorb to steroids including cholesterol, fatty acids such as palmitic and stearic acids, and various fatty acid derivatives. It is believed the attachment of these lipophilic viruses to cholesterol occurs via van der Waal's forces, whereas attachment to the fatty acids and their derivatives involves ionic forces (36). In some instances the adsorption is irreversible. Cholesterol, palmitic, stearic, and other fatty acids, all of which are known to serve as receptors for NDV (27), are present in the lipoprotein inhibitor prepared from chicken lung and may contribute to the inhibitory activities of this macromolecule. All of the well-characterized hemagglutination inhibitors of myxoviruses contain sialic acid, upon which the basis for their biological activity resides (17,19). Removal of 40% of the sialic acid in the lipoprotein inhibitor caused a 10-fold loss in its HAI activity, supporting its classification as a Francis type inhibitor. The immunodiffusion test revealed a single band with the delipidized antigen only. Both of the lipid-bearing antigen preparations gave negative tests. The exact cause for this was not established. Hypothetically, this could result from the masking of antigen determinants in the protein by lipid so that they were unavailable to react with antibodies in the absence of any antilipid antibody formation, or simply the inability of the complete lipoprotein to diffuse in the aqueous menstruum owing to its hydrophobic characteristics. Although only a single precipitation band was detected even with the delipidized antigen, it was rather broad, indicative of antigenic impurity. Important to the problem of whether this lipoprotein inhibitor is a "natural" receptor for the virus on chicken lung may be the observation that it will inhibit viral infectivity. Many inhibitors of myxovirus hemagglutination have been described (13,17,19) but few have been reported to retard infection by the virus. Ovine ca,-glycoprotein will inhibit both HA and plaque formation by NDV (6).

v3-fos

2018-04-03T01:20:30.520Z

1971-06-01T00:00:00.000Z

22659926

s2

Microorganisms in Unamended Soil as Observed by Various Forms of Microscopy and Staining A light-diffraction microscope was modified to allow sequential viewing of the microorganisms in a soil smear by transmitted, reflected, and reflected-polarized incandescent light and by reflected ultraviolet light. Observations were also made by conventional incandescent and ultraviolet transmitted-light microscopy. All results for the various forms of bright-field microscopy with stained and unstained soils were in agreement, but they differed from the results obtained for two types of ultraviolet-fluorescence microscopy. The latter proved to be nonspecific for in situ soil microorganisms. Capsule-like areas were noted surrounding many of the resident microbial cells of soil when viewed by the various forms of bright-field microscopy. These areas could not be stained or removed by a variety of treatments, but they apparently often did take up stain after in situ soil growth had been initiated. It was concluded that these areas are not capsules but may represent a structural component of nonmultiplying microbial cells in soil. Several microscopic methods are available for viewing the total in situ microbial flora of soil. These include bright-field viewing (6,23) and color infrared photography (5) of nonstained soil, bright-field microscopy of stained soil preparations (1,7,8,11,12,21,23,26,31,32,35), and ultraviolet-fluorescence microscopy of soil stained with unconjugated (3, 4, 9, 10, 15-17, 24, 25, 30, 33, 36) and conjugated (20) fluorochromes. It has been commonly accepted that these methods selectively demonstrate microorganisms in soil preparations, and that they are in agreement with each other as to the types and numbers of microorganisms observed. The present study examines the validity of this conclusion for certain of these methods. Also, information is presented on a capsule-like area surrounding many of the soil microorganisms, as observed in situ by various techniques of bright-field microscopy but which is not detected by ultraviolet fluorescence microscopy. MATERIALS AND METHODS Suspensions of non-air-dried soil were prepared by shaking 1 to 2 g of soil in 25 ml of distilled water for 10 min and by subjecting 1 g of soil in 25 ml of distilled water to 1 min of sonic treatment at 25 to 50% of 1 This research was authorized for publication as paper no. 3939 in the journal series of the Pennsylvania Agricultural Experiment Station on 11 March 1971. maximum power in a Biosonic IL oscillator. In both instances, the larger soil particles were allowed to settle for a short period before smears were prepared from the supernatant fluids. For Pronase treatment of soil, 0.1 g of non-airdried sieved soil was mixed with 3 ml of 0.003 M potassium phosphate buffer (pH 7.4) containing 3 mg of Pronase (Calbiochem, 45,000 proteolytic units per g) in a 25-ml Erlenmeyer flask with a rubber stopper. The flask was incubated stationary at 38 C but was shaken by hand before each sampling for preparing smears. For toluene treatment of soil, 2 g of soil plus 25 ml of distilled water and 1 ml of toluene were shaken for 4.5 hr at 30 C. Samples for smears were removed at 30 min and 4.5 hr. Lysozyme-ethylenediaminetetraacetic acid (EDTA) treatment of soil was as described by Lichstein and Oginsky (18). Eight grams of soil in 40 ml of distilled water was sonically treated for 2 min at 30% of maximum power and then allowed to settle for 10 min. The supernatant fluid was used for the trials, and the lysozyme (K and K Laboratories, Inc., Plainview, N.Y.) levels ranged from 0.25 to 9.0 mg per ml. The Anthony, Tyler, and Leifson capsule stains were those described in the Manual of Microbiological Methods (27). Dark field was not required for the Strugger (30) acridine orange fluorescence procedure, nor was it necessary to disperse the aqueous suspension of stained soil in immersion oil. To obtain high resolution and color correction for all three primary colors, a Zeiss 100 X apochromatic objective with iris diaphragm, allowing numerical apertures of from 0.8 to 1.32, was used for all micros-copy. Usually, its NA was set at 1.32, and the condenser NA was 1.4. Bright-field microscopy of stained soil smears usually utilized a conventional transmittedlight microscope with the above apochromatic objective, but comparisons were also made with the following modified microscope. The reflected-light microscope described by Casida (6) was further modified to allow alternative viewings of microorganisms by transmitted incandescent light, reflected incandescent light diffraction with and without polarization, and reflected ultraviolet light. These modifications utilized a Leitz HBO 200 ultraviolet source (normally used with a Leitz Ortholux microscope), with connected switching arrangement to allow entry of incandescent light from the side but with the 6-v incandescent source removed. The 12-v incandescent source attached to the rear of the base of Casida's reflected-light microscope was detached, and the above ultraviolet source was added and aligned. The 12-v Zeiss incandescent source was then aligned where the 6-v Leitz source had been. The latter 6-v source plus the Leitz condenser was mounted above the gliding stage of the reflected-light microscope to allow transmitted-light microscopy. The aperture and field diaphragms were not stopped down, except in the case of reflected incandescent light diffraction microscopy where the aperture diaphragm was stopped down completely, and the field diaphragm was partially stopped down when decreased flare or increased resolution was desired. The NA of the objective diaphragm was reduced only when excessive flare was encountered. Mineral fragments were detected by reflected polarized incandescent light. A polarizer was mounted in the head of the microscope, and an analyzer was laid over the stopped-down aperture diaphragm. Rotation of the analyzer did not extinguish vision for the mineral fragments. RESULTS Stained preparations. Smears of unamended soil were stained with phenolic Rose Bengal (2) and observed by bright-field transmitted-light microscopy at 1,000to 1,250-fold magnification. The soil materials appeared red or they were almost colorless due to lack of affinity for the stain. The soil bacteria were easily discerned as darker-red to purple-red coccoid and coccoid-rod cells, frequently surrounded by a distinct unstained capsule-like area. The latter was more easily observed in those areas of the preparation in which a slight excess of stain had caused negative staining Preparations negatively stained with Anthony's capsule stain (and with the Tyler modification) clearly demonstrated wine-red bacteria surrounded by a colorless capsule-like area (Fig. 1). The soil materials were a purple to purple-red. As noted above, the capsule-like area did not stain with the Anthony, Leifson, or McKinney capsule (or cyst) stains. To test the Anthony procedure, vegetative cells and cysts of Azotobacter chroococcum were mixed with soil, and smears were prepared. Examination of these smears revealed staining of the cysts as described by Socolofsky and Wyss (28). A similar trial with McKinney's (19) Alcian Blue stain for A. chroococcum and A. vinelandii added to soil showed red-stained cells surrounded by blue capsules. Attempts were made to remove or alter the capsule-like areas by pretreatment of the soil smears before applying Anthony's stain. After treatment, the smears were washed and blotted dry before staining. Significant changes in the capsule-like area did not occur with any of the following treatments: (i) 1 min at room temperature for 95% ethanol, 90% acetone, formaldehyde, ethyl acetate, glacial acetic acid, pyridine, and chloroform; (ii) 1 min at room temperature for 1% aqueous solutions of EDTA, citric acid, oxalic acid, urea, FeCl3, K2Cr2O7, and AgNO3; (iii) various combinations of tris(hydroxymethyl)aminomethane buffer, sucrose, lysozyme, and EDTA; (iv) Chlorox, 20 min at room temperature; (v) 6 N NaOH, 20 min at room temperature; (Vi) 1 N H2S04, 30 to give slightly increased cell numbers and then caused some lysis of the cells. In contrast to these findings, the capsule-like areas surrounding some of the indigenous soil organisms seemed to disappear, or become markedly reduced in size, by shaking 2 g of soil for 22 hr at 30 C in 50 ml of sterile Heart Infusion Broth (Difco) in a 250-ml Erlenmeyer flask. During this incubation, large purple-staining rods with and without endospores became apparent, as did smaller reddish-purple to purple-staining coccoid and rod-shaped cells lacking the capsulelike area. Soil bacteria which had not initiated growth were still present in high numbers, with their wine-red cells and capsule-like areas, but they appeared smaller than the multiplying soil organisms. However, their total diameters for cell plus capsule-like area of approximately 1.0 to 1.4 ,um (the cell itself measuring approximately 0.5 to 0.8 um) were approximately equal to the widths of many of the multiplying cells lacking the capsule-like area. Bright-field microscopy of unstained preparations. Alternation between the transmitted-and reflected-light sources on Casida's reflected-light microscope (6) allowed a comparison of individual microbial cells as observed under both conditions of microscopy. By reflected light with the aperture diaphragm closed, the coccoid and coccoid-rod cells diffracted light as described by Casida, but the capsule-like areas were not visi-ble. These areas often could be detected, however, by partially stopping down the field diaphragm in the head of the microscope. An objective numerical aperture of 1.32 on the apochromatic oil objective was used for these comparisons, and polarized light in conjunction with the reflected light microscope was used as an additional means for detecting artifact mineral fragments in the preparations. Thus, all forms of bright-field microscopy examined were in agreement concerning the bacterial cells observed and the capsule-like areas surrounding the cells. Ultraviolet fluorescence microscopy. Addition of reflected ultraviolet light and transmitted incandescent light to Casida's reflected-light microscope allowed a comparison of the various forms of bright-field microscopy with the unconjugated fluorescein isothiocyanate (FITC) fluorescence methods of Pital et al. (24) and Babiuk and Paul (3) and the acridine orange fluorescence procedure of Strugger (30). Observation of soil by the unconjugated FITC procedure revealed large, fluorescing rod and coccoid bodies which appeared similar to the published photographs of Pital et al. However, these objects were not visible by either transmittedor reflected-light bright-field microscopy. Reversing the order, FITC-stained in situ soil bacteria detected by the bright-field microscopy techniques did not fluoresce under ultraviolet light Strugger's acridine orange fluorescence proce-dure for in situ soil bacteria, with both transmitted and reflected ultraviolet light, also did not agree with the results of reflected-and transmitted-light bright-field microscopy. Only a small percentage of the objects fluorescing under ultraviolet light demonstrated a morphology by the bright-field microscopy procedures which could be attributed to soil bacteria. Some of the objects which had fluoresced blue-green under ultraviolet light appeared under bright-field microscopy as highly irregular forms with jagged edges, whereas other objects were amorphous. In several instances, the objects observed by fluorescence microscopy actually were portions of larger unidentifiable structures. Sometimes, part of a soil protozoan cell fluoresced blue-green; in other instances, the entire protozoan cell fluoresced. What appeared under ultraviolet light to be chains of strongly fluorescing large bacterial rods proved by bright-field microscopy to be partially degraded actinomycete or fungal hyphae with many nonfluorescing soil bacteria adhering to the hyphal surface. The total numbers of fluorescing objects with acridine orange were one to several orders of magnitude less than the microbial cells observed by the various forms of bright-field microscopy. Also, at least a part of the soil material did not fluoresce in any manner. Regardless of these results, however, autoclaving of the soil before staining prevented the occurrence of the bluegreen fluorescence. Also, the fluorescence characteristics described by Strugger and others did occur for a Sarcina strain newly isolated from food when it was mixed with soil previous to staining with acridine orange. These added cells were easily detected in the soil by both brightfield and fluorescence microscopy, with some of the cells fluorescing blue-green and the rest fluorescing orange-red DISCUSSION The methods of bright-field microscopy evaluated in this study are in agreement for stained and unstained preparations of unamended soil. They reveal coccoid and coccoid-rod cells approximately 0.5 to 0.8 ,um in width, which appear as various intensities of a reddish hue regardless of whether stains are present or the particular stains used. Most of the cells were surrounded by a clear non-staining capsule-like area having a total width of approximately 1.0 to 1.4 ,um. It would appear that the cells do not stain or take up only small amounts of stain and that they naturally exhibit a reddish hue, which is more apparent when viewed with an apochromatic objective corrected for red. The latter observation could explain the red image for bacteria in unstained soil recorded by Aero Ektachrome Infrared photography in conjunction with an apochromatic objective (5), if this type of photography should tend to intensify the red color. The capsule-like area was visible in unstained and stained soil with all forms of bright-field microscopy tested, although it was more easily detected with apochromatic objectives and negative staining. It was not observed during fluorescence microscopy. Phase-contrast microscopy was not used because of its inherently lower resolution and its characteristic formation of halos of light surrounding the cells. Although not mentioned or discussed in their studies, other workers, using bright-field microscopy of stained soil, have published photographs showing this capsule-like area, e.g., Conn (8). Casida (6) stated that the outer boundary of cells residing in soil was not detected by his light diffraction microscope but that it was visible when growth occurred in the soil and the light diffraction capability of the cells was lost. The present study notes that this outer cell boundary can be seen in light diffraction microscopy when the field diaphragm is partially stopped down to reduce flare and the objective diaphragm is not stopped down (which decreases resolution). For both stained and unstained soil, alternating observations of cells with this method and with transmitted-light bright-field microscopy revealed that the above outer cell boundary is the same as the capsule-like area. These observations and observations in the present study that coccoid and coccoid-rod cells which have initiated growth in soil apparently totally stain (including the capsule-like area) and that, even though the cells may elongate during growth initiation, their widths often are equal to the widths of the capsule-like areas in nonmultiplying cells indicate a possible major cytological difference in the resident cells as they occur dormant in unamended soil and as they are forced by soil amendment or root growth (6) into a type of growth more closely resembling that of laboratory-grown cultures. The capsule-like area observed for resident cells in soil apparently is not a capsule, at least as occurs for laboratory-grown cultures. It was not removed or stained by any of the methods tried, which included capsule stains. Also, encapsulated cells of laboratory cultures examined separately and after addition to soil did not yield lightdiffraction colors (6) as do resident soil organisms. Lastly, as a laboratory type of growth is initiated in soil, this area of the cell does not disappear but often becomes stainable and indistinguishable from the rest of the cell. It should not be concluded from the above that resident organisms in soil do not possess capsules; there could be a thin capsule layer which was not detected in these studies. The microscopy, appearance, and growth behavior of resident soil organisms somewhat resemble those of a cyst and its germination. However, the lack of cyst staining by the procedure of Socolofsky and Wyss (28) and the failure to remove chemically the outer boundary of the cell would predict that, if they are cysts, they must be protected in some manner or they are structurally different from the Azotobacter cyst. The latter might be predicted by the fact that laboratory-grown Azotobacter cysts do not yield light diffraction colors with Casida's microscope. Nevertheless, having the characteristics of a cystlike stage associated with much of the resident soil microflora, if this should actually be the case, would be of considerable interest. The cells would be desiccation-, phage-, and radiation-resistant (14,29,34) and would have almost no endogenous activity, but they would, nevertheless, be able to utilize exogenous substrates without delay and at a lower respiration rate than encountered for vegetative cells (22,29). No ready explanation is available for the results obtained by both procedures of ultraviolet fluorescence microscopy. Obviously, they do not agree with any of the methods of bright-field microscopy with which they were compared. The fluorochromes do not seem to differentiate the more dormant resident soil bacteria, but they do fluoresce with various other types of soil microbial life, such as protozoa, and with various inanimate soil materials. The latter would be possible if certain soil materials should be coated with proteinaceous material, for which FITC has an affinity, or nucleic acids, which can fluoresce blue-green with acridine orange. The fact that partially degraded actinomycete and fungal hyphae fluoresced as if they were chains of bacterial rods would tend to support the latter concept. Addition of a laboratory culture of a Sarcina strain to soil before acridine orange staining presented no problem as regards bright-fieldmicroscopy cell detection or ultraviolet-fluorescence vital staining. Thus, the Strugger effect occurred for laboratory cultures, even when superimposed on soil. The validity of this vital staining characteristic, however, has been questioned (13) and, therefore, the lack of blue-green fluorescing objects for autoclaved soil in the present study or for "sterilized" soil (30) may not be a valid criterion for the efficacy of acridine orange as a fluorescence vital stain for soil.

v3-fos

2017-06-28T23:31:56.908Z

1971-07-15T00:00:00.000Z

6241712

s2

Hereditary osteopetrosis in aberdeen-angus calves. I. - Pathological changes Twenty-three Aberdeen Angus calves with a congenital mandibular defect, brachygnathia inferior with impacted molars and a generalized bone disorder, osteopetrosis, were encountered in Aberdeen Angus herds in the United States. Two calves were necropsied and the heads of 4 others were examined. Facial and cranial bones were dense and thickened and all foramina were hypoplastic or aplastic. The cranial base, vertebrae, and long bones were solid and lacked marrow cavities. Microscopically, the cranial base, vertebrae, long and phalangeal bones had fewer blood vessels and a disturbed relationship between bone formation and bone resorption. Gross and microscopic finding in bovine osteopetrosis were similar to those in human congenital osteopetrosis. Congenitally anomalous calves may be brought to the attention of the veterinarian for several reasons: his obstetrical or remedial surgical skills, or his opinion as to the cause. If it is environmental, the owner may be able to alter herd management to prevent reoccurrence; if it is hereditary, he may alter the herd breeding program. The latter is involved in the recent occurrence of a generalized osteologic defect in 3 A be y deen Ang2as herds. Gross and microscopic changes of the defect are reported here. I. -MATERIALS AND METHODS The deformed calves were reported in connection with a previously described long-term study of the nature and cause of congenital defects in domestic animals (Huston and Wearden, 1958;Leipold and Huston, 1968). Herd histories were obtained by personal visits and mail inquiries. Although 23 calves in 3 herds were reported with the defect, only 7 were received for clinical or pathological examination. Three from Herd 1 were examined on the ranch; 2 and the partially autolyzed head of the third were subsequently transported to Kansas State University for necropsy. Later, frozen heads from 3 other calves also were examined. One calf from Herd 3 was examined on the ranch and transported to the University for clinical but no pathologic examination. Heads and bones were sawed in half; frozen specimens were thawed in 10 % neutral buffered formalin. One half was examined radiographically and histologically; the other half was macerated except for the autolytic head which was entirely macerated. The following tissues were taken for routine histologic examination: cerebrum, cerebellum, spinal cord, pituitary, thyroid, parathyroids, adrenal glands, liver, kidney, spleen, and lymph nodes. Tissues were fixed in 10 % neutral buffered formalin, embedded in paraffin, and sectioned at 6 : micra. In addition to routine staining with hematoxylin and eosin (H et E), special stains, PAS, Alcian blue and Mason's trichrome, were used. Histologic sections were taken from several long bones, phalangeal and carpal bones, vertebrae, mandibles, base and vault of the cranium. Longitudinal and transverse sections of the bones were made at various levels. For comparison similar sections were taken from 2 Aberdeen Angus stillbirths. Sections were decalcified in 20 % formic, sectioned and stained routinely with H et E, Alcian blue, Mason's trichrome, and van Gieson's stain. II. -RESULTS A. -Herd histories Herd i was extremely well-managed and comprised 950 registered and grade breeding Aberdeen Angus cows. The cows were inseminated artificially commencing December 15 while on a diet supplying 15 lb. TDN and 1 . 25 lbs. protein daily. In March, bulls were turned in to breed any nonpregnant cows. In the early ig6o's, an abnormal calf was born that may have been the first with osteopetrosis. Five more were born in 19 6 7 and 5 in 19 68. All but I were born dead. Fetal age of the first 7 of those 10 were 253 , 251 , 2 68, 27 6 a6q, 250 , and 272 days, respectively, an average of 2 6 2 days or about 3 weeks premature. Seven of the I calves were by i bull; the remaining 4 by 4 other bulls. Herd 2 was a well-managed herd of 350 registered and grade A be y deen Angus cows. One abnormal calf was born in 19 6 7 and 3 in 19 68. All were by a single bull and all but one were from daughters of another bull. Herd 3 included 90 grade Aberdeen Angus cows, half calving in the fall and half in the spring. In 19 68, 7 calves with abnormal jaws were born 3 to 6 weeks premature; all were by a single bull and from daughters of another bull. In 19 6 9 one abnormal was from a sister of the 19 68 abnormal, sired by another bull. B. -Gross pathological changes 1° Sheletal system The external features of the 4 calves seen in their entirety, 3 from Herd i and one from Herd 3 , were similar. All calves were small; the two necropsied weighed 14 and 22 kilograms. The mandibles were immobile, up to 4 cm. shorter. The mouth was open and the tongue protruded ( fig. I , a). The incisor teeth were not completely erupted, and the molar teeth were irregular and impacted. The cranial cavity was smaller than normal and each had bony closure defects of I to 3 cm. in diameter between frontal and parietal bones ( fig. I , b). All facial and cranial bones were thicker ( 0 .6 to 0 .8 cm.) and denser than normal. Transversely rising folds occurred on the internal cranial vault and were particularly marked at the frontal bones and orbital wings of the sphenoid: the basisphenoid bones either lacked nutritive foramina or were stenotic; one specimen already had an ossified sphenoidal-occipital synchondrosis ( fig. 2 , a). The infraorbital, supraorbital, major palatinal, and ethmoidal foramina were either poorly indicated, some ended blindly after a short distance, or were absent. None of the mandibles had any indication of mental foramina ( fig. i, a). The bony accommodation of the optic chiasms was irregular and smaller than normal, and the optic canals were narrow ( fig. 2 , a). Mandibles were markedly compressed longitudinally, and medio-laterally resulting in malocclusion. The mandibular rami were incomplete and did not articulate normally with the squamous temporal bone ( fig. I , b). The intraalveolar border was extremely short and the molar teeth were misplaced and impacted. Toothbuds of 2 incisors and 2 molars were horizontal and lateral to the other teeth. All long bones were tough to cut but broke easily under moderate lateral pressure. Upon longitudinal section, the long bones had a solid &dquo; bone within bone &dquo; appearance due to lack of marrow cavities. Both the epiphyses and metaphyses contained dense endochondral bone ( fig. 2 , b). Radiographically all the vertebrae had a sandwich-like, compressed appearance. In longitudinal section, the vertebrae appeared compact, and the wedges of endochondral bone were readily discernible. ' 2° Other systems The central nervous system accommodated the osteologic defects. The cerebral hemispheres were rectangular and the cerebellum was compressed ( fig.y , a). Other internal organs were normal. C. -Histopathological changes 1 0 Skeletal system The nasal, frontal, temporal, parietal and maxilla bones were denser than normal with evidence of deposition of bone on the outer surface but no bone removal from the inner surface. Pockets of marrow and small foci of fine connective tissue were interspersed throughout these bones. Median and paramedian sections of the cranial base had irregular intersphenoidal and spheno-occipital synchondroses. From both synchondroses, apices of wedge-shaped endochondral bone extended into solid cortical bone. The endochondral bone wedges consisted of calcified matrix and osteoid, occasional islands of resting chondrocytes and a few small foci of marrow. The sparsity of the vascular supply was striking. The growth plates of the vertebral bodies were irregular due to broad spicules of hypertrophied chondrocytes. Wedges of endochondral bone extended from the growth plates and their apices joined in the approximate center. The sides of the wedges were surrounded by dense cortical bone. Evidence of bone resorption was absent. The long bones had similar microscopic features. The articular surfaces were normal. The epiphyses consisted of primary spongiosa. The epiphyseal plates were irregular due to spicules of hypertrophied chondrocytes protruding towards the metaphyses ( fig. 3 , a). Islands of the hypertrophied chondrocytes were present in metaphyses. Calcified matrix arising from the epiphyseal plates was dense and persisted throughout the endochondral bone core. Marked osteoid seams were formed around the calcified cartilegenous martrix ( fig. 3 , b). The metaphyses contained calcified cartilagenous matrix, osteoid, and osseous tissue. Some osteoclasts were observed but they appeared inactive. Few penetrating vessels were observed. Marrow spaces were limited, visible blood vessels were few and dilated and the hematopoietic tissue was sparse. Numerous areas of potential pockets of marrow cavities were replaced by a network of fine connective tissue. Cross-sections of bone in the metaphyseal and diaphyseal areas revealed narrowing of marrow spaces by cartilagenous matrix and osteoid, and by surrounding encroaching cortical bone ( fig. 3 , b). Upon transverse and longitudinal sections, the middle of the diaphyses had dense compacted cortical bone which contained a few foci of marrow and loose connective tissue. The center of the diaphyses contained a narrow core of endochondral bone. Signs of bone resorption and remodelling were lacking. Longitudinal sections of phalangeal bones had a wedge of endochondral bone, the apex of which met distally with dense cortical bone in a V-shaped line. 2° Other systems The central nervous system had petechial hemorrhages in the cerebrum and cerebellum. A few smaller blood vessels, specifically dural vessels over the cerebellum, were calcified. The parathyroid glands were small but not histologically different from parathyroids of normal neonatal calves. The liver, spleen, and lymph nodes contained numerous foci of extramedullary hematopoiesis. Other tissues were normal. III. -DISCUSSION This defect has recently been diagnosed twice in the Aberdeen-Angus breed: once in the United States (T HOMPSON , 19 66) and once in Canada (L EIPOLD et al., 1070 ). Because there are some common ancestors in the Saskatchewan (Canada) cases and the present ones, the title &dquo; hereditary osteopetrosis &dquo; is appropriate. The genetic study will be presented in a following paper. Osseous anomalies comprise a considerable part of the whole spectrum of congenital defects in cattle. They may be restricted to certain regions of the body or may be generalized. The defect described here is generalized and is readily recognizable by such gross features as brachygnathia inferior and impacted molars, and lack of bone marrow cavities. Some congenital osteologic defects affecting bovine jaws are restricted to the jaws; others are generalized. Hereditary brachygnathia inferior resulting from a disparity in length of the jaws has been described in several breeds; agnathia, a hereditary lethal, also has been noted (L AUVERGNE , zg68). However, no similar case in Aberdeen-Angus has been reviewed by L AUVERGNE (rg68). The nearest condition (No. 5 8 of the catalogue) has appeared in Milking Sho y tho y ns in the United States, New Zealand and Great Britain under the name of &dquo; impacted molars &dquo;. The bones of those cattle were neither radiographed nor examined grossly. Whether the two congenital diseases (brachygnathia inferior with malformed, impacted molars) and that described here are the same remains to be clarified. Gross and microscopic lesions described in the present paper correspond to those encountered in human congenital osteopetrosis. Although, the condition appears to be an imbalance between bone formation and bone resorption, the basic mechanism is obscure. Injecting parathormone and vitamin D failed to improve a child affected with congenital osteopetrosis (F RASER et al.,19 68).

v3-fos

2018-04-03T03:23:47.351Z

1971-03-01T00:00:00.000Z

3158117

s2

Isolation and identification of psychrophilic species of Bacillus from milk. Forty isolates from 97 raw milk samples (heated to 80 C for 10 min and stored at 4 to 7 C for 3 to 4 weeks) were sporeforming, aerobic, gram-positive or gram-variable, rod-shaped bacteria. Fifteen isolates that were identified had characteristics similar to species of Bacillus, except that they had lower growth temperature ranges, were gram-variable, and were somewhat different in sugar fermentations. Four isolates grew well within 2 weeks at 0 C, but they grew faster at 20 to 25 C. These psychrophilic sporeforming bacteria, the importance of which is discussed, are considered to be variant strains of mesophilic bacilli adapted to low temperatures. Antarctic soil. The first report of psychrophilic sporeforming bacteria in food was that of Grosskopf and Harper (Abstr., J. Dairy Sci. 52:897, 1969), who reported isolating some from milk. We have found species of Bacillus capable of growing at 7 C or less to be present in 25 to 35% of raw milk samples. This paper reports their isolation and identification. MATERUILS AND METHODS Samples. Ninety-seven raw milk samples were collected from several areas of California. The samples were refrigerated during transit to the laboratory and tested immediately. The samples (200 ml of each) were held at 80 C for 10 min in a water bath, cooled, stored at 4 to 7 C, and examined for microbiological spoilage after 3 to 4 weeks. Media. The following media (from BBL) were used: Trypticase soy agar (TSA), Trypticase soy broth (TSB), nutrient broth, nutrient agar, and AK sporulation medium. Sporulation was determined with each of the above media, with nutrient agar plus 0.05% MgSO4 and 0.05% MnSO4 and with soybean agar (17). Microscopic examination. Cell morphology, cell arrangement, cell size, motility, presence of spores, and presence of cell granules were determined by phase microscopy. Stained mounts were used for determining the Gram reaction and spore formation (18). Identification tests. Isolates were identified accord-ing to Bergey's Manual. Analyses for biochemical and microbiological characteristics were done by the procedures of Smith et al. (17). Measurement of growth. For determining ability to grow at different temperatures, the isolates were streaked on plates of TSA, and the plates were incubated at predetermined temperatures from 4 to 50 C. Before the streaking, the plates were preheated or precooled to the incubation temperature. Plates were discarded if colonies did not develop in 1 week. Isolates that grew at 4 C were tested for growth within 2 weeks at 0 C in TSB in an antifreeze-water bath thermostatically controlled at 0 i: 1 C. Experiments for determining specific growth rates were conducted in a G76 gyratory water-bath shaker (New Brunswick Scientific Co., New Brunswick, N.J.) adjusted to predetermined temperatures (5 to 35 C) and an agitation rate of 120 rev/min. A 250-ml Erlenmeyer flask containing 50 ml of TSB was inoculated with 0.2 ml of culture (grown at 10 C for 2 to 3 days in TSB), and the culture was transferred periodically (inoculum, 0.1 ml) until it reached the steadystate of growth (13,14). Growth was determined by periodically measuring optical density at 600 nm with a Beckman spectrophotometer (model DB) with 5-cm cuvettes. RESULTS About 25 % of 50 milk samples collected in the summer spoiled in 3 to 4 weeks in the refrigerator (4 to 7 C). This percentage was about 35% for 47 samples collected in the winter. Portions of the spoiled samples were plated on TSA, and the plates were incubated at 21 C for 2 to 3 days. Colonies were transferred to TSA slants, incubated at 21 C, and stored in a refrigerator. Forty cultures isolated from morphologically different colonies by the above procedure were found to be sporeforming, aerobic, gram-positive Minimal (except 0 C) and maximal growth temperatures were determined on Trypticase soy agar. Tests at 0 C were in Trypticase soy broth in an antifreeze-water bath at 0 1 C. bSpore formation was tested in a variety of media. c Lowest temperature tested. or gram-variable, rod-shaped bacteria. They grew in refrigerated milk and produced off flavors, including rancid, fruity, and sour. Fifteen isolates that were morphologically different and indicated by preliminary experiments to have low minimal growth temperatures were selected for further study and identification. These isolates could be grouped into two different groups on the basis of minimal growth temperatures (Table 1). Those in group A were able to grow at temperatures as low as 0 C, whereas those in group B had minimal growth temperatures of 5 to 7 C. More recent experiments in broth have indicated a doubling time of about 30 hr at 0 C for the isolates of group A. Although the isolates of group A grew at 0 C within 2 weeks, they did not sporulate well at any temperature tested (3 to 25 C). In contrast, the isolates of group B, which had higher minimal growth temperatures, sporulated well at each temperature tested. The growth kinetics of one isolate (RH3) of group A are shown in Fig. 1. Growth was slow at 5 C, in comparison to higher temperatures, and growth did not occur at 35 C. Specific growth rates and doubling times at different temperatures for this isolate are given in Table 2. The doubling times at 5 C for the other three isolates of group A (TS3, TS4, and RH22) were 6.75, 6.75, and 9.8 hr, respectively. The biochemical and microbiological characteristics of those isolates of group A did not match completely any of the species of Bacillus described in Bergey's Manual. All four of these isolates were gram-variable and had growth temperature ranges considerably below those specified for species of Bacillus. One isolate, RH22, was considered to be a variant of B. subtilis since the few sporangia observed were not definitely swollen, the protoplasm of young cells grown on glucose agar were not vacuolated, growth on glucose or soybean-agar was good, and the organism hydrolyzed starch, produced nitrites from nitrates, liquefied gelatin rapidly, and grew in broth containing 4% NaCl. However, with ammonium salts as the source of nitrogen, this organism failed to ferment manitol, sucrose, lactose, or glycerol, and it did not grow in broth containing 7% NaCl. The characteristics of another isolate, RH3, indicated that it was a variant strain of B. circulans, though there was good growth on soybean-agar and acid was produced from each of the above four carbon sources or arabinose or xylose. Two isolates, TS3 and TS4, resembled B. coagulans in biological and microbiological tests. They produced acid from glucose and from each of the above six carbon sources. DISCUSSION Psychrophilic bacteria have been defined by Ingraham and Stokes (10) as bacteria that grow well at 0 C within 2 weeks and by Witter (22), based partly on a standard method for determining psychrophilic bacteria (1), as bacteria that grow at a relatively rapid rate at 7.2 C, i.e., that lorm visible colonies on plates at this temperature in 10 days. Foster et al. (6) defines them as bacteria that grow relatively rapidly at 1.7 to 10 C, the temperature normally used in commercial holding and distribution channels. Witter (22) suggested that a generation time of about 15 hr or less at 7.2 C would be a reasonable requirement for organisms that are to be called psychrophiles, and Ingraham and Stokes (10) suggested a generation time of 48 hr or less at 0 C. Two of the psychrophilic sporeformers isolated from mud and studied by Larkin and Stokes (11) had generation times of 8.5 and 11.5 hr at 5 C, and Ingraham (9) reported generation times of 7 to 10 hr at 4 C for psychrophilic pseudomonads. Some investigators prefer to describe such bacteria as psychrotrophic rather than psychrophilic (12,21), and there seems to be merit in using some term that indicates that they merely are able to grow at low temperatures rather than that they are cold-loving. In any event, all of the isolates we studied grew under refrigeration at 7 C or less, but they grew faster at higher temperatures (25 to 35 C). Those in group A (Table 1) were able to grow within 2 weeks at 0 C and had generation times of about 30 hr at 0 C and 6.5 to 9.8 hr at 5 C. Of the isolates we studied, at least these four fit each of the above definitions proposed for psychrophilic bacteria. The important difference between the bacteria we isolated and known characteristics of mesophilic bacilli is that those we isolated were able to grow at lower temperatures. Mesophilic bacilli normally have not been found to grow below about 8 C, and 10 C often has been found to be their minimal growth temperature (5,10,19,22). We suspect that the organisms we isolated are variants of mesophilic bacilli adapted to lower growth temperature ranges (2,7,16). Simultaneously with this adaptation, there apparently developed a tendency toward poor sporulation that was particularly evident in the isolates of group A. We did not attempt to reverse this adaptation, but Grosskopf and Harper (Abstr., J. Dairy Sci 52:897, 1969) reported that the psychrophilic sporeforming bacteria they isolated could be adapted to higher growth temperature ranges and that they then were unable to grow at low temperatures. Spoilage of pasteurized milk and milk products often results from the growth of heat-sensitive, gram-negative, nonsporeforming bacteria that enter products after pasteurization. In regard to this, failure of the isolates of group A to form spores readily on laboratory media and the fact that they were gram-variable are important. Possibly some instances have occurred in which gram-variable bacteria such as those we isolated have been mistaken for heat-sensitive species, e.g., Pseudomonas, Alcaligenes, or Aerobacter. The doubling times of the four isolates of group A at low temperatures are not greatly different from those of some species ofPseudomonas known to be important in the spoilage of foods (4,8,9,20,22), but the isolates of group B grew more slowly. In regard to this, it is important that the organisms of group A were found in only four of 97 samples of raw milk. Obviously, it would be a mistake to relax efforts to prevent nonsporeforming psychrophilic bacteria from getting into milk and dairy products after pasteurization. However, as attempts are made to extend the shelf-life of fluid dairy products, psychrophilic sporeforming bacilli will become a greater potential problem. This is also important in regard to the use of dairy products in other foods, the development of aseptic filling, and the probability of a shift toward "sterilization" of milk and fluid milk products.

v3-fos

2020-12-10T09:04:11.471Z

1971-08-01T00:00:00.000Z

237229543

s2

Isolation of Psychrophilic Bacteriophage-Host Systems from Refrigerated Food Products Thirty-eight bacteriophage-host systems were isolated from 22 of 45 refrigerated food products examined under psychrophilic conditions. Isolates were obtained from ground beef, pork sausage, chicken, raw skim milk, and oysters, whereas no isolations were made from liquid egg whites and processed meat products. Thirty of the 38 psychrophilic bacterial hosts were gram-negative rods, and 27 of these were classified within the genus Pseudomonas; three were members of the family Enterobacteriaceae. The remaining eight were gram-positive cocci, which were tentatively classified as Leuconostoc. Plate counts of psychrophilic bacteria were greater than 2.2 × 105/ml (g) in all but one sample which contained phage, whereas phage titers ranged from less than 100 to 6.3 × 106 plaque-forming units/ml (g). Phage isolates showed limited host ranges usually attacking only those hosts upon which they were isolated. Of eight phages tested against 13 cultures of known identity, one showed lytic action, and this was against strains of P. fragi. Thirty-eight bacteriophage-host systems were isolated from 22 of 45 refrigerated food products examined under psychrophilic conditions. Isolates were obtained from ground beef, pork sausage, chicken, raw skim milk, and oysters, whereas no isolations were made from liquid egg whites and processed meat products. Thirty of the 38 psychrophilic bacterial hosts were gram-negative rods, and 27 of these were classified within the genus Pseudomonas; three were members of the family Enterobacteriaceae. The remaining eight were gram-positive cocci, which were tentatively classified as Leuconostoc. Plate counts of psychrophilic bacteria were greater than 2.2 X 105/ml (g) in all but one sample which contained phage, whereas phage titers ranged from less than 100 to 6.3 x 106 plaque-forming units/ml (g). Phage isolates showed limited host ranges usually attacking only those hosts upon which they were isolated. Of eight phages tested against 13 cultures of known identity, one showed lytic action, and this was against strains of P. fragi. Bacteriophages specific for psychrophilic species of the genus Pseudomonas have been reported (5,13,14). Isolations of these phages from food products have been made by Billing (2) and Delisle and Levin (5). Reports that streptococcal phage cause failure of acid development in the manufacture of cultured dairy products were first introduced in the mid-thirties. No literature reports were observed, however, regarding the occurrence of phages in association with their food spoilage hosts within refrigerated processed foods. The purpose of this research was to determine the prevalence and concentrations of bacteriophage present within refrigerated foods. Isolated bacterial hosts were characterized as to their biochemical activities and were taxonomically classified. MATERIALS AND METHODS Bacteriophage enumeration. The agar layer method as described by Adams (1) was utilized. Plates were incubated at 20 C for 15 to 20 hr, after which plates showing 30 to 500 plaque-forming units (PFU) were counted with the aid of a Quebec colony counter. Isolations of phage-host systems. All samples of ground beef, pork sausage, chicken parts, oysters, and processed meat products were purchased at local markets. Eleven-gram portions were blended for 2 min ' Contribution from the Missouri Agricultural Experiment Station. Journal series no. 7081. 2 Present address: General Foods Corp., Battle Creek, Mich. in 99 ml of phosphate-buffered distilled water, and the homogenate was plated with Standard Methods agar (19). Fresh, raw skim milk and egg whites were obtained from the Department of Food Science and Nutrition. Samples (11 ml) were diluted in 99-ml water blanks and then plated. Plates from all samples were counted after incubation at 10 C for 5 days (psychrophilic bacteria count/ml). Twenty milliliters of the blended mixtures was centrifuged for 15 min at 15,000 X g. Membrane filtration (0.45-jAm pore size) of the supernatant fluid followed. Skim milk samples were sterilized by adding 0.5 ml of chloroform to 9.5 ml of skim milk. Egg white, diluted 1:100, was passed consecutively through membrane filters of 3.0-, 1.2-, and 0.45-,Am pore diameter. These preparations were labeled "food sample filtrates" and were used as sources of phages. Representative isolated colonies were picked from plates on which psychrophilic bacteria of the foods had been enumerated. Colonies were chosen which differed in appearance under ultraviolet or incandescent light, or both. These were inoculated individually into tubes ofTrypticase soy broth (TSB) and incubated at 20 C for 20 to 24 hr. One milliliter of each culture was transferred to a single tube containing 9 ml of TSB. To this was added 1 ml of the food sample filtrate from which the culture was isolated. After incubation for 10 to 12 hr, the presence or absence of phage, active against each isolate, was determined by the double-layer method of plating. Isolates which were lysed (discrete plaques) were twice restreaked to insure purity and then transferred to Trypticase soy agar (TSA) slants on which they were stored. All isolates of the phage were thrice purified by picking single plaques with a toothpick into soft agar 220 containing the host. Dilutions of this suspension were plated to obtain isolated plaques. Phage titers in the food sample filtrates were determined by using, as indicators, strains of the isolates from the respective foods. Identification of host isolates. All bacterial isolates were separated into two groups on the basis of the Gram stain reaction. Those which were gram-negative were further classified according to the results of the following tests: (i) colony morphology and pigmentation on TSA plates, (ii) motility by hanging drop mounts (17), (iii) type of flagellation by using the Leifson flagella stain (17), (iv) blood hemolysis on bovine blood-agar plates, (v) hydrolysis of casein on casein plates (3), (vi) hydrolysis of fat on spirit blue plates (6), (vii) production of fluorescent pigments on Pseudomonas-agar F (7), (viii) growth at various temperatures and modification of litmus milk in litmus milk tubes (8), (ix) breakdown of glucose in Hugh and Leifson stabs (9), (x) deamination of arginine in arginine broth tubes (8), (xi) type and extent of liquefication of gelatin in gelatin-agar stabs (15), (xii) reduction of nitrates in nitrate-peptone broth (15), (xiii) extent of acid production and the production of acetyl methyl carbinol in MR-VP broth (15), (xiv) presence of cytochrome oxidase by Kovac's oxidase test (10). Most of these tests were also used in the classification of the gram-positive cocci. Other tests used to identify this group of organisms were as follows: (i) catalase production (8), (ii) detection of cytochrome system by the pseudocatalase test (4), (iii) growth in the presence of 6.5% NaCl, 0.1% methylene blue, pH of 9.6, at 10 and 45 C (8). With the use of Bergey's Manual of Determinative Bacteriology and Microbiological Methods (3), these isolates were classified. Those organisms characterized as Pseudomonas were further subdivided into groups by the scheme of Shewan et al. (16). Host range of phage isolates. The routine test dilution (RTD) was determined by the procedure of Adams (1) for all phages used in host range studies. Seven phage isolates were tested against 13 Pseudomonas cultures of known identity: five strains of P. fragi, three strains of P. fluorescens, two strains of P. aeruginosa, and one strain each of P. ambigua and P. putrefaciens. Dried TSA plates (10 cm diameter), seeded individually with these cultures, were spotted with each of the eight phages at their routine test dilutions. Plates were incubated at 20 C for 24 hr after which the susceptibility of the cultures to lysis was recorded. All 38 phage isolates were tested for host range against each of the 38 host isolates. Plates (15 cm diameter) containing dried TSA were inoculated with 2 ml of a log-phase culture. The excess was poured off, and plates were allowed to dry at room temperature. A drop of the RTD of each phage, dispensed from a tuberculin syringe equipped with a 27-gauge needle, was placed on a designated area (1 through 38) of each dried, seeded plate. After incubation for 24 hr at 20 C, the plates were examined for areas of near confluent lysis. These experiments were repeated twice. RESULTS AND DISCUSSION Isolation of phage-host systems. Forty-five food samples yielded 216 bacterial isolates when plated at 10 C. As many as nine isolates were subcultured from some samples. More than one isolate of the same strain may have been picked due to the inability to differentiate organisms on the basis of colony morphology and fluorescence, the two criteria used. Thirty-eight of the 216 isolates were lysed by phages present within their respective food sample filtrates. Twenty-eight of these were taxonomically different, and the remaining 10 formed five pairs which were taxonomically indistinguishable by the criteria used. Both members of each pair were from the same food sample. However, crossreactions observed in the host range studies, discussed later, indicated that three of these pairs were definitely different, that one pair was probably different, and that one pair was the same. Phages and their bacterial hosts were isolated from approximately 50% of the food samples (Table 1). This percentage, however, was likely biased by the unequal number of samples of each product examined. No phages were obtained from either egg white or luncheon meats, but all samples contained less than 3,000 psychrophilic bacteria per g. Phages were isolated only from products with high bacterial counts, and, except for one sample of pork sausage, all positive samples had psychrophilic bacteria counts in excess of 2.2 X 105/ml (g). Phage titers in the food sample filtrates (Table 1) ranged from undetectable levels at a 1:100 dilution up to 6.3 X 106 PFU/ml. Evidently the added step in the isolation procedure, which involved enriching of cultures with the food sample filtrate, increased phage numbers in some samples so that after incubation a detectable concentration was present. Only one phage for each host was isolated from each filtrate, even though in some instances different plaque morphologies suggested that more than one phage was present. Thus, when the food sample filtrates were titered, the counts may have been due to more than one specific phage. Phage purification (plaque picking) was performed to eliminate contaminating phages which might have been present; this produced pure phage suspensions. Identification of host isolates. Thirty-eight bacterial isolates were characterized. Thirty of these were gram-negative rods, whereas the other eight were gram-positive cocci. Twenty-seven of the former were classified as Pseudomonas by using the determinative scheme of Shewan et al. (16). All were oxidative. The six strains which produced diffusible pigment on Pseudomonas agar F were placed in group I, and the remaining 21 fell in group II. The three other strains of gramnegative rods were classified into the family Enterobacteriaceae without further differentiation as to genus. Comparison of the two groups of Pseudomonas showed definite differences. Group I was much more biochemically active with all isolates hydrolyzing fat and specific proteins, whereas a lower percentage of the isolates within group II showed this ability. Their optimum temperature was 21 C or lower; thus, these isolates showed truly psychrophilic, rather than mesophilic, temperature ranges of growth. However, all of these organisms were isolated from plates incubated at 10 C. It was somewhat surprising that 27 of these 30 isolates were members of the genus Pseudomonas even though this genus constitutes a large percentage of the psychrophilic isolates from refrigerated foods (21). Phages for other genera apparently were not present. All eight isolates of gram-positive cocci were shown to be very similar in their biochemical activities. They occurred in chains, dividing in one plane. Their colonies on blood-agar were typical of streptococci in appearance but showed no hemolysis. They were slightly catalase positive (8), but the test of Deibel and Evans (4) indicated that pseudocatalase produced the reaction. Because acid production was not observed in litmus milk and the cultures were nonhemolytic, these isolates were not considered to be members of the genera Aerococcus (20) or Streptococcus. Their growth on blood-agar under aerobic conditions was not typical of Pediococcus, nor was their chain formation. They fermented glucose and grew in 6.5cr NaCl, in broth (pH 9.0), and at 10 but not at 45 C. Therefore, they were tentatively classified as an unknown species of Leuconostoc. Host range studies. Seven of the phages isolated from the food samples (phages psl, wy, i, d, f, y2, and gb3) were tested for ability to lyse 13 cultures of known identity. The purpose was to gather additional information as to the identity of the hosts found in the food samples. Only phage i lysed any of these bacteria, and it lysed strains 7, P27, and KI of P. fragi. The hosts of three other phages Psl, wy, and y2, were identified as P. fragi, but these phages failed to lyse any of the P. fragi cultures. Phage strain f, from P. fluorescens, failed to lyse any of three known strains of the same species. Some Pseudomnonas and Xanthomonas phages have been shown to react across species and even generic lines (15,18), whereas other phages of Pseudomonas were species to strain specific (11,12). Eleven of our Pseudomonas cultures, representing four of the five species, are common food contaminants. Therefore, it was not unlikely that these 11 strains would react with the isolated phages. The phages tested may well have been strain specific, and, since their homolgous strains were not present, lysis was not observed. The 30 gram-negative hosts and their respective phages were cross-reacted to determine host ranges ( Table 2). All phages produced confluent lysis on their homologous hosts, but few attacked any other bacterial strain. The greatest number of phages which showed cross-reactivity, 3, was found within the B series, all isolated from a single, ground beef sample. Based on biochemical studies, only hosts B2b and B5 appeared alike, and these were believed to be different strains as shown by phage susceptibility (phage typing). There was little cross-reactivity between phages and bacteria isolated from different samples, although some phages showed the ability to lyse isolates from other samples, viz. k8 on B4; b2a on I; i on B2a; u3 on B2a, B2b, and V3; and v3 on U3. Based on taxonomic data, hosts B2b and I appeared to be the same species (from different samples); this was further substantiated by the host range studies. These tests verified other taxonomic data, showing that practically all the bacterial isolates were at least different strains. Phage b2a lysed cultures B2a, B2b, B5, and I. These isolates were all classified within Group II of Shewan et al. (17). Tentative taxonomic classification of three of these four isolates as P. fragi had been made independently. Nine other gramnegative isolates were also classified as P. fragi but were not lysed by phage b2a. Phage b4, besides lysing its homologous host B4, showed confluent lysis on culture K8. These hosts were found to belong to Group I. No phage was shown to cross-react between groups. Of the phages to the gram-positive cocci, only phage d4 cross-reacted, lysing host M2. Host range studies were performed before the bacterial hosts were characterized; therefore, all isolates of gram-positive cocci were tested with phages specific for gram-negative hosts, and vice versa. As would be expected, no cross-reactions were observed between these two groups. In conclusion, it is clear that phage-host systems exist in refrigerated food products. It appears that both phage and host are present as food contaminants, and that these phages enter either as prophages within lysogenic hosts or as free, virulent phages. At present, work is in progress to determine what effect these phages have upon their food spoilage hosts within foods. These phage-host systems may significantly influence the shelf life of refrigerated food products.

v3-fos

2020-12-10T09:04:17.478Z

1971-08-01T00:00:00.000Z

237234160

s2

Analysis of Staphylococcal Enterotoxin B by the Polyacrylamide Electrophoresis Technique The electrophoretic mobility of enterotoxin B was investigated through the use of the disc electrophoresis technique. Ideal patterns were developed with a 7.5% acrylamide gel system (pH 4.3). The toxin can be separated and identified from other complex proteins such as serum or suspect samples of foods by this technique. The technique can be used as an assay method for the toxin as well as to elucidate physical changes in the toxin due to temperature. The method should not be considered exclusive for enterotoxin B. provide a means of separating molecules, thereby identifying and classifying them. If one molecule is so peculiar or specific that it can be differentiated from all others, it can also be used to identify the organism from which it comes. The present study deals with a very specific compound elaborated by certain microorganisms, staphylococcal enterotoxin B. Until recently, very little electrophoretic work has been done with enterotoxin B because the Tiselius (20) moving boundary method is cumbersome; most zone electrophoretic methods were found to be impractical, and known or "standard" enterotoxin was not available. For optimum results, consideration must be given to the isoelectric point and molecular weight of the material being studied as well as to all conditions under which the run is to be made. Staphylococcal enterotoxin is no exception to these considerations. Hibnick and Bergdoll (10) using the Tiselius technique reported the isoelectric point of enterotoxin at pH 8.6 and a molecular weight of 24,000 i 3,000. More recently Wagman et al. (21) reported a molecular weight of 35,300. Bergdoll (2) reported unsuccessful attempts to fractionate enterotoxin with paper electrophoresis. Thatcher et al. (19) also used paper as a support medium and pointed out the ability of filter paper to absorb enterotoxin and their inability to elute enterotoxin from it. They also investigated the use of "gold-beater's skin," parchment, and nylon fabric and found them impractical. Therefore, most workers, Bergdoll and co-workers (2, 3), Casman and Bennett (4), Baird-Parker and Joseph (1), and Dalidowicz et al. (6), have used starch as a support medium. With the use of acrylamide-gel as a support medium, as introduced by Raymond and Weintraub (14), and the technique developed by Davis (7) and Ornstein (11), an ideal system became available for zone electrophoresis. Frea et al. (9) used disc electrophoresis and the method of Davis (7) in their studies on purification of staphylococcal enterotoxin B, but consideration was not given to basic parameters for optimum results. Their patterns and results are not ideal and should be considered only a first approach inasmuch as it would be difficult to obtain further information by their method. In the present study, consideration is given to optimum conditions for the electrophoretic analysis of staphylococcal enterotoxin, and these conditions are presented. MATERIALS AND METHODS Partially and highly purified staphylococcal enterotoxin B was kindly supplied by E. J. Schantz, Fort Detrick, and prepared by the method of Schantz et al. (17). The lyophilized enterotoxin was dissolved in sterile, distilled water to give a concentration of 5 mg/ml. Portions of this were added to the sample gel. Disc electrophoresis procedures and methods were those of Davis (7) and were based on the theory of Ornstein (11). Amounts of enterotoxin, as indicated in the figures, were added to the sample gel. On the basis of isoelectric point and molecular weight, the gel buffer system of Reisfeld et al. (16) for basic proteins was used for optimum results and is similar to the method of Denny et al. (8) for enterotoxin A. The method was first referred to by Schantz et al. (17) as work done by this author but not reported in detail at that time. A constant current of 5 ma per tube was applied for 30 min at room temperature, unless other-214 ELECTROPHORESIS OF ENTEROTOXIN B wise noted. Gels were removed from tubes under cold water and stained with 0.5%o Amido Schwartz in 7% acetic acid for at least 1 hr; excess stain was removed electrophoretically in an alcohol-acetic acid bath consisting of 500 ml of methanol, 500 ml of water, and 100 ml of acetic acid. A fixed and stained disc gel pattern was used as template for cutting out sections of unstained gel for subsequent elution of toxin for testing biological activity by injecting monkeys on a micrograms per kilogram basis. Elution was carried out overnight in 0.5 ml of saline at 5 C, and microgram amounts were recovered. Larger amounts (milligrams) of staphylococcal enterotoxin were recovered by using slabs of acrylamide by the method of Raymond et al. (13) and by using the elution-convection method of Raymond and Jordan (12). One milliliter of a 5 mg/ml solution of enterotoxin was layered in the premolded slot of the acrylamide slab. Electrophoresis was carried out in a formate buffer (pH 3.5) for 6 hr with 120 ma across the gel. Running tap water was used for cooling. Elution was carried out with the same buffer and coolant and a current of 112 ma for 5 hr. A 2-ml amount of a 5 mg/ml solution of highly purified enterotoxin B was heated in a boiling water bath for 5 and 10 min. Samples (20,liter) were taken for electrophoretic analysis. Twenty-microliter samples of the highly purified enterotoxin were also heated for 5 min at various temperatures from 30 to 90 C and again used for electrophoretic analysis. Figure 1 represents a variety of different preparations of staphylococcal enterotoxin B run over a period of several years. Since individual tubes may have been run at different times under slightly different conditions of temperature, time, and buffer ionic strength, the known toxin band may be at different distances from the origin. When working with unknowns, a known toxin was always run for comparison of bands. As can be seen, the number of bands varies from one to five, with the exception of 13A, which contains no bands and thus no protein. Of the toxins tested and illustrated in Fig. 1, it would appear that D2, 21, and 23 are homogenous and contain no impurities and are, therefore, the most highly purified material tested. However, when higher concentrations of enterotoxin are used (including D2, 21, and 23), additional bands may be seen (Fig. 2). Sample SC (Fig. 1) contains a large amount of toxin but also many other bands. Other samples, as can be seen, contain various amounts of toxins and impurities or polymers of the toxin. RESULTS AND DISCUSSION To prove that the heavy band in the figures assumed to be enterotoxin was in fact enterotoxin, an electrophoretic run was made with the method stated above except that the time was increased to 55 min to achieve greater separation between bands. One gel was stained and destained as rapidly as possible and then used as a template for cutting unstained gels. These small selected areas of cut gels were elu*td, and the eluted material was injected intravendis,W into monkeys at a dose of 2.2 to 3.3 /.ig/kg. "Five of six monkeys gave positive responses. There were no deaths and no response in controls injected with eluates from control gels. Sufficient material from the lighter or minor bands could not be recovered from small disc gels. Thus, the question still remained as to whether the other bands were impurities or polymers of the enterotoxin. To test further the biological activity of the major and more especially minor bands, the elution-convection method of Raymond and Jordan (12) was used with a larger quantity of enterotoxin. This is more of a preparative method in contrast to the analytical disc electrophoresis method. Sufficient material was obtained from one of the minor bands as well as of the heavy band to allow bioassay in monkeys. Three at the 125-,ug level. It is evident from these results and the level of toxin used by Frea et al. (9) that they were not using optimum conditions for their electrophoretic procedure. When sharp or ideal patterns are obtained, as in Fig. 2, it is possible to use these patterns as a standard for comparison with unknown quantities for assay purposes. Electrophoretic results can be obscured and misleading unless optimum conditions are used. Figure 3 further illustrates this point. In this experiment, tube 1 was run under optimum conditions given above, but tube 2 was run by the methods of Frea et al. (9), and the results were similar to theirs. It should also be noted that they failed to mention the band at the origin, as well as the faint band near the bottom of the tube. There are other diffuse bands present in their figure and, because of the conditions under which their run was made, none of the bands is sharp or ideal. These diffuse bands are important for they can contain biologically active material and can be better defined and isolated if optimum conditions are used. Their selection of the basic gel-buffer FG. 2. Disc electrophoretic patterns from a single batch of staphylococcal enterotoxin B. Microgram amounts added to each gel are indicated in figure. Anode and origin are to the left. positive responses from the major band, and two of three monkeys gave positive responses at the same dose level from the minor band. No deaths occurred in any monkeys. It is thus apparent that both the heavily and lightly stained bands contain biological activity even though they have different electrophoretic mobilities. Additional work should be done at higher dose levels to determine whether different biological activity is associated with the different bands alone or in combination. Since only emesis has been recorded in these few experiments with no deaths, the location of the lethal factor or cause of death in some cases as seen by other workers, Silverman et al. (18), has not been determined. The method can therefore be used to test for the presence or absence of toxin as well as for its purity and, hopefully, for a better understanding of the biological activity of the various factors of different electrophoretic mobilities. In most cases, the appearance of impurities or polymers or, in any case, a nonhomogenous elecphoretic pattern depends on sample size. Thus, as material is diluted, impurities disappear. This is shown in Fig. 2. If a sample of 4 to 5 ,ug is used, only one band is seen. At the 10to 20-;Lg level, (7), Ornstein (11), and Gerloff (M.S. Thesis, Univ. of Wisconsin, 1963) when the isoelectric point of the toxin is 8.6 is a poor one because these two figures are too close for practical purposes. If the pH of the buffer system is not sufficiently different from the isoelectric point of the material being studied, either migration will not occur or it may not proceed in the desired direction. It is only possible to isolate and elute fractions when extremely sharp or discrete bands are present. Diffuse bands lead to overlapping of factors as well as reduction in concentration of material needed, and they are not improved with time since diffusion increases with time, especially at room temperature. Frea et al. Figure 4 shows how bands became more diffuse with longer electrophoretic runs and, in fact, can even be lost. The faint band one third down in the first tube becomes quite diffuse in the second tube and lost in successive tubes. With proper conditions, it is possible to use the technique to further advantage. It was noted when enterotoxin was heated to boiling temperature the solution became turbid, and samples gave no bands after electrophoresis (Fig. 5). It can also be seen that the toxin band is partially changed after 5min at 70 C and completely gone after 5 min at 80 C. This is also probably due to the toxin coming out of solution and forming a precipitate. Only overall changes as determined by electrophoresis were determined. The changes in biological activities have not been determined but should be. It would be of interest to determine whether biological activity is lost when staining quality of the protein is also lost. It is not believed that the toxin has been destroyed after 5 min at 80 C, since Read and Bradshaw (15) found that inactivation took place with longer exposure to higher temperature than used here. However, since there are changes in the electrophoretic bands at lower temperatures (Fig. 5), the method can be used to detect changes not previously considered. The technique can be used not only to follow purification of the toxin, but also to study inactivation or degradation such as by heat when coupled with assay in monkeys. One of the greater practical problems in research on staphylococcal enterotoxin B has been the separation and identification of the toxin in the presence of other mixtures of proteins such as foods. To show the ease with which enterotoxin can be separated from protein, 5 mg of toxin per ml was mixed with 1 ml of chicken and 1 ml of beef serum, respectively, and 0.05 ml of this ma- tures and that polyacrylamide disc electrophoresis can be used to advantage in the study of this toxin. Although only type B enterotoxin was used 955 in this study, the principles involved indicate that the procedures and methods will hold for other 953 toxin types and has been used by Denny et al. (8) in their studies on enterotoxin A. terial was added to sample gels and electrophoretic runs were made (Fig. 6). It should be noted that with the basic protein-gel buffer system of Reisfeld et al. (16), which is optimum for enterotoxin, serum fails to migrate into its usual 25 to 30 components and remains confined in an area close to the spacer gel, whereas the enterotoxin migrates through and beyond it. Preliminary studies were undertaken to show the usefulness of the technique in identifying enterotoxin in suspect food samples. A field sample of potato salad from a food poisoning episode in Indiana was tested by placing small amounts of the salad on the spacer gel and covering the sample with sample gel. The results indicate that several proteins were present because several bands were present. One very faint band was located in the same position as the major enterotoxin band when compared with a known enterotoxin control tube. As has been pointed out previously (Fig. 2), 1 ,ug of enterotoxin in approximately 20 ,uliters is about the lower limit of the technique. Thus, one problem in working with the field sample is concentration of the enterotoxin to a level that would be detectable in the small volume required for the test. This is not peculiar to the electrophoretic technique but applies to other techniques, as pointed out by Casman and Bennett (5), and can be overcome through their various extraction and concentration procedures. 952 It has been shown that, by proper selection of all conditions and methods, enterotoxin can be easily separated from complicated protein mix-

v3-fos

2018-04-03T02:52:03.381Z

1971-01-01T00:00:00.000Z

21901970

s2

Identification of a pea component stimulatory for heat-stressed putrefactive anaerobe 59-123 spores. Pea extract contains a factor which improves recovery counts of heat-stressed putrefactive anaerobe spores in a complex medium up to threefold. The factor is heat-stable and nondialyzable. Most of the active principle is found in the precipitate which forms during storage of pea extract at 4 C. The precipitate disperses upon heating, is high in starch content, and retains activity after extraction with organic solvents and water. Treatment of pea extract with alpha-amylase results in complete destruction of the active principle. These observations indicate that starch is the factor in pea extract responsible for increased recovery counts of heat-stressed putrefactive anaerobe spores. Pea extract contains a factor which improves recovery counts of heat-stressed putrefactive anaerobe spores in a complex medium up to threefold. The factor is heat-stable and nondialyzable. Most of the active principle is found in the precipitate which forms during storage of pea extract at 4 C. The precipitate disperses upon heating, is high in starch content, and retains activity after extraction with organic solvents and water. Treatment of pea extract with a-amylase results in complete destruction of the active principle. These observations indicate that starch is the factor in pea extract responsible for increased recovery counts of heat-stressed putrefactive anaerobe spores. In 1937, Curran and Evans (2) reported that spores of Bacillus species surviving a damaging heat treatment are more exacting in their nutritional requirements for germination and outgrowth than before treatment. This observation has since been extended to include spores of Clostridium botulinum (1,4,9,10,13) and putrefactive anaerobe (PA) 3679 (5,(11)(12)(13). Thus, when determining the thermal resistance of spores, careful selection of a recovery medium is important if underestimates of survivor numbers are to be avoided. In studies with anaerobic sporeformers, which are by nature quite fastidious, many substances have been used to enrich recovery media. Popular among these additives are peas and pea extract (1,3,5,12,13). Although the enrichment value of peas has been well established the active component has not been identified. Therefore, a study of the factor from peas stimulatory for heat-stressed PA spores was undertaken with the hope that pea extract might be replaced in recovery media by a commercially available chemical. In addition, a knowledge of the nature of the stimulant may lead to a better understanding of the metabolism and early stages of heat inactivation of anaerobic sporeformers. MATERIALS AND METHODS Test organism. A suspension of PA 59-123 spores was obtained from the National Canners Association, Washington, D.C., and was stored at 4 C. Vegetative cells present in the spore suspension were inactivated by adding an equal volume of absolute ethanol and incubating for 1 hr at 25 C (6). Spores were recovered by centrifugation. Serial dilutions of stock suspensions were made in sterile water so that 0.2 ml of a heatstressed suspension yielded between 30 and 100 colonies on unenriched recovery medium. Heat stressing was effected by immersing 10-ml quantities of the spore suspension, contained in a screw-cap tube (20 by 150 mm), in boiling water for 8 min followed by rapid cooling in an ice bath. Preparation and assay of pea extract. Pea extract was prepared as described by Andersen (1), placed in either glass dilution bottles or dialysis tubing, and autoclaved for 15 min at 121 C. The response of heatstressed spores to various concentrations of the test material was measured in PA 3679 Agar. The composition of PA 3679 Agar was 10 g of tryptone (Difco), 5 g of dextrose (Fisher Scientific Co.), 1 g of yeast extract (Difco), 3 g of beef extract (Difco), 0.5 g of sodium thioglycollate (BBL), 1.25 g of K2HPO4 (Fisher Scientific Co.), 8.5 g of lonagar (Colab) and 1 liter of deionized water. This medium is T Best Agar, described by Wheaton and Pratt (12), without the soluble starch. For assay purposes, each plate contained 10 ml of medium, spores, and sterile deionized water or test substances, or both, in a total volume of 12.2 ml. Plates were incubated at 37 C for 60 hr in Case-Anaero Jars (Case Laboratories, Inc., Chicago, Ill.) containing an atmosphere of 90% nitrogen and 10% carbon dioxide (Matheson Co., Inc.). Before incubation, jars were successively (three times) exhausted to 26 inches (66 cm) of vacuum and flushed with the gas mixture to a slight positive pressure. To determine the relative stimulatory activities of pea extract and its fractions, all preparations were adjusted with sterile water to the volume of the original pea extract. The degree of stimulation was on March 21, 2020 by guest http://aem.asm.org/ Downloaded from STIMULATORY PEA COMPONENT measured as the fold increase in recovery count caused by 1 ml, or fractions thereof, of the test substance over the count obtained with the same heat-stressed spore suspension on unenriched PA 3679 Agar. Fractionation of pea extract. Dialysis sacs (Fisher Scientific Co.) containiing 40 ml of pea extract were suspended in open flasks and autoclaved for 15 min at 121 C. Dialysate was obtained from pea extract by one of two procedures. Agitated dialysis was carried out in 2-liter quantities of sterile deionized water for 15 hr at 4 C. Sacs were transferred to a second 2-liter quantity of sterile deionized water and dialyzed for an additional 6 hr. Dialysate was concentrated to 40 ml by flash evaporation, sterilized by membrane filtration (MilliporeCorp., Bedford, Mass.), and assayed for stimulatory activity. The endofraction was recovered aseptically and likewise assayed. Alternatively, dialysate was obtained by vacuum dialysis. A white, flocculant precipitate developed in pea extract during storage at 4 C. The precipitate was removed from 20 ml of extract by centrifugation at 20,000 X g for 10 min and washed twice at 4 C with 20 ml of each of the following: acetone, anhydrous ether, 85% methanol, 95% ethanol, and water. The extracted precipitate was suspended in 20 ml of sterile water, heated for dispersion, and assayed for stimulatory activity and starch content. Starch determination. Starch concentrations were determined by a modification of the method described by McCready and Hassid (7). For standard curve construction, known amounts of com starch (Stein, Hall & Co., Inc., New York, N.Y.) were permitted to hydrate for 10 min in 1 ml of water. A 5-ml quantity of 1 N NaOH was added, and the mixture was incubated at 55 C for 5 min. Excess NaOH was neutralized with 0.5 N HCI. The volume was diluted to 100 ml with boiling water, and the temperature was maintained at 90 to 100 C for 5 min. A 3-ml sample was added to 96 ml of water and developed with 1 ml of iodine reagent (0.2% iodine, 2% potassium iodide). Absorbance at 650 nm was measured by using a Beckman DB spectrophotometer. Starch hydrolysis. Starch in fresh pea extract was hydrolyzed by the addition of 40 ,ug of a-amylase (Nutritional Biochemicals Corp., Cleveland, Ohio) per ml of extract. The reaction mixture was incubated at 25 C for 30 min. Disappearance of starch was followed by testing with iodine reagent. The reaction was terminated by autoclaving at 121 C for 15 min. Boiled enzyme added to pea extract and untreated pea extract served as controls. ca-Amylase-treated and untreated pea extracts were assayed for the ability to increase recovery counts of heat-stressed PA 59-123 spores on PA 3679 Agar. spore suspensions on PA 3679 Agar by approximately 75% (Fig 1). Inclusion of pea extract in the medium increased the count of heat-stressed spores by as much as threefold. Concentrations of pea extract greater than 20 % in PA 3679 Agar had no additional effect. Unheated spores showed no response to pea extract. RESULTS AND DISCUSSION The stimulatory component of pea extract was stable to boiling for 1 hr and autoclaving. Dialysis of pea extract did not diminish its activity (Table 1). Dialysate failed to improve recovery counts, suggesting that classical germinants such as L-alanine, glucose, and salts are not responsible for the pea extract effect. The precipitate which formed in pea extract during storage at 4 C is high in starch content and contains most of the stimulatory activity. Small amounts of starch and stimulatory activity remained in the supernatant. The precipitate retained 85%XO of its starch content and 73% of its stimulatory capacity after washing with a variety of organic solvents and water. None of the washings possessed the ability to improve recovery counts of heat-stressed PA 59-123 spores. The stimulatory activity of pea extract was compared with that of corn starch (Stein, Hall & Co., Inc.) and soluble starch (Difco). Maximal recovery counts were achieved with 0.15%o corn starch, 0.25% soluble starch, or 20% pea extract (0.12% pea starch) present in PA 3679 Agar. Incorporation of combinations of suboptimal levels of pea extract, corn starch, and soluble starch into the recovery medium resulted in an additive effect up to a maximal count. The maximal count achieved with combinations of starches did not exceed the count obtainable with optimal additions of the individual starches. Starch in fresh pea extract was completely hydrolyzed by the action of at-amylase. Pea extract treated with ca-amylase possessed no ability to improve recovery counts of heatstressed PA 59-123 spores on PA 3679 Agar (Fig. 2). Pea extract to which boiled enzyme had been added and untreated pea extract contained high levels of starch and stimulated recovery counts of heat-stressed spores approximately threefold. Glucose and maltose, hydrolysis products of starch, had no effect on recovery counts when added to PA 3679 Agar. Thus, starch appears to be the sole component of pea extract responsible for increased recovery counts. Starch (8,9,10), charcoal, or serum albumin (8, 10) are known to improve survivor counts of heat-stressed Bacillus and Clostridium spores. These additives are thought to act by adsorbing inhibitors from the medium (10). The nature of the inhibitor has not been established conclusively, but long-chain, unsaturated fatty acids (4) or their oxidation products (11) have been implicated. In the present study, slightly higher levels of corn starch and soluble starch than of pea starch were required for maximal stimulation. This discrepancy may be due to an underestimate of the amount of starch in pea extract or to the fact that pea starch contains a relatively high proportion of amylose (14) which is more active than amylopectin in improving recovery counts. Alternatively, extraction and drying procedures may render commercially available soluble and corn starches less effective.

v3-fos

2014-10-01T00:00:00.000Z

1971-01-15T00:00:00.000Z

5138706

s2

Theoretical aspects of crossbreeding SUMMARY Methods of utilising breeds and bleed crosses in animal production are discussed, taking account of both genetical and economic aspects. The theoretical principles for breed and breed cross comparison are analysed, but most emphasis is given to methods of improvement of existing crosses. A new synthetic breed is likely to have higher genetic variation, and reach a higher selection limit than the pure breeds from which it originates. However, it may take many years for the synthetic to surpass the best available purebred under continuous selection. Returns obtained in early years have more monetary benefit than those obtained later, for they can earn interest and incur a smaller risk element, so that a synthetic of use only in later years is unlikely to be cost-effective. Despite the flexibility in maintaining several alternative breeds, these need to be continually selected if they are to remain competitive, so better returns may be obtained by exerting more pressure on the best available present material. It is unlikely on theoretical grounds that cross testing schemes such as reciprocal recurrent selection have much to offer for breed cross improvement in large animals where growth and carcase traits are important. INTRODUCTION Crossbreeding has been an established practice for centuries in the domesticated animal species. Breeders have had many objectives: crosses have been made every generation to obtain any benefits there may be from heterosis or from the particular merits of the individual breeds as maternal or paternal parents. Alternatively the crosses have been used to form new populations with desirable characters from each of the parental breeds with, perhaps, increased variability to enable more rapid progress from later selection. The theoretical basis of crossbreeding has been studied extensively to enable us both to understand the genetic mechanism underlying heterosis and to design breeding programmes to utilise it. There are two essentially separate aspects of crossbreeding, although they can not be considered entirely independently of each other. The first includes the choice of breeds and method of utilising them in crosses, if necessary, in order to maximise present economic performance. For example, we may wish to know whether breed cross A X B is superior to A x C or to A as a single breed, when all productive and maternal traits are considered. The second area of breed utilisation is concerned with improvement over a period of a few generations. We would like to know which breeds or crosses to choose now and use in a selection programme so as to maximise economic merit over the next 10 or 20 years. The extreme examples occur with corn or poultry breeding, using a cross of inbred lines. The breeder may have the best two-way cross on the market at present, but could find difficulty improving it. There is some suggestion that breeding programmes in corn are moving back from an inbreeding and crossing scheme towards programmes in which selection is practised every generation. In the large animal context we are more concerned with whether to form new breeds with, perhaps, enhanced variation, or whether to use the best available at present. In a recent review Dic K Exsorr (ig6g) discussed the experimental information required for a rational choice of breeds, but was primarily concerned with immediate performance. Although I shall briefly discuss the theoretical framework on which such decisions should be made, I will give more emphasis to the problem of maximisation of future performance which has not, I believe, been investigated adequately in the context of breed utilisation. Unfortunately the analysis is bound to be somewhat speculative, for we generally lack adequate information on genetic parameters within different breeds and crosses in most practical situations. However it is possible to set out some of the conditions under which new cross populations might respond faster and further than their parent breeds. The analysis has not been taken very far, but hopefully it will provide a few pointers, and I shall give more attention to the arguments on which decisions should be based, rather than to conclusions in any specific instance. For the purpose of this discussion the term breed will refer to any closed population from which members can be identified by phenotype or pedigree. A breed may have been kept distinct from other breeds under consideration for only a few generations, so that, for example, Canadian and Dutch Holstein cattle may be viewed as separate breeds for this purpose. I shall also make considerable reference to productive and maternal traits. In the class of productive traits are included growth and carcase characters of animals for slaughter for meat and milk production in a dairy breed. Maternal traits include litter number, conception rates, milk production in suckler herds and perhaps even adult body size, in so far as it affects breeding costs. In effect, the genes for productive traits are contributed by both parents in a cross, those for maternal traits are expressed only in the dam. The other term to be defined is synthetic, which will be used for any new breed cross which is maintained as a new population, breed or &dquo; gene pool &dquo;. CROSSBREEDING AND PRESENT PERFORMANCE In principle, the utilisation of crossbreds to obtain maximum performance at the present is simple. It is necessary only to find the most efficient purebred or crossbred combination, taking account of both productive and maternal traits. There may, however, be considerable difficulties in actually finding the best cross combination, especially when there are specific heterotic relationships between pairs of breeds and when there are important genotype by environment interactions. In these situations it may be necessary to test a large number of combinations. Otherwise good predictions of merit may be possible from pure line performance in some standard environment. Monv ( 19 66) discussed criteria for evaluating crossbreds. He defines a non-linear relation between maternal performance and economic merit, but we shall simplify this here to linearity. Consider a cross of breeds A (sire) and B (dam) with productive performance P A , P B and heterosis P!B, and for the dam breed a maternal performance R,. The economic merit, E, is or in a three-way cross A x (B X C) it is, approximately, Here K, x and y are appropriate constants. Of these K includes fixed costs and does not affect comparisons between breeds. Examples of the values of x and y are given by M OAV ( 19 66) for pigs, and these can be modified to correspond with the formulation used here. Let E be the excess of returns over variable costs, measured in pounds sterling per pig of 100 kg live weight marketed. Letting P be the feed conversion efficiency (kg feed per kg gain) then x = 3 . 1 , and letting R be the number of pigs marketed per sow per year then y = 0 . 21 , where R has a mean of about 1 6. These figures are for integrated operations, and they may not reflect present economic conditions, but should serve as an example. These formulae illustrate some important, if somewhat obvious, points. Unless there is a large amount of interaction, P AB , specific to particular breed combinations, the sire breed with highest performance on productive traits should be used, for we are assuming here that many dams are mated per sire, or that AI is used, so that the sire breed contributes a very small proportion of total maintenance costs. In the dam breed both productive and maternal traits have to be considered, and the weightings x and y determine how much should be given to each. These same weightings can be used for calculating indices for selection within breeds. We see that the fixed crossing scheme takes full advantage of any heterosis for productive traits in a two-way cross, and for maternal traits also in the three-way cross. In cattle or sheep a high proportion of animals may have to be bred pure to provide replacements in the dam breed. If a proportion, q, of the animals marketed are pure breds of the dam breed, and I -q are crosses, the average merit becomes so that productive performance in the dam breed becomes relatively more important. If a new synthetic breed is made from the cross of the A and B breeds the overall merit becomes There is a loss of half the heterozygosity for productive traits, but a gain in the maternal traits. With a rotational crossing scheme on two breeds the average merit, taken over successive crosses, includes 2 !3 of the heterosis between the breeds for productive and maternal traits, but is otherwise the same as for the synthetic. This discussion will not be carried further here. Reference should be made to the papers of Dicx!RSOrr ( 19 6 9 ), M OAV ( 19 66) and F!wso!r and J AKUB EC (1970). CROSSBREEDING AND FUTURE PERFORMANCE In making decisions about breed or breed cross improvement in future years we face problems at two levels. We have to estimate the potential genetic progress and compare these rates of progress with alternative schemes. In addition we should consider the costs of these schemes and relate these to their potential economic benefits. Most geneticists have occupied themselves with measurement of response, considering economics only when designing a selection index to give optimum weight to the traits. I feel we need to go further than this and will attempt to do so after some discussion of the relevant genetic theory. Imagine that on the basis of our breed and breed cross testing programme we find that the breed cross A, X B l is most efficient. Therefore, unless there are specific interactions between these breeds, A, is the best available for productive traits and B, is good for both productive and maternal characters. We now have several options open for improving the cross, although some of them may not seem very promising. These are: (a) form a synthetic from the A, X B, cross; (b) select solely within the breeds A, and B l ; (c) initiate rotational crossbreeding between A, and B l ; (d) form a synthetic sire or dam breed; and (e) maintain alternative sire or dam lines. The options are not mutually exclusive not do they cover the whole range of possible programmes, but they give some indication of the main direction of selection effort. We shall consider them in turn. A. -Form synthetic from A 1 X B i cross. A new breed could be formed and maintained and marketed as a pure breed but this is unlikely to be useful. There is an initial loss of half the heterosis between the breeds for productive traits, which later increases as the synthetic becomes inbred, and a loss of half the maternal advantage of breed B, over A l . Secondly, it has been shown by SMITH ( 19 6 4 ) and MoAV and HILL ( 19 66) that greater progress for overall merit is made if separate sire and dam lines are maintained, with selection in the sire line (or breed) made solely for productive traits and in the dam line for an index of productive and maternal traits. This advantage may be small in species such as pigs in which important maternal traits all have low heritability so that little pressure should be imposed on them. In a dual purpose beef and dairy cattle system there may be considerable advantages in maintaining separate breeds. In the dam, or dairy breed, most selection effort has to be applied to milk production, and selection for beef characteristics can only be undertaken with minor weighting in the milk progeny test, or by performance testing prior to the progeny test. In either case the rate of response for traits relevant to beef production is much smaller than could be achieved in a beef breed used solely as a sire in crosses. In the beef breed intense selection can be practised on a performance test, using a short generation interval. Imagine, for example, that a pure Holstein could currently outperform any cross with the Holstein on some intensive management systems. Yet after a few years of selection either in a beef breed or in a separate strain of Holsteins, crosses to this breed or strain could be superior for beef traits, so that cross matings in excess of requirements for dairy breed replacement should be made. There may be an increase in variability in the A, X B, cross relative to the parent lines so that response is enhanced. However there are more appropriate means of forming synthetics with the aim of increasing variation, and these are discussed later. B. -Select within A i and B l breeds. In this way we retain, at least in the short term, the heterosis and other desirable properties of the cross combination. The main issue in this scheme is the mode by which selection should be practised: whether it should be based on pure line or on cross performance using some scheme such as reciprocal recurrent selection. For traits determined primarily by additive or completely dominant genes it has been shown theoretically that the rate of improvement in the cross and the selection limit are approximately the same in pure line and reciprocal recurrent selection schemes, providing that the same intensity of selection is practised in each system (HILL, i 97 o). But it is unlikely that any improvement scheme using cross testing could be operated in large animals with the same intensity and generation interval as in schemes for within breed selection, except perhaps in programmes to improve milk production using progeny testing. If there is overdominance faster rates and higher limits can, of course, be achieved with reciprocal recurrent selection. An indication of whether this might be possible can be obtained from the genetic correlation of pure and cross performance. If this is close to unity there will be no advantage in the short term in selecting for cross performance directly. However, it is conceivable, in theory at least, that an initial programme of pure line selection would reduce later gains with . reciprocal recurrent selection when both breeds have approximately the same gene frequency so that there is selection towards the equilibrium frequency. In large animals the traits of major importance include growth rate (and feed conversion efficiency), carcase quality (or simply degree of fatness), milk yield and milk quality, and reproductive traits. Of these carcase and milk quality typically show little heterosis, growth rate and milk production moderate heterosis, and the reproductive traits exhibit rather more. One can conjecture therefore that at most only a small proportion of the variance for all these traits, with the possible exception of fertility and litter size, for example, are contributed by overdominant genes. Breeding programmes with selection on pure line performance can therefore be continued with safety. Whilst there appears to be little place for selection programmes based on cross performance in a two way cross structure they could be more relevant for improving the reproductive performance of the B X C mother in the three-way cross A X (B X C). But although each breed in the dam side of the cross contributes only i /q. of the genes for the productive traits in the final crossbred animals it also contributes only i /z to the maternal performance of B X C. The relative index weightings which should be applied to maternal and productive traits in these breeds B and C are therefore almost the same as should be used in the single dam breed of a two way cross. In pigs the economic weightings for food conversion efficiency and carcase quality are so high, and the heritability of litter size is so low that most selection pressure should be devoted to these productive traits in the dam breeds. Thus even in a three-way cross a reciprocal recurrent selection programme would seem unjustified. Similarly, inbreeding schemes used to generate between line variation within the chosen breeds can not be effective relative to programmes utilising constant selection for the highly heritable traits. C. -Rotational crossbreeding of A 1 and B r . In a rotational crossbreeding scheme each breed contributes to the cross to the same extent on average, both as a sire breed and as part of the dam combination. Therefore selection pressure has to be put on the same traits, both productive and maternal, in each of the two (or more) parent breeds, so that specialised sire and dam lines can not be developed. We must then expect to make less selection progress in the rotational crossbred than in a fixed crossing scheme such as A, X B I' where different programmes can be used for the two breeds. If we have available other breeds A,, A 3 etc. which are only slightly poorer than A, as sire breeds, these could be crossed with A, to form a synthetic and yet retain general heterosis in the cross. Similarly other dam breeds B,, B 3 could be crossed with B I to form a synthetic sire line. These are likely to be more attractive alternatives than making a synthetic from the cross A, X B,. The new synthetic breeds could be useful if they show greater genetic variation thanthe pure breeds, so that after a few years of selection their merit will reach and then surpass that of A, or B i , and could then be substituted in the cross. JAMES ( 19 66) has discussed procedures for selecting animals from among several populations, but only in the context of maximising the present performance of the synthetic. If there is information available on heritabilities in the breed A, and the synthetic A Ix2' say, it is simple to predict the time needed before it surpasses A,. However this could be many years in a practical situation. For example, assume that in beef breeds the trait, weight to 400 days, has a standard deviation of 40 kg and that A 1 exceeds A Ix2 by 10 kg (in breeding value since heterosis within the sire line is not of interest). In an efficient breeding programme with selection only on males and rapid replacement of females an annual response of 1 6 h 2 kg per year can be made. So if the heritability in the synthetic was, say, 5 0 % and in the pure breed it was 40 % and both were continuously selected, it would take zo /(i6 X o.i) or at least six years for the new breed to catch up. Some years would also be needed to establish and multiply the synthetic and obtain the necessary estimates of genetic parameters. It is usually difficult or expensive to obtain accurate estimates of heritability, and it is unlikely in many situations that estimates of differences in heritability between synthetics and pure breeds could be obtained with sufficient precision that practical decisions could be taken using them. It is possible to make some theoretical predictions of differences in genetic variance, but these too suffer from severe limitations. The simplest situation is where breeds A l and A 2 , say, are essentially randomly selected but distant by several generations from a common base. Assume there is additive gene action, and the additive variance in the synthetic (or in the foundation population) is aa. If the populations have been inbred by an amount F, the expected within-population variance is (i -F)aa, and the variance between populations is 2 F C2 . In a sample of size two from a normal distribution the first ranking individual is, on average, 0 . 5 6 standard deviations superior to the mean of the two. If h 2 is the heritability in the foundation or in the synthetic population, and the phenotypic variance is assumed to be altered, the synthetic will take about o.g6!2F/tFA generations to reach the better pure line when both are under continued selection. For example, if F = 0.2, i = 1 . 0 (averaged over sexes) and h 2 = 0 . 4 , the synthetic is expected to take 2 .8 generations to reach the better pure line, or 7 years for our beef cattle example with the 2 , 5 year generation interval. After that period, assuming there had been no change in variance through selection or further inbreeding, the synthetic would gradually become increasingly superior. In other cases predictions of variance in the synthetic are essentially speculative, although one or two useful relationships are known. Let q i and q 2 be the frequency of some gene in lines A l and A 2' and 7j be the mean frequency. Then so the mean heterozygosity at this locus and variance if the genes act additively is at least as high in the synthetic as in the average of the two parental lines. More generally, J ACKSON and JAMES ( 1970 ) have shown that, with additive effects, the variance within the synthetic is given by 2 !o-2B -! o-2 w , where a 2 B is the genetic variance between populations and 6 z W the genetic variance within populations, assumed to be the same in each. At loci showing complete dominance the additive variance is higher in the synthetic when the mean frequency of the recessive allele is greater than o.5, otherwise it is less (L ERNER , rg 5 q.). But at such loci most additive variance is expressed when the recessive frequency is high, so that averaged over all loci the synthetic will probably have higher variance. If the parent lines and synthetic are selected in closed populations of the same size for a long period of time the selection limit is expected to be higher for the synthetic than for the mean of the two pure lines. This relationship holds for both additive and completely dominant genes at all frequencies but the effects of linkage and epistasis are being ignored. However we are making the basic assumption that the traits under selection are influenced by a large number of loci, so there are only small differences in mean gene frequencies between the alternative populations. If there are wide differences in mean initial frequency the synthetic could have higher initial variance than the best line, yet never catch up with it under continued selection. But this would seem unlikely, especially as one population may have genes segregating which are absent from another. In general however, we lack concrete evidence and have an unsound basis for making practical decisions. In the Institute of Animal Genetics in Edinburgh a relevant experiment with Drosophila melanogaster has been started by I,or!z-Fm·rJur,. Response to selection for sternopleural bristle number is being measured in two populations (Kaduna and Pacific) from different locations which have been maintained in cages in the laboratory for many years, and in synthetics formed from crosses between them. The initial performance of the two populations is almost exactly the same, but Pacific shows rather higher genetic variance and has responded somewhat more rapidly to selection. The cross shows no significant heterosis. With selection started from the Fi generation the synthetic has advanced at a rate intermediate between that of the parent lines. After allowing six generations of random mating without selection after the cross the heritability was estimated in another sample of the synthetic. Although a higher heritability value was obtained from the offspring-parent regression at this time, the subsequent selec-tion response was no faster than in the parent lines. This result is rather hard to interpret, for one would expect an increase in genetic variance in F 2 and later generations if there was negative linkage disequilibrium between the populations making the cross, but this should be accompanied by greater subsequent response. These results are as yet preliminary and the experiments are small. Nevertheless it is clear that the synthetic has little or no more additive genetic variance than the parent lines, which suggests that essentially the same loci are segregating in the two populations. More definitive conclusions will be possible when selection limits are reached. Unlike our domestic species these populations have no history of selection, so we should be cautious about making inferences from the D!oso!'At7a work. E. -Maintain alternative sire or dam lines. In addition to selecting in our chosen breeds A i and B l , selection could be continued alongside in other populations, although their merit may be less at present. Of course the synthetic could be one of these. If rather different criteria were chosen for selection in these populations the programme would be much more flexible in that alternative breeds could be substituted as market demand and economic conditions change. The main disadvantage of this kind of scheme is that these potential substitute breeds have to be selected at almost the same rate as the ones already used, or they will gradually lag behind for the major traits and can never be utilised. Thus the breeding programme becomes much larger and more expensive. The same requirement has to be met for any breed which may be crossed into A i or B i in future years because it has some particularly valuable feature. Unless these breeds have performance near that of A i or B, the new synthetic A or B will be inferior. However there could be benefits from forming new synthetics if reproductive performance in A i or B, had deteriorated with inbreeding. If our objective is to maximise gain over a long period of time, yet our facilities for maintaining animals under selection are limited, we have two distinct options. A synthetic can be formed immediately and selected as a single population. Alternatively the separate populations can be maintained as smaller populations, and each selected for a period before crossing and reselecting as a single larger population. R OB E R T SON ( 19 6 0 ) and M ARUYAMA ( 197 o) have shown that the same limit is obtained in either case. However the average rate of response will be higher if the synthetic is made initially since the subpopulations will become inbred more rapidly. But in the short term, in generations at least, our best strategy is probably to select in the highest ranking available breed or population. ECONOMIC ASPECTS Attempts have been made recently to evaluate breeding programmes in monetary terms using, in effect, the discounted cash flow procedure commonly employed in management accounting. The principles of the technique were first used in a genetic context by PouTous and V ISSAC ( 19 6 2 ) and subsequently by S OLL E R , B A x-Arrarr and PnsT!xrrnx ( 19 66). I shall give these in outline, and discuss their implications on alternative breed and cross bred improvement programmes. Returns and costs incurred in any year are discounted back to some base, perhaps the year at which a decision is made to build a new testing station, or perhaps merely to the year at which a selection decision is made. For example, with an interest rate of 8 %, £ 100 invested now would realise £ io8 next year, £ ioo x (i.o8) 1 the following year and so on. Thus £ io8 earned next year is equivalent to having only 10 0 now, or £ I obtained next year is worth £ i /i.o8 = £ 0 . 92 6 now, and £ i earned 5, 10 or 20 years later is equivalent to £ 0 .68, £ o.q.6 and £ 0 . 21 earned now. With such an approach we can compute the aggregate benefits of selection response which are both permanent (at least in terms of changes in the traits) and cumulative. We can calculate either an overall &dquo; profit &dquo; or the investment yield, which is the interest rate at which the scheme would just break even. Widely different programmes can be compared, or the returns from minor changes in selection procedure, involving relatively small extra expenditure, can be evaluated. Of course many simplifying assumptions need to be made, and it is difficult or impossible to take account of unforeseen changes in economic conditions. Such risks can be hedged to some extent by adopting discount rates considerably in excess of current interest rates. For example an estimated yield of 20 % evaluated over a period of only 15 years might be considered necessary before undertaking a programme. Especially when high discount rates are used the returns made in early years are weighted very heavily; it is this property of the procedure which has most relevance to our discussion of crossbreeding, for with large animals any programmes undertaken are likely to be of a long-term nature. Consider the merit of maintaining synthetics or other substitute breeds of lower initial performance, but with the hope that they will eventually surpass the present superior population. No returns are obtained from this synthetic until the nucleus herd has reached the level of that of the superior breed, itself under selection, and until the population has been multiplied and progeny marketed. We considered earlier an example with beef cattle where the synthetic would require 6 years to catch up. We have to add to this, say, 2 years for bulls to mature and have progeny by A.I. and another 2 years before progeny are slaughtered, making a total of 10 years in all. At 10 years the discount factor is o.q6 if the rate is 8 %, and o.z6 if it is 20 %. Further, the extra returns after this period come only from the increased gain of the synthetic over the original breed, although only one selected population, the synthetic, now has to be maintained. Using the same arguments it becomes ditficult to justify maintaining several pure breeds or strains as potential substitutes. These must be selected at rates near those of the current commercial populations if they are ever likely tobe competitive, whether or not the objectives in the schemes are exactly the same. The costs of maintaining and selecting these populations will inevitably be considerable. Our rather simplified arguments lead us, therefore, to the conclusion that almost all our attention should be devoted to improving the breeds or crosses which are currently best. However a breeding organisation or country committing itself to such a scheme is vulnerable to a change in consumer demand or an exhaustion of genetic variance. But no scheme runs entirely in isolation, for there are competitors or other countries running similar programmes. These offer the best potential source of new variation! LIMITATIONS In conclusion a few comments should be made about the limitations of the analysis. In the first place it has been idealistic, and has by-passed many practical difficulties and limitations imposed by existing breeding systems, and by breeders' and farmers' prejudices. For example there may be resistance to use of what is clearly the best breed, or there may be legislation, as in Britain, to prevent the use of crossbred bulls. Even within the theoretical framework many simplifying assumptions have been made. In particular, interactions have been ignored both at the genetic level, between loci, and at the applied level, between environments. Nor has any general solution been given, but this is not possible with our current state of knowledge. There is clearly considerable need for greater understanding of the genetics of the major quantitative traits in our domestic species. Re!u pour publication en octobre 1970 .

v3-fos

2020-12-10T09:04:16.669Z

1971-09-01T00:00:00.000Z

237230575

s2

High Aflatoxin Production on a Chemically Defined Medium Aspergillus parasiticus ATCC 15517 produced 28 to 30 mg of aflatoxin per 100 ml of a medium containing sucrose, asparagine, and salts in stationary and shaken cultures. In the absence of asparagine in the medium, the toxin yields fell drastically, and the thin-layer chromatograms of the chloroform extracts of the cultures indicated the total absence of aflatoxin G1 and the presence of new intense blue and green fluorescent bands having RF values lower than aflatoxins. Initial pH was critical and had to be around 4.5 for good growth and high toxin production on this medium. Optimum concentrations of KH2PO4 and MgSO4·7H2O in the medium were much lower than those normally used in fungal growth media. medium. Optimum concentrations of KH2PO4 and MgSO4.7H10 in the medium were much lower than those normally used in fungal growth media. Complex and synthetic media have been recommended for the production of aflatoxin by Aspergillus (1,3,5,6,20,21). The yields of aflatoxin are generally low in synthetic media normally used for fungal growth. Aflatoxin production is high in crude media or in synthetic media supplemented with crude extracts (5,9,18). Detailed investigations on the pathway of aflatoxin biosynthesis and the various factors affecting toxin production have been hampered by the lack of a suitable chemically defined medium supporting high yields of toxin. It is the object of this paper to describe the development of a chemically defined medium giving high yields of aflatoxin. MATERIALS AND METHODS Culture conditions. A. parasiticus ATCC 15517 (formerly known as A. flavus) was used throughout the study and was maintained as a soil culture. Five-to-7 day-old spores, obtained from a bottle containing 50 ml of glucose-peptone-agar, were distributed equally to five 500-ml Erlenmeyer flasks containing 100 ml of sterile basal medium. These flasks were incubated at 26 i 1 C on a rotary shaker or as stationary cultures for 8 days unless otherwise stated. All of the chemicals used were of British Drug House Ltd. analytical reagent grade, and asparagine was obtained from Sigma Chemical Co. Unless specified otherwise, the initial reaction of all the media used was pH 4.5. Sodium hydroxide and distilled hydrochloric acid were used for readjusting the pH. In some experiments, the media were freed from traceclement impurities by alumina treatment (10). The experiments were performed in duplicate, and the results are reported as averages. The difference in aflatoxin production between duplicate flasks was generally less than 10%. Assay. The media and mycelia were separated, and wet and dry weights of the mycelium were determined. Aflatoxins were extracted with chloroform, separated by thin-layer chromatography on Silica Gel G by using toluene-isoamyl alcohol-methanol (90:32:3; reference 17), eluted with methanol, and estimated by measuring the absorption at 363 nm (15). Since the amounts of aflatoxins B2 and G2 were very low, aflatoxins B, and B2, as well as GI and G2, were generally measured together. RESULTS The effect of removing trace impurities from AM and GAN media by treatment with alumina and the effect of supplementation with asparagine were studied. Either alumina treatment or the addition of asparagine increased the toxin yields on both media (Table 1). Higher yields were obtained by the addition of asparagine to alumina-treated media. In another set of experiments, a nutrient medium containing the components of SH medium and a number of amino acids and B group vitamins was used. This did not give high yields of aflatoxins (Table 2). However, as a result of chance observation, it was found that, on removal of the precipitate formed by dissolving the salts required for 1 liter of medium in 50 ml of doubledistilled water, this medium yielded 26 mg of toxin per 100 ml. Asparagine was necessary for good growth and high toxin yields. The presence of other amino acids and vitamins was not obliga-tory for toxin production, and essentially the same results could be obtained with SH medium if the precipitated salts were removed. SL medium which contained low concentrations of inorganic salts supported the formation of 28.5 mg of aflatoxin per 100 ml (Table 2). In the absence of asparagine, aflatoxin G1 was totally absent and aflatoxins B1 , B2 , and G2 were present in small amounts; in addition, intense blue and green bands having RF values lower than aflatoxins were observed on thin-layer chromatograms. The bands were not noticed when asparagine was present in the medium. A detailed study of the nature of these fluorescent bands and the effect of other salts on their formation is under investigation. The effect of initial pH of the medium on growth and aflatoxin production is shown in Table 3. An optimal initial pH of 4.5 assured good growth and high toxin production. KH2PO4 and MgSO4 7H20 were essential for growth and aflatoxin formation. The optimal levels of these salts for toxin production were 750 and 350 mg per liter, respectively. An increase in their concentration to 10 and 2 g per liter, as in GAN or AM media, drastically reduced toxin yields ( Table 2); calcium chloride at concentrations as high as 200 mg per liter did not have any effect on aflatoxin production in stationary cultures. Maximum yields in SL medium were obtained by 8 days in the case of stationary cultures, and there was a continuous increase up to 10 days in the case of shaken cultures (Table 4). MgSO4*7H20 per liter a SH was synthetic high-salts medium; SL was synthetic low-salts medium. b The precipitate formed on dissolving the salts required for 1 liter of medium in 50 ml of doubledistilled water was removed, and then the other components of the medium were added. c Precipitate was not removed; the salts were added to a sufficiently large volume of the medium so that no precipitate was formed. (13) reported that aflatoxin formation on a casein substrate was high at extreme (mg/100 mI) acidic or alkaline pH values (pH 2 and 9.5, respectively). It has also been reported that afla-G Total toxin yields on a Czapek-Dox medium increased several-fold when the pH was changed from 7.4 1.00 18.00 to 4.0 (11). With SL medium, toxin production 8.60 28.44 was maximal at a pH 4.5 and was reduced at 7.14 24.97 lower or higher pH values. These data are con-0 ditions which are obviously unfavorable for high salts edium.yields of afiatoxin (1,5,12,19). DISCUSSION The increase in yields obtained on alumina treatment of the media suggest the presence of inhibitory impurities in AM and GAN media (Table 1). At levels normally used in fungal growth media, KH2PO4 inhibited toxin production. A similar inhibition of streptomycin production by phosphate has been reported and has been ascribed to the inhibition by phosphate of some phosphatases involved in streptomycin biosynthesis (7,14). Since the biosynthetic pathway of aflatoxin is not very well understood, it is not possible to state if a similar mechanism is responsible for the effect observed. The effect of asparagine is not due simply to the presence of excessive concentrations of nitrogen, since replacing asparagine by an equimolar amount of nitrogen led to a considerable decrease in toxin yields. Preliminary experiments indicated that aspartic acid could replace asparagine effectively. The initial pH of the medium is another im-

v3-fos

2019-04-03T13:05:58.950Z

1971-01-01T00:00:00.000Z

91468905

s2

Aluminium and acidity in Finnish soils In the present study an attempt was made to study by statistical methods the proportion of Al of the exchange acidity of 298 soil samples of various kind, and to what extent the titratable nonexchangeable acidity in these soils is connected with Al, when Al soluble in Tamm’s acid oxalate was used as its indicator. Unbuffered N KCI replaced Al only from soil samples with a pH less than 5.3 in 0.01 M CaCl2 . In this part of the material, Al corresponded, on the average, to one third of the exchange acidity of mineral soil samples, and to 16 per cent of that of organic soils. The amount of Al was usually the higher the lower the soil pH, but the correlation was close only in the group of clay soils. Titratable nonexchangeable acidity was estimated as the difference of the amount of acidity neutralized at pH 8.2 and the corresponding amount of exchange acidity replaced by unbuffered KCI. In 100 clay soil samples it was, on the average, 12.0 ± I*3 me/100 g, in 42 samples of silt and loam soils 8.8 ± 1.8 me/100 g, in 99 sandy soils 8.9 ±l.l me/ 100 g and in 57 organic soils 49.1 ± 6.8 me/100 g. There was no correlation between titratable nonexchangeable acidity and the clay content within various soil groups. In the clay soils exalate soluble Al alone explained 78.3 %, in the silt and loam soils 59.8 %, in the sandy soils 6.5 %, and in the organic soils 0.6 % of the variation in titratable nonexchangeable acidity. Taking into account the content of organic C increased the rate of explanation only to 82.1 % in clay soils, to 84.1 % in silt and loam soils, to 83,1 % in sandy soils, and to 63.7 % in the organic soils. Further, adding the soil pH increased the rate of explanation 5.8 to 9.6 per cent units in various soil groups, but considering of oxalate soluble Fe did no more distinctly increase the part of variation explained, except in the organic soils. Regression equations were calculated for the relationship of these variables. According to the partial correlation coefficients and to the the relative importance of oxalate soluble Al in explaining the variation in titratable nonexchangeable acidity was in the clay soils higher than even that of organic G content, but in the other mineral soil groups it was less important than both C content and pH; in the organic soils even oxalate soluble Fe appeared to be slightly more important. Modern concepts of soil acidity emphasize the role of aluminium. The exchange acidity replaced on leaching with unbuffered solution ofa neutral salt is claimed to be mainly due to monomeric trivalent aluminium ions (Coleman et al. 1959, Jackson 1963, Chernov 1964), at least in soils low in organic matter (Schwertmann 1961). According to recent opinions, aluminium is connected also with titratable but nonexchangeable part oi the total acidity which is neutralized first at a higher pH. This »pH-dependent acidity» which is Titratable nonexchangeable acidity was estimated as the difference of the amount of acidity neutralized at pH 8.2 and the corresponding amount of exchange acidity replaced by unbuffered KCI. In 100 clay soil samples it was, on the average, 12.0 ± I*3 me/100 g, in 42 samples of silt and loam soils 8.8 ± 1.8 me/100 g, in 99 sandy soils 8.9 ±l.l me/ 100 g and in 57 organic soils 49.1 ± 6.8 me/100 g. There was no correlation between titratable nonexchangeable acidity and the clay content within various soil groups. In the clay soils exalate soluble Al alone explained 78.3 %, in the silt and loam soils 59.8 %, in the sandy soils 6.5 %, and in the organic soils 0.6 % of the variation in titratable nonexchangeable acidity. Taking into account the content of organic C increased the rate of explanation only to 82.1 % in clay soils, to 84.1 % in silt and loam soils, to 83,1 % in sandy soils, and to 63.7 % in the organic soils. Further, adding the soil pH increased the rate of explanation 5.8 to 9.6 per cent units in various soil groups, but considering of oxalate soluble Fe did no more distinctly increase the part of variation explained, except in the organic soils. Regression equations were calculated for the relationship of these variables. According to the partial correlation coefficients and to the the relative importance of oxalate soluble Al in explaining the variation in titratable nonexchangeable acidity was in the clay soils higher than even that of organic G content, but in the other mineral soil groups it was less important than both C content and pH; in the organic soils even oxalate soluble Fe appeared to be slightly more important. Modern concepts of soil acidity emphasize the role of aluminium. The exchange acidity replaced on leaching with unbuffered solution ofa neutral salt is claimed to be mainly due to monomeric trivalent aluminium ions (Coleman et al. 1959, Jackson 1963, Chernov 1964, at least in soils low in organic matter (Schwertmann 1961). According to recent opinions, aluminium is connected also with titratable but nonexchangeable part oi the total acidity which is neutralized first at a higher pH. This »pH-dependent acidity» which is usually supposed to be chiefly caused by weak acidic groups of organic matter, may be partly attributed to positively charged hydroxy-Al polymers. These complexes may be sorbed as surface coatings on soil particles, or they may block interlayer spaces of 2:1 clay minerals (Rich and Obenshain 1955, Barshad 1960, Clark 1964b, Coleman et al. 1964, Schwertmann and Jackson 1964, de Villiers and Jackson 1967. Also part of the exchange sites of organic matter may be countered by fixed Alor Al-hydroxy ions (Keränen 1946, Schnitzer and Skinner 1963, Clark 1964b, Schnitzer 1965, Pionke and Corey 1967, McLean and Owen 1969. These forms of aluminium are not exchangeable with a neutral salt, but they are proton donors or OH acceptors, which will increase the consumption of base when acid soils are titrated. Hydroxy-Fe polymers are supposed to react in a similar way. In the present work an attempt is made to study with statistical methods to what extent titratable nonexchangeable acidity in various kind of Finnish soils is connected with aluminium, when aluminium extracted by acid ammonium oxalate is used as its indicator. Attention is also paid to oxalate soluble iron, and to the contents of clay and organic carbon of the soils. The titratable nonexchangeable acidity is taken to correspond to the amount of acidity neutralized at pH 8.2 minus the exchange acidity which is replaced by unbuffered KCI. Preliminary studies on aluminium in exchange acidity are also reported. Material and methods The material consists of 298 samples of various kind of soils, collected from different parts of Finland. In order to get very acid soils, also virgin samples were included. Both surface layers and deeper horizons were sampled. According to the particle size composition, 100 of the samples were clay soils containing at least 30 % of the fraction less than 2 p. in diameter. There were 42 samples of silt and loam soils, 99 samples of sandy soils, or fine sand, sand and till soils, and 57 samples of organic soils which represented both peat soils and mull and mor layers. The samples were air-dried and ground to pass the 2 mm sieve. Thus, particularly till soil samples lost a large part of their coarser components, and therefore, differ from the original soils. The base consumed on leaching the soil samples with N KCI-triethanolamine, buffered to pH 8.2, was taken as an estimate of the titratable acidity. The exchange acidity was displaced by unbuffered N KCI. In both cases, a 10 g sample ofmineral soil, or a 2 g sample of organic soil was shaken for one hour in 20 ml of the extracting solution and centrifuged. The soil was then washed with four 20 ml-portions of the extractant, and an aliquot of the combined extract was titrated. A 1 in the unbuffered KCI extract was determined by the fluoride method (Yuan 1959), and the sum of exchanged Ca and Mg was estimated with versenate titration. A 1 and Fe were extracted with Tamm's acid ammonium oxalate. The ratio of soil to solution was 1 to 20, and the period of extraction was two hours. A 1 was determined by the aluminon method, and Fe by the sulfosalicylic procedure, after organic matter in the oxalate extract was destructed by ignition. Organic C was determined by wet combustion and iodometric titration. Soil pH was measured in 1 to 2.5 suspension in 0.01 M CaCl 2 . Results The groups of soil samples are characterized by data in Table 1. The pH values range from 3.3 to 7.5, but there are only a couple of samples with a pH higher than 6.7. The very acid clay samples were from uncultivated postglacial soils rich in acid salts. The low mean pH-value of the group of sandy soils is due to the fact that it contains more samples of virgin soil than the other groups of mineral soils. Typically, the mean pH of the organic soils is even lower. The three groups of mineral soils do not markedly differ in their contents of organic C or oxalate soluble Al. The content of oxalate soluble Fe tends to be somewhat higher in the clay soils and organic soils than in the groups of the coarser mineral soils. On an average, exchange acidity is highest in the organic soils. This may be partly due to the lower ratio ofextraction in these soils as compared with that in the mineral soils. In the mineral soils, the acidity replaced by unbuffered KCI is less than 10 me/100 g, even in samples with a pH value below 3.5. The proportion of Al in exchange acidity of these soils was studied only superficially. With the method used no Al was found in the KCI extract of soils with a pH higher than 5.3. There were two clay soils with pH 5.3 which contained almost 0.1 me Al/100 g. About two thirds of the clay samples, and silt and loam samples were more acid than pH 5.3. To this part belonged 85 % of the sandy soils, and 95 % of the organic soils. Data for these samples are recorded in Table 2. One half of these organic soil samples did not contain KCI-extractable Al. This was true also with one fourth of the clay soils and silt and loam soils, but only with one fifth of the sandy soils, with a pH value less than 5.3. The highest amounts of exchangeable A 1 were found in some very acid postglacial clay soils and in some virgin peat soils. Though the average content of exchangeable A 1 is equal in the clay soils and in the organic soils, the proportion of A 1 of the exchange acidity (AI + H), or of the cation exchange capacity (AI -f-H + Ca + Mg in the KCI extract) is, on the average, distinctly lower in the organic soils than in the clay soils. Also in the other mineral soils, exchangeable A 1 corresponds to an, averagely, distinctly higher part of the exchange acidity than in the organic soils. Other factors being equal, usually the amount of exchangeable A 1 tends to be the higher the lower the soil pH is. The regression is curvilinear, though often the part below pH 5 in water or pH 4 in N KGI does not markedly deviate from a linear relationship. In the samples listed in Table 2, the amount of exchangeable AI as me/100 g is in the clay samples closely correlated with the soil pH (total linear correlation coefficient r = -o.9l***), far less closely in silt and loam soils (r = -o.66***) and sandy soils (r = -o.sB***) and even more poorly in the organic soils (r =-o.4B***). The correlation between the proportion of A 1 of the exchange acidity and pH was marked only in the clay soils (r = --o.Bo***). Elimination of the effect of organic C did not increase the correlation between pH and the proportion of A 1 in exchange acidity. Titratable acidity neutralized at pH 8.2 was very high in the peat samples, usually between 60 and 120 me/100 g. In the other organic soils it was lower, but in every case markedly higher than the corresponding exchange acidity. Thus, even their difference, titratable nonexchangeable acidity, is high in the organic soils (Table 1). In the mineral soils this part of soil acidity ranges from 0.3 to 27 me/100 g. There is no significant difference between the mineral soil groups in this respect, though the mean value of clay soils tends to be somewhat higher than the means of the coarser mineral soils. The relation of titratable nonexchangeable acidity to other soil properties was first studied by calculating total and partial linear correlation coefficients between these variables. As could be expected, no correlation was found between titratable nonexchangeable acidity and the clay content within the various groups. The other results are recorded in Table 2. In the clay soils titratable nonexchangeable acidity is surprisingly closely correlated with oxalate soluble Al (r = o.B9***), and even in the silt and loam samples this relation is distinct (r = o.77***). In the sandy soils it is almost insignificant (r = 0.25*), and in the organic soils there is no correlation. Elimination of the effects of organic C, pH, and oxalate soluble Fe tends to decrease this correlation in clay soils and silt and loam soils, but to increase it in the other soil groups. Though titratable nonexchangeable acidity is relatively closely correlated with the organic C content in all soil groups, taking into account the variation in oxalate soluble Al results in the clay soils in a low partial correlation coefficient, r = o.42***. In the silt and loam soils only a slight decrease is found, but in the sandy soils and organic soils no effect is detectable. Elimination of the effect ofoxalate soluble Al also decreases the closeness of correlation between titratable acidity and pH in the clay soils, but increases it in the sandy soils. Both in the clay soils and in the silt and loam soils, elimination of the effect of oxalate soluble Al reduces the correlation between titratable nonexchangeable acidity and oxalate soluble Fe to zero. Partial correlation coefficients between titratable nonexchangeable acidity and each of the variables studied indicate that after the elimination of the effect of the three other variables, in clay soils the relation is relatively closest with oxalate soluble Al. In the silt and loam soils the content of C and pH appear to be more important. This is more distinctly the case with the sandy soils, and in the organic soils even oxalate soluble Fe seems to deserve more attention than Al in this respect. Calculation of the coefficients of determination and multiple determination shows that in the different soil groups the following percentage of variation in titratable nonexchangeable acidity may be explained by oxalate soluble A 1 and the other variables: The relationship between titratable nonexchangeable acidity as me/100 g (x 1), the content ofoxalate soluble A 1 mmol/100 g (x 2), organic C % (x 3), pH (x 4), and oxalate soluble Fe mmol/100 g (x 5) conforms to the following regression equations: In the clay soils = 0.78 x 2 + 0.87 x 3 2.65 x 4 0.12 x 6 + 15.56 The coefficient of multiple correlation is R = o.94***, and the standard error of estimate is S = 2.45. In the silt and loam soils x x = 0.19 x 2 + 1-80 x 3 2.60 x 4 + 0.19 x 5 + 14.06 R = o.97*** and S = 1.90 In the sandy soils The relative importance of these four factors affecting titratable nonexchangeable acidity may be compared on the basis of the following values of standard partial regression coefficients or (ä-coefficients: The rank of these four variables in order of importance is the same as it is according to the coefficients of partial correlation (Table 3). Table 3. Total and partial correlation coefficients for the relation between titratable nonexchangeable acidity (1), oxalate soluble A 1 (2), organic G (3), pH (4) Discussion The terms used for the characterization of different parts of soil acidity vary, as do also methods of their determination. Unbuffered N KCI is nowadays again employed for the replacement of acid cations at »the soil pH», and titratable acidity is often measured with BaCl 2 -triethanolamine at pH 8.2. In this work, KCI was used instead of BaCl 2 even in the latter case, and it is likely that the results differ to some extent from those which could have been obtained with BaCl 2 . Apparently, in most soils with a low anion exchange capacity, titratable nonexchangeable acidity is more or less equivalent to »the pH-dependent cation exchange capacity» or the difference in the CEC e.g. at pH 8.2 and at the pH of soil. Usually, it is mainly attributed to weak acidic groups of organic matter, but also to some extent to the proton dissociation of exposed OH-groups of clay particles. Yet, according to Bolt (1961) the exchange capacity of montmorillonite and illites remains practically constant in the pH range from 4 to 8, and deViLLiERS and Jackson (1967) proved that no pH-induced CEC was present in kaolin and vermiculite. These authors claim that the »latent acidity» of clay minerals free of sesquioxide coatings is provided only by the deprotonation ofA!OH 2groups at edges of fixed interlayer hydroxy-Al units. This kind of polymeric hydroxy-Al interlayers are typical of chemical weathering of acid soils (Jackson 1963), and apparently, these intergrade minerals are not lacking in Finnish clays (Soveri 1956). Yet, it is likely that positively charged Al hydroxides as surface coatings contribute more to the pH-dependent CEC and titratable nonexchangeable acidity in our soils than as blocks of the interlayer spaces of 2:1 clay minerals. Tamm's acid ammonium oxalate is supposed to extract from soils free Al oxides and hydrous oxides. It is doubtful, whether the hydroxy Al polymers in the interlayer spaces of layer silicates will be dissolved to any significant degree (cf. Dixon andJackson 1962, Wiklander andAleksadrovic 1969). It is also uncertain, whether Al fixed by soil organic matter is completely released by this extract. On the other hand, it probably dissolves Al compounds which are not blocking exchange sites or acting as proton donors. Although the amount of Al in Tamm's acid oxalate extract would not be equivalent to these forms of Al, there apparently exists a relatively close correlation between them in the clay soils. In the other soils this correlation may be poorer, or then in these soils Al actually is less closely connected with titratable nonexchangeable acidity. In the samples of the clay soil group the clay content ranges from 30 to 95 per cent, but no correlation was found between it and titratable nonexchangeable acidity. On the other hand, the latter was surprisingly closely correlated with the content of oxalate soluble Al, even more closely than with the content of organic C. The partial correlation coefficient for the relation of titratable nonexchangeable acidity and the content of organic C, when the effect ofoxalate soluble Al was eliminated, was markedly lower than the corresponding total correlation coefficient. This may be taken to indicate that a marked part of the total correlation between titratable nonexchangeable acidity and organic C might be due to Al fixed by organic matter. In all mineral soil groups, but particularly in the clay soils, oxalate soluble Fe appeared to be less important than A 1 in relation to titratable nonexchangeable acidity. This could be caused by the possibility that acid oxalate removed in dark only the most reactive part ofFe hydrous oxides (cf. Schwertmann 1964),and this part is not closely correlated with the amount of Fe which would contribute to soil acidity. On the other hand, the present results are in accordance with the findings of that Al hydroxide seems to cover negative sites of clays more effectively than Fe hydroxide. In the organic soils the correlation of both oxalate soluble Al and Fe with titratable nonexchangeable acidity was insignificant or very low, and Fe seemed to be more important than Al. Because this group was small and heterogeneous, it is obvious that a larger material and more thorough studies are needed before any conclusions may be drawn on the mutual contribution of Al and Fe to titratable nonexchangeable acidity in various kind of organic soils. It is likely that, at least, a small part of the titratable nonexchangeable acidity even in Finnish soils originates from anion exchange. Sulphate or phosphate ions or other acid radicals sorbed by positively charged Al and Fe hydroxides may be replaced by OH ions. On the basis of the present data, it is not possible to estimate the contribution of this exchange to the consumption of base when these soils were titrated. The relatively high phosphate sorption capacity of our soils (Kaila 1959(Kaila , 1963 points to the possibility that this will not be quite insignificant. Though further studies are necessary to elucidate more thoroughly the role of A 1 in acidity of Finnish soils, it is apparent that A 1 not only as monomeric trivalent ion, but probably to a much larger extent as polymeric hydroxy Al, increases the amounts of lime needed to amend our soils.

v3-fos

2018-04-03T05:49:10.828Z

1971-02-01T00:00:00.000Z

22291608

s2

Method for Studying Particle Size and Infective Potential of Infectious Bovine Rhinotracheitis Virus Aerosols A technique is described for estimating the number of potential respiratory infectious loci of aerosolized infectious bovine rhinotracheitis virus. The Anderson (1) aerodynamic particle sizing sampler was used in the past decade to collect and size bacterial particulates. Airborne particles are impacted directly on nutrient agar surfaces in six stages of aerodynamic sizes. Because of localized drying of the cell monolayer at the impaction site, it is implausable to collect virus particles directly on a cell monolayer. The useful-ness of the Andersen sampler was extended to virus by Jensen the surface and assayed the for plaque-forming (PFU). Mitchell a gelatin collection surface which was melted and assayed. procedures an estimate of PFU for each size category of the sampler but do not provide information as to the number of particulates involved. A single particle may contain many PFU but produce only one locus of infec-tion. Alternatively, many particles may each contain few PFU, and thus a large number of loci be produced from a small quantity of These differences result not from variations in the but from dissimilarities in the manner of these study of response relationships of A technique is described for estimating the number of potential respiratory infectious loci of aerosolized infectious bovine rhinotracheitis virus. The Anderson (1) aerodynamic particle sizing sampler was used in the past decade to collect and size bacterial particulates. Airborne particles are impacted directly on nutrient agar surfaces in six stages of aerodynamic sizes. Because of localized drying of the cell monolayer at the impaction site, it is implausable to collect virus particles directly on a cell monolayer. The usefulness of the Andersen sampler was extended to virus collection by Jensen (3), who washed the agar surface and assayed the washings for plaque-forming units (PFU). Guerin and Mitchell (2) and Wolfe et al. (4) proposed a gelatin collection surface which was melted and assayed. These procedures provide an estimate of PFU for each size category of the sampler but do not provide information as to the number of particulates involved. A single particle may contain many PFU but produce only one locus of infection. Alternatively, many particles may each contain few PFU, and thus a large number of loci could be produced from a small quantity of virus. These differences result not from variations in the virus but from dissimilarities in the manner in which it was aerosolized. A knowledge of these parameters is desirable in the careful study of dose response relationships of airborne infection. A technique capable of providing an estimate of the number of infectious particles and hence an estimate of the number of potentially infected loci in the lungs is presented here. The technique consists of collecting the aerosol by jet impaction on agar-solidified tissue culture media. After collection the agar is inverted on a monolayer of susceptible cells thus forming an overlay. Two strains in infectious bovine rhinotracheitis (IBR) virus were utilized in developing this sampling technique. Strain "V" is a vaccine strain originally obtained from Armour-Baldwin Laboratories. This particular strain was useful because it was a readily available attenuated virus and produced consistant plaque characteristics in testing the technique. The other strain, LY 985-3, was isolated from a field case of IBR at Utah State University. Specific viral suspensions were aerosolized into a test chamber of 2 ft3 with a deVilbis clinical atomizer and equilibrated for 1 min. The virus laden particulates were collected from the test chamber by using an aerodynamic sizer model 30 (AIR, Inc., Logan, Utah). Molded glass collection plates filled with 27 ml of tissue culture media solidified with 1.5% Noble Agar (Difco) were used. The agar surface may be conditioned by storage for several days or by inverting the opened dishes on a sterile rack for a few hours before use in the sampler. This conditioning removes the surface water which tends to distort the impaction pattern. Water removal also assists in applying the agar surface to the monolayer without slippage. Transfer of the agar from the collection plates to the cell monolayer is facilitated by cutting a ring in the agar }j inch from the collection plate wall. A no. 11 surgical blade fitted with a gauge may be used for this purpose. The peripheral ring of agar is pulled from the corner of the dish while inverted. The agar disc is then held vertical and pried away from the glass plate with a sterile spatula. About one-third of the disc can be separated from the glass without danger of the agar falling. The spatula is then carefully inserted under the agar as the plate is turned back to horizontal. The agar layer may then be transferred to the cell monolayer in a petri dish from which the liquid media have been aspirated. Any air trapped under the agar must be removed and can be done by stroking the agar with the spatula or a sterile swab. Plaques appear as necrotic areas in the cell APPL. MICROBIOL. monolayer when viewed with the low power microscope. The use of a petri dish clamp on a mechanical stage facilitates scanning the areas to be counted. The results of aerosols generated from two virus suspensions are shown in Table 1. The distribution of counts among the six stages reflects the particle size distribution of the aerosol. The LY 985-3 suspension had a virus titer approximately 10 times that of strain "V." Precautions against including areas of damaged cells in the plaque count should be observed. The immediate areas of the agar edge and bubble areas can be removed from consideration in the plaque count by use of counting masks made by punching holes of known size in self-adhesive paper stock. The masks can be placed on the petri dish over randomly selected areas that are not otherwise affected by the factors mentioned. For example, since the impaction areas of each stage of the sampler are approximately 7.07 square inches, a circular mask 1 inch in diameter represents 11.1 % of the collection surface. Therefore, the average count of 1-inch masks should be multiplied by 9.0 to obtain an estimate of the number of positive impact areas per stage containing at least one PFU. Characteristics of the model 30 aerodynamic sizer permit more than one particle to be collected on a specific impact area. This may result in a cluster of microplaques in one impact area. Such areas merge into single plaques with time and should be counted as one plaque. This estimate may then be transformed to a particle count estimate by use of a conversion table supplied with the instrument. This technique, therefore, estimates in each of six size categories the number of potentially infectious particles in the respirable air of experimental animals.

v3-fos

2018-04-03T05:16:29.650Z

1971-05-01T00:00:00.000Z

42204762

s2

Elutriation and Coulter Counts of Tetrahymena pyriformis Grown in Peanut and Cottonseed Meal Media Growth of Tetrahymena pyriformis W has been used to evaluate nutritional quality of peanut and cottonseed meals. An efficient elutriation method is described for separating cells of this organism from particulate matter left in the substrate (enriched with basal medium) after 4 days of incubation. After elutriation the cells can be counted with a Coulter counter by using calibration procedures which are presented. Elutriation and Coulter counting provide a rapid and efficient method of measuring the growth response of T. pyriformis W. Utility of the method is demonstrated by agreement between Coulter counts and visual counts of the cells and by demonstration of a linear response of cell numbers to substrate nitrogen. formnis W has been used to assess relative nutritional quality, growth being measured by microscopic counting of the cells, and is described in recent reviews by Celliers (9), Reynolds (21), and Evans and Bandemer (12). Microscopic cell counting to assess the growth response is very tedious, time-consuming, and subject to error particularly in the range of relatively high concentrations of the cells, food, and other particles. Several investigators have stressed the need of a quantitative method of separating the cells to allow automatic counting and to allow the criteria for protein quality to be based on parameters such as weight gain or nitrogen balance (W. R. Fernell and G. D. Rosen, Abstr. Proc. Nutr. Soc., vol. 13, p. xviii, 1954) measured in terms of protein synthesis (13, 1; W. R. Fernell and G. D. Rosen, Abstr. Proc. Nutr. Soc., vol. 13, p. xviii, 1954), dry weight (24), or total nitrogen (24) in addition to cell yield. This is a report of a quantitative method of separating preserved T. pyriformis W cells without microscopically visible damage from particulate substrates, especially peanut and cottonseed meals, and the development of a method for electronically counting the cells of various sizes in each population using a Coulter counter. MATERIALS AND METHODS Assay. The procedure was essentially that previously described (28), with some exceptions. The peanut and cottonseed meals were fed to the test organism in the same form as they were fed to animals in feeding tests and were ground to pass a 40-mesh screen. The residual oil and any fatty acids left after processing were not extracted before microbiological assay, although it is known that some free fatty acids inhibit the growth of this test organism (16). The aqueous suspensions of the test substances were prepared to contain approximately 5 mg of nitrogen per ml after adjustment of the pH to 8.2 and were then analyzed for total nitrogen (3). The analyses of homogeneous test substances, e.g., casein, were made on duplicate subsamples of 3 ml each. The analyses of heterogeneous substances, e.g., the oilseed meals, were made on duplicate composites each prepared by transferring 1.0, 0.75, 0.50, 0.40, 0.25, and 0.1 ml of suspension with a bacteriological pipette into a macro-Kjeldahl flask. Each composite then consisted of 3 ml estimated to contain ca. 15 mg of total nitrogen. The composites were made in this manner to include sampling error similar to that possible in the assays. The components of the basal medium were not lyophilized. The various stock solutions for this basal medium were prepared separately by using the methods of Baum and Haenel (5) with some modifications. The stock solution of the vitamins was prepared at COUNTS OF TETRAHYMENA PYRIFORMIS W soluble starch) were mixed and stored frozen at 2.5X final concentration. A 20% solution of soluble starch was prepared as required. For each assay, 4 ml of solution or suspension of test substrate of known total nitrogen concentration, 1 ml of the mixed vitamins, 1 ml of 20% soluble starch (Difco), and 4 ml of mixed solution of the salts, purines, and pyrimidines were autoclaved in a 2-oz bottle with a screw-cap at 120 C for 15 min. After cooling, each bottle was inoculated with two drops (approximately 0.03 ml each) of actively motile cells of T. pyriformis W from a composite of two 3-day 10-ml proteose peptone broth cultures. The bottles with loose caps were placed in special racks at a slope of 150 to the horizontal to insure a high surface to volume relationship of the culture medium. The inoculated cultures were incubated for 4 days to ensure that comparisons were made on cultures of motile organisms of near maximal population (14), at 24.9 +: 0.5 C at 60 to 90% relative humidity. Sampling for counts was made from single cultures. Elutriation of the cells from the particulate substrates. A suitable subsample (usually 1 ml) of each culture of known substrate nitrogen content mixed on a shaker was transferred to 4 ml of potassium phosphate preservative (28) in a 12or 15-ml graduated, conical centrifuge tube with a screw-cap or a tightly fitting plastic cap. The preserved culture is later referred to as a subsample. The tube was shaken during addition of the culture and for several minutes afterwards to avoid clumping of the cells. The elutriation apparatus is shown in Fig. 1. A stirring magnet (3 by 10 mm, Teflon-covered) was inserted in the tube of preserved subsample, saline (0.9% NaCl plus 0.1% formalde-hyde) was added to bring the volume to ca. 10 ml, and the suspension was mixed with a magnetic stirrer. The suspension was viewed with a hand lens (Aspheric Cataract Reader, 50 mm in diameter, 20 diopters, Combined Optical Industries Ltd., Bath Road, Slough, England). After extraneous particles settled from the upper part of the suspension and the cells looked clean, part of the suspension was drawn off through the plastic tube with the syringe in the continuous pipetting outfit (Becton, Dickinson, and Co., Rutherford, N.J.) and transferred through a 100-mesh and then a 200-mesh (sieve opening, ca. 0.074 mm) stainless-steel screen, each held in a stainless-steel membrane-holder (Millipore Corp., Bedford, Mass.) into a volumetric flask of suitable size for the final dilution desired for counting. The dilution was usually 1: 200. The elutriation process was repeated until the extraneous particles left in the centrifuge tube were washed free from cells, a clean tube was substituted, and the apparatus was washed well with saline-preservative. This saline was also transferred into the volumetric flask. Coulter counting of the cells. For Coulter counting, the cells are suspended in an electrolyte. The suspension of cells is drawn under vacuum through an aperture of known size in a tube which has an immersed electrode within it and another outside of it. These electrodes cause an electric current to flow through the aperture. Each cell passing through the aperture displaces an equal volume of electrolyte causing a modulation (or resistance) in the electric current. This modulation is detected by the instrument as a signal or pulse. The pulses are electronically amplified and automatically counted. volumes. An upper threshold setting is chosen to screen out counts of particles above a predetermined volume, and a lower threshold is selected to screen out counts of particles below a selected volume. Therefore, the dimensions of cells of various sizes and shapes grown in several substrates, including casein, were measured microscopically with a calibrated micrometer. The volumes of the cells were then calculated as prolate spheroids, since the predominant type was pyriform, or as spheres, even though the round types are not quite spheres. Cell dimensions and counter settings. Of numerous cells measured, the smallest were almost round, approximately 15 to 20 ,m in diameter. The largest cells, pyriform, were 30 by 70, 28 The ranges of the calculated volumes of the cells, assumed to be primarily prolate spheroids and secondarily spheres, are given in Table 1 with the settings of the Coulter counter (model B, aperture 140 Mm) required for counting these volumes. The particles are counted according to volume and not as to shape. Even if the cells were rectangular, the volume of the largest measured would be included for counting. Each time the instrument was repaired, it was recalibrated, and the "zero" of the threshold settings was checked frequently. A model B Coulter counter, described by Barnes et al. (4), was used with a tube with an aperture of 140 M4m and without the size distribution plotter. It was calibrated with ragweed pollen, diameter predominantly 20.3 MAm, to obtain a constant for determining the volumes of particles that would be counted at stated thresholds and settings of the instrument. The method of calibration is described in the manual supplied by the manufacturer of the instrument. The settings were chosen to include the range of the volumes of the smallest, the predominant size, and the largest cells measured. Two-milliliter subsamples of the diluted cell suspension were counted. At least three readings were made and averaged for each of the three ranges of cell volumes. The recorded total cell count is the sum of these means. The electrolyte used in the counting assembly was 0.9% NaCl plus 0.1%,O formaldehyde, filtered through a membrane (Millipore Corp.) of 4-,Mm porosity and counted to be sure it was substantially free from particles the same size as the particles to be counted. The functioning of the instrument was checked periodically by testing the zero settings of the thresholds, by recalibrations with the ragweek pollen, and by counting the particles in weighed samples of pecan pollen (Hugh Graham Laboratories Division, Hollister-Stier Laboratories, Dallas, Tex.). The pollens were suspended in the electrolyte at known volume and counted at the same settings used for the T. pyriformis cells. The diameter of the pecan pollen was reported to be predominantly 45 to 50 ,m (48,032 to 65,888 Mm3), and the particles were considered to be "essentiallv mono-sized." It was expected that these particles could be counted at the same instrument settings that were selected for counting the cells of the test organism. As shown in Table 2, this was true. If blocking of the aperture occurred too frequently for one subsample, another elutriated subsample was poured through a 200-mesh stainless-steel funnel from the volumetric flask into the beaker for counting. If another subsample was not available, the one partly used for counting was filtered in the same manner into another beaker, stirred well, and counted. No corrections were made for coincidence counting. Growth response. The growth response was evaluated by computing the linear regression coefficient for the regression of numbers of organisms per unit of medium, without the restriction that the line of response must pass through the origin. RESULTS Elutriation of the cells. During development of the elutriation method, its adequacy for quantitatively transferring the cells from the particulate substrates to another medium for counting was checked by microscopic examination of rinsings of the stainless-steel screen after elutriation of numerous samples. Few, if any, cells were found in the rinsings. The adequacy of the method was also checked to determine if extraneous particles of sizes similar to the cells were being transferred into the suspensions to be counted. Microscopic examinations of numerous cell suspensions diluted for counting showed that they were virtually free from extraneous particles that might be counted as cells. The number of cells lost during elutriation was negligible. This is shown by examples of counts of cells not elutriated and elutriated from proteose peptone broth in Table 3 and from casein in Table 4. Six subsamples were elutriated in about 1 hr, and usually two sets of six subsamples could be partitioned in 1 day. This included setting up parts of the equipment, washing the necessary parts at the end of the runs, and diluting the elutriated cells for counting. The time for the 21,1971 on May 7, 2020 by guest http://aem.asm.org/ Downloaded from procedure could be reduced by making the apparatus automatic with solenoid valves and timing devices, by the addition of saline under pressure, and by the removal of the cell suspension under vacuum. The volumetric flask was chosen so that the cells were diluted properly for counting without further transferring. By trial, a 1:200 dilution of the original sample was usually suitable. Cells were stable in cultures preserved in phosphate buffer with 6% formaldehyde (28) for 2 years before elutriation. Growth response evaluated by visual and Coulter counts. Results of visual and Coulter counts of the test organism grown in several substrates and results of replicated automatic counts are shown in Table 3. The automatic counts tend to be lower than the visual ones at the higher test substrate nitrogen concentrations assayed but check fairly well at different dilutions and volumes of subsamples. Previous studies of assays of various protein concentrations (28) and of numerous cottonseed meals (unpublished data) showed that their relative nutritive value could be determined by comparing the growth response of the organism, determined by visual counts, at various levels of nitrogen of the test substance up to about 1 mg per ml of final medium, the range in which the response was linear. This is illustrated in Fig. 2 with results obtained with a very good quality meal and a moderately good one, before and after heat damage. In developing the elutriation and Coulter counting methods, a cottonseed meal and a peanut meal were assayed at various nitrogen concentrations up to ca. 2 mg per ml of final medium, and the growth response was determined by both types of counts. The results (Fig. 3) were linear up to at least 1 mg of nitrogen per ml. The visual counts of cells in diluted substrate were made within a few days of sampling to avoid possible evaporation of the liquid, since it was not feasible to make up the volume just before counting. For these two meals, preserved subsamples for Coulter counts were stored diluted with the phosphate buffer preservative in 25-ml volumetric flasks. Just before elutriation, each suspension was quantitatively transferred to a centrifuge tube. Therefore, for these two meals, one more transfer was required than for subsequent assays. For the regression lines shown in Fig. 3, the slope b for the cottonseed meal is 0.5769 for the visual counts and 0.4608 for the Coulter counts. For the peanut meal in this same range, b is 0.6947 for visual counts and 0.5193 for the Coulter counts. The statistics for all of the data for each of these two meals are shown in Table 5. The probability for the correlation between the nitrogen levels and the growth response for each type of counts for both meals is > 99.9. Although for each meal b is somewhat greater for the visual than for the Coulter counts, it seems feasible that the latter could also be used in determining the nutritional index. Two more peanut meals were assayed with the Coulter counts spot-checked by visual counts (Fig. 3). The efficacy of the elutriation and automatic counting methods was tried on five more peanut meals before using the methods to determine the Oval svmbols, xy. relative nutritive value of a series of treated and untreated meals with the growth response determined by Coulter counting only. As shown in Fig. 4, the response was again linear in a meal nitrogen range of as high as ca. 1 mg per ml of final medium. These results indicate that the growth response of T. pyriformis W to peanut and cottonseed meals at various meal nitrogen contents of at least 1 mg per ml of final medium, evaluated by Coulter counting of cells elutriated by the described method, can be used to demonstrate differences between meals. A study is to be reported on the successful use of this assay by the described procedures to predict for higher Table 4. Waithe (30) reviewed reports of other investigators who tried to obtain undamaged, washed suspensions of protozoa from nonparticulate media for physiological studies, but no report is known of an adequate method of separating Tetrahymena or other protozoa from a particulate medium. In addition, no report was found giving details of Coulter counting of a population of Tetrahymena of various sizes. Elutriation of the cells from particulate substrates. Attempts were first made to partition the cells from other particles in the media by methods similar to those suggested by Albertsson (1). Two water-soluble polymers, arabinogalactan (kindly supplied by Stein, Hall and Co., New York, N.Y.) and sodium carboxymethyl cellulose, were tried at various concentrations. Both were satisfactory in removing the relatively large meal particles, but the lightest-weight extraneous particles of various sizes remained in the phase with the cells. The most dilute concentrations of the polymers used were the best for partitioning the cells, so the potassium buffer of the basal medium and then 0.9% aqueous sodium chloride were tried. Both were satisfactory for the purpose. The latter plus 0.1 % formaldehyde is suitable for use in a Coulter counter. In the buffer and in the saline, the meal particles in a mixed suspension settle faster than living or preserved cells in separatory pyriform funnels and conical centrifuge tubes but not in straight-sided vessels, such as cylinders or ordinary test tubes. The funnels of various sizes, especially 125 ml, are satisfactory for large volumes of culture. Volumes of the cells. The volumes given are within the ranges of those reported for T. pyriformis W calculated as prolate spheroids by Cashland and Johnson (8) and Reynolds and Wragg (22). These volumes are also within the ranges of other strains whose volumes were calculated by other methods by Thompson (Ph.D. Thesis, Univ. of Alabama, 1960), Thormar (29), and James and Read (14), and for an unidentified strain during different phases of growth but with the method of volume calculation not reported by Summers, Bernstein, and James (26). The dimensions of width and length are within those given for strain W by R0lle (23) and for 16 strains of T. pyriformis, including W, by Loefer (19). Method of Coulter counting. The method of using the instrument had to be devised because of the scant information found in the literature. There are a few reports of Coulter counting of T. pyriformis G, precooled to 0 C, with "a capillary tubesize 100 ,um," Byfield and Scherbaum (6, 7) counted T. pyriformis GL and WH-14, and Wille and Ehret (32) and Szyszko et al. (27) reported counts of T. pyriformis using a model B but did not give details of the method used. Coincidence counting. The results were not corrected for coincidence counting. For the assays of peanut and cottonseed meals, this apparently was not necessary under the conditions of this study. It is not feasible to determine the coincidence error per se for every type of sample counted at various cell-volume ranges and substrate nitrogen levels because of the amount of time required to assay, preserve, and elutriate enough subsamples and then make dilutions and counts to calculate the correction required. The method of averaging at least triplicate counts for each of the three cell-volume ranges and using the sum of the means for the final count should reduce this error to a minimum. Each of these nine counts of the cells grown at a particular nitrogen concentration is made on a different sample of the cell suspension of high dilution. Actually, determining the final counts at various substrate nitrogen concentrations is in effect determining the counts at various dilutions of the original sample. The results of final Coulter counts of various dilutions and sizes of subsamples of the organism grown in proteose peptone broth, peanut meal, and cottonseed meal (Table 3) and the good checks of final counts of replicate subsamples of the organism grown in the higher levels of casein during several years ( (31) made counts of T. pyriformis W in duplicate with a model B instrument without the distribution plotter and found they usually agreed within 0.5% when the average counts were corrected for coincidence. These investigators did not describe the method they used for counting or for the coincidence correction. Discrepancies between the two types of counts are probably not due to changes in temperature at the aperture (4), since the model B has built-in automatic compensation for such changes (Coulter Manual for the Model B Coulter Counter, 5th ed. 4-67, 420100201, Coulter Electronics, Inc.), or of changes in the temperature of the cell suspensions which were counted at room temperature regulated by air conditioning. Degkwitz and Selle (10) found discrepancies due to variations in ambient temperatures in a laboratory without air conditioning. Anderson, Petersen, and Tobey (2) observed anomalies in the determination of cell number by Coulter counting of Chinese hamster ovary cells due to the failure of daughter cells to separate after mitosis. However, this is not true for the T. pyriformis W assays incubated for 4 days. Only an occasional pair of dividing cells was found during visual counting of each assay of more than 50 substrates, including casein (Teunisson, unpublished data), each assayed at six or more nitrogen levels. Discrepancies would more probably be due to sedimentation of cells in the more dense suspensions during counting, but more study is required on this aspect of the problem. The method has not yet been tried for separating living cells, but it seems reasonable to assume that it could be used for this purpose. Since T. pyriformis W is negatively geotrophic and grows best with aeration, the actively motile cells swim near the surface of stationary medium, and the less motile cells can be easily suspended by gentle shaking of the container and could also be removed from the upper portion of the diluted suspension. A method of harvesting Tetrahymena cells without the deleterious effect of centrifuging is desirable. This is especially so for cells grown in Tween (15), for cell extraction procedures, and for enzyme and cytological studies (11). The procedure might also be used to separate other organisms from particulate substrates for similar purposes; in cell culture studies, e.g., to separate trypsinized cells from fibrous tissues; or to determine the microbial populations in food slurries, soil suspensions, washed fibers, etc., without adding nutrients or inhibitory substances from the test substrates. Other possible uses for the elutriation procedure. In addition to cell yield, other parameters of growth response can be determined on cells elutriated from particulate substrates. For the assay of nutritional quality of oilseed meals or of other proteinaceous substances with T. pyriformis W, measurement of cell nitrogen or dry weight produced would be desirable.

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2020-12-10T09:04:11.248Z

1971-05-01T00:00:00.000Z

237230326

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Preliminary Observations on the Pathogenesis of a Virulent Strain of Newcastle Disease Virus in Chickens Development of Newcastle disease, after experimental and natural infection with the virulent strain VLT of Newcastle disease virus, and its growth and distribution in some selected tissues as assayed by the enumeration of plaques are reported. The plaque technique developed by Dulbecco (1) has been extensively used in studies of Newcastle disease virus (NDV; references 2, 6-8). Despite its potential value in yielding precise determination of virus, the technique was little utilized in studies of the pathogenesis of the disease in the domestic fowl or in epizootiological investigations. Earlier workers (3,4,9) have reported successful isolation and enumeration of different strains of NDV in embryonated eggs from various tissues after experimental or natural infection of chickens. In this paper we describe the development of Newcastle disease (ND) in chickens after experimental and natural infection and present data on proliferation of the virus in some selected tissues, as enumerated by the plaque technique. The virulent strain VLT of NDV, isolated during 1968 from an outbreak of highly fatal ND among chickens at Talamara, Lebanon, was plaque purified and used in its third chick embryo passage level for these experiments. Virus stock was prepared by inoculating with approximately 104 plaque-forming units (PFU) in 0.1 ml of seed virus into the allantoic cavity of 9-day-old chick embryos. Infected allantoic fluid was stored in 1-ml amounts at -60 C. Primary chick embryo cell cultures were prepared from minced 9-day-old decapitated chick embryos subjected to repeated trypsinization. The cells were grown in Eagle's minimum essential medium (MEM) containing 5% calf serum and 8% Tryptose phosphate broth. Approximately 5 x 106 cells per 2-oz prescription bottle were seeded, and monolayers were overlaid after infection with 6 ml of overlay medium, which consisted of Hanks balanced salt solution without phenol red, 0.5 j' lactalbumin hydrolysate, 1.0 Noble agar (Difco), 3 %-y, horse serum, 1.5% of 1:1,000 dilution of neutral red, 5% of 4.4%,, sodium bicarbonate solution, 100 units of penicillin and 100 ,ug of streptomycin per ml. Monolayers were used for virus assays 3 days after seeding (10). Seventy White Leghorn six-week-old chicks were divided into seven groups of 10 each. They were caged and placed in a room in which birds were never kept before. To avoid the risk of transmission of infection through feed and water supplies, each group was provided with its own feed and water. One group was maintained as contact controls, and the birds in the remaining groups were inoculated intramuscularly (pectoral muscles) with 0.5 ml of serial 10-fold dilutions of VLT strain of NDV. Chicks were observed twice daily. At postmortem examination, brain, spleen, trachea, and lung tissues were removed aseptically with separate, sterile instruments to avoid cross contamination from dead chickens as well as from sick ones killed by cervical dislocation. A 10%, (w/v) suspension of tissue was made in MEM containing 1,000 units of penicillin and 1,000 ,ug of streptomycin per ml. Tissue suspensions were kept frozen at -60 C until tested. All end points were calculated by the Reed and Muench method (5). The embryo-propagated virus stock had a titer of 109.6 50% chicken lethal doses per 0.5 ml, indicating that the virus strain is highly virulent for susceptible chickens. The incubation period varied from 3 to 4 days, but the majority of birds showed symptoms on the third day. Respiratory symptoms with rales were pronounced in almost all birds. Most birds also showed typical nervous symptoms. Death generally oc-946 curred within 2 to 4 days after the onset of the symptoms. Torticollis and lateral movement of the head were commonly observed. A few birds developed paralysis of both legs. The gross pathological lesions consisted of extensive involvement of the proventricular submucosa and intestinal follicles. Severe hemorrhagic necrotic lesions adjacent to lymphoid plaques were also common. Infection spread easily to the contacts as signs appeared 4.8 days after probable exposure (Table 1). A summary of virus titers in various tissues is presented in Table 2. The VLT strain multiplied extensively in the tested tissue, namely, spleen, trachea, brain, and lungs. Virus titers varied between 10-3 and 108.1 PFU/g of the brain tissue, indicating the relationship between concentration of virus in the brain and occurrence of nervous symptoms. Large amounts of virus were present in the tested tissues even on the day the chickens first showed symptoms, indicating that generalization of NDV in chickens probably occurred before the onset of clinical signs. Virus may therefore be excreted 1 or 2 days preceding the appearance of clinical signs. From the practical standpoint, it seems that probably the trachea is more suitable than other tissues for virus recovery. Bird 1848, which had a moderate quantity of virus in its trachea but not in the spleen, brain, or lung, remained apparently healthy, whereas its cagemates died on the sixth day. In bird 1844, which almost recovered after showing respiratory and nervous signs (paralysis of extremities), a high concentration of virus was found in the trachea and brain when killed on the 10th day. It is possible that such birds can become effective carriers. From our study it can be concluded that the strain VLT is highly pathogenic for 6-week-old chickens. In our experience it spread readily and multiplied extensively in various tissues after experimental and natural infection. Critical organs, damage of which reflects the occurrence of symptoms, were the brain, trachea, and lungs. The virus seemed to generalize before the onset of clinical signs, and we consider that it could disseminate during this period. It has been observed that some birds can recover and may possibly become carriers after infection. The po-947 NOTES tential value of the plaque technique in primary chick embryo cell culture for the study of pathogenesis of ND has been indicated.

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2020-12-10T09:02:23.753Z

1971-10-01T00:00:00.000Z

237232966

s2

Substitution of Milk for Serum in the Production of Human Leukocyte Interferon The presence of serum in suspensions of Sendai-induced human leukocytes is necessary for the synthesis of significant amounts of interferon. Very little interferon is obtained from serum-free suspensions. Cow's milk or milk casein can substitute for serum in the production of high yields of human leukocyte interferon. Suspensions of human leukocytes require high serum concentrations to yield substantial quantities of interferon (1, 7). Because it is often undesirable to have interferon preparations contaminatedwith the complex and ill-defined components of serum, attempts have been made to prepare interferon in the absence of serum. Strander (5) reported that a combination of serum albumin and a high concentration of a dipolar ionic buffer such as N-tris(hydroxymethyl)methylglycine (Tncine) can be used as a substitute for serum in the production of potent preparations of human leukocyte interferon. In the present report, the use of milk or milk casein as a serum substitute is described. MATERIALS AND METHODS Viruses. The Sendai strain of parainfluenza 1 virus was grown in chick embryos, and the Indiana strain of vesicular stomatitis virus (VSV) was passed in U cells. Details of the procedures have been described previously (6,7). Cells. A continuous line, "U," of human amnion cells was grown in Roux bottles. Leukocytes were stored at 4 C in the presence of 0.5% (w/v) ethylenediaminetetraacetate and purified by treatment with NH4Cl by using a procedure which has been described previously in detail (6,7). No serum was used during the purification. Interferon production. Purified leukocytes were suspended at a concentration of 107 cells per ml in Eagle's minimal essential medium (MEM) buffered with 3 mg of Tricine per ml instead of phosphate. The cell suspensions were supplemented with 5%o human serum (inactivated by heating at 56 C for 30 min) or with one of the other additives, and volumes of 100 to 400 ml in 2,000-ml round flasks were incubated in water baths at 37.5 C. The flasks were closed with loose foil covers, and the cells were kept in suspension by means of magnetic stirrers. Interferon production was induced by the addition of a priming dose of 100 units of human leukocyte interferon per ml, followed 1 hr later by the addition of 300 hemagglutinating units of Sendai virus per ml of suspension. Samples of the suspensions were stored at 4 C before assay. Interferon assay. Cells were removed by centrifugation, and the supernatant fluids were dialyzed against glycine-HCl buffer (pH 2) to destroy remaining virus. After back-dialysis to pH 7.3, the interfering activity in each preparation relative to that in the research standard for human interferon was estimated by plaque reduction of VSV in U-cell cultures. The results of all assays are given in terms of the unit which has been assigned to the research standard preparation 67/87 (3). Milk. Cow's milk was purchased locally and sterilized by autoclaving at 115 C (0.72 kg per cm2) for 15 min. The color of the milk became light brown during the autoclaving, but no precipitate was formed. The pH was adjusted to 7.3 before use by the addition of 1 N NaOH. Casein was precipitated from milk by acidification with dilute HCI to pH 4.6 or was sedimented by centrifugation for 90 min at 80,000 X g (Spinco 40 rotor). The resultant casein pellet was dispersed in a volume of MEM equal to the original volume of milk from which the pellet was obtained. In what follows, this suspension has been termed "casein preparation." The supernatant solution, called whey, remaining after centrifugation was used without further treatment or, following the acid-precipitation procedure, after a readjustment of the pH to 7.3. Protein concentrations were measured with both the biuret and Lowry methods with each sample, and, since the results were in good agreement in all cases, the values given are the average of the two numbers. Other materials. Solutions of 20% human albumin, suitable for injection, were prepared by the Finnish Red Cross Blood Transfusion Service. Tricine was purchased from Calbiochem, Los Angeles, Calif. RESULTS Serum substitutes. Although many substances have been tested as possible substitutes for serum in interferon production by human leukocytes, in previous studies only ascitic fluid and serum albumin were found to be active in this respect, and the latter required a high concentration of a dipolar ionic buffer such as Tricine to give titers of interferon approaching those obtained in suspensions containing serum (5). It has now been found that the addition of milk to serum-free leukocyte suspensions consistently results in the production of high yields of interferon upon induction with Sendai virus. Skim milk (0.05% fat) is as effective as partly skimmed (2.5 % fat) or whole milk (3.9% fat); thus, it was used throughout the study. Sterilization of milk by autoclaving does not decrease its activity. Concentrations of milk in the 10 to 20%o range gave optimal yields of interferon (Fig. 1). In comparison, the minimum concentration of serum which can be used to obtain consistently optimal yields is 5%;, (unpublished data). The stimulating effects of serum and milk are not additive. In a typical experiment, interferon yields of 10"5, 104.0, and 104 l units per ml were obtained in suspensions containing 5% serum, 10% milk, and 5%o serum plus 10% milk, respectively. Milk fractions. As a preliminary step in determining the active principle in milk, skim milk was separated into two fractions by either highspeed centrifugation or acid precipitation of the casein component. The whey and casein preparations were both tested in leukocyte suspensions. Whey, added in concentrations ranging from 5 to 45% (v/v), stimulated interferon production by a factor of two to five above that in unsupplemented suspensions. Yields of interferon did not differ significantly at different whey concentra- tions in this range and were all approximately 10 times less than those obtained in the presence of 10% milk. The addition of casein preparations resulted in the production of as much as 10 times as much interferon as the whey when added in concentrations covering the same range. Optimal yields were obtained by the use of 10 to 20 % of the casein preparation (Fig. 2). It should be emphasized that the concentrations given with respect to casein refer. in all cases, to the amounts of the casein preparation, prepared as described above, which were added to the leukocyte suspensions. No differences in activity between casein preparations obtained by the centrifugation or the acid precipitation methods were observed. Concentrations of protein in the casein preparations were determined and were found to range from 15 to 20 g/liter in different preparations. The protein concentrations of milk and human serum, determined at the same time, were 30 and 70 g/ liter, respectively. Thus, the total amounts of protein added to leukocyte suspensions by the addition of 5% serum, 10% milk, or 20% of the casein preparation are roughly the same. Comparative effects of serum substitutes. The results of many experiments were pooled, and the data are given in Table 1. It is clear that milk and casein, at optimal concentrations, are almost as effective as serum in stimulating interferon production and are considerably better than albumin as serum substitutes. A small, but significant, amount of interferon is produced in the absence of any of these supplements. With respect to the low activity of albumin, we found that increasing the concentration of Tricine above 3 mg/ml did not increase the effectiveness of albumin as a serum substitute, as has been reported by Strander (5). The activity of albumin in the presence of 3 mg of Tricine per ml in both studies, however, is roughly equivalent. The reasons for the inconsistent results obtained when the buffer concentrations were increased cannot be explained. Kinetics of interferon production. Samples were removed from leukocyte suspensions at intervals after induction with Sendai virus and assayed for interferon production. The results of three experiments have been pooled to give the results shown in Fig. 3; it can be seen that interferon synthesis DISCUSSION The use of milk or casein in the preparation of human leukocyte interferon has some advantages over the use of serum. Not only is it much more economical for mass production of interferon, but the product is also likely to be more suitable for certain purposes. For example, although the use of milk or casein does not result in the production of a crude product with a clearly higher activity per milligram of total protein, the use of casein may simplify the purification of the interferon protein. Also, human serum contains viral antibodies; thus the use of casein could avoid possible problems associated with the presence of these antibodies in interferon preparations. It seems evident that most, if not all, of the activity of milk resides in the casein fraction. The small amount of activity detected in the whey could result from either lactalbumin or some other whey proteins or from some casein which was not removed from the whey during the fractionation procedures used in this study. At present, it is not possible to explain how casein can substitute for serum so effectively, especially since so little is known about the active components of serum and the mechanism of serum action is not understood (2, 7). Strander could only conclude that the serum albumin molecule, or something firmnly attached to it, was in all likelihood the most critical of the required serum components but that it alone could not account for all of the activity of serum (7). It is intriguing that a protein like casein, which is chemically and biologically different from all of the proteins present in serum (4), possesses full activity in stimulating human leukocytes to produce interferon. It is, however, well known that both albumin and casein act as carrier proteins for a variety of other molecules. It might be that one such bound molecule is, in fact, responsible for stimulating interferon synthesis in both cases. Both the fact that the effects of milk and serum are not additive and the observation that the kinetics of the production are the same in the presence of serum and milk or casein are compatible with the premise that the same mechanism is involved in each case. Further information on the stimulation of interferon production in Sendai-induced human leukocytes may be obtained by the use ofpurified casein components. LITERATURE

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2020-12-10T09:04:11.777Z

1971-12-01T00:00:00.000Z

237234862

s2

Heat Resistance of Spores of Marine and Terrestrial Strains of Clostridium botulinum Type C Resistance to heat of spores of marine and terrestrial strains of Clostridium botulinum type C in 0.067 m phosphate buffer (pH 7.0) was determined. The marine strains were 6812, 6813, 6814, and 6816; the terrestrial strains were 468 and 571. The inoculum level equaled 106 spores/tube with 10 replicate tubes for each time-temperature variable. Heating times were run at three or more temperatures to permit survival of some fraction of the inoculum. Survivors were recovered at 85 F (30 C) in beef infusion broth containing 1% glucose, 0.10% l-cysteine hydrochloride, and 0.14% sodium bicarbonate. D values were calculated for each fractional survivor end point after 6 months of incubation. Thermal resistance curves were constructed from the D value data. D220 (104 C) values for spores of 468 and 571 equaled 0.90 and 0.40 min, respectively. The corresponding values for spores of 6812, 6813, 6814, and 6816 were 0.12, 0.04, 0.02, and 0.08 min. The z values for the thermal resistance curves ranged from 9.0 to 11.5 F (5.0 to 6.2 C). The classical paper of Bengston (2) is the only comprehensive report concerning the heat resistance of spores of Clostridium botulinum type C. Bengston studied spores of seven type C strains. Three strains were from fly larvae (Lucilia caesar or L. sericata), two from chickens showing symptoms of "limberneck," and one from the stomach contents of a horse reportedly dead from botulism poisoning; one nontoxigenic strain was included which Bengston had isolated by a singlecell technique from the prototype toxigenic culture. Spores of each strain were produced in a cooked-meat medium and heated in the same medium in which they were formed. Tubes were inoculated, flame-sealed, and completely submerged in a "boiling"-water bath. Spores showing the greatest heat resistance (strain from L. sericata) at an inoculum reportedly between 105 and 106 spores per tube survived heating for 60 min, but not 90 min. The purpose of the present study was to compare the heat resistance of spores of marine and terrestrial strains of C. botulinum type C. MATERIALS AND METHODS Suspensions. Terrestrial strains 468 and 571 and marine strains 6812, 6813, 6814, and 6816 were used. T,he sources, rmiethod of preparation, and standardization of spore suspensions were as previously presented (5). Aqueous suspensions were standardized to contain 107 spores/ml, based on preheating at 160 F (71 C) for 15 min. The standardized suspensions were stored unheated at 34 F (1.1 C). Heat resistance determinations. Ten-tube replicate sets were inoculated for each time-temperature combination. Sterile screw-cap tubes (16 by 125 mm) were inoculated with 0.1 ml of suspension (106) viable spores, and then 0.9 ml of heat-sterilized S0rensen's buffer (0.067 M, pH 7.0) was pipetted into each tube. Each set of tubes was heated simultaneously. For temperatures of 205 F (96 C) and below, a thermostatically controlled water bath with auxillary stirrer and insulated cover was used. Prior to heating, the screw caps were firmly tightened and sealed with a waterproof, heat-resistant tape. The tubes were placed in a metal holder, weighted down, and completely submerged in the bath. For temperatures of 215 F (101 C) and above, thermal death time retorts were used (4). The correction factor for the come-up time, taken from previous heat resistance studies, was considered to be 1.2 min. Various time-temperature combinations were run to obtain survivors in some fraction of the replicates. Survivors were recovered with beef infusion broth containing 1%glucose, 0.10% L-cysteine hydrochloride, and 0.14% sodium bicarbonate. The medium was prepared as previously described (5), dispensed in 200-ml quantities, and autoclaved at 250 F (121 C) for 15 min. The L-cysteine hydrochloride solution (20%) was heat-sterilized, and the bicarbonate (10%) was membrane-sterilized. Each was added aseptically to the medium immediately before use; then heatsterilized 1 N sodium hydroxide was added to adjust the medium to pH 7.0 to 7.2. About 10 ml of the re-1030 a D values are based on arithmetic averages of the data in the preceding tables. Value from extrapolation of thermal resistance curve based on data obtained at lower temperatures. covery medium was poured into each heated tube, and then stratified with melted Vaspar. The tubes were incubated at 85 F (30 C) and examined for turbidity and gas at periods up to 6 months. D values (time in minutes at a specified temperature to cause a 10-fold reduction in count) were calculated for each fractional survivor end point. The formula of Stumbo (6) was used: t log Alog S where t is corrected heating time (minutes), A is the population per tube times the number of replicates, and S is the number of positive tubes, presuming that giowth originated from one surviving spore in each positive tube. Thermal resistance curves for each strain were constructed by plotting the average D values on the logarithmic axis versus temperature on the linear scale on semilogarithmic graph paper. RESULTS AND DISCUSSION Determinations of thermal processes for foods involve calculations in degrees Fahrenheit; hence, the actual heating temperatures used are shown in degrees F with conversion to the nearest degree C. Tables 1 through 3 show the survivor data and D values of spores of the six type C strains studied. Where heat-resistant survivors were recovered, growth was usually detectable within 2 weeks after incubation. Occasionally, there was a marked lag, sometimes ranging up to 1 month, before turbidity and gas were seen in some tubes. Such lags were commonly associated with the longer heating times or higher temperatures used and apparently reflect the slow recovery of severely heat-damaged spores. The presence of type C toxin was confirmed Table 4 compares t spores of the terrestria summarizes the z val thermal resistance data heat resistance of spo: terrestrial strain (468) to that of spores of the most resistant marine strain (6812). Based on the limited number of cultures studied, spores of marine strains appear to be much more heatsensitive than spores of most terrestrial strains. In general, the data presented suggest that spores of both marine and terrestrial type C strains possess a resistance to heat which is intermediate between that shown by spores of C. botulinum type A or proteolytic type B (3) and that shown by spores of type E (1).

v3-fos

2020-12-10T09:04:17.509Z

1971-05-01T00:00:00.000Z

237229877

s2

Application of Immunofluorescence to Studies on the Ecology of Sphaerophorus necrophorus Sphaerophorus necrophorus (bovine liver abscess isolates) antiserum was fractionated and labeled with fluorescein isothiocyanate. The fluorescent-antibody (FA) conjugate proved to be species-specific and facilitated the detection of S. necrophorus cells in liver abscesses, viscera, and ruminal contents of cattle. Brightly fluorescing S. necrophorus cells were observed in experimentally inoculated soil incubated anaerobically at 37 and 4 C for 8 and 10 months, respectively. When incubated under moist conditions (80% water holding capacity) at 37 C, the test organism survived in both sterile and unsterile soil for as long as 8 weeks. Results strongly support the feasibility of using FA techniques concurrent with cultural methods for rapid detection of S. necrophorus infections. Sphaerophorus necrophorus is a highly pleomorphic, gram-negative, obligate anaerobe which can produce necrotic, gangrenous, or purulent lesions in man and animals (2,7,8). Its ability to induce hepatic abscesses in cattle has caused considerable economic loss through the condemnation of infected livers (15). Moreover, its consistent association with enzootic foot-rot infections in cattle and sheep has led to the growing suspicion that this pathogen could survive in soil (10,12). Knowledge of the ecology of this pathogen is meager despite its apparent widespread distribution probably because convenient methods for its detection are not available. This study was undertaken to explore the feasibility of applying immunofluorescence, concurrent with cultural methods, to investigate certain aspects of the ecology and survival of S. necrophorus. MATERIALS AND METHODS Organism and media. Ten bovine liver abscesses were obtained from freshly slaughtered animals and brought to the laboratory for study. S. necrophorus was isolated from each abscess by the decimal dilution technique of Calkins and Schrivner (5). The isolates were grown in fluid thioglycolate medium and cooked meat broth or streaked on blood-agar plates (5% defibrinated bovine blood) and incubated at 37 C under a 5% CO2 plus 95% N2 or H2 gas phase. Identification of S. necrophorus isolates was verified by morphological, cultural, pathogenicity, and antibiotic sensitivity tests (4,8,9). For inoculum and antigen preparations, 24to 48-hr cultures were used. In each case, retrospective checks of the purity of the culture were made on 5% bovine blood-agar. Preparation of conjugated antiserum. Washed S. necrophorus cells were suspended in 0.3% formolized phosphate-buffered saline (PBS, pH 7.1) and adjusted to a turbidity equivalent to that of Brown's opacity tube no. 8 (Burroughs Weilcome and Co., London). Since there were no detectable variations among the 10 S. necrophorus isolates, their growth was pooled into three batches of antigen. Young rabbits (four per batch of antigen and one control) were bled before injection. Preimmune serum was used as a control and for verifying the absence of antibodies against S. necrophorus. An initial dose of 0.5 ml of a 1:1 antigensodium alginate (Colab, Weston, Ontario) mixture was injected subcutaneously into four sites of each rabbit's abdomen. This was followed by 10 intravenous injections spaced every other day for 4 weeks with doses increasing from 0.1 to 2.0 ml. Final bleeding was done after a 10-day pause from the last injection. Antibodies against S. necrophorus were detected by agglutination and diffusion techniques. The latter technique was also applied to both intact cells and crude cell fractions. Cells were broken by ultrasonic disruption for 10 min. Crude cell wall fraction was sedimented after spinning the disrupted cells at 18,000 X g for 10 min. The 18,000 X g supernatant constituted the cytoplasmic fraction. Antiserum was fractionated by two methods, the first involving direct ammonium sulfate precipitation (14) and the second procedure employing rivanol (2-ethoxy-6,9-diaminacridine lactate) to obtain a chromatographically pure gamma globulin in solution (1). The gamma globulins obtained by both fractionation methods were conjugated with fluorescein isothiocyanate (FITC; reference 14). Acetone-fixed smears were stained with fluorescent antibody (FA) for 30 min in a moist chamber, washed with two changes of PBS, and mounted in buffered 809 glycerin (pH 7.1). Stained preparations were observed with a Reichert Zetopan provided with ultraviolet illumination (14). FA staining reactions of bacterial cells were rated from 0 (no fluorescence) to 4+ (intense fluorescence with clearly defined outlines). Specificity of conjugates. FA conjugates prepared by the two fractionation methods were tested against smears of various organisms from stock cultures, foodstuffs, infected tissues, intestinal tracts of rabbits and poultry, and soils. Direct smears of the last three materials were also stained with the FA conjugates and the conjugated preimmune serum. A supplemental test to prove the specificity of the conjugates was done by employing the inhibition test (13) which involved treatment of S. necrophorus smears with the homologous unlabeled antiserum before FA staining. Cultures showing positive reaction were compared with the test organism by immunodiffusion techniques and by disc gel electrophoresis (16). Distribution of S. necrophorus in animal viscera and ruminal contents. Portions of the liver, gall bladder, spleen, mesenteric lymph node, small intestine, and ruminal content of five steers with liver abscesses and five apparently healthy steers were collected at the time of slaughter. The specimens were brought to the laboratory and processed aseptically for cultural and FA tests. Cultures containing FA-reactive cells were further screened to confirm their identity. Direct smears of the tissues and ruminal contents were examined similarly by the FA technique. Soil studies. A freshly sampled pasture soil was passed through a 2-mm sieve and dispensed into screwcapped test tubes. Each tube contained 5 g of fresh soil. The soil contained 4.0%7 oxidizable organic matter and had a pH value of 6.6. Soil sterilization was achieved by autoclaving the tubes at 126 C for 20 min. A two-part experiment was devised to evaluate the effect of incubation time and temperature on the antigenic reactivity of the test organism. The first part consisted of two sets of tubes incubated at 37 and 4 C. One set was kept anaerobic under a CO2 plus N2 atmosphere; to the other set about 1-cm layer of water was maintained above the soil surface. At monthly intervals, for 10 months, duplicate tubes were removed from each set and smears from diluted samples were subjected to direct FA examination. In the second part of the experiment, autoclaved and unsterile soils were inoculated with the test organism and the moisture content was adjusted to 80% of the water-holding capacity (WHC). Both inoculated soils and the corresponding uninoculated controls were partitioned into four sets and incubated as follows: set 1, 37 C, N2 plus CO2 atmosphere; set 2, 37 C, air; set 3, 22 C, air; set 4, 4 C, air. Tubes were removed from each set at 0, 3, 5, 8, and 16 weeks and examined by FA procedures. A short-term study was also undertaken to assess the relative survival of S. necrophorus in both sterile and unsterile soils. Samples were removed weekly and processed by cultural and FA methods for the presence of viable and fluorescing S. necrophorus cells. In a parallel experiment, heat-sterilized glass slides were buried in duplicate jars of inoculated sterilized and unsterile soils and incubated at 37 C. At weekly intervals, slides representing each treatment were removed and stained with the antibody conjugate and acridine orange (6). RESULTS AND DISCUSSION FA staining. S. necrophorus cells isolated from bovine liver abscesses reacted strongly with the labeled homologous antiserum. Figure 1 illustrates the typical intense fluorescence exhibited by actively growing S. necrophorus rods. Direct smears from 50 bovine liver abscesses obtained at different times invariably contained pleomorphic bacilli showing staining reactions of 3 + to 4+. A representative smear from an abscess (Fig. 2) demonstrates a cluster of fluorescing filaments among nonspecific fluorescing pus cells. The occurrence of nonspecific fluorescence by tissue cells and inert materials was of little consequence since the specific fluorescence of the test organism was quite intense and of a different color (bright apple green) than that of other materials (yellow, orange, red, or dull yellow-green). Absorbing the conjugates with dried liver powder (13) appeared to reduce the intensity of nonspecific reactions in tissue smears. Similarly, pretreating the smears with a gelatin preparation (3) before FA staining was effective in decreasing nonspecific fluorescence in soil smears and, to some extent, in tissue smears. The agglutination titer of the antiserum was found to be 1:1,600. Five distinct precipitin bands were produced between the wells of the antiserum and the cytoplasmic fraction (18,000 x g supernatant fluid) by double agar-diffusion technique. Reactions between the antiserum and intact cells revealed three clear bands, whereas the crude cell wall fraction (18,000 X g pellet) elicited only one weak band. This observation suggests that S. necrophorus antigens prepared from disrupted cells might prove a better way of eliciting antibody response than using whole cell antigens. Antibody conjugates derived from the two serum fractionation methods were both satisfactory and did not exhibit any obvious difference in staining the test organism. The rivanol fractionated antiserum, however, revealed fewer nonspecific reactions than antiserum precipitated directly with ammonium sulfate. Specificity test. None of the heterologous bacterial species subjected to FA staining crossreacted with the S. necrophorus-conjugated antiserum (Table 1). Fusiform bacilli cultures derived from a case of bovine foot-rot and a chronic foot infection showed strong staining reactions. These isolates were found to possess morphological, biochemical, and pathogenic characteristics assigned to S. necrophorus species. Furthermore, results from a comparative gel diffusion test and disc gel polyacrylamide electrophoresis suggested the presence of certain antigens common to the foot isolates and the test organism. Thus, the S. necrophorus-conjugated antiserum appeared to be species-specific. Griffin (11) recently demonstrated similar FA reactions between S. necrophorus strains and suggested that there is more than one serotype within the species. Drastic reduction of specific cell fluorescence in the inhibition test provided additional evidence for the specificity of the conjugated antiserum used in the study. analysis of the anaerobically maintained inoculated soils indicated that S. necrophorus could retain its antigenic reactivity, as evidenced by fluorescing cells, for 8 and 10 months at 37 and 4 C, respectively. Unsterile soil produced a similar result (Table 3), although aerobic incubation at higher temperatures (37 and 22 C) effected a progressive reduction of the cell fluorescence after 8 weeks of incubation. The fluorescence of cells kept aerobic at 4 C and anaerobic at 37 C appeared to be maintained up to the last sampling date (16 weeks). When cultural and acridine orange staining methods were used as adjuncts to the FA technique, S. necrophorus was found to survive under both sterile and unsterile soil conditions for as long as 8 weeks (Table 4). Cells in unsterile soil, however, became progressively shorter starting at the 2nd week so that by the 8th week their length had decreased to about onehalf to one-third that of cells inoculated to autoclaved soil. Coccoid forms of the test organism also appeared more numerous in the unsterile than the sterile soil. This morphological reaction might be a survival mechanism against an apparently competitive environment. The relatively prolonged survival in soil of S. necrophorus even under aerobic maintenance might be due to anaerobic pockets occurring in the lower portions of the soil column and the relatively high soil moisture content (80% WHC). In a subirrigated swamp pasture, Marsh and Tunicliff (12) demonstrated the occurrence of foot-rot infection in sheep for 10 months but not for 20 months after the area has been heavily seeded with infectious materials. Thus, the persistence of this pathogen in soil, although transitory, should not be overlooked particularly where recurrent foot-rot infections occur. Recent work (unpublished data) in this laboratory has shown the effectiveness of FA techniques in detecting S. necrophorus on the feet of both healthy and foot-rot-infected animals in various feedlots.

v3-fos

2020-12-10T09:04:16.720Z

1971-10-01T00:00:00.000Z

237235308

s2

Enumeration and Identification of Bacillus cereus in Foods An egg yolk-polymyxin medium (KG) for rapid enumeration of Bacillus cereus is described. The test is presumptive in that differentiation of B. cereus (and closely related organisms) from other species is based on the formation of turbidity in the agar surrounding the colonies of the cereus group organisms. The medium is formulated to encourage sporulation and release of free spores for serological confirmatory tests within the 24-hr incubation period. The production of turbidity in egg yolk and free-spore production by 25 strains of B. cereus on KG agar were measured. The recovery of food poisoning strains of B. cereus inoculated into nonsterile food slurries was assessed. A comparison of KG agar and mannitol-egg yolk-polymyxin-agar indicated that the two media were comparable in their abilities to recover low levels of B. cereus from naturally contaminated foods. Since KG agar enhances spore formation by B. cereus, thus permitting early serological testing, its use in screening food products is advocated. The role of Bacillus cereus as an etiological agent of foodborne disease was described by Hauge (7,8). Since then, other investigators have reported outbreaks and frequencies of B. cereus food poisoning (1,11,14,15). Oddly, until 1970, there was not a single report of a well documented outbreak of B. cereus poisoning in the United States. To date, there have been no outbreaks of this illness reported in England (B. Hobbs, personal communication). In light of the rather high frequency of occurrence in Hungary (14), for example, it is quite probable that the infrequency of outbreaks in England and the United States reflects a lack of attention or examination of suspect foods rather than absence of occurrence. One possible explanation for this is that until recently a selective medium for B. cereus had not been described. At present, most media employed for enumeration of B. cereus contain egg yolk. Thus, the primary differentiation of B. cereus from other Bacillus species depends on the detection of phospholipase C activity in cultures of the former organism. This stems from the early work of Colmer (4) and Chu (2), who described the presence of lecithinase in cultures of B. cereus. Donovan (5) devised a peptone-beef extract-2.5% egg yolk medium which also contained polymyxin-lithium chloride for the inhibition of gram-positive and gram-negative bacteria, re-spectively, and trisodium citrate to facilitate assessment of the egg yolk reaction. More recently, Mossel et al. (12) described a similar medium (MYP) which omitted the lithium chloride and added D-mannitol and phenol red for differentiation purposes. These authors successfully employed this medium for the detection of B. cereus in various food products. They suggested the employment of further biochemical tests, e.g., anaerobic dissimilation of glucose, hydrolysis of gelatin, reduction of nitrate, and profuse growth on chloral hydrate-agar for the identification of presumptive isolates of B. cereus. In a survey of selected dry food products in retail distribution, Kim and Goepfert (10) employed the MYP agar of Mossel et al. and reported that mannitol utilization by colonies appearing on this medium was not significantly helpful in eliminating non-B. cereus organisms and, indeed, made interpretation somewhat difficult when plates were held for 40 hr at 32 C. Moreover, the usefulness of acetylmethylcarbinol (AMC) production, NO3 reduction, and starch hydrolysis was found to be somewhat questionable. Preliminary experiments in this laboratory suggested that a serological test employing spore antigens might serve as a rapid confirmatory test for presumptive B. cereus isolates and prove valuable in epidemiological investigations of food 581 poisoning outbreaks. Such an approach necessitated the development of a selective (and differential) medium that would support the formation of free spores within the shortest possible period of time. MATERIALS AND METHODS Cultures. The strains of B. cereus employed in this study and their sources are listed in Table 1. Cultures were maintained on nutrient agar slants in screw-capped tubes at room temperature. Production of turbidity in egg yolk and free-spore production. The degree of egg yolk turbidity and free-spore formation by B. cereus on the various media was assessed as follows. A loop was used to streak from agar slant cultures onto the surface of the test medium. Since these slant cultures varied in age from 1 week to several months, the inoculum consisted overwhelmingly of spores rather than vegetative cells. The test medium plates were incubated at 35 to 37 C for 16 to 24 hr. Organisms capable of eliciting turbidity on the medium evidenced this by formation of an opaque zone in the otherwise translucent pink-red medium. After incubation, the size of the zone of precipitate surrounding the isolated colonies was measured and recorded on a scale of 1 to 4+ (see footnote a, Table 3). A small portion of the center of the colony was picked, and this growth was emulsified in a drop of distilled water on a slide. The free-spore population was estimated to the nearest 10%0 after examination of the smear under dark-field illumination at X1,250. Composition of KG medium. The final composition of the egg yolk-polymyxin (KG) medium as it evolved during this study was as follows. The basal medium was prepared separately and consisted of 0.1% peptone, 0.05% yeast extract, 0.0025%0 phenol red, and 1.8% agar. The pH of the medium was 6.8. The basal medium was autoclaved at 121 C for 20 min. After sterilization and cooling to 50 C, 100 ml of sterile, concentrated egg yolk emulsion (Oxoid) and sufficient polymyxin B sulfate (Pfizer Co. Inc., Brooklyn, N.Y.) to result in a final concentration of 10 ,g/ml were added to 900 ml of basal medium. The medium was poured into petri dishes, allowed to solidify, and stored in a manner to eliminate excess surface moisture. The complete medium can be stored for 1 week at 4 C before use. Longer storage is not recommended. Food samples. The food samples employed in RESULTS AND DISCUSSION During an earlier study (10), it was noted that many colonies that appeared on MYP agar plates evidenced a restricted (1 +) zone of precipitate beneath the colony. The result was that many colonies were picked and subjected to extensive biochemical testing to ascertain that they were not weakly lecithinase-positive B. cereus. Additionally, two lecithinase-positive strains of the polymyxa-macerans group of Bacillus were also isolated. Various other species of Bacillus, e.g., B. laterosporus, B. thuringinesis, B. mycoides, and B. anthracis, have been reported to possess phospholipase C activity (16). Therefore, it was desirable to have a rapid confirmatory test for B. cereus organisms isolated from selective agar plates that employed the production of turbidity in egg yolk-agar as a differential system. Serological tests for species-specific spore antigens appeared to be suitable for this purpose (Kim and Goepfert, unpublished data). These tests necessitated the presence of free spores. The first step was to determine whether a suitable population of free spores was present in colonies growing on MYP agar. Three colonies from each of 10 different MYP agar plates (representing different food samples) were examined after 24, 48, and 72 hr at 35 C. No free spores and very few endospores developed in the colonies within that period. Various substances [Gsalts (3), 0.05% MnCl2, and 0.05 % sodium citrate] were added alone and in combination to MYP medium in an effort to enhance sporulation. None of the addends had an adverse effect on the lecithinase reaction of the test strain B. cereus T, but free spores could not be detected after 24 hr at 35 C. Since the MYP medium contains a substantial amount of peptone (1 %), the possibility existed that the medium was too nutritionally endowed, i.e., favorable to vegetative cell growth and not conducive to sporulation. Consequently, a basal medium containing polymyxin B, egg yolk emulsion, agar (1.5 %), and phenol red was formulated. Phenol red was incorporated solely to facilitate observation of the zone of turbidity in the egg yolk medium. The basal medium alone and with various additions was tested for the ability to support ample lecithinase production and free-spore formation by the test strain B. cereus T ( Table 2). Initially it appeared that lecithinase production and sporulation were mutually exclusive or only weakly compatible. Substances which stimulated lecithinase production (1% peptone) seemed to prohibit sporulation. This effect seemed not to be attributable to glutamate inhibition of sporulation, a phenomenon well documented by other workers (6), since the addition of 0.2% sodium glutamate did not affect the sporulation rate. Substances alone and in combination (MnCl2, sodium citrate, ethylenediaminetetraacetic acid, and glucose) that supported sporulation seemed to preclude lecithinase production (or activity). Phosphorochloline (0.1 %) was added to the plating medium to determine whether the end product of lecithinase activity would inhibit sporulation. This did not appreciably influence free-spore formation. Finally, the incorporation of low levels of peptone and yeast extract into the basal medium resulted in lecithinase production and the formation of free spores by B. cereus T. Increasing the agar concentration from 1.5 to 1.8% enhanced free-spore formation by B. cereus T within the 24-hr incubation period at 37 C. Twenty-five additional strains of B. cereus and the two lecithinase-positive polymyxa-macerans strains were streaked from pure cultures onto the complete medium. After incubation for 24 hr at 37 C, the plates were examined for evidence of turbidity around the colonies, and a small portion of a single colony of each strain was removed to a slide for examination by microscope. Preliminary experiments had disclosed that spore formation was greatest in that area of the plate where growth was heaviest. In the series of experiments reported here, the estimation of freespore population was performed on colonies that were well isolated from the area of heavy growth. It is recognized that examination of a single colony does not yield data on the spore-forming performance of other colonies on the same plate, but the examination of a colony that was well isolated would appear to represent the minimum sporulating capacity under these given conditions. For this reason, examination of a single colony was accepted as representative. The data in Table 3 indicate that the KG medium was sufficient to encourage turbidity production and free-spore formation by most of the strains within the 24-hr period of incubation. It was possible to detect turbidity formation by the colonies quite readily after 16 hr at 35 to 37 C. It was noted previously ( Table 2) that MnCl2sodium citrate mixtures had a stimulatory effect on free-spore formation by B. cereus T. These substances were tested for their ability to enhance free-spore formation by some of the strains that did not form free spores readily on KG agar incubated at 35 to 37 C for 24 hr. This was accomplished by placing one drop of a solution containing 0.1% MnCl2 and 0.1% sodium citrate (MC) on a 16-hr colony growing on KG agar. In addition to this, isolated colonies on the same plate that did not receive the solution were examined after 48 hr on KG medium ( Table 4). The administration of MC solution to the colonies enhanced the free-spore formation by each strain except C-14 and 27 Benz. Incubation of untreated colonies for 48 hr resulted in increased free-spore production by all strains except 27 Benz. This strain exhibited a low level (-10%) of free-spore production only in colonies treated with MC and incubated for 48 hr. Untreated colonies at both 24 and 48 hr were observed to contain less than 10% endospores. The reasons for the failure to enhance free-spore formation by 27 Benz are not known. The success of the MC treatment in enhancing free-spore production within 8 hr after application indicates that in the great majority of cases serological analyses can be initiated (and completed) on the second working day. It is suggested that MC not be applied routinely to colonies appearing on KG agar but that it be administered only if initial examination by microscope reveals a lack of spore formation. Efforts to incorporate MC into the KG agar and thus eliminate the cumbersome process of addition directly to the colonies have failed because of the inhibition of the lecithinase reaction by this solution. It should also be noted that the lecithinase reaction of the two polymyxa-macerans strains was inhibited on the KG agar under the test conditions employed. This would indicate an increased specificity for B. cereus organisms. To determine whether food materials would affect the isolation and recognition of B. cereus on KG medium, a series of trials employing food materials was undertaken. Initially, a suspension of spores of B. cereus T was prepared, diluted in peptone, and inoculated into autoclaved 1:10 aqueous suspensions of several food materials. A dried potato product, gravy mix, and seasoning mix were employed. The recovery and free-spore production data are presented in Table 5. At this level of inoculum (1.3 X 103/ml), it appeared that the sterile food slurry had little or no effect on the recovery of B. cereus T. However, free-spore production may have been influenced by the food constitutents of the slurry even though these were diluted significantly by the plating procedure. Five additional strains of B. cereus (all isolated from food poisoning outbreaks) were then inoculated into nonsterile 10% aqueous slurries of four food prototypes. The recovery rate of these strains on KG agar was calculated by comparing the KG agar counts with nutrient agar counts made on the cell suspension serving as the inoculum for the slurries ( Table 6). The recovery of the five strains on the KG medium ranged from 63.8 to 276.7% of the organisms inoculated into the food prototypes. This efficiency of recovery of relatively high numbers of B. cereus prompted a pilot study to determine the effectiveness of the KG medium in recovering low numbers of B. cereus from naturally contaminated retail food products. Six food prototypes were chosen, and 10-g samples were aseptically transferred to 90 ml of 0.1% peptone-water. Samples (0.1 ml) of the 10-l, 10-2, and 10dilutions were surface-plated in duplicate on KG agar and MYP agar. Representative colonies of lecithinase-producing organisms that appeared on KG agar were picked after incubation at 35 to 37 C for 22 hr for examination by microscope and estimation of free-spore populations ( Table 7). B. cereus colonies were observed on both MYP and KG agar plates from 21 of 26 food samples. Three samples yielded colonies on MYP agar but not on KG agar. Two samples were positive on KG agar but not on MYP agar. Fourteen samples yielded higher B. cereus counts on KG agar, eight samples were higher on MYP agar, and in four samples identical counts were obtained on both media. Based on these data, it seems safe to assume that the two media were comparable in terms of ability to recover low numbers of B. cereus. Examination of colonies appearing on KG agar from 19 of the 26 samples showed that 15 of the 19 had produced a sufficient number of free spores to be detectable by microscope. Unfortunately, the plates containing the free-spore "negative" colonies were not kept an additional 24 hr and reexamined. Moreover, these colonies were not treated with MC solution to enhance sporulation. Thus, it is not known whether these treatments would have affected detectable freespore formation. In addition, it is not known whether (i) these organisms were asporogenic or oligosporogenic mutants (unlikely), (ii) sporulation was influenced by food constitutents on the plate (likely), or (iii) these were normally slow sporulating cultures. The results indicate that KG agar is as efficient as MYP agar in recovering and presumptively identifying B. cereus organisms from food products. Lecithinase reactions by known B. cereus cultures and food contaminants are comparable on each medium. Nygren (13) has postulated that lecithinase-negative mutants of B. cereus are not pathogenic. However, in light of recent studies on the role of lecithinase in food poisoning by Clostridium perfringens (9), it appears too early to judge the pathogenicity of lecithinasenegative B. cereus strains. For this reason, we concur with the suggestion of Mossel et al. (12) that lecithinase-negative egg yolk turbiditypositive colonies of appropriate morphology be picked for confirmatory testing. Based on our earlier experience with biochemical tests [e.g., anaerobic glucose dissimilation, nitrate reduction, acetylmethylcarbinol production, and gelatin and starch hydrolysis (10)], we cannot agree that these time-consuming and often erratic tests are the confirmatory methods of choice. Preliminary data in this laboratory have indicated that serological reactions involving certain spore antigens are more rapid, sensitive, and specific than biochemical tests. Therefore, it would be extremely beneficial to have a plating medium that would not only indicate presumptive B. cereus colonies but also induce these organisms to sporulate within the 24-hr incubation period. The KG medium as presently formulated, although not 100% effective in inducing free spores detectable by microscope, has achieved the desired result in the majority of trials. It is hoped that the existence of two comparable, easily prepared and interpreted selective media for the isolation of B. cereus will prompt those laboratories currently not examining food samples or suspect food poisoning vehicles for these organisms to do so. Only in this manner will the necessary data be generated to ascertain whether this organism is the cause of a significant number of foodborne outbreaks in this country. Current knowledge about C. perfringens food poisoning, compared to its known role in foodborne disease 15 years ago, is an outstanding example of what a systematic, scientific approach can add to our knowledge of public health hazards.

v3-fos

2020-12-10T09:04:11.113Z

1971-09-01T00:00:00.000Z

237234608

s2

Chemical Nature of Malty Flavor and Aroma Produced by Streptococcus lactis var. maltigenes Mature skim milk cultures of Streptococcus lactis var. maltigenes were steam distilled at low temperature under reduced pressure. Ethyl ether extracts were prepared from the distillates and analyzed by gas-liquid chromatography and mass spectrometry. Twenty of 31 components detected in the culture distillates were identified positively and 11 tentatively, whereas 10 of 19 components detected in the heated skim milk control were identified positively and 9 tentatively. Among components detected in the culture distillate, but not detected in the heated skim milk distillate, and which have not been previously identified in milk cultures of the organism were phenylacetaldehyde and phenethanol. Quantitative analyses of the volatiles entrained from milk cultures of several strains of S. lactis var. maltigenes revealed a probable relationship between variation in the character of the aroma of the cultures and the alcohol/aldehyde ratio. The conversion of leucine to 3-methylbutanal by Streptococcus lactis var. maltigenes was first implicated as being primarily responsible for the malty flavor and aroma produced in milk cultures (2). Later work revealed that the organism possesses transaminase and decarboxylase systems which mediate the conversion of several amino acids to the corresponding aldehydes (4,5,10). These observations along with more recent analyses of culture volatiles (6) suggest that the malty character may be an expression of several components in addition to that of 3-methylbutanal. The present investigation provides further information concerning the flavor and aroma produced in milk by S. lactis var. maltigenes. MATERIALS AND METHODS Cultures of strains of S. lactis var. maltigenes from the collection described by Gordon et al. (1) designated Ml, RM2, M3, and P25 were maintained in sterile reconstituted antiobiotic-free nonfat dry milk medium. Transfers were made every third day by using 1% inoculum. Cultures were incubated at 30 C until coagulation occurred (18 to 20 hr) and were then stored at 2 C. Culture for distillation was prepared in 37. 8 solids-nonfat) heated at 96 C for 1 hr in stainless-steel cans. The milk was cooled to 30 C, inoculated with 1% active culture, and incubated for 18 hr. Seventy-five-liter quantities of culture Ml and a heated skim milk control (acidified to pH 5.2 with 10% phosphoric acid) were steam distilled at a pressure of 2 mm of Hg (3). Approximately 10% of the culture volume was recovered as distillate. This was extracted with peroxide-free ethyl ether (11) for 24 hr in a conventional liquid-liquid extractor (8.5 by 60 cm extraction chamber). The extracts were dried over anhydrous sodium sulfate and fractionally distilled to remove the excess ether. Components of the extracts were separated by gas-liquid chromatography (GLC) on two columns of differing polarity and tentatively identified by coincidence of relative retention times with those of known compounds. The columns were 305 cm by 3.175 mm outer diameter stainless-steel packed with 20% diethyleneglycol succinate on 80 to 100 mesh, acid-alkali-treated Celite 545, and uncoated, 100 to 120 mesh Poropak Q and were temperature programmed as follows: 60 C for 15 min, then 4 C/min to 200 C; 4 C/min from 100 to 210 C, respectively. Nitrogen was the carrier gas (25 cc/min and 20 cc/min measured at the starting temperature), and the separated components were detected by flame ionization. To achieve greater resolution of components prior to mass spectral analysis, the extracts were chromatographed on 91.44 m by 0.254 mm open tubular columns coated with either Apiezon L or butanediol succinate and temperature programmed as follows: 60 C for 15 min, and then 4 C/min to 200 C; 125 C for 10 min, and then 4 C/min to 200 C; respectively. Quantitative analyses of the volatiles produced in skim milk by different strains of S. lactis var. mal-+ tigenes were made by an on-column trapping GLC technique (7) as employed by Morgan et al. (8). A + Varian-Aerograph (model 1200) gas chromatograph equipped with a hydrogen flame detector and a column (366 cm by 3.175 mm outer diameter) packed with 20% 1,2,3-tris-(2-cyanoethoxy)propane on 80 to 100 mesh, acid-alkali-washed Celite 545 was used for the analyses. The column was operated isothermally at 55 C and adequately separated the volatile + constituents. To obtain quantitative data, the GLC recorder response for each sample component was compared to that obtained for graduated concentrations of the pure compound in skim milk. A subjective evaluation of the aroma and flavor + produced in skim milk culture by strains of S. lactis var. maltigenes and by addition of mixtures of + authentic compounds was made by a laboratory panel of three members who were familiar with the malty + aroma. A 10-member trained panel was employed in + determination of the average flavor threshold (9) in + both skim milk and water of pure compounds found to be associated with the malty cultures. + Helium, at a flow rate of about 1 cc/min, served as the carrier gas. The column effluents were directed into the ionization chamber of an Atlas CH4 mass spectrometer operated at 1.5 X 10 6 mm of Hg.A 20-ev source was employed as a chromatographic detector and the 70-ev source provided the mass spectra. Spectra were recorded from mass 25 to 250 in 2 or 4.5 sec with a Honeywell Visicorder (model 1508). The identification of components was considered positive when confirmed by mass spectral analysis. RESULTS AND DISCUSSION In the summary of the volatiles identified in the extracts of the distillates from the malty culture and the heated skim milk (Table 1), it will be noted that certain aldehydes, alcohols, and esters were detected only in the extract obtained from the culture distillate. Along with 2-methylpropanal, 2-methylpropanol, 3-methylbutanal, and 3-methylbutanol, all of which have been previously detected in milk cultures of this organism (8), phenylacetaldehyde, phenethanol, and several simple esters were detected. Since the conversion of several amino acids including phenylalanine to the corresponding aldehydes by resting cells of the organism has been demonstrated (6) and since a phenylacetaldehyde-like note is occasionally detected in the aroma of malty cultures, the detection of phenylacetaldehyde in a vacuum distillate of the culture employed in this study was not unexpected. The alcohols present in the culture distillate undoubtedly resulted from a reduction Although appreciable ethanol, other alcohols, and probably some volatile acids were produced in the culture, it is doubtful that esters detected in the extract of the distillate were produced by the organism. None of these esters has been detected in analyses of malty cultures by the entrainment on-column trapping GLC technique (9) and ester-like notes are not detectable in the aroma or flavor of mature malty cultures. It appears, therefore, that the esters detected in the present study were formed during the extraction of the culture distillate or concentration of the extract. The subjective evaluation of the aroma over 24-hr-old skim milk cultures of four strains of S. lactis var. maltigenes is shown in Table 2. Although each of these strains produced titratable acidities in excess of 0.6% expressed as lactic acid and possess an active a-keto acid decarboxylase system (Table 3), they represent extremes in respect to these criteria (1), i.e., strains Ml and RM2 produced a more pronounced malty aroma and attained an appreciably higher titratable acidity than did M3 and P25. The quantitative analyses of the volatile compounds entrained from the mature skim milk cultures as determined by GLC are shown in Table 3. Variation in the quantities detected are apparent and appear to be related to the flavor and aroma of the cultures. Since the average flavor threshold of the aldehydes is much lower than that of the corresponding alcohols (Table 4), it appears that the decreasing intensity of the aroma observed in cultures RM2, Ml, and M3, respectively, is due to increases in the alcohol/aldehyde ratios (ethanol/acetaldehyde, 2-methylpropanol/2-methylpropanal, 3-methylbutanol/3methylbutanal). This loss of intensity in flavor and aroma is undoubtedly related to an aldehyde to alcohol conversion as exemplified by the relatively weak aroma of strain M3. Strain Ml had a moderately intense but smooth aroma which would be indicative of a balance between the alcohols and aldehydes. The aroma of strain RM2 possessed a harshness which suggested a high aldehyde content or a low alcohol/aldehyde ratio. Although strain P25 produced a relatively large amount of acetaldehyde, it produced only traces of 2-methylpropanal and 3-methylbutanal. Despite the low conversion of aldehydes to alcohols, the aroma of the culture possessed but little malty character. Qualitatively and quantitatively, the volatiles entrained from this culture resemble those from a number of other weakly malty or nonmalty cultures of S. lactis examined previously (8). Threshold values of the authentic compounds (Table 4) indicate that all of the compounds (except ethanol and acetone) detected by GLC in the volatiles entrained from the cultures are probably essential to the characteristic aroma developed in mature cultures. This does not contradict the earlier hypothesis that 3-methylbutanal is the primary odor constituent in immature cultures of S. lactis var. maltigenes (2) or the early stages of the malty defect as might be detected in producer samples of raw milk (6). The parameters employed in the GLC analyses of the volatiles entrained from the culture did not permit detection of higher boiling compounds such as phenylacetaldehyde and phenethanol. These compounds undoubtedly contribute to the flavor and aroma of cultures of the organism, especially in view of their low flavor threshold in water and skim milk ( Table 4). To evaluate further the flavor character of the compounds in the GLC analyses of entrained culture volatiles, a synthetic flavor formulation was prepared by using the quantitative data for the MI culture. Milk containing the mixture of flavor compounds possessed a distinct malty flavor and aroma although it lacked the overall character of mature MI culture. The flavor character could be easily manipulated by varying concentrations of aldehydes and alcohols and by adding phenylacetaldehyde and phenethanol. The aroma variations were quite similar to those observed during different stages of growth of the organism. Although the flavor and aroma of S. lactis var. maltigenes cultures is usually described as malty, other terms such as burnt, caramel, and "grapenuts" have been employed. In view of the potentially desirable flavors from these cultures, and their similarity to volatile yeast metabolites, it is possible that this organism could be utilized in the production of natural flavors for food products. LITERATURE CITED

v3-fos

2020-12-10T09:04:11.776Z

1971-12-01T00:00:00.000Z

237230257

s2

Parameters of Rumen Fermentation in a Continuously Fed Sheep: Evidence of a Microbial Rumination Pool The feed and feces of a continuously fed sheep were analyzed for carbon, hydrogen, and nitrogen, with oxygen as the remainder. The daily feed-feces weight difference was used as the reactant in an equation representing the rumen fermentation. The measured products were the daily production of volatile fatty acids (VFA), CH4, CO2, and ammonia. The carbon unaccounted for was assumed to be in the microbial cell material produced in the rumen and absorbed before reaching the feces. The ratio of C to H, O, and N in bacteria was used to represent the elemental composition of the microbes formed in the rumen fermentation, completing the following equation:C20.03H36.99O17.406N1.345 + 5.65 H2O → C12H24O10.1 + 0.83 CH4 VFA + 2.76 CO2 + 0.50 NH3 + C4.44H8.88O2.35N0.785 microbial cells absorbed With C arbitrarily balanced and O balanced by appropriate addition of water, any error is reflected in the H. The H recovery was 98.5%. The turnover rate constant for rumen liquid equilibrating with polyethylene glycol (PEG) was 2.27 per day. Direct counts and volume measurements of the individual types of bacteria and protozoa in the rumen were used to calculate the total microbial cell volume in the rumen, not equilibrating with it. The dry matter in the rumen (582 g) and the nitrogen content (12.05) of the microbes in the rumen were estimated, the latter constituting 85% of the measured N in the rumen. Calculations for rumen dry matter and nitrogen turning over at the PEG rate introduce big discrepancies with other parameters; a rumination pool must be postulated. Its size and composition are estimated. Arguments are presented to support the view that dry matter and some of the microbes, chiefly the protozoa, do not leave the rumen at the PEG rate. One experiment with the same sheep fed twice daily showed significantly less production of microbial cells than did the continuous (each 2 hr) feeding. Analysis of the microbial cell yield suggests that, on the basis of 11 mg of cells per adenosine triphosphate molecule, a maximum of six adenosine triphosphate molecules could have been formed from each molecule of hexose fermented. microbial cells absorbed With C arbitrarily balanced and 0 balanced by appropriate addition of water, any error is reflected in the H. The H recovery was 98.5%. The turnover rate constant for rumen liquid equilibrating with polyethylene glycol (PEG) was 2.27 per day. Direct counts and volume measurements of the individual types of bacteria and protozoa in the rumen were used to calculate the total microbial cell volume in the rumen, not equilibrating with it. The dry matter in the rumen (582 g) and the nitrogen content (12.05) of the microbes in the rumen were estimated, the latter constituting 85% of the measured N in the rumen. Calculations for rumen dry matter and nitrogen turning over at the PEG rate introduce big discrepancies with other parameters; a rumination pool must be postulated. Its size and composition are estimated. Arguments are presented to support the view that dry matter and some of the microbes, chiefly the protozoa, do not leave the rumen at the PEG rate. One experiment with the same sheep fed twice daily showed significantly less production of microbial cells than did the continuous (each 2 hr) feeding. Analysis of the microbial cell yield suggests that, on the basis of 11 mg of cells per adenosine triphosphate molecule, a maximum of six adenosine triphosphate molecules could have been formed from each molecule of hexose fermented. The steps in analyzing a microbial ecosystem can be formulated as (i) describing the kinds and numbers of organisms concerned, (ii) identifying what they do, and (iii) observing how fast they do it. A complete description is kinetic, involving the rates of component processes and of the whole. Steps i and ii have been practiced extensively at I Present address: Department of Experimental Statistics, Bio numerous laboratories over the world, and many rumen microbial species have been identified (2,4). Their activities in the rumen have been inferred from the characteristics of the pure cultures and in a few instances (14,15) have been investigated experimentally. The rates of many rumen activities have been measured in pursuance of step iii. One criterion for the completeness and precision of a kinetic analysis of an ecosystem is the magnitude of the discrepancy between measured component rates, algebraically summed, and the measured rates of the total. This determination is necessary also for computer simulations. For the rumen, this involves measurement of microbial activities and their integration with host functions, in particular with the amount and kind of material disappearing during passage of food, and the turnover rates of the materials in the rumen. Such an analysis of the rumen is attempted, based on the continuous fermentation model (13). Since constancy of the rumen increases its resemblance to the model, the sheep was fed at frequent evenly spaced intervals. For comparison, some measurements were made on the same animal fed twice daily. MATERIALS AND METHODS Experimental animal. From February until December, 1969, a 55-kg Corriedale wether with a permanent rumen fistula was fed from an automatic feeder set to supply 90 g of air-dried alfalfa pellets at 2-hr intervals. In January, 1970 the same daily ration [976 g (dry wt) from the same batch of alfalfa] was fed in two portions of 540 g each at 7:30 and 19:30. Water was available at all times. Rumen contents were obtained through a glass tube (20-mm inside diameter) to which suction was applied. Prior to sampling, the contents were thoroughly mixed. Rumen liquid and small particles were obtained by inserting the perforated tube previously described (11) (27), and ash by conventional techniques. The pH was estimated with indicator paper and checked with a Beckman Zeromatic SS-3 pH-meter. Elementary analyses of feed and feces for carbon, hydrogen, and ash were performed by Galbraith Laboratories, Inc. Polyethylene glycol application and analysis. Polyethylene glycol (PEG) was used as a marker for several measurements of the rumen turnover rate constant in the continously fed animal and for one measurement in the twice daily system in January, 1970. PEG (10 g in 200 ml of water) was added to the rumen at 9:15 for the 2-hr feeding, and samples of rumen fluid were collected after 2, 3, 4, 6, 7, and 8 hr. For the twice daily feeding, the same amount was given immediately before feeding. Rumen samples were centrifuged and the PEG in the supernatant fluid was analyzed by the technique of Hyden (16). Rumen volume and rate of passage (turnover) were obtained from the linear regression equation for the ln PEG concentration against time. Measurement of fermentation activity. Volatile fatty acids (VFA) were determined by gas chromatography (Aerograph model 600-D, FFAP column) of rumen fluid supernatant (30,000 X g) passed through a 0.45-,um membrane filter (Millipore Corp.) and acidified with orthophosphoric acid. Production rates of VFA and ammonia were determined by the zero-time method (3,13) with carbon dioxide in the gas phase. Methane production was estimated by incubating a sample of rumen contents anaerobically for 1 hr in a stoppered container in the water bath (39 C). The gas produced was allowed to escape into a syringe, measured, and analyzed with a thermal conductivity gas chromatograph provided with a silica gel column. For rates of carbon dioxide production from organic C (not bicarbonate), the carbon dioxide above the sample was displaced with N2 just before the start of the measurement. At the end of the experiment, the culture was killed with sulfuric acid, inserted through the rubber stopper, which also released all CO2 from bicarbonate. The acid was added to a control before incubation. Control and experimental tubes were equilibrated at room temperature, and the excess gas was allowed to escape into the measuring syringe. The carbon dioxide remaining in solution when the initial and final gas volumes were measured was estimated from the solubility coefficient of carbon dioxide at room temperature and added to the measured excess gas. Microbial counts. Samples of rumen contents were preserved by adding one volume of 8% formaldehyde. This 2X dilution was further diluted 40X, a little crystal violet was added, and the bacteria were counted in a Petroff-Hauser counting chamber under oil immersion at X 1,000 magnification. Protozoa were identified according to Dogiel's monograph (4). Individuals of different species were counted separately as follows (25): 1 ml of a formaldehyde-diluted sample was further diluted to 12 ml with 10 ml of water or Lugol's iodine solution and 1 ml of glycerol. The solution was mixed by syringe, and 0.05 ml was placed on a microscope slide. The largest particles of hay were removed with a thin pin, and the sample was covered with a cover glass. All protozoa in the drop were counted. The rumen microbes were assumed to have a specific gravity of 1.1 and to contain 10% dry matter. An average nitrogen content of 10.5% was assumed for the bacteria (12) and 8% for the protozoa. RESULTS The results of proximate and elemental analyses of feed and feces are collected in Table 1. The nitrogen content (2.41 %) of the alfalfa. VOL. 22, 1971 pellets was close to that reported for 15 % protein (2.4% N) alfalfa (Dehydrated Alfalfa, Assay Report, 3rd ed. American Dehydrators Ass., Kansas City, Mo. 64112). The mineral percentages given in that report were used to predict the ash expected in our material. A value of 6.45% ash was calculated, as compared to the found value of 6.94% for our alfalfa sample. The usual analytical values for ash differ from the actual mineral content because some elements, e.g. sulfur, take up oxygen in the process of ashing. From the mineral analyses of alfalfa (American Dehydrators Ass.), the amount of oxygen taken up during ashing was estimated to be 25% of the final ash weight. The corrected ash value used was 4.84%. The oxygen value in Table 1 was the difference between 100% and the summed percentages of C, H, N, and corrected ash. The results of all PEG experiments to determine the turnover rate constant and the volume of the rumen contents, calculated from the PEG results, are shown in Table 2 for the 2-hr feeding experiments. The results of these experiments on the 2-hr regime were pooled, and a linear regression was calculated from the ln of all values of PEG concentration against time, giving the first order relationship in Fig. 1 leaving the rumen in 10.6 hr was equal to the volume in the rumen; the rumen PEG volume turned over 2.27 times per day. At the initial concentration of 2.65 g/liter, 10 g of PEG was contained in 3.77 liters. This has been used as the average volume of the rumen liquid into which PEG diffused. PEG does not enter the microbial cells or the ruminant cells lining the gut. We have assumed that ingested plant cells are killed in the rumen, permitting PEG to enter. In the insoluble plant materials, PEG probably does not reach the same concentration as in the liquid, but since the extent of PEG exclusion from nonviable material is unknown and its neglect introduces relatively little error (see below for its estimation), it is assumed in this study that PEG equilibrates with all nonliving material but not with living particulate matter. It is also assumed that, because of the The counts of protozoa in the particle (LSP) material remov sampling tube, and in the total are shown in Table 3 for the 2-hr bacterial counts are tabulated ir the size and number of the micr LSP material, their volume (Table 5) at 197.6 ,uliters/ml of Table 5 summarizes also the resu bial counts for the 12-hr feed containing volume is 802.4 ulit total rumen volume is 4.7 liters. The calculated microbial dry contents was 2.17% (w/v). The ( rumen fluid supernatant was 1.3 content was 0.41% (w/w dr measured dry matter in the total was 12.38%, standard deviatior This parameter, measured 21 1 month period, showed no consis change. With 2.17 and 1.33% d microbial cells and the dissolved particulate material amounts t( If it totally excluded PEG, the ( volume is wrong by this percent error is less to the extent that PE solids. The samples of liquid rumen removed from within the samp] 3 and 4) differed in dry matter ( deviation 0.84%) from the total (12.38%). The average percent. ammonia) in the pooled dry matt( LSP material was 3.855% as com for the total rumen contents. In Table 6 Fifteen similar measurements of the rate of methane production gave values ranging from 16 20 24 0.51 to 1.01, for an average of 0.74 mmoles per 100 g per hr or 0.83 moles/day in the entire ylene glycol con-rumen esults of aU°ll -Zero-time rate values for production of ammonia in incubated whole rumen contents were eding system,1aU 0.44, 0.54, 0.30, and 0.50, for an average of 0.445 mg sllysteim,th mmoles per 100 g per hr, equal to 0.50 moles of COPI,aly in the ammonia per rumen per day. id, had the same The rates of formation of the volatile fatty liquid and small acids are shown in Table 7. Initially, the proximate analyses of feed and ,ed through the feces (Table 1), with assumed chemical composirumen contents, ton for each component, were used in chemical feeding, and the equations to describe the fate of material disap- Table 4. From pearing during passage of feed through the sheep obial cells in the on the 2-hr regime. The summed values repwas estimated resented the estimated feed and fecal composition. rumen contents. The large amount of "other carbohydrates," delts of the microtermined by difference, was of unknown composiling. The PEG-don. Further, this method assumed elemental ,ers/ml, and the compositions for each component as the basis for calculating the total composition. Use of the weight in rumen direct elemental analysis of the total material to dry matter in the express the initial and final composition seemed 3 % (w/v); its N more reliable. y matter). The The daily feed-feces difference in the amounts rumen contents of each element ( (20) and assumed values of 9.39% N and 4i45' t standard 5.19% ash, an oxygen content of 32.22% was 4*45%,s'tandr calculated for microbial cells. The 9.39% N value agrumen contents was estimated from an assumed nitrogen content age of N (nonof 8% for the protozoa and 10.5% for the bacteria er ofthe sampled (12), the relative quantities of each being estipared to 2.445% mated from Table 5 In equation 2 the ash-free microbial cell dry weight is 110.5 g/day or 116.5 g including ash, containing 11.0 g of N. This represents the cells digested and absorbed. Any undigested and unabsorbed microbial material would not be detected as feed-feces difference. If it is assumed that the feed nitrogen is totally convertedinto either microbial cells or ammonia, the 4.69 g of N in the feces represents the microbial cell N produced but not absorbed. Addition to the 11 g of microbial N of equation 2 gives 15.69 g of total microbial N formed, corresponding to 167 g (dry weight) of cells per day, with a digestibility of 69%. The microbial N leaving the rumen at the PEG rate is 15.69 g per day, or 6.9 g per turnover. The dissolved nonammonia N in the entire rumen was 0.20g. Of the 4.7 liters of total rumen contents, 582 g (12.38%) was dry matter containing 14.2 g of N (2.445%), of which 14.0 g was not dissolved and was assumed to be chiefly microbial. With 6.9 g of microbial N and 0.2 g of dissolved N leaving the pool with PEG, 7.1 g of N remained in the rumen as part of the "rumination pool" (13). From the direct count estimates, the 185.5 mg of protozoa per ml of rumen contents (Table 3) and the 102.3 mg of bacteria (Table 4) amount to 6.97 g of protozoal N and 5.06 g of bacterial N, or a total of 12.03 g of microbial N in the entire rumen, of which 5.13 constituted a rumination pool. Both methods of calculation indicate a sizable retention of microbes in a rumination pool. The values for dry matter also indicate a rumination pool of considerable magnitude. The dry matter leaving daily includes the 369 g recovered as feces, the 116.5 g of microbial cell material not recovered in the feces (equation 2), the 57 g of VFA absorbed (calculated from Table 6), and the 43 g of minerals not recovered in the feces (Table 1), for a total of 586 g per day. With 582 g of dry matter in the entire rumen contents, a turnover rate of only once per day would supply the 586 g leaving daily. Such a rate indicates that the total rumen dry matter cannot turn over at the rate (2.27 turnovers per day) found with PEG. On the assumption that the only way dry matter and microbes leave the rumen is with the PEG pool, the amount in the rumen at any one time, leaving with PEG, is 586/2.27, or 258 g. The dry matter (324 g) remaining in the rumination pool does not leave with PEG, but through comminution during digestion, mixing, and rumination continuously contributes small particles to the The 6.9 g of microbial N leaving with PEG, with a volume of 679 ml, makes the volume of material leaving with PEG a total of 4.44 liters. The 258 g of dry matter in this volume gives a dry matter of 5.84% (w/v). In Table 9, the results of experiments with the 2-hr and the 12-hr feeding ( Table 5) are collected. Comparisons show that under the 12-hr feeding regime the rumen volume was larger, its dry matter less, and the turnover slower. Also, the concentration of both bacterial and protozoal cells was smaller. The estimated total microbial N supplied under the 2-hr feeding system is almost double that with the feed given twice daily. DISCUSSION Most of the values measured in the sheep are similar in magnitude to those reported by others. Rumen fluid turnover rate constants and rumen fluid volume for the 2-hr and 12-hr feeding agree with the estimates of Hogan et al. (8) and Hyden (17), respectively. VFA and ammonia-N concentrations are in good agreement with the estimates in the literature (5,8,23). The zero-time rates of VFA production are similar to the results obtained with a radiotracer technique in sheep fed continuously with approximately the same daily amounts of feed (6,8,23). The ammonia production estimates are within the ranges reported by Pilgrim et al. (23). Hungate (13) reported CH4/CO2 ratios of 0.22 to 0.50 for various grazing animals as comparedto 0.30 in the present trial. Protozoal N yield per day for the 12-hr feeding experiment agrees with the estimate of Reichl (26) for similar feeding conditions. Weller et al. (28) found in sheep fed once daily that 50 to 82% of the total nitrogen in the feed was assimilated into microbial cells. The value in our experiments is 83%. The 2-hr and 12-hr comparisons in Table 9 provide only a rough approximation of the effect of frequent feeding; the retention of microbes in the rumination pool of the 12-hr system cannot be estimated from the available data. But the results do indicate a significant superiority of frequent feeding for production of a large microbial cell crop. TIhere are some discrepancies in the nitrogen assumptions, calculations, and measurements. It was assumed that all feed N was converted either to ammonia or microbial cells. The method for measuring the N in rumen dry matter did not include ammonia, yet the nonammonia N by analy-a LSP, liquid-small particle fraction. b Rest of the protozoa assumed to be in the rumination pool. e Includes the total nitrogen in the rumen. sis was 2.445% or a total of 14.2 g in the estimated total volume (4.7 liters) of the rumen. Yet, by estimates from direct counts, the rumen contained only 12.05 g of microbial N. Only 0.20 g of this discrepancy can be accounted for as dissolved N. The 15.69 g total microbial N formed and the 7 g N in the ammonia produced amount to a total of 22.69 g of N per day as compared to the 18.83 g per day in the feed-feces difference. This increase (3.86 g) over that in the feed can be due to production of ammonia in the rumen from sources other than feed nitrogen. Urea N entering the rumen in the saliva is converted to ammonia. The total liquid volume leaving the rumen at the PEG rate is 2.27 X 4.44 liters, or 10.06 liters per day. This is approximately the volume of saliva expected per day. If all of the nitrogen (chiefly urea) in the saliva (17.6 meq/liter) (18) is converted to ammonia in the rumen, it amounts to 2.4 g per day. In addition, the urea N diffusing into the rumen from the blood can supply as much as 7.5 g of N per day to the rumen (9). The total possible rumen N supplied from all of these sources amounts to 28.73 g. Since 7 g appeared as ammonia, a maximum of 21.73 could theoretically have been assimilated into microbial cells, as compared to the 15.69 g of equation 2. If this additional N were synthesized into microbial cells, absorbed, and metabolized by the host, the N recycled from saliva and blood would not appear in equation 2, being assimilated into the host or excreted as urea. Thus the N balance in equation 2 is not a check on microbial N assimilation. But in such additional microbial cells, also, the H, derived from the feed, would not be recovered. The good H balance in equation 2 is not consistent with extensive microbial assimilation from recycled N. The balance for H atoms in Table 8 is much better than expected, considering the errors and estimates involved, particularly since any errors in C and 0 would be reflected in a discrepancy in H. The evidence for such a large rumination pool in this animal was unexpected. The rumen contents seemed fairly homogeneous when removed with the large-bore glass tube and the particles were small as compared to those in animals consuming hay. But the evidence from the direct estimate of microbial N by microscopic examination, as well as from measured total N in the rumen both indicate that the rumen nitrogen (chiefly microbial) did not turn over at the PEG rate. The rumen dry matter content also cannot turn over at the PEG rate and at the same time give only the amount of dry matter recovered in the feces. Digestive activity posterior to the fore-stomach could hardly account for the disappearance of the 324 g of dry matter in the rumination pool and disappearance of this amount would be inconsistent with the feed-feces difference. If all of the rumen microbial N (12.05 g) turned over with PEG, a total of 27.35 g per day would leave the rumen. This excess over the 15.69 g of microbial N produced per day indicates that the net average rate of passage or turnover rate of the microbes is 15.69/27.35 X the PEG rate, or 1.25 per day. It is doubtful that this average passage rate. for microbial N applies equally to all species Some bacteria such as Bacteroides succinogenes, adhering to plant particles, may be delayed in passage more than loose-floating cells such as Streptococcus bovis. The slower rate for microbial turnover as compared to PEG can account for a discrepancy between measured turnover rates of the rumen and the rates of growth observed in vitro for some of the large rumen protozoa. Polyplastron multivesiculatum (10; Coleman, Proc. Soc. Gen. Microbiol., vol. 61, p. iv., 1970) and Ophryoscolex purkynei (19) do not divide in vitro more often than once per day. Division each 7.3 hr is required to maintain their concentration in a continuous system turning over 2.27 times per day. But if, due to their high specific gravity or to a tendency to remain with the particles not passing with PEG, their average passage rate was only 0.69 per day, a division each 24 hr would be sufficient to maintain their numbers in the rumen. Inspection of Table 3 shows that the concentrations of Ophryoscolex, Polyplastron, Ostracodinium, and Eudiplodinium were considerably greater in whole rumen contents than in the LSP pool sampled. This is consistent with a turnover rate slower than PEG. Since these large protozoa contain about 65% of the total protozoal nitrogen, such retention could be sufficient in magnitude to account for a considerable part of the slow rumen microbial passage as compared to the LSP pool. For rapid and complete fermentation of food and production ofa maximal microbial crop within a limited time, retention of microbes in a rumination pool has the same advantages as cell feedback in a continuous culture (24). A larger population of microbes is retained in the rumen than could be held there if the total rumen contents turned over at the PEG rate. This may be an important factor in the success of the rumen fermentation of fiber. It may also explain the preponderance of starch-digesting protozoa in ruminants well adapted to a grain ration. The retained population of protozoa is so large that it ingests and thereby sequesters from S. bovis and other amylolytic bacteria much of the starch consumed; the bacteria cannot grow explosively as they do in hay-fed animals suddenly given grain. The latter do not contain the high concentration of protozoa (14). In batch culture, Bauchop and Elsden (1) found cell yields of Streptococcus faecalis to be 19 to 23 g/mole of glucose or 11.7 to 14 g/100 g of glucose added. Hungate (12) in continuous cultures of Ruminococcus albus found an average dry cell yield of 26.3 g/100 g of cellobiose used. Hobson and Summers (7) obtained values as high as 47 g/100 g. The dry cell yield for the 2-hr system is 27.3 g/100 g of feed-feces difference. This agrees with the estimates of Hume (9) for sheep on a 3hr feeding interval. If all of the 20.03 g atoms of C in the feed-feces difference is assumed to be hexose (3.34 moles), the yield of microbial cells for the 2-hr feeding is 50 g of microbes/mole of hexose. This is near the top of the values reported by Payne (22). The 1.345 g atoms of N in the feed-feces difference indicates that about 5 g atoms of the feed C was in protein (assumed to contain 16% N and 52.5% C). This leaves 15 g atoms of C in hexose, equivalent to 2.5 moles. The cell yield then becomes 67 g/mole of hexose used, equivalent to 6 adenosine triphosphate (ATP) molecules per mole of hexose, if 11 mg cells are derived from one ATP molecule, the value indicated in the extensive review by Payne (22). Two further factors influence this figure: (i) the actual value is less because the protein was fermented along with the carbohydrate, although less efficiently for cell production, and (ii) the actual value may be more because part of the feed carbohydrate is assimilated into cell material, diminishing the extent to which it can supply ATP. A high ATP yield may result from the greater number of ATP-yielding reactions possible in mixed as compared to pure cultures.

v3-fos

2018-04-03T03:18:06.443Z

1971-04-01T00:00:00.000Z

2018549

s2

Lactic acid utilization by the cutaneous Micrococcaceae. Human cutaneous staphylococci and micrococci utilized lactic acid as an energy source on a minimal medium. Propionic acid was not utilized, but l(+)-lactic acid and pyruvic acid could replace ld-lactic acid as a substrate. Selected strains of cocci were inhibited more by the l(+) and d(-) forms of lactic acid than the balanced ld form, particularly at pH 5.6. With proper dilution of substrate, lactic acid was utilized by selected strains in the presence of 10 mug of oleic and palmitic acids per ml. Numerous dietary and other salivary-derived carbohydrates are available to the facultative oral bacteria as energy sources, whereas simple sugars are absent from the secretions of the epidermis (3). This study was initiated to determine whether lactic acid has a role in the nutrition of the predominent gram-positive facultative cutaneous bacteria. Cutaneous samples were obtained by using the swabbing techniques previously reported (5). Gram-positive catalase-producing cocci were separated into groups of staphylococci and micrococci by the methods of Baird-Parker (1). Nutrient agar slants (Difco) were used to maintain the isolates. Isolation and enumeration of skin bacteria were done with Trypticase Soy Agar without glucose (BBL) and a minimal medium with the following composition per liter: NH4Cl, 1.0 g; K2HPO4, 7.0 g; KH2PO4, 3.0 g; MgSO4. 7H20, 0.2 g; MnCl2, 10 mg; FeCl3*6 H20, 1 mg; NaCl, 1.0 g; CaCl2, 1 mg; and yeast extract (Difco), 0.5 g. The final pH was 7.0. The minimal medium was also prepared with a pH of 5.6 by using 0.5 g of K2HPO4 and 9.5 g of KH2PO4 per liter. Solid minimal medium was prepared by adding 1.5% Noble agar (Difco). Selection of lactic acid-utilizing bacteria was made by plating skin swabbings on the Trypticase and minimal agars with and without the addition of 0.25% optically balanced LD-sodium lactate (Sigma Chemical Co., St. Louis, Mo.). Filter-sterilized pure L(+) and D( -)-lactic acids (Mann Research Laboratories, New York, N.Y.) were added to media after sterilization. Sodium pyruvate, sodium propionate, and sodium acetate were sterilized with media. All media were autoclaved at 121 C for 15 min. Sodium oleate and palmitic acid (Sigma) were each dissolved in 95% ethanol and added to media after sterilization. Lactic acid was measured colorimetrically (2) by measuring the quantity of substrate depleted from broth which was compared to controls to express the amounts of substrate utilized. Acetylmethylcarbinol production was determined with 5% alcoholic alpha naphthol and KOH-creatine reagents. Cultures were incubated in 125 by 16 mm screwcapped tubes under stationary conditions or shaken on a metabolyte water bath (New Brunswick Scientific Co.) at 200 rev/min. All experiments were conducted aerobically at 34 C. Optical density of broth cultures was measured with the Bausch & Lomb Spectronic-20 colorimeter. Inoculum was washed off nutrient slants in phosphate-buffered saline and adjusted to an optical density of 0.5 at 620 nm. A 0.05-ml quantity of this cell suspension was used to inoculate 4 ml of broth. When cutaneous samples were plated on Trypticase Soy Agar with and without 0.25% LD-lactate, little or no differences in total aerobic counts were observed on these two media although the size of all the colonies on the lactate-containing medium was generally larger. By using minimal medium, the numbers of bacteria from each of 18 samples were consistently greater and colony size was larger on minimal lactate agar when compared to basal minimal medium (Table 1 and Fig. 1). Bacterial populations from some sites did not grow on minimal medium unless lactate was added. Four additional samples from the scalp, forehead, groin, and toeweb were plated on minimal media containing 0.2% LD-lactic, L(+)lactic, pyruvic, and propionic acids. This experiment indicated that pyruvic and L(+)-lactic acid could replace LD-lactate as substrates for the skin flora, but propionic acid was not utilized. Since the media did not contain significant amounts of lipid, the cutaneous lipophilic diphtheroids were APPL. MICROBIOL. FIG. 1. Effect of medium composition on enumeration and detectioni of lactic acid utilizationi by cutaneous bacteria. Top: Trypticase Soy Agar; left, basal medium; right, with 0.25% LD-lactic acid. Lower: minimal agar; left, basal medium; right, with 0.25% LD-lactic acid. All media were pH 7.0. Plates were incubated for 4 days at 34 C and represent a 1:500 dilution of a swab rinse from thle toeweb. either absent or grew as pin-point colonies. When observed, they were counted on Trypticase Soy Agar as part of the total count which was the reason that total counts from some samples were much higher than those on minimal lactate medium. Neither the number nor the colony size of the diphtheroids increased on the lactate medium. The lactate-utilizing bacteria were essentially all coagulase-negative staphylococci or micrococci. Forty of these strains were isolated from minimal lactate medium and transferred to the minimal lactate broth to confirm lactate utilization. Certain strains were selected for further study. Six staphylococcal strains were tested for utilization of LD-lactic, pyruvic, acetic, and propionic acids at pH 5.6 ( Table 2). Lactic and pyruvic acids were utilized and acetylmethylcarbinol was produced by the strains in these broths. Acetylmethylcarbinol was not produced in basal, acetic, or propionic broths. Utilization of more than 50% of the LD-lactic acid indicated that both isomeric forms of lactate were used to some extent. When 10 jig of oleic or palmitic acid per ml was added to minimal medium at pH 5.6 containing 0.1 % LD-lactic acid, the substrate was also utilized from 50% to 98% by four staphylococci and five micrococcal strains. At pH 5.6, 0.1% L(+)and D(-)-lactic acids were inhibitory to the strains in minimal medium. At 0.025%, nine strains of staphylococci and micrococci were stimulated by L(+)-lactic acid, and the substrate was utilized ( All media (pH 7.0) were incubated for 4 days. Total counts were made on Trypticase Soy Agar without glucose. Samples were taken from seven normal adult subjects over a period of 4 months. concentration to 0.05% inhibited the growth of five strains. The results-of this study have some dermoecological implications. The cutaneous cocci utilized lactate at pH 5.6, the commonly recorded pH of many skin sites (4), in the presence of oleic and palmitic acids which are abundant in epidermal lipids (6). Utilization of L(+)-lactic acid is also of interest because it is the enantiomer of lactate formed from muscular metabolism (7). More detailed metabolic studies are in progress.

v3-fos

2018-04-03T01:54:16.679Z

1971-12-01T00:00:00.000Z

31386315

s2

Heat resistance of spores of marine and terrestrial strains of Clostridium botulinum type C. Resistance to heat of spores of marine and terrestrial strains of Clostridium botulinum type C in 0.067 m phosphate buffer (pH 7.0) was determined. The marine strains were 6812, 6813, 6814, and 6816; the terrestrial strains were 468 and 571. The inoculum level equaled 10(6) spores/tube with 10 replicate tubes for each time-temperature variable. Heating times were run at three or more temperatures to permit survival of some fraction of the inoculum. Survivors were recovered at 85 F (30 C) in beef infusion broth containing 1% glucose, 0.10% l-cysteine hydrochloride, and 0.14% sodium bicarbonate. D values were calculated for each fractional survivor end point after 6 months of incubation. Thermal resistance curves were constructed from the D value data. D(220) (104 C) values for spores of 468 and 571 equaled 0.90 and 0.40 min, respectively. The corresponding values for spores of 6812, 6813, 6814, and 6816 were 0.12, 0.04, 0.02, and 0.08 min. The z values for the thermal resistance curves ranged from 9.0 to 11.5 F (5.0 to 6.2 C). The classical paper of Bengston (2) is the only comprehensive report concerning the heat resistance of spores of Clostridium botulinum type C. Bengston studied spores of seven type C strains. Three strains were from fly larvae (Lucilia caesar or L. sericata), two from chickens showing symptoms of "limberneck," and one from the stomach contents of a horse reportedly dead from botulism poisoning; one nontoxigenic strain was included which Bengston had isolated by a singlecell technique from the prototype toxigenic culture. Spores of each strain were produced in a cooked-meat medium and heated in the same medium in which they were formed. Tubes were inoculated, flame-sealed, and completely submerged in a "boiling"-water bath. Spores showing the greatest heat resistance (strain from L. sericata) at an inoculum reportedly between 105 and 106 spores per tube survived heating for 60 min, but not 90 min. The purpose of the present study was to compare the heat resistance of spores of marine and terrestrial strains of C. botulinum type C. MATERIALS AND METHODS Suspensions. Terrestrial strains 468 and 571 and marine strains 6812, 6813, 6814, and 6816 were used. T,he sources, rmiethod of preparation, and standardization of spore suspensions were as previously presented (5). Aqueous suspensions were standardized to contain 107 spores/ml, based on preheating at 160 F (71 C) for 15 min. The standardized suspensions were stored unheated at 34 F (1.1 C). Heat resistance determinations. Ten-tube replicate sets were inoculated for each time-temperature combination. Sterile screw-cap tubes (16 by 125 mm) were inoculated with 0.1 ml of suspension (106) viable spores, and then 0.9 ml of heat-sterilized S0rensen's buffer (0.067 M, pH 7.0) was pipetted into each tube. Each set of tubes was heated simultaneously. For temperatures of 205 F (96 C) and below, a thermostatically controlled water bath with auxillary stirrer and insulated cover was used. Prior to heating, the screw caps were firmly tightened and sealed with a waterproof, heat-resistant tape. The tubes were placed in a metal holder, weighted down, and completely submerged in the bath. For temperatures of 215 F (101 C) and above, thermal death time retorts were used (4). The correction factor for the come-up time, taken from previous heat resistance studies, was considered to be 1.2 min. Various time-temperature combinations were run to obtain survivors in some fraction of the replicates. Survivors were recovered with beef infusion broth containing 1%glucose, 0.10% L-cysteine hydrochloride, and 0.14% sodium bicarbonate. The medium was prepared as previously described (5), dispensed in 200-ml quantities, and autoclaved at 250 F (121 C) for 15 min. The L-cysteine hydrochloride solution (20%) was heat-sterilized, and the bicarbonate (10%) was membrane-sterilized. Each was added aseptically to the medium immediately before use; then heatsterilized 1 N sodium hydroxide was added to adjust the medium to pH 7.0 to 7.2. About 10 ml of the re-1030 Value from extrapolation of thermal resistance curve based on data obtained at lower temperatures. covery medium was poured into each heated tube, and then stratified with melted Vaspar. The tubes were incubated at 85 F (30 C) and examined for turbidity and gas at periods up to 6 months. D values (time in minutes at a specified temperature to cause a 10-fold reduction in count) were calculated for each fractional survivor end point. The formula of Stumbo (6) was used: t log Alog S where t is corrected heating time (minutes), A is the population per tube times the number of replicates, and S is the number of positive tubes, presuming that giowth originated from one surviving spore in each positive tube. Thermal resistance curves for each strain were constructed by plotting the average D values on the logarithmic axis versus temperature on the linear scale on semilogarithmic graph paper. RESULTS AND DISCUSSION Determinations of thermal processes for foods involve calculations in degrees Fahrenheit; hence, the actual heating temperatures used are shown in degrees F with conversion to the nearest degree C. Tables 1 through 3 show the survivor data and D values of spores of the six type C strains studied. Where heat-resistant survivors were recovered, growth was usually detectable within 2 weeks after incubation. Occasionally, there was a marked lag, sometimes ranging up to 1 month, before turbidity and gas were seen in some tubes. Such lags were commonly associated with the longer heating times or higher temperatures used and apparently reflect the slow recovery of severely heat-damaged spores. The presence of type C toxin was confirmed Table 4 compares t spores of the terrestria summarizes the z val thermal resistance data heat resistance of spo: terrestrial strain (468) to that of spores of the most resistant marine strain (6812). Based on the limited number of cultures studied, spores of marine strains appear to be much more heatsensitive than spores of most terrestrial strains. In general, the data presented suggest that spores of both marine and terrestrial type C strains possess a resistance to heat which is intermediate between that shown by spores of C. botulinum type A or proteolytic type B (3) and that shown by spores of type E (1). points by the intraperi- [ues from a plot of the 6. Stumbo, C. R. 1948. A technique for studying resistance of a. Figure 1 contrasts the bacterial spores to temperatures in the higher range. Food res of the most resistant Technol. 2:228-240.

v3-fos

2018-04-03T02:00:07.310Z

1971-01-01T00:00:00.000Z

25518740

s2

Characteristics of antimicrobial resistance of Escherichia coli from animals: relationship to veterinary and management uses of antimicrobial agents. Five-hundred fifty-five (555) isolates of Escherichia coli were obtained from fecal specimens of a representative number of animals from five farms in the United States. Antibiotic exposure of the selected herds was determined by an epidemiological survey of these farms. The incidence of multiple resistance in the E. coli isolates was higher in herds exposed to continuous feeding of antimicrobial agents (84.8%) than in a herd not receiving antimicrobials (15.7%). The most common resistance configuration observed was the triple pattern of dihydrostreptomycin (DS), sulfonamide (SU), and tetracycline (TE). The second most frequent pattern consisted of four resistances: ampicillin (AM), DS, SU, and TE. The frequency of transfer factors was much higher in multiply resistant organisms from the herds exposed to antimicrobial medicaments. The E. coli isolates were relatively efficient in fostering and transferring heterologous resistance factors. AM resistance factors occurred more frequently in herds which were exposed to feed levels of penicillin (27.9%) than in those that were not (6.4%). The phenomenon of the transfer of multiple antibiotic resistance by conjugation was first reported in Japan (13) in 1959 to 1960 and in Great Britain in 1962. Between 1962 and 1965, English investigators (1)(2)(3)5) published several papers on the subject and showed that Salmonella typhimurium strains from human and animal sources which had resistance to several important antimicrobials [tetracycline (TE), streptomycin (SM), ampicillin (AM), and sulfonamide] could transfer all of these resistances to sensitive E. coli strains. Likewise, resistant E. coli could transfer its resistance to sensitive E. coli. More recently, other investigators (9-11) obtained evidence that infectious drug resistance was prevalent among nonpathogenic Escherichia coli from humans, calves, pigs, and fowl. It is possible, therefore, that these bacteria, which formed the bulk of the aerobic enterobacterial flora of the alimentary tract, were a reservoir of infectious resistance from which animal pathogens could IPresent address: Animal Health Division, Agricultural Research Service, U.S. Department of Agriculture, Hyattsville, Md. 2 Present address: Bacterial Diseases Branch, Center for Disease Control, Atlanta, Ga. become resistant and also potential donors upon ingestion of R factors to human intestinal flora. The overall use of antibacterial drugs in livestock rations for various purposes supplies a continuing pressure to maintain the process of resistance transfer. A survey was deemed necessary to determine how widespread these infectious resistant bacteria were in animal populations in the U.S. It was also important to determine the relationship between the use of antimicrobial agents in feed and the occurrence of multiply resistant organisms which contained transfer factors. A traceback system was used to select each farm for the study. A continuous supply of Salmonella isolates was obtained from the Diagnostic Services, Animal Health Division, National Animal Disease Laboratory at Ames, Iowa. Antibiograms were determined for each isolate received. Alternate farms of origin of multiply resistant and completely sensitive Salmonella, and the willingness of the farm owner to cooperate, were used as criteria for selection of premises. A cooperative agreement was established between the Food and Drug Administra-tion, Center for Disease Control, and U.S. Department of Agriculture, which provided a mechanism for obtaining samples from the farms of origin of the traceback cultures. This arrangement provided for the epidemiological support necessary to the study. MATERUILS AND METHODS Selective media were utilized to determiine whether any Salmonella isolates could be obtained from each fecal sample collected. No Salmonella isolates were detected from any of the animals examined on any of the farms. To isolate E. coli, rectal swabs were inoculated onto MacConkey agar plates which were then streaked on the premises and incubated overnight prior to return to the laboratory. Three typical lactose-fermenting colonies were picked to Triple Sugar-Iron-Agar slants and incubated at 37 C overnight. Strains giving typical E. coli reaction were further screened on Simmons citrate agar and adonitol and were checked for motility and for production of ornithine decarboxylase and oxidase. The antimicrobial susceptibilities of confirmed E. coli were tested by the technique of Bauer et al. (4), except that dihydrostreptomycin (DS) was substituted for SM; studies in our laboratory demonstrated equivalent zone sizes against E. coli. The following antimicrobials were used in the testing system: AM, DS, cephalothin (CEPH), sulfamethoxypyridazine (SU), colistin (CL), chloramphenicol (CHLOR), furazolidone (FU), neomycin (NEO), TE, and nalidixic acid (NA). All strains resistant to two or more antimicrobials, including resistance to either tetracycline or ampicillin, or both, were examined for transferable resistance. The R-factor transfer procedure and media used were those of Schroeder et al. (8) with slight modifications. Four media were used: I, MacConkey agar; II, Mac-Conkey agar plus 25,ug of NA per ml; III, MacConkey agar plus 25 ,ug of NA and 4 ,ug of TE per ml; and IV, MacConkey agar plus 25 ,g of NA, 10,ug of AM, and 10 ,ug of dicloxacillin per ml. To test for R factors, equal volumes (0.5 ml) of overnight cultures in Mueller Hinton broth (MHB) of the donor E. coli and recipient non-lactose-fermenting E. coli (NAresistant strain of K-12F-, courtesy of H. Williams Smith) were mixed and incubated at 37 C for 2 hr. At the same time, MHB tubes were inoculated with recipient alone and with a multiply resistant Salmonella control alone and were incubated for the same time interval. After 2 hr, all cultures were streaked onto plates of media I and II, and 0.1-ml portions were spread onto plates of media III and IV. For results to be valid, the Salmonella control strain was required to grow on all four plates and recipient control strain only on plates I and II. In conjugated mixtures, both recipient and donor must have been present on plate I and only recipient on plate II. Typical colonies of the recipient strain appearing on plates III or IV, or both, were picked and tested for antimicrobial susceptibility. Table 1 outlines the major species of animals tested on each farm. Farms 1, 3, and 4 utilized a continuous program of medication with antibiotics. Farm 2 was primarily a cattle operation utilizing a management system which included the administration of antimicrobials during periods of "stress" to the animals. Farm 5 utilized three separate locations for the production of swine, calves, and dairy cattle. The swine were receiving continuous low levels of oxytetracycline in the feed. The calf operation utilized nitrofurazone at therapeutic levels (e.g., administered as one dose at the time of birth). RESULTS The dairy cows were receiving no continuous medications, but two cows had been treated for mastitis the previous 2 weeks before the test. These two cows were the source of 40% of the multiply resistant strains isolated from this herd. Animals exposed to continuous levels of antimicrobials (farms 1, 3, 4) had a much higher percentage of resistant organisms and had a frequency of occurrence of multiple resistance substantially greater than animals on the other farms ( Table 2). Of the E. coli isolates from the swine populations of farms 1, 3, 4, and 5, 67 to 95.9% were multiply resistant. The cattle population on farm 2, receiving intermittent antimicrobial pressure, yielded 38.4% multiply resistant E. coli. In contrast, the calves and dairy cattle on farm 5, receiving no continuous antimicrobial pressure, had substantially lower frequencies of multiple resistance, 19.0 and 15.7%, respectively (Table 2). Correspondingly, the incidence of sensitive organisms was higher in the animals which were not being exposed to continuous levels of antimicrobial agents. Table 3 clearly illustrates that as the antimicrobial pressure was reduced there was a corresponding drop in multiply resistant strains. In Table 4 can be seen the ratio of homologous resistances (resistant factors in fecal flora which are the same as the antimicrobial being used in the feed) to the heterologous resistances (resistant factors not related to the antimicrobial used in the feed). When an antimicrobial was used in the feed, the percentage of isolates resistant to it was substantially higher than when the antimicrobial did not appear in the feed. Multiple resistance was also correspondingly high in these herds and showed a definite correlation to the content of the antimicrobial in the feed. A striking exception to this general tendency was the high incidence (65.0%) of DS-resistant organisms ( Table 5). There was no evidence that this drug had been used on any of the farms for the previous 6 Table 4) that AM resistance occurred more frequently when penicillin was used in the feed (27.9%) than when it was not used in the feed (6.4%). This finding appears to be in contrast to an earlier report (6) which indicated that penicillin in feed may have little influence on the buildup of AM-resistant, gram-negative organisms. Of the 491 isolates from animals exposed to antimicrobials in feed, 394 (80%) were resistant strains and fell into 39 different antimicrobial patterns. In contrast, of the 64 isolates obtained from animals not exposed to antimicrobials in feed, 14 (15.6%) were resistant strains falling into 10 different antimicrobial resistance patterns. Table 6 illustrates the distribution patterns of singly and multiply resistant isolates. The most commonly occurring resistance patterns were those organisms resistant to three and four antimicrobials. The least common were organisms resistant to five and six antimicrobials. The occurrence of one and two resistance factors was equally prevalent. Nearly 70%O of all isolates contained three or four resistance factors. Following in sequence of occurrence were two resistances (12%7), five resistances (11 %), single resistances (6%) and six resistances (1 %). In Table 7 are presented the frequency and rank in which six of the antimicrobials were commonly included in resistance patterns and which occurred with additional resistances. NEO was found to be the most dependent on other resistance factors. It never occurred as a single resistance and thus was most likely to appear in a multiply resistant pattern. On the other hand, DS apparently has less dependency on other R-factors. It occurred more often as a single resistance than did any of the other antibiotics. The nine most common and frequently occurring resistance patterns found in the study are presented in Table 8. DS, SU, TE was the most frequently occurring pattern, followed by the DISCUSSION Antimicrobials have received widespreadK popularity as constituents of animal feeds in the United States. They are used at low levels for growth promotion purposes, at median levels for, disease prevention, and at therapeutic levels for the treatment and control of disease. One of the: five farms surveyed in this study (farm 5) utilized' a growth promotion level (50 g/ton) of oxytetracycline in the swine ration. Three other farms (1,, 3, 4) were utilizing a combination of drugs (peni-cillin, chlortetracycine, and sulfamethazine) at a combined level of 250 g/ton. This level of drugs is commonly used for disease prevention. In. comparing the percentage of multiply resistant isolates from these farms (86% for farms 1, 3, and 4 versus 76% for farm 5) there apparentlywas little difference in the levels of resistant organisms stimulated by the use of either ofthese dosage levels. The cattle on farm 2 were receiving a dosagelevel of chlortetracycline (350 mg per head perday) and sulfamethazine (350 mg per head per day) which might be considered disease prevention levels. These drugs were used on an intermittent basis, and the level of multiply resistant organisms was much lower in this group of animals (38.4%). The calves on farm 5 received a single therapeutic dose of nitrofurazone at birth. The incidence of multiply resistant organisms on March 21, 2020 by guest http://aem.asm.org/ Downloaded from found in these animals was also comparatively low (19.0%) and comparable to that found in the herd of dairy cows receiving no antimicrobials in feed (15.7%). Forty per cent of the multiply resistant isolates from these animals could be traced to two cows which had recently been treated for mastitis. These findings indicate that the level of drug may not be the major factor in determining the amount of resistance that will develop. The continuous antimicrobial pressure seems to have a greater influence. This was true for both the level of resistance and the percentage of transfer factors that was observed. This work also confirms the earlier work of Walton (12), who found a definite association between the types of drugs supplied to the animals and the isolation from their feces of strains of E. coli resistant to these drugs and capable of transferring this resistance. The high incidence of DS resistance, even though this drug was not being used on the premises investigated, cannot be explained with the information available from these farms. One can only speculate about the origin of such a high incidence of resistance. It is well recognized that DS is commonly utilized as a therapeutic agent for many diseases of domestic animals. The drug may have been used therapeutically on these farms. Specific inquiries regarding such exposures were included in the questionnaire but could have failed to verify this fact. Another possibility would be the dependence of DS on the transfer factors of other antibiotics for its continual influence on resistance. This does not seem likely, since it was shown (Table 7) to be the least dependent upon the cross-transfer of R factors. However, recent work by Datta et al. (7) may provide some insight into this observation. They found that TE exerted strong selection, not only for TE resistance but also for multiple resistance, significantly increasing the frequency of resistance to AM, SM, CHLOR, and sulfonamide. A higher incidence of ampicillin resistant organisms was observed in herds which were receiving feed additive penicillin. The combination of three drugs (penicillin, chlortetracycine, and sulfamethazine) was substantially more effective in producing AM resistance of the E. coli isolates from swine than was oxytetracycline alone (86 of 307 strains versus 6 of 110 strains). This occurrence could be explained, and in fact might be expected, since both AM and penicillin are susceptible to the action of penicillinase, and penicillin would induce this enzyme among gram-negative enteric flora. However, since the use of both sulfonamides and TE may be associated with the emergence of heterologous resistance to AM, the role of penicillin per se can not be confidently ascertained. The observation of a substantially higher frequency of transmissible resistance among multiply resistant strains that come from animals exposed to antimicrobials invites further speculation. Presumably, a selective advantage is conferred to One 1 31 1 4 1 0 0 0 23 2 63 6 Two 3 53 2 40 2 0 2 1 36 3 142 12 Three 16 159 3 158 0 1 2 8 139 0 486 42 Four 62 80 15 77 3 4 0 13 67 1 322 28 Five 17 26 16 26 0 0 0 20 25 0 130 11 Six 3 3 1 3 0 0 2 3 3 It is probable that humans and animals ingest small and continuing doses of enteric organisms carrying resistance determinants; in the absence of antimicrobial selective pressures, such strains may conjugate with indigenous flora but no selective advantage accrues to their offspring. However, if a population of strains resistant to drugs being administered is present, and these strains carry transfer factor at a much higher frequency, as the data show, then the likelihood of transfer of resistance determinants to "outsiders" is increased; such transfers would confer a survival advantage to a recipient and its progeny which now carry the combined resistances of initial donor and recipient. This postulated mechanism may account for the appearance of heterologous resistance in animals exposed to agents such as sulfonamides and TE. Another area for concern was the inordinately high incidence of resistance observed in the swine herds. One herd yielded 100% resistant isolates, and the other herds ranged from 89% to 95% resistant isolates. A logical question would be whether these levels of resistance could be tolerated in high-density production procedures, especially if an infectious disease outbreak occurred. This is a particularly interesting question when one considers that the majority of the isolates were multiply resistant and contained viable transfer factors and that one of the major causes of early swine mortality and morbidity is colibacillosis. The selection of an effective therapeutic agent under these circumstances could become very difficult. The results of this survey suggest that a high incidence of resistant organisms does exist in animals being exposed to continuous levels of antimicrobial drugs. R factors were common in these organisms, and multiple resistance to three or more antimicrobials was usually prevalent. Serious consideration must be given to the desirability and future acceptability of producing livestock in an environment comprised primarily of highly resistant microorganisms.

v3-fos

2018-04-03T01:46:39.415Z

1971-03-01T00:00:00.000Z

30892163

s2

Listeria monocytogenes in nature. Samples from 12 farms were examined during two successive spring and early autumn seasons. L. monocytogenes was isolated from vegetation or soil taken from 11 of the 12 farms and from 6 of the 7 nonagricultural sites. A total of 27 strains were isolated from the 19 sites. The organism was not isolated from any of the autumn collections. The wide geographical distribution of listeric infections involving diverse species of animals (7) suggests common sources of Listeria monocytogenes shared by man and animals. The epidemiology of listeriosis is perplexing, and the habitat of L. inonocytogenes is obscure. Seeliger (15) has commented on the resemblances of the biochemical and cultural characteristic of L. monocytogenes to some plant-soil inhabitants and has speculated "that there may well be a primary saprophytic life of Listeria" in which event the epidemiology and epizootology of many listeric infections would be more comprehensible. The purpose of this investigation was to implicate the soil-plant environment as a reservoir of L. monocytogenes. In previous preliminary studies (18), L. monocytogenes was isolated from vegetation in an agricultural area where listeriosis was rare. In the present study, vegetation at the same sites was sampled during two spring and two autumn seasons of successive years. Soil was cultured from the same sites during the second year. To obtain information on the presence of L. mnonocytogenes in a different setting, uncultivated vegetation was examined from sites within a small area of another county. A nonagricultural, residential, suburban community with no known listeric infection was selected to minimize the possible role of livestock in Listeria dissemination. The aims were: to note possible seasonal variation in the frequency of occurrence of the organism, to observe difference or similarity of strains recovered on repeated sampling, to compare plant isolants with the strains isolated from surrounding soil, and to observe similarity or difference in isolants from nature and L. niionotogenes isolated from infections. MATERIALS AND METHODS Plant samples and soil were obtained in Hanover County, Va., from 12 different farms scattered throughout the county and separated by as much as 50 km. One sample per farm was collected in April 1967 from dead and decayed corn or soybean plants as described elsewhere (18). The same farm was visited during September 1967, April 1968, and September 1968 when plant samples again were obtained as near as possible to the original site. These specimens were collected from portions of the plant 50 cm above the ground. Some of the plants collected in auturnn were still green, some were partially green, and some were completely brown. Also, at the time of the last three collections, a total of about 20 g of surface soil was collected to a depth of 2 to 3 cm obtained near the base of the plants being sampled. Plant material also was collected from a nonagricultural, partially developed, suburban residential location (2 to 3 km2 in area) in Henrico County. With one exception (day-lily leaves), the specimens collected here were wild grasses growing either in or at the edge of a wooded area, along the banks of a brook, or in open lots. Seven sites were selected, and each site was visited in April and again in September 1968. The April samples were dead plants which had remained standing during the winter; the September samples were still green. The vegetation was placed in large jars with Brain Heart Infusion (BHI; Difco) as previously described (18); and held at 4 C over a period of 3.5 months (1967) to 5 months (1968) with samples of the liquor taken every 20 to 25 days (1967) to 30 days (1968). Previous experiences (Welshimer, titipuiblished data) in culturing vegetation without prior and prolonged cold enrichment were unrewarding; therefore, all specimens were held at least 20 days before subculturing for Listeria. The culture of the liquor obtained April 1967 has been described in detail (18) and modified as follows for the 1968 specimens: BHI (5 ml) was inoculated with 0.05 to 0.1 ml of the liquor and incubated overnight at 37 C. This culture was handled in two ways. (i) It was streaked on several plates of blood-agar base containing I % glucose (TBG; Difco) and at the same time streaked on Mc-Bride Listerica agar (14). Each plate was streaked by a different spreading technique to ensure good distribution of colonies. (ii) The BHI ctLlture was placed in the dark at 20 to 25 C. If L. monocytogenes was not recovered from the initial plating of (i), the BHI cultures were held for 2 weeks before subculturing on plates of TBG agar and McBride agar. The TBG plates were incubated 37 C, and colonies were examined microscopically at 24 and 48 hr by the oblique lighting method as described by Gray (6). The Mc-Bride plates were inoculated with 4 loopsful of the BHI cultures, incubated at 37 C for 2 days, placed at 20 to 25 C for 1 to 2 days, and examined by oblique lighting for small intensely blue colonies. Soil samples (15 g) were placed into 100-ml Erlenmyer flasks containing 50 ml of BHI adjusted to pH 7.4. These samples were held at 4 C and subcultured over a period of 3.5 months as described above for vegetation. Colonies suspected of being L. monocytogenes were picked, streaked on TBG agar plates, and incubated for 18 to 24 hr. Typical colonies of catalase-producing, grampositive, evenly staining, small rods, motile at 20 to 25 C, were tested on the following carbohydrates: glucose, salacin, lactose, trehalose, esculin, rhamnose, maltose, melezitose, mannitol, and sucrose. These carbohydrates were held at 37 C and observed for 8 days for reactions characteristic of L. monocytogenes (8,15). Organisms with the morphological, cultural, and biochemical reactions of L. monocytogenes were tested serologically in Richmond by macroscopic tube agglutination tests with rabbit antisera developed against the major serotypes and then forwarded to Utrecht where, in addition to repeating the biochemical and cultural reactions, an extensive analysis was made of each strain by using monospecific antisera developed against the individual H and 0 antigen factors of the various serotypes of L. monocytogenes. Pathogenicity of the isolants was based on the response of mice (Rockland Farm SW) weighing 16 to 18 g, intraperitoneally inoculated with suspensions of 2 X 109 L. monocytogenes. Isolants failing to kill sets of mice in 3 weeks were considered avirulent. At this dose, the mice either died within 5 days or survived the 3-week observation period. The dead mice were autopsied and the liver and spleen were cultured. Isolants were streaked on sheep and rabbit bloodagar with 5% blood in Tryptose-blood-agar base, incubated at 37 C, and observed at 24 and 48 hr. After mixing and holding the soil or vegetation in BHI at 4 C for the first 18 to 24 hr, some of the broth was streaked on eosin-methylene blue plates. RESULTS L. monocytogenes was not recovered from vegetation or soil collected from the agricultural area (Hanover) during September 1967 or September 1968. L. monocytogenes was not recovered from vegetation collected from the smaller suburban area (Henrico) during the single September collection, but it was isolated from the spring collections in both areas. Eight strains of L. monocytogenes were isolated from vegetation at 7 of the 12 sites in the spring of 1967, and 9 strains were isolated from vegetation of 9 of these sites 1 year later. L. monocytogenes was isolated from vegetation at three sites where none was found the previous year. Only at one site were the organisms isolated the first year and not the second year. Specimens simultaneously collected from soil and the adjacent vegetation provided contrasting results: L. monocytogenes was isolated from soil at four sites, three of which also yielded Listeria from the vegetation; however, the soil strain and the vegetation strains were antigenically distinct. A virulent soil strain was isolated from a site where previously no Listeria were isolated. Of the 12 farms examined, L. monocytogenes was recovered from vegetation, soil, or both at each of 11 sites either in April 1967 or 1968. L. monocytogenes was isolated in the spring from six of the seven nonagricultural sites. The usual H and 0 antigens of L. monocytogenes with an additional 0 factor, XV, were found in 17 strains. This factor previously was encountered (Donker-Voet, unpublished data and designated as "XV" in L. monocytogenes W-Li 93/65 received by one of us (D-V.) from H. Seeliger, Wurzburg, originally isolated by H. E. Larsen, Copenhagen, from chicken feces. Strains with factor XV closely resembled subtypes of type 4 L. monocytogenes; e.g., in addition to H factors A, B, C and 0 factors III and XV, seven strains contained 0 factors V, VII, and IX as does type 4a; five of the strains contained factors VI and IX; and five strains contained factors V, VI, and IX, thus resembling 4ab and 4e but lacking factor VII or VIII associated with these serotypes. One strain, with the unusual combination of factors I and IX, was indeed anomalous. The nine remaining Listeria strains were type la or lb; two strains were avirulent. Factor XV strains were all avirulent. Mice inoculated with the virulent strains presented the hepatic lesions and overwhelming septicemic response characteristic of mice inoculated with human animal strains of Listeria. All strains were tested on both rabbit and sheep blood-agar. The virulent strains hemolyzed sheep blood-agar, but the avirulent strains did not. Rabbit blood was hemolyzed by all of the virulent strains and six of the avirulent strains. The avirulent strains which hemolyzed rabbit blood produced a darkening or alpha-like hemolysis on sheep blood. The method of subculturing the refrigerated specimens collected in 1968 resulted in earlier detection of L. monocytogenes than previously accomplished (18). Of the 19 positive isolations of L. monocytogenes in 1968 from the different specimens, 11 were obtained from specimens cultured after refrigerating for 1 month, and 8 isolations were obtained after refrigerating the specimens VOL. 21, 1971 WELSHIMER AND DONKER-VOET for 2 to 5 months. The four soil strains were isolated after 1 month of refrigeration. Nine of the isolations were obtained from BHI subculture of the refrigerated liquor held at 20 to 25 C for 2 weeks after the initial overnight incubation at 37 C (method ii). AU of the plant specimens obtained in the agricultural area during the spring gave lactosepositive colonies on eosin-methylene blue-agar. The number varied from 3 to 4 colonies per plate to uncountable numbers. No Escherichia coli was observed, for these were all Klebsiella-Enterobacter types of colonies. Only one sample of soil from the same 12 sites contained any lactose-positive colonies. At the nonagricultural sites, lactosepositive colonies were found in two of the seven plant samples. Autumn specimens were comparable to spring specimens with respect to the numbers and distribution of Klebsiella-Enterobacter type colonies. No E. coli colonies were observed in any of the 52 specimens examined during 2 years. DISCUSSION Decaying moist vegetation favors the support of L. monocytogenes, for none was isolated from the green or recently dead vegetation collected in early September although the same plant growth yielded Listeria in the spring after standing over winter. The dryness of the surface soil may explain the absence of organisms from that source in the autumn collection; experimental studies (13,17) have shown that survival of Listeria in soil is influenced by moisture content. The morphology, colony appearance, biochemical reactions, growth characteristics, antigenic composition, and mouse response to inoculation with the virulent plant-soil strains of Listeria were indistinguishable from L. monocytogenes strains isolated from infected humans and animals. The avirulent strains likewise were similar except for the presence of factor XV in many of the strains and the absence of beta-hemolytic activity on sheep blood-agar. These properties should not exclude the strains from the species, for the organisms do possess other H and 0 factors associated with L. monocytogenes (5). In the absence of less definitive antigenic analysis, some of these strains might have been placed within the group 4 serotypes as was the case in an earlier report (18). Bojsen-M0ller (2) found that 12% of the strains of L. monocytogenes which he isolated from human feces grew without hemolysis on a variety of blood-agar media. infection is important, with the animal gut acting as a reservoir of the agent. In our studies, the absence of E. coli indicates that there was no marked fecal contamination of the soil at the time of sampling; however, it does not exclude the possibility of earlier deposition with death of the less hardy E. coli. The isolation of Listeria from the suburban community indicates that the organism is not peculiar to agricultural pursuits nor restricted to association with farm animals in the area, but one can not exclude the possible role of birds and feral animals such as squirrels, chipmunks, and oppossums. The ability of Listeria to multiply at low temperature, its ability to survive for long periods in soil (13,17), and its recovery from decaying vegetation implies a saprophytic existence wherein the plant-soil environment may serve as a reservoir. Rather than attribute the presence ofListeria in nature solely to past contamination with animal feces to the exclusion of an independent role as a free-living organisms, one might liken Listeria to the Klebsiella-Enterobacter organisms which extensively exist as free-living forms on plants and soil yet inhabit the gut of man and animals and under appropriate circumstances produce disease.

v3-fos

2018-04-29T17:05:05.331Z

1971-01-01T00:00:00.000Z

13825943

s2

Effect of Light Quality and Light Intensity and Various Sugars on the Sexual Expression and Some Observations on the Red Pigment in Equisetum Gametophytes The purposes of this study were to explore what effect light quality and light intensity and various sugars have on the sexual expression of Eguisetum gametophytes and to determine some characteristics of the red pigment in Eguisetum gametophytes. Light quality and light intensity were employed to seek the presence of a morphogenetic factor involved in the sexual expression of Eguisetum gametophytes. The gametophytes were grown in mass culture in petri dishes and singly in test tubes under red light and white light or under high light intensity and low light intensity. Light quality was employed when the Eguisetum species was Eguisetum hyemale. When the species was Eguisetum arvense, light intensity was employed. A higher percentage of antheridial gametophytes in mass culture in petri dishes than singly in test tubes under either red light or high light intensity would indicate an interaction among the gametophytes due to a diffusible substance and thus would indicate that light quality or light intensity was probably involved in a mechanism which activated a morphogenetic factor determining the sex of Eguisetum gametophytes. In mass culture in petri dishes, the morphogenetic factor activated by either red light or high light intensity would influence the sexual expression of other gametophytes in the same petri dish; whereas, singly in test tubes the gametophytes are isolated from each other so that any morphogenetic factor that is produced could not influence the sexual expression of the gametophytes. The factor would probably be some sort of diffusible substance comparable to the antheridogens of ferns. The results were ambiguous. Mannitol, sucrose and glucose we~e added separately to Bold's basal medium to determine what effect these sugars have on the sexual expression of Eguisetwn gametophytes. The results were ambiguous. The red pigment often associated with Eguisetum antheridial production was isolated by column chromatography and some characteristics were determined using visible light spectrophotometry. It is definitely not rhodoxanthin, as reported, but could not be identified other than to be a carotenoid. This study showed that Eguisetum gametophytes are a difficult system with which to experiment. It seems the sensitivity of Eguisetwn spores to light quality and light intensity can vary. INTRODUCTION Eguisetum, a vascular cryptogam, has a life cycle of two alternating generations, the sporophyte generation and the gametophyte generation. The sporophyte generation bears the strobili that shed the spores. The gametophyte generation, the sexual phase, bears the gametangia (archegonia and antheridia) which form the gametes (egg and sperm). Uncertainty over the nature of sexuality in Eguisetum gametophytes is seen in the morphology textbooks. Some authors (Bold, 1957;Campbell, 1913) have considered them to be unisexual. Other authors (Eames, 1936;Foster and Gifford, 1957;Haupt, 1953) have considered them bisexual. Hauke (1967) believes that Eguisetum gametophytes have the potential to be bisexual, but most gametophytes are unisexual and those that are bisexual are postmature and were once unisexual. There is only one species of Eguisetum which has bisexual gametophytes from the beginning (Hauke, 1963 and. Factors other than genetic seem to play an important role in the sexual expression in Eguiseturn gametophytes. Several authors (Campbell, 1913;Schratz, 1928;Walker, 1931;Eames, 1936;Williams, 1938;Haupt, 1953;Scott and Ingold, 1955;and Doyle, 1970) have reported crowded and unfavorable conditions increase antheridial formation in Eguisetum gametophytes. Factors such as nutritional deficiencies, hormonelike substances, and the accumulation of waste products may influence sexual expression in Eguiseturn gametophytes. It may be that a hormonelike substance may control sexual expression in Equisetum gametophytes. Already antheridogens that control the forma-tion of antheridia have been found in fern gametophytes (D8pp, 1950;NHf, 1956NHf, , 1959NHf, , 1960NHf, and 1961Voeller, 1964). Because fern gametophytes are much like those of Eguisetum in that they are both photosynthetic, dorsiventral and terrestrial, substances comparable to those in ferns may be operating in Equisetum. Recently Hauke (1971) has shown that light quality affected sexual expression in Eguisetum hyemale gametophytes and that light intensity affected the sexual expression in Eguisetum arvense gametophytes. One purpose of this study was to further explore the effect of light quality on sexual expression in Eguisetum hyemale gametophytes and of light intensity on Eguisetum arvense gametophytes. Hauke (1971) has shown that sucrose, a sugar, also affects the sexual expression in both of these species of Eguisetum. Another purpose of this study was to determine what effect sugars have on these species of Eguisetum gametophytes. Glucose, mannitol and sucrose were the sugars chosen for study. If the effect of these three sugars were the same, the effect would be assumed to be osmotic. The effect would be considered nutritional if mannitol showed no effect and the other sugars did. If only one of the sugars produced an effect, this would indicate some special morphogenetic effect. A red pigment has been observed in Eguisetum gametophytes (Eames, 1936;Chatterjee and Ram, 1968). The red pigment is seen under the microscope in chromoplasts in cells adjacent to antheridia, so that it seems the red pigment is related to the sexual expression of Eguisetum gametophytes. The final purpose of this study was to determine some characteristics of this red pigment. METHODS AND MATERIALS Spores ~ inoculation of cultures. Eguisetum gametophytes were grown from spores on Bold's basal medium agar (BBMA) prepared f rom stock solutions (Bold, 1967 Cheesecloth was used to regulate light intensity but did not affect 1 ight quality. Two layers of cellophane "K" 210 FC Red from DuPont were The gametophytes were allowed to grow about six weeks before they were harvested to determine the percentage of antheridial gametophytes that formed. The percentage of archegonial gametophytes formed was not determined because of the difficulty in observing archegonia in Eguise-.E!fil gametophytes. Antheridial gametophytes were however identified without difficulty by observing antheridia under a dissecting microscope. Whenever there was any doubt, the gametophyte was observed under a microscope using lOOX magnification to eliminate any uncertainty. Those gametophytes that were not identified as antheridial gametophytes were considered either archegonial or neutral. containing the red pigment was treated in exactly the same manner as the original acetone homogenate and the resulting residue was taken up again in 3 ml of light petroleum ether and chromatogrammed as before, but using a column of aluminum oxide (activity of 2). The red band was eluted and the eluate was treated again in exactly the same manner as the original acetone homogenate and the dried purified residue was taken up again in 3 ml of light petroleum ether. An absorption curve was determined between 300 nm and 700 nm using a Cary recording model 15 spectrophotometer. This procedure is an adaptation from that of Jagels (1970) and Foppen (1969 Table 3. The first set of experiments showed that sucrose and glucose had no effect on the formation of antheridial gametophytes but that mannitol stimulated antheridial production. In the second set of experiments the results showed mannitol inhibited antheridial gametophyte development, but glucose had no effect. The results from the third set of experiments showed glucose and sucrose increased the formation of antheridial gametophytes, but mannitol had no effect. The fourth set of experiments showed that glucose had no significant effect on the formation of antheridial gametophytes. In the fifth set of experiments the results showed that glucose, sucrose and mannitol all increased antheridial gametophyte formation. Finally the results from the last set of experiments showed that mannitol inhibited the formation of antheridial gametophytes, but glucose had no effect. It must be noted that contamination might have been a factor in these results, and sucrose showed no effect on the sexual expression of Eguisetum hyemale gametophytes under 1,500 ergs/cm 2 /sec but did under 5,000 ergs/cm 2 /sec. The results from experiments using light intensities of 15,000 and 20,000 2 ergs/cm /sec were not interpreted because the spores used in these experiments were either stored at -10 C in glycerol or obtained outside in freezing temperatures during the winter which might have affected the physiology of the spores. 2 The results under 1,500 ergs/cm /sec suggested the effect of glucose and sucrose is not nutritional because neither sugar showed an effect. 2 The effect of these two sugars under 5,000 ergs/cm /sec could not be determined to be either nutritional or osmotic because the effect of mannitol was inconsistant. In summary light intensity may have interacted with the various sugars or may have no effect, but at any rate the author interpreted the results both ways. These experiments would have been repeated until a conclusion had been obtained, but there were no longer available viable spores. In repeating these experiments, light intensity should be kept constant and the sugars should be added to the agar medium in equal molarity concentrations, rather than equal weight to volume concentrations. The results of various sugars on sexual expression in Eguisetum arvense gametophytes showed that the effect of mannitol, which produced an effect under high light intensity, is osmotic and not nutritional because mannitol is not metabolizable by many plants, but sucrose, which produced no effect, is. It appears light intensity interacts with this osmotic effect of mannitol because mannitol showed the effect under high light intensity but not under low light intensity. The ef f ect of glucose is at least partly osmotic because glucose is also, like mannitol, a monosaccharide and not like sucrose, a disaccharide, but the effect is only partly osmotic because glucose had a greater effect than mannitol and some other additional factor must be involved in the effect of glucose. Because glucose showed a much greater effect than mannitol, this additional factor may be a direct morphogenetic one. It is interesting to note that Wollersheim (19S7) observed a higher percentage of archegonial gametopbytes using Eguisetum fluviatile with a medium supplemented with glucose, but the author observed instead a higher percentage of antheridial gametophytes using Equisetum arvense. It appears a different mechanism is involved in the effect of glucose on these two species of Eguisetum. These experiments were done only once because there were no longer available viable spores from Eguisetum arvense. In further experiments sugars should be added to the nutrient medium in equal molarity concentrtions rather than equal weight to volume concentrations. Red pigment isolated f!:2m Eguisetum. The absorption spectrum of the red pigment in light petroleum ether from both Eguisetum gametophytes and Eguisetum sporophytes indicated that the pigment was a carotenoid but not rhodoxanthin. Rhodoxanthin in light petroleum ether has a different absorption spectrum than the red pigment in the same solvent. The red pigment has absorption peaks at 4S2, 47S and SOS nm in light petroleum ether and rhodoxanthin has peaks at 4S6, 487 and S21 nm in the same solvent (Karrer and Jucker, 1950). Lippmaa (1926a, b and c) had reported the red pigment to be rhodoxanthin in a few Eguisetum sporophytes. He had apparently identified and named the pigment rhodoxanthin just by looking at a few drawings by Schimper (189S) of chromoplasts containing this red pigment in Eguisetum telmatia sporophytes, for he gave no indication in his article of having extracted the pigment from Eguisetum. The red pigment cannot be considered another els-trans form of rhodoxanthin because unlike rhodoxanthin the red pigment was not reduced by NaBH 4 • This red pigment also has a different partition coefficient value than rhodoxanthin in light petroleum and 953 methanol. The partition coefficient value for the red pigment was determined to be 0:100. Quackenbush (1965) has determined the partition value in hexane and 953 methanol to be 55:45 for rhodoxanthin. The red pigment could not be identified for the author could not find a red pigment in the literature that had an absorption in light petroleum ether similar to the absorption spectrum of this red pigment in the same solvent. It is possible the red pigment had not yet been identified. Nuclear magnetic resonance spectrum and infrared absorption spectrum would be the next steps to take in identifying the pigment, but because of the scope of the problem, amount of pure pigment required and equipment required, these steps were not taken. Under the microscope the . red pigment is found in chromoplasts in cells adjacent to the antheridia. It is possible the red pigment is related to the sexual development of Eguisetum antheridial gametophytes. CONCLUSIONS This study showed that working with Equisetum gametophytes is difficult. However~ this study did show that glucose promoted an increase in number of antheridial gametophytes in Eguisetum arvense and that the red pigment observed in Eguisetum is not rhodoxanthin as reported in the literature but some other red carotenoid not yet identified.

v3-fos

2020-12-10T09:04:11.469Z

1971-12-01T00:00:00.000Z

237232256

s2

Effect of Newcastle Disease on Serum Copper, Zinc, Cholesterol, and Carotenoid Values in the Chick Experimentally induced Newcastle disease virus infection of chicks, with a mortality index of 48%, was accompanied by increased concentrations in serum of copper and cholesterol and decreased concentrations of zinc and total carotenoids. These changes distorted, or were superimposed upon, the rhythmic variability in the normal serum concentrations of each of these moieties. Changing values for copper, zinc, and cholesterol became apparent before any overt signs of disease. Experimentally induced Newcastle disease virus infection of chicks, with a mortality index of 48%, was accompanied by increased concentrations in serum of copper and cholesterol and decreased concentrations of zinc and total carotenoids. These changes distorted, or were superimposed upon, the rhythmic variability in the normal serum concentrations of each of these moieties. Changing values for copper, zinc, and cholesterol became apparent before any overt signs of disease. Infectious illnesses appear to influence the concentration of many normal constituents of serum. Of the trace elements studied, copper values generally increase, whereas those of zinc tend to decrease during bacterial or viral diseases (8; W.R. Beisel and R.S. Pekarek, Int. Rev. Neurobiol., in press). The reaction of serum cholesterol, on the other hand, is often variable in the presence of infection in that a depressed concentration has been observed during bacterial pneumonia, cholera, tuberculosis, viral infections, hepatitis, and malaria in man (1,6). Increased cholesterol values have been reported during tuberculosis in man or Escherichia coli bacteremia in dogs, with normal values being maintained during gram-positive or gram-negative infections in man, or experimental yellow fever in monkeys (1,7). Serum carotenoids tend to decrease during Newcastle disease virus (NDV) infection in the chick (12) and during active tuberculosis in man (4,9). Earlier studies have shown that the chick, infected with NDV, constitutes an excellent model for experimental attempts to relate biochemical responses resulting from infection with fluctuations resulting from daily biological rhythms (14,15). The present report describes the effects of a standardized NDV infection in chicks on serum copper, zinc, cholesterol, and carotenoid concentrations. MATERIALS AND METHODS To insure adequate numbers for selection, approximately 500 White Leghorn male chicks were obtained at one day of age and housed in an isolated air-conditioned room with constant lighting; a commercial chick diet and water were offered ad lib. This regimen continued for 28 days and served to deplete the birds of any parental immunity to NDV. Body weights were taken weekly and only chicks meeting their genetic potential curve for full normal growth were used in the experiment. Two days before the date set for NDV inoculation, at 8:00 AM, the chicks (28 days old) were divided into two study groups: a group to serve as noninfected controls and another to be infected experimentally with NDV. At the same time, five chicks were selected at random from each group and blood samples were obtained from them by cardiac puncture; the chicks were then discarded. The serum was separated and frozen for later analysis. This procedure was repeated at 3:00, 8:00, and 12:00 PM daily for 48 hr. After this period of preinoculation control collections, all chicks in the group designated for infection were inoculated (at 8:00 AM) with 0.1 ml of a 10-3 dilution of a Grun/ Rutgers strain of NDV that showed an embryo lethal dose of 50%0 equal to 10-9 per 0.1 ml in 10-day-old embryos. Seventy inoculated chicks were reserved in separate cages to monitor disease progression and mortality. Blood samples continued to be taken from five control and five infected chicks at the above sampling times for the next six days; only living birds were sampled, and all were discarded after the samples were obtained. At the end of the NDV incubation period, 72 hr postinoculation, care was taken to insure that the five infected chicks selected for bleeding manifested a range of clinical findings characteristic of the NDV syndrome at the time of sampling (13). In the analyses of the tissues, each sampling period and treatment were equally represented in any one run. Standard reference tissues and synthetic reference standards were also included. Total carotenoids were determined by the method of Bessey et al. (2), cholesterol was determined according to Bowman and Wolf (3), and the trace metals were determined by atomic absorption spectrophotometry (8). Analyses of variance were performed as in Snedecor and Cochran (11). The average serum concentration for each moiety was determined from all samples from noninfected control chicks; this was ascribed a value of 100%'I and was used as the reference point for plot-ting the percentage changes of control and infected groups at each sampling time. RESULTS At the end of the active involvement stage, mortality in the NDV reference group was 48%, confirmiing that the level of NDV involvement in this trial was severe (13). Table 1 presents a statistical summary of the data analyzed over days postinoculation to show the effect of NDV on the serum components studied. Figure 1 depicts the sequential changes in serum cholesterol, carotenoids, zinc, and copper in control and NDV-infected chicks over the course of the entire observation period. Each point on the curve represents the average of 10 control chicks up to the time of inoculation and five control and five infected chicks thereafter. Time of inoculation is represented by the vertical dashed line. Cholesterol. Cholesterol concentrations in the controls over the 8-day period averaged 139 ±i 16 mg/I00 ml ( standard deviation) butvaried each day by as much as 27% of this value; the observed fluctations did not conform to a precisely repetitive rhythm throughout each 24-hr period. Early in the incubation period in the NDV-infected chicks, cholesterol concentrations tended to become somewhat lower than those of the control group, but by 55 hr postinoculation and thereafter values were consistently higher ( <0.05 to <0.01). Carotenoid. Carotenoid values in the control chicks averaged 483 86 ,ug/100 ml; they also showed nonrepetitive variability when compared to clock hours. There was a slight but sustained trend here toward the development of higher carotenoid concentrations throughout the 8 days of study. In the NDV-infected chicks, the carotenoid values began to decline soon after inoculation and concentrations continued to be lower than in control birds throughout the subsequent observation period ( <0.05 to <0.01). Zinc. Zinc values in the noninfected control chicks fluctuated up to 45% of the 8-day average of 166 38 ,ug/100 ml within each 24-hr period, with troughs generally occurring at 3:00 or 8:00 PM and peaks at 8:00 AM or 12:00 AM. Very soon after inoculation with NDV, zinc values became depressed (<0.01) below those of the noninfected control birds. Zinc concentrations continued to be depressed (<0.01) throughout most of the incubation period, returned transiently to control levels during the initial stages of overt disease (active involvement), and then dropped significantly (<0.01) during the last 48 hr of the study. DISCUSSION The present data indicate that an NDV infection (48% mortality index) of growing chicks is associated with increased values for serum copper and cholesterol and depressed values for serum zinc and carotenoids. These changes are similar to those reported to accompany a variety of bacterial or viral infections in mammalian species (1,2,4,(6)(7)(8)(9). The use of closely spaced serial collections in the present study allowed a correlation of biochemical changes with the timing of different stages of the NDV infectious process. For example, during the incubation period (0 to 72 hr), the depression in zinc and the increase in copper concentrations, and to a lesser extent total cholesterol, were true infection-related effects since dietary intake of the chicks remained normal at this time. The depression of total serum carotenoids, on the other hand, could have been influenced by the reduction of food intake which began 72 hr after inoculation with the virus. Feigin et al. (5) observed in man that attenuated Venezuelan equine encephalomyelitis virus could alter the periodic circadian rhythm of whole blood amino acid concentrations, could markedly alter their absolute mean concentrations, or could do both, depending on the time of day that the attenuated virus was given as a vaccine. Rapaport et al. (10) found that daily periodicity in the urinary excretion of tryptophan metabolites was maintained in patients with Rocky Mountain spotted fever even though the absolute amounts excreted were greatly increased. The present study suggests that infection-related influences on both periodic rhythms and on deviations from a normal range of concentrations may vary from substance to substance during the same time period in a single infection. The present data also emphasize the influence of periodicity on interpretation of results obtained from samplings of blood and other tissues. The diurnal oscillations observed here have a large magnitude of change, e.g., serum copper varies as much as 75 % in 24 hr, to illustrate the size of potential error. If the concentration of a substance normally undergoes rhythmic changes during the course of an investigation, a set of values obtained at a single point in time cannot be employed as an acceptable control sample. Moreover, the diurnal patterns of the oscillations indicate that serum copper in normal chicks possesses a classical circadian rhythm; yet in the same milieu at the same time, zinc, carotenoids, and cholesterol apparently lack a definite pattern. This would suggest that diurnal changes of all serum constituents are not circadian or that some are more susceptible than others to Zeitgeber inputs. The significant changes in patterns caused by NDV at various periods of the disease cycle would lend support to the latter possibility.

v3-fos

2016-05-04T20:20:58.661Z

1971-01-15T00:00:00.000Z

29754208

s2

The design of beef breed comparisons regards the station tests the accuracy became very low as compaired to the expected. In contrast to that the accuracy of the field tests was in good agreement with expectation. It was concluded that the large variance component between bulls at the stations and also the low repeatability of a station test in commercial herds are caused by a non-genetic correlation between daughters within groups. This accounts for about 2/3 of the intraclass correlation between half sibs at the stations. As regards the station tests the accuracy became very low as compaired to the expected. In contrast to that the accuracy of the field tests was in good agreement with expectation. It was concluded that the large variance component between bulls at the stations and also the low repeatability of a station test in commercial herds are caused by a non-genetic correlation between daughters within groups. This accounts for about 2/3 of the intraclass correlation between half sibs at the stations. Les corrélations entre les performances de reproduction (taille de la portée à la naissance et au sevrage, poids de la portée à 60 jours) de 1 140 truies de race Large White et les performances d'engraissement (gain moyen quotidien de 30 à 100 kilogrammes et indice de transformation) et de carcasse (longueur, épaisseur du lard dorsal, % de morceaux nobles et % de morceaux gras) de 3 de leurs descendants issus de la même portée et contrôlés dans les stations de contrôle de la descendance ont été estimées. Les corrélations phénotypiques intra-verrat et station sont très faibles dans l'ensemble et non significatives. Trois coefficients dépassent cependant légèrement le seuil de signification (p < 0,05) : entre la longueur de la carcasse et la taille de la portée à la naissance (r = -0,09) et au sevrage (r = -0,10) et entre le pourcentage de morceaux nobles et le poids de la portée à 60 jours (r = 0,11). Les corrélations génétiques accentuent ces tendances sans qu'il soit possible de leur attribuer une valeur définitive. In planning the comparison of several breeds of cattle for use as beef crossing sire lines it is necessary to decide how many bulls (N) per breed and how many progeny (n) per bull to measure. Assuming that the breeds are to be compared for a single major trait, the following seven factors need to be taken into account : h' : the intrabreed heritability of the trait. r : the repeatability or accuracy of the progeny test. a : the phenotypic standard deviation for the trait. d : the true difference between breeds that it is desired to detect. Y : the probability that a true difference of d will be detected. p i the probability level at which it is desired to test the significance of a difference. B : the number of breeds to be compared. If h' is presumed known, and a value is specified for r, n becomes fixed. It then becomes possible to use standard experimental design theory to find the minimum value of N which satisfies a given difference, phenotypic standard deviation, probabilities and number of breeds. N must be found iteratively. A computer program was written to find N for a range of values of the various factors, and the results are presented in a table. The use of the table can be illustrated as follows : There breeds are to be evaluated, chiefly for growth rate, which has a heritability of .3 and a phenotypic coeflicient of variation of a bout 10 per cent. If the required repeatability of the progeny test is .8, then 22 progeny per bull are needed. To have a probability of .8 of detecting a difference of 2 per cent of the mean at the .05 level will require 11 bulls per breed, or an experiment of 3 x 11 x 22 = 726 cattle in total. With a relaxed significance level of .10, 7 bulls per breed will suffise, requiring an experi-

v3-fos

2020-12-10T09:06:50.931Z

1971-06-01T00:00:00.000Z

237229435

s2

Production of Ochratoxins in Different Cereal Products by Aspergillus ochraceus The effects of temperature and length of incubation on ochratoxin A production in various substrates were studied. The optimal temperature for toxin production by Aspergillus ochraceus NRRL-3174 was found to be around 28 C. Very low levels of ochratoxin A are produced in corn, rice, and wheat bran at 4 C. The optimal time for ochratoxin A production depends on the substrate, ranging from 7 to 14 days at 28 C. Ochratoxin B and dihydroisocoumaric acid, i.e., one of the hydrolysis products of ochratoxin A, were produced in rice but at levels considerably lower than ochratoxin A. No ochratoxin C was produced in rice at 28 C. When added to rice cereal or oatmeal, the toxin was found to be very stable over prolonged storage and even to autoclaving for 3 hr. The effects of temperature and length of incubation on ochratoxin A production in various substrates were studied. The optimal temperature for toxin production by Aspergillus ochraceus NRRL-3174 was found to be around 28 C. Very low levels of ochratoxin A are produced in corn, rice, and wheat bran at 4 C. The optimal time for ochratoxin A production depends on the substrate, ranging from 7 to 14 days at 28 C. Ochratoxin B and dihydroisocoumaric acid, i.e., one of the hydrolysis products of ochratoxin A, were produced in rice but at levels considerably lower than ochratoxin A. No ochratoxin C was produced in rice at 28 C. When added to rice cereal or oatmeal, the toxin was found to be very stable over prolonged storage and even to autoclaving for 3 hr. The discovery of toxicity produced in corn by Aspergillus ochraceus (10) led to the isolation of three chemically related toxic metabolites, i.e., ochratoxins A, B, and C (6). Ochratoxin A has been shown to be toxic to a variety of laboratory animals (8,16). Although natural outbreaks of this kind of food intoxication have not been reported, A. ochraceus has been readilv isolated from many cereal and other food products. The occurrence of this toxin in poor-grade corn (14) and moldy wheat (13) has been demonstrated. In addition to A. ochraceus, A. sulphureus, Penicilliunm viridicatum (12), and A. melleus (M. Lai, G. Semeniuk, and C. W. Hesseltine, Phytopathology 58:1056, 1968) also have been shown to produce ochratoxin A. Studies have been published on the detection and estimation of the ochratoxins (1,4,11) and also on their microbiological and toxicological properties (2,5,8). Adequate control measures can not be determined until more is known about the conditions under which the toxin is produced. This study was undertaken to determine the effects of incubation time on total ochratoxin A production, the occurrence of other ochratoxins in the substrate, and the effects of various substrates on toxin formation. The stability of ochratoxin A in food stored under different conditions was also studied. MATERIALS AND METHODS Organism. The culture used for ochratoxin production throughout this study was A. ochracetus NRRL-3174 kindly supplied by C. W. Hesseltine, Northern Regional Research Laboratory, USDA, Peoria, Ill. The culture was grown on potato-dextrose-agar slants at room temperature and stored at 5 C. Culture. The substrate, either 20 or 30 g, was placed in a 500-ml Erlenmeyer flask, soaked with an equal amount of tap water for 2 hr, and autoclaved for 20 min at 121 C. Flasks were inoculated with spores from 1-to 2-week-old slants. The flasks were incubated in the dark and shaken once a day to break the mycelial mass. Five temperatures (4,15,20,28, and 37 C) and four foods (wheat bran, corn meal, polished rice, and bleached flour) were studied. Methods used for extraction, analysis, and preparation of secondary standards have been described previously (1). Toxin stability. To study the stability of the toxin in cereals, 18 ,ug of purified ochratoxin A in 0.5 ml of sterilized, distilled water was added to 10 g of food. The toxin-containing food was transferred to a plastic bag, flushed twice with nitrogen, and vacuum-sealed. These were stored at 4 and 28 C for various times, and two assays of 5 g each were made for individual samples. To determine the stability of the toxin to autoclaving, 40 ,ug of ochratoxin A in 0.5 ml of sterilized, distilled water was added to 100 g of cereal in a 500-ml Erlenmeyer flask. Either no water, 50 ml of distilled water, or 50 ml of 20% acetic acid was added to each flask. The samples were autoclaved at 121 C for 0.5, 1, and 3 hr, respectively. Similarly, two analyses of 50 g each were made for individual samples after autoclaving. 1032 RESULTS Effect of temperature and substrate. Two flasks were harvested for each substrate and temperature at various times over a period of 2 weeks, except in the 4 C experiments which were harvested after 21 and 28 days of incubation. Table 1 shows the results of incubation at 4 C; only slight growth of the fungus was observed at this temperature. The amount of toxin formed is very low; about 3 ,ug of ochratoxin A was produced after 28 days of incubation. This is a contamination level of slightly over 100lig/kg. Table 2 shows the effect of temperature on ochratoxin A production in polished rice and wheat bran over a 2-week incubation period. Toxin production at 15 C is very slow; however, 1 to 2 mg of toxin is present after 2 weeks. This is a final toxin level of 30 to 60,ug/g of substrate. The optimum temperature for ochratoxin A production was found to be 28 C. At this temperature, ochratoxin A was found at levels of 1.5 to 1.8 mg/g of substrate after 7 to 14 days of incubation. Ochratoxin A formation at 37 C was considerably lower than at 28 C. The effects of substrate on ochratoxin A production are shown in Fig. 1. At 28 C, chopped corn was the best substrate for production of ochratoxin A. Polished rice and wheat bran yielded comparable amounts of toxin, but slightly longer incubation was required for maximum production. Bleached flour did not support the production of large amounts of toxin. This may be due to inadequate aeration since the flour formed a paste after sterilization. The occurrence of ochratoxins other than ochratoxin A was studied in rice incubated at 28 C. Standards of ochratoxin B, ochratoxin C, and one of the hydrolysis products of ochratoxin A, i.e., dihydroisocoumaric acid, were prepared b Numbers indicate Centigrade temperature. D, experiment carried out in the dark; LD, flasks exposed to light during the day. c Per cent of ochratoxin A remaining. for quantitation of these compounds in the rice extracts. The results of this experiment are shown in Table 3. No ochratoxin C was found in any of the extracts. The amount of ochratoxin B was about 25 times less than the amount of ochratoxin A formed ( Table 2). The dihydrocoumarin was present in even lower concentrations. Stability of ochratoxin A. Table 4 shows the stability of ochratoxin A in cereal products. The toxin was very stable when stored in darkness at 4 or 28 C for 1 week. After 12 weeks, about 45% of the toxin was still recoverable from these samples. Although pure ochratoxin A is lightsensitive (7), the effects of light on toxin added to the cereal seemed to be slight, with similar levels in samples stored in the dark and those stored in light and dark. Autoclaving contaminated samples for up to 3 hr failed to destroy completely the toxin (Table 5). It is of interest that the toxin was more readily destroyed in dry cereal than in the presence of water or even 20% acetic acid. Examination of thin-layer chromatography plates from the autoclaved samples revealed new fluorescent spots; however, the toxicity of these substances is not known. DISCUSSION The optimum temperature for ochratoxin A formation by A. ochraceus appears to be very similar to that for aflatoxin formation, 28 C. The major difference is that there is considerable ochratoxin A formation at 15 C, whereas only small amounts of aflatoxin are formed at temperatures in the range of 10 to 15 C (3,15). Very slow formation of ochratoxins appears to be occurring at 4 C. This fact could be of significance in the storage of grains at low temperatures. Although the amount of toxin present after 4 weeks of incubation at 4 C is very low, continuing production might occur over longer periods. The finding that lower yields of ochratoxin A are obtained at 37 C could be due either to rapid degradation ofthe toxin at this temperature or decreased toxin formation by the organism. Evidence for the former case might be seen in the presence of considerable toxin after 9 days of incubation at 37 C but a rapid decrease after 3 more days of incubation. These optimal and inhibitory temperatures are certainly dependent upon the strain and species of mold used to produce ochratoxins. Penicillium viridicatum shows a considerably lower range and optimal temperature profile than A. ochraceus (A. Ciegler, personal communication). The toxin distribution study indicated that ochratoxin A was the primary toxin produced by A. ochraceus. Ochratoxin B and dihydroisocoumaric acid are present at considerably lower levels and are also much less toxic than ochratoxin A. Ochratoxin C which is about as toxic as ochratoxin A is apparently not produced in rice by the isolate used. The optimum incubation time for ochratoxin A production was found to be dependent on the type of substrate used, varying from 7 to 14 days. Schindler and Nesheim found that maximum production of ochratoxin A in shredded wheat occurred after 19 to 21 days at 22 C (9). Although the use of shredded wheat may have affected toxin production, the lower incubation temperature (22 C) would slow toxin production, thus requiring a longer incubation period for maximum yield. The toxin stability studies revealed that ochratoxin A is a very stable compound which can persist in foods even after 3 hr of autoclaving. This would indicate that removal of ochratoxins from food products might be very difficult, and the best protection would be to prevent toxin formation through satisfactory handling procedures.

v3-fos

2017-07-29T04:24:53.599Z

1971-01-15T00:00:00.000Z

8132828

s2

Blood groups and polymorphic proteins in cattle and swine HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. BLOOD GROUPS AND POLYMORPHIC PROTEINS IN CATTLE AND SWINE Bent Larsen The results from studies of genetic linkage among 10 blood group systems and 7 loci controlling protein polymorphism in cattle are presented and a close linkage of the bovine A blood group system to the haemoglobin types and a close genetic relationship of the 0 :-, 13-, and IC-casein is found. Further a close relationship of the J blood group system to the p-lactoglobulin types in cattle is discussed and the possibility of a loose linkage between the loci controlling transferrin and serum amylase polymorphism is mentioned. Together with the linkage groups among cattle blood and protein polymorphisms, the linkage groups known in pigs and sheep are mentioned in relation to the possible future use of linkage to marker loci in animal breeding. During the 30 years since systematic studies of blood groups in farm animals were initiated by means of isoimmune antibodies, the subject of immunogenetics and the more recent field of biochemical genetics have developed greatly and a number of gene controlled polymorphisms have been discovered and elucidated. In animal genetics it is of great interest to be able to identify genes at certain loci by means of relatively simple serological and biochemical methods. Also animal breeding programs have benefited from the discoveries of these polymorphisms. Identification of breeding animals, parentage control and in cattle the diagnosis of monozygotic twins and freemartins by means of blood grouping and the more recently discovered polymorphic protein systems in many countries are incorporated into the breeding schemes. As a direct tool in selection programs, these polymorphisms have not proved successful so far. The hope that the immunoof biochemically detectable polymorphisms should be of direct influence upon characters selected for in animal breeding has not been justified by results which were of significant value in selection programs for large farm animals. This however, may not mean that polymorphic systems will not be of any further value for animal breeding in the future. Thus analyses of the data on blood groups and polymorphic protein systems have revealed evidence for the first linkage groups within some of the polymorphic systems known to-day in farm animals. Also the great expansion of the number of known polymorphic systems, in particular polymorphic enzymes, going on at the moment may contribute to mapping out the chromosomes which in turn may result in the discovery of linkage of serologically or biochemically detectable marker locito characters, which it is of value either to remove or preserve in our domestic animals. Before presenting some of the studies going on in this field I shall mention some of the polymorphic systems known in cattle and pigs. BI,OOD GROUP SYSTEMS IN CATTLE AND SWINE The blood group factors or red cell antigens are identified by means of specific antisera or so-called blood type reagents, mainly prepared from isoimmune antisera. The formation of antibodies against the red cell antigens is achieved by planned series of immunizations, and from the resulting antisera, specific reagents are prepared by appropriate antigen-antibody absorptions and tests for unity (NEI MAN N-SO R ENSEN, 1958). The technique employed in the typing procedure for red cell antigens depends on the nature of the antibodies formed and the animal species to be typed. As indicated below the haemolytic test is exclusively used for the typing of cattle red cells, while three different test methods may be employed for swine (A NDR E-SE N , 19 6 3 ; H OJN Y and H RADECK Y, 19 68). The term blood group system denotes a genetic system controlling the inheritance of one or more blood group factors. In table I the well known blood group systems in cattle and pigs and the factors they control are listed (see S TOR -MON T 19 6 2 , A NDR ESE N 1 9 63, HOJN Ý et al., i 9 66). In cattle 12 systems are inter-nationally recognized and in pigs 15 systems have been described. As it appears from the table, the number of factors belonging to the various systems range from one to approximately 30 . With the exception of the Oc substance of cattle and the closely serologically related 0 of pigs and r (0) in sheep which appear as recessive characters (ST ORMON T, r 95 i; S PRA G U E, 195 8), the blood group factors known to-day in farm animals are inherited as dominants to their absence. For the systems where only a single factor is known this implies that two alleles can be recognized, e.g. the allele giving rise to the blood group factor in question and the so-called « silent o allele &dquo;-&dquo; (das), not forming a known antigen on the red cells. This type of system is called &dquo; open &dquo; in contrast to a &dquo; closed &dquo; system where the silent allele is not found. Thus the systems comprising more than one factor may form &dquo; closed &dquo; or &dquo; open &dquo; genetic systems controlling a number of multiple alleles. As indicated in table i the cattle R'S' system appears as a two allele closed system and in domestic cattle of Bos taurus origin the FV appears as a three allele closed system. In the more complex systems, such as the bovine B system, a number of alleles each of which controls the presence of one or more blood group factors, the so-called &dquo; phenogroups & d q u o ; , and the ,,-&dquo; gene are known. With rare exceptions these phenogroups are transmitted from parents to offspring as units. In pigs the blood group factors tend to form closed systems more frequently than in cattle and other animal species. As indicated in table i six systems are recognized as two allele and three as multiple allelic closed systems. POLYMORPHIC PROTEIN SYSTEMS IN CATTLE AND SWINE Not only on the red cells but also on the leucocytes and blood platelets antigenic factors are present and the study of these has more recently been taken up in farm animals. However, also proteins and enzymes in the blood, milk and other body fluids exhibit gene controlled variations, some of which can be detected by serological methods, for instance by means of inhibition tests or immunodiffusion. The majority of the polymorphic protein systems known in farm animals to-day have, however, been detected by electrophoresis in a supporting medium of starch gel as introduced by SMITHIES in 1955 . By electrophoresis in starch gels, which are prepared by heating a suspension of partially hydrolysed starch in a suitable buffer, the separation of the amphoteric protein molecules takes place on the basis of differences in charge and in molecular size. After completion of electrophoresis the gel may be stained or incubated in order to identify the components of the protein under study. In table 2 a number of polymorphic protein systems which have been studied in cattle and pigs are indicated (for ref. see A S C HAFF E NBUR G, 19 68, and HESSEL-HOLT , i 9 6 9 ). Like the blood group factors, the components of the polymorphic proteins are controlled by genes dominant to their absence. To each other the alleles are non-dominant, which is referred to as codominance. Because a silent allele is not present in any of the systems indicated in table 2 , the genotype can be established directly from the phenotype. The polymorphisms observed by starch gel electrophoresis are due to variation in the amino acid sequence in the polypeptide chains, and in recent years much work has been devoted to studies on proteins at the molecular level to provide a chemical basis for the variation exhibited by polymorphic proteins. Thus it has been shown that the difference between the bovine haemoglobin components A and B is due to the p-polypeptide chain of the haemoglobin molecule, where one glycine and two lysine residues in the f3A chain have been replaced in the p B chain by one serine, one histidine and one aspartic acid residue (H UISMAN , i 9 66). Also the polymorphic proteins of the cow's milk have been subjected to physicochemical and structural studies and the nature of the chemical differences between the p-lactoglobulin A and B components was found to be due to two aspartic acid and two valine residues in the P-Lg l being substituted by two glycine and two alanine residues in the f3-Lg B component (GoRDOrr et al., ig6i; P W z et al., ig6i). Such studies are under way also for the casein polymorphisms of cow's milk. APPLICATIONS OF BLOOD GROUP AND POLYMORPHIC PROTEIN SYSTEMS During the last 10 to 15 years the blood groups and some of the polymorphic protein systems have been used in many countries for the identification of breeding animals, in parentage control and for cattle in the diagnosis of twins. This application has been and is still of great value for the breeding schemes of large farm animals since it is the only way to keep the necessary standard of the pedigree records and to make sure that progeny tests really are made with offspring from the parents whose ability is to be judged. Also in experiments with cattle twins it is valuable to be able to verify the morphological diagnosis of zygosity by tests for blood groups and polymorphic proteins. The data on blood groups accumulated from investigations of this kind have first of all been used for the identification of the alleles present in the systems of the various breeds and for the characterization of the breeds by means of the frequencies of the polymorphic components. Since only systems showing variation are of value, these studies have contributed greatly to the knowledge about the usefulness of the various polymorphic systems for genetic studies within a given breed. Thus for example in Danish Landrace pigs the B, C and 0 blood group systems are useless because no variation occurs and the same is the case for the bovine haemoglobin system in Holstein Friesian cattle and many other domesticated cattle breeds. Besides comparative and population genetic studies a number of investigations have been devoted to elucidating the possible relationship of loci controlling blood groups and polymorphic proteins to characters of economic importance for animal production. The studies in this field have been greatly stimulated by the pioner work of BRIBES and coworkers in chickens (BRIBES and A LLAN , 10 6 1 ), where heterozygosity at the B locus was observed to be related to superiority in viability and egg production. Thus a number of studies on the relation of the well known blood group systems and some of the polymorphic protein loci to productive characters have been carried out in cattle and pigs, indicating that some of these marker loci may be associated with production ability (MITSCII!Rr,ICII et al., 1959 ; NEI-MANN-SOR!NS!N and R OB E R TSO N , I g6 I ; RE ND E L , I g6 I ; J AMI E SON and RO B ERTSO N , I g67; BRUM et al., I g68; JE NS E N et al., I g68). In particular certain alleles at the bovine B blood group locus have repeatedly been found to be related to milk and butterfat production, but also an effect of other loci has been observed. SMITH ( 19 6 7 ) has described the conditions under which the use ot specific marker loci in selection programs may be of value. He points out that the value of marker genes depends on the amounts of the total genetic variation controlled by a marker locus in relation to the heritability of the character under study, and also to the form of selection practised. He concludes that for characters with high heritability and when normal selection is effective only little may be gained by including marker loci. However, for characters with low heritability or when indirect selection on relatives must be used, then the additional information obtained by marker loci may add significantly to the possible rate of improvement. The possible effects of marker loci in relation to production so far disclosed in farm animals are small and without further studies may not be used in improvement schemes. Apart from serum amylases, however, polymorphic enzymes have so far not been included as marker loci in studies in relation to productive traits. With the rapid development in the discoveries of gene controlled variation in various enzymes, some of which play an important role in the animal body, it is, therefore, still possible that valuable relations between these marker loci and the quantity or quality of animal products may be found. The increase in the number of known loci showing gene controlled variation has more recently focused attention on the possibility of genetic linkage among the immunoor biochemically detectable systems in farm animals. Analyses of this kind as well as those mentioned above have been greatly facilitated by computers now available in many laboratories and because of the development of computer programs which can handle the problems to be studied. Using the data on bovine blood groups and polymorphic proteins which have been accumulated during some years we have recently completed an analysis for genetic linkage among 10 blood group systems and polymorphic protein systems, the results of which are summarized in table 3 . The details about the methods applied and the data have been published elsewhere (I,ARS!N, 1970 ). The figures in table 3 indicate the recombination frequencies for which linkage can be excluded between the various systems compared. Thus for the comparison of the A system to the p-lactoglobulin (p-I,g), significant evidence was obtained to the effect that these two loci are not located closer to each other on the same chromosome than corresponds to at least 13 per cent of recombination. For loci where linkage has been disclosed an estimate of the recombination frequency (4) is given in table 3 . In agreement with previous analyses it appears from table 3 that the A blood group system is closely linked to the haemoglobin types, the three casein polymorphisms, Ksi -Cn, p-Cn and r.-Cn form a system of closely linked genes and that the J blood group system shows a close relationship to the p-lactoglobulin types. The families relevant to the study of the linkage groups just mentioned are shown in table 4 . In this table the families are grouped according to the most probable phase of the sire. From the table it appears that between the A and the Hb systems only 3 possible recombinants are observed out of a total of 175 offspring. In the comparison of the three casein systems only the parental types are found in our data. Also in a study made by GROSC!,aun! et al. ( 19 64, 1965) only parental types of offspring were found, while H INES et al. (ig6g) observed some apparent recombinants in the as j -Cn and p-Cn comparison as well as in the «si-and x-Cn comparison, but not between the p-Cn and x-Cn types. Thus a very close linkage of the genes controlling the casein polymorphisms has been established. The data in table 4 relevant to elucidating the relationship of the J blood group substance and the p-lactoglobulin types show about q. per cent of recombination, while H INES et al. (ig6g) in a similar study observed 20 per cent of recombination. Furthermore, in our data a disturbed segregation of the p-lactoglobulin types in the families relevant to the linkage studies was observed, while the total data showed a perfect i : I segregation of the P-Lg. This may suggest that the observed relation of the p-Lg is not directly to the J substance or cellular J, but to a factor or genetic system related to or influencing the appearance of the J substance in the plasma and on the red cells. The correct interpretation of this relationship may, therefore, await a better understanding of the genetics underlying the J factor and the closely related Oc substance. From table 3 it is seen that 25 per cent of recombination can be excluded between the Tf and Am loci. However, a possible but non-significant indication for a loose linkage of these two loci was observed. In the case of linkage being present between the transferrin and amylase loci, the recombination frequency is estimated to be about 37 per cent. The remainder of the total r 4 g comparisons indicated in table 3 did not reveal any significant dependency and as a matter of fact moderate to loose linkage has been excluded, indicating that these systems are fairly well distributed on the entire set of autosomes in cattle. In table 5 the linkage relations of blood groups and biochemical polymorphisms known to-day in farm animals are indicated. Besides linkage relations in cattle just mentioned, four examples of close linkage have been disclosed in pigs. For the C and J blood group systems the recombination frequency is estimated to 5.5 per cent (A NDR E S E N , ig68), and the other three groups show even closer linkage. A NDRESEN ( 19 68) showed to what extent linkage among blood group and polymorphic protein loci in pigs can be excluded and presented data indicating a possible but non-significant loose linkage between the B blood group system and the prealbumin system in pigs. In sheep the genetically controlled low (K L ) and high (K h ) potassium level in the red cells is closely related to the M blood group system. Animals having the dominant gene K for low potassium content in homozygotous state seem to be negative for the M factor (M-/M-), heterozygote KL /K! possibly also is heterozygotous for the M factor (M M /M ' ) and individuals being homozygous for high potassium level (K h /K h ) appear to be homozygous for the M blood group factor also (M M /M M ). So far the search for linkage of monogenetic characters in farm animals has been devoted mainly to studies of the mutual relations of serologically and biochemically detectable marker loci. However the relationship of these marker loci to morphological and pathological characters with known inheritance needs our attention. Such studies are under way and recently I M r,nH ( 1970 ) presented evidence for the Tf locus being associated to a lethal factor in a strain of pigs. In cattle M AIJALA AND I ,mr DST xoM ( 19 6 4 ) investigated the possible linkage of blood group systems to the occurrence of hairless calves in connection with a prolonged gestation period, presumed to be caused by a recessive lethal gene. They found that the lethal was not closely linked to any of the blood group systems studied. With regard to morphological characters we have had an opportunity to test a family where a bull of the Danish Black and White dairy cattle, being heterozygous for the black coat colour, was mated to cows of Red Danish dairy cattle breed. From the offspring in this family it could be concluded that the locus controlling the black and red colour in cattle is not closely linked to the B, M, R'S', Tf, and Am loci. The results from the linkage studies on blood groups and polymorphic protein loci in cattle and swine show that apart from the linkage relations mentioned in table 4 and the possibility of a Tf-Am relationship in cattle and a B-Pra relation in pigs the loci so far studied in these two animal species are well distributed over the entire number of autosomes. This result alone is important because it suggests that a search for linkage is more likely to be successful whenever additional loci are included. The ultimate objective is a more complete mapping of the chromosomes to which also cytogenetic studies may contribute for the localization of the various loci in the chromosomes. Thus it seems possible that the contribution of immunogenetics and biochemical genetics to animal breeding programs may be extended in the future. Re(u pour publication en septembre 1970 . Les groupes de linkage du Porc et du Mouton sont également envisagés en relation avec une future utilisation des gènes marqueurs en sélection animale.

v3-fos

2018-03-27T17:47:55.988Z

1971-01-01T00:00:00.000Z

16257889

s2

The application of chemical criteria to biological classification in the eighteenth century. THE INVESTIGATION of animal and vegetable substances, traditionally a part of the chemist's general inquiry into the properties of natural objects, was increasingly pursued in the eighteenth century. Some of these materials were important economically in the various arts of dyeing and tanning, agriculture, and the manufacture of soaps and glues, and of course they continued to supply pharmacy with much of its materia medica. Medical interests were a strong incentive to the concentrated study of these substances. The men who conducted this chemical research, several of whom were physicians,2 believed that great advances in medicine would result. In particular it was thought that this would provide the key to the understanding of the animal economy, for in the nutrition of animals the basic vegetable foods were altered in such a way that they could be incorporated within the body. This was interpreted as the conversion of vegetable into animal substances. It seemed that this process of animalization, as it was called, could be investigated through a comparative chemical examination of animal and vegetable substances. The parts of animals and plants were subjected to the old technique of distillation in closed vessels, they were treated with various reagents, and attention was given to their remarkable alterations in the natural processes of fermentation and putrefaction. As the work progressed, moves were made to establish the position, in fact false, that animal and vegetable substances were chemically distinguishable. This conclusion, in turn, seemed to be important for biological classification. Ambiguous living forms, especially marine organisms, had puzzled natural historians since antiquity. Morphological and physiological criteria had failed to establish whether they should be classified as plants, animals or intermediates. In the eighteenth century, and after, attempts were made to arrive at the truth through the application of chemical tests, which appeared to offer a valuable means of distinguishing animals and plants. Application of Chemical Criteria to Biological Classification in the 18th Century In the eighteenth century the cruciferous plants were frequently referred to in accounts of the chemical properties of animals and plants. They became a standard exception, preventing a sharp distinction between the two kingdoms onthesupposition that volatile alkali was an exclusively animal product. Their chemical properties led to their description as 'animal plants'.'3 Their analysis was pursued all the more vigorously because of their anti-scorbutic properties, especially the Cochlearia officinalis or scurvy-grass.'3 Boerhaave also knew that it was impossible to make a complete separation of plants and animals from their spontaneous natural alterations. For while some plants fermented to produce acids, they generally putrefied like animals. He said that every vegetable, provided it was soft and succulent and put in heaps, would soon generate foul-smelling volatile alkaline salts. Plants acquired a cadaverous taste like putrefied urine and were converted to a greyish pap, just like the gangrene of decayed animals."o So far there seemed to be an agreement that the chemical composition of living matter, whether animal or vegetable, was very similar. A different interpretation however was given by Beccari in a memoir'5 which described his discovery of gluten, a vegetable protein, in wheat flour. As has recently been pointed out, Beccari seemed strangely unaware of current opinion.'6 Beccari, a physician who held the chair in medicine, and later in chemistry, at the University of Bologna, was interested in nutrition. He showed that wheat flour contained two different substances, which could be separated by kneading in a current of water. As the water carried away the amylaceous fraction, a tenacious, gluey material remained. The glutinous component putrefied within a few days like a corpse, emitted a foul odour, and left a black substance which he likened to rotten meat. When the gluey part of wheat flour was distilled, foetid, volatile alkaline products collected in as great a quantity as was extracted from hartshorn. Beccari might have argued that this further demonstrated the resemblance of plants and animals. Instead he proposed the opposite. Contrary to existing information, he maintained that organic substances fell into two divisions, whichcorresponded to the two kingdoms, because of their different behaviour in distillation and spon-12 J. B. M. Bucquet, Introduction a l'Etude des Corps naturels, tires du Regne vcgftal, Paris, 1773, 2 vols., vol. 1, p. 422. Bucquet wrote that this term was coined by the 'older chemists'. The name was used by G. F. Rouelle to describe haricots, partly because of the great quantity of volatile alkali which they gave in distillation. He regarded the cruciferous plants as transitional between the plant and animal kingdoms for the same reason. Rhoda Rappaport, 'G. F. Rouelle: an eighteenth-century chemist and teacher', Chymia, 1960, 6, 95. Is In 1783-4 a prize was offered by the Societ6 Royale de M6decine for the analysis of cruciferous plants. P. F. Tingry, 'Analyse de quelques plantes cruciftres', Mem. Soc. Roy. de Med., 1787, 5, 341-414. 14 Boerhaave (n. 9), vol. 2, pp. 199-203. The fermentation of an organic substance would have been sufficient to indicate its vegetable nature, according to Boerhaave, since he believed that no animal experienced this change, ibid., vol. 2, p. 115. However he thought this process only affected some plants, ibid., vol. 2, p. 202. In another work he made a different statement, remarking that fermentation affected nearly all plants, except a few which putrefied: Dr. Boerhaave's Academical Lectures on the Theory of Physic, London, 1742-6, 6 vols., vol. 1, p. 187. 16 'De Frumento', De Bononiensi Scientiarum et Artium Instituto atque Academia, 1745, 2, part I, 122-27. This was a report of Beccari's discourse. For a translation of the chief passages see Eliot F. Beach, 'Beccari of Bologna, the discoverer of vegetable protein', J. Hist. Med., 1961, 16, 354-73. 16 F. R. Jevons, 'Boerhaave's teaching in relation to Beccari's identification of gluten as an "animal" substance', J. Hist. Med., 1963, 18, 174-75. 25 D. C. Goodman taneous alteration. He stated that natural putrefaction with alkaline products occurred only in animals, never in vegetables, which underwent an acidic fermentation instead. Further animal substances in distillation gave foetid, volatile alkaline extracts, while vegetable substances only yielded acids. Therefore the gluey part of wheat flour was an animal substance,17 present in a plant. The other amylaceous part behaved, on the other hand, like a typical vegetable substance. In this way, ignoring the properties of Cruciferae and vegetable putrefaction, Beccari concluded that in one and the same flour there were two substances of entirely different nature, apparently belonging to two different kingdoms. Crude generalizations of the type proposed by Beccari were attacked by Venel, a physician who was soon to become professor of chemistry at the University of Montpellier. He remarked: 'It is always surprising to see errors, which a single experiment ought to eradicate, persist and spread '.18 He was thinking of the current doctrine that vegetable and animal substances could be distinguished by their distillation products. The prevalence of this false assertion was, he said, particularly strange in view of the analyses carried out earlier in the century by L6mery and other chemists of the Academie des Sciences. He said that their results proved that the appearance of volatile alkali in distillation, far from being an exclusively animal phenomenon, was one of the most common and general results for plants. Therefore he could not accept Boerhaave's divisionl9 of the plant kingdom into those which gave acids predominantly in distillation and others, the cruciferous group, which gave alkalis. Nevertheless he thought the Cruciferae were unusually like animals, because he had extracted a jelly from turnips which seemed exactly like the jelly of animal lymph or hartshorn. So he called Cruciferae 'gelatinous plants' and put them in a separate chemical class. 20 Venel wrote most of the chemical articles for the Encyclopedie. In one article, he defined a vegetable substance as any body coming from the vegetable kingdom. He said this could apply to an entire vegetable, its organized parts such as its roots or flowers, its non-organized juices, or any product given by these in chemical art. This last category included volatile alkali, which was therefore a vegetable substance. Precisely the same characteristic, the production of volatile alkali by distillation, would later be used by Berthollet to define an animal substance, a clear indication of the continuing confusion. Volatile alkali was sometimes called 'animal alkali'." 26 Application of Chemical Criteria to Biological Classification in the 18th Century In addition to conflicting statements of analytical results, there were other difficulties which led the eighteenth-century chemists to be cautious in their conclusions on the constituents of the various kingdoms. There was the old objection that the technique of analysis by fire was faulty on the grounds that this generated new substances not originally in the plant or animal.23 Also it was pointed out that the isolation of a material from a member of a particular kingdom did not imply that this material was essential to that kingdom. It might have been accidentally imported from another kingdom. So it was supposed that the phosphorus found in animals was brought by vegetable foods,24 and that the common salt obtained in the analysis of organic substances was of extraneous mineral origin.26 The same difficulties applied to volatile alkali. It was stated that chemistry was not yet advanced enough to provide the answers, but that a probable hypothesis was that volatile alkali was essential only to animals, the debris of which carried it accidentally into the plant kingdom.26 Further developments occurred in the work of Berthollet, who also had a medical training. He said that from the time that he had began to take an interest in chemistry he felt the importance of analytical studies on animal and vegetable substances, since this would give an understanding of nutrition, the chemical action of medicines, and other changes in animals.27 This led him to undertake a comparative study of substances from the two kingdoms. He already inclined to the view that general distinctions could be made, and accepted the crude divisions28 of the type made by Beccari. But he was dissatisfied with existing chemical tests, since he said these altered the substances under investigation, and so could only give an imperfect knowledge of their constituents. As Venel and others had proposed before, he thought it would be better to replace dry distillation by tests with solvents, and from these he selected nitric acid.229 He studied the effects of nitric acid on silk. He said the reaction produced a fatty substance which no material of vegetable origin gave when similarly treated.30 Wool, a This was forcibly expressed in the article 'V6g6tal', (n. 21), stating that the immediate principles composing plants could only be isolated by the use of various solvents in succession. 24Baume (n. 22), vol. 2, p. 52. The reverse of this, that phosphorus entered plants accidentally from animals, was suggested in 'Acide phosphorique', Encyclopedie methodique: Chimie, Pharmacie et Metallurgie, Padua, 1786-c. 1807, 6 vols., vol. 1, p. 218. Baum6 said that the division of nature into three kingdoms by the natural historian was not recognized as exact by chemists, because vegetables and animals were made up of common chemical principles. He therefore preferred to speak of these collectively as 'organised bodies', ibid., vol. 1, xvi. 26 P. J. Macquer, 'Kingdoms', A Dictionary of Chemistry, trans. J. Keir, London, 1771, 2 vols., vol. 1, p. 363. This article also stated that while chemistry could separate the minerals from organic bodies, the differences in the latter were not clear, and that these were due to quantitative variations in constituent principles which were common to plants and animals. See also the articles 'Jelly' and 'Mucilage'. The former was described as the principal animal substance and supposed to be derived from the closely similar vegetable mucilage. D. C. Goodman skin, tendons and hair all gave the same fat or oil with nitric acid. He concluded that this was a distinctive animal characteristic. But he was more concerned with the gaseous products of the reaction. He thought that Priestley had treated animal substances with nitric acid without an adequate consideration of the source of the gases evolved. Berthollet was convinced from his own experiments that nitrogen was produced in abundance when animal substances were treated with concentrated nitric acid at room temperature, and that the nitrogen was coming from the animal substance, not from the acid.31 In support of this he argued that the liberation of the nitrogen preceded the decomposition of the acid. He said that, although in reactions with zinc, nitric acid could be decomposed to nitrogen, this must not be confused with the reaction with animal substances in the cold. He stated that no vegetable substance behaved in this way. Instead of nitrogen they produced a mixture of fixed air and nitrous gas. Therefore animal substances were distinguished by their constituent nitrogen. This would explain the appearance in their reactions of volatile alkali, the composition of which he had just discovered. In terms of the new nomenclature, which he assisted in formulating, volatile alkali became ammonia, a compound of azote (nitrogen) and hydrogen, the new elements of Lavoisier's chemistry. So when an animal substance was dry distilled or putrefied, Berthollet explained that its nitrogen combined with hydrogen from water to generate ammonia. Berthollet defined an animal substance as one which gave volatile alkali in distillation.32 This included parts of certain plants such as the gluten of wheat and the seed of the mustard plant. Like Beccari, Berthollet regarded these as animal substances which were mixed with the other vegetable parts of the plant. Apart from nitrogen, Berthollet thought that phosphoric acid was peculiar to animal substances.-3 He said this acid was detected in animal charcoals, and accounted for their incombustible nature compared to vegetable charcoals, which were easily burned. He thought that the phosphorus which Marggraf had found in plants must be due to their animal parts.3' This was the same logic which he had applied to the source of volatile alkali in the distillation of plants. The effect was to maintain the separation of organic substances into two distinct classes. The result of Berthollet's work was to establish nitrogen as the characteristic element of animal matter. It also led to the conception that animal substances were 32 Berthollet, 'Suite des recherches sur la nature des substances animales, et leurs rapports avec les substances v6g6tales', Mem. Acad. Sci., 1785, pp. 331-349. At the same time Scheele also believed that animal substances had a characteristic reaction with nitric acid; unlike vegetable substances, they gave vitiated air. The Collected Papers of Carl Wilhelm Scheele, trans. L. Dobbin, London, 1931, p. 274. Berthollet's interpretation of the reaction was opposed by Keir, who thought that the nitrogen originated in the nitric acid, since the other mineral acids extracted none from animal substances. In fact the nitrogen was produced from nitrogenous organic substances, through a reaction with nitrous acid, generally present in nitric acid. In modem terms, amino-groups of a-amino acids, present in plants and animals, react with nitrous acid to produce nitrogen. The nitrogen comes from both the nitrous acid and the organic material. 32 Berthollet (n. 31), p. 333. " Ibid., p. 348. " Ibid. Berthollet argued that the urine of cows and camels was alkaline because these animals fed on plants which contained little animal substance, that is little phosphoric acid. It is true that the urine of vegetarians is alkaline. 28 Application of Chemical Criteria to Biological Classification in the 18th Century more complicated than vegetable substances. In his early papers, the former contained nitrogen, phosphoric acid, and a peculiar oily principle; the latter contained none of these. Later he weakened these absolute qualitative distinctions, stating that the differences were quantitative: animal substances contained much more nitrogen and much more hydrogen (this formed the oil) than the vegetable class.36 Berthollet's views influenced Lavoisier, whose new chemistry interpreted organic substances as compound radicals joined to oxygen. The compound radicals of animal substances contained the elements carbon, hydrogen, azote, phosphorus and sulphur.36 Vegetable substances were less complicated, their compound radicals generally consisting of carbon and hydrogen only. He said that these were the true elements of plants, common to all, and that any other elements were peculiar to the particular plants in which they were detected.37 This applied to cruciferous plants which contained azote, and others which had phosphorus. He said these approached the complexity of animal substances, but their quantitative composition distinguished them from the animal kingdom, since the two extra elements were present in much smaller quantities.38 Although he thought that azote was present in many vegetables, the low content reduced its importance and it failed to qualify in Lavoisier's system as an important element for plants.39 It was not difficult to proceed from this position to the false view that trace elements were inessential.40 The most elaborate investigation of animal and vegetable substances in the eighteenth century was due to Fourcroy. As a medical student he was interested in the applications of chemistry, which he believed would one day revolutionize medicine. He said that a major research problem for the chemist was a study of animalization, the process by which essential vegetable foods were converted into the parts of the animal body; once this was solved, the animal economy would be fully understood. He thought the best way to approach this would be a comparison of substances from the two kingdoms, discovering their differences and then inquiring into the causes of these differences.4' At the start of his research Fourcroy was impressed with a striking analogy between these substances, through his discovery of albumen in plants.42 Albumen was well known in eggs as a viscous, white liquid characterized by a remarkable coagulability by heat. Fourcroy now showed that a substance with the same properties43 was I' Berthollet, kle'mens de l'Art de la Teinture, Paris, 1791, 2 vols., vol. 1, pp. 131-35. Here he indicated how a knowledge of animal and vegetable substances could lead to an understanding ofthe processes of dyeing wool, silk, cotton and linen. " He added elsewhere that more experiments were necessary to discover the differences, which he thought must exist between animal and vegetable albumen, in spite of their close analogy. 'Albumine', Encyclopedie Methodique: Chimie, vol. 2, p. 18. 29 D. C. Goodman present in cruciferous plants. When the juices of the horse-radish, cabbage or cress were placed in bottles in a warm water-bath, the familiar white flakes of coagulated albumen were deposited. Like animal albumen it generated ammonia during putrefaction, or when it was distilled. This would explain the peculiar chemical properties of Cruciferae, though he thought that albumen was present in all green plants. Fourcroy's interpretation of this result was different from that given by Beccari for gluten. He regarded it as a further argument for classifying animals and plants in a single organic kingdom, separate from the inorganic minerals." The existence of a common albuminous substance was a further example, he said, of the chemical similarity already noticed in vegetable oils and animal fats; vegetable mucilages and animal jellies; gluten and fibrine. It seemed to him that chemistry had confirmed the analogies which anatomists and physiologists had detected in structures and functions. However Fourcroy soon changed his mind. He wrote that the analogies must not be stressed too much, since animal and vegetable substances showed more differences than resemblances.45 This applied to the albuminous, fibrous and mucilaginous substances, the immediate principles46 of plants and animals. He said albumen was more abundant in animals. Wheat gluten stretched to many times its original length and imitated a membrane, but he said muscle fibre was more elastic and far more abundant in animals. It was after all reasonable, he said, that the moving parts of animals should be made of a peculiar substance, absent in vegetables which lacked mobility.47 But the greatest differences involved the remote principles or elements. He agreed with Berthollet that the principal difference was the much greater quantity of nitrogen in animal substances. This determination was based on his use of Berthollet's nitric acid test. Fourcroy found that most nitrogen was liberated in this way from fibrous muscular matter, albuminous matter gave less and the gelatinous group of animal matters extracted from skin, tendons and cartilage gave least.48 He concluded that gelatinous animal matter49 approached vegetable substances, which generally produced little or no nitrogen with nitric acid. The process of animalization therefore appeared to be due to an increase in the nitrogen content, and Fourcroy considered the sequence gelatine, albumen, fibrine to 44Fourcroy (n. 42), 3, 253-54. 30 Application of Chemical Criteria to Biological Classification in the 18th Century represent an increasing scale of animalization. The removal of nitrogen from these animal matters should convert them to their original vegetable state, and he thought this was shown in the formation of vegetable oxalic acid in their reactions with nitric acid. Conversely the addition of nitrogen to vegetable substances should cause their animalization,50 and the mechanism of this would lead to a better understanding of the animal economy. But animalization was more than a simple addition of nitrogen. Fourcroy said the process also involved changes in the quantities of other elements.51 Phosphorus was abundant in animals; Scheele had found phosphates in bone, and Fourcroy had isolated them from urinary calculi. Plants contained less phosphorus, and he suspected that this was not essential to them, but was merely a foreign ingredient taken from the earth by their roots.52 The same remarks applied to the role of sulphur in the two kingdoms. He agreed with Berthollet that there was much more hydrogen in animals, since their materials in distillation gave more water and oil than plants. He added that there was less carbon in animals, since, compared to plants, less carbon dioxide was produced in dry distillation. In fact, he said: 'Carbon in animals plays nothing like the role it has in the vegetable economy'." For while carbon accumulated in plants, it escaped continuously in the respiration of animals; it was a transitory, unimportant element for animals. Fourcroy concluded that animal substances had a more complicated composition than vegetables. He conceived of plants as chemical instruments which began the organization of crude inorganic matter, synthesizing this into food for animals." He said they were intermediates in the scale of being, between minerals and animals. The general result of this synthesis was a compound of carbon, hydrogen and oxygen, and sometimes a little nitrogen. But here the place of nitrogen in the plant kingdom was not clearly delineated; its importance, which does not depend on its quantitative presence, was not yet understood. Sometimes Fourcroy referred to nitrogen, together with phosphorus and sulphur, as an inessential accessory;55 elsewhere he resorted to the imprecise statement that plants were made up of three or four elements.6 On the other hand the more elaborate animal substances were at least quaternary compounds of carbon, hydrogen, oxygen and much nitrogen, and besides these there were variable quantities of phosphorus and sulphur. The emphasis was on the quantitative differences of the elements. Fourcroy believed these caused marked variations in chemical properties, which permitted the character- thought. Cuvier considered that the possession of more functions in animals compared to plants, as in mobility and sensation, required a more complicated chemical composition. Therefore animals contained the extra element nitrogen, which was only present in plants by accident. Like Fourcroy he thought animals got rid of their excess carbon by respiration, while their nitrogen accumulated. G. Cuvier, Le Regne animal, 2nd ed., Paris, 1829-30, 5 vols., vol. 1, pp. 18-21. c 31 D. C. Goodman ization of an organic substance as animal or vegetable. He remarked that there was now a much larger number of distinguishing tests, and he proceeded to describe these in greater detail than had been attempted before.57 The action of heat was a well-established distinguishing test, but he said it was not before understood. The phenomena were quite different for animal substances because of their more complicated composition. Animal liquids tended to coagulate; animal solids emitted abundant vapours with the familiar foul odour, due to hydrogen, compounds of nitrogen, phosphorus and sulphur. During their thermal decomposition, animal solids exhibited a twisting and agitation, which Fourcroy interpreted as an indication of irritability and resistance to destruction. This produced an animal charcoal, denser, more adherent, and in smaller quantity than vegetable charcoal. Above all, animal charcoal was much more difficult to burn to an ash, because of the abundance of phosphates, and the smaller quantity of carbon. Therefore in contrast to the easy conversion of wood to ashes, hours of strong heat with agitation were required to incinerate blood or muscle.58 The prime animal characteristic of plentiful nitrogen was clearly indicated in distillation, as Berthollet had already said, in the formation of ammoniacal products such as the crystalline carbonate of ammonia. Fourcroy thought that animals putrefied in a distinctive manner. Their decay was more rapid and more marked than plants. He explained that this was another consequence of elaborate composition. The presence of more elements in greater quantities in animal compounds created multiple attractions which made them less stable. The slightest changes in temperature and moisture were sufficient to destroy the equilibrium and decompose the animal substance into simpler volatile compounds ofhydrogen with nitrogen, phosphorus and sulphur; these produced the insupportable odour. A new distinguishing test proposed by Fourcroy was the reaction with water. He said the effect of warm water on animal solids was most familiar in the cooking of meat, which through changes in colour, taste, smell and hardness became edible. The cooking of vegetables produced smaller changes, and besides they could be eaten raw. The differences were more striking still after a prolonged digestion in water. Most animal matter was then converted to a fatty substance like spermacetti, and ammonia was generated; but vegetables blackened and carbonized. He said the process of cooking was not understood, but he attributed the differences to the greater amounts of hydrogen and nitrogen, and the lower carbon content of animal substances. Finally Fourcroy described the reactions of animal compounds with acids and alkalies. Again he supposed that the peculiar effects, not shown by vegetables, were due to a more complicated composition. Sulphuric acid decomposed animal substances, producing a peculiar fat and generating ammonia. Nitric acid turned animal compounds yellow, liberated much nitrogen, produced a peculiar fat and prussic acid.59 He thought that alkalis acted more powerfully on animal substances, which 67 Fourcroy, 'Matieres animales', (n. 45), pp. 318-43. 68 For further details see Fourcroy's article 'Cendres', Encyclopidi Mithodique: Chimie, vol. 3, p. 123. ' Fourcroy thought that prussic acid was one of the most distinctive products of reactions with animal substances. But soon it was to be described as one of the immediate principles of plants, present in bitter almonds, prunes and other fruits. Vauquelin, 'Exp6riences qui demonstrent la pr6sence de l'acide prussique tout form6 dans quelques substances v6g6tales', Ann. Chim., 1802-3, 45, 206-12. 32 Application of Chemical Criteria to Biological Classification in the 18th Century soon softened. This explained the medical use of caustic alkali in the treatment of tumours. On the other hand vegetable tissues were little affected. These tests did not carry the specificity which Fourcroy claimed. They were insufficient to identify animal substances uniquely, since various vegetable materials behaved in the same way, as Fourcroy himself knew. In his description of the distillation of animal substances he said that the formation of particles of ammonium carbonate had long been recognized as a characteristic product of this operation; but he was forced to add that the same phenomenon occurred in the distillation of vegetable extracts, Cruciferae, and mushrooms.60 The tests were particularly inadequate for gluten, which Fourcroy regarded as vegetable. He said it was 'like no other vegetable matter'.' A tenacious, fibrous material with an odour, which he likened to sperm, it was found by Fourcroy to have a remarkable conformity with animal substances in its chemical reactions. He had to admit that in a strong fire it behaved just like an animal fibre, swelling, moving and burning like feather or horn, and emitting a foetid odour. In dry distillation gluten produced much ammonium carbonate and some prussic acid. The disagreeable smell was exactly like that accompanying animal distillations, and he said this could lead one to confuse them. The residual charcoal, like those of animal origin, was difficult to incinerate. Further striking analogies with animal materials were exhibited in its identical mode of putrefaction in warm, moist air and in its reaction with nitric acid. Additional conflicting instances occurred in the albuminous substances, which Fourcroy had found widespread in the plant kingdom. He had searched in vain for chemical tests which would distinguish these from animal albumen. He said vaguely that albumen was more abundant in animals, but it soon became apparent through the work of Vauquelin, his student and colleague, that not even this quantitative distinction could be rigidly maintained. Vauquelin investigated the milky juice of the Carica papaya or tropical pawpaw,62 which interested him on account of its medical employment as an anthelmintic. The results astonished him. The dried juice in water putrefied with an animal odour and deposited white flakes. When heated the flakes crackled like roasted flesh, and fat droplets appeared; no residue remained. From the reactions of the juice with acids, its behaviour in distillation, and its coagulation by heat, Vauquelin concluded that the pawpaw contained a substance which was most like albumen. He thought the juice also contained a little fibrine and much phosphate; it seemed to differ from blood only in the absence of a colouring ingredient. He remarked: 'This teaches us that nature has also given to certain types of plants the faculty of forming compounds similar to those produced in the animal machine, which must however put us on our guard when it is a question of pronouncing if a material belongs to vegetables or animals.'" There was nothing unusual in the mere presence of an albuminous substance in the pawpaw, since, as Fourcroy had shown, many plant juices contained this; but he 33 D. C. Goodman added: 'What is surprising is . . . the abundance and its purity in the pawpaw, in which one finds nothing having the properties of vegetables; and if this substance was coloured like the albumen of the blood ... one could easily confuse the one with the other.'" Here was a complete plant juice behaving exactly like an animal substance. Vauquelin also found much nitrogenous matter in the Salsola,65 tobacco66 and belladonna.67 His analyses opposed Fourcroy's chemical separation of animal and vegetable substances, and his conclusions approached the opinion expressed earlier in the eighteenth century that the chemical analogies of the two kingdoms were more impressive than their differences. The latter could not be stated with precision; the exceptions prevented the establishment of two mutually exclusive classes. APPLICATIONS IN BIOLOGICAL CLASSIFICATION In the eighteenth century there were two principal areas in which chemical evidence was employed for the purposes of establishing the place of ambiguous organisms in the scale of nature. The biological groups concerned were the cryptogamia and the zoophytes, an Aristotelian designation referring to living forms which appeared to have properties in common with both animals and plants, and which comprised a heterogenous group, whose classes were not distinguished until the nineteenth century. Amongst the cryptogamia there were certain algae with properties which surprised even those who were most inclined to accept them as plants. The Oscillatoria was found to have the animal faculty of independent motion. The texture of various algae was membranous and likened to the vesicles of the lungs68 or the tissue of the peritoneum;69 they were also gelatinous, and one type so resembled frog-spawn that it was called Batrachosperma. 70 In the middle of the century descriptions71 were given of the green algae which collected in the warm waters of spas, and they were classified as plants; but this later became a matter for argument. An Austrian physician72 reported that he had seen signs of animal movement in the green creature found in the waters at Carlsbad. He was therefore inclined to transfer it from the plant to the animal kingdom, and he supported this proposal with the results of a chemical analysis. The distillation of the green substance produced volatile alkaline salt; the residue was phosphorescent and contained no potash. He regarded these products as typically animal. It represented an application of the current crude generalization which described volatile alkali as an exclusively animal product. A similar argument with the opposite conclusion was employed by Georgi,73 a German pharmacist who became a professor of chemistry at St. Petersburg. He thought that chemistry could decide the status of the ambiguous algae, on the grounds that most plants behaved differently from most animals in distillation and alteration. Ignoring the exceptions, he applied this generalization to the Confervae. He heated some specimens and obtained resins which gave a pleasant smell in burning; he detected no foul animal odours. He distilled Confervae in a retort and collected an acidic phlegm; but no ammoniacal salts, and barely a trace of volatile alkali. The residue was easy to calcine and produced an abundant ash. He concluded that analysis had shown the Confervae to be of a vegetable nature, not containing anything animal. A series of chemical tests on the algae were next performed as a result of Priestley's description of green matter, which he was surprised to find on the inside of phials of water, in which he had been studying the growth of sprigs of mint.74 He doubted that the green matter was a plant, since it seemed to have no form. Moreover it appeared abundantly even in tightly corked vessels of water, so that seeds or animalcules floating in the air could not be the cause. Therefore he said that green matter was neither animal nor vegetable, but 'a thing gui generis'. He had observed bubbles of the pure air which plants generated in water, but at first he thought these were coming from the water itself, not from the green matter. He corrected this after he was convinced by the microscopic studies of some friends that green matter was after all a plant. He thought it was a conferva, and called it 'water moss'. It is interesting that the purification of the air was not regarded as an exclusive property of plants. Fontana75 said botanists had been deceived by the green organisms found in stagnant water. They were animals since they were oviform and in motion. The dephlogisticated air which they produced simply showed that animals as well as plants could prepare this gas. This influenced Ingenhousz, who had originally regarded Priestley's green matter as a plant,76 since his own experiments in photosynthesis had shown that all green plants in sunlight emitted dephlogisticated air. He studied the organism every day for more than three years, but still he confessed that its behaviour baffled him. The microscope showed a series of transformations. At first the greenish particles were round and clearly moving, so he had no doubt they were insects. Then these became imprisoned in a gelatinous crust, which he said was the state in which Priestley had observed the organism. Some weeks later the crust was found to have transparent fibres, which had a propagating motion like a worm. Ingenhousz collected dephlogisticated air throughout these changes, but agreed with Fontana that the production of this air was no proof of the vegetability of an organism.77 It seemed to him that green matter in its 71 I. G. Georgi, 'De Confervae Natura, Disquisitio Chemica', Acta Acad. Sci. imp. Petropolit., D. C. Goodman changes alternated between the animal and vegetable kingdoms.78 Further, he said the debris of green matter generated the filamentous Conferva rivularis of water reservoirs and the Tremella nostoc. He therefore thought the vegetability of these two species was now also in doubt.79 He conjectured that the insects of green matter had formed them. The filaments of this conferva also contained round corpuscles, which when released by cutting the filaments acquired motion within a few days. He said this showed they were insects full of life. It seemed to be a zoophyte, like coral. Ingenhousz was already convinced from the appearance of motion that Priestley's green matter had to be put in the animal kingdom. He then turned to chemical analysis for confirmation of his conclusion; but he remarked: '. . . only a weak argument can be drawn from chemical analysis, a fallible conjecture, in judging if a substance is animal or vegetable.'80 This was because the products of distillation were not peculiar to a particular kingdom. Nearly all animal substances gave an alkaline principle; plants sometimes gave acids and sometimes, as in the Cruciferae, volatile alkali. Without giving the details he said that green matter, the Conferva rivularis and the Tremella nostoc all behaved like animal substances, presumably in distillation; but he insisted: '. . . I repeat this analysis alone could not serve as a demonstration'."8 Chemistry played a more important role in Senebier's discussion of Priestley's green matter. He thought that analysis was still imperfect since it generated products not originally in the specimen. Nevertheless it provided useful information and he was optimistic that future improvements would assist plant physiology. He particularly wanted a rigorous chemical analysis of aquatic and cryptogamous plants. 82 His chemical experiments were assisted by Tingry, his teacher and professor of chemistry at Geneva.83 Senebier distilled green matter" and collected an ammoniacal liquor; the abundant charcoal was calcined to an ash which contained potash. Alcohol extracted a typically vegetable resin. It was true, he commented, that green matter had given ammonia by distillation, but so did the Cruciferae, and no one suspected that these were animal. Besides he continued: 'It is possible that this ammonia is due to the debris of numerous animalcules, ffies and butterflies which have perished in the green matter'.85 He said that if he had not solved the problem, this was the path to follow. The results of the analysis, together with the production of oxygen and the loss of green colour in the dark, showed, he said, that green matter was a true plant, a species of conferva. At the end of the century the animality of Confervae was again proposed in a joint 78 Ibid., vol. 2, pp. 9-10. 79 36 Application of Chemical Criteria to Biological Classification in the 18th Century study86 by Girod-Chantran, a naturalist, and Lacroix, the renowned mathematician. This involved a combination of observations with the microscope and numerous chemical tests, in which the influence of Berthollet and Fourcroy is apparent. The nitric acid test was applied to the Conferva bullosa; it gave azote. With a byssus, nitric acid produced the yellow coloration that Berthollet had noticed with silk. Digestion of Confervae in water was tried; one specimen became more viscous, another putrefied with an ammoniacal odour. The conferva was heated with caustic soda; ammonia was liberated. Various Confervae, Byssus and Tremella were distilled or heated in air; animal odours were observed, ammoniacal products collected and the ashes examined. The co-workers stressed the large quantities of the latter, their difficult incineration, their large lime content, and occasionally their complete absence of vegetable potash. These observations convinced Girod-Chantran that these organisms had been 'usurped by botanists'. 87 He wrote: '. . . all these considerations show that the time is not far offwhen these aqueous products, not having any type of fruit, till nowclassed with the plants, will rise in the scale ofbeing, and be placed withthepolyps, above the plants'.88 The Soci6t6 Philomathique requested Vauquelin and others to repeat these chemical experiments. It was stated that the commissioners confirmed the results, and made some additions.89 According to deCandolle, who was also asked by the Societe to evaluate this research, Vauquelin came to the opposite conclusion, that the specimens were vegetable.'0 The importance of chemistry in reaching a decision was particularly emphasized by Decandolle: 'Let us see if the older view of Confervae as plants is more admissible than this of their animality. The examination of their chemical nature and of their mode of life and structure must resolve this question'.91 Referring to Vauquelin's analyses, he said that Confervae gave very little ammonia in distillation. Also Giron-Chantran and Lacroix had mistaken potash in the ashes for soda. Finally the large quantity of the ashes proved, not their animality, as these workers suggested, but on the contrary their vegetability, as indeed Fourcroy had maintained. Decandolle therefore rejected their animality or intermediate nature and pronounced them true plants and put them with the algae. However the animal nature of algae continued to be asserted. Blumenbach92 put D. C. Goodman with an animal odour, which he compared to rotten oysters. Nitric acid produced prussic acid, which Fourcroy had mentioned as an animal characteristic. Its behaviour in distillation also convinced Bory that this species was remarkably animalized. The continual transfer of the algae from one kingdom to another indicated the difficulties which these organisms present in classification. It also showed that the chemical tests were inconclusive. The varying interpretations which were given, particularly to the detection of ammonia, revealed the uncertainties concerning nitrogenous matter in the plant kingdom. The same difficulties arose with the fungi, which in the eighteenth century were candidates for all three kingdoms."4 Mushrooms were without flowers. leaves or roots, and apparently without seeds. They fed on decaying matter and grew at a remarkable rate. Their texture was compared to flesh or muscle. Their infusions in water appeared to some observers to contain moving insects. These peculiarities led to their exclusion from all the kingdoms and the creation of a special kingdom to contain them. Necker"5 called this the 'Regnum mesymale' or intermediate kingdom, placed between plants and minerals. Willemet"6 suggested a new class of 'Pseudo-zoo-lithophytes'. Mushrooms were also known to have peculiar chemical properties."7 The analyses had medical interest, since the causes of mushroom poisoning were sought, as were reliable means for distinguishing noxious and nutritious species. In distillation LUmery" reported surprising quantities of ammoniacal products, and said this could confuse mushrooms with animals. The analysis of fungi was often described as animal. The mushroom was said to be more alkalescent than any other plant and likened to animal food.9 Nitrogenous substances compared to albumen and gluten were extracted from mushrooms, and the accompanying odours likened to fish or burning horn. 10 Bouillon-Lagrangel0l analysed truffles and mushrooms, employing tests suggested by Berthollet and Fourcroy. Nitric acid turned the truffle yellowish and nitrogen was liberated. He commented that this distinguished it from vegetables. In water the truffle exhibited a filamentous texture, which he compared to animals. When the water was heated, a flocculent precipitate appeared, due to the coagulation of albumen. He left a paste made with truffles and water in the air; after a few days this decayed with an odour like cheese, and much ammonia was produced. Distilled truffles left a residue that was difficult to incinerate and contained phosphoric acid. Mushrooms gave the same results. On the basis of these chemical experiments Bouillon-Lagrange concluded: '. . . the truffles must be distinguished from vegetables and form a special class under the title of Animalized plants.'102 Apart from the fungi and algae, the zoophytes continued to present problems in classification for the eighteenth century, and here the introduction of chemical arguments was most apparent. The group consisted of sponges, gorgonians (invertebrates with branching forms remarkably like vegetation, as in the sea-whip, sea-fan, sea-willow and sea-plume), corals, and the so-called corallines, a confused class which included coelamate and coelenterate invertebrates and also calciferous algae. They were variously described as spongey, stony, horny and ligneous. At the beginning of the century Tournefort regarded these as marine plants and put them in a class with the algae and fungi."* Their evident porous texture was seen by him to indicate the mode of nutrition. He said that when the branches of some specimens were lit, they burned with a smell of horn or birds' feathers and left little residue, which led him to conclude that volatile ammoniacal salts were present. 14 There was no question here of considering animality. Tournefort had already decided that corals and corallines were plants, and his description ofthe combustion phenomena simply implied that animals and plants were chemically alike, as the chemists were then asserting. The same was true of Marsigli's discussion. He relied on chemistry to establish that stony corals and madrepores were really organic and not petrifications. His famous observation of expanding and contracting forms in coral, fresh from the sea, had convinced him that he had seen the flowers of the coral plant. He added: 'Chemical analysis also shows us in an indisputable manner that these stony vegetations really are plants; when fresh the same constituents can be extracted from them as from terrestrial plants and animals. '105 The importance of chemical evidence for this natural historian was apparent in his simple statement that: 'Chemical analysis must terminate the question so often asked, that is, if coral is or is not a plant. '106 He distilled fresh coral and obtained alkaline liquors, pungent ammoniacal products and bituminous deposits. He made no remark that any of these products was peculiarly animal. Instead he compared the analysis with that of another specimen of coral, which had been out of the sea for over a year. The distillation of this no longer gave alkaline phlegm or bitumen, and he concluded that this was typical of marine plants, which lost their constituents when they were taken away from their food D. C. Goodman Lemery that chemically there was a single organic kingdom, distinguishable from the minerals. His analysis of the sponge confirms this; he saw nothing to conflict with his belief that the sponge was a plant from the large quantities of ammoniacal salts which appeared during distillation. 108 A different interpretation of the chemical analysis of marine zoophytes was soon proposed by Peyssonnel, a physician and botanist, who consequently classified sponges, corals and madrepores as animals.1" He regarded the milky juice of coral as the blood of the insects which produced it. The smell of rotten fish in the putrefaction of coral was caused by the death of the insects. He supposed the insects were housed in the bark, and believed this was confirmed by distillation, which exhibited the same oils, phlegms and salts as were extracted from human skull, hartshorn and other animal parts. More consideration was given to the theory that coral was made by insects after Trembley's discovery ofthe water polyp in 1739. It was then confirmed by the microscope that what Marsigli had taken to be flowers in coral were actually the tentacles ofpolyps. Attention then turned to the corallines and gorgonians. The natural historian who was most widely credited with the demonstration of their animality was John Ellis, who based his arguments on observations with the microscope, and especially on chemical evidence. Ellis said that the gorgonians such as the sea-fan were like shrubs with roots, stems and a network of branches. Their forms had misled many into regarding them as plants, a conclusion which he said was falsified by chemical analysis: 'The chemical Experiments, that have been made on these Bodies are a strong Proof, if there were no other Demonstrations, of their being animal. I need not mention any other to the curious, than the great Quantity of volatile Salts, that may be extracted from them and the strong Smell they yield, when burnt, of roasted Oysters.' 110 Ellis could not accept an intermediate nature for these organisms, which Linnaeus supposed in classifying them as zoophytes. Ellis therefore wrote to Linnaeus insisting that any suggestion of vegetability in the stem of gorgonians was excluded on chemical grounds."11 Similarly Ellis said that chemistry showed that the sponge was an animal.112 He was particularly concerned with establishing the true nature of the corallines, which like corals had a porous texture. His interest in these originated in a collection of specimens which he had been sent. He had preserved these and made landscapes with them. 108 Marsigli (n. 105), pp. 55-56 and pp. 60-63; also his 'Lettre touchant quelques branches de Application of Chemical Criteria to Biological Classification in the 18th Century This delighted Stephen Hales, who asked Ellis to acquire more corallines to make a landscape for the Princess Dowager of Wales, and this culminated in Ellis' attempts to classify them.113 He likened their slender, jointed branches to moss and admitted that at first, like the botanists, the forms had deceived him into the belief that corallines were plants. But his doubts grew when their texture under the microscope seemed unlike plants. He travelled to Sheppey to study live specimens. His suspicions were confirmed; some specimens exhibited polyps with contracting tentacles. He thought there was also conclusive evidence from chemistry; he wrote: 'These differ from Sea-plants in Texture, as well as Hardness, and likewise in their chymical Productions. For Sea-Plants, properly so called, such as the Algae, Fuci, etc., afford in Distillation little or no Traces of a volatile Salt: Whereas all the Corallines afford a considerable Quantity; and in burning yield a Smell somewhat resembling that of burnt Horn, and other animal Substances: Which of itself is a Proof that this Class of Bodies, tho' it has the vegetable Form, yet is not intirely of a vegetable Nature." 14 Ellis was apparently unaware that the interpretation of the chemical evidence was not as straightforward as he had presented it. In the argument which followed Ellis simply tried to persuade his opponents that the chemical phenomena were really as he had described them. Pallas, the natural historian and traveller, had advanced similar chemical evidence in support of the animality of various zoophytes. For example he referred to an analysis on a species of Alcyonaria carried out by his friend S. G. Gmelin."65 But he left the corallines until the end of his book, and said they were not really zoophytes, but plants and ought to be left to the botanists. Apart from their nodulous structure and fructification, which he compared to Fuci and Confervae, Pallas said the chemical behaviour of the corallines distinguished them from the zoophytes. He had neither the time nor the opportunity to perform the tests himself; instead he maintained that others had observed no animal oil or volatile salt, and that the odour in burning was more like vegetables."16 Ellis took up the challenge and elaborated his chemical evidence to persuade Pallas that he had put the corallines in the wrong kingdom. In a letter"17 to Linnaeus he described an experiment he had made at the Society for the Encouragement of Arts, Manufactures and Commerce, which he said had convinced the members present of the great difference in odour accompanying the combustion of plants and animals. The Society was investigating the nature of orchell, a lichen employed by dyers to produce a red colour, and had invited members to bring specimens for the next meeting. This was attended by Ellis who noticed that one sample inscribed as orchell was really a coralline. He told the others that while the rest of the samples were vegetable lichens, this was an animal coralline. Then, he recalled: '. . . in order to convince the Society of the difference, I called for a lighted candle, and having first set fire to the Lichen Roccella, it yielded the same smell that burnt vegetables usually Ibid 41 D. C. Goodman do; but when the Coralline ... was burnt, it filled the room with such an offensive smell like that of burnt bones, or hair, that the door was obliged to be opened, to dissipate the disagreeable scent, and let in fresh air.'118 To deal with Pallas' other objection that corallines gave no animal oil or volatile salts in distillation, Ellis sought the co-operation of Peter Woulfe,"19 a London chemist and the inventor ofthe Woulfe bottle, an improvement in the apparatus for performing distillations. Confident that Woulfe would provide accurate results, Ellis gave him a specimen of Corallina officinalis, fresh from the coast near Harwich. Woulfe placed the sample in a retort, fitted to a receiver, and applied a gentle heat for eight hours, collecting the distillate. This was continued at increasing temperatures for two successive periods of six hours. The first fraction contained a little volatile alkali, but its presence was much more noticeable in the subsequent fractions, when ammoniacal salts crystallized; oil with an animal smell also appeared. Woulfe remarked that if the distillation had been hurried the ammoniacal salts would have been missed. Ellis said that if Pallas tried this analysis, with the requisite care, he would change his opinion. He gave the same advice to Baster, a Dutch naturalist who had argued for the vegetable nature of corallines from their structural resemblance to Confervae. As a further proof he invited his opponents to treat Confervae, fuci or lichens with acid; nothing would happen, he said, whereas corallines would effervesce as their calcareous covering dissolved. 120 Ellis believed that the calcareous exterior itself revealed the true nature of the corallines, since he thought lime was only produced by animals.'2' He was following Linnaeus, who told Ellis that for this reason he had never doubted that the corallines were animal.'22 Ellis and Linnaeus were wrong, since lime is also produced by marine plants. This was soon demonstrated by Cavolini,123 who macerated marine fuci and showed that they effervesced with acids. This falsified Linnaeus' canon 'omnis calx a vermibus', and Cavolini felt free to assert that corallines were plants. Some of the corallines examined by Ellis were polypiferous invertebrates, and he had correctly concluded that they were animal, though for the wrong reasons; others however were calciferous algae, in which no observer could find polyps. The chemical arguments on which Ellis relied so much were naive and inconclusive, since they ignored the available information on plant analyses, though as has been seen Ellis was not alone in this. Ironically the Corallina officinalis, whose analysis by Woulfe he had particularly valued as a proof of animality, is not an invertebrate animal, but a calciferous alga. The chemical discussion of the corallines was taken a stage further and related to

v3-fos

2020-12-10T09:04:17.168Z

1971-09-01T00:00:00.000Z

237230440

s2

Bacteriology of Dehydrated Space Foods The initial bacteriological requirement established in 1964 for space foods by the U.S. Army Natick Laboratories are: a total aerobic plate count (≤ 10,000 per g), a total coliform count (≤ 10 per g), fecal coliforms (negative per gram), fecal streptococci (≤ 20 per g), coagulase-positive staphylococci (negative in 5 g) and salmonellae (negative in 10 g). Of the space foods and prototypes tested during 1968 and 1969, 93% complied with the total aerobic plate count, 98% had less than 1 coliform per g, and 99% were negative for fecal coliforms; 88% complied with the streptococci requirement; 100 and 98% were negative for staphylococci and salmonellae, respectively. Nineteen food samples which did not comply (as indicated parenthetically by actual counts per gram) with the requirements were (i) total aerobic plate count: beef soup and gravy base (18,000), chicken soup and gravy base (57,000), spaghetti with meat sauce (12,100 and 14,000), sugared coffee (> 300,000), chocolate ice cream cubes (20,000), and each of four samples of chocolate candy (12,000 to 61,000); (ii) coliforms: two out of three vanilla milk drinks (16 and 127) and one beef hash bar (14); (iii) fecal coliforms: one sample of chicken soup and gravy base positive; (iv) fecal streptococci: two samples of peanut cubes (40 and 108), coconut cubes (75), chicken soup and gravy base (2,650), beef soup and gravy base (33), and five out of six flavored milk drinks (23 to 300); (v) salmonellae: one each of chicken and beef soup and gravy base were positive. The initial bacteriological requirement established in 1964 for space foods by the U.S. Army Natick Laboratories are: a total aerobic plate count (.10,000 per g), a total coliform count ( <10 per g), fecal coliforms (negative per gram), fecal streptococci ( < 20 per g), coagulase-positive staphylococci (negative in 5 g) and salmonellae (negative in 10 g). Of the space foods and prototypes tested during 1968 and 1969, 93% complied with the total aerobic plate count, 98% had less than 1 coliform per g, and 99% were negative for fecal coliforms; 88% complied with the streptococci requirement; 100 and 98% were negative for staphylococci and salmonellae, respectively. Nineteen food samples which did not comply (as indicated parenthetically by actual counts per gram) with the requirements were (i) total aerobic plate count: beef soup and gravy base (18,000), chicken soup and gravy base (57,000), spaghetti with meat sauce (12,100 and 14,000), sugared coffee (> 300,000), chocolate ice cream cubes (20,000), and each of four samples of chocolate candy (12,000 to 61,000); (ii) coliforms: two out of three vanilla milk drinks (16 and 127) and one beef hash bar (14); (iii) fecal coliforms: one sample of chicken soup and gravy base positive; (iv) fecal streptococci: two samples of peanut cubes (40 and 108), coconut cubes (75), chicken soup and gravy base (2,650), beef soup and gravy base (33), and five out of six flavored milk drinks (23 to 300); (v) salmonellae: one each of chicken and beefsoup and gravy base were positive. The microbiological requirements for dehydrated space foods were established in 1964 in an effort to protect the astronauts from food poisoning (7). The stress factor was an important consideration in the establishment of these requirements since stress is known to alter resistance to infection. Simulation of stresses encountered during space flight such as high altitude (3)(4)(5)(6), irradiation (10), emotional strain (14), cold (12,13), heat (12,15), and sonic stress (M. A. Jensen and A. F. Rasmussen, Bacteriol. Proc., p. 149,1962) have been shown in the laboratory to alter the resistance of the animal host to microbial infections and intoxication. This paper presents production experience accumulated over a 2-year period with dehydrated (freeze-dried) space foods and demonstrates the attainability of the microbiological requirements established for these foods. MATERIALS AND METHODS Rehydratable space foods. Typical rehydratable products which are used for the Apollo feeding systems are shown in Fig. 1 Bite-size space foods. In addition to the rehydratables, dehydrated bite-size foods (Fig. 2) are used on the Apollo missions. These products are eaten by depositing them directly into the mouth. Preparation of slurry. A 25-g amount of the dehydrated space food sample was aseptically transferred to a sterile blendor cup and blended in 225 ml of Butterfield's (8) chilled sterile buffered water (SBW) for 2 min. This slurry constituted a 1:10 dilution and contained the equivalent of 0.1 g of food sample per ml. Hereafter this dilution shall be termed extract A. Extracts were maintained at no greater than 5 C and were used within 1 hr as prescribed in the following tests. Media. All media were purchased from Difco Laboratories, Detroit, Mich. Total aerobic plate count. Ten milliliters of extract A was transferred into 90 ml of SBW, giving a final dilution of 1:100. One ml of the 1:100 dilution was transferred into each of five petri plates and poured with plate count agar. Plates were incubated at 35 C and counted after 48 hr. The total number of colonies on the five plates should not exceed 500. Total coliform count. A 1-ml amount of extract A was transferred into each of 10 petri plates and poured with Violet Red Bile (VRB) agar. Solidified plates were overlaid with 5 ml of VRB agar. Plates were incubated at 35 C, and typical coliform colonies (dark red, 0.5 mm or more in diameter) were counted after 18 to 24 hr. The total number of typical colonies on all 10 plates constitutes the total coliform count per 1 g of food and should not exceed 10. attached to the outside of each package is ant antiseptic which is added to the package after the food has been consumed. FIG. 2. Bite size space foods. Fecal coliform count. Each typical VRB colony was transferred into phenol red-lactose broth fermentation tubes. Tubes were incubated at 35 C for 18 to 24 hr. Two loopfuls (3 mm diameter) of broth from each positive tube (displaying acid and gas) were transferred into an EC broth fermentation tube, which was incubated at 45.5 i 0.2 C for 24 hr. Both temperature and time are critical for this differential test. Hence, incubation was carried out in a constanttemperature bath and monitored with a certified Bureau of Standards thermometer or equivalent. EC tubes displaying gas production were considered positive for fecal coliforms. A single EC-positive culture constituted rejection. Fecal streptococci count. A 1-mI amount of extract A was transferred into each of 10 petri plates and poured with KF Streptococcus Agar. Plates were incubated at 35 C for 48 hr, and all red or pink colonies were counted. The total number of typical colonies on all 10 KF plates constitutes the fecal streptococci count per 1 g of food and should not exceed 20. Coagulase-positive staphylococci. Fifty milliliters of extract A (equivalent to 5 g of food) was transferred into 200 ml of cooked meat medium with NaCl. The medium was prepared overstrength by adding 31.25 g of cooked meat and 24.5 g of NaCI to 200 ml of distilled water. The final concentration of cooked meat and NaCl in 250 ml was 12.5 and 10%1c, respectively. The cooked meat medium was incubated at 35 C for 24 hr. A 0.1-ml amount of cooked meat medium was spread on each of two plates of Vogel and Johnson (VJ) agar. VJ plates were incubated at 35 C and examined after 24 and 48 hr. Two or more representative colonies which reduced tellurite were transferred to Brain Heart Infusion (BHI) tubes and incubated at 35 C for 24 hr. The remainder of each colony was emulsified in 0.2 ml of BHI. Then 0.5 ml of coagulase plasma was added, mixed, and incubated in a 35 C water bath for 4 hr. If these tubes were negative, the coagulase test was then repeated with the 24-hr-old culture. A single coagulase-positive colony constituted rejection. Salmonella. One hundred milliliters of extract A (equivalent to 10 g of food) was transferred into 100 ml of double-strength lactose broth and incubated at 35 C for 24 hr. A 25-ml amount of the lactose broth culture was transferred into 225 ml of each of selenitecystine broth and TT broth base [modified tetrathionate broth containing Brilliant Green (1:100,000)] and incubated at 35 C for 18 to 24 hr. Each enrichment culture was streaked on one plate each of three selective media: Brilliant Green Sulfadiazine (BGS) agar, Bismuth Sulfite (BS) agar, and Salmonella-Shigella (SS) agar. BGS and SS plates were incubated for 24 hr and BS plates for 48 hr at 35 C. Two typical colonies were picked from each plate and inoculated on Triple Sugar-Iron (TSI) agar and Christensen's urea (CU) agar slant. All slants were incubated at 35 C for 24 hr. CU slants were observed Negative in 10 g periodically for 4 to 6 hr. If the culture showed a urease-positive reaction, the respective colony was Salmonella-negative, and the test was ended. Positive TSI agar tubes associated with a urease-negative reaction constituted a presumptively positive Salmonella culture. Transfers from positive TSI slants were typed against Salmonella 0 and H polyvalent antisera. Positive reactions constituted confirmed presumptively positive Salmonella in the test sample. Presumptively positive TSI cultures were further confirmed through reactions in the following: dulcitol (+), malonate (-) broths, lysine decarboxylase broth (+), KCN broth (-), and indole broth (-). A single confirmed positive Salmonella culture constituted rejection. RESULTS The microbiological requirements (Table 1) established for dehydrated space foods were attainable as evidenced by the microbiological data presented in Table 2. Of the foods analyzed in 1968 and 1969, 93 %O had total aerobic plate counts of less than 10,000 per gram, 98%o had less than 1 coliform per gram, and 99% were negative for fecal coliforms per gram; 88% had less than 20 fecal streptococci per gram; 100%ac were negative for coagulase-positive staphylococci; and 98% were negative for salmonella. The microbiology of 19 foods which did not comply with the microbiological requirements for dehydrated space foods during 1968 and 1969 is presented in Table 3. With the exception of some of the chocolate cubes and the peanut cubes, these items were prototype foods which were being tested for space flight. There was no correlation between total aerobic plate counts and other bacterial indicators, except in the case of chicken and beef flavored soup and gravy base. Both soups, in addition to having relatively high total aerobic plate counts, were positive for Salmonella and exceeded the fecal streptococci requirement. The chicken soup was also positive for fecal coliforms. There was only a 30% correlation between the presence of coliforms an i fecal streptococci in the 19 foods presented in Table 3. For example, of the 10 foods which contained fecal streptococci, only three contained coliforms. The higher recovery rate of fecal streptococci may have been due to the greater resistance of streptococci to drying, heat, and other food manufacturing processes. DISCUSSION The microbial indexes (Table 1) were selected to measure the sanitary conditions during space food production and to detect the presence of foodborne pathogens and organisms that may indicate the presence of enteric pathogens. The methods employed to recover and enumerate the microorganisms are generally accepted by microbiologists worldwide and are modifications of standard methods recommended by many authorities (1,2,11,16). All of the microorganisms tested for, with the exception of coagulase-positive staphylococci, were detected in at least one of the space food prototypes ( Table 3), indicating that the methods employed were suitable. Although it is possible that staphylococci were not present in the foods tested, it is also possible that 10% sodium chloride (NaCl) was too inhibitory, particularly to injurel staphylococci. It has been demonstrated, for example, that thermally injured cells of Staphylococcus aureus were salt-sensitive and unable to grow in Trypticase Soy Agar containing 7.5% NaCl (9). Allowing for this possibility and to improve the recovery of staphylococci, the procedure has been modified by incubating the food sample in Tryptic Soy Broth (TSB) for 3 to 4 hr before the addition of 10% NaCl to the medium. Iandolo and Ordal (9) reported that the recovery of thermally injured cells of Staphylococcus aureus was completed within 4 hr in Trypticase Soy Broth incubated at 37 C and that thereafter growth in 7.5% salt-agar equalled the untreated controls. The attainability of the microbiological requirements established for dehydrated space foods is evidenced by the high percentage of foods which complied with these requirements ( Table 2). The excellent microbiological quality of these space foods was achieved by purchasing and properly storing good quality raw products, by processing these foods in special laminar airflow and conventional clean rooms (class 100 and 10,000, respectively), and by observing good manufacturing practices and sanitary operating procedures. The microbiological requirements for space foods are under continual surveillance and will continue to be amended as warranted by new knowledge and the state of the art. Process-and product-specific studies are still needed to determine clearly the etiological significance of the organisms tested for, as well as organisms such as Clostridium perfringens, Bacillus cereus, and others that are less well known, including viruses, rickettsia, vibrios, and mycoplasma. ACKNOWLEDGMENTS This work was supported by NASA and the U.S. Air Force under Order Numbers NASA T-25041 G and Air Force AM 6-40061. We thank Mary Klicka for kindly providing us with photographs of space foods.

v3-fos

2019-04-01T13:16:29.528Z

1971-01-01T00:00:00.000Z

89468524

s2

The milk production of reindeer cows and the share of milk in the growth of reindeer calves The milk quantities obtained from the reindeer cows in the present study are probably, due to technical difficulties in the milking, lower than the actual values. For this reason the figures on the milk producing ability, and the connection between the milk yield and other characteristics of the cpw or its calf, are bound to be uncertain. The general composition of reindeer milk is in fair agreement with results reported by other researchers (Tables 1, 4 and 5). Individual differences in the milk producing ability could clearly be detected. The correlations between the milk quantity and the constituents were usually not significant. The association between the dam’s spring weight and the protein percentage was significant (r = —0.47*»). The range of the calves’ weights at birth was to a large degree mainteined during the entire period of growth (Table 9). The weight and growth of the calves was clearly dependent on the weight or on the size of the dam. The results obtained did not indicate any connection between the milk production characteristics of the dams and the growth of the calves (Tables 11 and 12). The spring weight of the dam has a more decisive influence on the growth of the calf than the birth weight of the latter. The results obtained seem to indicate that in the breeding for growth capacity, selection on the basis of the weight or size of the dams os of conclusive importance. In spite of the individual variations existing in the milk producing ability of reindeer cows, the importance of the quantity and composition of the milk seems to be covered by the genetically determined growth factors. As the size of reindeer can be determined more easily than the weight, the combined measure, (body length -fchest girth), seems tobe a more suitable basis for evaluation. Since 1965 the reindeer breeding studies, started on the initial of the ReindeerKeeper’s Association in 1962 at Askankangas in Puolanka (Varo 1964), has been continued at the Reindeer Experimental Region Kaamanen in Inari. During the summer 1967, in the period June Ist to Sept. 15th, the subject of study was the milk production ability of reindeer cows and the possibly existing variation between dams in this respect, as well as the influence of these factors on the development of the reindeer calves. During the experiment the weather conditions were relatively good. Precipitation and temperature were below average, which apparently resulted in a decreased number of mosquitoes and gadflies. Work among the reindeer was thus rather easy. general composition of reindeer milk is in fair agreement with results reported by other researchers (Tables 1, 4 and 5). Individual differences in the milk producing ability could clearly be detected. The correlations between the milk quantity and the constituents were usually not significant. The association between the dam's spring weight and the protein percentage was significant (r = -0.47*»). The range of the calves' weights at birth was to a large degree mainteined during the entire period of growth (Table 9). The weight and growth of the calves was clearly dependent on the weight or on the size of the dam. The results obtained did not indicate any connection between the milk production characteristics of the dams and the growth of the calves (Tables 11 and 12). The spring weight of the dam has a more decisive influence on the growth of the calf than the birth weight of the latter. The results obtained seem to indicate that in the breeding for growth capacity, selection on the basis of the weight or size of the dams os of conclusive importance. In spite of the individual variations existing in the milk producing ability of reindeer cows, the importance of the quantity and composition of the milk seems to be covered by the genetically determined growth factors. As the size of reindeer can be determined more easily than the weight, the combined measure, (body length -f-chest girth), seems tobe a more suitable basis for evaluation. Since 1965 the reindeer breeding studies, started on the initial of the Reindeer Keeper's Association in 1962 at Askankangas in Puolanka (Varo 1964), has been continued at the Reindeer Experimental Region Kaamanen in Inari. During the summer 1967, in the period June Ist to Sept. 15th, the subject of study was the milk production ability of reindeer cows and the possibly existing variation between dams in this respect, as well as the influence of these factors on the development of the reindeer calves. During the experiment the weather conditions were relatively good. Precipitation and temperature were below average, which apparently resulted in a decreased number of mosquitoes and gadflies. Work among the reindeer was thus rather easy. In order to catch the reindeer, small pasture fences had to be erected. When fencing new pasture areas one sometimes encountered such difficult conditions of terrain that cloth fences had to be used. This was also why the entire herd escaped in the middle of July and was lost for over a week. Later, the reindeer were herded in a 4 km 2 wide area, situated 10 km from the experimental region. The construction of a small fence, however, took so long that the herd again scattered. In spite of continuous efforts barely one half of the experimental animals could eventually be caught. This misfortune naturally seriously affected the reliability of the results. The observed quantities of milk should be regarded with great caution also because the reindeer cows were not used to such close contact with human beings as was required in the milking process. Greatly depended on the nature of cow whether the animal did or did not give milk. Review of the literature The literature on the milk yields of reindeer is very limited. The Norwegian research worker Skjenneberg (1965) notes that the amount produced varies between 100 and 750 g per day, the most favourable lactation period being July-August. On the other hand, information on the average composition of reindeer milk is plentiful. Table 1 shows the values, as percentages, given by different researchers. The values are reported either as averages or as changes during the lactation period. (Tables 2 and 3). The breed in question is considerably larger than our own. According to the present writer, R.M. Varo (1964), the average birth weight of calves, born in spring 1963, was 5.80 kg, the heaviest calf weighing 7.45 and the smallest one 4.70 kg. A small but statistically significant difference between the sexes was noted. The calves by cows of different ages did not significantly differ in weight. Own studies Material and methods. The reindeer cows of the experiment were mainly born in 1963 (Varo 1964). Some otherreindeer, also younger ones, were likewise included. In the spring 1967 a total of 33 calves were born, of these two died immediately after the calving and two dams with calves disappeared. Thus the material consisted of 29 calf-cow pairs. Later, during the course of the experiment, three calves had to be delivered to the State Game Research Institute and one calf was lost in an accident. Originally the intention was to do 3-4 milking rounds by milking the dams in their calving order. In this way the milk samples would have been obtained after approximately the same period after calving. This plan could, however, not be fully realized because the reindeer managed to escape. The collection of samples and the weighing of the animals was done by two students, H. Kullberg and H. Varo. The cows to be milked, two animals each day, were in the evening separated from their calves and kept in a 2 X 3 m enclosure. The animals were given water, moistened lichen and Peura-fodder. The milking took place the following morning and evening. The calves were weighed during the same day. The milk quantities obtained were combined and from the mixture a sample of approx. 80 ml. was taken and put into a plastic flask containig formalin. The samples were kept in a nearby cold brook and were weekly sent for analysis to the Department of Dairy Science of Helsinki University. With the aid of Peurafodder the reindeer were easily caught, but the milking often took quite long. Occasionally a cow had to be released because it had damaged its developing horns. Results The milk production of the cows. The milk quantities obtained varied between 47 and 206 ml per milking day. In order to make the values comparable they were corrected to correspond to the amounts of milk obtained 30 and 60 days after calving. The new values obtained in this vay varied between 54 and 207 ml and between 47 and 202 ml for samples collected 30 and 60 days, respectively, since the calving. The corresponding averages were 99.8 and 96.9 ml. For reasons given above connected with the milking, it is probable that the milk quantities obtained are lower than the real ones of reindeer cows. The general composition of reindeer milk in the present material is given in Tables 4 and 5 (Luhtala, Rautiainen & Antila 1968). Table 5 shows that the solids and fat percentages steadily increase during the experimental period, whereas the protein percentage is at its lowest in the middle of the period. In order to detect the possible individual differences in the milk production ability of reindeer cows an analysis of variance was carried out. As the milk samples were obtained at different stages of the nursing period, the measure of production capacity used was the milk yield deviation of the individual cow from the general average curve. The F-values and the coefficients of repeatability are collected in Table 6. Table 7 shows all correlations between weight in spring, milk yield and milk composi- tion. It appears that the contents of fat and protein are independent. The correlations between milk yield and composition were not significant and were always negative. Likewise the correlations between the cow's weight in spring and the other caracteristics were in most cases nonsignificant. Only the correlation for the protein content was significant, r = -0.47 (P < 0.01). The weight development of the calves. The calves included in the experiment were born between May 6th and 23rd. The birth weight was, on an average, 5.63 kg, the standard deviation being 0.74 kg. The lightest calf weighed 4.2 and the heaviest 7.5 kg. Female calves weighed, on an average, 5.28 and male calves 5.93. Table 8 shows the development of the weights of the calves during the summer period. The weight of 3 month old calves in a few cases had to be evaluated on the basis of their earlier development and the overall average growth capacity. The individual differences with regard to the weight development of the calves were detected by means of a similar analysis of variance as in the matter of the milk production of the cows. The calculations were thus done on the basis of the deviations of the weights from the means at different ages. Table 9 shows that the ranges in weight at birth have to a considerable extent been mainteined during the experiment, although less so in the males than the females. When evaluating the dependence of the growth of the calves on the weight of the dams in spring, the weight differences caused by sex have been levelled. As all dams were weighed at the same time, April 24-25, 1967, the weights in spring are not altogether comparable on account of the differences in the stages of pregnancy. Moreover, the closeness of the weighing date to the calving may have increased the correlations. The correlations calculated using the weight of the dams in autumn (1967) probably give a more accurate picture of the association. Table 10 shows the dependence of the weight of the calves on the weights of the dams in spring and autumn, on the dam's body length, chest girth and on the two latter measures combined. Taking into account the inaccuracy of the spring weights of the cows when evaluating the dam, the combined measure seems quite suitable. The magnitude of the coefficients indicates that in addition to the genes also the embryonic period and the ability of the dams to take care of the calves may have had its effect on the correlations. Table 11 shows the correlations between different milk characteristics of the dam and the growth rate of the calf. The large negative values are probably due to the fact that the correlations between the weights of the calves and the spring weights of the dams are high and positive (Table 10), while those between the weights of the dams and the milk traits are negative (Table 7). Moreover, it should be noted that already when the calves were less than one month old they consumed other nutrients beside milk, among other things Peura-fodder, which is rich in protein and trace elements. If the influence of the spring weight of the dams is eliminated from the above correlations, the coefficients get higher and become positive, with the exception of the correlations concerning the protein percentage. The values are, however, not significant, as the 5 % level is at 0.37 (Table 12). When evaluating the correlations concerning the weights and growth of the calves (Table 13), it may be noted that the influence of the birth weight on the later weights all the time decreases. The influence of the birth weight on the growth is negligible. Alunperin oli tarkoituksena suorittaa 3 -4 lypsykierrosta siten, että emät olisi lypsetty vasomisjärjestyksessä. Näin olisi maitonäytteet saatu suunnilleen yhtä pitkän ajan kuluttua vasomisesta. Tästä jouduttiin kuitenkin tinkimään porojen karkaamisen takia. Näytteiden keruun suorittivat ylioppilaat H. Kullberg ja H. Varo. Lypsettävät vaatimet, joita oli kaksi kunakin päivänä, erotettiin illalla vasoistaan n. 2 X 3 m:n suuruiseen aitaukseen. Eläimille annettiin vettä, kostutettua jäkälääja Peura-rehua. Lypsy tapahtui seuraavana aamuna ja iltana. Vasat punnittiin saman päivän aikana. Saadut maitosaaliit yhdistettiin ja seoksesta otettiin formaliinia sisältävään muovipulloon n. 80 mhn näyte. Maitonäytteet säilytettiin läheisessä kylmässä purossa ja lähetettiin viikottain Helsingin Yliopiston maitotalouslaitokselle analysoitaviksi. Peura-rehun avulla porot saatiin helposti kiinni, mutta itse lypsy venyi usein hyvinkin pitkäksi. Silloin tällöin oli vaadin päästettävä vapaaksi sen loukattua kehittyvät sarvensa.

v3-fos

2020-12-10T09:04:11.224Z

1971-02-01T00:00:00.000Z

237231646

s2

Effects of Oxyamylose and Polyacrylic Acid on Foot-and-Mouth Disease and Hog Cholera Virus Infections Two interferon-inducing polycarboxylates were tested for antiviral activity on foot-and-mouth disease (FMD) virus infections in mice, guinea pigs, and swine. Polyacrylic acid, given intraperitoneally, had a protective effect on infection by FMD virus administered in the peritoneal cavity of mice and in the foot pad of guinea pigs. Chlorite-oxidized oxyamylose (COAM) was effective in mice at a dosage of 2 mg/kg. Swine were not protected against naturally transmitted FMD by 120 mg/kg of COAM nor by polyacrylic acid. Swine were not totally unresponsive to COAM since it delayed symptoms of hog cholera. Interferon was not detected in the serum of COAM-treated swine. With FMD virus, an example was found of activity of interferon inducers in experimental hosts and lack of activity in a natural host. Synthetic polycarboxylic acids, or carboxylate polymers, are a new class of antiviral compounds which induce transient levels of interferon in the blood of the intact animals. They protect it against the effects of a virus challenge administered up to many weeks later. Pyran copolymer (14), polyacrylic acid (6,9,10), and chloriteoxidized oxyamylose (COAM; 2, 4) have been studied in detail. These compounds appear to offer particular interest for the prophylaxis against viral disease in veterinary medicine. As the complex of polyinosinic and polycytidylic acids (poly I :C; 12,15) and other antiviral ribonucleic acid (RNA) species, they are much more active when given before exposure than for cure of viral infections. Unlike the antiviral RNA species, they display prolonged antiviral activity. An acceptable compound for prophylaxis in animals will probably belong to the long-acting group of interferon inducers, such as the carboxylate polymers, rather than to the short-acting group, such as poly I :C. Furthermore, experience with the longacting compounds in naturally occurring viral disease of large animals could provide more realistic information with regard to their possible application in man than may be derived from experimental infection of small laboratory animals. The broad host range of foot-and-mouth disease (FMD) virus and its importance in natural hosts (1) seem to make FMD virus infection an ap-propriate model for testing the activity of interferon-inducing polycarboxylates. The purpose of this study was to ascertain whether polyacrylic acid and COAM have a protective effect in experimental rodents and in swine, a natural host. Polyacrylic acid was found to be effective in mice and in guinea pigs but not in swine. Similar findings were obtained in mice and in swine with COAM, a highly active, potentially more useful polycarboxylate which became available more recently (2,4). The results of this investigation also led us to examine interferon induction and protection against hog cholera in swine by COAM. MATERIALS AND METHODS Animals. Conventional guinea pigs and swine were used. Pregnant pathogen-free CFLP mice were obtained from Carworth Europe Laboratories, Huntingdon, England. Viruses. FMD virus, type (h Vienna, was obtained from G. Kubin, Vienna, Austria. It was passaged three times in cattle and four times in guinea pigs, from the lesions of which it was harvested for use in the experiments on guinea pigs. FMD virus, type 01 Lausanne, was obtained from G. Moosbrugger, Basle, Switzerland, and passaged once in cattle. Then it was passaged three times in suckling mice and harvested from infected muscles for use in the experiments on mice. Another sample was passaged three times in swine, and the virus was harvested from the lesions for use in the experiments on swine. With the adaptive passages, the type 01 provoked early lesions, appearing nearly simultaneously in all infected guinea pigs and swine, and rapid death in mice infected under 10 days of age. Unlike mice, guinea pigs and swine generally recovered from FMD virus infection. The Behring 65 strain of hog cholera virus was obtained from Behring Laboratories, Marburg, West Germany, and passaged three times in swine. The virus stock was defibrinated viremic blood. All swine succumbed to infection with this virus. Swine interferon and FMD virus antibody assays. The continuous porcine kidney cell line IB-RS-2 (5) and primary swine kidney cells were grown and used for assay of swine interferon as described for human and rabbit cells and interferons (7). The methods were plaque reduction and yield reduction of vesicular stomatitis virus (VSV). Neutralizing antibody to FMD virus was assayed by mixing equal volumes of doubling dilutions of swine serum and 100 median tissue culture infective doses of swine-adapted FMD virus, as mentioned above. The mixtures were kept for 1 hr at 37 C. End points of neutralizing antibody activity were measured by the suppression of FMD virus cytopathic effect in IB-RS-2 cells. Antiviral compounds. Polyacrylic acid and COAM were prepared as previously described (4,9). They induced interferon in mice (4, 10) and protected mice against viral infection as described (2,6). The polymers were dissolved at appropriate concentrations in phosphate-buffered saline and injected intraperitoneally in volumes of 1 ml in guinea pigs, 0.1 ml in suckling mice, and at concentrations of 10 or 30 mg/ ml in swine. For subcutaneous injection of COAM in swine, a concentration of 30 mg/ml was used. Statistically significant delay of mortality in mice infected with FMD virus was taken as an index of antiviral activity, rather than indefinite survival. Although indefinite survival was observed in treated mice, it was a less sensitive criterion of antiviral activity. Similarly and unless otherwise noticed, the criterion for protective activity in guinea pigs and in swine was delay in the appearance of lesions. RESULTS FMD virus infection and polyacrylic acid. Suckling mice, 5 to 7 days of age and weighing 4 or 5 g, were injected intraperitoneally with 20 mg of polyacrylic acid per kg. This dosage did not cause mortality, growth inhibition, or other apparent ill effects during an observation period of 3 weeks. Eighteen hours after injection of the compound, FMD virus was administered through the same route. With the higher virus inocula, mice started succumbing on the second day after virus administration. Older mice could not be used because of their resistance to the virus. In all experiments, mortality due to FMD virus was delayed in polyacrylic acid-treated mice. The protective effect of 20 mg of polyacrylic acid per kg against different virus challenge doses is recorded in Fig. 1 Protection experiments were also performed in guinea pigs. These animals did not die after virus administration in the plantar pad, but they did develop typical lesions as seen in the natural hosts. The guinea pig model provides the best approximation in small animals of the infection that develops in naturally susceptible large animals (1). Guinea pigs weighing 500 g were injected intraperitoneally with 5 or 20 mg of polyacrylic acid per kg. After 18 hr, FMD virus was administered in the plantar pad. Both dosages of the compound were found to protect the animals against generalization of the infection. A representative experiment is shown in Table 1. The appearance of lesions at the injection site was also delayed. However, a number of guinea pigs died as a result of the toxicity in this animal of protective doses of polyacrylic acid. Swine were chosen to test the activity of polyacrylic acid against naturally transmitted FMD in a naturally susceptible host. Twelve pigs weighing between 16 and 20 kg were given 30 mg of polyacrylic acid per kg intraperitoneally. Six were exposed after 18 hr and six after 7 days to swine having FMD. All treated swine developed symptoms at about the same time as the untreated, simultaneously exposed, control group. Three additional swine treated 3 days before exposure also showed no effect. The dosage of polyacrylic acid given was the highest which was not followed by toxic effects resulting in death (or visible illness) during an observation period of 3 weeks. Thus, unlike mice and guinea pigs, swine were c Per group of four, number of animals which died due to toxicity of polyacrylic acid. Toxicity was similar in guinea pigs which did not receive FMD virus. not protected at all against FMD virus by polyacrylic acid administered intraperitoneally. FWM virus infection and COAM. COAM and a number of related compounds derived from natural polysaccharides are markedly less toxic than pcolyacrylic acid (2,4). This and other properties make these newly described compounds more interesting for use as antiviral agents. COAM was found to protect mice against FMD virus when used under conditions as described for polyacrylic acid. The activity of the compound against a small virus dose was studied in detail. Three litters of eight mice were given COAM intraperitoneally, 10 to 300 ,ug, or 2 mg per kg to 60 mg per kg per suckling mouse of 5 g. After 18 hr they received one LD5o of FMD virus by the same route. Mortality was delayed in all mice which had received COAM. The pooled results are given in Fig. 2. The protection given by the lowest dosage, 2 mg/kg, was significant. Swine used for protection experiments weighed between 17 and 25 kg, with an average of 20 kg. In initial experiments, it seemed that COAM- treated swine were unusually susceptible to death caused by handling stress. However, deaths also occurred in untreated animals submitted to frequent or energetic experimental handling. The number of deaths in COAM-treated animals became negligible when handling stress was reduced ( Table 2). The animals showed no gross ill effects after intraperitoneal or subcutaneous administration of 30 mg/kg and 120 mg/kg of COAM for observation periods of 3 weeks. Higher dosages were not tested. At autopsy, fibrin deposits were found on the peritoneum of a majority of intraperitoneally treated animals. No or minimal local lesions were found after subcutaneous administration of COAM. COAM-treated and control animals were infected through contact with pigs which had received FMD virus intramuscularly (day 0) and developed a com lete picture of FMD on days 2 and 3. Control animals showed symptoms shortly afterwards (Table 2). Twelve pigs were given 30 mg of COAM per kg intraperitoneally 7 days and 18 hr before exposure. They developed lesions simultaneously with the control animals. With 120 mg of COAM per kg, there was no significant difference between treated and untreated animals with regard to the time that the first foot lesions appeared and proceeded to all extremities (Table 2), the number and extent of lesions, and temperature measurements. At autopsy on day 10, lesions were found on snout, tongue, and heart to the same extent in treated and untreated animals. The appearance of virus-neutralizing antibodies was examined on day 10. Pre-exposure sera had titers of less than 2. Postinfection sera had titers between 2 and 16, with a geometric mean titer of 3.8 in COAM-treated animals. There was no difference between the different COAM-treated groups. In untreated animals, postinfection sera had titers of 2 to 8, with a geometric mean of 3.6. Thus no difference in antibody response between COAM-treated and control animals was noted. COAM at a dosage 60 times greater than the effective dose in mice had no measurable effect against FMD in swine. Search for interferon in COAM-treated swine. Conceivably, the lack of anti-FMD effect of polycarboxylates in swine could be due to failure to induce interferon in these animals. Interferon induction by COAM in swine was therefore investigated. A male and female pig, each weighing 20 kg, received 120 mg of COAM per kg intraperitoneally. Blood samples were taken after 0, 6, 12, 18, 24, and 36 hr. This schedule was adopted since mice have highest titers of circulating interferon 18 hr after intraperitoneal injection of carboxylate polymers (4,10,14). Serum was stored at -20 C and assayed for interferon by VSV plaque and yield reduction on primary swine kidney cells and on IB-RS-2 cells. After 18 hr of pretreatment with doubling dilutions of serum starting at 1:8, cells were washed and challenged with VSV. There was no plaque reduction or yield reduction of VSV by the test sera compared with control sera taken at 0 hour or with untreated controls. The VSV-swine cell system was sensitive to interferon for the following reasons. (i) Primary swine kidney cells, treated for 3 hr with 10,ug of poly I:C (PL-Biochemicals) per ml (12) and 30,ug of diethylaminoethyl (DEAE)-dextran per ml (11), and then washed and further incubated for 18 hr, released a factor in the medium which suppressed plaque formation of VSV in the same types of cells when used at a 1:8 dilution in an interferon assay; this factor had properties of interferon. (ii) A much lower concentration of poly I :C, 0.1 jAg/ml, induced cellular resistance to VSV; DEAE-dextran was needed for the effect. (iii) Recently, primary swine kidney cells and IB-RS-2 cells were found sensitive to swine interferon when FMD virus was used for assay (17). On the other hand, there is no record of VSV being insensitive to interferon in primary cultures of normal cells. It is concluded that swine did not react with detectable serum interferon to COAM. Hog cholera and COAM. The marked discrepancy between the effects of COAM on FMD virus infection in small rodents and in swine also led us to investigate protective effects in a different viral infection of swine. Hog cholera was chosen as another infectious system. The virus is of major importance in swine, the only animal in which it provokes symptoms. Its properties are entirely different from those of the FMD rhinovirus, and although unclassified it shares many biophysical and biological characteristics with rubella virus (16). Natural exposure was not deemed advisable in the present experiment since this resulted in symptoms arising over a prolonged period in different animals and difficulty of comparison between test and control groups. Swine weighing 20 kg were randomized, and 8 received 120 mg of COAM per kg intraperitoneally. After 18 hr, the COAM-treated and eight control pigs were given intramuscularly 1,000 LD50 units of hog cholera virus, as previously determined in vivo. The maximum rectal temperature was reached on days 3, 4, or 5 in control animals. This temperature was attained in none of the test animals until 6 days after virus administration. The average temperature curves are charted in Fig. 3. Diarrhea was first noticed in control animals on days 2 and 3 and in COAM-treated animals on days 5 and 6. Skin lesions appeared in control animals on days 5 and 6 but not before day 7 in treated animals. On day 8, the first swine died: they were two control animals. There was, however, only a very slight difference in average time of death: 10 from treated and untreated groups by munofluorescence in PK-15 cells (13). DISCUSSION Polyacrylic acid and COAM prote against mortality caused by FMD v lowest dosage of COAM given, 2 mj effective. It was similar to the lowest effe in vaccinia and mengovirus infections administration schedule of the compo the virus was as previously adopted for ( infections (2,4,6,14) and may there been less than optimal in the case of F infection. Since the susceptibility of mici virus decreased drastically after 10 da in our assay system, the duration of the action could not be determined. The activity of interferon-induci: carboxylates in FMD virus infection i expected. FMD virus has shown good to interferon in cells from all species te including mice (18) and swine (17). potential inducer of interferon used t against FMD virus was yeast RNA. A per kg per day in multiple injections, i symptoms in guinea pigs (3), and a I effect was also evidenced in mice and c; Recently, microgram amounts of the stranded synthetic RNA, poly I :C, were protect mice against FMD virus infectic Surprisingly, swine did not react tc given in amounts up to 60 times greater effective dose in mice. COAM, 120 mg, 18 hr and 1 week prior to natural exposure to pigs injected with FMD virus, did not delay or attenuate the symptoms of disease. Polyacrylic acid, a plastic polymer less likely than COAM to undergo breakdown in vivo (2), also lacked activity when given under comparable conditions. A major difference between the experiments in swine and in mice was that swine received the compound intraperitoneally and virus by contact, EA whereas mice were given the compound and the virus successively in their peritoneal cavity. In the latter situation, interference by residual COAM or polyacrylic acid with viral adsorption or penetration or direct interaction with the virus might have occurred. This phenomenon was 7 8 demonstrated in vitro with other viruses, and it may account for part of the in vivo antiviral effect k ntra- (2,9). The virus dosage effect observed (Fig. 1) is ngan.st compatible with such an explanation. However, . treated polycarboxylates also protect mice when given gearance of by another route than the route of virus advwine; -----ministration (2,14), although less effectively than when the compound and the virus are both given intraperitoneally. The experiments with guinea pigs prove that direct imanimals may be protected by the intraperitoneal route against FMD virus introduced by a parenteral route. Unlike treated swine infected by contact, treated guinea pigs infected by way of the cted mice foot pad showed protection against the developrirus. The ment of FMD lesions. g/kg, was The lack of detectable interferon after COAM ,ctive dose administration in swine could be related to its (2). The failure to modify FMD in this animal. However, )unds and polycarboxylates impart antiviral protection in other viral many situations in which circulating interferon is -fore have not found after administration. Only the higher MD virus doses induce demonstrable interferon, whereas e to FMD long-term protection is also given by the lower Lys of age doses. The smallest interferon-inducing dose of protective polyacrylic acid in mice is 20 mg/kg (10). Mice are protected against vaccinia lesions with 2 mg/ ng polykg (6). Among antivirally active chlorite-oxidized is not unoxypolysaccharides, the chemical group to which sensitivity COAM belongs, only three out of seven induced -sted, also demonstrable interferon (4). With polyacrylic The first acid, we were unable to demonstrate interferon o protect production in rabbits and in rats. Vaccinia lesions kt 350 mg were inhibited in rabbits, and polyoma virus init delayed duced fewer tumors in rats after treatment with protective the polymer (8). Sellers (personal communication) attle (19). found an antiviral effect of pyran copolymer in double-FMD virus infection in guinea pigs, but serum found to taken after pyran administration did not protect )n (18). guinea pig cells. The absence of detectable inter-COAM feron, therefore, has limited value in explaining r than the the lack of anti-FMD effect of polycarboxylates /kg given in swine. In late general unresponsiveness of the interferon system. As has been shown, the interferon system is induced in swine cells by double-stranded RNA, and swine leukocytes produce interferon when exposed to phytohemagglutinin, a weak inducer in other species (17). FMD virus is sensitive to swine interferon in homologous cells (17). The experiment with hog cholera indicates that COAM exerts protective activity in swine in certain viral infections. In each of the treated animals, temperature rise, diarrhea, and skin lesions were delayed when compared to untreated swine. The effect was limited, and there was no survival of significant duration. The effect of polycarboxylates on FMD virus infection of mice and guinea pigs and their lack of effect in swine are exemplary of the hazard of extrapolating observations obtained with experimental infection of unnatural hosts to naturally occurring disease. To our knowledge, this is the first example of activity of interferon inducers in the experimental host and lack of activity in a natural host.

v3-fos

2017-07-29T04:24:53.860Z

1971-01-15T00:00:00.000Z

15055703

s2

The effect of size and type upon the efficiency of milk and beef production in cattle Partly on the basis of their investigation and of synthetising data of the professional literature the authors have analyzed the correlation and effects of the body size and of some other grading characters of cattle in relation to the production of products by whole populations. According to their examinations the efficiency of size of the individual production is not always characteristic for the trend in the productivity of the total population. Thus e.g. the selection aiming at a larger daily gain, which is advantageous for the feed conversion of the individual, may lead to the development of a cow stock of a large body weight, which may decrease the quantity of beef (calfs) and of milk which can be produced on the identic quantity of nutrients. Examining the relation of body size and milk-yield in populations producing under the same environmental conditions -in contradiction with most earlier examinations&mdash; within the breed there is no conspicuous correlation between the two characters according to the more recent results of researches. Similarly a substantial reciprocity -within the breed&mdash; between the body size of parents and beef-production of their progenies cannot be demonstrated either. The probable cause of this is that within the breed the different live weights are not separated genetically. On the ground of all these over a certain limit no meritorious and rational increase of beef&mdash;, nor the milk production can be expected from the increase of body size. in relation to beef-production. The beef production of cattle and particularly its efficiency is a function of a very large number of characters. The selection aiming at the improvement of grading characters typical for the individual (daily growth rate, feed convention, carcase percentage, beef-conformation, beefquality, etc.) do not enhance unequivocally the increase of the beef producing capacity of the total population. The latter is determined by the maintenable cow number and fertility indices in the first place. The selection at milk-production can be with great difficulties brought in harmony practically with the simultaneous improvement of the beef production, moreover the increase in the milk yield per cow in case of dual-purpose breeds does not allow alone in itself that milk&mdash; and beef production structures corresponding to the market demands be formed. The breeding method suggested by the authors seems useful for a simultaneous rational increase of beef production relative to milk. The main point of this is the performance of utility cross-breeding with early maturing beefbreed bulls in sexually early maturing cow populations of the dairy type. The born bulls are marketed after fattening, while heifers of beef breed paternity are fertilized at young age -similarly with beef bulls&mdash; and after one month following the calving is marketed for slaughter as a young cow. After the beginning of full rotation -with the calving of the beef-purpose heifers&mdash; the live weight of the produced young beef animal marketable after one dairy cow grows with 4 q per cent, against an additionnal 14 per cent starch-equivalent and concomittently improves also the quality of the beef considerably. If this starch equivalent surplus would be allotted to beef-cattle keeping, the beef output would lag considerably behind that level which can be attained by the current calving of beef-purpose heifers. Beyond this in case of special given conditions and demands, the keeping of beef-cattle may however likewise come into the foreground. In the future the more extensive use of specialised types (milk-and beef), the faster breedand type-change and the more wide-spread employment of combinative cross-breeding will have to be taken into consideration more extensively also in cattle breeding. Efficiency of production in cattle-breeding is essentially more intricate than it is in general presumed. This is due mainly to the fact that efficiency and size of the individual production are not always the same as the trend in the productivity of the total population. This became evident in poultry raising and led e.g. to the necessity of separating -in broiler production&mdash; the male line from the female line. The first was put into the service of increasing the individual meat productivity, while the second line served the purpose of economic reproduction, utilizing at the same time the appropriate heterosis effect. The diverging interest between the integrated and specialized poultry breeders has a similar background. In cattle breeding, particularly the milk and beef production performed with the same population raises similarly a large number of unelucidated problems. Obviously this resulted in the divergences of opinions being very considerable, both in the question of type and body weight and as regards the direction of optimum utilisation, even in the so-called specialized breeding aimes (specialised beef-and dairy breeds). This situation is complicated by the need that the economically producing cow must have not only a considerable degree of efficiency in order to convert the feed into butterfat and milkprotein and to produce economically calves corresponding to the purposes of fattening but must likewise stand the test in the up-to-date mechanised establishments. The rentability of milk-production from the point of view of the cow is relatively well reflected by the quantity of milk produced, concentration of milk (W I TT, 6 3 ; HORN, 29 ; B IR 6, 4 ; IVICDANI!L, 49 ; H OFMANN , 2 8; etc.), by the milk-yield related to live weight, which seems the safest index of food conversion for the practice (SucxAN!K, 56; D OHY and L UDROWSYY , 17 , etc.) and even beyond this by the milk-production per one day of life related to the live weight, which is perhaps the most pregnant indicator for the efficiency of milk-production (D O II Y , 1 6). According to results of earlier researches (K RIZ E N EC ZKY , 3 8; G OW E N , 2 6; T UR -N E R , 5g; I,AUrR!cIIT and Do RIN G, 41 ) the conception became wide-spread that 100 kg increase of the cow live weight involves an about 300 to 6 00 kg additional milkproduction. This finding was subjected to revision by a few research workers (JoIIANSSO N , 37 ; W II ,x, YOUNG and COLE, 6 2 ). From more recent examinations in which the authors studied the correlation between live weight and milk yield, in populations of large numbers, producing under the same environmental conditions (e.g. CLARK and TouCHBERRY, I 2; Boz6 and DUNA Y , 7; VASILJEV and STARTZEV, 6I; V AN V!ECK, 60; M ONOENOKV and V SJAKIH , 5 I ; B R E I TE NS TE IN and F I E DL E R , 9 ; CsOmOs, I q.; $r,sAI!D, 23 ) the conclusion can be drawn, that a considerable increase in the milk-yield of cows cannot be hoped -for from an increase of live weight. It seems furthermore as if the concentrated milk-producing types would -related particularly to a live weight unit&mdash; easier produce economically. The outstanding relative productions, which are attained by concentrated milk producing breeds and individuals, specially related to the live weight unit, are pointing to this fact. In this connection I may point to the Danish Jersey, Finnish Ayrshire breeds, as also to the cow stocks of New-Zealand, etc. (Table I). The hungarian results seem to confirm similarly that types producing a more concentrated milk -specially under conditions of large-scale farming&mdash; can easier and with more security hold their leading position, than breeds or crossbreed populations producing more but less concentrated milk. While indices of the efficiency of milk production are relatively well clarified, those referring to beef production or to a combination of the two are far more contradictory. This applies particularly to the integrated beef producing capacity of one population. The cause of this is that while in some phases of fattening good many indices or parameters have a positive effect, with regard to the total beef production of the population, the effect is negative. Already during the Congress at Edinbourgh HORN ( 31 ) has presented a report on examinations concerning the milkand beef production produced by Hungarian Fl a ckvieh cows belonging to different weight categories, from the same nutrient quantity. The data have clearly shown already at that time that with growing cow live weight the total beef production of cow categories from 5 50 to 8 50 kg comes -just contrary to expectations&mdash; into a negative phase, particularly in relation of the more valuable young beef. On the ground of these and other examinations it appears that it is more than probable, that the most economically producing cattle type may be fundamentally different from that which is qualified the best by the traditional conception. In connection herewith it is particularly interesting to emphasize the daily growth-rate as one of the most important individual parameters of beef-production. Thus, e.g. a breed selection aiming at a large daily gain in the interest of beef production is undoubtedly advantageous from the point of view of food conversion during fattening. At the same time however such a breeding aim -specially when fattening aims at a large final weight&mdash; may lead in general to heavier types which mature later. The general opinion keeps a certain positive correlation in evidence between the live weight of parents and the weight gains of progenies. Parameters characteristic for some breeds point also into this direction, as well as results obtained during the different crossbreeding experiments (HORN, DurraY, Boz6, 35 ; S ZUR O MI , 5 8). Though this correlation -concerning its tendency&mdash; is reliable by all means, still a reciprocity within breeds between live weight of the parents and weight gain of their progenies could not be established: B AR -A NAN and L EVI ( 2 ), B RINKS et al. ( 10 ), D UNAY ( 20 ), S ZUROMI (5 7 ), Max!ov! (q. 7 ). This is probably due to the weight-categories not being sepa y ated genetically within the different breeds. The growing body weight related to a given feed growing area or to the same feed quantity, diminishes the number of produceable calves and contrary to the expectation reduces the beef-producing capacity related to the whole population. The loss resulting herefrom may be occasionally larger than the profit which can derive from the utilization of cows of larger live weight so Nuch the more as according to our today's knowledges the most tMt!o!M!'MM'!'Mg'/ac! of beef production is the numbe y of calves which can be produced on the feed-unit (Fig. I ) . Considerations of those shortly outlined above led to the set of experiments which we have conducted already hitherto and are going to perform also further on with the view to find out how during the utilization of populations of different body weight and type, the relation and profitability of butterfat, milk protein and beef production develop on the same quantity of feed. A very intricate mass of facts must be synthetized, before certain grades could be assigned to the different populations. At the same time we must share the opinion of those C OLE et al., I3 ), according to whom for instance neither in the field of beef-production do exist unequivocally ideal breeds. We would like to point out a few contradictions in this connection. The capacity of a high daily gain which is a fundamental character of economic beef-production, is in many cases manifested more superiorly in dairy breeds of a larger body (e.g. U.S.A. or Canadian Holsteins), than is specialised beef-breeds (CoI,! et al., I3 ; L iNDH t, 4 6). In the same way the early maturity which in the earlier period of fattening usually result in a more favourable weight-gain and simultaneously in a faster rotation of the stock, impairs the rentability of beef production in case of fattening to a heavier weight. Neither the fattening to a heavier weight is a practicable road of rendering the beef production profitable, since almost twice as much feed is required per live weight unit, when fattening goes-on to 500 -6 00 kg weight than quantity needed for fattening to 200 -30 o kg weight (N A G Y , 5 2 ; I,E NSC xow; ST UNZ , OTTO, 43, etc.). The carcass weight percentage is likewise regarded as an important beefproduction index. At the same time less developed respiratory and digestive organs are associated with a favourable carcass percentage. Therefore from the point of view of carcass weight !eycentage, exactly those types are !ushed into the background which have the highest feed-uptake capacity and may be the best converting animals mainly in relation of milk-but also of beef production. This statement emphasizes similarly the necessity of a certain revision of the blocky and short, classical beef-cattle type, J OBST ( 3 6). We do not wish either to deal here with the beef quality in more detail, we point only to the heavier weight being in general accompanied with coarser fibres. Thus it could occur that in different taste and qualification tests (FLOCK, zq.; E DGAR , 21 , etc.) the beef of precisely those breeds proved the best which fro.m fattening and meat industrial points of view, obtain the lowest grading in general. Quality of the beef is a function far more of feeding, degree of fatness, age and state prior to slaughterage of the animal, then of the breed. Considerable importance is attributed also to the beef-conformation, which are decisive particularly in the selection of sires, of dual-purpose breeds, though beef-conformation is in no close correlation with either the daily growth-rate or with some other characters (B A I, IK A, I; DU M O N T, I(!; HARING, 27 ; I,A N GI,ET, GRAV!RT, Ros!NIIAxN, 40 ). At the same time the beef-conformation judged as favourable and the increasing width-measures may lead to the tainted inheritance of calving difficulties. The shortly outlined characters determine jointly the beef-producing capacity of the individuals and their value for the meat industry. On the other hand in relation to whole cattle populations the beef-producing capacity of a stock is determined by the question: with which stock more milk and beef can be produced from the same nutrient quantity? From such point of view several kinds of possibilities are offering themselves, commencing from the classical dualpurpose breeds to the specialized breeds, in some cases with the inclusion of utility-crossing. In order to illustrate the effect of individual grading characters, displayed on production related to population, let us present five cattle populations, having different individual grading characters used in our evaluations (Tabl. 2 ). In our days one of the most serious problems in cattle breeding of Europe is undoubtedly to create a differencial balance of milk-and beef production for the good of beef. This has biological reasons in part, but national economy causes in the first place. The presently still almost generally prevailing breeding tendency sees the breeding ideal in dual-purpose breeds which after certain possibilities are made use off leads lawfully to the f ormation of f milk sur!luses and to beef sho y tage. This may be traced back to a fundamental rentability interest of the breeder being linked with the increase of milk-yield per cow. Having in view that the uptake capacity of the milk market is limited it is comprehensible that an increase of milk-yield per cow is associated with the reduction of the cow-numbers. On the other hand this has grave consequences, since the number of calves is a limiting factor which basically is a function of cow number and fertility (HORN, 32 ; C ZAK 6, 15 ; K R6GER , 39 ; B ED 6, 3 ). This in part turns the attention increasingly to the development of beefcattle keeping (S CHNI TTE N , 5 4 ) and raises on the other hand the necessity of breeding methods such as commercial crossing, possibilities of inducing twin-calvings (E DW A RD S, 22 ; C ARM A N , II ) which previously have been barely dealt with by researchers. In our experiments, where we performed commercial cross-breeding with He y e f o y d and Charolais bulls in a &dquo; dairy Hunga y ian brown &dquo; population, of 50 per cent Je y sey generatio, -beyond the considerable improvement of beefconformation and marketability&mdash; an about 6 percent surplus appeared also in beef-production (HORN, D UNAY , Boz6, 35 ), which though regarded in itself, is important, still does not yet solve the formation of such a ratio of milk-and beef-production, which corresponds to the market demands. To increase the beef production (Boz6, D UANY , DE AK , 8) in dairy-type stocks commercial crossbreeding is performed with Hereford bulls, extend to all heifers and to 30 per cent of the cow-stock. The born heifer of beef-bull paternity is fertilized at young ( 14 -15 months old) age with a He y eford bull and after claving (in about 24 -25 months of age) followed by one month fattening, the once calved young cow is marketed for slaughterage. According to calculations, related to 100 cows, with this method, the output of valuable young beef can be increased with about 44 per cent -against about 14 per cent surplus of starch equivalent, compared to the result when we would not have carried out in the stock the commercial cross-breeding and would not have made the heifers of beef-bull paternity to re-calf again (Table 3 ). This method -to which the results published by M!scAr, (5 0 ) and Ros-T OVZ E V (5 3 ) are the nearest&mdash; enables us to rationally increase the calf-number produceable by the population and through this the beef production per one cow of the population. The question may be raised in connection herewith, wether it would not be more purposeful to manage one-purpose beef cattle on the surplus feed required for the re-calving of beef-purpose heifers? According to our calculations, illustrated on figure 2 , the young fattening-animal-live-weight production of the population is less than the beef production realizable in the case of re-calving of the meat-purpose heifers. It should be noted however 'that Ithe beef-increase produceable by means of re-calving the beef-purpose heifers cannot exceed the already mentioned 40 to 5 0 percent, therefore for a satisfaction of a higher claim than this the management of beef-cattle seems justified, provided adequate conditions are given. This methods provides possibilities of an increased beef production without augmenting the number of stalls in the dairy farm, which require heavy investments, so that through this the rentability of the production of basic materials for fattening can be largely improved. It is advisable to follow this breeding procedure in the firts place with using early maturing populations. This sexual F IG . 2 . -Young beef producing ability % of cow populations of equal milk production by using different breeding methods (A population = roo %). early maturity is necessary also for the reason because -in addition to the speeding-up of rotations&mdash; only thus can be attained that development, growth of the heifer and the building-up of pregnancy should fall into one period and the building-up of the foetus, from the point of view of weight gain, should not come into an inactive stage. Thorough further investigations are required to allow the establishment of that type of cattle which according to special given conditions, is the most productive. It should be carefully considered that under certain conditions the female line is to be separated from the male line, not only in the dairy stocks but within beef cattle as well. In this way the production linked to the female sex (milk yield, calf production, early maturity) may take place with favourable conversion, while the characters connected with fattening (weight-gain, slaughter value) may be ensured through the male lines. It would be advisable to liberate the minds of prejudice and revise our breed-targets form time to time. It may be that in spite of the slow generation interval we must prepare ourselves in cattle breeding to a faster type&mdash; and breed change, in the same way which is already common in plant production but is an every increasingly spread phenomenon in other branches of animal production as well. During this course we shall have to make use also of those gene reserves which have been disclosed somewhere in the world. Jointly with an intensive purebreeding at a high level, this process will push the combinative crossbreeding likewise into the foreground. Reçu pour publication en di c embre z g 7 o. RÉSUMÉ L'INFLUENCE DE LA TAILLE ET DU TYPE SUR LE RENDEMENT EN LAIT ET EN VIANDE CHEZ LES BOVINS A partir de leurs propres recherches et de la synthèse des résultats de la littérature spécialisée, les auteurs ont analysé l'incidence de la taille corporelle et d'autres critères de jugement individuel des bovins sur la production totale au niveau de la population. Il est possible, sans grandes difficultés, de sélectionner en vue de la production laitière et d'améliorer en même temps la production de viande et, d'autre part ce n'est pas à partir de la seule augmentation du rendement laitier individuel dans les races mixtes qu'on pourra ajuster les productions de lait et de viande aux besoins du marché.

v3-fos

2020-12-10T09:04:10.761Z

1971-10-01T00:00:00.000Z

237229143

s2

Effect of Controlled Atmosphere on Growth of Mold on Synthetic Media and Fruit Growth of seven spoilage molds on agar plates at several temperatures in both controlled atmosphere (CA) and in air was studied. Each mold responded somewhat differently to CA at each temperature; however, there were some general tendencies. The lag phase was generally increased by CA and, in some cases, was substantially extended when incubation was just above the minimum growth temperature. The mycelial structure of molds seems to be different when grown in CA than when grown in air. With only two exceptions of 24 holding conditions, the maximum amount of mycelia was always less in CA than in air. Spore development varied with each mold at each temperature; generally, it was considerably less in CA than in air. CA storage of cherries above 34 F (1 C) did not retard mold infection to any extent; at 34 F, mold growth was inhibited and storage life was extended several days as compared to air storage. CA storage of strawberries at 34 F resulted in a mold-free product after 7 days of incubation, whereas the air-stored berries were slightly infected. However, when mishandled berries showing some mold growth were stored at 34 F, CA did not stop further mold growth. Controlled atmosphere (CA) is defined as an environment with elevated carbon dioxide and decreased oxygen levels as compared with air. It has been used in conjunction with refrigeration to extend the storage life of certain fresh food products (6). CA gas composition may be adjusted by several means. The earliest method took advantage of the natural respiration of the commodity in storage (3). However, several weeks were generally required to develop the desired atmosphere. Faster methods of obtaining the desired CA composition involved the use of dry ice or compressed CO2. In recent years, CA generators (Whirlpool Corp., St. Joseph, Mich.) have been developed which burn natural gas and instantly deliver a CA of the desired composition. The extension of storage life of food held in CA is partially attributed to inhibition of the microbial flora (2). There have been a number of studies on growth of bacteria and fungi in CA (6), but few studies have been made on mold growth in CA developed by generators (1, 5; C. W. Hastings, M.S. Thesis, Univ. of Illinois, Urbana, 1967). The purpose of this research was to compare the growth of selected spoilage molds in CA with that in air on a synthetic medium and on cherries and strawberries at several temperatures. MATERIALS AND The synthetic agar medium was of the following composition: dextrose (anhydrous), 4.0 g; peptone (Difco), 1.0 g; agar (Difco), 1.5 g; water, 100 ml. Agar plates were inoculated by dispersing mold from a stock culture in 5 ml of sterile 0.01% Triton X-100 solution. Then 0.1 ml of inoculum was spread over the poured agar surface with a bent glass rod. Each inoculated plate was placed, uncovered, within an individual plastic box to prevent cross-contamination. The plastic box, 4.25 inches (10.8 cm) by 4.25 inches by 1.5 inches (3.8 cm), was covered and sealed except for inlet and outlet ports (19/64 inches in diameter) for gas flow at opposite ends. The boxes were placed in refrigerated incubators and connected to a gas manifold by vinyl tubing. The gas, either CA or air, was pumped in order through a cotton sterilizing filter, a copper tube for cooling to incubation temperature, and a water trap to remove condensed water from the saturated (100% relative humidity) atmosphere. Air had been humidified before the pump, and CA was obtained with a high relative humidity. The gas was then forced through another cotton filter before passing through the manifold to each plastic box. Outlet gas was led outside the incubator via another manifold. Mold growth measurements. The time required for appearance of definite growth was noted. This growth was considered to be aerial mycelia rather than thin, thread-like mycelia on the surface. Storage was terminated when there seemed to be no apparent increase or change in the mold. The number of spores on the plate at termination was determined by plate count. The entire mycelial mat, including the agar, was blended with 100 ml of 0.01% Triton X-100 solution in a 300-ml cup (Omni-Mixer, Sorvall, Inc., Norwalk, Conn.). Dilutions were pour-plated on the synthetic medium described above and incubated at 75 to 80 F (24 to 27 C). The counts were expressed as the number of spores per plate. This expression assumed that outgrowth of mycelial fragments would not make a significant increase in the number of colonies. For those molds (Mucor, Rhizopus, Penicillimn and Aspergillus) whose spores could be counted directly with an improved Neubauer AO Spencer Bright Line counting chamber, this was shown to be true at the 95% confidence level. The dry weight of the mycelia was determined from the homogenate used to make the spore count. All of the homogenate, except for the 0.5% that had been removed for plating, was melted in boiling water and vacuum-filtered through a tared Whatman no. 1 ifiter paper on a Buchner funnel. After rinsing with hot water, the paper and mycelia were air-dried overnight at 150 F (66 C) before weighing. Fruit storage. Bing cherries of good quality were purchased from local stores. The cherries were placed in sealed boxes (same as used for the plates) and continuously flushed with CA or air. The boxes were held at 60, 45, and 34 F (16, 7, and 1 C). They were examined for degree of infection and breakdown. Strawberries were shipped directly from California. The berries were ripe and quite soft upon arrival, with many exhibiting gray mold growth. The poorest berries were discarded. As one treatment, the strawberries were placed in plastic boxes for storage at 60, 40 (4 C), and 34 F and continuously flushed with CA or air. As .a second treatment, the berries were held overnight at warm room temperature so that the mold growth was barely evident before the experiment was started. Then the experiment was performed as the first treatment, except that storage temperatures were 40 and 34 F. Replication. In every experiment, there were two plastic boxes of each inoculated plate or fruit at each condition. All synthetic media experiments were duplicated and some were replicated as many as four times. The fruit experiments represent one trial. RESULTS AND DISCUSSION Pure culture study. The time at which definite growth occurred and the extent of final growth as indicated by spore count per plate and total mycelial weight are given for each mold at several temperatures in Table 1. P. expansum. As shown in Fig. 1, CA holding caused a lag in the appearance of mold growth as compared with air storage. This lag period increased with decreasing storage temperature. Spore counts at termination of growth were (Fig. 2). However, as storage temperatures approached 34 F, the number of spores that developed in CA was about one-twentieth that found in air. Mycelial development (Table 1) showed little difference between air and CA over the temperature range of 70 to 45 F. However, growth at 34 F in CA was two-thirds that in air, and the submerged mycelia at 34 F in CA had an intense orange coloration. A. niger. CA holding of A. niger caused a delay in initial appearance of growth by 2.5 days over the temperature range of 70 to 55 F (13 C; Table 1). The mold did not grow in either air or CA at 50 F (10 C). The spore count at termination of growth in air at 70 F was nearly 1,000 times more than in CA, but at 55 F the spore counts were equal. Mycelial weights in air and CA were equal at 70 F, but at 60 and 55 F growth in air was twice as great as in CA. M. hiemalis. The time for initial appearance of growth at 70 F was only 1 day in both CA and air ( Table 1). The times were also equal at 60 F. However, at 45 and 34 F, it took about 1 to 2 days longer in CA. Spore counts were similar in air and CA from 70 to 45 F, but at 34 F there appeared to be a greater count in the CA sample Mycelial weight was greater from 70 to 45 F in air than in CA (Table 1). However, at 34 F there was better growth in CA than in air. Microscopic examination showed that the aerial mycelia in CA were deformed, very thick, and highly branched as compared with those in air storage. R. oryzae. The increase in time for initial appearance of growth resulting from CA holding was about 1 day in the temperature range 70 to 55 F ( Table 1). The mold did not grow at 50 F. At storage temperatures of 70 and 60 F, there were about 1,000 times more spores produced in air than in CA. At 55 F, which was near the minimum growth temperature, there was a greater development of spores in CA than air. The dry mycelial weight was always a little greater in air than in CA at the several storage temperatures (Table 1). Microscopic examination showed that the mycelia produced in CA were thicker and rougher, more deformed, and more highly branched than those in air. Many "immature" sporangia were noted in the CA samples. C. herbarum. This mold showed high sensitivity to CA storage. Growth in air gave a dense mycelial mat with typical dark green color. Growth in CA was poor with only slight green color. Initial appearance of growth was delayed in CA, and the delay was increased with decrease in storage temperature (Table 1). At 45 F, the lag in CA was at least 6 days, and, in several replications, the mold did not grow at all at this condition. At termination of holding at 50 and 45 F, there were about 50 times more spores and 10 times more mycelia in air than in CA. Alternaria sp. CA holding caused a delay in the initial appearance of growth, and the delay increased with decrease in storage temperature. After 50 days of incubation, the mold had not grown in CA storage at 34 F. At 70 F, there was little difference in spore count between air and CA storage. At temperatures below 70 F, there was about 10 times more spore development in air than in CA. Mycelial weight development at 70 F was nearly equal for air and CA storage. At 45 F, mycelial development in air was about 10 times greater than in CA, and, at 34 F, the mold did not grow in CA (Table 1). F. bulbigenum. CA holding caused a delay in appearance of growth by several days compared with that caused by air storage. Spore counts were equal for air and CA samples at 60, 50, and 45 F ( Table 1). The mold did not grow at 34 F in either air or CA, even with extended incubation time. Mycelial weight determination showed that slightly greater development occurred in air than in CA. Fruit storage. Cherries held in air at 60 F showed mold within 3 days, but the CA samples were still mold-free; after 5 days, several molds as well as yeasts were growing on the CA-stored cherries. After 12 days at 45 F, the air-stored cherries had a musty off-odor and several cherries were slightly infected with Penicillium and yeast; however, in CA there was little infection and the cherries were of firmer body, better odor, and brighter color. After 3 weeks at 34 F, storage in air resulted in some mold development but in CA there was none. Although CA cherries showed some bacterial and yeast growth, they had better color and odor than the air-stored cherries. In treatment 1, strawberries at 60 F were highly infected within 1.5 days in either air or CA storage. At 40 F, the air-stored berries were visually infected by gray mold within 4 days and the CA berries within 5 days. After 7 days of storage at 34 F, berries in air were infected to a slight degree, whereas the CA-stored berries were free from mold, of brighter red color, and had greener caps. In treatment 2, the objective was to observe the degree to which CA could stop or inhibit growth after it had been initiated. At 40 F, mold continued to grow well for about 1.5 days and then the growth rate decreased in both air and CA. After 7 days at 40 F, the berries in both atmospheres were spoiled but the CA samples showed less mold growth. After 7 days at 34 F in both air and CA, mold growth was greatly reduced as compared with storage at 40 F but neither sample was acceptable. This study shows that the effect of CA on growth and development of seven spoilage molds is quite variable and, for the most part, is temperature-related. Of most interest in this work is the interaction effect of CA and storage temperature. The effectiveness of the CA was usually increased as storage temperature was reduced. Thus, for control of mold growth, it is evident that CA should generally be used with the lowest storage temperature that would be acceptable for the specific product.

v3-fos

2020-12-10T09:04:11.848Z

1971-01-01T00:00:00.000Z

237232718

s2

Lipoprotein Inhibitor of Newcastle Disease Virus from Chicken Lung A lipoprotein inhibitor of Newcastle disease virus was obtained from chicken lung tissue by means of dilute alkaline extraction procedures. The inhibitor was further purified by ammonium sulfate fractionation, isoelectric precipitation, and density gradient centrifugation. The purified lipoprotein inhibited active Newcastle disease virus hemagglutination at a concentration of 2.0 μg/ml which represented a 30-fold purification over the original extract. Infection of chicken embryo fibroblasts by Newcastle disease virus was also inhibited by the purified lipoprotein, the degree of inhibition depending upon the inhibitor-to-virus ratio. Chemical analysis of the purified inhibitor provided a composition of 72% lipid, 26% protein, and 3% carbohydrate, although some compositional variation was observed from one preparation to another. The chloroform-soluble lipids were shown to contain 40 to 50% phospholipid and 10 to 20% cholesterol; of the fatty acids recovered from the saponified lipoprotein, 39% was palmitic, 22% oleic, and 17% stearic. Careful analyses of large quantities of the inhibitor revealed a small (0.84%) but significant content of sialic acid. Removal of sialic acid from the lipoprotein by means of digestion with neuraminidase produced a sharp diminution in inhibitory properties. A delipidized form of the inhibitor was obtained by ether extraction, and this material produced a single broad band of precipitate in gel immunodiffusion tests. A lipoprotein inhibitor of Newcastle disease virus was obtained from chicken lung tissue by means of dilute alkaline extraction procedures. The inhibitor was further purified by ammonium sulfate fractionation, isoelectric precipitation, and density gradient centrifugation. The purified lipoprotein inhibited active Newcastle disease virus hemagglutination at a concentration of 2.0 ,ug/ml which represented a 30-fold purification over the original extract. Infection of chicken embryo fibroblasts by Newcastle disease virus was also inhibited by the purified lipoprotein, the degree of inhibition depending upon the inhibitor-to-virus ratio. Chemical analysis of the purified inhibitor provided a composition of 72% lipid, 26% protein, and 3% carbohydrate, although some compositional variation was observed from one preparation to another. The chloroform-soluble lipids were shown to contain 40 to 50% phospholipid and 10 to 20% cholesterol; of the fatty acids recovered from the saponified lipoprotein, 39% was palmitic, 22% oleic, and 17% stearic. Careful analyses of large quantities of the inhibitor revealed a small (0.84%) but significant content of sialic acid. Removal of sialic acid from the lipoprotein by means of digestion with neuraminidase produced a sharp diminution in inhibitory properties. A delipidized form of the inhibitor was obtained by ether extraction, and this material produced a single broad band of precipitate in gel immunodiffusion tests. The intensive past studies on the inhibitors of myxovirus hemagglutination and infectivity have focused primarily on those inhibitors present in sera (13,17,19). Among the most active of these inhibitors are those glycoproteins classified as Francis or alpha inhibitors, the activity of which is associated with the sialic acid portion of the molecule. Removal of the sialic acid with neuraminidase causes a loss of inhibitor activity. The beta and gamma class inhibitors of serum are resistant to neuraminidase and have generally been less well characterized than those of the Francis type. In contrast to the ambitious investigations of myxovirus inhibitors on serum, tissue inhibitors, excepting those present on erythrocytes, have been largely ignored. Hirst (15) was the first to demonstrate that perfused ferret lung would first adsorb and then release influenza virus. A similar behavior of mouse, hamster, rat, guinea pig, rabbit, and chicken lung toward human and swine influenza virus, Newcastle disease virus, and mumps virus (34) has been described. It was an interesting observation that chicken lung did not adsorb Newcastle disease virus as well as the other influenza viruses and mumps virus. Investigation of the myxovirus inhibitor in mouse lung has indicated that it is easily extracted by low molarity bicarbonate solutions (4,23). No detailed chemical characterization of this solubilized inhibitor has yet been attempted; however, it was partially purified by differential centrifugation and Sephadex G-200 filtration, indicating its macromolecular nature. It was resistant to trypsin at 65 C for 30 min; however, it was sensitive to periodate and receptor-destroying enzyme, suggesting that it is a Francis-type inhibitor. Laucikova (20) has described a lipoprotein inhibitor for influenza virus which she extracted from chicken embryo chorioallantoic membranes. The chemical composition of the inhibitor varied somewhat from one preparation to another but was approximately 55% protein, 18% lipid, and 2% carbohydrate. Extraction of the lipoprotein with ether destroyed the inhibitor, indicating its dependence upon lipid for its antiviral activity. Periodate treatment, exposure to receptordestroying enzyme, or active virus also destroyed the activity of the inhibitor (21), supporting its classification as a sialic acid-dependent, Francistype inhibitor. The extraction of ox brain with a mixture of chloroform and methanol liberated an inhibitor of influenza virus hemagglutination (31). It was described as a mucolipid consisting of as much as 20% sialic acid but no evidence was presented concerning the relation of the sialic acid to antiviral activity nor was the inhibitor described further in chemical terms. The research reported here was initiated to determine whether inhibitors of Newcastle disease virus (NDV) infection and hemmagglutination were present in chicken lungs. Since the lung is a primary tissue invaded by this virus, it was felt that the isolation of an inhibitor might be, simultaneously, an isolation of a natural receptor site for NDV. A unique lipoprotein inhibitor has been partially purified from chicken lungs and characterized in chemical terms. Its antiviral activity, as an inhibitor of viral hemagglutination and infectivity, has also been described. MATERIALS AND METHODS Unless stated to the contrary, all procedures were performed at 4 C. Purification of lipoprotein inhibitor. Approximately 25 g of minced, washed chicken lungs were homogenized in 100 ml of 0.001 M NaHCOs adjusted to pH 7.5 (26) with the aid of a Sorvall Omnimixer. After 10 min, this homogenate was diluted by the addition of 500 ml of cold bicarbonate solution. The diluted homogenate was clarified by centrifugation at 12,100 X g for 10 min. These conditions were employed for all future clarifications and for the collection of (NH4)2SO4 and isoelectric precipitates. The clarified bicarbonate extract was adjusted to 20% saturation by the addition of solid, reagent-grade (NH4)2SO4, held for 24 hr and clarified by centrifugation. The supematant was adjusted to 50% (NH4)2S04 saturation by the addition of more ammonium sulfate and again held for 24 hr before being clarified by centrifugation. The precipitate was dissolved in 0.15 M phosphate-buffered saline (pH 7.2) which was 0.01 M with respect to ethylenediaminetetraacetic acid (EDTA). This solution was dialyzed against distilled water to remove the ammonium sulfate. The dialyzed 20 to 50% ammonium sulfate fraction was adjusted to pH 5.0 by the dropwise addition of 1.0 M CH3COOH and held for 24 hr. The precipitate was collected by centrifugation and dissolved in 0.15 M phosphate-buffered saline containing 0.1 M EDTA (pH 7.5). The redissolved isoelectric precipitate was placed on top of a 20 to 50% sucrose gradient prepared at room temperature with the aid of a Beckman density gradient former. The sucrose solution was prepared in 0.15 M phosphate-buffered saline (pH 7.5) containing 0.1 M EDTA. The gradient was subjected to 90,000 X g for 90 min in the SW25 head of a Spinco model L preparative ultracentrifuge. The top band (1.130 density) was collected, diluted by the addition of an equal amount of the buffer solution, and recentrifuged on another 20 to 50% sucrose gradient. The single band was collected and dialyzed against distilled water before chemical analysis. It was dialyzed against 0.15 M phosphate-buffered saline (pH 7.2) before hemagglutination inhibition (HAl) and plaque inhibition tests. Chemical analyses. Protein determinations were performed by the method of Lowry et al. (24). Total hexose was determined by the method of Scott and Melvin (33) adapted to smaller volumes. A slight modification of the Elson and Morgan procedure (8) was used to quantitate hexosamine, and the thiobarbituric acid and diphenylamine methods of Warren (35) and Saifer and Siegel (32) were applied to the determination of sialic acid. The sum of hexose, hexosamine, and sialic acid is reported as total carbohydrate. Total lipid was measured by the gravimetric procedure of Folch et al. (11). Twenty milliliters of chloroform-methanol (2:1) at room temperature was added to 20 mg of lyophilized inhibitor which was extracted for 1 hr with magnetic stirring before being filtered upon sintered glass. The insolubles on the glass filter were extracted with an additional 20 ml of solvent. Distilled water was added to the filtrate in a biuret until the supernatant aqueous phase cleared. The lower chloroform phase was collected and dried completely under vacuum at room temperature before being weighed. Cholesterol was determined by the procedure of Zlatkis (37), and phosphorous, in the chloroform-methanol extract, was measured by Bartlett's method (3). Phosphorus was converted to phospholipid by assuming it represented 25% of the phospholipid. Fatty acids were determined as their methylated derivatives by gas chromatography (28) after saponification and petroleum ether extraction. For this analysis, 10 mg (dry weight) of lyophilized inhibitor sample was saponified with 2.5 ml of 2.5 M KOH in 85% methanol for 18 hr at 55 C. Unsaponified materials were extracted three times with 5 ml of petroleum ether, and the aqueous phase was adjusted to pH 2.0 with 5 M HCl. Fatty acids were extracted by two 5-ml applications of petroleum ether which were evaporated under vacuum and then dried over anhydrous Na2SO4. Methylation was accomplished by adding 1.0 ml of boron trifluoride-methanol reagent (25) to the dried product, followed by heat at 60 C for only 2 min. A petroleum ether extract of this mixture was washed with water before being evaporated to dryness. The methylated fatty acids were taken up in heptane and separated by a Barber-Coleman, series 5,000 gas chromatograph operated with helium gas carrier at 190 C and a 20% ethylene glycolsuccinate column on a gas pack (60 to 80 mesh). The flow rate was 80 ml/min, and the column effluent was analyzed by means of a hydrogen flame detector and paper strip recorder. Virus and inhibitor titration, tissue culturing, and plaque inhibition. NDV was used throughout this study and was perpetuated in the chorioallantoic cavity of 9or 10-day-old chick embryos, with harvesting 48 hr after inoculation. Chick embryo fibroblast (CEF) cultures were prepared from 9-or 10-day-old embryos by the method of Hoskins (16) and grown in plastic tissue culture plates (60 by 15 mm) in Eagle's growth medium (7), con-taining 200 units of penicillin and 200 ug of streptomycin per ml. Newborn calf serum was added to a final concentration of 10%. Monolayers were usually complete after 48 hr of incubation at 39 C under 5% CO2. For the plaque inhibition test, CEF cultures with complete monolayers were rinsed twice with 5 ml of cold (4 C) Hanks balanced salt solution (BSS) (14), lacking phenol red, and placed at 4 C. The lipoprotein inhibitor was diluted twofold in Hanks BSS containing either 96 or 180 plaque-forming units (PFU) of NDV per ml. These mixtures were added to the CEF cultures and held at 4 C for 1 hr to allow virus attachment. Thereafter, the excess fluid was removed and overlay agar was added. The overlay medium was composed of Eagle's medium with 5% calf serum and 0.7% Noble special agar. After 72 hr of incubation at 39 C, neutral red agar (12% Noble special agar, 0.25% lactalbumin hydrolysate, and 0.04% neutral red in Hanks BSS) was added, and plaques were counted. Triplicate plates were prepared for each concentration of inhibitor. The hemagglutination (HA) titer of NDV was established as described by Hoskins (16). The titer of the hemagglutination inhibitor (HAI) was also determined by Hoskin's procedure (16). HAI titer is reported in terms of micrograms of inhibitor protein per milliliter which will inhibit 4 HA units of NDV at 4 C. Neuraminidase digestion. Top band inhibitor from the sucrose gradient was dissolved in 0.08 M phosphate-buffered saline (pH 6.1) at a level of 4.4 mg/ml. Neuraminidase from Clostridium perfringens type IV (Sigma Chemical Co., St. Louis) was added to a final concentration of 287 ,ug/mL. The solution contained 0.02% sodium azide to retard microbial growth. This mixture was incubated at 37 C for 45 hr with constant shaking. Appropriate controls were employed in which lipoprotein was incubated under the assay conditions without the presence ofneuraminidase. This digestion procedure is essentially that of Ada et al (1). At the end of the digestion period, the samples were dialyzed against distilled water; the dialyzates were then lyophilized and analyzed for sialic acid. The digested inhibitor and control were assayed for HAI activity after dialysis. Preparation of lipoprotein antigen, immunization, and serological testing. The lipoprotein fraction resulting from the isoelectric precipitation at pH 5.0 was dissolved in 0.01 M phosphate buffer at pH 7.0 and adjusted to 2.0 mg of protein/ml. The HAI titer of this solution was 17.5 ,g. Twenty-two milliliters of this preparation was applied to a carboxymethyl cellulose (CMC) column (70 by 1.9 cm) equilibrated with the same buffer but containing 0.02% NaNs as a preservative. The column was developed by batch elution at room temperature with 0.01 M phosphate buffers (pH 7.0 and 8.0), 0.20 M phosphate buffer (pH 9.0), and 2.0 M phosphate buffer (pH 9.5) containing 1.0 M NaCl. All buffers contained 0.02% NaN, . The column was adjusted to a flow rate of 10.0 ml/hr, and fractions were collected with the aid of a Beckman model 132 fraction collector. The first elution peak, which contained the inhibitor, was dialyzed against distilled water, lyophilized, and redissolved in 0.15 M phosphate buffer (pH 7.2). The inhibitor solution (330 pg of protein/ml) was injected intravenously into a rabbit with an irregular schedule over a period of 1 month, consuming approximately 2.0 mg of inhibitor before a capillary fluid precipitation test became positive. This antigen was also used in the immunodiffusion test. A delipidized sample of the antigen was prepared by dissolving the lyophilized CMC, peak 1 product in 0.15 M NaCl to a final concentration of 330 pug of protein/ml. Four milliliters of this solution was added dropwise, with vigorous mixing, to 50 ml of an ethanol-diethyl ether solution (1:3) at -21 C (10). After 4 hr at this temperature, the precipitate which formed was collected by centrifugation (-21 C) and washed once with approximately 25 ml of diethyl ether (-21 C). The precipitate was dissolved in 0.13 M tris(hydroxymethyl)aminomethane (Tris) buffer (pH 8.6) containing 0.06 M sodium lauryl sulfate and was then dialyzed against this buffer lacking the detergent before use in the immunodiffusion experiment. A third antigen used in the immunodiffusion test consisted of the top band sucrose gradient purified inhibitor. The gel immunodiffusion experiment was performed essentially as previously described (2). The CMC peak 1 antigen was tested at 430,ug of protein per ml and lesser concentrations. The delipidized peak 1 antigen and the undelipidized top band gradient purified antigen were tested at 430 pug of protein per ml and 1.7 mg of protein per ml, respectively. The gel plates were cut with a commercially available cutter (Consolidated Laboratories, Chicago, Ill.) which spaced the center antiserum well 7.5 mm from the peripherally located antigen reservoirs. The serological reaction was developed at room temperature, and observations were made daily until the precipitation band diffused into a broad zone. Table 1 illustrates the purification scheme used to isolate the lipoprotein inhibitor of NDV. The biological activity of all inhibitor fractions are reported as micrograms of inhibitor protein per milliliter which will inhibit 4 HA units of NDV at 4 C. The original, clarified bicarbonate homogenate of chicken lung had an HAI titer of 62.5 ,ug/ml. This value was quite constant from preparation to preparation, presumably because each lot represented a pool of several lungs. The fraction precipitating between 20 and 50% ammonium sulfate saturation improved the HAI activity by approximately fourfold. This fraction had an HAI titer of 17.5 ,ug/ml. The 0 to 20% fraction generally had a titer of 95 4g/ml. Fractions recovered in the range 50 to 75 % and greater than 75% ammonium sulfate saturation had titers in excess of 200 ,ug/ml. Ammonium sulfate fractionation removed hemoglobin from the inhibitor fraction. Isoelectric precipitation at pH 5.0 produced a material with very good HAI activity (i.e., VOL. 21,1971 HAI = 9 ,ug/ml). This product was poorly soluble in ordinary buffers unless stirred overnight in the cold. Sucrose banding of this material produced three bands (Fig. 1) the uppermost of which, at a density of 1.130, contained the inhibitor (HAI activity = 2.0 ,ug) in a 31-fold purification from the lung extract. The lowest band in the sucrose gradient appeared to consist of aggregated materials. RESULTS At each stage in the purification scheme, protein, carbohydrate, and lipid analyses were performed to provide insight into the chemical nature of the inhibitor. Table 2 presents a summary of these data, from which it can be seen that, as purification of the inhibitor progressed, the percentage of protein decreased and the percentage of lipid increased significantly. The purest preparation consisted of 3.6% carbohydrate, 26% protein, and 72% lipid, the sum of which account for the entire molecule. On the basis of the high lipid content, the molecule is referred to as the lipoprotein inhibitor hereafter. More definite expressions of the carbohydrate and lipid content of the lipoprotein inhibitor are presented in Table 3. Of the carbohydrate, 1 to 2% was in the form of hexose, about 1% in the form of hexosamine, and 0.82 to 0.86% in the form of sialic acid. Chloroform-soluble lipids were analyzed for cholesterol and phospholipid which accounted for 10 to 20% and 40 to 50% of these lipids, respectively. Of the fatty acids recovered (Table 4), palmitic acid was the most abundant (39.3%), followed by oleic acid (22%) ( Table 5). In the first experiment utilizing 96 PFU of virus, plaque reduction by as much as 64% was accomplished by 3.0 mg of inhibitor per ml. When the virus load was increased to 180 PFU, 3.0 mg of inhibitor per ml reduced the plaque count by only 14%, and lower concentrations were noninhibitory. Digestion of the lipoprotein inhibitor with neuraminidase for 45 hr reduced its sialic acid content from 0.84 to 0.37% and lowered its HAI activity from 2 to 20 ,ug/ml, a 10-fold increase in activity. During the digestion period 40% of the silaic acid of the inhibitor was removed. Inhibitor incubated without enzyme did not change in HAI activity nor sialic acid content. A typical elution patternof theinhibitorfraction on carboxymethyl cellulose is presented in Fig. 2. The first elution peak corresponded with the void volume of the column and had an HAI titer of 9.0 jig of protein per ml. This represented a twofold purification over the isoelectric precipitation stage. Later elution peaks were not active against NDV virus. The CMC-purified fraction was chosen as the antigen for hyperimmunization of a rabbit. Figure 3 presents the results of the Ouchterlony test after 24 hr. Precipitation was noted only when the delipidized antigen was tested against the antiserum. A rather broad band was formed. The untreated immunizing antigen failed to produce a precipitate even though it did react in the fluid VOL. 21,1971 capillary test used to establish antibody activity in the rabbit serum. Undelipidized sucrose gradient purified material also failed to exhibit precipitation with the antiserum. Upon observation for longer periods of time, the precipitation band broadened but could not be resolved into definite minor components. DISCUSSION The purification scheme for isolation of the lipoprotein inhibitor was devised by utilizing typical biochemical fractionation procedures and noting the improvement in HAI activity on a protein basis. Bicarbonate solutions are often used in the initial stages of cell membrane preparation (4,23,26,34). Laucikova (20,21) used bicarbonate extraction and isoelectric precipitation at pH 5.1 to separate an inhibitor of influenza virus HA from chick embryo chorioallantoic membrane extracts. Her inhibitor was composed of only 18% lipid and 55% protein and is thus quite dissimilar from the lipoprotein inhibitor described herein. Our effort to utilize diethylaminoethyl (DEAE) cellulose in the purification scheme was unsuccessful; the lipoprotein inhibitor bound so tightly to the DEAE that it could not be eluted. Philipson lipoprotein inhibitor of enteroviruses upon ionexchange chromatography. Blumenfeld (5) has reported complete retention of cell membrane sialoproteins by DEAE, so this problem is not unique to this inhibitor. On the other hand, chromatography of the active material on CMC at neutral pH demonstrated that inhibitor activity was not retained on the column but eluted with the void volume. Some noninhibitory materials were retained on the column, and this procedure was employed in the preparation of the inhibitor antigen. Because of the inability to use ion-exchange chromatography to great advantage, we attempted purification by sucrose gradient density centrifugation. Apparently, this method has not been used previously in the purification of viral inhibitors. Nevertheless, this method revealed that our isoelectrically precipitated product could be fractionated into three components, with approximately fouror fivefold improvement in purification. The lipoprotein inhibitor stratified at a density of 1.130. The precise chemical composition of the NDV lipoprotein inhibitor varied somewhat from one preparation to another (Table 3). This is a typical problem in lipoprotein chemistry (10,18,20), the causes of which are not entirely known. Nevertheless, it is obvious that the inhibitor is not chemically similar to fowl immunoglobulins (22), neither is it chemically similar to fowl interferons (9) although it will prevent cell infection like the latter. It does not, in fact, have the same chemical composition as the influenza inhibitor from chicken chorioallantoic membrane described by Laucikova (20,21). The plasma membrane from chick embryo fibroblast cultures has a composition of 25% protein, 64 to 71 % lipid, and 7 to 8% carbohydrate, very similar to that of the lipoprotein inhibitor (29). This suggests that the inhibitor is a portion of the cell membrane and adds significance to its description, for it could represent a natural receptor of the virus on chicken lung cells. The major fatty acids present in the lipoprotein inhibitor are shown in Table 4 to be palmitic, oleic, stearic, linoleic, and arachidonic. These fatty acids have been reported to be in highest concentration in the influenza virus and in-calfkidney and chick-embryo host cells (18). It has been suggested that host lipid components are incorporated into the influenza virus particles at some time during their synthesis (12). Future work will attempt to determine whether lipid incorporated into Newcastle disease virus during infection resembles the lipid obtained as lipoprotein inhibitor from host tissue. Newcastle disease, influenza, herpes simplex, and vaccinia viruses have all been described as lipophilic viruses (27) based on their ability to adsorb to steroids including cholesterol, fatty acids such as palmitic and stearic acids, and various fatty acid derivatives. It is believed the attachment of these lipophilic viruses to cholesterol occurs via van der Waal's forces, whereas attachment to the fatty acids and their derivatives involves ionic forces (36). In some instances the adsorption is irreversible. Cholesterol, palmitic, stearic, and other fatty acids, all of which are known to serve as receptors for NDV (27), are present in the lipoprotein inhibitor prepared from chicken lung and may contribute to the inhibitory activities of this macromolecule. All of the well-characterized hemagglutination inhibitors of myxoviruses contain sialic acid, upon which the basis for their biological activity resides (17,19). Removal of 40% of the sialic acid in the lipoprotein inhibitor caused a 10-fold loss in its HAI activity, supporting its classification as a Francis type inhibitor. The immunodiffusion test revealed a single band with the delipidized antigen only. Both of the lipid-bearing antigen preparations gave negative tests. The exact cause for this was not established. Hypothetically, this could result from the masking of antigen determinants in the protein by lipid so that they were unavailable to react with antibodies in the absence of any antilipid antibody formation, or simply the inability of the complete lipoprotein to diffuse in the aqueous menstruum owing to its hydrophobic characteristics. Although only a single precipitation band was detected even with the delipidized antigen, it was rather broad, indicative of antigenic impurity. Important to the problem of whether this lipoprotein inhibitor is a "natural" receptor for the virus on chicken lung may be the observation that it will inhibit viral infectivity. Many inhibitors of myxovirus hemagglutination have been described (13,17,19) but few have been reported to retard infection by the virus. Ovine ca,-glycoprotein will inhibit both HA and plaque formation by NDV (6).

v3-fos

2020-12-10T09:04:17.021Z

1971-02-01T00:00:00.000Z

237230818

s2

Characterization of Growth Stimulants in Corn Steep for Lactic Streptococci The production of acid in milk cultures of lactic streptococci was stimulated by the addition of corn steep liquor. Separation by ion-exchange and paper chromatography indicated the presence of four major stimulatory components in the corn steep. Some variation was noted in the response of the lactic streptococci to the individual stimulatory components. The four components were further purified by paper and column chromatography. One of the four stimulatory components stained positively on chromatograms with ninhydrin. The remaining stimulatory components were detectable only by bioautography. All four components were unstable to acid hydrolysis and absorbed ultraviolet light between 230 and 275 nm in aqueous solution. The stimulatory components did not contain pentoses, suggesting that they were not nucleotides or nucleosides; however, they might be purine or pyrimidine bases. The production of acid in milk cultures of lactic streptococci was stimulated by the addition of corn steep liquor. Separation by ion-exchange and paper chromatography indicated the presence of four major stimulatory components in the corn steep. Some variation was noted in the response of the lactic streptococci to the individual stimulatory components. The four components were further purified by paper and column chromatography. One of the four stimulatory components stained positively on chromatograms with ninhydrin. The remaining stimulatory components were detectable only by bioautography. All four components were unstable to acid hydrolysis and absorbed ultraviolet light between 230 and 275 nm in aqueous solution. The stimulatory components did not contain pentoses, suggesting that they were not nucleotides or nucleosides; however, they might be purine or pyrimidine bases. Metabolic activity of lactic streptococcus starter cultures in milk can be stimulated by the addition of various extracts of plant and animal origin (5, 6, 7, 8, 9, 11, 12; W. Orme-Johnson HII, Ph.D. Thesis, Univ. of Texas, Austin, 1964). The stimulatory activity of these complex substances has been generally attributed to their being sources of readily available nutrients (10). Many of the stimulants have not been identified; however, portions of the stimulatory components in pancreas and liver extract have been characterized as peptides (8, 9; W. Orme-Johnson III, Ph.D. Thesis). Nucleic acid derivatives in pancreas extract (7) and in tomato juice (2) have also been identified as stimulatory components. The stimulation of milk cultures of lactic acid bacteria by corn steep (a by-product from the manufacture of corn starch) was first reported by Kennedy Acid production. Acid production by the cultures in 10% reconstituted nonfat milk solids (NFMS) was measured by continuously monitoring the pH by using the method described by Gilliland and Speck (4), except that the inoculated milk was dispensed in 19-ml quantities into sterile glass tubes (1.7 by 15 mm) containing 1 ml of the desired concentration of test material or 1 ml of sterile water. The inoculated milk was incubated in a water bath at 32 C. Bioautographic analysis. Stimulatory zones were located on paper chromatograms by a bioautographic method similar to that reported by Speck et al. (11). The medium for each bioautographic assay consisted of three components: 160 ml of 4% agar, 160 ml of 20% reconstituted NFMS, and 5 ml of 20% aqueous litmus. All components were sterilized separately by autoclaving for 15 min at 121 C. The components were tempered to 45 C, mixed together on a magnetic stirrer, inoculated with 1% of the test organism, and poured into a Plexiglas plate (14 by 60 by 2.5 cm). (The Plexiglas plate was rinsed thoroughly with ethanol followed by sterile distilled water before pouring the bioautograph medium.) Strips of the chromatograms (2 cm in width) to be assayed were placed on the surface of the solidified medium beside a strip from a control chromatogram (no sample) and incubated at 22 C. Stimulatory zones were observed through the bottom of the plate as white areas (reduced litmus) on a blue background appearing within 4 to 8 hr. Cation-exchange chromatography. A 10-ml sample of corn steep was passed through a column (2.2 by 41 cm) of the cation-exchange resin [AG5OW-X8 (H+), 100 to 200 mesh (BioRad)]. The sample was washed through with distilled water at a rate of 1 ml per min until a negative Molisch test for sugar was obtained. The column was then eluted with 6.5 bed volumes of 2 N NH40H. Excess NH40H was removed from the eluate by repeated dilution and evaporation as described by Cogan et al. (2). The NH4OH-free eluate was concentrated at 42 C under reduced pressure to 10 ml and passed through a sterile 0.45-MAm membrane filter. A portion of the material was stored at 3 C and the remainder lyophilized. Paper chromatography. Stimulatory components were isolated by using descending paper chromatography on sheets (46 by 37 cm) of Whatman 3MM chromatography paper. The chromatograms were developed for 15 hr with one of the following solvent systems: (I) n-butanol-acetic acid-water (4:1:1), (II) n-butanol-acetic acid-water (250:60:250), and (III) n-propanol-water (7: 3). Stimulatory components were located by bioautographic assay of marker strips cut from the chromatograms. Marker strips were also stained with 0.25% ninhydrin in acetone or scanned for ultraviolet light-absorbing zones under a 254-nm emitting lamp. Zones on the remaining portion of the chromatograms which corresponded to the stimulatory zones on the bioautographed marker strips were excised and eluted in distilled water for 1 hr. The aqueous solution was then concentrated to 5 ml under reduced pressure at 42 C and stored at 3 C. Gel chromatography. The components eluted from the chromatograms were applied to a column of G-25 Sephadex (1.3 by 33 cm; Pharmacia) equilibrated at room temperature with 0.1 N NH40H. The column was eluted with 0.1 N NH40H at rate of 0.6 ml per min at room temperature. Fractions of 2 ml were collected and assayed for absorbance at 260 nm by using a Beckman DU spectrophotometer. Acid hydrolysis. Evacuated and sealed glass vials containing the samples in 6 N HCl were heated at 110 C for 24 hr. The HCl was removed by evaporating the samples to dryness and rediluting to 4 ml. Corn extraction. Twenty grams of whole corn of the same grade used for corn starch manufacture was ground to 20 mesh in a Wiley mill, added to 200 ml of distilled water at 50 C, and agitated in a shake water bath at 50 C for 1 hr. The extract was filtered through a Whatman no. 4 filter paper and centrifuged for 20 min at 5,800 X g; the supernatant was concentrated to 3 ml at 42 C under reduced pressure and stored at 3 C. RESULTS The production of acid by single-strain lactic streptococci in miLk was stimulated by corn steep ( Table 1). The amount of stimulation was recorded as the difference in the time required for the culture to reach pH 5.0 in the control milk and in the milk containing 0.05 % corn steep. The amount of stimulation for four of the five cultures tested ranged from 0.9 to 2.0 hr, whereas S. lactis HI, which produces acid quite slowly in milk, was stimulated for 11.1 hr. The active components in corn steep were absorbed on cationic-exchange resin, and the active material was eluted from the column with NH40H. A portion of the lyophilized eluate (0.6 g) was dissolved in 6.6 ml of distilled water and applied to 22 sheets of chromatography paper. After development with solvent system I, marker strips cut from each sheet were assayed by bioautographic analysis. The results ( Table 2) revealed stimulatory zones for S. lactis AC2 at RF values of 0.18, 0.29, 0.40, and 0.54, which were designated A, B, C, and D, respectively. Bioautographic assays were also conducted with other single-strain lactic streptococci and mixed-strain commercial Cheddar cheese starter cultures. S. lactis Cio and S. cremoris C3 responded to the four stimulatory components observed with S. lactis AC2. S. cremoris AC,, responded to zones corresponding to B, C, and D and, in addition, responded to a different stimulatory zone having a slightly higher RF (0.58) than D. Mixed strain commercial cheese starter cultures MD and MU responded to zones having RF values similar to those of components A, B, C, and D, whereas cultures VT3 and VT7 responded to zones having RF values comparable to components B and D. In addition, VT7 responded to a zone at RF 0.73. Components A, B, C, and D were selected for further isolation and characterization. The zones containing the stimulatory components were eluted from the paper with distilled water and concentrated to 5 ml, and each was applied to 16 additional sheets of chromatography paper. The chromatograms for components A and B were developed with solvent system II; solvent system III was used for components C and D. Bioautographic analysis of marker strips from these chromatograms revealed a single stimulatory zone for each component. The RF values were as follows: 0.35 for A, 0.33 for B, 0.56 for C, and corresponded to a ninhydrin-positive zone on the chromatogram. Attempts to visualize the active zones with reducing agents, sulfuric acid ashing, or iodine were unsuccessful. Because of the lack of a chemical visualization method, the bioautographic assay was used to locate the active components to be eluted from the chromatograms. Sephadex gel chromatography was employed to remove impurities eluted from the chromatography paper along with the stimulatory material. Preliminary investigations indicated that the stimulatory components eluted from chromatograms absorbed ultraviolet light at 260 nm; therefore, the eluate from the Sephadex column was monitored at 260 nm. One major peak was observed for each component (A, B, C, and D) along with several minor peaks. Analysis of the fractions by spot assay (similar to the bioautographic assay) indicated that the tubes containing the fraction under the major peak in each case contained the stimulatory activity. Paper controls were prepared for each component. For these controls, equal amounts of paper (based on weight) were excised from blank paper chromatograms which had been developed in the appropriate solvent systems used in the second paper chromatographic separation of the stimulatory components. The paper controls were eluted, concentrated, and passed over the Sephadex column in the same manner as used for the stimulatory material. For each paper control, the tubes corresponding to the stimulatory fraction from the respective component were pooled and treated in a manner similar to that of the stimulatory components. These samples were lyophilized and used for further characterization. Aqueous solutions (0.75 mg/ml) of each stimu- latory component were heated at 100 C for 10 min and then assayed for stimulatory activity. The results indicated that the components were resistant to boiling. The acid hydrolysates of the stimulatory components and equivalent amounts of unhydrolyzed components were applied to chromatography paper strips and developed with solvent system I. The unhydrolyzed samples exhibited stimulatory zones having RF values comparable to those observed in previous bioautographic assays. However, the stimulatory components were apparently not stable to acid hydrolysis in that no stimulatory zones were observed for the hydrolyzed samples. No ninhydrin-positive zones were evident on the chromatograms of the hydrolyzed samples. The ultraviolet-absorption spectra of the stimulatory components revealed absorption in the 220-to 300-nm range. The maximal and minimal wavelengths in 0.1 N HCI and 0.1 N NaOH for each component are presented in Table 3. The spectra of the stimulatory components were suggestive of nucleic acid derivatives; however, they did not coincide closely with the spectra which have been reported for the common nucleic acid derivatives. Analyses of the components with orcinol ferric chloride reagent (1) revealed the absence of pentoses, thus eliminating the possibility that the components are nucleosides or nucleotides, or both. In an attempt to determine the origin of the stimulatory material, an aqueous extract of ground corn was chromatographed on paper with solvent system I, and the resulting chromatograms were bioautographed with S. lactis AC2 as the test culture. The results revealed stimulatory zones at RF values of 0.15 and 0.37. These were similar to the RF values for components A and C, indicating that at least two of the stimulatory components originated in the corn. DISCUSSION The measurements of stimulation in this study were based on the ability of the test culture to produce acid or to lower the oxidation-reduction potential of the growth medium. No attempt was made to correlate these measurements with growth of the organisms. Kennedy and Speck (6) reported that corn steep stimulated the growth as well as the acid production by L. casei. The results of the present study involving stimulation of single-strain lactic streptococci and multiplestrain Cheddar cheese starter cultures indicated some variation among cultures with respect to the amount of stimulation and to the number of stimulatory components in corn steep. The results, however, indicate that the components designated as A, B, C, and D were the major stimulatory components for the lactic streptococci. It was not possible to locate the stimulatory components A, B, and C on paper with chemical detection methods. Only component D was located in this manner; it stained weakly with ninhydrin. This ninhydrin zone remained associated with the stimulatory zone during the second paper chromatographic separation. Since they were not stained with ninhydrin, stimulants A, B, and C apparently do not contain free amine groups or else were present in amounts below the detection limits of ninhydrin. None of the stimulatory components exhibited ultraviolet light-absorbing or fluorescing zones on the paper chromatograms, suggesting that they were not nucleic acid derivatives or that they were not present in sufficient quantities for visualization. Heimbuch et al. (5), with L. casei as the test culture, isolated a stimulatory component from corn steep which possessed a pentose and exhibited an ultraviolet light-absorption spectral maximum at 265 nm and minimum at 245 nm. They postulated that the factor could be a nucleoside and further showed that several known nucleic acid derivatives were slightly stimulatory to L. casei. Zuraw et al. (12) studied the stimulatory effect of corn steep liquor on L. casei and S. lactis and isolated three stimulants for L. casei. The purified stimulants were not stimulatory for S. lactis and possessed ultraviolet light absorbtion maxima and minima between 230 and 290 nm. One of the stimulatory compounds was identified as phenylalanine. The other factors were postulated as being free aromatic amino acids or peptides containing aromatic amino acids. In the present study, the isolated stimulatory substances retained their activity after boiling but lost activity when acid-hydrolyzed. If the stimulatory components were peptidic as has been shown for some materials that stimulate the streptococci (8,9,10), the hydrolyzed product may or may not contain stimulatory components. Stimulant D, which stained with ninhydrin before hydrolysis, was not stimulatory after hydrolysis and did not reveal additional ninhydrin-positive zones. Most amino acids are resistant to acid hydrolysis, although a few are partially destroyed. The ultraviolet absorption spectra and other results obtained for stimulant D are indicative of an aromatic amino acid. However, further characterization studies are needed before making a definite classification. Components A, B, and C had ultraviolet absorption spectra similar to D but did not stain with ninhydrin. Since these spectra are characteristic of nucleic acid derivatives, a pentose determination was employed in an attempt to implicate nucleotides or nucleosides, but the results were negative. Purine and pyrimidine bases would not be eliminated by these results. Component D could also fit into this group. Certain nucleic acid derivatives are known to be stimulatory for lactic streptococci (3,7). Vitamins could also be involved, since some exhibit ultraviolet light absorbance in the range found for the stimulants. Such compounds on paper chromatograms might be detected by bioautography and not by chemical tests if they were active in catalytic amounts. Knowledge of the origin of the stimulatory components found in corn steep is desirable for evaluating its use as a food additive. Two of the components apparently originated in the whole corn. The remaining components may have been synthesized during the fermentation that occurs in the steeping process used in the manufacture of corn starch or they may be hydrolysis products of some nonstimulatory component of corn.

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2020-12-10T09:02:21.738Z

1971-06-01T00:00:00.000Z

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Brine Shrimp (Artemia salina L.) Larvae as a Screening System for Fungal Toxins Concentrations resulting in 50% mortality, determined with brine shrimp (Artemia salina L.) larvae exposed to known mycotoxins for 16 hr, were (μg/ml): aflatoxin G1, 1.3; diacetoxyscirpenol, 0.47; gliotoxin, 3.5; ochratoxin A, 10.1; and sterigmatocystin, 0.54. 4-Acetamido-4-hydroxy-2-butenoic acid γ-lactone gave no mortality at 10 μg/ml. Used as a screening system involving discs saturated with solutions of known mycotoxins, the larvae were relatively sensitive to aflatoxin B1, diacetoxyscirpenol, gliotoxin, kojic acid, ochratoxin A, rubratoxin B, sterigmatocystin, stemphone, and T-2 toxin. Quantities of 0.2 to 2 μg/disc caused detectable mortality. The larvae were only moderately sensitive to citrinin, patulin, penicillic acid, and zearalenone which were detectable at 10 to 20 μg/disc. They were relatively insensitive to griseofulvin, luteoskyrin, oxalic acid, and β-nitropropionic acid. The disc screening method indicated that 27 out of 70 fungal isolates from foods and feeds grown in liquid or solid media produced chloroform-extractable toxic material. Examination of toxic extracts by thin-layer chromatography for 17 known mycotoxins showed that the toxicity of eight isolates could be attributed to aflatoxin B1 and B2, kojic acid, zearalenone, T-2 toxin, or ochratoxin A. Nine out of 32 of these fungal isolates grown in four liquid media yielded toxic culture filtrates from at least one medium. Chemical tests for kojic, oxalic, and β-nitropropionic acids showed the presence of one or two of these compounds in filtrates of seven of these nine isolates. The disc screening method indicated that 27 out of 70 fungal isolates from foods and feeds grown in liquid or solid media produced chloroform-extractable toxic material. Examination of toxic extracts by thin-layer chromatography for 17 known mycotoxins showed that the toxicity of eight isolates could be attributed to aflatoxin B1 and B2, kojic acid, zearalenone, T-2 toxin, or ochratoxin A. Nine out of 32 of these fungal isolates grown in four liquid media yielded toxic culture filtrates from at least one medium. Chemical tests for kojic, oxalic, and ,B-nitropropionic acids showed the presence of one or two of these compounds in filtrates of seven of these nine isolates. In the search for hitherto unknown toxic fungal metabolites, evaluation of toxicity on the basis of mortality or pathological symptoms, such as kidney or liver damage in higher animals, is laborious and puts severe restrictions on the number of fungal isolates and culture conditions that can be tested. Besides, it is possible that the toxicity observed in animal experiments is due to the presence of one or more of the many known fungal metabolites, viz., known mycotoxins (3,10) or commonly occurring compounds such as oxalic and kojic acids. Toxicity of feedstuffs inoculated with Aspergillus spp. was attributed in some instances to oxalic acid production (21), and many of the filtrates and extracts of fungi isolated from Japanese fermented foods contained kojic and ,B-nitropropionic acids (12). From a practical view point, it may therefore be advantageous to (i) test fungal isolates for toxicity with one or more easily manageable screening systems, (ii) screen the toxic extracts by chemical screening techniques for the presence of known toxins, and (iii) test those cultures, whose toxicity cannot be explained, against higher animals. It must be borne in mind, however, that the screening system used may not be sensitive to some fungal metabolites that are toxic to higher animals. In this paper, we describe the use of brine shrimp larvae as a screening system for toxic fungi and investigate their sensitivity to some known mycotoxins. This system has the advantage that brine shrimp eggs are commercially available. Active larvae can be obtained within 1 to 2 days, and maintenance of live cultures is not required. The organism has been widely used, e.g., for quantitation of the potency of anesthetics (15), evaluation of toxicity of dichloro-diphenyltrichloroethane (11) and other insecticides (13), of heavy metal salts (7) and antibiotics (8), and for testing the inhibitory effects of carcinogens on the hatching of the eggs (6). Brown et al. (5) described a bioassay involving brine shrimp larvae for aflatoxin B1. Under the conditions of this test, 0.5 jug/ml in artificial seawater resulted in mortality of over 60%. The larvae were also relatively sensitive to ochratoxin A and highly sensitive to an acetone extract of Fusarium tricinctum (4). These data suggested that the HARWIG AND SCOTT brine shrimp might be a suitable screening system for testing fungi for toxicity. MATERIALS AND METHODS Culture. Most of the fungi tested had been isolated from foodstuffs (20). Some cultures which were implicated in death and illness of animals had been isolated by J. Forgacs, Good Samaritan Hospital, Suffern, N.Y. Others were isolated from naturally moldy wheat containing ochratoxin A (18) and from other cereals. Cultures were reisolated from sterile soil in which they had been stored and grown on fresh potato-dextrose agar for about 2 weeks; spores were then suspended in 0.05% aqueous polyoxyethylene sorbitan monooleate. The majority of the cultures were tested for toxicity after growth in liquid media. One milliliter of a suspension containing 106 spores was added to 24 ml of yeast extract (2%)-sucrose (15%7) medium (YES), Czapek-Dox broth (CD), CD supplemented with 0.5% yeast extract (CD + YE), and Sabouraud's maltose broth (SM) in 250-ml Erlenmeyer flasks. Cultures were incubated for 6 to 9 days at 25 C. Some isolates of Aspergillus candidus and A. glaucus from moldy wheat were tested for toxicity after 2 weeks of growth on 6 g of moistened and autoclaved wheat at 37 and 30 C, respectively. Fusarium and Trichothecium isolates were cultured on 3 g of moistened, crushed corn in addition to YES under conditions described previously for Fusarium isolates (17). Filtrates and extracts. Mycelial mats were separated from the culture liquids and frozen in liquid nitrogen. Culture liquids were filtered with 0.45-,Am Nalgene filter units (Nalge, Rochester, N.Y.). Mycelial mats and culture filtrates were stored at -10 C. Just before testing, culture filtrates were adjusted to pH 6.5 with 5 N NaOH. Mycelial mats and fungal cultures that had been grown on grains, and also some of those grown on liquid media, were extracted by immersion in boiling chloroform for 10 min; the chloroform fraction was removed with a pipette and the cultures were further extracted by dispersion in another portion of chloroform with a high-speed mixer (Silverson Machines Ltd., London, England). The two extracts of each culture were combined and reduced to 1 ml under nitrogen. The toxicity of each filtrate and extract was evaluated by the disc screening method described below. Larvae. Methods involving the use of brine shrimp larvae for testing fungi for the production of toxic secondary metabolites (Higgens et al., Eighth Intersci. Conf. Antimicrob. Ag. Chemother.. Abstr., 1968, p. 18) were followed with minor modifications. Brine shrimp medium (BSM) was composed of 3.0 g of dipotassium glycerophosphate; 30.0 g of NaCl; 0.3 g of CaD12-2H20; 0.5g of MgSO4-7H20; 1.5g of MgCl2-6H20; 0.8 g of KCl; 0.1 g of MgBr2-6H20; 6.0g of glycine; and deionized water to make 1,000 ml of BSM (C. E. Higgens, personal communication). The pH of the medium was adjusted to 6.5 prior to autoclaving for 15 min at 121 C. For each experiment, 100 to 200 mg of brine shrimp eggs (Connecticut Valley Biological Supply Co., Southampton, Mass.) was placed in 100 ml of BSM contained in a 750-ml Erlenmeyer flask and shaken at 140 rev/min on a rotary shaker for about 30 hr at 30 C. Larvae were separated from egg cases and eggs by means of a medicine dropper after they had started to cluster. The concentration of larval suspensions was adjusted to about 200 larvae/ml. Eggs were stored over CaSO4 at room temperature. The percentage of larvae emerging from eggs thus stored remained generally satisfactory over a period of several months; in some batches, however, hatchability was low for unknown reasons. The toxicity of these mycotoxins was evaluated by means of the disc screening method or assay method, when possible in two independent experiments with different batches of brine shrimp eggs. Most toxins were dissolved in methanol to obtain concentrations of 1,000, 500, 100, and 10 ,g/ml. Sterigmatocystin was dissolved in acetone; oxalic and,-nitropropionic acids were dissolved in water, and the pH was adjusted to 6.5 with 1 N NaOH. In the assay method, mycotoxin solutions were diluted 100-fold with BSM to obtain final concentrations of 10, 5, 1, and 0.1 ,ug/ml. Disc screening method. Blank paper discs (7 mm diameter; BBL) were saturated with a solution of toxin or fungal extract in organic solvent (about 20 Aliters/disc), the solvent was allowed to evaporate, and e,ach disc was placed in a 0.5-ml well of a FB-48 Linbro Disposo Tray (Winley-Morris, Montreal). When aqueous solutions of toxins and culture filtrates (about 30 1liters/disc) were tested, discs were placed directly in the well. Toxicity of each solution was evaluated in triplicate. Two drops (about 0.1 ml) of a suspension of larvae (containing 20 to 40 larvae) were added to each well. Trays were incubated at 30 C for about 16 hr. Mortality was determined by counting the immobile (dead) larvae under a stereoscopic microscope, killing the living larvae with heat or Formalin, and then counting the total number. Mortality in controls was determined simultaneously with each screening test. Natural mortality associated with discs saturated in noninoculated media or water averaged 3 and 1%7, respectively; that of discs soaked in chloroform or methanol, which was allowed to evaporate, averaged 2%. Chloroform extracts of quantities of corn meal and wheat, equal to that used for culturing some of the fungi examined, gave a mortality of 6 and 0.3%, respectively. Toxicity of filtrates and extracts was rated as follows: 0 to 9%at 1012 APPL. MICROBIOL. VOL. 21,1971 BIOLOGICAL SCREENIN mortality, nontoxic (NT); 10 to 49% mortality, slightly toxic (ST); 50 to 89% mortality, toxic (T); 90 to 100% mortality, very toxic (VT). Assay method. Of each mycotoxin solution to be tested, 4.8 ml was pipetted into a 10-ml beaker, and 4 drops (0.2 ml) of a larval suspension containing about 30 larvae was added to each beaker. Each concentration was tested in quadruplicate. Larvae were incubated at 30 C for about 16 hr. Mortality was determined on the basis of mobility, as viewed under a Quebec colony counter. Simultaneously run controls consisted of BSM containing 1% (v/v) of solvent. Natural mortality in 1 % methanol solutions averaged 5% and ranged from 0 to 17%; in 1% acetone the mortality averaged 1%N. Concentrations giving rise to 50% mortality (LC5, values) were estimated from the weighted regression lines of the probit mortality on the logarithm of the concentration, as fitted by the maximum likelihood solution (9). The toxicity of some chloroform extracts was evaluated with the assay method on a lipid weight basis. The chloroform was removed under nitrogen from the remaining portions of the extracts. Residues were dissolved in 1 ml of methanol. A 0.2-ml portion of this solution was diluted 100-fold with BSM and tested as described above. The weight of the lipid residue from another 0.2-ml portion was determined after removal of the methanol. Thin-layer chromatography. Thin layers (0.25 mm) of silica gel (Adsorbosil 5, Applied Science Laboratories, Inc., State College, Pa.) were activated at 100 C for 2 hr. Five Aiters of T or VT filtrate, or T or VT chloroform extract (concentrated to 0.5 ml), were spotted on the thin-layer chromatography (TLC) plate together with several known mycotoxins (17). Plates were developed 15 cm in the solvent systems toluene-ethyl acetate-(90%) formic acid (6: 3: 1) and benzene-methanol-acetic acid (24:2:1). Chromatograms of T and VT extracts were examined under ultraviolet light and after spraying and heating with an acidic methanolic solution of p-anisaldehyde (17). This method was used for examination of extracts for aflatoxin B1, B2, G1, and G2, aspertoxin, citrinin, diacetoxyscirpenol, gliotoxin, luteoskyrin, nivalenol, nivalenol acetate, ochratoxin A, patulin, penicillic acid, sterigmatocystin, T-2 toxin, and zearalenone. Kojic acid and ,B-nitropropionic acid were detected by spraying with 0.1% of diazo blue B salt in methanolwater (1:1) followed by brief exposure to ammonia fumes; they formed mauve and orange spots, respectively, at average RF values of 0.15 and 0.51 in the first solvent system. Amounts were estimated by visual comparison with standards (10 mg of kojic acid/ml and 2 mg of j-nitropropionic acid/ml). Kojic acid was confirmed by spraying with ferric chloride solution after repeat TLC; all filtrates were also tested with this reagent in test tubes. Oxalic acid. T and VT filtrates suspected to contain oxalate were treated with Moir's calcium acetate reagent (14). The precipitate was purified and checked for reduction of 0.02 N potassium permanganate on heating in 2 N sulfuric acid (14). RESULTS Sensitivity of brine shrimp larvae to known mycotoxins. The data in Tables 1 and 2 show that the larvae are sensitive to aflatoxin B1, aflatoxin G0, diacetoxyscirpenol, gliotoxin, kojic acid, ochratoxin A, rubratoxin B, sterigmatocystin, stemphone, and T-2 toxin. Toxicity from these toxins would have been detected if they had been present in the chloroform extract of fungal cultures at a concentration of 10 to 100 ,g/ml or more. The larvae were less sensitive to citrinin, patulin, penicillic acid, and zearalenone. These would have been detectable only if present in the chloroform extracts at a concentration of about 500 sglml or more. Griseofulvin and luteoskyrin had no toxicity at the concentrations tested. The 4-acetamido-4-hydroxy-2-butenoic acid y-lactone caused no deaths when tested at 10 ,g of BSM per ml with the assay method. 3-Nitropropionate and oxalate were toxic at high concentrations only. Of the toxins tested, diacetoxyscirpenol and sterigmatocystin were more toxic to the larvae than aflatoxin B1 and G1. The LC50 values obtained for aflatoxin G1 and gliotoxin in two different experiments ( Table 2) indicated good reproducibility with this method. Toxicity of culture filtrates and extracts. Table 3 shows, for most isolates, only those media from which the most T or VT filtrates and extracts were obtained. Many isolates grew well in YES, SM, and CD + YE but only poorly in CD. Some isolates grew a thick mycelial mat on several media but proved T or VT in one medium only. In some instances, poorly developed mycelial mats yielded T or VT extracts. YES yielded T or VT filtrates and extracts more frequently than the other media. Nine out of 32 isolates produced, in at least one medium, culture filtrates that were either T or VT. Of the 70 isolates, 27 showed toxicity in chloroform extracts obtained from at least one medium. The data in Table 4 confirmed the high toxicity of the Fusarium and Trichothecium extracts observed with the disc screening method. Known mycotoxins detected. Mycotoxins detected by TLC are recorded in Table 3. f,-Nitropropionic acid was present in two T SM filtrates at a concentration of approximately 1 mg/ml (isolates no. 53 and 69); a lesser amount (about 0.25 mg/ml) was detected in a NT filtrate (isolate no. 50), out of several selected at random. No ,B-nitropropionic acid was detected in any of the T or VT mycelial extracts. Concentrations of kojic acid, when present in the T or VT filtrates, ranged from approximately 3 to 10 mg/ml; about 0.2 to 0.5 mg/ml was present in several of the corresponding mycelium extracts. No kojic acid was detected in the ST or NT filtrates (ferric-chloride test). The toxicity of four filtrates (isolates no. 50, 51, 53, and 69) can be explained by the presence of kojic acid. The presence of oxalate in culture filtrates from isolates no. 47 and 49, which contained 3.6 mg/ml and 6.1 mg/ml, respectively, may partially account for their toxicity. Isolate no. 48 produced little oxalate. In view of the low sensitivity of the larvae to oxalate, factors other than oxalate alone may have been responsible for the observed high mortality in this filtrate. No toxins were identified in the filtrates of no. 52 and 68. The toxicity of some chloroform extracts could be attributed to afiatoxin B1 and B2 (no. 35), kojic acid (no. 50, 53, and 69), zearalenone (no. 57), T-2 toxin (no. 58 and 59), and ochratoxin A (no. 62). In the remaining 26 extracts that were either T or VT no known mycotoxins were detected. Isolation of "TLC-pure" unknown toxin from a Fusarium spp. Most of the toxicity of the chloroform extract of a YES culture of isolate no. 59, after initial separation into seven fractions by preparative TLC, lay in the T-2 toxin fraction and a more polar fraction. The latter fraction contained a substance with an R, value half that of T-2 toxin in the toluene-ethyl acetate-formic acid solvent system. The material behaved the same as T-2 toxin toward the anisaldehyde spray (17) and was highly toxic to the brine shrimp larvae after further purification by TLC. Microacetylation with acetic anhydride and pyridine yielded a spot with the same Rp value in two solvent systems as the spot obtained by acetylation of T-2 toxin. The unknown toxin is possibly a desacetyl T-2 toxin (2). DISCUSSION Brine shrimp larvae were found to be convenient test organisms for toxic fungal metabolites. Larvae were readily obtained in large numbers, and their low natural mortality in controls involving media and chloroform made it possible to test crude fungal filtrates and extracts. As the natural mortality in 1 % methanol and acetone solutions was low, these solvents can be used to help dissolve the less water-soluble components of lipid extracts. In most experiments in which the assay method was used, a straight-line relationship between probit mortality and logarithm of the concentration of mycotoxin could be demonstrated. Two failures (not reported) to establish this relationship may be attributed to variable natural mortality as observed in some control solutions. This may have resulted from toxicity associated with the glassware used and may be avoided by the use of disposable containers. Disposable containers were used in the disc screening method, and natural mortality was less variable. LC50 values were determined by using 1015 VOL. 21,1971 1016 HARWIG AND SCOTI the maximum likelihood solution (9), which corrects for natural mortality. When the amount of toxin available is ample, the use of a greater number of concentrations and replicates would be preferable to reduce variation in LC50 values. As is probably the case with any one organism, brine shrimp larvae showed a wide range of sensitivity to different mycotoxins. The order of sensitivity of the brine shrimp larvae to many of the toxins tested was comparable to that of zebra fish larvae (1). Both systems were found highly sensitive to sterigmatocystin. The results indicate that most of the known mycotoxins tested (e.g., aflatoxins B1 and G1 and diacetoxyscirpenol) could have been discovered with the brine shrimp system, whereas a few of the toxins tested (e.g., luteoskyrin and 4-acetamido-4hydroxy 2-butenoic acid 'y-lactone) would have remained undetected. The use of additional biological screening systems may reduce the likelihood of this occurrence. Even with this one system, however, a number of filtrates and extracts were found to possess toxicity that could not be explained by the presence of the known mycotoxins looked for. It is unlikely that this finding is chiefly due to "false positives" resulting from sensitivity of the larvae to commonly occurring fungal constituents. Isolates that grew equally well on two different substrates proved T or VT in one substrate but NT in the other. This suggests that nonspecific toxicity from common fungal constituents is low. To evaluate the toxicity of a certain isolate, it may be advisable to determine the amount of lipid residue responsible for the mortality, as was done for three Fusarium and one Trichothecium isolate (Table 4). Since a large proportion of the lipid residue from these isolates probably consisted of nontoxic storage lipids and structural lipids, the toxicity of the actual toxin in these extracts is likely to be high. The high toxicity of isolate no. 59 (YES) could be attributed to the presence of two toxins in the lipid residue, T-2 toxin and a related compound.

v3-fos

2020-12-10T09:04:10.755Z

1971-11-01T00:00:00.000Z

237231946

s2

Growth Factor Requirements of Ruminal Cellulolytic Bacteria Isolated from Microbial Populations Supplied Diets With or Without Rapidly Fermentable Carbohydrate The predominant cellulolytic ruminal bacteria isolated from microbial populations supplied diets containing cellulose as an energy source and essentially devoid of amino acids or rapidly fermentable carbohydrates were shown to require branched-chain acid(s) for growth. The predominant cellulolytic ruminal bacteria isolated from microbial populations supplied diets containing cellulose as an energy source and essentially devoid of amino acids or rapidly fermentable carbohydrates were shown to require branchedchain acid(s) for growth. In ruminants consuming natural forage, ruminal cellulolytic bacteria require branchedchain fatty acids for growth (1,2,3,8), and these acids appear to be formed mainly by degradation and deamination of branched-chain amino acids from dietary protein by some of the noncellulolytic bacteria and ciliate protozoa (6,7,9). However, diets essentially devoid of amino acids have been shown to support large numbers of cellulolytic bacteria which require branched-chain acids for growth and the rumen was shown to contain these acids (20). This suggested that branchedchain acids secreted as protein into the rumen by the host animal or biosynthesized mainly from carbohydrate by noncellulolytic microorganisms are sources of the branched-chain fatty acids. The cellulolytic bacteria previously isolated (20) were from steers fed diets which contained 12.8%7, starch (19), and readily fermentable carbohydrates, such as starch, have been suggested to modify the ruminal population to satisfy the strict growth requirements of cellulolytic microorganisms (10). To test the role of readily fermentable carbohydrates as precursors for growth factors, we isolated 65 strains of ruminal cellulolytic bacteria from 10-8 dilutions of steer or in vitro fermentor contents and determined their nutritional requirements ( Table 1) with methods previously described (20). The cellulolytic bacteria from samples in which wood pulp was the sole carbohydrate source (19) were of similar type, were present in greater numbers (18), and had nutritional requirements similar to cellulolytic bacteria obtained from steers fed starch and wood pulp (18) or starch, glucose, and wood pulp (17). These results suggest that animal diets containing readily fermentable carbohydrate(s) are not needed to maintain large numbers of cellulolytic bacterial with branched-chain fatty acid requirement. In the present study, four Ruminococcus strains isolated from a protozoal-free in vitro culture, supplied with 17.7 g of a biuret-wood pulp diet (19) twice daily for 2 weeks, required branchedchain fatty acids for growth. This indicates that branched-chain acids can be synthesized directly by other bacteria or that branched-chain amino acids are synthesized and then degraded by other bacteria to form the fatty acids (13,15). The conditions required for optimal growth of 27 strains of ruminococci and 4 strains of Bacteroides succinogenes isolated from the steers in the present study are similar to those for strains of ruminococci (1, 2, 8) and B. succinogenes (3,8) isolated from ruminants fed natural diets. Of the ruminococci, nine required branched-chain acids, nine were stimulated by branched-chain acids, and nine required or were stimulated by clarified rumen fluid, whereas none was shown to require both branchedand straight-chain acids. As expected (3), all four B. succinogenes strains required both branchedand straight-chain fatty acids. The growth factor(s) present in clarified rumen fluid, which was required for the growth of 29% of the ruminococci and 37% of unidentified bacteria, was not identified. However, the unidentified bacteria which grew in clarified rumen fluid were not tested in the straightplus branched-chain medium and might require a combination of straightand branched-chain fatty acids. Basal medium contained cellobiose, Casitone (vitamin-free), minerals, hemin, cysteine, sulfide, and B vitamins as previously indicated (20). Clarified rumen fluid growth factor requirement is indicated when optimal growth occurs in basal medium plus rumen fluid but not in basal medium or basal medium supplemented with branched-chain acids or straight-chain acids. A branched-chain fatty acid requirement is indicated if the strain grows in basal medium supplemented with branched-chain fatty acids and with clarified rumen fluid but does not grow in the basal medium or basal medium supplemented with straight-chain fatty acids. In preparing media with fatty acids, the basal medium was supplemented with acids in one-tenth the amount previously described (20). In addition, a branched-plus straight-chain fatty acid medium was prepared which contained the following acids (millimoles/100 ml of medium): isobutyric, 0.013; 2-methyl-butyric, 0.013; isovaleric, 0.013; acetic, 0.8; propionic, 0.13; ni-butyric, 0.065; and caproic, 0.006. b Percentages in columns, left to right, are based on the study of 13,16,14,13,6, and 3 strains, respectively. Within these groups were six, five, seven, six, two, and one strains of ruminococci, respectively, and zero, one, zero, one, one, and one strain of Bacteroides succinogenes, respectively. The ruminococci and B. succintogenies, but not the unknown cellulolytic strains, were tested for a combined branchedand straight-chain fatty acid requirement for growth. For this reason, the four Bacteroides strains which required the branched-plus the straight-chain fatty acids are listed with those which require clarified rumen fluid. None of the ruminococci required the combined acids for growth. The nutritional data from this study, although indicating that branched-chain fatty acids are formed by a mixed rumen population fed a diet devoid of amino acids and rapidly fermentable carbohydrates, should not be interpreted to suggest that dietary fatty acid(s) addition could not be stimulatory to animal production. Branchedchain fatty acids have been reported to increase total microbial synthesis (11,16) and nitrogen retention in ruminants (17), although a beneficial effect has not always been attained (5,14,17).

v3-fos

2018-04-03T00:55:57.131Z

1971-01-01T00:00:00.000Z

27363580

s2

Dry-Heat Resistance of Bacterial Spores Recovered from Mariner-Mars 1969 Spacecraft The dry-heat resistances of 70 bacterial spore isolates recovered from Mariner-Mars 1969 spacecraft were determined and expressed as D values (decimal reduction times). Fifty per cent of the spore isolates had D values of 60 min or less at 125 C. Of organisms with D values greater than 60 min, four were selected for a study of the effect of sporulation medium and suspension menstruum on dry-heat resistance. Both sporulation medium and suspension menstruum were found to affect significantly the dry-heat resistance of the bacterial spores tested. It is imperative that an understanding of the dry-heat resistance of microorganisms on spacecraft that require sterilization, i.e., landers for an extraterrestrial life-detection mission, be acquired (6). An important step toward obtaining this necessary knowledge for the determination of a sterilization cycle for such a spacecraft was made by the prelaunch recovery of spore isolates from the Mariner-Mars 1969 flybys. During the Mariner-Mars 1969 Microbiological Moni-toring Program at Cape Kennedy, Fla., 70 spore isolates were recovered from the craft and their dry-heat resistance was tested. In addition, samples of environmental fallout were subjected to dry-heat testing. This program served to further the knowledge of the dry-heat resistance of microorganisms accumulating on ffight hardware during assembly. A similar program is planned for the Mariner-Mars 1971 mission. A major difficulty in defining the dry-heat resistance of microbial populations in spacecraft assembly areas rests in the inability to work with a natural population because of the low population densities present (8). Hence, a study of the heat resistance of organisms from such areas requires that the organisms be cultured to produce sufficient numbers for testing. It has been observed that the D value [a D value (or decimal reduction time) is the duration of exposure at a given temperature necessary to reduce a microbial population by 90%0] for a microorganism may vary according to the microorganism's inherent thermal resistance and environmental influences acting on that resistance (7). Of these environmental influences, the chemi-I Present address: Resources Planning and Control Corporation, El Segundo, Calif. cal milieu of the organism prior to dry-heat treatment is of importance in determining its dry-heat resistance (1). Therefore, a separate study was undertaken to define the effect of certain sporulation media and storage menstrua on the dry-heat resistance of bacterial spores isolated from Mariner 1969. MATERIALS AND METHODS Spore recovery and selection. Bacteria were collected from Mariner 1969 by the swab-rinse method, as defined by NASA (5). The samples were heatshocked at 80 C for 15 min prior to quadrant streaking on Trypticase Soy Agar (TSA; BBL). Colonies were removed from the TSA plates and, from these, 70 organisms capable of sporulation in a synthetic sporulation medium (SSM; 4), were selected for dry-heat resistance testing (phase I). Four isolates capable of satisfying the criteria of D values at 125 C (D125 c) in excess of 60 min when sporulated on SSM and of 95% or greater sporulation in both SSM and TAM sporulation agar (Difco; supplemented with 80 ,g of CaCd2 per mg and 20 ,ug of MgSO4 per mg) were chosen for further study (phase II). Culture and sporulation technique. Isolated colonies were removed from TSA streak plates, added to Trypticase Soy Broth (TSB; BBL), and incubated at 37 C until visible turbidity occurred. For phase I testing, 2 ml of the TSB suspension was inoculated into a flask containing 250 ml of SSM. (Phase II included inoculation onto a TAM agar plate.) The flask (or plate) was then incubated at 37 C until spores constituted 95% or more of the cells (as determined by microscopic examination of a stained preparation) at which time the spores were harvested. (For the TAM-grown isolates, harvesting consisted of washing the spores from the agar surface with sterile distilled water. The SSM spore cultures were harvested by centrifugation.) The suspension was centrifuged (10,400 X g for 15 min), resuspended, and washed eight times in sterile distilled water. After the final washing, the spore pellet was resuspended and divided equally to form both 95% ethanol and sterile distilled water suspensions which were then stored at 4 This facility provided temperature and humidity control by circulating air through a system of heating and cooling coils. The system used a water spray and cooling coil to assure that air leaving the cooling coil was at saturation. The air then underwent a reheat function to provide air at the specific temperature and humidity conditions required for the room. After the equilibra- tion period, the coupons were placed on trays in a dryheat oven and exposed to 125 C (+0.5) for designated time intervals. The oven was a mechanical convection oven modified by installing removable sliding trays into the door (Fig. 1). To minimize temperature fluctuations in the oven, the trays were individually removed and replaced without opening the door. Thermocouples were mounted at 23 oven locations (with an additional thermocouple exposed to ambient conditions), and the leads were connected to a 24-point recorder for continuous temperature monitoring during an experiment. After a 10-min temperature come-up time, coupons inoculated with TAM-grown spores were removed and assayed at 30-min intervals, whereas those with SSM-grown spores were removed at 1-hr intervals. This variation in assay intervals was established because of the differences in heat resistance that resulted from sporulation on the two different media. The Mariner 1969 Microbiological Monitoring Program included a study to determine the heat resistance of the microbial burden collected on stainlesssteel strips exposed in the Assembly Operations (AO) Building and the Explosive Safe Facility (ESF), at the Air Force Eastern Test Range (AFETR), Cape Kennedy. Upon collection, the strips were exposed to given temperatures (125 or 115 C) for designated intervals, at which time groups of eight were removed from the dry-heat oven and subjected to a microbiological assay. Assay procedure and data handling. After a designated heat exposure of the spore isolate, the three coupons were removed from the dry-heat oven tray with sterile forceps and placed individually into three flasks each of which contained 20 ml of sterile 0.1% peptone water. The flasks, partially immersed in an aqueous solution of 0.1 % Tween-80, were then treated in an ultrasonic bath for 12 min at 25 kHz. After this treatment, 10-fold serial dilutions were made in sterile 0.1% peptone water. Dilutions were then plated in triplicate by the pour-plate method by using TSA. a D125c value is duration of exposure at 125 C necessary to reduce a microbial population by 90%. b 0, no significant effect on dry-heat resistance; +, significant effect on dry-heat resistance (P < 0.01). Plates were incubated for 48 hr at 32 C, and the dilutions yielding 30 to 300 colony-forming units per plate were counted and recorded. The resulting data were subjected to a computerized linear regression analysis which generated a survivor curve and calculated the reciprocal of the slope of the curve (or D value) and the 95% confidence limits about the D value. An analysis of variance and Duncan multiple-range tests were performed on phase II data (2). Strips collected in the AO and ESF areas were aseptically placed into a flask containing 50 ml of sterile TSB. All flasks were incubated at 32 C for 48 hr and examined for growth. Results were recorded as growth or no growth. Isolate identification. Identification of the phase II isolates was performed as described by Bergey's Manual (7th ed.). Figure 2 shows a cumulative relative frequency distribution of D125 c values (expressed in minutes) for the Mariner 1969 isolates sporulated in SSM and suspended in 95% ethanol prior to heat testing. The graph gives a measure of the likelihood that bacterial spores accumulating on spacecraft surfaces under Mariner 1969 assembly conditions would not exceed a specified D value. For example, 20 to 30% of the spores accumulating on the spacecraft were found to have Di2zc values of 30 min or less; 49 to 57%, 1 hr or less; and 86 to 90%, 3 hr or less. In general, the survivor curves for those spores with D125C values greater than 180 min were not satisfactorily explained by linear regression analysis; i.e., the resulting R (SQ) terms were quite low. [R (SQ) refers to the measure of the proportion of total variation about the mean explained by linear regression.] Identification of the four phase II isolates (see above) indicated that isolate number 1 was Bacillus cereus, whereas isolates 2, 3, and 4 were classified as B. licheniformis. Identification was not pursued to the subspecies level; however, because of differences in heat resistance of isolates 2, 3, and 4, it was believed that more than one variety of B. licheniformis was tested. Four D125c determinations were made for each phase II isolate test condition (a total of 64 heat-resistance tests). An analysis of variance was performed on the data to determine the significance of the sources of variation present in the experiment (Table 1). No effect on D125 c due to replication was noted, thus indicating the ability to repeat significantly D value estimates for an organism handled in a specified way. Differences in strain, sporulation medium, and suspension menstruum were all seen to affect D values. A strain-sporulation medium interaction that affected heat resistance was also noted; i.e., for all four organisms tested a significantly higher D125 c value was observed when the sporulation was performed with SSM rather than TAM (P < 0.05; Table 2). Table 3 summarizes the media-menstruum effects on the four Mariner isolates. The far right-hand column of this table indicates a significantly higher D125 c value for the ethanol versus the water-suspended spores; however, this observation did not hold for all strainmedia combinations. For example, the ethanol and water suspended-TAM cultured isolates, with the exception of isolate number 4, had a similar D125 c- Table 4 shows the results of determinations of thermal extinction points of environmental fallout strips from the AFETR. No growth was observed from the assay of strips exposed to 125 C for 90 min. However, growth was still present after 60 min of exposure, indicating made to compare quantitatively the dry-heat resistance exhibited by fallout strip populations with that of media-sporulated organisms. In general, more than one type of microorganism was present on the fallout strips, whereas purified spore suspensions were formed from the SSM and TAM cultures. In addition, the low number of viable microorganisms detected on the fallout strips (see footnote Table 4) made questionable any expression of the data in terms of D values. The U.S. Public Health Service at Phoenix, Ariz., has classified and dry-heat tested 103 Mariner 1969 bacterial spore isolates (9). Spores were cultured by using TAM agar and found to have substantially lower heat resistance than the 70 SSM-cultured spores discussed in the present study. Such findings would be in agreement with the sporulation medium effects on heat resistance reported above. Work by the Phoenix group (M. Favero, personal communication) suggests that spores harvested from SSM retain a dry-heat resistance more closely approximating their natural dry-heat resistance, i.e., resistance before culturing, than do spores cultured on TAM. RESULTS AND DISCUSSION The present study of the dry-heat resistance of bacterial spores recovered from the surfaces of flight craft provides some of the information necessary for the formulation of a dry-heat sterilization cycle for unmanned spacecraft. In addition to a knowledge of the dry-heat resistance of microorganisms on such a craft, a thorough understanding of other factors must be acquired, e.g., the level of microbial contamination and the temperature profile of the craft (3).

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1971-05-01T00:00:00.000Z

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Detection of Sulfa Drugs and Antibiotics in Milk A disc assay method for testing sulfa drugs and antibiotics in milk was developed wherein Bacillus megaterium ATCC 9855 was used as the test organism and Mueller-Hinton agar was used as the test substrate. Incubation was at 37 C for 4 to 5 hr. The test procedure is an improvement over the Bacillus subtilis-Antibiotic Medium No. 1 method, as described in Standard Methods for the Examination ofDairy Products, in that it is sensitive to eight sulfa drugs and to bacitracin without a significant change in sensitivity to eight other antibiotics commonly used for mastitis therapy. Grade "A" Pasteurized Milk Ordinance any 6-month sulfa commonly used along with antibiotics The Grade "A" Pasteurized Milk Ordinance of the Food and Drug Administration (4) requires that producers' milk or commingled milk be tested for antibiotics at least four times during any consecutive 6-month period. Although sulfa drugs are commonly used along with antibiotics to treat bovine mastitis, the test procedure used (1) is generally insensitive to sulfa drugs. Chemical methods are available for detecting sulfa drugs in milk; however, the analytical procedures involved are far more complex than the antibiotic test now in use for routine regulatory testing (3,5). We believed it would be desirable to develop a procedure for routine regulatory testing that would be useful in detecting sulfa drugs as well as the antibiotics commonly used to treat mastitis, provided that the test developed was not more complex than the Bacillus subtilis method described in Standard Methods for the Examination of Dairy Products (1). This paper reports the development of a testing procedure that meets this requirement. MATERIALS AND METHODS Preparation of assay plates. Three sporeforming and two nonsporeforming organisms were studied as candidate test organisms for sensitivity testing. These were B. subtilis ATCC 6633, B. cereus ATCC 11778, B. megaterium ATCC 9855, Sarcina lutea ATCC 9341, and Escherichia coli ATCC 11229. B. subtilis spores were obtained from Difco Laboratories, Inc. Spore suspensions of B. cereus and B. megaterium were prepared by growing the cells in AK Sporulation Medium No. 2 (BBL) in 6-oz prescription bottles incubated for 48 hr at 35 C. Suspensions of S. lutea and E. coli were prepared daily from slants of Mueller-Hinton agar that had been incubated for 18 to 20 hr at 32 C. Three successive transfers were made before the cells were harvested and used. Growth from sporeforming or vegetative cell cultures was washed from the agar with phosphate-buffered distilled water and centrifuged at a relative centrifugal force of 5,000 for 15 min at 3 C, and the centrifugation-washing process was repreated three times. Spore crops were stored in buffered dilution water at 4 C until used. All inocula (vegetative cells or spores) were adjusted in optical density to give a final concentration of about 5 X 104 per ml of agar. The inoculum was added to the agar at 50 C, the inoculated agar was mixed gently by swirling to avoid air-bubble formation, 4 ml of agar was pipetted into 90-mm inside diameter plastic petri dishes, and the agar was distributed over the dish by swirling the dish. Fresh agar was prepared for each day of testing, and the spores, when used, were not heat-shocked. Preparation and testing of milk samples. Farm-bulktank or dairy-storage-tank raw milk was used for this study. Discs containing a sulfa derivative or an antibiotic were prepared by weighing the inhibitor under test and suspending it in distilled water. From this, an appropriate amount was added to milk, and twofold serial dilutions were made of the inhibitor in milk. One-tenth mililiter of milk containing the inhibitor was added to 0.5-inch (12.7-mm) blank discs (Carl Schleicher and Schuell Co.), and the discs were placed on the inoculated solidified agar for inhibitory testing. Temperatures of 25, 32, 35, and 37 C were used for various incubation times. Zone measurements were made with a vernier caliper with the plates illuminated from the back by fluorescent light. Zones, 15 mm in diameter or larger, were recorded as positive (disc diameter is 12.7 mm). For routine testing, any zone should be recorded as positive providing the proper controls are run. The 15-mm criterion was used in this study because it gave a zone that could not be mistaken even by a relatively untrained analyst. All minimum sensitivities reported were obtained in at least two trials. RESULTS AND DISCUSSION Eight sulfa drugs and nine antibiotics were selected for study, and the inhibitor testing procedure listed in Standard Methods for the Ex- amination of Dairy Products was used to establish the sensitivity of the procedure now in general use for regulatory testing to the inhibitors selected (Table 1). We believe that the general insensitivity of the standard method to sulfa drugs was the result of the medium (Antibiotic Medium No. 1) containing p-aminobenzoic acid or folic acid or both. Since B. subtilis produces bacitracin, it is insensitive to it. Mueller-Hinton agar has been widely used for the detection of sulfa drugs, and this medium was selected to test the sensitivity of five species of bacteria to the 17 inhibitors under study ( Table 2). In all cases, incubation was at 37 C for a previously determined incubation time that gave optimal zones with the test organism. S. lutea and B. megaterium were more sensitive to the test inhibitors than were the other three organisms tested. B. megaterium was selected for further study inasmuch as a sporeformer is preferable to a nonsporeformer for routine testing because it can be purchased in readyto-use form and stored until used. This would make the test easier to standardize between laboratories. Heat shocking the spores of B. cereus, B. subtilis, and B. megaterium did not enhance sensitivity or significantly reduce assay time. Since 37 C incubation is not commonly used in milk testing laboratories, incubation temperatures of 35, 32, and 25 were studied to determine whether they might be as useful as 37 C ( Table 3). In general, the sensitivity of the procedure increased with increased incubation temperature, and 37 C was the temperature of choice. Standard Methods for the Examination of Dairy Products recommends that all milks be heated to 82 C for 2 to 5 min to avoid reporting false-positives resulting from natural inhibitory substances in raw milk. Since this is part of the testing procedure and would affect apparent sensitivity if any of the inhibitors were sensitive to heat, all 17 inhibitors under study were assayed in milk before and after heating when incubated at 25, 32, 35, and 37 C. In no case was the apparent sensitivity of the test changed by more than one step in a twofold dilution series after the milk containing the inhibitors had been heated. This variation is normal for this procedure when used for repetitive testing of milk containing an inhibitor. Unlike the results of Marth,Alexander,and Hussong (2) in studies of the effect of heating milk on apparent sensitivity of assay, the heating technique of 82 C for 3 min had no effect on penicillin. This may be due to the heating techniques used since Marth et al. steamed their test milks for 7 min before testing. With penicillin, penicillinase discs are used for identification in that the antibiotic is inactivated by the enzyme. Similarly, we tested 0.5inch (12.7-mm) discs impregnated with 50 ,ug of p-aminobenzoic acid for their usefulness in identifying an inhibitor as a sulfa drug. These discs were made by adding the acid in aqueous solution to the disc, followed by drying the disc at 40 to 44 C. These discs inactivated the inhibitory properties of all sulfa drugs studied in concentrations of at least 5 ,g of sulfa derivative per disc. Accordingly, we believe this technique is useful in identifying an unknown inhibitor as a sulfa drug. From the alternatives studied, we believe the following to be most useful for the detection of sulfa drugs and antibiotics in milk: (i) add B. megaterium ATCC 9855 spores to Mueller-Hinton agar at 50 C to give a final spore concentration of 5 X 104 per ml of agar; (ii) dispense 4 ml of inoculated agar into a flat-bottom petri dish of about 90-mm inside diameter and agitate the dish so that the agar will cover the dish surface; (iii) allow agar to solidify on a level surface; (iv) touch edge of 0.5-inch (12.7-mn) filter disc to milk sample to wet disc by capillary action, and place disc on surface of inoculated agar; (v) incubate plates at 37 C for 4 to 5 hr; (vi) examine for zones of inhibition; and (vii) identify zones using conventional techniques as described in Standard Methods for the Examination of Dairy Products (1) including heating the milk at 82 C for 2 to 5 min to detect natural inhibitors. Discs containing p-aminobenzoic acid may be used to identify inhibition from sulfa drugs. We believe that the substitution of the B. megaterium-Mueller-Hinton procedure for the one now in common use for testing milks offers the advantage of sensitivity to sulfa derivatives and to bacitracin. This is accomplished without significant change in sensitivity to the other antibiotics tested and without making the test procedure more difficult to perform. For these reasons, we feel that the procedure described merits consideration as a standard method for the regulatory testing of antitiotics and sulfa drugs in milk.

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1971-02-01T00:00:00.000Z

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New method of isolating Salmonellae from milk. The use of a cotton gauze swab and subsequent culture of the swab was found to be a more sensitive method for isolating Salmonella from liquid milk than the revised procedure of North. The swab method was found to be as sensitive as the North procedure for recovering Salmonella when incubated at 37 C but more sensitive when incubated at 43 C. Incubation of the swab cultures at the elevated temperature of 43 C gave good results when Salmonella was present at levels as low as one per liter. Swabs exposed to milk contaminated with 100 Salmonella per liter remained positive even when subsequently washed for 2 hr in noncontaminated milk. Bismuth sulfite agar and Brilliant Green sulfadiazine agar were equally effective for isolating Salmonella from broth cultures; use of both media resulted in maximal isolations. The use of a cotton gauze swab and subsequent culture of the swab was found to be a more sensitive method for isolating Salmonella from liquid milk than the revised procedure of North. The swab method was found to be as sensitive as the North procedure for recovering Salmonella when incubated at 37 C but more sensitive when incubated at 43 C. Incubation of the swab cultures at the elevated temperature of 43 C gave good results when Salmonella was present at levels as low as one per liter. Swabs exposed to milk contaminated with 100 Salmonella per liter remained positive even when subsequently washed for 2 hr in noncontaminated milk. Bismuth sulfite agar and Brilliant Green sulfadiazine agar were equally effective for isolating Salmonella from broth cultures; use of both media resulted in maximal isolations. In 1965 and early 1966, an interstate outbreak of gastroenteritis occurred involving 29 laboratory-confirmed cases of Salmonella newbrunswick (2). Epidemiological and laboratory evidence implicated instant nonfat dried milk as the source of infection. S. newbrunswick was isolated from shelf samples of the product and from the milk drying plant in which it was produced. A study of the plant indicated an environmental situation that allowed continual perpetuation of salmonellae within the plant; it was assumed that the initial source of salmonellae was the raw milk supply. As a result of the S. newbrunswick epidemic, a protocol for the examination of milk for salmonellae was recommended by members of a joint committee from the Center for Disease Control, the Food and Drug Administration, and the Division of Environmental Engineering and Food Protection (13). It is a revision of the procedure of North (11). Julseth and Deibel (6) have shown that in the case of nonfat dry milk, nine salnoneilae per liter of reconstituted milk can multiply to a level sufficient to cause disease. Thus it appears that a method of analyzing milk should be sufficiently sensitive to detect fewer than 10 salmonellae per liter of milk. The purpose of this study was to evaluate the recommended method of analyzing raw whole milk for salmonellae and to develop a procedure more applicable to field conditions. MATERIALS AND METHODS Preparation of Salmonella inocula. S. typhimurium isolated in this laboratory from a frozen dessert product was used throughout this study. The inoculum was prepared from a culture grown in Trypticase Soy Tryptose (TST) broth [15 g of Trypticase Soy (BBL) and 13 g of Tryptose Broth (Difco) per liter] for 18 to 24 hr at 37 C. After incubation, 1 ml of the culture was transferred to 8 ml of fresh TST broth, and the inoculated TST was incubated at 37 C for 4 to 6 hr. The growth was quantitated by reading turbidity at 500 nm on a Spectronic-20 colorimeter (Bausch & Lomb). The culture was diluted to a 40% transmission with TST broth, and plate counts on MacConkey agar (Difco) showed this to contain 5 X 108 salmonellae per ml. Final dilutions to obtain the desired inoculum for tests were made in normal saline. Milk. Raw milk used in this study was obtained from a local dairy which produces grade A milk. Total bacterial and coliform counts were determined on the raw milk prior to each experiment by making appropriate dilutions of milk in normal saline, inoculating 0.1 ml of each dilution to duplicate plates, and spreading the inoculum on the plates with a sterile glass rod. Tryptone glucose extract (TGE) agar (Difco) was used to determine total counts. The plates were read after 24 hr of aerobic incubation at 37 C. The coliform count was defined in this study as the number of lactose-fermenting colonies present on MacConkey agar after 24 hr of aerobic incubation at 37 C. Conventional method. The currently accepted method of isolating salmonellae from liquid milk is a modification of the procedure of North (11). The method consisted of adding 20 mg of Brilliant Green per liter of liquid milk. After 24 hr of incubation at 37 C, a loopful of this pre-enrichment was streaked to Brilliant Green agar (Difco) containing 80 mg of sodium sulfadiazine per liter of agar (BGS) and bismuth sulfite agar (BS, Difco). At the time of plating, 10 ml of the milk was subcultured to 100 ml of tetrathionate broth (Difco) containing 10 (TET), and this enrichment was streaked after 24 hr of incubation at 37 C to BGS and BS. Swab culture method. Moore (8) found that suspending a cotton gauze swab in the flowing sewage in a sewer for 1 to 3 days with subsequent culture of the swab was a useful technique for isolating salmonellae. This method was modified in this study for isolating salmonellae from liquid milk. For laboratory evaluation, the swab, which was a piece of cotton gauze 4 ft by 6 inch, was folded (eight times), tied in the middle with a wire, and suspended in a liter of milk. The milk was then stirred on a magnetic stirrer at about 100 rev/min for 10 min. The swab was removed from the milk, placed in 150 ml of TET broth, and after 24 and 48 hr of incubation a loopful was streaked to BGS and BS. Serial transfer experiment. Thirteen beakers, each containing a liter of raw milk, were used. The second beaker was inoculated with the test organism. The gauze swab was suspended in the first beaker (uninoculated), stirred on a magnetic stirrer for 10 min, and then passed sequentially through the remaining 12 beakers in the same manner. This was to simulate a condition in which a swab in a flowing stream of milk would be exposed temporarily to contaminated milk (beaker 2) and washed in noncontaminated milk for a prolonged period (the remaining 11 beakers). After removal from the last beaker, the gauze swab was cultured by the procedure described above. Secondary enrichment. Subcultures, after 1 week of incubation at room temperature, were made in several of the experiments. The initial tetrathionate enrichment broths, after the first culturing, were left at room temperature for 1 week, and then 1 ml was transferred to 9 ml of fresh tetrathionate enrichment broth (secondary enrichment). The latter was incubated for 24 hr, and then a loopful was streaked to BGS and BS. Isolation and identification. The BGS and BS plates were incubated at 37 C: the BGS for 24 hr and the BS for 48 hr. Three salmonella suspect colonies were picked from each positive BGS or BS plate and transferred to triple sugar iron (TSI, Difco) agar slants. All TSI cultures having typical salmonella reactions after 24 hr of incubation were subjected to serological and, when indicated, biochemical tests. Details of the procedures followed the techniques described by Galton, Morris, and Martin (3). Statistical analyses. Statistical analyses were conducted with probabilities based on exact binomial confidence limits. Probabilities of 0.05 or less were considered significant. RESULTS The efficiency of the conventional method for recovering salmonellae from raw liquid whole milk was evaluated by inoculating known numbers of S. typhimurium into the milk, and the results indicated that an inoculum of over 1,000 organisms was required to recover salmonellae by this method. These data indicated that the sensitivity of the conventional method was in- adequate for routine surveillance of milk for salmonella contamination. Subculturing a primary broth to a secondary broth has been shown to be advantageous for isolating salmonellae in a procedure where a lactose broth is subcultured to a selective broth (12) and in a procedure where tetrathionate broth is subcultured to another tetrathionate broth (5; G. K. Morris, J. G. Wells, and C. G. Dunn, unpublished data). Therefore, it was decided to evaluate the usefulness of a secondary tetrathionate enrichment inoculated (1 ml to 9 ml) from the primary tetrathionate broth utilized in the conventional method. Also, since the cotton gauze swab described by Moore (8) for sampling sewer effluent would be very applicable to field conditions for sampling raw milk, it was decided to evaluate in the laboratory the efficiency of this swab for isolating salmonellae from milk as compared with the conventional method. The results of the comparisons indicated that the swab culture technique was approximately equivalent to the conventional method for isolating salmonellae ( Table 1). The secondary tetrathionate enrichment of the conventional method yielded the best results, followed by the primary tetrathionate broth, whereas no isolates were obtained by plating the pre-enrichment. A secondary enrichment also appeared to be advantageous when using the swab culture technique. Total bacterial and coliform counts determined on the raw milk prior to each experiment indicated that the sensitivity of the method was influenced by these counts. The total counts ranged from 9,000 to 172,000 per ml, and the coliform counts ranged from less than 10 to 4,000 per ml. A more sensitive test for Salmonella was observed in the milk with lower bacterial counts. Serial transfer experiments were conducted to determine whether salmonellae were retained by the gauze swab after washing in noncontaminated milk. After 2 hr of serial washing (12 beakers of milk at 10 min each), salmonellae were recovered from the swab even when the swab was inoculated with as few as 100 salmonellae prior to washing. There have been reports of an increased sensitivity in the isolation of salmonellae from various type samples by utilization of the elevated incubation temperature of 43 C (1, 4, 10). All of the milk in studies previously discussed were incubated at 37 C. Incubation temperatures of 37 and 43 C were compared by utilizing the swab culture technique and a secondary enrichment ( Table 2). There were more recoveries made at the 43 C incubation temperature at the inoculum levels of 1,000, 100, and 10 salmonellae per liter. In addition, at 43 C frequent recoveries were made from samples at the level of one salmonella per liter, whereas there were no recoveries made at this level at the 37 C incubation temperature. There was not a statistically significant increase in the recovery rate at either temperature between the primary and secondary enrichment, but recoveries obtained by 43 C incubation were greater than those at 37 C by both primary and secondary enrichment (P < 0.01). Since incubating at 43 C increased the sensitivity of the swab culture technique, an experiment was conducted to determine whether the swab culture technique was more sensitive than the conventional method at this temperature. The results indicated that the swab culture technique was superior (P < 0.001) to the conventional method for isolating salmonellae (Table 3). A comparison was made of the method currently recommended, the conventional method incubated at 37 C, and the method of choice as indicated by these studies, the swab culture technique incubated at 43 C ( Table 4). The swab culture technique incubated at 43 C was superior to the conventional method incubated at 37 C (P < 0.01). The conventional method yielded salmonellae from 10 of 24 samples, whereas the swab culture recovered salmonellae from 18 of 24 samples. In addition, two recoveries by the latter procedure were made at the one salmonella per liter level, whereas no recoveries were made at this level by the conventional method. The BS agar and BGS agar were equally effective for isolating salmonellae in this study. However, recoveries on the two media were not on May 4, 2020 by guest http://aem.asm.org/ Downloaded from always made from the same sample. Recoveries on the BS agar alone were frequently made from samples with low salmonella inocula. The raw milk analyzed in these experiments appeared to contain natural flora that was highly adapted for fermenting lactose, and with plating medium such as BGS, which contains lactose as a differential agent, the indicator system in the plate was frequently overwhelmed by these lactose fermentors, causing an acid condition over the entire plate. Under these conditions, small numbers of salmonella colonies did not give the typical alkaline reaction. BS agar does not contain lactose as a differential agent; thus small numbers of samonella colonies were not overlooked because of these lactose fermentors. BGS agar, however, was advantageous in that it suppressed these coliforms better than BS agar. DISCUSSION Efficient methods of isolating salmonellae from raw milk are necessary to facilitate surveillance of the milk industry for salmonella and to aid in investigating salmonella outbreaks in which milk products are implicated as the source. Milk products are still a problem as a source of salmonella as indicated by U. S. Department of Agriculture data reported in the Center for Disease Control Salmonella Surveillance Report (14). During 1969, salmonellae were isolated from 44 of 1,697 product samples of dry milk and from 89 of 196 environmental samples. The raw milk from which this milk was manufactured must be considered as a possible source of contamination. Although workers have recovered salmonellae from nonfat dried milk and other milk products with relative frequency, there has been little success in isolating salmonellae from the raw milk when attempts were made to trace the contamination back to its source. The conclusions from these investigations usually are that the problem is one of perpetuation of salmonella in the plants rather than contaminated raw milk supplies. Although perpetuation in the plant may be a major part of the problem, the raw milk cannot be ruled out as a source of contamination without a sufficiently sensitive method of detecting salmonella, especially at very low levels. Contaminated milk from one cow when combined with milk from hundreds of other cows in a bulk tank truck would be diluted to the point that the number of salmonellae present would be extremely low, possibly in the range of 1 to 10 salmonellae per liter. We have shown in this study that the conventional method is inadequate to detect this level of salmonellae. To make a recovery by the conventional method, the presence of 103 salmonellae per liter of raw milk was frequently required. In contrast, the swab culture technique at 43 C recovered salmonellae from liter quantities of raw milk containing 1 to 10 salmonellae. These results indicate that the swab culture technique has an advantage over the present method for isolating S. typhimurium from raw milk. In addition, larger quantities of milk can be examined by the swab technique than by the conventional method. Salmonellae were recovered from the swab inoculated with 100 organisms after 12 successive washings of 10 min each while stirring on a magestic stirrer (2 hr total washing time). This indicates that a swab can be suspended in a flowing stream of milk at a milk drying plant or suspended in milk delivery trucks; thus large quantities of milk can be examined with one swab. In contrast, only 1-liter samples can be conveniently examined by the conventional method. Additionally, many problems are encountered when shipping refrigerated quantities of liquid milk to the laboratory for examination, whereas swabs are easily shipped and require no refrigeration if shipped in tetrathionate broth. The secondary enrichments appeared to yield a better recovery of salmonellae than the initial enrichments with both the conventional method and the swab culture technique. This difference appeared to be greater for the swab culture technique at 37 C than at the 43 C incubation temperature, indicating that the secondary enrichment is of little value when using the swab culture technique at 43 C. The secondary enrichment would appear to be most advantageous when samples are held at room temperature for a period of time before examination (such as one might expect under field conditions). Incubation of the swab culture in tetrathionate at 43 C was found to be superior to incubation at 37 C. Other workers have observed success with the elevated temperature of 43 C for isolating salmonellae from sewage by using selenite broth (4) and feeds and poultry litter by using selenite-Brilliant Green-sulfapyridine broth (1). McCoy (7) found that tetrathionate broth incubated at 43 C was lethal to salmonellae. No evidence of such toxicity was noted in this study nor in other studies in this laboratory involving the isolation of salmonellae from fish meal and pork sausage by using tetrathionate broth (9,10). The use of duplicate plating media was advantageous in this study. Duplicate media, even if they were the same medium, would probably increase the chances of making a recovery, but it appears advantageous to use BS in conjunction with BGS in examining raw milk. Since raw milk 238 APPL. MICROBIOL. on May 4, 2020 by guest http://aem.asm.org/ Downloaded from contains a large number of lactose fermentors which tend to upset the acid base balance in a medium that contains lactose, a medium that does not contain lactose as a differentiating agent such as BS is advantageous. BGS agar, however, was advantageous in that it suppressed coliforms better than BS agar. The results of this study indicate that: (i) sampling raw milk with a cotton gauze swab and subsequent culture of the swab is a more sensitive method of isolating salmonellae than the conventional method, (ii) salmonellae are retained by the swab even after extensive washing, (iii) an incubation temperature of 43 C is better than 37 C for isolating salmonellae from the swabs, and (iv) both BGS agar and BS agar should be used as plating media. ACKNOWLEDGMENT We thank W. Jere Housworth, Center for Disease Control, for conducting the statistical analyses. ADDENDUM IN PROOF Subsequent to the completion of this study, 45 shipments of raw milk arriving at commercial milk plants were examined by the conventional method incubated at 37 C and the swab culture technique incubated at 43 C. S. typhimurium was recovered from one of these shipments. The isolation was made by the swab culture technique, but not by the conventional method.

v3-fos

2020-12-10T09:04:11.247Z

1971-06-01T00:00:00.000Z

237231007

s2

Toxicity to Chicks of Aspergillus and Penicillium Species Isolated from Moldy Pecans Isolates of Aspergillus chevalieri, A. flavus, A. ochraceus, A. repens, and Penicillium funiculosum and complexes of P. citrinum-P. implicatum isolated from moldy pecan meats were toxic to chicks. In the spring of 1967, a mold problem in stored pecans (Carya illinoensis Koch) was brought to our attention. The pecans involved were harvested in the fall of 1966 and stored in the shell for 4 months at 16 C under relatively humid conditions (>80%). The pecans appeared to be in very good condition externally. When the shells were cracked open, however, clouds of spores arose and fungal growth was evident on the meats. Analysis by thin-layer chromatography revealed that the pecan meats were contaminated with 75 ppb of aflatoxins (B. Doupnik, Jr., unpublished data). Subsequent to this finding, a report was published on aflatoxins in pecans (8). Because of recent concern over mycotoxins as public health hazards in foods and feeds, we conducted a study to determine the mycoflora of moldy pecans and the toxigenicity of the most prevalent fungi to chicks. Most of the pecan samples investigated were submitted to us by Ray Worley (Department of Horticulture, Georgia Coastal Plain Experiment Station, Tifton, Ga.). The remaining samples were obtained from various sources by the authors. Upon receiving a given sample, pecans were soaked (with shell intact) for 3 min in a 0.5 % (w/v) solution of sodium hypochlorite. The shells were then cracked open, and the meats were removed aseptically. The meats were cut into halves, soaked for 3 min in 0.5 % (w/v) sodium hypochlorite, and plated, two halves per petri dish, on either warm rose bengal-streptomycin-agar (9) or high salt-malt-agar (3). After 5 days of incubation at 28 C, fungal colonies growing from the pecan meat halves were enumerated and identified. Several of the Aspergillus and Penicillium spp. were identified by Dorothy Fennell (Northern Regional Research Laboratory, Peoria, Ill.). Two or three isolates of each of the 10 most prevalent fungi isolated from the pecan meats were screened for toxigenicity to chicks. Diets for the toxicity tests were prepared as previously described (4,5). Each isolate was grown singly in 2,800-ml Fernbach flasks containing 500 g of moist, autoclaved, cracked corn at room temperature (approximately 27 C). The flasks were shaken daily to reduce mycelial matting of the corn. After incubation for 2 weeks, each culture (isolate) was dried at 50 C for 15 hr, ground, and singly mixed with a 36% protein supplement (6:4, w/w) to form a diet. Each diet, representing a single isolate, was then fed ad lib to a group of chicks. Each group consisted of 10 1-day-old Babcock B-300 cockerels. A control group of chicks received sterile corn similarly treated and mixed. Water was provided ad lib to all groups. Initial body weights and body weights of surviving chicks were recorded and averaged for each group at 7 and 14 days of age. Each isolate was classified, according to its toxic effect, at the end of the 14-day screening period as follows: none, reduced growth rate, or lethal ( Table 1). The reduced growth rate classification arbitrarily included those isolates which depressed gains 20 /,; or more when compared to the controls. Data on gross lesions and histopathology were not taken as this was primarily a study to determine if toxigenic fungi were present in unshelled pecans. The diets of those isolates producing toxic effects (either reduced growth rate or lethal) were analyzed for aflatoxins and ochratoxins by using the method of Eppley (6). Only the diets of the two toxic isolates of A.flavus contained detectable amounts of aflatoxins, and only the diets of the three toxic isolates of A. ochraceus contained detectable amounts of ochratoxins. The toxigenicity of the other toxic isolates then is presumably due to other mycotoxin(s). Tests were also conducted to determine whether autoclaved, moist pecan meats would support ochratoxin and aflatoxin production by known toxigenic isolates of A. ochraceus and A. flavus. We found that autoclaved, moist pecans would support ochratoxin and aflatoxin production under our experimental conditions. Autoclaved pecans have previously been reported to support aflatoxin production (8) but not ochratoxin production. The only previous report on toxigenic fungi isolated from pecans is by Lillard et al. (8). Their report was related to a mold problem involving pecans in bakery products and dealt specifically with A. flavus group fungi (A. flavus and A. parasiticus). Although they undoubtedly encountered other fungi during their mycofloral assays, no mention was made of them. To our knowledge, this is the first report of the isolation of toxic isolates of A. chevalieri, A. ochraceus., A. repens, and P. funiculosum and complexes of P. citrinum-P. implicatwn from pecans. All of the above species, however, have previously been reported to be toxic to experimental animals. Isolates of A. flavus, A. ochraceus, and A. repens have been reported to be toxic to chicks and other animal species by several workers (1,2,4,10,11). Isolates of A. chevalieri and an isolate ofP. funiculosum have been reported to be toxic to mice (1,7,11). Both of the species involved in the P. citrinwn-P. implicatum complex have been shown to be toxic to rats and mice (7,12). Both species have also been shown to produce citrinin, a potent mycotoxin (12). We did not analyze our toxic diets for citrinin. It is apparent from this study that pecans can support the growth of fungi which may be toxigenic. Only limited information is available on the proper moisture and temperature conditions for storing unshelled pecans (13), and less is known concerning the relationship of these factors to fungal invasion of meats of unshelled pecans. In view of the possible health hazards, research to determine the proper moisture and temperature conditions for safe storage of unshelled pecans is of vital concern to the pecan industry.

v3-fos

2020-12-10T09:04:11.240Z

1971-12-01T00:00:00.000Z

237229956

s2

Minimal Growth Temperature, Sodium Chloride Tolerance, pH Sensitivity, and Toxin Production of Marine and Terrestrial Strains of Clostridium botulinum Type C Minimal growth temperatures of four marine and two terrestrial strains of Clostridium botulinum type C were determined in a laboratory culture medium, fortified egg meat medium (FEM), and in ground haddock. The inoculum equaled 2 × 106 viable spores per tube with five-tube replicate sets. The spores were preheated in aqueous suspension at 71 C for 15 min prior to inoculation to reduce toxin carry-over. Similar results were obtained in both substrates. Both the marine and the terrestrial strains grew at 15.6 C, but only the terrestrial strains grew at 12.8 C. None of the strains grew at 10 C during prolonged incubation. The sodium chloride tolerance and the pH sensitivity of the marine and the terrestrial strains were determined at 30 C. The basal medium consisted of beef infusion broth. The inoculum level equaled 2 × 106 unheated spores per replicate. Growth was inhibited at salt concentrations from 2.5 to 3.0%. The terrestrial strains were more pH-sensitive than the marine strains. Whereas the terrestrial strains failed to grow below pH 5.62, three of the marine strains grew at pH 5.10, but not at pH 4.96, during extended incubation. One marine strain grew at pH 5.25, but not below. FEM and proteose peptone-Trypticase-yeast extract-glucose medium permitted the production of high levels of botulinum toxin among four media tested. Toxin produced by the marine and terrestrial strains showed no increase in toxicity after incubation with trypsin. Michener and Elliott (13), who reviewed the minimal growth temperatures of various microorganisms, found no evidence of growth of Clostridiwn botulinum type C at temperatures below 10 C. Tanner and Oglesby (20) observed growth from a vegetative-cell inoculum in a laboratory culture medium at 15 C, but not at 10 C. From a spore inoculum, they noted growth and toxin production at 20 C, but not at 15 C. However, Tanner, Beamer, and Rickher (18) obtained growth from a spore inoculum in peas and in asparagus at 10 C, but they were unable to detect growth at 5 C, the next lower temperature tested. Castell (5) obtained type C growth in ground lobster and ground cod fillets at 25 C, although he was unable to demonstrate growth in either substrate at 3 C during 3 months of incubation. Beerens, Sugama, and Tahon-Castel (2) failed to obtain growth of two type C strains in a laboratory culture medium at 6 C. Although definite proof is lacking, it appears that all C. botulinum type C strains tested for psychrophilic ability have been isolated from terrestrial sources. Because type C exists in marine environments with botulinum types known to possess low-temperature growth ability, and because of cultural similarities to those types, it was considered that marine strains might show psychrophilic growth characteristics (16). Therefore, the principal objective of this research was to compare the minimal growth temperatures of marine and terrestrial strains of C. botulinum type C. In addition, work was conducted to determine their sodium chloride tolerance, pH sensitivity, and toxin-producing ability in various culture media. MATERIALS AND METHODS Type C strains. The origin of the terrestrial and the marine strains was previously described (16). The terrestrial strains are numbered 468 and 571; the marine strains are designated 6812, 6813, 6814, and 6816. Sporulation and standardization of suspensions. Spores of each strain were produced in FEM medium (Difco Egg Meat Medium fortified with additions of 1% yeast extract, ammonium sulfate, and glucose). As already described (16), aqueous suspensions were prepared and standardized by a deep-tube technique with beef infusion agar (BIA). The recovery medium was supplemented with 0.1% L-cysteine hydrochloride and 0.14% sodium bicarbonate by aseptic additions immediately before use. Colony counts were made after 24 to 48 hr of incubation at 30 C. Low-temperature growth studies. FEM, autoclaved at 121 C for 15 min, was used in 20-g quantities in screw-cap tubes (20 by 150 mm). A fish substrate was prepared from fresh haddock fillets (Melanogramus aeglefinus). The fillets were finely ground and distributed in 10to 12-g amounts in screw-cap tubes (16 by 125 mm). Air pockets in the substrate produced during filling were removed by centrifuging at a low speed. Tubes for incubation at 10 C and below were heated in flowing steam for 30 min; those for incubation above 10 C were autoclaved at 121 C for 15 min. After heating and cooling in cold tap water, the tubes were dried and held at 2 C for inoculation the next day. The inoculum level used equaled 0.1 ml/tube or 2 X 106 viable spores. Prior to inoculation, the spores were preheated at 71 C for 15 min. Five-tube replicate sets were inoculated per variable. The inoculated tubes and corresponding uninoculated controls were Vaspar-sealed and incubated at 15.6 C (60F), 12.8 C (55 F), lOC (50F), and7.8 C (46F). Incubated tubes of FEM medium and ground haddock were examined for gas production on a Monday, Wednesday, and Friday time schedule. After approximately 8 months of incubation at the highest temperatures showing no gas production, each replicate set was assayed for type C toxin and examined for growth by phase-contrast microscopy. The Vaspar was removed from each tube with sterilized cotton-tipped swabs. For the haddock substrate, the contents of each tube were blended with 90 ml of sterilized, prechilled sodium acetate buffer (0.5 M, pH 5.4). About 6 ml of the blend was transfered to a sterilized tube and held overnight at about 3 C; toxin was assayed the next day. For the FEM medium, 1 ml of the liquid portion was diluted with 9 ml of gelatin-phosphate buffer and the assays were run immediately. Duplicate white mice each were injected intraperitoneally with 0.5 ml of a sample. The mice were observed for botulinum symptoms up to 4 days before a sample was considered nontoxic. Sodium chloride and pH studies. The basal medium used in the sodium chloride and pH studies consisted of beef infusion (BI) broth. Disodium phosphate normally added to the medium was deleted. In the salt experiments, various concentrations of sodium chloride were dissolved in the basal medium on a weight basis. The medium was dispensed in 200-ml quantities in screw-cap bottles and autoclaved at 121 C for 15 min. Before use, sterile L-cysteine hydrochloride was added to the medium to a final concentration of 0.1%. Sterile 1 N sodium hydroxide was added to readjust the medium to pH 7.0 to 7.2. The balance. of the loss in weight due to autoclaving was restored to the nearest gram by the addition of sterile distilled water. In the pH experiments, the basal medium was adjusted from pH 6.0 to 5.0 at 0.2 pH unit intervals by the addition of dilute HCI. The medium was bottled and autoclaved as described above. The pH values cited in the tables represent determinations made after the incorporation of 0.1% L-cysteine hydrochloride. Five-tube replicate sets were inoculated per variable with 0.1 ml of unheated spore suspension (2 X 106 spores) per tube. About 10 ml of medium was poured aseptically into each inoculated tube. Each set of replicates was sealed with Vaspar and incubated at 30 C. Examinations for growth (gas and turbidity) were made daily for the first 2 weeks of incubation and then at less frequent times. Uninoculated control tubes, poured at the beginning of the experiment, were used to detect a possible change in pH of the medium during incubation; no pH changes occurred. Toxin studies. Cardella's medium (4) consisted of 40 g of Proteose Peptone (Difco), 20 g of Trypticase (BBL), and 10 g of glucose per liter; it was adjusted to pH 7.0 before sterilization. Jensen's medium (12) was slightly modified, consisting of 30 g of Lactalysate (BBL), 20 g of yeast extract (Difco), 3.5 g of sodium citrate, and 10 g of glucose. BI broth and FEM medium were described earlier. Jensen's medium, BI broth, and FEM medium were adjusted to pH 7.2 to 7.4, bottled, and autoclaved at 121 C for 15 min along with Cardella's medium. Toxin titrations were made at intervals during 1 month of incubation at 30 C. Serial 10-fold dilutions were prepared in gelatin-phosphate buffer. Duplicate white mice, weighing 18 to 20 g each, were intraperitoneally injected with 0.5-, 0.2-, and 0.1-ml volumes of the dilutions to obtain a minimal lethal dose (MLD) end point. Trypsin digestion. To determine the effect of trypsin on type C toxin, single tubes of FEM medium were inoculated for each type C strain and incubated at 30 C for 3 days. For trypsin digestion, 1 ml of the culture was mixed with an equal volume of 1.0% trypsin (Difco, 1:250) and incubated at 37 C for 1 hr. Serial 10-fold dilutions of the trypsinized and an untrypsinized culture were prepared and injected into mice as described above. RESULTS Tables 1 and 2 summarize the results of the miniimial growth temperature studies. FEM and ground haddock media gave similar results. Both the marine and terrestrial strains grew at 15.6 C, but only the terrestrial strains grew at 12.8 C. Type C toxin was verified in all of the tubes showing gas. The marine strains failed to grow at 12.8 C, and the terrestrial strains did not grow at 10 C or below during prolonged incubation. All inoculated tubes of FEM medium and ground haddock at the highest temperature showing no gas formation were examined microscopically for growth and assayed for botuLnum toxin by the injection of white mice. None of the tubes showed growth or. detectable toxin. The minimal growth temperature of strain 6816 in ground haddock was not determined, because the results in FEM medium showed that it had Table 1. When fewer than five tubes showed growth, the time of growth for each tube is given; where no range is shown, all five replicates showed gas production at the same time. a limiting temperature for growth similar to those of the other marine strains tested. The sodium chloride sensitivity of the marine and terrestrial strains is shown in Table 3. Vegetative-cell growth from a spore inoculum was shown by the development of turbidity and gas. Each strain grew in the presence of 2.0% salt, but only 468 and 6814 grew in 2.5% salt. None of the strains grew in 3.0% salt. Recovery of spores of each strain at the end of incubation from the 3.0% salt medium showed that some spore germination had occurred, although many spores were still refractile by phase-contrast microscopy. The limiting pH for type C growth is presented in Table 4. The terrestrial strains failed to grow belo'* pH 5.62. In contrast, three of the marine strains grew as low as pH 5.10, but not below. Strain 6812 was somewhat less pH-tolerant than the other marine strains. The effect of various media formulations on type C toxin production is shown in Table 5. In general, FEM medium gave the highest levels of toxin, although Cardella's medium was almost as good. During 1 month of incubation, the toxin remained relatively stable in each case. The pH of each medium at 3 days showed little change during 30 days of incubation. Jensen's and Cardella's media ranged from pH 5.6 to 5.8 at 3 days depending on the strain; BI broth showed pH 5.2 to 5.6, and FEM ranged from pH 5.8 to 6.5. The marine strains commonly gave a somewhat lower final pH than the terrestrial strains. The effect of trypsin on type C toxin was determined. The results were based on an MLD comparison; hence, a two-to threefold difference between a trypsinized and an untrypsinized titer was not considered significant. There was no conclusive evidence of activation with trypsin; with toxin from 468, an indication of possible inactivation with trypsin was observed (fourfold reduction). Mention should be made of the rapidity of mouse death after intraperitoneal injection with type C toxin. At high toxin levels, i.e., 100 MLD/ml and higher, deaths invariably occurred in less than 24 hr; at low toxin levels, deaths often occurred after 24 hr and up to 72 hr. No deaths beyond 72 hr were ever recorded. DISCUSSION Nonproteolytic strains of C. botulinum type B, type E, and type F are capable of growth and toxin production down to about 3 C (7,8,14). In contrast, it is generally accepted that type A and proteolytic type B are unable to grow at 10 C or below (13). Although type C coexists in a Based on an unheated inoculum of 2 X 106 spores per replicate and five replicates per variable. Range is shown in parentheses; whete no range is shown, all five replicates showed gas production and turbidity on the same day. Where fewer than five replicates showed growth, the time of growth for each tube is presented. Table 3. marine environments with the nonproteolytic botulinum types, neither marine nor terrestrial strains apparently possess psychrophilic growth characteristics. The minimal temperature for growth of C. botulinum type C is very near that accepted as limiting for growth of type A and proteolytic type B. The usual pattern that nonproteolytic strains of C. botulinum exhibit lowtemperature growth characteristics does not appear to be applicable to type C. It is usually recognized that about 10% sodium chloride (calculated as per cent brine concentration) is necessary to inhibit growth and toxin production of C. botulinum type A and proteolytic type B (1, 9,19). In contrast, type E growth is inhibited by a brine concentration of about 5.0% (15). Type C appears to be even less salt-tolerant than type E, its growth being inhibited by 3.0% salt. The lowest pH permitting growth and toxin production of C. botulinum is 4.7 to 5.0, based on numerous strains, inoculum levels, and culture media (11,15,21). Judged by the data presented here, there is no evidence to show that type C can grow at any lower pH than the other botulinum types. Cardella et al. (4) and Skulberg (17) reported that a proteose peptone-Trypticase-yeast extractglucose medium permitted the production of high levels of botulinum toxin by type C. Work presented here shows that both marine and terrestrial strains produce appreciable quantities of toxin in either Cardella's medium or FEM medium. Of course, Cardella's medium has the distinct advantage of being an aparticulate medium, in contrast to FEM. C. botulinum includes both proteolytic and nonproteolytic strains based largely on their ability or inability to digest coagulated egg albumin or meat particles. All available type A strains are reportedly proteolytic, whereas all type C, D, and E strains are nonproteolytic. Both proteolytic and nonproteolytic strains of types B and F are known, but most of the nonproteolytic strains are weakly proteolytic, as shown by the fact that they often hydrolyze gelatin. Until recently, it was generally accepted that only the nonproteolytic strains produced botulinum toxin that could be activated upon treatment with the proteolytic enzyme trypsin. The phenomenon of trypsin activation of botulinum toxin has been studied thoroughly since the first report by Duff, Wright, and Yarinsky (6) on type E toxin. It it commonly presumed that type E toxin is elaborated by the organism as a prototoxin (17). Since type E, unlike types A and B, lack strongly active proteolytic enzymes, the toxin is released by the organism principally in the prototoxin form that is commonly activated by trypsin. In contrast, toxin from the proteolytic strains usually does not show activation with trypsin except in very young cultures (3). The organism's cellular proteases presumably cause activation of the toxin in the same manner as trypsin causes activation of toxin of many nonproteolytic strains. It was recently shown that the proteolytic enzymes of some botulinum strains do not always activate the prototoxin; similarly, toxins produced by some nonproteolytic strains do not always show activation with trypsin. Iida (10) observed both situations in his studies. Two type C strains studied by Iida produced toxin that showed no significant indications of activation by trypsin. In this work, toxin produced by the marine and the terrestrial strains also showed no conclusive signs of activation when incubated with trypsin.

v3-fos

2019-03-28T13:43:10.307Z

1971-01-01T00:00:00.000Z

237231809

s2

Identification of a Pea Component Stimulatory for Heat-Stressed Putrefactive Anaerobe 59-123 Spores Pea extract contains a factor which improves recovery counts of heat-stressed putrefactive anaerobe spores in a complex medium up to threefold. The factor is heat-stable and nondialyzable. Most of the active principle is found in the precipitate which forms during storage of pea extract at 4 C. The precipitate disperses upon heating, is high in starch content, and retains activity after extraction with organic solvents and water. Treatment of pea extract with α-amylase results in complete destruction of the active principle. These observations indicate that starch is the factor in pea extract responsible for increased recovery counts of heat-stressed putrefactive anaerobe spores. Pea extract contains a factor which improves recovery counts of heat-stressed putrefactive anaerobe spores in a complex medium up to threefold. The factor is heat-stable and nondialyzable. Most of the active principle is found in the precipitate which forms during storage of pea extract at 4 C. The precipitate disperses upon heating, is high in starch content, and retains activity after extraction with organic solvents and water. Treatment of pea extract with a-amylase results in complete destruction of the active principle. These observations indicate that starch is the factor in pea extract responsible for increased recovery counts of heat-stressed putrefactive anaerobe spores. In 1937, Curran and Evans (2) reported that spores of Bacillus species surviving a damaging heat treatment are more exacting in their nutritional requirements for germination and outgrowth than before treatment. This observation has since been extended to include spores of Clostridium botulinum (1,4,9,10,13) and putrefactive anaerobe (PA) 3679 (5,(11)(12)(13). Thus, when determining the thermal resistance of spores, careful selection of a recovery medium is important if underestimates of survivor numbers are to be avoided. In studies with anaerobic sporeformers, which are by nature quite fastidious, many substances have been used to enrich recovery media. Popular among these additives are peas and pea extract (1,3,5,12,13). Although the enrichment value of peas has been well established the active component has not been identified. Therefore, a study of the factor from peas stimulatory for heat-stressed PA spores was undertaken with the hope that pea extract might be replaced in recovery media by a commercially available chemical. In addition, a knowledge of the nature of the stimulant may lead to a better understanding of the metabolism and early stages of heat inactivation of anaerobic sporeformers. MATERIALS AND METHODS Test organism. A suspension of PA 59-123 spores was obtained from the National Canners Association, Washington, D.C., and was stored at 4 C. Vegetative cells present in the spore suspension were inactivated by adding an equal volume of absolute ethanol and incubating for 1 hr at 25 C (6). Spores were recovered by centrifugation. Serial dilutions of stock suspensions were made in sterile water so that 0.2 ml of a heatstressed suspension yielded between 30 and 100 colonies on unenriched recovery medium. Heat stressing was effected by immersing 10-ml quantities of the spore suspension, contained in a screw-cap tube (20 by 150 mm), in boiling water for 8 min followed by rapid cooling in an ice bath. Preparation and assay of pea extract. Pea extract was prepared as described by Andersen (1), placed in either glass dilution bottles or dialysis tubing, and autoclaved for 15 min at 121 C. The response of heatstressed spores to various concentrations of the test material was measured in PA 3679 Agar. The composition of PA 3679 Agar was 10 g of tryptone (Difco), 5 g of dextrose (Fisher Scientific Co.), 1 g of yeast extract (Difco), 3 g of beef extract (Difco), 0.5 g of sodium thioglycollate (BBL), 1.25 g of K2HPO4 (Fisher Scientific Co.), 8.5 g of lonagar (Colab) and 1 liter of deionized water. This medium is T Best Agar, described by Wheaton and Pratt (12), without the soluble starch. For assay purposes, each plate contained 10 ml of medium, spores, and sterile deionized water or test substances, or both, in a total volume of 12.2 ml. Plates were incubated at 37 C for 60 hr in Case-Anaero Jars (Case Laboratories, Inc., Chicago, Ill.) containing an atmosphere of 90% nitrogen and 10% carbon dioxide (Matheson Co., Inc.). Before incubation, jars were successively (three times) exhausted to 26 inches (66 cm) of vacuum and flushed with the gas mixture to a slight positive pressure. To determine the relative stimulatory activities of pea extract and its fractions, all preparations were adjusted with sterile water to the volume of the original pea extract. The degree of stimulation was STIMULATORY PEA COMPONENT measured as the fold increase in recovery count caused by 1 ml, or fractions thereof, of the test substance over the count obtained with the same heat-stressed spore suspension on unenriched PA 3679 Agar. Fractionation of pea extract. Dialysis sacs (Fisher Scientific Co.) containiing 40 ml of pea extract were suspended in open flasks and autoclaved for 15 min at 121 C. Dialysate was obtained from pea extract by one of two procedures. Agitated dialysis was carried out in 2-liter quantities of sterile deionized water for 15 hr at 4 C. Sacs were transferred to a second 2-liter quantity of sterile deionized water and dialyzed for an additional 6 hr. Dialysate was concentrated to 40 ml by flash evaporation, sterilized by membrane filtration (MilliporeCorp., Bedford, Mass.), and assayed for stimulatory activity. The endofraction was recovered aseptically and likewise assayed. Alternatively, dialysate was obtained by vacuum dialysis. A white, flocculant precipitate developed in pea extract during storage at 4 C. The precipitate was removed from 20 ml of extract by centrifugation at 20,000 X g for 10 min and washed twice at 4 C with 20 ml of each of the following: acetone, anhydrous ether, 85% methanol, 95% ethanol, and water. The extracted precipitate was suspended in 20 ml of sterile water, heated for dispersion, and assayed for stimulatory activity and starch content. Starch determination. Starch concentrations were determined by a modification of the method described by McCready and Hassid (7). For standard curve construction, known amounts of com starch (Stein, Hall & Co., Inc., New York, N.Y.) were permitted to hydrate for 10 min in 1 ml of water. A 5-ml quantity of 1 N NaOH was added, and the mixture was incubated at 55 C for 5 min. Excess NaOH was neutralized with 0.5 N HCI. The volume was diluted to 100 ml with boiling water, and the temperature was maintained at 90 to 100 C for 5 min. A 3-ml sample was added to 96 ml of water and developed with 1 ml of iodine reagent (0.2% iodine, 2% potassium iodide). Absorbance at 650 nm was measured by using a Beckman DB spectrophotometer. Starch hydrolysis. Starch in fresh pea extract was hydrolyzed by the addition of 40 ,ug of a-amylase (Nutritional Biochemicals Corp., Cleveland, Ohio) per ml of extract. The reaction mixture was incubated at 25 C for 30 min. Disappearance of starch was followed by testing with iodine reagent. The reaction was terminated by autoclaving at 121 C for 15 min. Boiled enzyme added to pea extract and untreated pea extract served as controls. ca-Amylase-treated and untreated pea extracts were assayed for the ability to increase recovery counts of heat-stressed PA 59-123 spores on PA 3679 Agar. spore suspensions on PA 3679 Agar by approximately 75% (Fig 1). Inclusion of pea extract in the medium increased the count of heat-stressed spores by as much as threefold. Concentrations of pea extract greater than 20 % in PA 3679 Agar had no additional effect. Unheated spores showed no response to pea extract. RESULTS AND DISCUSSION The stimulatory component of pea extract was stable to boiling for 1 hr and autoclaving. Dialysis of pea extract did not diminish its activity (Table 1). Dialysate failed to improve recovery counts, suggesting that classical germinants such as L-alanine, glucose, and salts are not responsible for the pea extract effect. The precipitate which formed in pea extract during storage at 4 C is high in starch content and contains most of the stimulatory activity. Small amounts of starch and stimulatory activity remained in the supernatant. The precipitate retained 85%XO of its starch content and 73% of its stimulatory capacity after washing with a variety of organic solvents and water. None of the washings possessed the ability to improve recovery counts of heat-stressed PA 59-123 spores. The stimulatory activity of pea extract was compared with that of corn starch (Stein, Hall & Co., Inc.) and soluble starch (Difco). Maximal recovery counts were achieved with 0.15%o corn starch, 0.25% soluble starch, or 20% pea extract (0.12% pea starch) present in PA 3679 Agar. Incorporation of combinations of suboptimal levels of pea extract, corn starch, and soluble starch into the recovery medium resulted in an additive effect up to a maximal count. The maximal count achieved with combinations of starches did not exceed the count obtainable with optimal additions of the individual starches. Starch in fresh pea extract was completely hydrolyzed by the action of at-amylase. Pea extract treated with ca-amylase possessed no ability to improve recovery counts of heatstressed PA 59-123 spores on PA 3679 Agar (Fig. 2). Pea extract to which boiled enzyme had been added and untreated pea extract contained high levels of starch and stimulated recovery counts of heat-stressed spores approximately threefold. Glucose and maltose, hydrolysis products of starch, had no effect on recovery counts when added to PA 3679 Agar. Thus, starch appears to be the sole component of pea extract responsible for increased recovery counts. Starch (8,9,10), charcoal, or serum albumin (8, 10) are known to improve survivor counts of heat-stressed Bacillus and Clostridium spores. These additives are thought to act by adsorbing inhibitors from the medium (10). The nature of the inhibitor has not been established conclusively, but long-chain, unsaturated fatty acids (4) or their oxidation products (11) have been implicated. In the present study, slightly higher levels of corn starch and soluble starch than of pea starch were required for maximal stimulation. This discrepancy may be due to an underestimate of the amount of starch in pea extract or to the fact that pea starch contains a relatively high proportion of amylose (14) which is more active than amylopectin in improving recovery counts. Alternatively, extraction and drying procedures may render commercially available soluble and corn starches less effective.

v3-fos

2020-12-10T09:04:16.721Z

1971-08-01T00:00:00.000Z

237231367

s2

Action of Pseudomonas fragi on the Proteins of Pig Muscle Considerable salt-soluble protein degradation was observed in pork muscle inoculated with Pseudomonas fragi. During a 20-day incubation period at 10 C, the samples proceeded to rank spoilage or putrefaction. There was a large decrease in the salt-soluble protein fraction and a corresponding increase in nonprotein nitrogen. Disc gel electrophoretic patterns showed that breakdown of the salt-soluble proteins had occurred after incubation for 20 days. During incubation for 10 days at 10 C, P. fragi produced large amounts of extracellular proteolytic activity in ground pork. Most of the proteolytic activity appeared immediately after spoilage occurred. However, a significant increase in the ability to hydrolyze casein and a slight increase in the ability to hydrolyze denatured hemoglobin occurred prior to spoilage. Certain metabolic changes caused by bacterial growth in fresh meat which lead to spoilage have not been elucidated. In particular, there are conflicting reports concerning the role of muscle proteins in the spoilage process. According to Jay (12), low-temperature spoilage occurs in the absence of significant proteolysis. Several species of bacteria produced no alterations in the myofibrillar proteins of pork as determined by density gradient centrifugation, gel filtration, and electrophoresis (21). Metabolism of low-molecular-weight nonprotein molecules by spoilage microorganisms has been reported (14,18). A number of strains of Pseudomonas fragi capable of producing strong off-odors in fish muscle were shown by Castell and Greenough (7) to be nonproteolytic. These authors noted, however, that this might be the result of the method by which the cultures were selected and that proteolytic strains could have been missed. Although spoilage may normally be initiated by the utilization of low-molecular-weight compounds, there is evidence that proteolysis occurs with some bacteria. Protein breakdown in fishmuscle juice was observed (18) after spoilage by Pseudomonas species had become evident. Hydrolysis of sarcoplasmic proteins was reported by Hasegawa et al. (9,10). These workers reported that two species of bacteria commonly associated with meat spoilage, P. fragi and Leuconostoc mesenteroides, had the highest proteolytic activity (10). Also, Borton et al. (4,5) found that P. fragi caused an increase in the water soluble and nonprotein nitrogen content of pig muscle and a decrease in both the salt-soluble and insoluble protein content. The myofibrillar fraction of beef spoiled by a mixed flora was found to contain two new antigenic species (20). A number of workers (2,6,13) have observed that Pseudomonas organisms constitute the predominant group contributing to the spoilage of fresh meat. Thus, the present investigation was undertaken to determine the extent and nature of proteolytic activity by P. fragi. MATERIALS AND METHODS Sampling procedures, inoculation, and bacterial counts. A pure culture of P. fragi (ATCC 4973) was used in this experiment. Aseptic sample preparation, inoculation, and bacterial counts were the same as those reported by Hasegawa et al. (10). Extraction of proteins. The procedure for extracting sarcoplasmic and myofibrillar protein was based on that of Helander (11). Duplicate samples of ground pork were placed in cold extracting solutions and homogenized. Each sample was homogenized and extracted three times. Extraction of the salt-soluble proteins was difficult due to the high fat content of the ground pork (8 to 18%G). Soluble nonprotein nitrogen was obtained after precipitation of the water-soluble proteins with an equal volume of ice-cold trichloroacetic acid (20%, w/v). Chemical analyses. The nitrogen content of the protein and nonprotein nitrogen fractions was determined by micro-Kjeldahl analysis followed by nesslerization (17). Fat was determined gravimetrically on an ether extract of the dried tissue (1). Trichloroacetic acid-soluble peptides were assayed in water extracts by the biuret reaction as follows. The water-soluble protein was precipitated with trichloro-224 ACTION OF P. FRAGI ON PROTEINS acetic acid, and the filtrate was neutralized with potassium hydroxide. A 2-ml sample of the standard or analytical solution was mixed with 0.2 ml of concentrated biuret reagent (E. M. Reagents, Brinkmann Instruments, Inc., Westbury, N.Y.). Color was allowed to develop at room temperature for 30 min, and absorbance was read on a spectrophotometer at 545 nm. The tripeptide glycylglycylglycine (Mann Research Laboratories, New York, N.Y.) was used as a standard. The intensity of the biuret reaction varied depending upon the number of peptide bonds in a molecule and upon the constituent amino acids. Ammonia was determined in the nonprotein nitrogen fractions by the direct colorimetric method of Okuda et al. (22) as modified by McCullough (19). A Coming model 12 pH-meter was used to determine the pH on a 1-5 homogenate of meat with distilled water. Disc gel electrophoresis. Myofibrillar proteins were prepared from the residue after aqueous extraction of the meat sample. The basic disc gel electrophoretic system of Davis (8) was used. The spacer gel and the running gel were prepared by the method of Jolley et al. (15), both containing 7 M urea. Acrylamide was added in the form of Cyanogum. The gels were stained with a solution of 0.36% Buffalo Black NBR (naphthol blue black) in methanol-acetate-water (5:5:1, v/v) and destained electrically. Determination of spoilage. A panel of four judges was trained prior to the experiment. Two samples of pork muscle were presented to it. One of the meat samples was inoculated with P. fragi, and the other was an aseptic control. During spoilage of the inoculated sample, the judges became familiar with the off-odors associated with spoilage by P. fragi. For the experiment, the panel of four trained judges was presented with inoculated and aseptic coded meat samples. The tests were conducted in individual booths equipped with red lights to remove the effect of color differences. Each day during the incubation period, the panel was asked to determine the acceptability of the samples by odor evaluation. Extraction of proteolytic activity. The ground pork was extracted with 1.5 volumes of potassium phosphate buffer (0.1 M, pH 8.0) by stirring for 15 min and then centrifuging (7,500 X g, 30 min). The supernatant fluid was decanted through cheesecloth and dialyzed against potassium phosphate buffer (0.03 M, pH 7.5) for 20 hr at 4 C. Assay for proteolytic activity. A 1-ml amount of meat extract was incubated with 1 ml of substrate solution at 37 C for a period of 1 to 5 hr. During this time, the reaction mixtures were shaken automatically (180 excursions per min). The reaction was terminated by adding 2 ml of 5% (w/v) trichloroacetic acid. After 15 min, the precipitated protein was removed by filtration through Whatman no. 2 filter paper. Blanks were treated in the same manner, except the trichloroacetic acid was added immediately before the meat extract. All assays were performed in duplicate. Activities were expressed as the difference in absorbance (280 nm) of the trichloroacetic acid filtrates of the blanks and assay mixtures. The RESULTS Protein breakdown in spoiled muscle. The growth ofP. fragi and its effect on the nitrogenous components and on the pH of pork at 0, 8, and 20 days after inoculation are shown in Table 1. No significant changes were observed after 8 days in the myofibrillar, sarcoplasmic, or nonprotein nitrogen fractions. Considerable protein breakdown was evident after 20 days, at which time the amount of myofibrillar protein in the inoculated pork (5.3 mg of nitrogen per g) had decreased to about one-third of its initial value. Separation of the residual myofibrillar protein after 20 days from the water-soluble fraction required an increase in the relative centrifugal force from 1,400 X g to 7,500 X g. The quantity of sarcoplasmic protein in the inoculated pork did not change significantly during 20 days of incubation. However, the quantity of nonprotein nitrogen (13.9 mg of nitrogen per g) increased more than threefold. The increase of nonprotein nitrogen consisted principally of peptides (4.37 mg of nitrogen per g) and ammonia (4.43 mg of nitrogen per g). The high pH (7.99) of the spoiled pork after 20 days of incubation was presumably due to the high concentration of ammonia present. The results of disc gel electrophoresis of the myofibrillar proteins at 0, 8, and 20 days are presented in Fig. 1. The patterns for the inoculated pork show that disintegration of the myofibrils occurred during incubation. The initial patterns (A) from inoculated and aseptic pork are identical. After 8 days, the myofibrillar proteins from the inoculated pork showed less distinct, lighter patterns than those from aseptic pork (B). Complete breakdown of the pattern was observed after 20 days of spoilage (C). The normal pattern for the myofibrillar proteins was replaced by numer- ,tchange in Production of proteolytic enzymes by Pseudomonas fragi. In this experiment, sampling was carried out daily from 0 to 10 days. Bacterial numbers did not increase in the inoculated meat until after the second day (Fig. 2), with the maximum count being reached on day 6. Bacterial growth of the inoculated samples was considerably faster in this experiment than in the previous one. There was no bacterial growth in the uninoculated meat samples. The pork was considered ..... spoiled when the panel decided unanimously that it was unacceptable due to the off-odors liberated. The inoculated meat was first considered objectionable on the fifth day of incubation, which is considered as the onset of spoilage and :,-3 is indicated by an arrow in Fig. 2-4. The uninoculated meat did not spoil during the experiment. Results demonstrate production of one or more proteolytic enzymes in pork undergoing spoilage by P. fragi. Extracts of inoculated meat were Wcg8-J found to contain high proteolytic activity. Although hemoglobin was less susceptible to hydrolysis than casein, the production of proteolytic C activity followed a similar pattern with both substrates. Figure present. However, immediately after spoilage, a very high level of proteolytic activity was reached. None of the extracts of aseptic meat showed any increase in the ability to hydrolyze casein during l0000-the 10-day period of the experiment. The low level of proteolysis in the aseptic samples may be due to the residual activity of the muscle cathepsins at the neutral pH of the assay medium. Results using hemoglobin as a substrate are -5,000 shown in Fig. 4. Immediately after spoilage, there was a very large increase in proteolytic activity. Prior to spoilage, the inoculated meat extract showed a small increase in the ability to hydrolyze _ XI hemoglobin. This occurred between days 3 and pork by P. fragi. Similar degradation presumably went undetected in the present study because the loss of sarcoplasmic protein was compensated for by the release of water-soluble fragments from the myofibrillar protein fraction. The demonstration of proteolytic enzyme production in pork by P. fragi indicates the mechanism by which protein hydrolysis occurs. The appearance of proteolytic activity was associated with the onset of spoilage. The development of proteolytic activity before the onset of spoilage may be of considerable significance. Although the quantity of enzyme produced at this stage was relatively small, its concentration on the surface of the meat presumably influenced bacterial growth and the production of off-odors. Immediately after spoilage there was a large increase in proteolytic activity, as indicated by the greatly increased ability of the meat extracts to hydrolyze casein and hemoglobin. At this time, the bacterial proteolytic enzyme(s) were present in high concentration and penetrated deep into the meat. The total sample was then considered rankly spoiled or putrid. It is not surprising that maintenance of a large number of viable cells would necessitate utilization of the prime source of energy and nitrogen in the medium, that is, the proteins. Results suggest that the proteolytic enzyme(s) are extracellular because the method used to extract the proteolytic activity involved mild treatments, insufficient to disrupt bacterial cells. Liberation of extracellular proteolytic enzyme(s) by P.Jragi was previously reported (16). However, from the present data, it was not possible to decide whether the enzymes were secreted by living cells or released by autolysis of dead cells. Evidence to indicate that the living cell secreted the enzyme(s) has been obtained (T. R. Dutson, Ph.D. Thesis, Michigan State Univ., East Lansing, Mich., 1971). Electron micrographs have shown myosin breakdown to occur at a distance from the bacterial cells, whereas the cells themselves formed and released globules into the medium. The globules may have contained the proteolytic enzyme(s). However, further work is necessary to establish the method by which the enzymes are released into the medium.

v3-fos

2018-04-03T05:36:17.554Z

1971-05-01T00:00:00.000Z

43421078

s2

Initiation of staphylococcal growth in laboratory media. The effects of pH and NaCl concentration on the probability of aerobic growth initiation in Brain Heart Infusion broth at 30 C by five staphylococcal strains producing enterotoxins A, B, C, and D were studied in a factorial design experiment. Statistical analysis of the data indicated: (i) significant effects of pH, NaCl, and strain on the probability of growth; (ii) diverse effects of NaCl with various pH levels and strains; (iii) essentially a linear relationship between NaCl concentration and probability of growth initiation when data for all strains were pooled; (iv) the relationship between NaCl concentration and probability of growth initiation varies from linear to sigmoid, depending on the pH of the broth, when the statistically untreated (raw) data are plotted for each strain. Equations were derived which relate the decimal reductions of the number of cells of a staphylococcal population to the concentration of NaCl and pH of broth to which the population was exposed. From the equations, the probability that one cell is capable of initiating growth can be calculated. The impact of these types of studies on the development of realistic staphylococcal standards in foods is discussed. The effects of pH and NaCI concentration on the probability of aerobic growth initiation in Brain Heart Infusion broth at 30 C by five staphylococcal strains producing enterotoxins A, B, C, and D were studied in a factorial design experiment. Statistical analysis of the data indicated: (i) significant effects of pH, NaCl, and strain on the probability of growth; (ii) diverse effects of NaCl with various pH levels and strains; (iii) essentially a linear relationship between NaCl concentration and probability of growth initiation when data for all strains were pooled; (iv) the relationship between NaCl concentration and probability of growth initiation varies from linear to sigmoid, depending on the pH of the broth, when the statistically untreated (raw) data are plotted for each strain. Equations were derived which relate the decimal reductions of the number of cells of a staphylococcal population to the concentration of NaCl and pH of broth to which the population was exposed. From the equations, the probability that one cell is capable of initiating growth can be calculated. The impact of these types of studies on the development of realistic staphylococcal standards in foods is discussed. The effects of heat and radiation on bacterial destruction have been studied extensively, and probabilities for survival and growth, especially of Clostridium botulinwr spores, have been estimated. As a result of such studies, a standard heat treatment for low-acid canned foods has been established (18) which will reduce C. botulinum spores by a factor of 1012 (12 decimal reductions or 12D values). This 1:1012 probability of C. botulinwn spore survival is considered a minimum safety requirement. The effect of other preservation methods, such as acidification, drying, curing, and smoking, have not been expressed in terms of decimal reduction or inactivation values. If such values were known, it would be possible to estimate minimum standards for cured and smoked vacuum-packed foods that would achieve a safety level comparable to that of canned foods. Several researchers (2, 6, 10-16) have described the effect of NaCI and pH on Staphylococcus aureus; but quantitative data are still lacking. Some preliminary work which included C. botulinum has been published (6,17). The present study is concerned with estimating the probability of initiation of aerobic staphylococcal growth in broths. Similar studies of growth and enterotoxigenesis in various meats are published separately in the following note (8). MATERIALS AND METHODS Preparation of experimental broths. To prepare the broths, 37 g of dehydrated Brain Heart Infusion (BHI) broth (Difco) was dissolved in 700 ml of boiling distilled water. After the broth was cooled to room temperature, an additional 100 ml of water was added to make the basal medium. To provide the broths with various concentrations of salt, 80-ml portions were placed in beakers, and NaCl was then added in amounts of 0, 4, 8, 12, and 16 g per beaker. After the NaCl was dissolved, the pH was adjusted to the desired values by adding 0.5 N NaOH or 0.5 N HCI. The values were checked with a Beckman Expandomatic pH meter equipped with a Beckman H2 glass electrode. The broths were autoclaved in 100-ml volumetric flasks (15 min at 121 C under 15 psi of pressure) and then cooled. The volume was brought to 100 ml with sterile distilled water. Overnight BHI broth cultures of these strains were inoculated into 25 ml of BHI broth containing 0.25% Tween 80. The fresh cultures were incubated at 37 C on a reciprocal shaker for 4 hr. The cultures were then centrifuged, the cells were washed once with saline, and concentration of cells was adjusted to an optical 934 STAPHYLOCOCCAL GROWTH IN LABORATORY MEDIA density (OD) of 0.3 to 1.0 at 660 nm, by using a Spectronic-20 colorimeter (Bausch & Lomb). Nine tubes each containing 9 ml of broth were prepared from each type of experimental broth. A 1-ml amount of the cell suspension was added in the first tube, and 10-fold serial dilutions of the suspension were added to the other tubes. Three portions of 2 ml each were transferred with a sterile syringe from each of the nine tubes to 2-ml screw-cap vials. The caps were put on loosely and the vials were placed in 2-lb coffee cans. In each can, there was a vessel containing a brine of the same NaCl concentration as the broth. The cans were closed with plastic lids and placed in the aerobic incubator at 30 C where they were left for 20 days. Every other day, vials with growth (turbidity) were removed and recorded. From the presence or absence of growth in the 27 vials prepared for each salt-pH combination, the most-probable number of cells which had initiated growth was calculated by the technique of Fisher and Yates (5). The number of staphylococcal cells present in the cell suspension used as inoculum was determined by plating on cow blood-agar (BHI base) in duplicate. This number was always between 107 and 9 X 107. Statistical methods. The experiments, arranged in a factorial design (19), involved five staphylococcal strains, five NaCl concentrations (1,4,8,12, and 16%), and six pH values (4.7, 5.1, 6.1, 7.0, 7.8, and 8.9). For the statistical evaluation of the effects of strain, pH, NaCl, and their interaction upon staphylococci, the logarithm (log) of the ratio RI/RG was used for each broth and strain condition, where RI is the number of cells in the inoculum and RG is the number initiating growth. This log represents the number of decimal reductions of a staphylococcal population resulting from its exposure to a particular broth environment. The probability that one cell will initiate growth in such an environment can be calculated from the formula P = 1/antilog [log (RI/RG)] = RESULTS Statistical analysis of the data indicated the following effects and interactions. (i) Strain, pH, and concentration of NaCl all significantly affected the size of the log decrease of the staphylococcal population. (ii) The effect of NaCl varied with pH levels and by strains. (iii) When data for all strains were pooled, NaCl concentration and size of log decrease were related linearly. (iv) When the raw data for each strain were plotted, the relationship between NaCI concentration and log decrease of the population varied from linear to sigmoid, depending on the pH of the broth (Fig. 1). The above four observed effects and interactions apply also to the probability of growth. From the factorial and multiple regression analysis, the following five equations were derived for the five staphylococcal strains used, .47, respectively. Approximate 95% confidence contours for a specified log reduction can be obtained by using Ye i 2 SE. Using the equations, response curves have been constructed for each strain relating pH, NaCl, and log reductions. In Fig. 2 the curves indicate the combinations of pH and NaCl which will reduce populations of strains S-6, 137, 243, and 472 by 1, 2, 3, 4, 5, and 6 logs. The range of NaCl and pH combinations which will decrease populations of all five strains by 3, 4, 5, and 6 logs is indicated in Fig. 3. It is obvious from the figure that the response of the various strains to NaCl concentrations is becoming more uniform (narrow) as the pH approaches values between 7 and 8. This more uniform response of strains exposed to optimum pH is also apparent in Fig. 1. Response curves and the approximate 95% confidence limits for 6-and 3-log reductions of strain 243 are presented in Fig. 4 for comparison. As indicated in this figure, there is a zone of pH and NaCl combinations which will result in 3to 6-log reductions, whereas the other two zones combinations are likely to result only in large (6-log) and in small (3-log) reductions of populations of strain 243. DISCUSSION The effect of various environmental conditions on staphylococcal growth in culture media and in foods has been studied extensively (2,7,(9)(10)(11)(13)(14)(15), and certain limited standards for staphylococci in foods have been established (1,4). The present study is one of a series aiming toward the accumulation of data which eventually will help in establishing realistic staphylococcal standards. To obtain basic information, we have studied the effects of two important factors, NaCl and pH, on the log reduction of staphylococcal populations inoculated in Brain Heart Infusion broth. The probability that one cell can initiate growth can be calculated from the derived equations which relate NaCl concentration and pH of the medium to log reductions of a staphyllococcal population exposed to this environment. Thus, for a broth at pH 6.0 with 5% NaCl, the log decrease for the strain S-6 incubated aerobically is 1.69, the antilog is 49, and the probability of initiating growth is Y9 or 2.04%. On the basis of data reported previously (7), the same strain incubated anaerobically in the same broth will have a log decrease of 2.92, and the probability of initiating growth is only 0.12%. The effects of aerobiosis (present findings) versus anaerobiosis (7) on the combinations of pH and salt which will decrease populations of strains 137, 243, S-6, and 472 by 3 and 6 logs are further compared in Fig. 5. In general, there is close agreement between the present findings and the data published previously on the effects of NaCl and pH on staphylococcal growth, indicating that the experimental techniques are comparable. The findings of the present and earlier studies are summarized as follows. (i) There is a decreased rate of growth of food-poisoning staphylococci when exposed to media with concentrations of NaCl increased from 0 to 20% (7,10,13,14). (ii) Increasing the NaCl concentration of a broth requires more concentrated inocula for initiation of growth (14). (iii) Higher concentrations of NaCl and extreme pH values prevent growth, or delay it, or diminish the total amount, depending on concentration of inoculum and time of incubation (6). (iv) Smaller concentrations of NaCl are required to inhibit initiation of staphylococcal growth at pH values remote from optimum (12). (v) There is better staphylococcal growth aerobically than anaerobically. The magnitude of the effects of the two conditions varies with strain and, at some limited pH values, appears to be reversed. (vi) The effects of pH and NaCl on staphylococcal growth vary with the strain and the type of medium used (2,(14)(15)(16). (vii) Different investigators have used different staphylococcal strains, inocula, and media (2,7,10,11,(13)(14)(15)(16). Consequently, minor disagreements in present and past findings in the reported values of upper NaCl concentrations and of pH limits permitting staphylococcal growth are understandable. Certain limited standards for staphylococci in foods have been established (1,4). These standards tolerate 0 to 1,000 staphylococcal cells per g of food. However, they vary with agency and with state and country and are based on research data not analyzed statistically. A suggested tolerance of less than 1,000 cells/g probably represents a contamination level that can easily be met in most commercial food preparations. Any NaCl-pH combination (level) that will reduce the probability of growth initiation by a factor of 106, therefore, seems reasonable. The problem then is to find what combinations of NaCl and pH will have such an effect with a probability of safety of over 95%. Some results concerning only strain 243 are presented in Fig. 4. In this figure, any combination of NaCl and pH outside the outer curve has a 95 % probability of giving at least a 6-log decrease in the number of staphylococcal cells. Such calculations may be biased, of course, because we have extrapolated results from our data to an area where we do not have experimental data. In the experiments, our most concentrated inoculum was 9 X 107 cells/ml, and here we are talking about log decreases in the range of 6 + 2 SE (7.83 to 8.78 logs) for all five strains used. This was the reason that decimal reductions due to the effect of 16% NaCl were not considered in the derivation of the equations. The maximum inoculum used was not enough to measure the full effect of 16% NaCl concentration on the initiation of staphylococcal growth in broths. The present data can only indicate, with 95% confidence limits, the combinations of NaCl and pH which will decrease populations of all five strains by 6 logs, less 2 SE (6 logs -2 SE = 3.22 to 4.17, depending upon strain). To obtain more realistic results, the present studies should be supplemented not only with other studies in which concentrated inocula have been used but also with studies in which appropriate food items serve as culture media. The use of such media will eventually allow the development of equations based on quantitative characteristics of foods and permit accurate predictions of the probability of initiating staphylococcal growth. The results of the first series of these types of experiments have been reported (8). Studies on a third food factor, nitrite, have been interrupted until further data are accumulated on the reactions and stability of nitrite in laboratory media and foods. ACKNOWLEDGMENTS This investigation was sponsored by the Food Protection and Toxicology Cetnter of the University of California and was supported by Public Health Service grant FD 00103 from the Food and Drug Administration.

v3-fos

2017-07-29T04:24:52.146Z

1971-10-15T00:00:00.000Z

9521720

s2

Hereditary osteopetrosis in aberdeen-angus calves. II. — Genetical aspects SUMMARY Evidence is presented to indicate that congenital osteopetrosis in 3 herds of Aberdeen-Angus cattle may be inherited as a recessive autosomal trait. INTRODUCTION A premature stillborn purebred Aberdeen-Angus calf with a short lower jaw and protruding tongue was the first of 23 such calves born in three herds during the i 9 6o's. Detailed pathologic examination revealed osteopetrosis, a disturbed relationship between bone formation and resorption (LEIPOLD et al.,I97I ). A similar pathologic condition was described in 2 other herds of the same breed (T HOMPSON , i 9 66 ; LE Iror,n et al., i 97 o). To be presented here is the evidence pertinent to the inheritance of bovine osteopetrosis. I. -MATERIALS AND METHODS The abnormal calves were reported in conjunction with a previously described Foa g '-temi study of the nature and causes of congenital defects in animals (H USTON and WEnxDE!,. m958;;. ;. L EIPOLD and H US TO N , 19 68 a). Pertinent information was secured by personal visits to the farms and by mail inquiries. Genetic analyses follow the rationale described by Moxxorr ( 19 6 2 ) for man, with certain simple modifications for differences in cattle family structure. Usually full-sib families of cattle are very small and relatively infrequent while three-fourths-sib families are large and frequent. A three-fourths-sib family often includes several full-sib families. Furthermore, several threefourths sib families may be part of a larger paternal half-sib family. For a recessive trait, a single proband in a three-fourths-sib family identifies the common parent but cannot indicate which of the parents of the other parent (usually the proband's maternal grandparents) contributed a recessive allele. A second proband usually identifies that common grandparent. Thus, examination of three-fourths-sib families with two or more affected calves provides a basis for testing segregation ratios, after appropriate adjustment for exclusion of families containing fewer than two affected progeny. However, when several three-fourths-sib families all are part of a half sib family, a subfamily containing a single affected progeny furnishes useful information. The conditional probability of each offspring being affected was computed under specified assumptions. These were summed and compared with the observed number by an approximate x 3 statistic (L E I POLD and H UST ON, 19 68 b). Complete ascertainment was assumed. II. -RESULTS A. -Herd I In this artificially inseminated herd of about 950 registered and grade Angus cows calving annually, the herdsman thought the first osteopetrotic calf (fig. i, no. i) may have been born in the early ig6o's. In the calving season from September 19 66 through March 19 6 7 , no abnormals were noted. In the calving season a year later, 5 abnormals (fig. i, nos. 2 to 6) occurred among 939 calves. At last report in the next year, 5 more have been born (fig. i, nos. 7 to II ). As reported elsewhere (I, W ror, D et al., I g 7I ), 7 of the I calves' average gestation was 2 6 2 days. All but one were born dead ; the single live calf was carried 2 68 days and killed shortly after birth. Close relationships among the calves are shown in figure i. Of the 4 calves of recorded sex, two were males and two, females. All calves but nos. q., 7 and his maternal half-niece 8 were related through both parents to either bulls A or B. In pedigrees containing 6 ancestral generations, bulls A and B had 3 common ancestors, two of which were father and son. The coancestry of A andB through that ancestral pair was . 007 ; through the remaining common ancestor, . 001 . ' Though 4 affected calves descended from bull G, none of his several thousand progeny was affected. 2 ). Another calf was from an unrelated dam. C. -Herd III In this herd of 8 0 -95 grade Angus cows, 8 abnormal calves were born, most 3 to 6 weeks premature. Seven sired by Bull Y were born in 19 68 and one by a registered bull of unknown ancestry was born in ig6g. From 19 6 5 to 19 6 7 , Bull Y mated to 35 to 45 cows yearly produced normal calves. In 19 68, he produced the abnormal calves. Some of Bull Y's mates likely were daughters of a previous herd sire who sired no abnormals when mated to ten of his own daughters. Bull Y was colaterally related to both Bulls A and B in Herd I. The sire of the other abnormal calf produced 29 other normal calves. III. -DISCUSSION The data of Herd I provide the most precise evidence concerning inheritance. Because normal parents produced only a few abnormal progeny and many normal progeny, the trait seems to behave as a recessive trait. The 50 matings in table i yield evidence appropriate for testing a recessive hypothesis. The probability of each of the 50 matings yielding an affected offspring was computed under the following conditions. Affected animal no. i ( fig. i) was excluded as a proband because the herdsman's recollection of that calf was uncertain. Affected animals 2 to 6 were included in table i and served as probands as follows : no. 2 for ancestors C and A ; no. 3 , ancestors D, A, B ; no. 5 ancestors F, E ; no. 6, ancestors E and B. For families involving A or B, only those compound families with two or more affected were included. For families involving E, only those having i affected from daughters of B ; or 2 affected, one each sired by D or F, or two sired by F, were included. For families involving F, only those with one or more affected were included. The single proband families involving affected animals nos. 2 and 4 were included as probands though they contribute no information to the segregation ratio. Under those conditions, the expected number of abnormal animals in table i was 9 . Only 5 were reported, leaving a deficiency of 4 affected animals ( X 2 = 2 . 50 , . 20 > P > .io.)The deficiency may have resulted from sampling, incomplete ascertainment or incomplete penetrance. Other evidence is consistent with recessive inheritance. Bulls A and B were related and thus might have received copies of the same mutant gene. Though the dams of animals no. 4 and no. 7 were thought to have been related to ancestors of A and B, their recessive genes could have been identical in state rather than by descent, a circumstance requiring a somewhat higher frequency of the gene in the breed or an older mutation. The evidence in Herd II accords suitably also, except for the single « unrelated » dam. She could have received her copy of the mutant gene from the same ancestral source as animals of Herd I since that source could have been a more remote ancestor than appeared in the owner's pedigree. The limited evidence from Herd III also accords with recessive inheritance. The coancestries of Bull Y with Bulls A and B were oii and.00 4 respectively. The cases from Saskatchewan, Canada (I,W Po!,D et al., 1970 ), descended from a son of bull A and were produced by sire-daughter matings. Because the deficiency in number of affected calves in table i was fairly large, evidence of embryonic death was sought in breeding records. The number of inseminations/conception for mates producing abnormal calves was slightly, but not significantly, higher than characteristic for the herd. The affected calves also were calved somewhat, but not significantly, later in the season than characteristic for the herd. However, available evidence is inadequate to achieve greater resolution on this point. Re!u pou y publication en août 1971.

v3-fos

2018-04-03T03:36:24.874Z

1971-12-01T00:00:00.000Z

7365822

s2

Effect of Newcastle disease on serum copper, zinc, cholesterol, and carotenoid values in the chick. Experimentally induced Newcastle disease virus infection of chicks, with a mortality index of 48%, was accompanied by increased concentrations in serum of copper and cholesterol and decreased concentrations of zinc and total carotenoids. These changes distorted, or were superimposed upon, the rhythmic variability in the normal serum concentrations of each of these moieties. Changing values for copper, zinc, and cholesterol became apparent before any overt signs of disease. Experimentally induced Newcastle disease virus infection of chicks, with a mortality index of 48%, was accompanied by increased concentrations in serum of copper and cholesterol and decreased concentrations of zinc and total carotenoids. These changes distorted, or were superimposed upon, the rhythmic variability in the normal serum concentrations of each of these moieties. Changing values for copper, zinc, and cholesterol became apparent before any overt signs of disease. Infectious illnesses appear to influence the concentration of many normal constituents of serum. Of the trace elements studied, copper values generally increase, whereas those of zinc tend to decrease during bacterial or viral diseases (8; W.R. Beisel and R.S. Pekarek, Int. Rev. Neurobiol., in press). The reaction of serum cholesterol, on the other hand, is often variable in the presence of infection in that a depressed concentration has been observed during bacterial pneumonia, cholera, tuberculosis, viral infections, hepatitis, and malaria in man (1,6). Increased cholesterol values have been reported during tuberculosis in man or Escherichia coli bacteremia in dogs, with normal values being maintained during gram-positive or gram-negative infections in man, or experimental yellow fever in monkeys (1,7). Serum carotenoids tend to decrease during Newcastle disease virus (NDV) infection in the chick (12) and during active tuberculosis in man (4,9). Earlier studies have shown that the chick, infected with NDV, constitutes an excellent model for experimental attempts to relate biochemical responses resulting from infection with fluctuations resulting from daily biological rhythms (14,15). The present report describes the effects of a standardized NDV infection in chicks on serum copper, zinc, cholesterol, and carotenoid concentrations. MATERIALS AND METHODS To insure adequate numbers for selection, approximately 500 White Leghorn male chicks were obtained at one day of age and housed in an isolated air-conditioned room with constant lighting; a commercial chick diet and water were offered ad lib. This regimen continued for 28 days and served to deplete the birds of any parental immunity to NDV. Body weights were taken weekly and only chicks meeting their genetic potential curve for full normal growth were used in the experiment. Two days before the date set for NDV inoculation, at 8:00 AM, the chicks (28 days old) were divided into two study groups: a group to serve as noninfected controls and another to be infected experimentally with NDV. At the same time, five chicks were selected at random from each group and blood samples were obtained from them by cardiac puncture; the chicks were then discarded. The serum was separated and frozen for later analysis. This procedure was repeated at 3:00, 8:00, and 12:00 PM daily for 48 hr. After this period of preinoculation control collections, all chicks in the group designated for infection were inoculated (at 8:00 AM) with 0.1 ml of a 10-3 dilution of a Grun/ Rutgers strain of NDV that showed an embryo lethal dose of 50%0 equal to 10-9 per 0.1 ml in 10-day-old embryos. Seventy inoculated chicks were reserved in separate cages to monitor disease progression and mortality. Blood samples continued to be taken from five control and five infected chicks at the above sampling times for the next six days; only living birds were sampled, and all were discarded after the samples were obtained. At the end of the NDV incubation period, 72 hr postinoculation, care was taken to insure that the five infected chicks selected for bleeding manifested a range of clinical findings characteristic of the NDV syndrome at the time of sampling (13). In the analyses of the tissues, each sampling period and treatment were equally represented in any one run. Standard reference tissues and synthetic reference standards were also included. Total carotenoids were determined by the method of Bessey et al. (2), cholesterol was determined according to Bowman and Wolf (3), and the trace metals were determined by atomic absorption spectrophotometry (8). Analyses of variance were performed as in Snedecor and Cochran (11). The average serum concentration for each moiety was determined from all samples from noninfected control chicks; this was ascribed a value of 100%'I and was used as the reference point for plot-on March 17, 2020 by guest http://aem.asm.org/ Downloaded from ting the percentage changes of control and infected groups at each sampling time. RESULTS At the end of the active involvement stage, mortality in the NDV reference group was 48%, confirmiing that the level of NDV involvement in this trial was severe (13). Table 1 presents a statistical summary of the data analyzed over days postinoculation to show the effect of NDV on the serum components studied. Figure 1 depicts the sequential changes in serum cholesterol, carotenoids, zinc, and copper in control and NDV-infected chicks over the course of the entire observation period. Each point on the curve represents the average of 10 control chicks up to the time of inoculation and five control and five infected chicks thereafter. Time of inoculation is represented by the vertical dashed line. Cholesterol. Cholesterol concentrations in the controls over the 8-day period averaged 139 ±i 16 mg/I00 ml ( standard deviation) butvaried each day by as much as 27% of this value; the observed fluctations did not conform to a precisely repetitive rhythm throughout each 24-hr period. Early in the incubation period in the NDV-infected chicks, cholesterol concentrations tended to become somewhat lower than those of the control group, but by 55 hr postinoculation and thereafter values were consistently higher ( <0.05 to <0.01). Carotenoid. Carotenoid values in the control chicks averaged 483 86 ,ug/100 ml; they also showed nonrepetitive variability when compared to clock hours. There was a slight but sustained trend here toward the development of higher carotenoid concentrations throughout the 8 days of study. In the NDV-infected chicks, the carotenoid values began to decline soon after inoculation and concentrations continued to be lower than in control birds throughout the subsequent observation period ( <0.05 to <0.01). Zinc. Zinc values in the noninfected control chicks fluctuated up to 45% of the 8-day average of 166 38 ,ug/100 ml within each 24-hr period, with troughs generally occurring at 3:00 or 8:00 PM and peaks at 8:00 AM or 12:00 AM. Very soon after inoculation with NDV, zinc values became depressed (<0.01) below those of the noninfected control birds. Zinc concentrations continued to be depressed (<0.01) throughout most of the incubation period, returned transiently to control levels during the initial stages of overt disease (active involvement), and then dropped significantly (<0.01) during the last 48 hr of the study. DISCUSSION The present data indicate that an NDV infection (48% mortality index) of growing chicks is associated with increased values for serum copper and cholesterol and depressed values for serum zinc and carotenoids. These changes are similar to those reported to accompany a variety of bacterial or viral infections in mammalian species (1,2,4,(6)(7)(8)(9). The use of closely spaced serial collections in the present study allowed a correlation of biochemical changes with the timing of different stages of the NDV infectious process. For example, during the incubation period (0 to 72 hr), the depression in zinc and the increase in copper concentrations, and to a lesser extent total cholesterol, were true infection-related effects since dietary intake of the chicks remained normal at this time. The depression of total serum carotenoids, on the other hand, could have been influenced by the reduction of food intake which began 72 hr after inoculation with the virus. Feigin et al. (5) observed in man that attenuated Venezuelan equine encephalomyelitis virus could alter the periodic circadian rhythm of whole blood amino acid concentrations, could markedly alter their absolute mean concentrations, or could do both, depending on the time of day that the attenuated virus was given as a vaccine. Rapaport et al. (10) found that daily periodicity in the urinary excretion of tryptophan metabolites was maintained in patients with Rocky Mountain spotted fever even though the absolute amounts excreted were greatly increased. The present study suggests that infection-related influences on both periodic rhythms and on deviations from a normal range of concentrations may vary from substance to substance during the same time period in a single infection. The present data also emphasize the influence of periodicity on interpretation of results obtained from samplings of blood and other tissues. The diurnal oscillations observed here have a large magnitude of change, e.g., serum copper varies as much as 75 % in 24 hr, to illustrate the size of potential error. If the concentration of a substance normally undergoes rhythmic changes during the course of an investigation, a set of values obtained at a single point in time cannot be employed as an acceptable control sample. Moreover, the diurnal patterns of the oscillations indicate that serum copper in normal chicks possesses a classical circadian rhythm; yet in the same milieu at the same time, zinc, carotenoids, and cholesterol apparently lack a definite pattern. This would suggest that diurnal changes of all serum constituents are not circadian or that some are more susceptible than others to Zeitgeber inputs. The significant changes in patterns caused by NDV at various periods of the disease cycle would lend support to the latter possibility.

v3-fos

2018-04-03T05:30:57.476Z

1971-03-01T00:00:00.000Z

43181156

s2

Microorganisms of the San Francisco Sour Dough Bread Process A medium was developed which permitted isolation, apparently for the first time, of the bacteria responsible for the acid production in the 100-year-old San Francisco sour dough French bread process. Some of the essential ingredients of this medium included a specific requirement for maltose at a high level, Tween 80, freshly prepared yeast extractives, and an initial pH of not over 6.0. The bacteria were gram-positive, nonmotile, catalase-negative, short to medium slender rods, indifferent to oxygen, and producers of lactic and acetic acids with the latter varying from 3 to 26% of the total. Carbon dioxide was also produced. Their requirement for maltose for rapid and heavy growth and a proclivity for forming involuted, filamentous, and pleomorphic forms raises a question as to whether they should be properly grouped with the heterofermentative lactobacilli. Previous reports (3,8,9) describe the nature of the San Francisco sour dough process and also identify the naturally occurring yeast moiety of the system, Saccharomyces exiguus, as responsible for the leavening action in the dough. The present report deals with the isolation, apparently for the first time, of undescribed, closely related lactobacilli-type strains of bacteria from various sources of sour dough and some of their properties in pure culture. Evidence supporting the role of these bacteria in the souring action in these doughs is also described elsewhere (3,8). MATERIALS AND METHODS The medium developed for isolation of the sour dough bacteria from the doughs and used for pure culture broth studies is shown below. For convenience it is referred to as SDB (sour dough bacteria) agar or broth and was autoclaved before use. It contained the following: maltose, 2.0%; yeast extract (Difco), 0.3%70; fresh yeast extractives (FYE), 0.5 to 1.5%; Tween 80, 0.03% (3 ml of 10% solution/liter); and Trypticase (BBL), 0.6%7c. The pH was adjusted to 5.6 with 20% lactic acid or 1 N to 6 N HCI. The FYE were usually prepared by autoclaving a 20% suspension of commercial compressed bakers' yeast in distilled water for 30 min at 15 psi, allowing the suspension to settle overnight at 2 to 8 C, decanting, and further clarifying the supernatant by centrifugation. The extract prepared in this manner contained 1.5% solids and, if not to be used within a few days, was frozen or freeze-dried immediately. Alternately, the FYE could be prepared by autolyzing a 1: 1 suspension of compressed yeast in distilled water under Toluene for 3 days at 50 to 55 C, heating to boiling, and clarifying. For maximum cell yields in broth culture, a level of 1.5% FYE was used in the medium; otherwise, 0.5% sufficed. For isolation and enumeration of the bacteria from and in the doughs, 11 g of dough was blended with 99 ml of sterile 0.1 % peptone as described in the previous paper for the sour dough yeasts (9). These yeasts did not interfere with enumeration of the bacteria on SDB agar as they occurred in numbers 15o to Moo those of the bacteria, and the yeast colonies, generally one to two at the most per plate, were easily distinguishable. Spread plating of 0.1 ml portions of the appropriate serial dilution was used in preference to pour plates as it was found to provide better access to the stimulatory effects of gaseous CO2 . Incubation of SDB agar plates was generally for 2 days at 31 C in an atmosphere containing 25 to >90% CO2. Pure culture isolates were maintained on slants of SDB agar, transferred approximately once a month, and held at 13 C between transfers. Viability was retained better at this temperature than at 2 to 4 C. Isolates were made from each of five sources (bakeries) and were designated according to the source as strains L, P, C, B, or T. SDB broth cultures were inoculated with 0.1 to 1.0% of a fresh 18-to 36-hr broth culture, flushed with CO2, closed off, and shaken slowly during incubation at 30 to 31 C for a period of 1 to 2 days. Growth was estimated by turbidity or by cell volume determined on 459 KLINE AND SUGIHARA a 10-ml sample subjected to centrifugation at 1,500 X g in a tapered tube calibrated in 0.1-ml divisions. This centrifugal force seemed to suffice due to the tendency of the cells to assume various degrees of asymmetry and to clump in broth cultures not neutralized during growth. Plate counts gave inconsistent results, apparently due to this tendency to clump. Examination of the morphology of bacteria from agar plate colonies and broth cultures was routinely made on wet mounts by using a Zeiss phase microscope at 1,200X magnification. Lactic and acetic acid were determined essentially by the column chromatography method of Wiseman and Irvin (11) with sucrose-Celite as the adsorbent and alphamine red-R as internal indicator to follow elution of the separated acid bands which were then titrated to a cresol red end point with 0.01 N NaOH. The column was capped with a mixture of sodium sulfate, Celite, and ammonium sulfate which permitted direct addition of culture filtrates after acidification with sulfuric acid without further purification. Doughs were analyzed by preparing alkaline aqueous extracts similar to the procedure of Cole et al. (1). These extracts, after centrifugation and acidification, also could be added directly to the column. In both cases, the sample being analyzed was mixed vigorously with the capping material directly on the column before elution procedures were begun. RESULTS Nutritional (general). Microscopically, the sour dough bacteria are readily observed in slurries made from the doughs as slender rods, short to medium length, in numbers roughly 30 to 100 times those of the sour dough yeast cells. The latter were easily counted on conventional media (8,9) and numbered approximately 2 X 107 g of fully developed dough so that the bacteria appeared to be present in numbers of the order of magnitude of about 109. However, all attempts to isolate or grow these bacteria on conventional media of numerous sorts were unsuccessful. These included various plate count, milk, tomato, and orange juice, anaerobic, wort, soy, thioglycolate, coliform, and acetic acid bacteria agars as well as media used specifically for enumeration of lactic acid or other bacteria in flours and doughs (2). The development of the medium for successful isolation of the sour dough bacteria was based on simulating some of the conditions of the natural environment in the sour dough. Thus, maltose, which is essential, was provided as the carbohydrate source since the yeast S. exiguus, with which these bacteria coexist, does not utilize maltose. Maltose then becomes the principal carbohydrate available in the sour dough system which is formulated without addition of sugars. (Maltose is produced after the dough is formed due to the action of amylase on free starch.) Similarly, the need for FYE and stimulation of growth by CO2 was predicated on the fact that the system contains an active yeast. The use of Tween 80 is related to the dual consideration that this compound, or other unsaturated fatty acids, are stimulatory for many lactic acid bacteria and that flour contains about 1 to 1.5% of lipid, and twothirds of the fatty acids in this lipid are unsaturated (5). The adjustment of the pH of the medium to below 6 stems from the observation that the pH range encountered in the starter sponge, the natural vehicle for carrying these bacteria in the bakery, is limited to the narrow range of 3.8 to 4.5 (3). Other considerations in the development of this medium include the deletion of sorbic acid from some media commonly used for lactics (2), as this compound completely inhibits the growth of the sour dough bacteria at a concentration of 0.1% (at pH 5.6). The combination of nutritional factors necessary for successful isolation of the sour dough bacteria will thus be shown to include maltose, Tween 80, FYE, and an initial pH below 6; accordingly, the lack of success in isolating them on known media becomes understandable. The nutritional effects of some of these factors are described in more detail. Requirement for maltose. By using the SDB agar and the B source of sour dough, i.e., from bakery B, no bacterial growth was obtained when the following carbohydrates or substrates were substituted for maltose: xylose, arabinose, glucose, galactose, lactose, sucrose, raffinose, rhamnose, lactate, and ethanol. In the case of xylose and glucose, this was also true even when they were filter-sterilized. The same pattern was observed on three other sources of the bacteria or strains (Table 1), thus confirming the need by all strains for maltose, at least as far as initial isolation from b Counts on fully developed doughs plated out on maltose-SDB agar ranged from 70 X 107 to 300 X 107 per g of dough. the sour doughs was concerned. No difference in growth response with one strain was observed with maltose from six different commercial sources, minimizing the possibility that the growth requirement was contributed by an impurity in the maltose. The concentration of maltose required for heavy growth appeared to be unusually high, suggesting that a permeability or other effect might be involved. On agar plates, growth rate and colony size for one strain tested (B) were noticeably greater at 2.4% maltose than at 1.0%. In broth culture (Table 2), growth was slight at 0.10 to 0.25% maltose and increased sharply up to 1.0%. In other studies, additional slight increases in growth and acid production have been observed with increasing maltose levels up to 2.0%, the level adopted for the SDB medium. Studies in progress indicate, however, that some of the strains subcultured in pure broth culture may adapt slowly to glucose, but, again, a high concentration (>1.0%) is required for good growth and there is considerable variation among the strains in this respect. FYE. Without the use of any FYE solids, growth was very slow, requiring 4 to 6 days for colonies to achieve sufficient size to be enumerated, and irregular, both in rate of growth and shape of colonies. Increasing the level of other ingredients such as commercial dried yeast extract or casein hydrolysate (Trypticase) did not substitute for the FYE nor did addition of sodium acetate (0.05 to 0.30%) or vitamin B12 (2.5 Ag/ ml). A level of 0.5% FYE gave adequate growth for most purposes, such as enumeration of colonies by plate counts after 2 days of incubation, but growth was markedly further stimulated by increasing the level of FYE to at least 1.5 %. This is illustrated in Table 3 for two strains grown in broth where the cell volumes are 2 to 3 times greater at 1.5% FYE than at 0.5%. The components in the FYE essential for vigorous growth remain to be determined. Freeze drying and storage of the solids in air at refrigerator temperatures was a satisfactory way of preserving the growth-stimulating properties for at least several months. Tween 80. As tested on the B and L strains, no growth was observed on SDB plate agar after 2 days of incubation when the Tween 80 was omitted from the medium. With strain B, a level of 0.02% (2 ml of 10% Tween 80/liter) was necessary for good growth on SDB plate agar and 0.035 to 0.05% was necessary for heavy growth. Accordingly, the SDB medium was formulated with 0.03% Tween 80. No other unsaturated fatty acids or their esters were tested. Trypticase and commercial yeast extract. Trypticase at a 1.0% level and commercial dried yeast extract at a level of 0.3% were empirically included in early attempts to devise a medium for isolation of the bacteria and were present when the first weak growth was observed. Several commercial dried yeast extracts and autolysates were tested; they varied in their growth-stimulation properties although none of them produced good growth. However, since a rather high level of FYE solids is necessary for heavy growth, the commercial yeast extract was arbitrarily left in the medium for its minor contribution. Trypticase appeared to be more significantly stimulatory for growth, at least for the one strain tested (Table 4). Both the colony size and bacterial count increased up to a 0.6% Trypticase level which was then adopted for use in the SDB medium. Gaseous atmosphere. The bacteria appeared more or less indifferent to oxygen as they grew out just as well under anaerobic conditions, providing CO2 was present. Early studies, particularly before formulation of the SDB medium was completed, showed marked beneficial effects of CO2. Colonies developed on plate agars much more rapidly in the presence of 25 to >90% CO2 as compared to air and even the latter was more beneficial than a nitrogen atmosphere. However, with the completed medium the stimulatory effects of CO2 were found to be variable both from time to time for a single strain and to vary between strains. When CO2 was beneficial, a level of at least 25 % was frequently found to give maximum growth stimulation. Accordingly, for most studies, either a flush with CO2 or a level of 25 to >90% Co2 in the atmosphere was employed. Addition of bicarbonate as a source of CO2 was not considered due to the requirement of the medium for a low pH (< 6). General properties. The sour dough bacteria, in the stage of active early growth in either the dough or pure culture and before the total acidity is developed, appear as short to medium slender rods or very short chains with only a minor tendency to form bent or filamentous forms (Fig. 1, 2). However, they showed an unusual proclivity for assuming involuted, filamentous, and occasionally pleomorphic forms even in relatively young cultures (24 to 48 hr). On plate agars, when vigorous, they grow out as smooth, round, translucent colonies of about 1 mm or less diameter. They are gram-positive in the early stages of growth, nonmotile, and catalase-negative, as indicated by lack of gas evolution when a 10% solution of H202 is poured on the colonies on a spread agar plate. In the doughs, approximately 70 to 80% of the total organic acidity produced in the natural system is lactic; the remainder is acetic (3). The proportion of acetic acid is somewhat lower in pure cultures and appears to vary between strains. Gas is produced by all strains in pure culture and is virtually all absorbed by saturated Ba(OH)2, indicating that it is probably CO2. These bacteria are mesophilic and do not exhibit any unusual heat or salt tolerance. A detailed description of some of these properties follows. Heat-resistance and salt-tolerance. SDB broth cultures (12 and 24 hr) of three different strains (B, L, and T) were subjected to submersion in a bath at 60 C for 15 min in a manner which included a 3to 4-min come-up time. The 12-hr cultures had counts varying from 19 X 107 to 34 X 107 cells per ml and the 24-hr cultures, 150 X 107 to 160 X 107 per ml. No viable survivors were found in any instance at the greatest dilution tested (<106), suggesting that these bacteria do not possess any unusual thermal tolerance. Four strains (B, L, T, and C) were tested for growth in SDB broth containing 4 or 6.5 % NaCl. No development of turbidity was evident even after 4 to 6 days of incubation, suggesting no unusual salt tolerance. Salt at a much lower con- 13 -± +1 0 0.03 0.040.075.24.54.23.9 24 +2 +3+4 31 +3 +4 +4 0.09 0.14 0.15 0.14 4. Optimum temperature for growth. Three strains (L, B, and T) were tested for growth rate both on slants and in broths at temperatures varying from 13 to 45 C (Table 5). They all appeared mesophilic, growing out most rapidly at about 31 C, with growth being very slow at 13 and 37 C. No growth was observed at 45 C. Optimum pH for growth. As noted earlier, these bacteria exist in their natural habitat within the narrow pH range of 3.8 to 4.5; in developing an artificial medium for their growth, it was found necessary to adjust the starting pH of the media to below 6. The data in Table 6 suggest an optimum initial pH in unbuffered SDB broth of about 5.0 for the strains shown (T and L). Similar results were obtained with the B and C strains. The growth observed at pH 6.5 was very slow (virtually none at 24 hr) and may have resulted from the slow production of acid, dropping the pH to the more favorable zones. When growth was attempted on plate agars at pH 6.5, it was not only very slow but the colonies were highly irregular. The relatively slow growth in broth at pH 4.0 to 4.5 may be an artifact because significant turbidity and sediment develops in the uninoculated SDB broth acidified to this region, and it is possible that essential nutrients are being removed. Comparison of the effects of different acids used for initial pH adjustment on the growth of four strains (C, T, L, and B) showed HCI was in (Table 7). Acid and gas production. In the natural sour dough environment, as mentioned above, acetic acid quite regularly comprises 20 to 30% of the total acidity produced. We have also recently found this to be the case when the sour dough bread is made with pure cultures in place of the usual "starter." However, the proportion of acetic acid produced in pure culture may be somewhat lower and is also highly variable ( Table 8). The reason for this variability is not, as yet, apparent. All strains, however, produce approximately the same amount of total acidity. Substantial gas production, assumed to be CO2, was observed by the inverted tube method, but its evolution was delayed somewhat even after substantial growth was observed (Table 9). Qualitatively, there does not appear to be a good correlation between the proportion of acetic acid (Table 8) and the amount of CO2 produced. Table 9 also illustrates the slower growth of the L strain on the SDB medium. This has been irregularly observed although occasionally difficulty is even encountered in plating this strain out from the dough, and colonies that do appear may be irregular. DISCUSSION Slight differences were observed among the four strains (L, B, C, and T) studied in some detail. (P strain appeared the same as the L strain and was not examined further.) The differences were evident in colony appearance, degree of nutritional fastidiousness, i.e., how they grew in the SDB medium, tendency to form elongated or swollen forms, tendency to clump in broth culture, ability to adapt to glucose, and proportions of lactic and acetic acid produced. Thus, nutritionally, the T strain appeared to be the least fastidious and the L strain the most. The latter also exhibited the maximum tendency for clumping in broth and produced the lowest proportion of acetic acid. However, all had the following characteristics in common: gram-positive, nonmotile, slender rods (at times), catalase-negative, indifferent to oxygen, inhibited by sorbic acid; required low pH, Tween 80, and FYE for good growth; stimulated by CO2 on occasion, and produced lactic and acetic acids and CO2. These characteristics tend to group them with the lactobacilli, in particular with the heterofermentative type. However, their requirement for maltose and proclivity for forming involuted, filamentous, and pleomorphic forms might place them with Bifidobacterium or Actinomyces whose overlapping with lactobacilli is described by Moore and Holdeman OUGH BREAD PROCESS. 11 465 (6). Lactobacilli also, however, can be induced to assume odd shapes (4,7,10). Work is underway at another laboratory (under contract from us) to determine the genetic relationship of these sour dough bacteria to known species of lactobacilli. If our preliminary identification is confirmed, we would like to suggest these bacteria be designated officially as Lactobacillus sanfrancisco in honor of their unique role in this uniquely situated process. Another interesting aspect of this sour dough system containing these bacteria and certain yeasts is its self-protective nature, i.e., its incredible resistance to contamination by other microorganisms which has been maintained for decades. No doubt the acetic acid produced in the pH range of 3.8 to 4.5 may contribute to this protection. However, considering the finding that the only yeasts which appear to survive significantly in this system are cycloheximide resistant, we speculate that possibly these bacteria may produce related antibiotics. This postulate is currently under investigation by us.

v3-fos

2020-12-10T09:04:16.838Z

1971-11-01T00:00:00.000Z

237234422

s2

Fate of Carbon Passing Through the Glucose Pool of Rumen Digesta The metabolism of the free glucose pool in rumen digesta from sheep fed roughage rations was studied by adding an insignificant quantity of glucose as uniformly labeled 14C-glucose of high specific activity to in vitro incubation systems. In all experiments wherein only trace quantities of glucose were added to digesta, most of the 14C-glucose entered acetate. This was true whether label was presented either as a single dose or by continuous addition over a period of 2 hr. Digesta collected at all times after feeding either once daily or at hourly intervals gave similar glucose dissimilation patterns. If, however, a relatively large quantity of carrier glucose was added together with the tracer, the 14C-acetate: 14C-propionate ratio was reduced by a factor of about 10. Physical removal of most of the protozoa from digesta generally had little effect on the dissimilation of 14C-glucose added in tracer amounts, but in one experiment there was a decreased turnover of the free glucose pool and a marked reduction in 14C entering butyrate. The paucity of 14C entering propionate when only trace amounts of glucose were added to digesta suggests that this acid was largely formed from substrates whose carbon did not equilibrate with that in free glucose or with that in intermediates of free glucose metabolism. The metabolism of the free glucose pool in rumen digesta from sheep fed roughage rations was studied by adding an insignificant quantity of glucose as uniformly labeled '4C-glucose of high specific activity to in vitro incubation systems. In all experiments wherein only trace quantities of glucose were added to digesta, most of the '4C-glucose entered acetate. This was true whether label was presented either as a single dose or by continuous addition over a period of 2 hr. Digesta collected at all times after feeding either once daily or at hourly intervals gave similar glucose dissimilation patterns. If, however, a relatively large quantity of carrier glucose was added together with the tracer, the '4C-acetate: 4C-propionate ratio was reduced by a factor of about 10. Physical removal of most of the protozoa from digesta generally had little effect on the dissimilation of '4C-glucose added in tracer amounts, but in one experiment there was a decreased turnover of the free glucose pool and a marked reduction in 14C entering butyrate. The paucity of 14C entering propionate when only trace amounts of glucose were added to digesta suggests that this acid was largely formed from substrates whose carbon did not equilibrate with that in free glucose or with that in intermediates of free glucose metabolism. A number of studies have been published concerning the utilization of soluble carbohydrates by the mixed populations of organisms from the rumen (1,2,5,(11)(12)(13)(14)(15)(16)20). Without exception, however, massive amounts of carbohydrate were added to the system in comparison to the size of the carbohydrate pool which normally occurs within the rumen. Since, under these conditions, the fermentations become abnormal in that, for instance, lactic acid accumulation frequently occurs, it seems that the results of previous work may not reflect the fate of carbon arising from the degradation of complex plant polysaccharides and passing through the free carbohydrate pools within the rumen of normally fed animals. This report concerns the utilization of carbon passing through the free glucose pool in rumen contents from hay-fed sheep. MATERIALS AND METHODS Bacteriological. Incubations were done in vitro with samples of rumen digesta obtained from a mature sheep fed a daily ration of 750 g of wheaten hay chaff plus 250 g of lucerne hay chaff given as a single feed. In one experiment, digesta samples were obtained from a sheep fed the same ration, but provided to the animal in equal portions hourly throughout the day. Water was freely available at all times. Digesta sampling was as described by Walker and Forrest (18), and about 10 times the amount required for in vitro incubation was withdrawn from the rumen. This was thoroughly mixed and subsampled, and the excess was returned to the rumen. To 15 g of digesta was added 1 ,uCi of glucose-U-14C (300,MCi/fAmole) in 1 ml of solution. The air space above the reaction mixture was briefly flushed out with nitrogen, the vessel was sealed with a rubber stopper, and incubation was carried out at 39 C for 1 hr except where otherwise stated. Solids were removed from the digesta by squeezing through terylene voile, and 5 ml of the liquid was centrifuged at 20,000 X g for 10 min. The clear supernatant fluid was retained for determination of 14C in volatile fatty acids (VFA) and glucose and for determination of total and individual VFA. The packed cells were resuspended in 5 ml of water and 1 ml of 2 N H2SO4 was added. After centrifuging, the cell debris was washed twice with 0.5 N H2SO4 and finally suspended in 5 ml of water for determination of 14C in ceH constituents and of deoxyribonucleic acid (DNA) concentration. Chemical determinations. DNA was determined on duplicate l-ml samples of cell suspension by the method of Burton (3). Concentrations of individual VFA species in rumen fluid were determined after separation of the acids on a column of Dowex AG5OW X 12 (200 to 400 mesh, Radiochemical. All radioactivity determinations were done by liquid scintillation counting with a Triton-diphenyl oxazole-p-bis[2-(5-phenyloxazolyl)] benzene phosphor (10). Uniformly labeled 14C-toluene was used as internal standard. Glucose-U-_4C of high specific activity was obtained from the Radiochemical Centre, Amersham, England. Glucose and VFA in 1 ml of rumen fluid were separated on ion-exchange resin as described above, collecting 1.3to 1.4-ml fractions. A 1-ml portion of each fraction was used for determination of radioactivity. '4C in cell debris was determined by mixing 1 ml of the washed debris suspension with 5 ml of Tritonphosphor. Virtually all of the solid material in the suspension was solubilized by the phosphor. 14C in protein and polysaccharide was determined after hydrolysis of cells from 2 ml of debris suspension in 0.4 ml of 30%7a KOH for 3 hr at 100 C. After cooling, 0.4 ml water and 2.4 ml of absolute ethanol were added to precipitate polysaccharide which was removed by centrifuging. The precipitated polysaccharide was washed three times with 70r'7 ethanol, dissolved in hot water, and quantitatively transferred to counting vials for 14C determination. A sample of the supernatant fluid remaining after precipitation of polysaccharide was neutralized with perchloric acid and allowed to stand for several days at 4 C before before removing the precipitated KC104 by centrifuging. By using the cysteine-sulfuric acid method of Dische (4), it was found that the amino acid-containing supernatant fluid contained carbohydrate. Therefore, the neutral supernatant fluid was placed on a column of Amberlite CG 120 exchange resin (Na+ form, 350 to 400 mesh) measuring 10 cm long by 0.5 cm diameter and equilibrated with 0.2 N sodium citrate buffer (pH 2.0). The columns were then washed with three bed volumes of citrate buffer (pH 2.0) and the washings were dried, redissolved in 1 ml of water, and counted. Qualitative tests with ninhydrin showed a virtual absence of amino acids in this fraction and the radioactivity present was included with that for the precipitated polysaccharide. The amino acids were eluted from the ion-exchange resin with three bed volumes of 2 N NH40H and the eluates were dried, redissolved in 1 ml of water, neutralized with 0.4 N H2SO4, and counted. Radioactivity in this fraction was assumed to have arisen from cell protein. RESULTS Effect of glucose concentration on distribution of glucose carbon in VFA. Since preliminary results with 14C-glucose added in amounts such that the glucose added did not materially affect the size of the glucose pool had shown that most of the label recovered in VFA was present in acetate, whereas previous reports (2,11,20) indicated substantial conversion to propionate, the effect of normal and high glucose concentration was studied. Digesta (15 g) were incubated for 1 hr with I uCi (0.6 ,ug) of glucose-U-14C and with In the presence of carrier glucose, the recovered '4C in acetate and propionate was 60.2 and 24.9% of label in the VFA, whereas in the absence of carrier the corresponding values were 94.6 and 3.9%,,ve,. Diurnal variation in distribution of glucose carbon in VFA and cell material. Walker and Nader (19) have shown considerable variation in intraruminal metabolism of energy-yielding substrates during the day in sheep fed once daily. Such variations might be expected to be mirrored in the metabolism of carbon passing through the glucose pool. Accordingly, digesta samples were removed at intervals of 2 hr from a sheep fed once daily and allowed 4 hr to consume its ration. In all incubations, glucose-U-1IC was added without carrier. Figure 1 shows the distribution of recovered label between the VFA, C02(+ methane), and cell material. C02(+ methane) was calculated as being half of the total label entering acetic and butyric acids since both must arise by decarboxylation of pyruvate formed from the uniformly labeled glucose pool. Mean recovery of label was 87.5 + 6.4%, which was considered adequate since no attempt was made to assess radioactivity associated with the organisms left with the solids in the digesta after removal of the fluid. The proportion of "'C appearing in propionate rose, and that in acetate fell during feeding but readjusted rapidly after cessation of feeding. At all times, by far the largest portion of the 14C derived from the glucose pool appeared in acetate. The proportion of label appearing in butyrate did not alter appreciably. The molar proportions of VFA in the rumen contents used were 68:19:13 and 69:20:11 for acetate, propionate, butyrate at 2 and 20 hr after feeding. The labeling of cell material rose proportionately during the feeding period, and Fig. 2 most of the increase was due to the fo carbohydrate materials rather than prc radioactivity per unit of DNA present higher in carbohydrate than in protein. Effect of frequent feeding. Samples were removed just prior to delivery of a portion of feed to an animal fed hourly by an automatic dispensing device. Three such samples were taken followed by one 15 min after the consumption of a 1/24th portion of the daily feed, and all were incubated with glucose-U-'4C without added __4--* carrier. Table 2 shows that a fairly constant proportion of the label appeared in the VFA and that, compared to the sheep fed once daily, rather less appeared in acetate and more in the cell material. Again, of the radioactivity incorporated into cell material, most was in the carbohydrate 0 :' fraction (mean of 7.7%7 of recovered activity), a lesser amount being in protein (mean of 3.2% of recovered activity). Effect of removal of protozoa. The conversion of the bulk of the glucose carbon in acetate was recognized as corresponding to the reported activities of the rumen protozoa metabolizing soluble 16 20 sugars (1,8). Consequently, experiments were done to assess the effect of removal of protozoa from from 14C-rumen digesta. Protozoa were removed by squeez-?ep fed onice ing digesta collected 20 hr after feeding through dose withlout terylene voile and centrifuging the fluid at about r 1 hr. 500 X g for a few seconds. The solids were gently washed twice with clarified rumen fluid from the same animal, and the washings were discarded. The supernatant fluid from the centrifuged fluid was then added back to the solids, and the reconstituted digesta were thoroughly mixed and divided into two equal portions. Half of the protozoa from the centrifuged fluid were then added to one portion and an equal volume of clarified rumen fluid was added to the other. This procedure has been shown to remove about 90% of the protozoa and less than 10%/ of the bacteria in rumen fluid (unpublished data). Three such treatments were done on separate occasions. Incubations were as described above except that a shorter incubation period was used in two of the experiments. Results are given in Table 3. In the first experiment, glucose pool * . turnover was unusually slow when protozoa had 2 been removed and was accompanied by a de- 16 20 crease in 'IC appearing in butyrate. However, the second and third experiments showed no sigeim and cell nificant effect of protozoal removal on distribu-" 14C-glucose tion of 14C. A parallel determination of rate of once daily. total VFA production, done in conjunction with experiment 3, disclosed an 8%o fall due to removal of protozoa. shows that Two control experiments were done to compare ,rmation of untreated rumen contents with contents fully )tein, since reconstituted after undergoing all steps of the was much protozoa removal treatment. There were no differences in distribution of 14C between the of digesta VFA species, or in rate of total VFA production, indicating that the treatment used to remove protozoa had no deleterious effect on any of the organisms present. Continuous infusion of glucose-U-'4C in vitro. Since one particular organism or group of organisms may have contributed substantially to the metabolism of the glucose pool labeled by a single dose of 1C and since turnover of the pool appeared to be relatively rapid, there existed the possibility that the added glucose did not equilibrate with all of the free intracellular pools available. Therefore, an experiment was done with 200 g of digesta (collected 20 hr postfeeding) to which a priming dose of 2 ,Ci of carrier-free glucose-U-14C was added. Further 'C was infused at a rate of 12 ACi (in 12 ml) per hr while stirring the reaction mixture fairly rapidly. To assist in keeping pH relatively constant during incubation, sodium bicarbonate at a concentration of 0.2 mmoles/ml was included in the glucose solution infused. Samples (5 ml) of rumen fluid were withdrawn at 30-min intervals for 2 hr and promptly acidified with 1 ml of 1 N perchloric acid. After 2 hr, a 20-ml sample of fluid was obtained; 10 ml was treated with 2 ml of 1 N HC104 and 10 ml was placed in an ice-cold tube and centrifuged at 0 C. This last sample was used to attempt the estimation of specific activities of the total free glucose pool and the extracellular pool. Rate of total VFA production was linear over the 2-hr period, being 7.5 ,umoles per hr per ml of rumen fluid. Radioactivity in the total glucose pool rose over the first 60 min and remained constant thereafter. Accumulation of radioactivity in individual VFA species was linear (Table 4), and all of these factors show that steady-state conditions were established. Distribution of 14C in the individual VFA species again showed that most of the glucose carbon was converted to acetate carbon. In addition, there was five times as much activity in butyrate as there was in propionate (Table 4). Specific activities of glucose in the total and extracellular pools appeared to be the same, being 2,370 and 2,450 dpm/,g, respectively. However, no significant difference in total and extracellular pool sizes was observed, so the question of equilibration of all available pools with the added glucose remains unresolved. A repeat of the continuous infusion experiment gave substantially the same results (Table 4). Since attempts to demonstrate equilibration between intraand extracellular glucose pools directly were not successful, it was decided to try to assess the rate at which glucose entered the microbial cells and was converted to products. Rumen fluid and "4C-glucose without added carrier were pumped into a mixing cell and magnetically stirred together, and the effluent was led through a length of plastic capillary tubing marked at points corresponding to known times after mixing. Effluent was collected first from the point most distant from the mixing chamber into dilute H2SO4 and, thereafter, the capillary was cut at points progressively closer in time to the time of mixing (P. R. Monk, unpublished method). By this means, it was established that glucose was metabolized very rapidly. In one experiment, the label in acetate was 30% of that in glucose after 6.2 sec, rising to 106% in 123 sec; the disappearance of 14C glucose was first order and yielded a rate constant for glucose pool turnover of 0.32 per min. A second experiment showed label in acetate after 1.2 sec to the extent of 8 % of that remaining in glucose. In both experiments, label appeared in a compound, tentatively identified as lactate from the position of its elution from the ion-exchange resin column, in greater amount than in acetate at times close to mixing. At times further removed from the point of mixing, radioactivity in lactate decreased as that in acetate increased. DISCUSSION Previous investigators of the metabolism of carbohydrate by the mixed microbial population of the rumen have used very large quantities of carbohydrate substrate in comparison to the sizes of the free pools normally existing in rumen digesta (1, 2, 5, 11-14, 17, 19). Even when isotopically labeled substrates have been used, the concentrations of substrate were quite high. (15). The results reported in this paper clearly demonstrate a very large effect of substrate concentration on the distribution of glucose carbon among the VFA end products of fermentation. When only a trace amount of glucose was added to rumen contents (about 1/100th the pool size), glucose carbon appeared in acetate and propionate in the ratio 24:1. On the other hand, when glucose was added in sufficient quantity to increase the glucose pool size by about 300-fold, glucose carbon appeared in acetate and propionate in the ratio 2.4:1. No doubt, a high concentration of glucose leads to the rapid generation of large amounts of reducing power, the disposal of which is reflected in greater propionate production. In addition, relatively large concentrations of lactate transitorily accumulate when high concentrations of glucose are used and this substrate may preferentially give rise to propionate (17,20). The value of 2.4:1 for the ratio of radioactivity in acetate and propionate in the presence of excess glucose is close to other values obtained by using excess glucose-U-'4C and organisms from roughage-fed cattle of 3.1:1(2) and 5.2:1 (20). In pure culture, Selenomonas rwninantiwn has been shown to yield differing ratios of fermentation end products over a range of glucose concentrations between 50 and 5,000 ,ug per ml (7). However, in this case, decreasing glucose concentration resulted in an increase in the proportion of propionate relative to acetate, and lactate was always found in large quantity. The fate of carbon derived from the free glucose pool does not alter greatly during the day whether the animal is fed only once or 24 times daily. During feeding in the animal fed once daily, the proportion of glucose carbon reaching acetate falls by some 10%, whereas that reaching propionate rises slightly. Presumably, the presence of soluble carbohydrates in the feed raised the concentration in the free sugar pools in the rumen (15), and this is the nearest approach under natural conditions to the concentrations used in previous investigations on carbohydrate metabolism. It is apparent, however, that the small change in distribution of glucose carbon to individual VFA species occurring during feeding is quickly reversed upon cessation of feed intake. A rather more marked change observed during feeding once daily is the greatly increased incorporation of glucose carbon into reserve and possibly structural carbohydrate within the microbial cells. This phenomenon has previously been studied in this laboratory (17,19) by using chemical determinations and is confirmed by using isotopes. Carbon derived from the glucose pool appearing in microbial protein accounted for about 3% of the glucose metabolized when feed was offered hourly. Accepting Hungate's (8) estimate of a cell yield of 10%0 of the substrate fermented, 65% of this being protein, it would seem that, of the microbial growth associated with glucose fermentation, about half of the carbon entering cell proteins is derived from glucose itself. In one experiment where physical removal of protozoa showed a distinct effect, a relatively large amount of 14C was left in free glucose after 1 hr. In addition there was a lowered incorporation of 14C into butyrate when protozoa were removed, an observation in keeping with studies showing acetate and butyrate as the chief products of protozoal carbohydrate metabolism (1,8) and consistent with the finding of lowered butyrate concentrations in the VFA species in the defaunated sheep rumen (9). Since, however, two other experiments demonstrated no difference in 1'4C distribution due to removal of protozoa, it is not yet resolved whether the protozoa play a major role in the metabolism of free glucose. Intracellular glucose pool size appears to be too small to be measurable by taking the difference between extracellular and total pool sizes, so it has not been possible to determine whether equilibration of the added '4C-glucose with all available glucose pools was achieved. Nonetheless, the distribution of 14C among the VFA end products was the same whether label was added as a single dose or by continuous infusion over a 2-hr period, indicating that all pools available for equilibration with added glucose were being reached. Appearance of 1C in acetate was very rapid, again indicating fast transport of extracellular glucose into the cells and prompt metabolism. Rapid appearance of label in a compound which is probably lactate as well as in acetate suggests that intracellular glucose concentration would be very low under the normal rumen conditions of low extracellular glucose pool size. Overall, it seems reasonable to suppose that the metabolism of the added '4C-glucose was similar to that of glucose arising from feed component breakdown. The mean results of 15 experiments on distribution of 14C from the labeled glucose pool indicate the following equation for ruminal glucose utilization in roughagefed animals: 1.0 glucose --1.76 acetate + 0.04 propionate + 0.10 butyrate + 1.95 CO2 + 3.48 [2H]. The major feature of this equation is the relative paucity of propionate compared to that proportion normally present in the rumen. This suggests that most of the propionate formed arises from sources whose carbon does not equilibrate with that in the free glucose pool or with that of intermediates of free glucose metabolism. A further apparent anomaly is the excess of reducing power available. However, in terms of the total rumen fermentation, the excess of reducing power would be quite small since unpublished preliminary estimates based upon pool size and turnover rates suggest that less than 10% of the VFA produced, in experiments so far completed, arises from the free glucose pool. Further studies are in progress to evaluate the contribution of free glucose to the VFA produced at various times after feeding.

v3-fos

2016-05-04T20:20:58.661Z

1971-04-15T00:00:00.000Z

5328218

s2

Some points of view on the estimation and importance of genetic change in populations of dairy cattle SUMMARY Some results from a study on genetic changes in milk yield and fat percentage in the Fin-nish Ayrshire (ay) and Finncattle (rc) breeds are discussed. Moreover the economic importance of the genetic change is considered. The genetic change was estimated in 2 ways: (a) from the selection practised in artificial insemination (AI) in 1961-66 and (b) from the change in the progeny tests of individual AI sires over the period 1957-68. The following results were obtained and conclusions drawn: 1) The genetic gain in milk yield was in ay 0.80-0. 91 % /year and in rc 0.84-0.99 % /year. In kilograms this corresponds to some 35 and 31.5 kg, respectively. 2) With regard to fat percentage no noticeable genetic change could be detected in either breed. 3) As both methods used gave similar results it was concluded that the estimates can be regarded as fairly reliable. 4) In both breeds about one half of the increase in average milk yield noted over the period 1957-68 could be attributed to the breeding work. 5) The rate of interest on investments made in breeding was calculated to be 107 %. (The genetic gain achieved in one year was considered permanent, i.e. available also the following years. As costs those pertinent for AI, milk recording and Breed Societies were taken into account. On the return side the average genetic gain in milk yield for the two breeds (0.93 % /yr) was considered and multiplied by the appropriate economic production value). 6) The genetic gain in milk yield could from the level observed in the present study be increased to 1.4-1.5 % lyear. This would require: a) that the best 1-2 % of the bulls were approved as bull sires (in study 35 % in ay and 25 % in fe); b) that as sires of female replacement only 10-15 % of the bulls be approved (52 % in ay, 45 % in fe); c) that average generation interval be decreased from the observed 7.3 years to 5.3 years. 3) As both methods used gave similar results it was concluded that the estimates can be regarded as fairly reliable. 4) In both breeds about one half of the increase in average milk yield noted over the period 1957-68 could be attributed to the breeding work. 5) The rate of interest on investments made in breeding was calculated to be 107 %. (The genetic gain achieved in one year was considered permanent, i.e. available also the following years. As costs those pertinent for AI, milk recording and Breed Societies were taken into account. On the return side the average genetic gain in milk yield for the two breeds (0.93 % /yr) was considered and multiplied by the appropriate economic production value). 6) The genetic gain in milk yield could from the level observed in the present study be increased to 1.4-1.5 % lyear. This would require: INTRODUCTION A frequent question of debate in many countries with an advanced dairy cattle industry is whether increases in production should be attributed to improvements in breeding or to better feeding and management. Reliable answers to this question have so far been available for rather few dairy cattle populations. Research workers seem to have been more interested in developing new complicated procedures rather than in checking if the hitherto used ones have been effective in practice. In the following some results from a study on genetic changes in milk yield and fat percentage in the Finnish Ay y shire and Finncattle breeds will be given and discussed. Detailed results can be found in L INDSTR 6 M (ig6g) ( 1 ) and therefore only those of general interest will be presented here. Furthermore some consideration is given the economic importance of genetic change. MATERIAL AND METHODS The genetic change was estimated in two ways: 1) from the selection practised in AI breeding in the period 1961-66, 2) from the change in the progeny tests of individual AI sires over the period 1957-68. In 1) the genetic change was calculated from the selection differentials for the four paths along which genes are transmitted from one generation to the next (R ENDEL and RosEnrsoN, 1950). In (2) only AI sires represented over a time span of at least 6 years, with a minimum of 20 daughters in each proof, were used. The genetic change was obtained both by regression analysis techniques and least squares procedures (SMITH, 1962). RESULTS . Selection practised in z 9 6z-66 The main results are given in tables 1 -2 . With regard to milk yield it is seen that in both breeds the selection of bull dams has been rather effective. Also the dams of female replacements have in both breeds been clearly above average. On the other hand the selection of sires, especially of the next cow generation, has been fairly weak, particularly in Ayrshi y e. In Finncattle the selection of bull sires has been almost as efficient as that of bull dams. In Ay y shi y e, however, the selection differential of the sires of the next bull generation was some 3 . 5 °,o units lower than for the bull dams. With regard to fat percentage the selection differentials are on the male side in both breeds negative. When picking the dams more attention has obviously been paid to the fat percent, especially in Finncattle. On an average the observed differentials indicate a genetic gain of 0 ,8 0 % per year in Ayrshi y e and of 0 ,8 4 % per year in Finncattle with respect to milk yield, whereas no genetic change can be predicted for fat percentage in either breed. Cha-nge in progeny tests The main results are given in table 3 . With regard to milk yield the analyses indicate an annual genetic gain of o.8o to 0 . 91 % in Ay y shire and of o. 99 to i.ii % in Finncattle. With respect to fat percentage the analyses indicate that no genetic gain has taken place in either breed. Comparison o results Comparing the results obtained by the two methods we can conclude that, on the whole, the agreement is fairly good. It must be pointed out that the genetic change in a cow population resulting from the selection practised in a certain year will take place only some 3 years later. Thus the results calculated from the selection intensity in y 6r-66 should be compared to the observed genetic change in 19 6 4 -6 9 . Separate results for this latter period were not computed as this would have decreased the accuracy. However, judging from the least squares constants for the different years it would seem as if the gain in milk yield in Ayrshire after 19 6 4 and in Finncattle after 19 66 had been less than in the previous period. Consequently one can regard the two methods of calculation to have yielded similar results. Fat percentage In the following the discussion will be limited to milk yield. Some comments on the rather poor results for fat percent seem, however, justified. The main reasons for the observed results are probably : i) the main emphasis in the breeding programmes has been on an increase in milk production. Thus the negative genetic correlation between milk yield and fat percentage will have influenced the results, 2 ) the price relations between milk and fat percent have not been such as to rromote a striving for a higher fat content, 3 ) no age correction was earlier made when calculating the progeny tests for fat percent. This resulted in positive deviations for many bulls being in fact below average. Agreement with theoretical calculations With respect to milk yield how do the results of the present study compare with those theoretically obtainable? From table 4 can be seen that the selection of dams in both breeds agrees well with the theoretical results. On the other hand the observed selection differentials correspond to approval of 35 °!° of all bulls as bull sires in !lyyshiye and of 25 % in Finncattle, while the theoretical figure is 1 -2 %. With regard to the sires of female replacements the observed selection intensity indicates approval of 52 % of all tested bulls in Ayrshire and of 45 % in Finncattle, compared with the theoretical figure 10 -15 %. Thus it is evident that in the planning and use of the bulls one has been far from the optimum situation (SxJxavo!,D and LnNGxor,z, 1964). With regard to the sires of female replacements part of the weak selection intensity noted can be ascribed to the large use (c. 50 °o) of unproven bulls ( 3 -4 years of age) in order to satisfy the increasing demand for fresh semen. However, it must also be pointed out that if enough young bulls had been put into AI use a much heavier culling would later on have been possible. In the period 1954 -6 2 , on an average, only 10 bulls per year in Ayrshire and 6 bulls per year in Finncattle were taken into AI use. It is obvious that with such numbers the selection intensity can not be very high. It is interesting to note that this fundamental--proving enough bulls annuallyhas been neglected in almost all countries,. even in those practising progeny testing for many decades. Generation interual One of the main reasons for the rather modest gain observed in the present study is the long generation intervals. From table 2 can be seen that the bull sires in both breeds have been 9 . 3 -9 . 4 years and the bull dams over 8.6 years of age. These are among the highest figures noted in the literature. The age of the sires and dams of female replacements are more in accordance with other reports. However, the relevant question is what generation intervals can be achieved in practice with a good planning of the breeding activities. From table 5 can be seen that it should be possible to reduce the average generation interval from 7 . 3 to 5. 3 years. This would be of first rate importance as the genetic gain would be s!ed up by some 37 %. Imfiortance of genetic gain We can now return to the question of how much of the total increase in milk yield is genetic in origin. This can be seen from ( fig. i). In other words in both breeds about one half of the increase in average milk yield noted over the period 1957 -68 can be attributed to the breeding wo y k. Economically this means, e.g. at the 4 ooo kg herd level that the benefit per cow and year is 3 6-40 kg milk, i.e. 20-2 2 fmk. By considering the costs for AI breeding as investments (L INDHE , 19 68) we can calculate the rate of interest (table 6). Here we regard the genetic gain achieved in one year as permanent, i.e. available also in the following years (S KJER -vor,D, i 9 65). The costs are those for milk recording, AI breeding and the Breed Societies. In the calculation only one half of the total costs for milk recording were taken into account as the other half can be considered costs for the planning of feeding and management. From the total costs in the AI sector the technical costs, i.e. those required just to get the cows pregnant (needed whether natural service or AI is used), were subtracted. These costs made up some 5 6 % of the total costs. Finally from the Breed Society costs those not directly bearing on the enhancement of the breeding work were subtracted (some 24 °/,). We can conclude that, even if the genetic gain could have been larger in our breeds, the investments made in breeding yield a good interest.

v3-fos

2020-12-10T09:04:11.647Z

1971-04-01T00:00:00.000Z

237230941

s2

T-2 Toxin Production by Fusarium tricinctum on Solid Substrate A method has been developed to produce and purify gram quantities of T-2 toxin [4β, 15-diacetoxy-8α-(3-methylbutyryloxy)-12, 13-epoxytrichothec-9-en-3α-ol], a mycotoxin elaborated by a strain of Fusarium tricinctum isolated from toxic corn. After growing for 3 weeks at 15 C on 1,200 g of white corn grits, F. tricinctum NRRL 3299 elaborated at least 9.0 g of T-2 toxin, and 2.3 g of crystalline product was recovered. A lesser amount of toxin was produced on rice, but none was detected in wheat incubated at 20 C. The amount of toxin measured in white corn grits declined as the incubation temperature was raised to 20, 25, and 32 C. A method has been developed to produce and purify gram quantities of T-2 toxin [4fl ,15-diacetoxy-8a-(3-methylbutyryloxy) -12, 13-epoxytrichothec-9-en-3a-ol], a mycotoxin elaborated by a strain of Fusarium tricinctum isolated from toxic corn. After growing for 3 weeks at 15 C on 1,200 g of white corn grits, F. tricinctum NRRL 3299 elaborated at least 9.0 g of T-2 toxin, and 2.3 g of crystalline product was recovered. A lesser amount of toxin was produced on rice, but none was detected in wheat incubated at 20 C. The amount of toxin measured in white corn grits declined as the incubation temperature was raised to 20, 25, and 32 C. Outbreaks of toxicosis in animals ingesting moldy corn are accompanied by several signs (E. B. Smalley et al., Proc. 1st U.S.-Japan Conf. Toxic Microorganisms, in press). Although many species of fungi are usually isolated from toxic corn samples, Fusarium tricinctum is one of the more common isolates, and extracts from cultures of this fungus are more potent than those of other fungi selected from molded corn (6). F. tricinctum NRRL 3299 (strain T-2 from E. B. Smalley, University of Wisconsin) is one of the more toxic strains and produces three characterized mycotoxins: T-2 toxin, 4-desacetoxy T-2 toxin (J. R. Bamburg, Ph.D. Thesis, Univ. of Wisconsin, Madison, 1969), and a butenolide (9). The molecular structure of T-2 toxin, the principal component produced at low temperatures by the T-2 strain, is 43, 15-diacetoxy-8a-(3methylbutyryloxy) -12,13 -epoxytrichothec -9en-3a-ol (2). Its oral LD50 in rats is 4 mg/kg (3). Albino rats fed diets containing 5 and 15 Ag of T-2 toxin per ml for 3 weeks were severely stunted and developed inflammations of the skin around the nose and mouth. Microscopic examination of the liver of these animals showed small areas of focal change and cytoplasmic degradation. Rats receiving a diet containing 10 ,ug of T-2 toxin per ml for 8 months consumed approximately 20 times the single LD50 without apparent ill effects and without any indication of hepatoma development (8). Signs of metabolic disorder in large animals consuming T-2 toxin have not been observed, but, in a preliminary study (7), a 650-pound steer receiving daily I A preliminary report of this work was presented at the 70th Annual Meeting of the American Society for Microbiology, Boston, Mass., 26 April-i May 1970 (Bacteriol. Proc., 1970, p. 14). intramuscular injections of 30 mg of T-2 toxin lost weight during the study and died on the 65th day. Autopsy revealed evidence of general internal hemorrhage, a sign occasionally found in cattle after ingestion of moldy corn (5). The production of T-2 toxin and a purification process by which 100 mg was routinely recovered from 1 liter of medium have been described, and it has been reported that 1 kg of corn fermented with the T-2 strain may contain up to 1 g of toxin (J. R. Bamburg, Ph.D. Thesis). Because this toxin may be involved in moldy corn toxicosis, a method was sought for producing it in amounts sufficient to determine its effect on livestock consuming chronic and acute dosages. A simple procedure for obtaining this toxin in gram quantities is presented here. MATERIALS AND METHODS Preparation of inoculum. Conidia of F. tricinctum NRRL 3299 were produced by the fungus on yeastmalt (YM) agar incubated for 14 days at 25 C. The conidia were suspended in water by gently scraping the agar surface with a wire loop to give a turbid suspension. Production of T-2 toxin. Fembach flasks containing 300 g of white com grits (WCG), pearled wheat, or polished rice were autoclaved for 30 min. After the flasks were autoclaved, 2 ml of the conidial suspension and 100 ml of sterile water were added to each flask. Four flasks of the inoculated WCG, wheat, and rice were kept for 3 weeks at an incubation temperature of 20 C. In addition, flasks of WCG were incubated at 15, 20, and 32 C, also for 3 weeks. Extraction and purification of T-2 toxin. Each of four flasks of WCG fermented at 15 C was extracted with 1 liter of chloroform-acetone (85:15) by blending in a Waring Blendor for 2 to 3 min. The corn slurry was filtered on paper toweling, and the solvent was pressed out with a large spatula. Solids were returned to the blendor jar with a second liter of solvent, and the process was repeated for a total of three extractions. A portion of each extract was saved for quantitative analysis, and the combined extracts were reduced to about 75 ml of an oil-like residue (OLR) in a rotary evaporator. Two volumes of acetone was added to the OLR, and a gummy substance precipitated as the crude mixture dripped into the hexane. The gummy substance was separated from the toxin-containing solvent by decanting. (T-2 toxin precipitates in hexane but is quite soluble in acetone. To ensure that the toxin remains in solution, the volume of acetone in the solution should be 5 to 10%.) A greenish-yellow OLR continued to settle from the solution for several hours as the solvent was evaporated at room temperature. The solution was decanted at hourly intervals until it was almost free from OLR. The T-2 toxin crystallized and precipitated along with a small amount of OLR as the solvents evaporated from the clear solution standing in an open beaker. The toxin and OLR were dissolved in 10 ml of acetone to which 1 to 2 g of activated charcoal was added. After 30 min, the charcoal slurry on Whatman no. 1 filter paper was washed five times with 10-ml volumes of acetone; extraneous charcoal was removed with a second filtration before the acetone volume was reduced to 10 to 15 ml. Hexane was poured slowly into the toxic solution until it became slightly cloudy, and then a few drops of toluene were added to clear the solution. The T-2 toxin crystallized from the hexaneacetone-toluene left at room temperature for 18 hr. The toxin was dried at 80 C and weighed, and the purity was confirmed. A melting point determination (150 to 152 C), the presence of a single spot on thinlayer chromatograms with an RF corresponding to the standard T-2 toxin, and nuclear magnetic resonance spectroscopy were criteria for judging product purity. The gummy residue from the initial hexane precipitation was dissolved in 100 ml of acetone, and the T-2 toxin recovery procedure was repeated. Hexaneinsoluble residue remaining after the second precipitation was also dissolved in 100 ml of acetone, and the purification process was carried out for the third time. The residue remaining after the third precipitation and the oily substance present in the hexane-acetonetoluene solution from which crystalline toxin was recovered were kept for quantitative analysis. An estimate of the T-2 toxin produced on the various substrates fermented at given temperatures was obtained from a single extraction by using 1 liter of solvent for each flask of substrate. Although less than 75% of the solvent was recovered from the blended corn slurries, the toxin estimates are based on the 4 liters of solvent used in the extraction. It was assumed that each milliliter of solvent held the same quantity of toxin whether recovered or absorbed by the substrate. Crystalline toxin was obtained from the extracts by the described procedure, but only one precipitation of the OLR in hexane was carried out. Estimation of T-2 toxin by microbiological assay. The minimum amount of T-2 toxin required to inhibit APPL. MICROBIOL. Rhodotorula rubra NRRL Y-7222, a yeast sensitive to this mycotoxin (4), was 4 yg when applied on a filter paper disc (Schleicher and Schuell, no. 740E). Cells of R. rubra growing on YM agar at 25 C were suspended in YM broth and diluted to a reading of 50% transmittance at a wavelength of 600 nm. Onetenth milliliter of the yeast suspension was added to 6 ml of YM agar at 45 C before the agar was poured into a standard petri dish. Decimal dilutions of the chloroform-acetone (85:15, v/v) extracts were added to antibiotic assay discs at the rate of 0.1 ml per disc. Discs made from test extracts and standard discs containing 5 and 10 ,g of crystalline toxin were placed on the same petri dish. After a 30-hr incubation at 25 C, the inhibition zones around the discs containing the extracts and the standard discs were compared. Extracts from samples with the smallest amounts of toxin were concentrated before their fungistatic effects were compared with the standards. An estimate of the total amount of toxin in the fermented grain was based on the dilution, or concentration, of a test extract required to give an inhibition zone comparable to that of the standard and on the volume of solvent used in the extraction procedure. Estimation of T-2 toxin by thin-layer chromatography. Culture extracts were spotted on Silica Gel G thin-layer chromatographic plates along with 5, 10, 15, and 20 ;g of T-2 toxin. The chromatograms were developed with ethyl acetate-toluene (3:1). After the development, the plates were air-dried, sprayed with ;Jiters of an extract concentrated 10-fold did not give a visible spot at the RF of T-2 toxin. RESULTS AND DISCUSSION An estimate of the quantity of T-2 toxin extracted from WCG fermented with F. tricinctum NRRL 3299 incubated at 15 C and the amounts recovered or lost at various stages in the purification process are given in Table 1. The solvent recovered from 1,200 g of substrate after blending three successive times with 4-liter volumes of chloroform-acetone contained about 10 g of toxin, and more than one-fourth of it (2.77 g) was recovered as crystalline product. Nearly 95% of the toxin was in the first two extracts and only 5% was in the third extract. The relatively large quantity of toxin (40%) in the second extract was due to the absorption of nearly 30% of the solvent by the substrate during the first extraction. The quantity of crystalline toxin obtained from hexane-acetone decanted from the first, second, and third precipitations of the OLRacetone in hexane was 1.2, 1.1, and 0.45 g, respectively. Most of the toxin not reclaimed (68%) was retained by the gummy OLR remaining after the third hexane precipitation. A lesser amount of the unclaimed toxin (10%) stayed in the oily substance dissolved in the hexane-acetone-toluene from which the T-2 toxin crystallized and in hexane used to wash the product. Toxin not accounted for (22%) was probably lost during filtering and decanting or may be due to variations inherent in the assay methods. In Table 2 are given estimated quantities of T-2 toxin elaborated by the fungus growing on WCG, pearled wheat, and polished rice at 20 C and those produced on WCG incubated at 15, 20, 25, and 32 C. Apparently the amount of T-2 toxin produced by this fungus is influenced by temperature and by the nature of the substrate. Most toxin, about 9.0 g/l,200 g, was produced on WCG incubated at 15 C. At incubation temperatures of 20 and 25 C, the amount of toxin produced declined 50 and 85%, respectively. No toxin was detected in WCG incubated at 32 C as determined by thinlayer chromatography, and the yeast assay suggested the presence of only a small quantity of a fungistatic substance. In this study, T-2 toxin was not detected in the pearled wheat, but, in a preliminary investigation, 1.3 g of toxin was recovered from 1,200 g of pearled wheat when the incubation temperature was 15 C. Polished rice was a comparatively poor substrate for toxin production, and only a small amount was present in the extract. Incubation of WCG inoculated with the fungus at 15 C yielded gram quantities of T-2 toxin. Bamburg et al. (1) report 8 C as the best temperature for toxin production in a cornsteep liquorsoybean meal based (Gregory's) medium. The twofold increase in toxin quantities when the incubation temperature of the WCG was reduced from 20 to 15 C suggest that larger amounts may be produced at a lower temperature on WCG as in Gregory's medium. Strict precautions are advised when handling the toxin or culture extracts because severe inflammation occurs if either comes in contact with the skin.

v3-fos

2019-03-20T13:04:28.773Z

1971-10-01T00:00:00.000Z

237043326

s2

Characteristics of Antimicrobial Resistance of Escherichia coli from Animals: Relationship to Veterinary and Management Uses of Antimicrobial Agents Five-hundred fifty-five (555) isolates of Escherichia coli were obtained from fecal specimens of a representative number of animals from five farms in the United States. Antibiotic exposure of the selected herds was determined by an epidemiological survey of these farms. The incidence of multiple resistance in the E. coli isolates was higher in herds exposed to continuous feeding of antimicrobial agents (84.8%) than in a herd not receiving antimicrobials (15.7%). The most common resistance configuration observed was the triple pattern of dihydrostreptomycin (DS), sulfonamide (SU), and tetracycline (TE). The second most frequent pattern consisted of four resistances: ampicillin (AM), DS, SU, and TE. The frequency of transfer factors was much higher in multiply resistant organisms from the herds exposed to antimicrobial medicaments. The E. coli isolates were relatively efficient in fostering and transferring heterologous resistance factors. AM resistance factors occurred more frequently in herds which were exposed to feed levels of penicillin (27.9%) than in those that were not (6.4%). The phenomenon of the transfer of multiple antibiotic resistance by conjugation was first reported in Japan (13) in 1959 to 1960 and in Great Britain in 1962. Between 1962 and 1965, English investigators (1)(2)(3)5) published several papers on the subject and showed that Salmonella typhimurium strains from human and animal sources which had resistance to several important antimicrobials [tetracycline (TE), streptomycin (SM), ampicillin (AM), and sulfonamide] could transfer all of these resistances to sensitive E. coli strains. Likewise, resistant E. coli could transfer its resistance to sensitive E. coli. More recently, other investigators (9-11) obtained evidence that infectious drug resistance was prevalent among nonpathogenic Escherichia coli from humans, calves, pigs, and fowl. It is possible, therefore, that these bacteria, which formed the bulk of the aerobic enterobacterial flora of the alimentary tract, were a reservoir of infectious resistance from which animal pathogens could IPresent address: Animal Health Division, Agricultural Research Service, U.S. Department of Agriculture, Hyattsville, Md. 2 Present address: Bacterial Diseases Branch, Center for Disease Control, Atlanta, Ga. become resistant and also potential donors upon ingestion of R factors to human intestinal flora. The overall use of antibacterial drugs in livestock rations for various purposes supplies a continuing pressure to maintain the process of resistance transfer. A survey was deemed necessary to determine how widespread these infectious resistant bacteria were in animal populations in the U.S. It was also important to determine the relationship between the use of antimicrobial agents in feed and the occurrence of multiply resistant organisms which contained transfer factors. A traceback system was used to select each farm for the study. A continuous supply of Salmonella isolates was obtained from the Diagnostic Services, Animal Health Division, National Animal Disease Laboratory at Ames, Iowa. Antibiograms were determined for each isolate received. Alternate farms of origin of multiply resistant and completely sensitive Salmonella, and the willingness of the farm owner to cooperate, were used as criteria for selection of premises. A cooperative agreement was established between the Food and Drug Administra-tion, Center for Disease Control, and U.S. Department of Agriculture, which provided a mechanism for obtaining samples from the farms of origin of the traceback cultures. This arrangement provided for the epidemiological support necessary to the study. MATERUILS AND METHODS Selective media were utilized to determiine whether any Salmonella isolates could be obtained from each fecal sample collected. No Salmonella isolates were detected from any of the animals examined on any of the farms. To isolate E. coli, rectal swabs were inoculated onto MacConkey agar plates which were then streaked on the premises and incubated overnight prior to return to the laboratory. Three typical lactose-fermenting colonies were picked to Triple Sugar-Iron-Agar slants and incubated at 37 C overnight. Strains giving typical E. coli reaction were further screened on Simmons citrate agar and adonitol and were checked for motility and for production of ornithine decarboxylase and oxidase. The antimicrobial susceptibilities of confirmed E. coli were tested by the technique of Bauer et al. (4), except that dihydrostreptomycin (DS) was substituted for SM; studies in our laboratory demonstrated equivalent zone sizes against E. coli. The following antimicrobials were used in the testing system: AM, DS, cephalothin (CEPH), sulfamethoxypyridazine (SU), colistin (CL), chloramphenicol (CHLOR), furazolidone (FU), neomycin (NEO), TE, and nalidixic acid (NA). All strains resistant to two or more antimicrobials, including resistance to either tetracycline or ampicillin, or both, were examined for transferable resistance. The R-factor transfer procedure and media used were those of Schroeder et al. (8) with slight modifications. Four media were used: I, MacConkey agar; II, Mac-Conkey agar plus 25,ug of NA per ml; III, MacConkey agar plus 25 ,ug of NA and 4 ,ug of TE per ml; and IV, MacConkey agar plus 25 ,g of NA, 10,ug of AM, and 10 ,ug of dicloxacillin per ml. To test for R factors, equal volumes (0.5 ml) of overnight cultures in Mueller Hinton broth (MHB) of the donor E. coli and recipient non-lactose-fermenting E. coli (NAresistant strain of K-12F-, courtesy of H. Williams Smith) were mixed and incubated at 37 C for 2 hr. At the same time, MHB tubes were inoculated with recipient alone and with a multiply resistant Salmonella control alone and were incubated for the same time interval. After 2 hr, all cultures were streaked onto plates of media I and II, and 0.1-ml portions were spread onto plates of media III and IV. For results to be valid, the Salmonella control strain was required to grow on all four plates and recipient control strain only on plates I and II. In conjugated mixtures, both recipient and donor must have been present on plate I and only recipient on plate II. Typical colonies of the recipient strain appearing on plates III or IV, or both, were picked and tested for antimicrobial susceptibility. Table 1 outlines the major species of animals tested on each farm. Farms 1, 3, and 4 utilized a continuous program of medication with antibiotics. Farm 2 was primarily a cattle operation utilizing a management system which included the administration of antimicrobials during periods of "stress" to the animals. Farm 5 utilized three separate locations for the production of swine, calves, and dairy cattle. The swine were receiving continuous low levels of oxytetracycline in the feed. The calf operation utilized nitrofurazone at therapeutic levels (e.g., administered as one dose at the time of birth). RESULTS The dairy cows were receiving no continuous medications, but two cows had been treated for mastitis the previous 2 weeks before the test. These two cows were the source of 40% of the multiply resistant strains isolated from this herd. Animals exposed to continuous levels of antimicrobials (farms 1, 3, 4) had a much higher percentage of resistant organisms and had a frequency of occurrence of multiple resistance substantially greater than animals on the other farms ( Table 2). Of the E. coli isolates from the swine populations of farms 1, 3, 4, and 5, 67 to 95.9% were multiply resistant. The cattle population on farm 2, receiving intermittent antimicrobial pressure, yielded 38.4% multiply resistant E. coli. In contrast, the calves and dairy cattle on farm 5, receiving no continuous antimicrobial pressure, had substantially lower frequencies of multiple resistance, 19.0 and 15.7%, respectively (Table 2). Correspondingly, the incidence of sensitive organisms was higher in the animals which were not being exposed to continuous levels of antimicrobial agents. Table 3 clearly illustrates that as the antimicrobial pressure was reduced there was a corresponding drop in multiply resistant strains. In Table 4 can be seen the ratio of homologous resistances (resistant factors in fecal flora which are the same as the antimicrobial being used in the feed) to the heterologous resistances (resistant factors not related to the antimicrobial used in the feed). When an antimicrobial was used in the feed, the percentage of isolates resistant to it was substantially higher than when the antimicrobial did not appear in the feed. Multiple resistance was also correspondingly high in these herds and showed a definite correlation to the content of the antimicrobial in the feed. A striking exception to this general tendency was the high incidence (65.0%) of DS-resistant organisms ( Table 5). There was no evidence that this drug had been used on any of the farms for the previous 6 Table 4) that AM resistance occurred more frequently when penicillin was used in the feed (27.9%) than when it was not used in the feed (6.4%). This finding appears to be in contrast to an earlier report (6) which indicated that penicillin in feed may have little influence on the buildup of AM-resistant, gram-negative organisms. Of the 491 isolates from animals exposed to antimicrobials in feed, 394 (80%) were resistant strains and fell into 39 different antimicrobial patterns. In contrast, of the 64 isolates obtained from animals not exposed to antimicrobials in feed, 14 (15.6%) were resistant strains falling into 10 different antimicrobial resistance patterns. Table 6 illustrates the distribution patterns of singly and multiply resistant isolates. The most commonly occurring resistance patterns were those organisms resistant to three and four antimicrobials. The least common were organisms resistant to five and six antimicrobials. The occurrence of one and two resistance factors was equally prevalent. Nearly 70%O of all isolates contained three or four resistance factors. Following in sequence of occurrence were two resistances (12%7), five resistances (11 %), single resistances (6%) and six resistances (1 %). In Table 7 are presented the frequency and rank in which six of the antimicrobials were commonly included in resistance patterns and which occurred with additional resistances. NEO was found to be the most dependent on other resistance factors. It never occurred as a single resistance and thus was most likely to appear in a multiply resistant pattern. On the other hand, DS apparently has less dependency on other R-factors. It occurred more often as a single resistance than did any of the other antibiotics. The nine most common and frequently occurring resistance patterns found in the study are presented in Table 8. DS, SU, TE was the most frequently occurring pattern, followed by the DISCUSSION Antimicrobials have received widespreadK popularity as constituents of animal feeds in the United States. They are used at low levels for growth promotion purposes, at median levels for, disease prevention, and at therapeutic levels for the treatment and control of disease. One of the: five farms surveyed in this study (farm 5) utilized' a growth promotion level (50 g/ton) of oxytetracycline in the swine ration. Three other farms (1,, 3, 4) were utilizing a combination of drugs (peni-cillin, chlortetracycine, and sulfamethazine) at a combined level of 250 g/ton. This level of drugs is commonly used for disease prevention. In. comparing the percentage of multiply resistant isolates from these farms (86% for farms 1, 3, and 4 versus 76% for farm 5) there apparentlywas little difference in the levels of resistant organisms stimulated by the use of either ofthese dosage levels. The cattle on farm 2 were receiving a dosagelevel of chlortetracycline (350 mg per head perday) and sulfamethazine (350 mg per head per day) which might be considered disease prevention levels. These drugs were used on an intermittent basis, and the level of multiply resistant organisms was much lower in this group of animals (38.4%). The calves on farm 5 received a single therapeutic dose of nitrofurazone at birth. The incidence of multiply resistant organisms found in these animals was also comparatively low (19.0%) and comparable to that found in the herd of dairy cows receiving no antimicrobials in feed (15.7%). Forty per cent of the multiply resistant isolates from these animals could be traced to two cows which had recently been treated for mastitis. These findings indicate that the level of drug may not be the major factor in determining the amount of resistance that will develop. The continuous antimicrobial pressure seems to have a greater influence. This was true for both the level of resistance and the percentage of transfer factors that was observed. This work also confirms the earlier work of Walton (12), who found a definite association between the types of drugs supplied to the animals and the isolation from their feces of strains of E. coli resistant to these drugs and capable of transferring this resistance. The high incidence of DS resistance, even though this drug was not being used on the premises investigated, cannot be explained with the information available from these farms. One can only speculate about the origin of such a high incidence of resistance. It is well recognized that DS is commonly utilized as a therapeutic agent for many diseases of domestic animals. The drug may have been used therapeutically on these farms. Specific inquiries regarding such exposures were included in the questionnaire but could have failed to verify this fact. Another possibility would be the dependence of DS on the transfer factors of other antibiotics for its continual influence on resistance. This does not seem likely, since it was shown (Table 7) to be the least dependent upon the cross-transfer of R factors. However, recent work by Datta et al. (7) may provide some insight into this observation. They found that TE exerted strong selection, not only for TE resistance but also for multiple resistance, significantly increasing the frequency of resistance to AM, SM, CHLOR, and sulfonamide. A higher incidence of ampicillin resistant organisms was observed in herds which were receiving feed additive penicillin. The combination of three drugs (penicillin, chlortetracycine, and sulfamethazine) was substantially more effective in producing AM resistance of the E. coli isolates from swine than was oxytetracycline alone (86 of 307 strains versus 6 of 110 strains). This occurrence could be explained, and in fact might be expected, since both AM and penicillin are susceptible to the action of penicillinase, and penicillin would induce this enzyme among gram-negative enteric flora. However, since the use of both sulfonamides and TE may be associated with the emergence of heterologous resistance to AM, the role of penicillin per se can not be confidently ascertained. The observation of a substantially higher frequency of transmissible resistance among multiply resistant strains that come from animals exposed to antimicrobials invites further speculation. Presumably, a selective advantage is conferred to One 1 31 1 4 1 0 0 0 23 2 63 6 Two 3 53 2 40 2 0 2 1 36 3 142 12 Three 16 159 3 158 0 1 2 8 139 0 486 42 Four 62 80 15 77 3 4 0 13 67 1 322 28 Five 17 26 16 26 0 0 0 20 25 0 130 11 Six 3 3 1 3 0 0 2 3 3 It is probable that humans and animals ingest small and continuing doses of enteric organisms carrying resistance determinants; in the absence of antimicrobial selective pressures, such strains may conjugate with indigenous flora but no selective advantage accrues to their offspring. However, if a population of strains resistant to drugs being administered is present, and these strains carry transfer factor at a much higher frequency, as the data show, then the likelihood of transfer of resistance determinants to "outsiders" is increased; such transfers would confer a survival advantage to a recipient and its progeny which now carry the combined resistances of initial donor and recipient. This postulated mechanism may account for the appearance of heterologous resistance in animals exposed to agents such as sulfonamides and TE. Another area for concern was the inordinately high incidence of resistance observed in the swine herds. One herd yielded 100% resistant isolates, and the other herds ranged from 89% to 95% resistant isolates. A logical question would be whether these levels of resistance could be tolerated in high-density production procedures, especially if an infectious disease outbreak occurred. This is a particularly interesting question when one considers that the majority of the isolates were multiply resistant and contained viable transfer factors and that one of the major causes of early swine mortality and morbidity is colibacillosis. The selection of an effective therapeutic agent under these circumstances could become very difficult. The results of this survey suggest that a high incidence of resistant organisms does exist in animals being exposed to continuous levels of antimicrobial drugs. R factors were common in these organisms, and multiple resistance to three or more antimicrobials was usually prevalent. Serious consideration must be given to the desirability and future acceptability of producing livestock in an environment comprised primarily of highly resistant microorganisms.

v3-fos

2019-03-19T13:12:06.370Z

1971-05-01T00:00:00.000Z

237107673

s2

Growth of Aspergillus repens in Flue-Cured Tobacco In laboratory tests, flue-cured tobacco inoculated with Aspergillus repens was stored at 75, 80, 85, 87, and 95% relative humidity at 20 and 30 C. Samples were taken weekly for 4 weeks and evaluated for mold growth (colony count) and moisture content (MC). The weekly rate of fungus increase was slower at 20 C than at 30 C. Tobacco at 20 C with MC between 25 to 30% supported a slight to moderate increase in A. repens after 3 weeks of storage. However, tobacco at the same MC stored at 30 C was subject to rapid invasion by the fungus in as few as 1 to 2 weeks. Tobacco with MC above 30% stored at either 20 or 30 C became moldy in about 1 week. A mold index is proposed for evaluating populations of A. repens in tobacco. In laboratory tests, flue-cured tobacco inoculated with Aspergillus repens was stored at 75, 80, 85, 87, and 95% relative humidity at 20 and 30 C. Samples were taken weekly for 4 weeks and evaluated for mold growth (colony count) and moisture content (MC). The weekly rate of fungus increase was slower at 20 C than at 30 C. Tobacco at 20 C with MC between 25 to 30% supported a slight to moderate increase in A. repens after 3 weeks of storage. However, tobacco at the same MC stored at 30 C was subject to rapid invasion by the fungus in as few as 1 to 2 weeks. Tobacco with MC above 30% stored at either 20 or 30 C became moldy in about 1 week. A mold index is proposed for evaluating populations of A. repens in tobacco. In a survey to determine the fungi associated with damaged flue-cured tobacco, Nicotiana tabacum L., species of Aspergillus and Penicillium were found to be predominant (13). Aspergillus repens, the most prevalent, was isolated from 81 % of the samples, averaged 12,900,000 colonies per g of tobacco, and ranged from 0 to 120 million colonies per g. The official USDA definition of tobacco damage does not include a qualitative or quantitative determination of the microorganisms. Damage is defined as "the effect of mold, must, rot black or other fungal or bacterial diseases which attack tobacco in its cured state. Tobacco having the odor of mold, must, or rot is considered damaged" (10). Factors that determine whether microorganisms will grow in cured tobacco are probably temperature and moisture content, as has been reported for cereal grains (1,2), some forest seeds (7), and textiles (6). Moisture content is important in the handling, storage, marketing, manufacture, and preservation of flue-cured tobacco. Tobacco grades, however, do not reflect moisture content unless the U.S. Government grader believes the tobacco is too moist for safe storage for a few days. Such tobacco is graded "W" (wet), "U" (unsound), or "No-G" (No-grade; reference 10). It is advantageous to the farmer to market tobacco as moist as possible without risking apparent deterioration. Moisture content directly affects gross return to the grower but, above safe levels, also increases the danger of deterioration. Moisture content considered I Paper no. 3369 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, N.C. 27607. proper by the farmer during marketing differs from that considered proper by the buyer for storage and manufacture. Therefore, tobacco is redried immediately after purchase to about 11% moisture (wet-weight basis); tobacco at or below 12% moisture ages properly and is safe from deterioration by fungi for prolonged periods (4). After farm curing and until purchased by the consumer as a finished product, tobacco undergoes several changes in moisture content. In a 1967 survey, 100 samples of tobacco from 12 tobacco markets in two tobacco belts had moisture contents ranging from 12.6 to 30.2% (13); the mean was 19.6%. For aging, tobacco is dried to 10 to 11 % and stored for 1 to 3 years. During the manufacture of blended cigarettes in the United States, tobacco is remoistened to 16 to 20% for cutting and redried to 12 to 13%o for machine manufacture into cigarettes. Most American cigarettes are marketed with 12 to 13% moisture. A. repens, the predominant fungus isolated from damaged tobacco, was also isolated from nondamaged flue-cured tobacco in storage and at the market (12,13). The fungus was not isolated from green tobacco leaves before curing or from dried leaves immediately after fluecuring (14). Green plants sprayed in the field did not become infected by A. repens (11). These data indicate that the association between A. repens and tobacco is saprophytic and, when moisture content is sufficient for fungal invasion, some time between on-farm curing and marketing, A. repens becomes associated with the tobacco leaf. To determine the conditions for and the rate at which A. repens grows in tobacco at different moisture contents and to use this information to relate to tobacco in storage, inoculated fluecured tobacco was stored in the laboratory at different relative humidities and temperatures. Fungus increase and tobacco moisture content were measured weekly. A preliminary report has been published (R. E. Welty, Phytopathology 60:1319). MATERIALS AND METHODS Fungus source. The strain of A. repens (MQF-1) used was isolated from nondamaged tobacco offered for sale in a tobacco warehouse at Durham, N.C., in 1967. Tobacco source. Field grown tobacco with relatively little brown-spot disease (caused by Alternaria tenuis Nees) was harvested and flue-cured in the usual manner (4) at the Border Belt Tobacco Research Station, Whiteville, N.C. The cured leaves were selected at random from all stalk positions, compressed, shredded into 1-mm-wide ribbons of various lengths, and stored at 15.8 to 17.5% moisture content at room temperature (23 to 28 C) until used for the experiments. Test unit. For measuring fungus increase, 40 tobacco samples (one for each of five relative humidities X two temperatures X four sample intervals) of 10 g each were weighed in 9-cm diameter petri dishes, sterilized in an unpressurized steam chamber, inoculated, and placed in desiccators above saturated salt solutions. For moisture content determinations, 40 10-g samples of tobacco were weighed in tared stainless-steel dishes, sterilized, inoculated, and also placed in the desiccators. Weekly for 4 weeks a glass and a metal dish of tobacco were removed from each desiccator and evaluated for growth of A. repens and for moisture content. inoculation. A 1-ml amount of an aqueous spore suspension of A. repens containing 1 million spores/ml was atomized onto the tobacco in all dishes. This resulted in a calculated initial inoculum of 100,000 spores per g of tobacco. The spore suspension was made by washing, with 10 ml of sterile distilled water (plus 0.06 ml of Tween 80), the surface of a 10day-old test tube culture of A. repens on potato dextrose-agar. The spores were counted in a hemocytometer, and the suspension was diluted with sterile distilled water to obtain the standard spore suspension. (16). These salt solutions were previously found to produce moisture contents in the range encountered by flue-cured tobacco during marketing (15). Inoculated tobacco in glass and steel dishes was placed over each of the five saturated salt solutions and incubated at 20 and 30 C. Evaluation. Growth of A. repens and tobacco moisture content were determined after 1 week of storage and subsequently at three weekly intervals for each storage condition (five RH X two temperatures). The steel dishes were removed from each storage condition at random and dried by heating at 100 C for 16 hr in a ventilated oven (8). Moisture content (MC) was calculated from the weight lost and is expressed on a wet-weight basis. Populations of A. repens were determined by a method previously reported (13) but slightly modified. The glass dish containing 10 g of inoculated tobacco was blended in 500 ml of a sterile solution of 0.15% agar for 2 min. Further dilutions were made, and 1 ml of each dilution was placed in each of three petri dishes. Czapek's plus 6% NaCl agar (Cz+6) and weak tomato juice-agar (WTJ), melted and cooled to 52 C, were added to one and two dishes, respectively. The dishes were swirled to distribute the suspension and incubated at room temperature. After 2, 4, and 6 days, young colonies were counted, and, on the sixth day, the dishes were incubated until the fungal colonies could be identified. Colony counts were determined by multiplying the dilution factors 10-3 through 10-8 by the average number of colonies growing in the two petri dishes of WTJ agar determined on the sixth day. Dilutions below 1: 101 were not cultured. A single petri dish of Cz+6 was included in the test to confirm culture purity and to facilitate identification of possible contaminating species of other fungi, particularly of the genus Aspergillus. For some treatments, A. repens colonies become too numerous to count at lower dilutions. As a result, data used for the analysis of variance were based on the counts from paired dilution cultures that averaged 20 to 100 fungal colonies per dish. Fungal increase in this study is based on colonies per gram. This procedure has been used previously (13) to measure the kinds and amounts of fungi associated with tobacco. Experimental design and statistical analysis. The experiment was set up in a split plot design with the whole plot (RH) arranged as a randomized complete block design. The subplot treatment was storage time, and the experiment was replicated three times. The resulting data for both temperatures were statistically analyzed by using an analysis of variance. Colony counts were converted to logarithms (base 10) for the analysis. The antilog was used to tabulate the results. RESULTS AND DISCUSSION When stored tobacco was gaining moisture and an MC suitable for growth of A. repens was reached, increased growth of the fungus was directly related to time and temperature (Fig. 1,2). It was unfortunate that the tobacco was stored in various environments immediately after inoculation as the aqueous spore suspension increased the tobacco MC above the equilibrium MC maintained by some of the saturated salt solutions. This resulted in decreasing tobacco MC in some conditions (solutions removing moisture from the air around the tobacco, which in turn removed moisture from the tobacco) as fungus counts increased. These op- positely changing values make it difficult to locate a minimum MC for A. repens growth in stored tobacco. From the tobacco stored at the five RH levels at 20 C, the minimum MC for A. repens growth appears to be near 29% since it was not until this MC was reached that the fungus began to increase. This value probably is not accurate. It seems likely that temperature affected the growth of A. repens in tobacco more than MC. Other evidence (2) indicates that A. repens is capable of growth in materials at MC in equilibrium with RH of 71 to 75 % and data obtained in this study for 30 C support this contention. Perhaps if the incubation period at 20 C had been longer than 4 weeks, fungus growth in the tobacco at lower RH may have been observed. Likewise, data obtained at 30 C do not indicate precisely where the minimum MC for fungus growth occurs, but it appears to be between 20 and 25% (see 75% RH, Fig. 2). Despite the decreasing MC from 25.3 to 21.3%, colony counts increased steadily from 293 to 48,750. The rate A. repens grew in stored tobacco at 30C with MC between 25 and 30% (see 80% RH) indicated a rapid increase from 358 to over 1 million colonies per g from 1 to 3 weeks. After 3 weeks at 20 C, only a slight-to-moderate increase in the fungus occurred in tobacco in the same MC range (see 85% RH, Fig. 2). At either 20 or 30 C, tobacco with MC above 30% is likely to be thoroughly invaded by A. repens after 1 or 2 weeks. Statistical analysis of the log of mean colony counts of A. repens per g of tobacco at 20 C showed significant differences at the 0.05 probability level in counts between RH levels and also for storage times within RH (Fig. 1 and Table 1). The mean square value for the analysis of the colony counts at the five RH levels at 30 C ( Fig. 2, Table 1) was significant only at the 0.07 level, due probably to considerable variability in the data. The MC of stored tobacco at 75 % RH at 30 C was certainly less favorable for A. repens growth than MC at other RH. The growth of an organism in pure culture frequently follows a sigmoid or S-shaped curve. Although none of the storage conditions with the test intervals used in this study represent the entire typical growth curve, individual storage conditions supply portions of that curve. The maximum stationary phase of the sigmoid curve for log colony count (base 10) appears to be near 10 which would be equivalent to 10 billion colonies per g. As expected, the maximum stationary phase is reached sooner at the higher RH and at 30 instead of 20 C. If counts of log base 10 are the actual maximum population which A. repens reaches per g of stored tobacco, then count values below these might be used to evaluate tobacco condition in actual storage with respect to the amount of damage caused by A. repens. The log of the colony count (base 10) could be used as a mold index (MI). Since we usually do not culture dilutions below 10in routine tests for the numbers and kinds of fungi in a tobacco sample, the lowest possible mold index would be MI 3. Based on present information, the index for the maximum VOL. 21, 1971 population of fungus which might be expected in tobacco would be MI 10 or MI 11. Applying this scheme to data already published (13) on A. repens in 74 samples of damaged and 26 samples of nondamaged tobacco in storage, damaged tobacco had an average MI of 6 and a range from MI 3 to MI 8; nondamaged tobacco had an average MI of 3 and a range from MI 3 to MI 5. The following data were not presented in that report, but came from that study and are used here to prepare a frequency distribution for the amount of A. repens in each MI category occurring for the samples in each tobacco class. Although populations of A. repens did not reach MI 10 in any samples of stored damaged tobacco, the system does appear to have some usefulness. The reason MI 10 was not reached is probably because of competition with other storage fungi (in some samples as many as eight) or because the storage period was not long enough. As more tobacco samples are evaluated for populations of A. repens, they will be assigned an MI by the scheme just described. It is hoped that the system will provide a convenient measure for evaluating fungi in a tobacco sample of unknown storage history and also provide for convenient comparisons between samples. After 4 weeks of incubation, mold growth in tobacco was not visible at and below 85% RH at 20 C and at 75% RH at 30 C without 25 or 50 x magnification. Despite the absence of macroscopically visible mold below 87 % RH and 20 C, the dilution culture from 85% RH and 20 C yielded 431,500 colonies of A. repens per g of tobacco (Fig. 1, Table 1). Also, at 75% RH and 30 C, no mold was macroscopically visible and yet the count was 48,800 colonies per g. These examples indicate the subtle nature of growth of the fungus in stored tobacco and support the contention that visual examination of tobacco for mold growth without the aid of magnification is not a reliable indicator of tobacco condition and should not be used to predict storability. By the time fungal growth is evident to the casual observer, it is usually too late to initiate control measures. These data do not show why fungal populations decrease under certain storage conditions. Some field fungi that invade (9) and some bacteria present on (5) stored barley lose viability rather rapidly if grain is stored at MC just below the limit for their growth. If the seeds are dry, these same organisms will survive for several years (3) and if the seeds are at a high enough MC, these organisms will grow (1). It seems likely, therefore, that the disappearance of A. repens from tobacco in these test conditions might be due to storage at MC near or below their limit for growth and may be similar to the situation with microorganisms in stored barley. These results might also explain the association of A. repens with the leaf and how tobacco infested with the fungus becomes damaged. Whenever the MC of the cured leaf exceeds a minimum level (perhaps between 21 and 25%) and temperatures approach the optimum for the fungus (in this case near 30 C), populations of the fungus will probably increase and "damage" may occur. If tobacco MC fluctuates around the minimum required for growth, conditions are sometimes favorable for invasion by A. repens but not yet favorable for rapid growth. In this situation A. repens can be isolated from tobacco not yet damaged, as is true of marketed tobacco (13). If tobacco MC is below the minimum for growth of A. repens, as it is immediately after on-farm curing, A. repens cannot invade the tissue (14). The presence of A. repens in cured tobacco could be interpreted to indicate that at one time or another MC had exceeded the minimum to permit fungus invasion, regardless of the MC when the sample was taken. Christensen and Kaufman (2) found this to be true for stored cereal grain and reported "for each of the common species of storage fungi there is a minimum moisture content in grain below which it cannot grow, these minimum moisture contents have been determined fairly accurately for most of the common storage fungi growing on the starchy cereal seeds and on some of the oils seeds." Elsewhere (1) they stated "the number and kinds of fungi isolated from a given lot of grain can tell a good deal about how the grain has been stored." The situation for stored tobacco is likely to be similar to that for stored grain. Studies are now in progress to establish more precisely the minimum MC required for A. repens to grow in stored flue-cured tobacco alone and in competition with other storage fungi.

v3-fos

2020-12-10T09:04:16.723Z

1971-05-01T00:00:00.000Z

237232782

s2

Initiation of Staphylococcal Growth in Processed Meat Environments The probability of staphylococcal growth initiation in laboratory-made cured meats is investigated and compared with growth initiation in broths. The probability of staphylococcal growth initiation in laboratory-made cured meats is investigated and compared with growth initiation in broths. Genigeorgis et al. (4) developed equations to predict the probability that one staphylococcal cell will initiate growth aerobically in Brain Heart Infusion (BHI) broths of various pH values and NaCl concentrations at 30 C. The studies reported here tested the reliability of the developed equations in predicting the probability of staphylococcal growth in a variety of laboratory-prepared processed meats. Thirty-nine processed meats at different pH values and with various concentrations of NaCl and NaNO2 were utilized during the course of four experiments. The meats were prepared by homogenizing defatted ground pork loin or beef sirloin in a meat grinder with desired amounts of NaCl and NaNO2. After 24 hr of refrigeration, the pH of each meat was adjusted to low and high levels by the addition of 1.75% (w/w) glucono-6-lactone powder and NaOH (5 ml of 2% NaOH/100 g), respectively, and the meats were again homogenized. After this, they were cured for an additional 2 days and then pasteurized for 15 min (6 min to 121 C, 7 min at 121 C, and 2 min to room temperature), after which the meat juices were drained off. The tubes containing meats with the same brine concentration were placed in a can along with a solution of NaCl equal in concentration to that of the brine concentration of the meats. The cans were covered with plastic lids, and the cultures were then incubated aerobically for 7 days at 30 C. Samples of the various types of meats were then analyzed for pH, brine concentration, water, and nitrite according to procedures described previously (5). After incubation, the meats were homogenized with 10 to 20 ml of sterile distilled water, and portions were plated on blood-agar (BHI base) for staphylococcal and total plate counts. The remainder of the homogenate was centrifuged, and the supernatant fluid was lyophilized after dialysis and treatment with chloroform (2). The lyophilized samples were rehydrated with 0.6 ml of phosphate-buffered 2.8% saline (pH 7.2, 0.02 M) and then tested by the single-gel diffusion (6) and microslide (3) techniques for the presence and amounts of enterotoxins B and C. The results of the experiments are presented in Tables 1 and 2. Only the minimum numbers of cells which initiated growth have been included. The present findings indicate that the meat environments were more conducive to growth of staphylococci than were the BHI broths. Also, at the same brine concentration and pH, it took fewer cells to initiate growth in meats than in BHI broths. This was particularly evident in meats with high brine concentration and low pH. Staphylococcal growth was initiated in such meats even though the levels of inoculated cells were below the minimum limits predicted by the equations (4) as necessary for initiation of growth. As expected (6,7), elevated yields of enterotoxins B and C were obtained at high pH values coupled with low brine concentrations. All meats supporting enterotoxin production had good staphylococcal growth to over 4 x 107 cells per sample. Yet, 70 samples with 108 cells per sample had no enterotoxin B or C production. The highest yields of enterotoxins B and C were 70 and 20,gg per sample, respectively. The results obtained in these studies indicate that the equations derived from data based on previous studies with BHI broth (4) cannot reliably be used to predict the probability of initiating staphylococcal growth in processed meats. Experiments statistically designed to permit development of predictive equations derived from results obtained by direct staphylococcal inoculation of meats are now in progress.

v3-fos

2018-04-03T00:00:34.947Z

1971-12-01T00:00:00.000Z

8346082

s2

Microbial Transformation of Flavonoids The ability of a number of fungal spores, and in particular of resting vegetative mycelia, to transform naringin and naringenin was studied. In general, only hydrolytic cleavage of the sugar moieties of naringin to produce prunin and naringenin was observed. Two cultures, Penicillium charlesii and Helminthosporium sativum, also produced two unidentified flavonoid compounds but in very low yields. No transformation of aglycone was detected, although the compound was metabolized by some cultures when supplied as the glycoside prunin. A fluorodensitometric method was developed for the quantitative analysis of flavonoid compounds. Recent action by the U.S. Food and Drug Administration banned the use of cyclamates as synthetic sweeteners in foods. It has been shown by Horowitz (11) and Horowitz and Gentili (12) that the chalcone and dihydrochalcones corresponding to naringin and neohesperidin are intensely sweet and appear to be free from toxicity to laboratory animals (3). However, the sensation of sweetness from the dihydrochalcones is slow in onset but of lasting duration and also imparts a menthol-like quality. These latter properties are regarded as undesirable characteristics for use in food and beverages. During a study of the transforming potential of fungal spores, we investigated the ability of conidia to convert naringin, the bitter principle of grapefruit, to a sweetening agent lacking the undesirable properties of the dihydrochalcones. These studies were subsequently extended to include vegetative mycelia. We confined our investigation to naringin, which is available in commercial quantities, and did not investigate the use of other less readily available flavonoid compounds. I Presented in part at the 71st Annual Meeting of the American Society for Microbiology, 2 to 7 May 1971, Minneapolis, Minn. MATERUILS AND METHODS Microorganisms. Microorganisms were maintained on Difco Y-M agar slants at 4 C. To develop inoculum, a small portion of a slant culture was inoculated into 100 ml of YES broth (yeast extract, 2%; sucrose, 15%) in 300-ml indented Erlenmeyer flasks. The inoculated flasks were incubated at 28 C for 48 hr on a rotary shaker (250 rev/min, 5-cm stroke), and the mycelia were recovered by centrifugation and washed six times with 100-ml portions of 0.1 M phosphate buffer, pH 7.0. The washed cells were resuspended in 100 ml of buffer; 10-ml portions of the suspension were added to 90 ml of buffer plus 0.75% naringin in 300-ml Erlenmeyer flasks. For larger-scale experiments, 50 ml of suspended mycelium was added to 450 ml of buffer plus 0.75% naringin in Fernbach flasks. Naringin was purchased from Sunkist Growers, Inc., Ontario, Calif. Bioconversion flasks were incubated at 28 C on a rotary shaker at 250 rev/min. Spores. Spore suspensions were prepared as previously described (14). For bioconversions, sufficient spores were added to the flasks to give an inoculum of about 109 spores/ml. Extraction of transformation products. At selected times during the incubation period, 5-ml portions were removed and centrifuged, and a small volume of supernatant fluid was spotted directly onto thin-layer chromatography (TLC) plates. For quantitative analyses, the 5-ml sample was extracted twice with 4-ml portions of butanol; the solvent extracts were combined; and the volume was adjusted to 10 ml. Further dilutions were made with butanol when required. TLC. TLC plates, 20 by 20 cm, were coated (0.5 cm) with Silica Gel G-HR (Brinkmann Instruments, Westbury, N.Y.). After being spotted with measured volumes, plates were developed in ethylacetate-isopropanol-water (80:20:10, v/v) until the solvent front was 1 to 2 cm from the top edge. The plates were airdried, sprayed with a freshly prepared solution of 2% sodium borohydride in methanol, and then exposed to HCI fumes for 15 min (10). Flavanones gave a bluishred color. The plates were also examined for the yellow color often produced by chalcones, flavones, flavonols, isoflavones, and aurones and for the occurrence of fluorescent derivatives under ultraviolet light. Flavonoid compounds were also detected by spraying the plates with a 2% solution of AlCli,6H20 in ethanol to give bright yellow-green fluorescent spots under ultraviolet light. Compound isolation. Isolation of products was achieved by extraction from the supernatant fluid of two Fembach flaskswith two 1-liter portions ofbutanol. The extracts were combined, the solvent was removed by flash evaporation, and the residue was dissolved in 40 to 50 ml butanol; insoluble material was removed by centrifugation. The solvent solution was then added to a small amount of 60to 100-mesh Florisil (Sigma Chemical Co., St. Louis, Mo.), the solvent was evaporated, and the dry solids were placed on top of a Florisil column (5 by 50 cm). The column was developed with benzene followed by 10% increment additions of methanol until the column was finally irrigated with methanol only. The eluate was collected in 15-ml portions with a fraction collector. Flavonoid elution was determined by testing every tenth tube by TLC. The first fraction (I) came off the column with 10 to 20% methanol; the second fraction (II) with 30 to 70% methanol. The solids in fraction I were dissolved in ether, the insoluble material was removed by filtration, and the filtrate was added slowly to hot water. After cooling at 4 C, white needlelike crystals were recovered by filtration, recrystallized from water, and dried in vacuo. Fraction II was rechromatographed on a Florisil column to effect further separation of the two flavonoids present in this fraction, naringin and an unknown. The separation was still imperfect. The unknown flavonoid was separated from naringin by preparative TLC on Silica Gel G-HR with the solvent system, ethyl acetate-isopropanol-water (80:20:10, v/ v). Zones were located by covering most of the TLC plate with a clean glass plate and spraying only a narrow vertical strip with sodium borohydride followed by concentrated HCI. The zone containing the unknown was scraped off; the compound was eluted with methanol and precipitated by slow addition of water and cooling. A second precipitation from the same solvent system was effected, but crystals were not formed. The white solids gave only a single spot on TLC in several solvent systems. Densitometric assay. For quantitative densitometric analysis, the Silica Gel G-HR coating on the TLC plate was scored by a mechanical device to give 20 lanes, each 1 cm in width. For absorbance analyses, only every other lane was spotted; for fluorescence determinations, every lane except the two outer lanes was used. A Schoeffel dual-beam spectrodensitometer model SD 3000, equipped with a mechanically driven stage and attached to a Schoeffel density computer, SDC 300, and a Disc Instrument Corp. model 610 automatic printer, was used for all quantitative analyses. Absorbance analyses were conducted with the dualbeam facility and the instrument set at a wavelength of 530 nm, the slit width on the scanner at 1.5 mm, and the slit width on the substage at 1 mm with the gain at 1.5. The single-beam setting was used for fluorescence analyses; excitation was set for 310 nm and emission for 504 nm; gain was at 8 and the slit of the scanner was set at 2.5. Fluorescence maxima for the AlC13 reaction products, obtained by adding a few drops of A1Cl3 solution to the purified compounds in ethanol, were obtained with an Aminco-Bowman Spectrophotofluorometer. Absorbance maxima of the reaction products with sodium borohydride and HCI were obtained with a Beckman model DB spectroplotometer. RESULTS Spore studies. Initially, 14 spore strains representing six genera and 10 species were tested for their ability to act on naringin. These organisms were: Aspergillus candidus, A. niger (three strains), A. ochraceus (three strains), A. oryzae, Myrotheciwn verrucaria, Paecilomyces varioli, Penicillium charlesii, Rhzizopus sp., Trichoderma roseum, and T. viride. After incubation for periods up to 1 week, some of the spores converted naringin to two new flavonones but in concentrations too small to permit ready isolation and identification. Spores of P. charlesii NRRL 1887 appeared to be the most active. In an attempt to increase product yield, studies were made in which the mycelium rather than the spores of this latter organism was used. fijikuroi has been reported to transform flavanone to a variety of products (18,19). After 5 days of incubation on both naringin and naringenin, using washed mycelia of 10 strains, we were unable to detect any major by-products other than the starting compounds. However, strains NRRL 2633, 2634, 2635, and 3198 either partially or completely degraded the naringn with only slightly detectable quantities of naringenin being produced. A. candidus. Two strains of A. candidus (ATCC 20022 and CMI 16046) produce an antifungal flavonoid antibiotic, chlorflavonin: 3'-chloro-2', 5-dihydroxy-3,7, 8-trimethoxyflavone (17). We incubated washed mycelia of eight strains of this organism in a medium containing phosphate buffer, 0.75% naringenin, and 0.5% KCI. The naringenin was poorly soluble and was present 975 VOL. 22, 1971 suspended mostly as fine particles. After 5 days of incubation, no transformations to a potential chlorinated derivative were observed. The cultures were extracted with butanol and the extracted substances were tested against Mucor ramannianus NRRL 1839 for antifungal activity; none was observed. P. charlesii NRRL 1887. There was considerable variation in the rate of production of the two principal products by washed mycelia of this organism. In most experiments, there was an initial lag of 4 to 8 hr before the appearance of products; thereafter, the reaction went rapidly, peaking at about 16 hr; after this time, all flavonoid compounds were gradually degraded as determined by TLC. The two principal compounds produced were identified as: fraction I, 5,7 ,74'-trihydroxyflavanone (naringenin); fraction II, naringenin-7-glucoside (prunin). Production of these compounds indicated the enzymatic action of naringinase. Compounds were identified by the following criteria. Naringenin. Elemental analysis: C, 65.90%; H, 4.66%; oxygen by difference, 29.44% (theoretical, C, 66.17%; H, 4.45%; 0, 29.38%). The elemental analysis and molecular formula, C15H12* 05, were confirmed by high-resolution mass spectroscopy, which also gave an mle, 272.27. There was no depression on a mixed melting point analysis with authentic naringenin, 246.7 to 247.2 C (determined with a Mettler FP-1 apparatus; 15). The compound co-chromatographed with authentic naringenin in four separate solvent systems and gave identical nuclear magnetic resonance spectra. A bathochromic shift from 289 nm in ethanol to 328, 328, and 311 nm on addition of sodium acetate, NaOH, and AlCl3. 6H20, respectively, matched literature values for naringenin (13). These shifts also confirmed the presence of OH-groups in the 7 and 5 positions. The color reaction with sodium borohydride and concentrated HCl of the experimental and authentic compounds gave a bluish-red color with a maximum at 544 nm. Prunin In some experiments, naringn was acted upon by P. charlesii at a much slower rate (Fig. 1) so that it was possible to detect the presence of two additional substances, but at concentrations too low to permit isolation. Their lower RF values, 0.40 and 0.34, as compared with naringenin, prunin, and naringin (0.91, 0.72, and 0.51, respectively) on TLC plates (Silica Gel G-HR; ethyl acetate-isopropanol-water, 80:40:10, v/v; Fig. 2), indicate that they might be the corresponding flavones oxidized at positions 2 and 3 or flavan-4-ol. These compounds reacted with sodium borohydride and HCI to give a red color and with AlCl3 to give a yellow-green fluorescence under ultraviolet light, indicating that they were not chalcones. Growth of P. charlesii in a medium containing naringin resulted in rapid degradation of the flavonoid but did not eliminate the initial product lag observed when washed mycelia were placed in transformation media. Addition of 2% glucose plus the naringin to this latter medium resulted in repression of naringinase activity. Washed cells of P. charlesii neither degraded naringenin nor produced derivatives after 5 days had the same R. values (0.40 and 0.34) as those produced by P. charlesli NRRL 1887. No other compounds could be detected by the sodium borohydride or A1C13 reactions. After 4 days of incubation, degradation of the by-products occurred rapidly. Densitometric analyses. Two methods were developed for the quantitative densitometric analyses of the flavonoids. The first method involved the reaction of these compounds on a TLC plate after development with sodium borohydride and HCI fumes to form red to bluish-red derivatives (10). The absorption maxima of the pigmented derivatives of naringin, prunin, and naringenin are 528, 534, and 545 nm, respectively. A linear response was obtained for all three flavonoids between 0.25 and 3.5 ,ug when the densitometer was set at 534 nm (Fig. 3). However, the pigments formed were highly unstable, necessitating completion of the analyses within 20 min after color development. The second analytical method which involved spraying the TLC plates, after development, with a 1 to 2% ethanolic solution of AlCl3. 6H0 resulted in formation of a highly stable, yellowgreen fluorescent derivative. Excitation and emission spectra of the AlCI3 derivatives of naringin, prunin, and naringenin are shown in Fig. 4 and 5. A linear response was obtained for naringin of incubation when this aglycone was used in place of naringin. This later method should be applicable to the I tion of chalcones from flavanone as a result of opening the heterocyclic ring between the 1 and 2 positions. Horowitz and Gentili (U.S. Patent 3,087,821, 1963) have produced chalcones from naringin, prunin, and neohesperidin by the action of 20 to 25 % alkali followed by a reduction of the ethylenic double bond to yield the intensely sweet dihydrochalcones. It would appear desirable to effect opening of the heterocycic ring by a milder process, such as microbial action, to avoid the necessity of neutralizing 20 to 25% alkali to recover the chalcone. Although none of the fungi tested acted on the heterocyclic ring, it might be worthwhile to carry out a more extensive screen. An operational difficulty, however, is the vulnerability of the sugar moieties on ring A to microbial action, as we have observed in the present investigation. Horowitz and Gentili (12) have shown that the glycosyl or neohesperidosyl radical on ring A of certain flavonoid compounds is critical for the property of sweetness. All of the fungi examined that were capable of hydrolyzing naringin were able eventually to degrade the aglycone produced, but were unable to transform or degrade naringenin when supplied as the free aglycone. A similar observation was made by Krishnamurty et al. (16) for Butyrivibrio sp. C3 which could anaerobically degrade quercetin when supplied as the 3-rhamnosylglucoside (rutin) or the 3-rhamnoside (quercitin) but could not degrade the free aglycone. This inability to metabolize the free aglycones may result from nonpermeability into the cells since these compounds are relatively insoluble.

v3-fos

2020-12-10T09:04:11.246Z

1971-07-01T00:00:00.000Z

237233668

s2

Quick Counting Method for Estimating the Number of Viable Microbes on Food and Food Processing Equipment A rapid method for estimating the extent of microbial contamination on food and on food processing equipment is described. Microbial cells are rinsed from food or swab samples with sterile diluent and concentrated on the surface of membrane filters. The filters are incubated on a suitable bacteriological medium for 4 hr at 30 C, heated at 105 C for 5 min, and stained. The membranes are then dried at 60 C for 15 min, rendered transparent with immersion oil, and examined microscopically. Data obtained by the rapid method were compared with counts of the same samples determined by the standard plate count method. Over 60 comparisons resulted in a correlation coefficient of 0.906. Because the rapid technique can provide reliable microbiological count information in extremely short times, it can be a most useful tool in the routine evaluation of microbial contamination of food processing facilities and for some foods. Several methods are commonly used to determine the extent of microbial contamination on foods and equipment: standard plate count, spread plates, shake tubes, most probable number, and dilution to extinction. These methods share the disadvantage of the time required for the microbial cells to grow to visible concentrations, usually 24 to 72 hr. Attempts have been made to shorten the length of incubation of colony-counting procedures. Such modifications have included the Frost "little" plate (5), agar strips, roll, and oval tube methods. Although these methods have been accepted in certain areas of microbiology, they have not supplanted the standard plate count method with its reliance upon 24 to 72 hr of incubation. Microscopic methods of Breed and Brew (1-3), although applicable to milk, are difficult to apply with vegetables where food particles are difficult to distinguish from the bacterial cells. In addition, there is the limitation of being unable to distinguish living from dead cells. Frazier and Gneiser (4), reporting on use of the membrane filter, reduced the time to 8 to 18 hr of incubation; however, they concluded that only limited application of their method was feasible in routine examination of fresh and frozen vegetables. In the studies reported here, the use of membrane filters to concentrate microbial cells, plus the use of a vital stain, makes it possible to observe microcolonies which develop after 4 to 5 hr of incubation. MATERIALS AND METHODS Reagents. Janus B green (60 mg per 100 ml in distilled water) was filter-sterilized. The medium employed was standard plate count agar (Difco). Preparation of sample. Fifty grams of sample was aseptically added to 450 ml of sterile saline solution and shaken vigorously for 1 min. A 10-ml amount of this diluent was then filtered through an 8.0-,um membrane to remove larger particles of vegetable material and yet permit passage of the bacteria. A washing with sterile water followed. The diluent and wash water were caught in a 1-liter sterile vacuum flask. The entire contents of the flask were filtered through a sterile 0.45-jAm membrane, which was then cut into three strips which were incubated on a sterile absorbant pad pretreated with 2 ml of medium. The strips were removed at intervals of 4, 5, and 6 hr, fixed in an oven at 105 C for 5 min, and stained with Janus green, taking care not to dislodge the microcolonies. After drying in an oven at 60 C, the strips were rendered transparent by immersion oil and examined under various magnifications. An average count per field was obtained and converted to units per gram. A simple method of accomplishing this was to determine the number of microscopic fields, for the magnification used, on the total membrane. This was done by lining up the edge of one field with the edge of a grid mark on the corner of one of the squares of the membrane. The grid marks appear as a series of dots, and by scanning across a line, with the dots of the line as a guide, the number of fields along one side was counted. It can be determined mathematically that the relationship of the area of the microscopic field to one grid square is 14n2/l 1, where n = the number of fields along one side of the square. As there are 100 squares in the membrane covered, multiplying by 100 gives the number of fields on the membrane. The colonies in the first strip were usually easily identifiable; however, in cases where longer incubation was necessary, the examination was facilitated by using the second or third strip. RESULTS AND DISCUSSION Because the membrane filter can be rendered transparent by immersion oil, it was assumed that the entrapped, living bacteria could be identified microscopically provided they could be differentially stained from dead cells. Although some success was realized by counting individual cells, it was difficult to differentiate between particles of food and bacterial cells, especially cocci, even with magnifications of 1,000 to 1,250 X. Pre- liminary filtration through filters of larger pore sizes was attempted to remove larger food particles. Since the use of filters having a pore diameter of 1.2 Asm, as suggested by Nobile (6), resulted in the entrapment of most of the bacterial cells, larger pore filters were used. It was found that an 8.0-ptm membrane would filter out the larger particles of vegetable without trapping the bacteria; however, difficulty remained in differentiating between bacteria and vegetable matter. Attempts were then made to allow the bacteria to grow sufficiently to form microcolonies, which could be more readily identified microscopically. T'he membranes were prepared as previously described, but, before staining, the membranes were placed on absorbant pads saturated with a nutrient medium (glucose-tryptone-yeast extract), incubated at 30 to 35 C for various periods of time, stained, dried, rendered transparent, and exarmined microscopically. It was observed that microcolonies could be identified after 2 to 4 hr of incubation under magnifications of 800 to 1,000 X (Fig. 1) and after 4 to 6 hours when a magnification as low as 80 x was used (Fig. 2). During the staining procedure, many colonies were found to be washed from the filter surface. The problem was solved by application of mild heat (105 C) for 1 to 2 mmn to fix the colonies without adversely affecting the membrane. If the rapid method is to be of practical value in estimating microbial populations, the results must be comparable to results from established methods of bacteriological quantification. Comparisons were made between the rapid method and the standard plate count method by running parallel determinations on several series of samples of fresh and frozen vegetables and on 1.9 X 106 2.9 X 104 3.5 X 104 4.9 X 106 1.8 X 104 2.0 X 104 9.1 X 104 7.7 X 104 a Statistical analysis: x = 9.666 X 106; y = 9.019 X 106; Xx = 5.993 X 108; Zy = 5.592 X 108; n = 62 (TLC and TNC disregarded); r = 2xy nxy/[(Zx2 -nX2) (2y2 -ny2)Jl/2 = 0.906. b Too numerous to count. c Too low to count. equipment surfaces. Various amounts of contaminations of frozen vegetables were effected by varying the care taken in preparing the vegetables before freezing. Approximately 60 comparisons were made and analyzed statistically ( Table 1). The correlation coefficient of 0.906 indicated that the rapid or quick count method is statistically valid when compared to the standard plate count method. The value of the quick count method lies in the fact that, within a relatively short time, 4 to 5 hr, persons with minimal training can determine, by line-checks and equipment and product monitoring, whether a given product will meet specific microbial specifications when packed and frozen. The quick count method is not intended to replace the standard plate count method in all aspects of food microbiology, nor can it be expected to give the same degree of accuracy within a 4-hr period. Because it yields results within the VOL. 22, 1971 ranges of "low," "moderate," or "excessive" contamination, i.e., less than 50,000 cells per g, between 50,000 and 100,000 cells per g, and over 100,000 cells per g, it will serve as the rapid monitoring system for which it is intended. Tlhe quick count method has been used successfully for monitoring purposes by several food processing plants that cooperated in the study.

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2014-10-01T00:00:00.000Z

1971-01-01T00:00:00.000Z

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The effect of body size on production efficiency in cattle. Breed comparisons and inter-breed relationships The effect of body size on the production efficiency of different breeds of cattle is considered only after examining the more general problem of breed comparisons in relation to genetics, nutrition and production physiology. Genetic problems include what breeds and crosses to compare, how to set up a structured multi-breed population, and what breeding programme to follow with a view to practising . across-breed selection. The main nutritional problem is what feeding system to adopt without prejudicing the outcome of a breed comparison, and mention is made of the advantages and disadvantages of ad libitum feeding with a complete standard diet. The main problem of production physiology is defining optimal performance. Indirect assessment of optimal performance, if it is to be effective, should be based on inter-breed relationships among characteristics of economic importance. Several inter-breed relationships are discussed, and those involving mean mature body size are considered with reference to making breed comparisons for productive efficiency. Some examples are given of specific breed comparisons with the effect of body size included and excluded. crossing to compare the relative merits of different breeds of sire on the same dam breed, in all cases we end up with genetically different groups to be compared for productive merit. At this point we might pass straight on to the problems of making specific breed comparisons. But this would be evading the main issue. The outstanding genetic problem of breed comparisons, recently reviewed by Dickerson (ig6g), is what breeds to compare, what crosses to make and what breeding programme to pursue thereafter. The problem arises because of the very large number of breeds that could potentially be compared. There are at least 250 major breeds of cattle spread throughout the various countries of the world, and Mason (ig6g) in his dictionary of breeds lists more than twice as many minor breeds. So we have something approaching i o00 different breeds of cattle to compare. Testing all breeds might just about be possible under certain conditions, and later I will refer to the possibility of international testing. If, however, we were to make all possible first crosses, the number would amount to almost one million. If we then think of making triple or multiple crosses or back crosses, the number of possibilities rapidly approaches infinity. Thus, on the one hand, testing all crosses is quite impossible, while on the other hand, without testing, the odds against hitting upon say, an outstanding triple cross by chance are many million to one, although we might persuade ourselves that good sense or reasoning and some available information might reduce these odds to a few hundred to one. Not unnaturally, selection in practice goes for those few breeds reputed to have the best performance in terms of the criterion currently in fashion, as high growth rate appears to be at present, or high total milk yield. However, prior to making any choice, one question we should ask is this: given facilities for testing N animals for production efficiency, how should numbers be distributed within and between breeds in order to maximise the probability of finding a breed or breeds with high performance. At one extreme we can have all animals on test from the same breed, while at the other extreme we would include as many breeds as animals, that is, one animal per breed. For a given genetic objective, we must determine at what point between these extremes the best solution lies. Surprisingly often, the answer will be to use relatively few animals per breed and a large number of breeds. Before deciding what crosses to make or compare, it is important to consider another question&mdash;how the breeding programme is to be continued. If the full benefits of hybrid vigour are to be realised as an added rather than an alternative method of improvement, the narrow perspective of a closed random breeding structure may have to be largely abandoned. Geneticists warn against inbreeding within a breed as this leads to depression in performance. Similarly at the breed level, perhaps they should warn against a closed random breeding structure, however many crosses were originally involved in its construction. Briefly then, let us suppose that it is desirable to test the optimum number of breeds for the facilities available, and that it is desirable to maintain heterozygosity to boost performance, and that it is also desirable to maintain maximum genetic variation for selection to operate upon. Then in all three cases we reach the same conclusion that in general we should make use of a very large number of breeds&mdash;in practice usually the maximum number obtainable&mdash;with relatively few animals per breed. In addition we should operate a breeding system involving the continued crossing of breeds and of crossbreds&mdash;interbred in such a way as to maintain both heterozygosity and genetic variation. In such a structured multi-breed population, selection of sires or individuals could be practised in the normal manner but with this one very important proviso (one which will be referred to again later)&mdash;that the selection criterion operates uni f ormly across all the breeds and crosses. Crudely translated, this means the selection criterion should be so arranged that the animals that would be chosen by mass selection would tend to be broadly the same as those that would be chosen by selection within breeds and crosses. Selection criteria with this property can be constructed provided we have suitable information on inter-breed relationships. Breed comparisons and inter-breed relationships are still very much a research problem and there is a big distinction between selecting breeds for research and for commercial production. For example, it may not serve research interests at all well to pick the few breeds that are extreme for some characteristic. The most balanced and unbiassed assessment of a population always comes from a random selection. Provisionally therefore, one might advocate the study of a random selection of breeds and crosses as the first step towards understanding breed differences. I will refer again later to this research approach in the section on inter-breed relationships. NUTRITIONAL PROBLEMS Breed comparisons or any comparison of genotypes for any character but especially for body composition and efficiency of food utilisation depend on the feeding system adopted. Breed differences are thus undefined unless the feeding system is fully specified. The crux of the problem is that sufficient experiments cannot be done to determine breed differences in growth and development, lactation and food consumption separately in each of the very large number of different feeding systems possible. A way round this problem suggested by Taylor and Young ( 19 66) was to look for a general form of equation interrelating growth rate, weight, food intake and age, with individual or breed differences in growth and efficiency represented by different parameter values. These parameters would, by definition, be independent of any particular growth and food intake curve. They would contain all the relevant information on each breed's intrinsic efficiency characteristics. All other measures of a breed's efficiency would depend on the weight, or gain, or food intake, and on the age and age intervals used, and could therefore be given different values according to the choice of the experimenter. The same approach could be applied to lactation performance. We do not yet know any easy method of estimating these parameters in individual animals because of the impossibility of making the same animal grow simultaneously at two different rates on two different levels of food intake. If, however, genetically similar groups can be put on different levels of food intake -as in the case of a breed or a sire progeny group&mdash;then it is possible to get estimates of parameters for efficiency of maintenance and efficiency of growth and possibly efficiency of milk yield. But such a procedure requires quite elab )rate experiments to evaluate a breed's efficiency characteristics, and most people are naturally reluctant to undertake them. In the absence of such a full-scale experiment, the problem of what feeding system to adopt to obtain valid comparisons remains. Since any attempt at controlled feeding may prejudice a breed comparison, the only alternative left is to let each genotype decide its own food intake, that is, use an ad Libitum feeding system. In practice, however, an ad libitum feeding system, together with recording of individual food intake, is very difficult to operate. For if concentrates, hay, silage, sugar-beet pulp, etc., are all fed ad libitum then, quite apart from the labour of recording individual intake of each foodstuff, different breeds will consume different proportions of the different foodstuffs, and there is the difficulty of combining the different foodstuffs into a single acceptable figure. There are very many ways of doing it, each leading to a slightly different result when comparing animals or breeds for efficiency. COMPLETE STANDARD DIET To overcome these difficulties of ad Libitum feeding, A.B.R.O. has devoted a great deal of effort over the last five years or so to obtaining a complete standard diet for cattle. And A.B.R.O. is deeply indebted to British Oil and Cake Mills Ltd., as it then was, or U.K. Compound Feeds Ltd., as it now is, for the tremendous effort they put into developing such a pelleted diet. Details of this diet have been discussed by Gi B SON (ig6g). It essentially contains 30 per cent of halfinch straw as the necessary length and level of roughage to allow normal rumination and normal milk composition. The more obvious advantages of a standard complete pelleted diet are constant food composition for all animals, which, as we have seen, is especially relevant for ad libitum feeding; a reasonably constant basis of comparison from year to year and from season to season allowing the slow build up of comparable results overt the years; and the problem of when to change from one ration to another so that different breeds are treated alike is bypassed. Results obtained vy different institutes can be compared without any special difficulties over the interpretation of the nutritional units used. Another potential use of a standard complete diet is that co-operative testing could be extented to animal breeding institutes in different countries, so that breeds could be tested and compared prior to importation. Co-operative testing on a international basis may sound a little far-fetched but it is quite a feasible proposition. Exporting the complete diet, instead of importing the cattle, could considerably increase both the extent and effectiveness of breed comparisons, in addition to greatly lowering the cost. The main limitation of any standard complete diet is inherent in its very nature. It is only one diet out of many. Nutritionists will sooner or later claim that some other combined ration is much better. Breeders will say that the results are not applicable to their methods of rearing. Those are very valid criticisms, but they only amount to saying that a standard complete diet won't give all the answers&mdash;and of course, it would be naive to imagine that it or anything else ever would. The more practical limitations of the diet as used at present are that it does produce some cases of bloat in stalled animals, and some animals seem to go off food around calving. While, like many other things, it falls short of the ideal, it is nevertheless an extremely useful working tool for breeding experiments with cattle. I might add that the two main experiments with cattle being set up by the Animal Breeding Research Organisation are naturally both wholly designed around this complete standard diet and that both experiments include breed comparisons as part of their design. This section on nutritional problems in breed comparisons might be summed up as follows. To determine optimal feeding systems for any breed would require very extensive trials based on a series of controlled feeding levels, several of which might have to be unproductive. Since at present we do not know the optimum, and since any controlled feeding system other than the optimum may seriously prejudice the outcome of a breed comparison, the only alternative at present would seem to be an ad libitum system. In practice, to operate an ad libitum system satisfactorily, a standard pelleted diet is probably necessary. However, we must keep in mind that performance on an ad libitum system may not correspond to optimal performance, and that an ad libitum feeding system, on its own, can never provide us with the necessary information for determining what the optimal feeding system is. PRODUCTION PHYSIOLOGY AND THE ASSESSMENT OF PRODUCTIVE MERIT The third problem in breed comparisons is the assessment of productive merit. The relevance and accuracy of our assessment is closely related to and limited by the current state of knowledge on production physiology&mdash;the combination of reproductive physiology, the physiology of growth, meat physiology and lactation physiology&mdash;which shapes our decision on what characters should be observed, and how and when to measure them. To begin with, let us consider the following restricted problem: we are presented with two specific breeds and are also given a clea y ly specified basis for comparison, and are then asked to compare them and determine whether or not one breed is superior to the other. This is a straightforward problem, and the method of comparing breed means is well established. Reasonably accurate comparisons usually require that each breed is represented by a fairly large number of animals because of withinbreed variability. If we know the magnitude of the difference we are looking for, and also have an estimate of the within-breed variation, then the number can be evaluated. To detect a difference of 2 to 3 per cent in body weight, about 5 o animals per breed are required; whereas for 305 -day milk yield about 400 animals per breed are required. Such numbers immediately show that few institutes have facilities for comparing more than two or three breeds with this degree of accuracy. In practice, we tend to leave many of the questions unspecified until the experiment is over. We set up a breed comparison experiment, we observe various characteristics, body weights, growth rates, milk yields, or perhaps we slaughter the animals and do some carcase analyses. Then we start asking specific questions such as which breed has the higher growth rate between 6 months and i year of age; or which breed has the greatest milk yield for 305 day lactation excluding lactations less than 100 days; or the point at issue might be which breed has the highest dressing out percentage at a body weight of 450 kilograms. So long as the questions remain specific, then the breed comparison can be made without undue difficulty. However, if the two breeds were slaughtered at the same body weight, somebody is sure to object to the conclusion that one breed has, for example, better muscle-bone ratio than the other; and often it is too late to go back to find an answer to the same question on a constant age basis or any other basis asked for. The difficulty lies in the fact that the number of such questions is unlimited. Which breed turns out to be best will very frequently depend on the detailed condition laid down in the question. Comparisons are never absolute. They vary with nutrition, they vary with age, they vary depending on whether or not they are measured relative to body weight, and so on. It soon becomes apparent that the problem of &dquo; what to compare breeds for &dquo; is just as complicated and confused a problem as the previous problems of &dquo; what breeds to compare &dquo; and what feeding system to adopt. Presumably, however, some criterion for comparison must be decided upon. It is only moderately helpful to say that most people would accept that breeds should be compared for optimal performance, but it is at least a start. Were the optimum performance points known or observable or calculable for each genotype, then the problem would revert to a simple comparison of means. Hence one approach to the problem of breed comparisons and breed selection is to attack the physiological and nutritional side of growth, development and lactation of genotypes and breeds with the specific objective of determining the optimal pattern of feeding, optimal ration composition, optimal age at slaughter and optimal lactation cycle. In addition, since an essential ingredient in achieving optimum performance is reliability in reproductive performance, an animal's optimal performance would have to be qualified by a probability of achieving it. CURRENT RESEARCH ON OPTIMAL PERFORMANCE The idea that beef breeds should all be compared at the same weight is no longer advocated quite so strongly as it was a few years ago. A recent publication by the U.K. Meat and Livestock Commission (M.L.C., 1970 ) entitled the &dquo; High Cost of f Over f atness &dquo; advised producers to slaughter different breeds and crosses at different ages and weights, to obtain greater economic efficiency. J OANDE T and CnRTwxiGaT (ig6g) give tables of optimal ages and weights for slaughter based on maximum economic return for a number of different beef breed types comprising pure He y e f o y d and z types of He y e f o y d-B y ahman crosses. Optimal ages ranged from 17 up to 22 months and optimal slaughter weights ranged from 3 34 up to 400 kg. Differences in profit of up to £ I per animal were estimated depending on whether a breed was slaughtered at 400 kg or its optimal weight. The authors conclude that &dquo; The magnitude of these differences show the importance of adequately comparing breeds and crosses and of comparing them at the point of maximal efficiency for each &dquo;. FRANKS and C AR T WRI G H T (1969), in a similar study, also found considerable breed differences in optimal slaughter weight. It would be most interesting and informative to see carcase traits and body composition of different breeds and crosses compared at optimal slaughter weight. For milk production, most people would again agree that breeds should be compared on the basis of optimal performance. The optimal performance problem in the case of milk is that of determining the pattern of feeding for maximum efficiency, together with the lactation intervals for optimal lifetime performance. Some recent work by B ROSTER ( 19 6 9 ) shows that a higher rate of feeding according to yield in the early part of lactation leads to a higher overall performance, but research specifically aimed at determining optimal performance has a long way to go yet. INDIRECT ASSESSMENT OF OPTIMAL PERFORMANCE A partial solution to the problem of comparing breeds or genotypes for optimal performance is to use the technique of indirect assessment&mdash;the equivalent of indirect selection, to which the geneticist or animal breeder resorts when faced with the problem of not being able to measure what he really wants to measure. The technique is variously powerful, weak, or abused, because it is often very tempting to adopt a criterion for indirect selection without sufficient justification simply because it is relatively easy to measure and is inexpensive in terms of time and money. The important question we should keep asking is whether selection for the observed measurement is really likely to improve optimal performance. The average expectation from disturbing the natural equilibrium is probably a deterioration in optimal performance&mdash;this because any selection criterion will usually be genetically correlated with a large number of homestatically interrelated components of performance which are not usually observed but which perhaps should be. Most of the measurements at present used for indirect selection have not yet been adequately shown to be genetically correlated with optimal performance. The present climate of opinion is that improved criteria for comparison or selection will depend on more direct measures of efficiency of production. For example, in a performance test of beef sires for efficiency of growth, measuring both growth rate and food consumption might be recommended but with either no carcase assessment or, if included, at a fixed weight or age. I am not necessarily opposed to this, but the question must be asked whether in such circumstances the additional measurement of food intake increases the genetic correlation between the observed character and optimal performance. Unless this fact is first established, there are no real grounds whatsoever for introducing the scheme or using the character for comparing breeds. The accuracy of indirect assessment can be measured by the currently existing correlation between the observed measurement and optimal performance. Hence any other measured characteristic associated with the observed measurement but uncorrelated with optimal performance can be used to increase the accuracy of indirect assessment. This is part of the theory of selection indices. In other words, we should do what we can to avoid comparing genotypes or breeds for characteristics that are uncorrelated with optimal performance by excluding from our breed comparisons irrelevant characteristics that cloud the outcome. The question of what information is necessary to do this brings us to the subject of inter-breed relationships. This section on the assessment of productive merit might be summed up as follows: Specific breed comparisons present no problems provided the characters being compared are clearly specified. The major problem is &dquo; what to compare breeds for &dquo;. The theoretical answer is to compare breeds for optimal performance. We either do not know how to do this, or when we do, the testing procedure is considered&mdash;and perhaps rightly so&mdash;to be too elaborate and costly to be put into operation. In practice, the solution is to compare breeds by indirect assessment of optimal performance. It is then of prime importance to know the genetic correlation between the observed measurement or index and optimal performance, and equally important to recognise that its estimation must be based on inter-breed statistics. INTER-BREED RELATIONSHIPS Just as we need estimates of heritabilities and genetic correlations for determining effective methods of within-breed selection, so also for betweenbreed comparison and selection, we likewisse need to know phenotypic and genetic relationships at the between-breed level among all the characteristics of economic importance and any others we may be interested in. The most balanced and effective method of obtaining inter-breed relationships applicable to the present population of about i ooo breeds of cattle or to some sub-population, is, as was mentioned earlier, to use a random sample of breeds. Furthermore, just as there are formulae for finding the number of offspring per sire progeny group to give the best estimate of heritability, so a similar formula gives the number of animals required per breed to give the best estimate of an inter-breed regression. The number of animals per breed usually turns out to be quite small&mdash;from 2 to 10 animals per breed. Thus, even with fairly limited experimental facilities, an experiment involving 30 or more breeds could be carried out without undue difficulty. An experiment of this kind has recently been started by A.B.R.O., with the objective of estimating interbreed relationships involving food intake. Much data from many different experiments on many different breeds has accumulated in the literature on characteristics such as growth rate, body weight, body composition, milk yield, milk composition, conception rate, and so on. Most of the results are not strictly comparable, having been carried out in many different countries and environments and with different feeding systems and types of ration. However, a great deal of useful information on inter-breed relationships can be extracted statistically. Thus analyses can be restricted to breed differences obtained within experiments. In addition, mean values quoted for the same breed from different experiments can be used to obtain estimates of betweenbreed components of variance and covariance. The inter-breed relationship between mean birth weight and mean mature maternal weight was examined in this way using a sample of 1 6 7 breeds, and an inter-breed regression coefficient of o.74 ! 0 . 05 was obtained for birth weight on maternal weight. The birth weight of a breed does not appear to increase in proportion to dam weight but more slowly; in other words, heavier breeds tend to have relatively lighter calves. In a breed comparison therefore, mean birth weight should perhaps be expressed as a proportion of mature metabolic weight, a result equivalent to that found by DorrAr,D and RussE!,!, ( 1970 ) for sheep breeds. An analysis based on 15 8 d.f. between breeds and 2 8 3 d.f. for replicates within breeds, showed that butterfat percentage tended to decrease slightly as total milk yield increased, but not significantly. A similar analysis of the inter-breed relationship between total milk yield and mature body weight presented many difficulties. The genetic inter-breed regression appeared to indicate that heavier breeds tended to have a proportionately greater total milk yield or possibly a yield slightly more than proportionally greater. The main difficulty was associated with the confusion introduced among measures of total yield by variable lactation lengths. Another more general example of an inter-breed relationship is that between mature size and time taken to mature by different breeds or strains within a species (T AYLOR , 19 68). The relationship so obtained can be used to eliminate the effect of size from specific breed or sex comparisons. Two scale changes are required. Size at all immature stages had to be scaled in accordance with mature size. The corresponding age conversion is effected by dividing age by the 0 . 27 th power of mature body weight to produce a metabolic age scale. When these allowances are made for size differences, the growth and development of our domesticated species, breeds and sexes all tend to become approximately the same. Inter-breed relationships involving food intake would be of considerable interest, but the necessary information is difficult to obtain. However, by examining the available evidence, some probable form of relationship can be obtained. From the dependence of heat production on mature metabolic weight, it can reasonably be inferred that, in mature animals, genetic differences in food intake are proportional to genetic differences in mature metabolic weight. A suitable procedure for comparing the pattern of food intake of different breeds during their growing period would therefore be to scale food intake at immature stages by mature metabolic weight and convert age, as before, to a metabolic scale. The result of this procedure, as far as can be seen from available data, is to produce a transformed pattern of food intake during growth that tends to be approximately the same for different breeds. If, when suitably adjusted for size, most breeds tend to show a quantitatively similar pattern of growth and development and also of food intake, then most breeds must also tend to exhibit a similar pattern for any combination of these. In other words, we can provisionnally conclude that most breeds will tend to show approximately the same pattern of productive efhciency during growth. This general similarity must include similarity in optimal efficiency. It follows that there is unlikely to be any systematic association between the optimal efficiency of a breed and its mature size. This claim that efficiency is independent of body size has been made on many occasions; both B RODY and Kr,!IB!R made it in the 1930 's. More recently, within-breed studies of production efficiency involving growth rate and /or milk production have, in general, come to the same conclusion (HoovEN, MILLER, and PI, OWM A N , I(!E)H; K R ESS, H AU SE R and C HAPM A N , ig6g; W I I,SO N , G ILL OO LY , R UGH , T HO NI SON and Pu RD Y, ig6g). Suppose we accept it and conjoin it with the argument previously referred to concerning the accuracy of indirect selection. It then follows that once the effect of size has been removed, and provided observations are taken at comparable stages of development, then most measures of efficiency of production will become more closely correlated with optimal performance. Hence we must exclude size from all our breed comparisons of production efficiency; and, by looking at what remains, we may get a clearer idea of differences in optimal efficiency. A necessary comment at this stage may be that if the non-food cost per animal is proportional to the food cost per animal, then it can be ignored. If, however, part of the non-food cost per animal remains the same whatever the size of animal, then production efficiency when extended to include the total economic cost of production will obviously not remain independent of body size. I do not know what weighting factor should be given to this constant non-food component, and I do not intend to persue here the economic arguments in favour of large size and the counter-arguments in terms of more expensive testing and slower rate of genetic improvement. The remainder of this talk consists of looking at some examples of observed breed differences, before and after the effect of size has been eliminated. THE EFFECT OF SIZE IN SOME SPECIFIC BREED COMPARISONS Body weight and growth rate In the British MLC Beef Recording Scheme, body weight at 400 days is the criterion on which bulls are at present ranked. K W xENrry ( 1970 ) gives mean body weights at 200 and 400 days obtained from this scheme for bulls of a number of beef breeds and these are reproduced in Table I as 400 -day weight, growth rate in kilograms per day between 2 oo and 400 days, and also percentage growth rate over the same period. For 400 -day weight, Charolais tops the list. The correlation between 400 day weight and gain from 200 to 400 days is very high (o.g6), so that 400 -day weight is a reasonably good inter-breed measure of absolute growth rate. The ranking of breeds for per cent growth rate is shown in the last column. The Cha y olais ranks near the bottom; the Aberdeen Angus ranks highest. The correlation between 4 oo-day weight and per cent growth rate has the small insignificant value of 0 . 21 . A minor adjustment would have to be made to per cent growth rate to render it free from any association with breed size; but it is not clear precisely what this allowance should be since heavier breeds are slightly slower maturing but at the same time have a higher per cent growth rate because they are less less mature. For those interested in trends, ten years ago, the rank correlation between the relative frequency of demand for A.I. by bulls of these breeds (English MMB figures for z 95 8 /i 959 ) and absolute growth rate was virtually zero ( 0 . 12 ) but the correlation with percentage growth rate was high (o.61). Today, or rather for 19 68 /ig6g, there is virtually no correlation between frequency of demand and percentage growth rate. On the other hand, there is some correlation with growth rate in kilograms per day ( 0 . 34 ), and if Friesians were included this would probably be much higher. A demand for high percentage growth rate ten years ago has been replaced by the current demand for high absolute growth rate. The main question to be asked, however, is how close a measure of optimal meat production is 400 -day weight likely to be? Relative growth rate, being almost uncorrelated with breed size, is likely to give a better indirect assessment of efficiency of production than either absolute growth rate or 400 -day weight, which are both very highly correlated with breed size. But percentage growth rate and 400 -day weight are virtually uncorrelated so that 4 oo-day weight is likely to be an extremely poor inter-breed measure of optimal performance. The differences between breeds for percentage growth rate are very much less than for 4 oo-day weight, the coefficient of variation being only 5 . 1 per cent for percentage growth rate as compared with over 13 . 0 per cent for 400 -day weight. Breed differences in optimal performance are therefore more likely to be of the order of 5 per cent than 13 per cent. COMPARISON OF BEEF BREEDS FOR PROFITABILITY Both birth weight and food intake show inter-breed proportionality to mature metabolic body weight, and hence to each other. If, therefore, the feeding of a dam is the main cost of producing a calf, the cost of a calf should be proportional to its birth weight. Suppose the proportionality relationship were, say, one pound sterling per kilogram of calf birth weight. A Friesian calf with a birth weight of q.o kilograms would then cost about £ q.o, whereas a Jersey calf with a birth weight of 25 kilograms would then cost £ 25 . In Table 2 , this calf price differential of £ per kilogram birth weight may be compared with those required for equal profitability given by Kr!,x!Nrry ( 1070 ) for a variety of beef breeds and crosses and based on a realistic assessment of the probable effects of differences in growth rate and finishing weights of different breeds and their relationship to feed costs. Calf price differentials proportional to birth weight may not be so far wrong as average values for a variety of methods of rearing. The conclusion might therefore be drawn that when calf prices are proportional to birth weight, there are only small differences in profitability remaining among the beef breeds and crosses listed. This example is, of course, restricted to singleborn calves, and does not deal with the potentialities of calf production involving increased twinning or multiple egg transfer from large to small dams. But these more complex schemes apart, the general conclusion to be drawn from these examples is that whenever allowance is made for differences in body size, most breeds are very similar in performance and productive efficiency. We might conclude from this that breeds are also probably very similar in their optimal performance, with marginal superiority in a few cases, and that this margin may vary with conditions and type of rearing. Breed differences in efficiency of meat production appear to be relatively smaller than those for body weight or growth rate, and the same is true for milk production. We might rightly fear that larger and larger and more closely controlled experiments would be required to detect them, and unless existing facilities were correspondingly enlarged, the number of breeds or crosses that could be compared would be correspondingly reduced. However, obtaining highly accurate breed comparisons for productive efficiency may not be a worth-while objective in cattle improvement. There are very many situations where the overall genetic information return per animal could be greatly increased by foregoing such unnecessarily high accuracy. For example, genetic progress from selection will often be greater in a population containing many breeds and crosses than in an equivalent sized population containing only one or two. Or as we saw earlier, the best estimate of an inter-breed relationship is obtained with only 2 or 3 sires per breed and no more than 2 or 3 offspring per sire. I feel that there is great scope and many unexplored possibilities at the other end of the scale from the large experiment dealing with only one breed, and I look forward to the time when multi-breed experiments and herds are a commonplace with no-one very surprised to find 20 or more breeds represented and crosses galore. L'effet de la dimension corporelle sur l'efficacité de la production de différentes races bovines ne peut être étudié qu'après examen de la question plus générale des aspects génétique, nutritionnel et physiologique des comparaisons de race. L'aspect génétique concerne le choix des races et des croisements à comparer, la manière d'établir un ensemble structuré de plusieurs races et le programme de sélection à suivre en vue de réaliser les croisements entre races. Le principal problème nutritionnel est le choix d'un système d'alimentation qui nuise le moins possible aux comparaisons de race. Les avantages et inconvénients de l'alimentation ad libitum avec un aliment complet standard sont mentionnés. Le principal problème de physiologie de la production est de définir la performance optimum.

v3-fos

2020-12-10T09:04:11.222Z

1971-09-01T00:00:00.000Z

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s2

Microbial Penetration of Muslin- and Paper-Wrapped Sterile Packs Stored on Open Shelves and in Closed Cabinets Microbial penetration of sterile packs was studied using single-wrap (two layers) muslin, double-wrap (four layers) muslin, and two-way crepe paper (single layer) to wrap 20 gauze sponges (2 by 2 inch). These packs were stored in the central sterile supply departments of two hospitals and processed for sterility at predetermined intervals. Microorganisms penetrated single-wrap muslin as early as 3 days and double-wrap muslin and single-wrap two-way crepe paper in 21 to 28 days stored in open shelves. The time required for microbial penetration was at least twice as long when closed cabinets were used. Single-wrap muslin packs stored in sealed, impervious plastic bags remained sterile for at least 9 months. All sterile materials in pervious wrappers should be handled as little as possible and then only with extreme care and caution. Closed cabinets offer more protection than open shelves, and single wrappers are not recommended. There are reports in the technical literature describing the length of time sterile goods can be stored and still be considered sterile, but the safe storage times reported range from as short as 1 week to indefinitely (10,11,14,15,17). To add to the confusion, some reports discuss neither the wrapping material used nor conditions for storage in relation to the safe storage periods (10,17). Dyer et al. (8) reported the shelf life to be at least 60 days for cotton applicators wrapped in parchment and muslin when stored in closed cabinets. Alder and Alder (1) found that crepe and bleached kraft paper were more effective than calico or balloon cloth. Also, they found that approximately 50 and 100% of the test swabs wrapped in double and single layers of muslin, respectively, were contaminated after 13 to 14 days of storage; 10 and 30%, respectively, were contaminated within 1 to 2 days. These investigators concluded that packs wrapped in two layers of paper and enclosed in cartons should have a shelf life of at least 3 weeks. Nichols (13) found that packs double-wrapped in muslin stored and sealed in plastic bags remained sterile for 18 months. Fitzwater (9) compared the number of viable microbial particulates settling into open petri dishes on open shelves and in a normally closed cabinet in an operating room during set up and an operative procedure. Doors of the closed cabinet were opened 24 times during the evaluation. Fewer than one-tenth as many viable particulates settled in the closed cabinet than on the open shelves. The exterior surfaces of sterile packages become contaminated during storage. Several investigators have commented on the probability of transfer of this contamination to the contents when single-wrapped sterile packages are opened (2,5,12,15,18). Speers and Shooter (16) demonstrated that sequential unwrapping of doublewrapped packages substantially reduces contamination during removal of the sterile contents. Central supply personnel have no firm scientific data on which to base selection of the most effective wrapping material or establishment of conditions for storing sterile packs; hospital central sterile supply departments (CSSD) use many types of wrappers and storage conditions vary widely. Sterile packs wrapped in muslin are considered by most hospitals in the United States to be safe if used within 1 month after sterilization, an assumption probably based on the findings and recommendations of Perkins (14). In previous studies on length of uncontaminated storage of sterile packs, the materials in the packs examined for microbial contamination were generally cotton applicators or small metal or 432 glass objects (1,4,8,13,14). A deficiency of all of these studies was that only small portions of material inside the sterile packs were assayed for microbial contamination. The present study was designed to determine how long sterile packs of a size widely used in hospitals (15) would remain sterile when wrapped in single-wrap muslin, double-wrap muslin, and single-wrap two-way crepe paper (4,15) when stored on open shelves and in closed cabinets. MATERIALS AND METHODS Standard packs were used for studies of microbial penetration into the pack. Twenty [2 by 2 inch (5.08 by 5.08 cm)] 12-ply gauze sponges were arranged to form a pack with a surface area of 8 by 10 inches. The packs were wrapped with single-wrap muslin (two layers), double-wrap muslin (each two layers), or single-wrap (single layer) two-way crepe paper. A Kilit Ampule containing resistant bacterial spores was placed inside each pack as a check on the effectiveness of sterilization. All packs were autoclaved in a conventional steam sterilizer for 1 hr at 121 C. Cloth wrappers used for the standard packs were 140 thread-count muslin, unbleached, dyed green, laundered, and ironed at least 1 to 10 times before use. The paper wrappers used were commercially available two-way crepe paper (Dennison Wrap). Both types of wrappers were approximately 24 by 24 inches (61 by 61 cm). After sterilizing and drying, the packs were held overnight in the autoclave with the steam supply turned off to allow the packs to cool. The packs were then removed and placed in sealed, sterile 8-mil polyvinyl chloride (PVC) bags [25 by 35 inches (63 by 89 cm)]; transported to the CSSD of the two hospitals; removed from the PVC bags; and placed on shelves in the same areas that the hospitals' sterile supplies were stored. On the same day, three packs wrapped in each type of wrapper were chosen at random and transported back from the hospitals to the laboratory for an initial control assay to confirm that the packs were not contaminated during transportation. The remaining test packs were picked up in pairs at selected time intervals and transported back to the laboratory for microbiological assay. All packs returned from the hospitals were transported in sealed, sterile, 3-mil polyethylene bags [9.5 by 18 inches (20 by 46 cm)]. Relative humidity and temperature were monitored in the two CSSD throughout the study by using 7-day recording hygrothermographs. Calibration for accuracy of these instruments was checked at weekly intervals by using a sling psychrometer. Hospitals were used for this study to provide locations for storage of test packs under actual institutional rather than laboratory conditions. Hospital no. 1 is a 100-bed pediatric hospital; the CSSD of the hospital has four employees on the day shift on weekdays and one on each of the other shifts. Hospital no. 2 is a 350-bed hospital; the CSSD had 14 employees on the day shift on weekdays and at least one on all other shifts. Storage shelves with enclosed backs and sides, but with the fronts open, were used in hospital no. 1. The distance between shelves ranged from 11 to 14 inches (28 by 36 cm). No packs were less than 16 inches (31 cm) from the floor. Some shelving completely open on all sides and some shelving with enclosed backs were used in hospital no. 2. The shelves were spaced 10 to 13 inches (25 by 33 cm) apart, and no packs were less than 24 inches (61 cm) from the floor. In addition, closed metal cabinets measuring 36 by 78 by 18 inches (91 by 198 by 46; width by height by depth) also were set up in hospital no. 2 for this study. The shelves were arranged 9 inches (23 cm) apart; no packs were less than 13 inches (33 cm) to the floor. A silent electrical counter was installed to record the number of times the cabinets were opened and closed. Test packs were picked up at various times during the first week of the study: days 1, 2, 3, and 4 for study series 1 (hospital 1); days 1 and 6 for study series 2 and 3 (hospital 2); days 1 and 3 for study series 4 (hospital 1); and day 3 for study series 5 (hospital 2). Thereafter, weekly pick-ups were made in all study series. The first pick-up for study series 6 (hospital 2) was on day 7. At each pick-up, two sterile packs used as transportation controls were wrapped in singlewrap (two layers) muslin, transported to the hospital in sterile plastic bags, and then returned to the laboratory along with and in the same manner as the test packs. Series 1, 2, and 3 were done during cold months, and series 4, 5, and 6 were done during hot months. Immediately on arrival at the laboratory, all packs were processed inside a closed laminar-flow hood (40 by 20 by 40 inch (102 by 51 by 102 cm) ]. The hood was decontaminated before each daily use with 70% ethanol and purged and dried for at least 30 min at a rate of three air changes per minute (ac/min) through an ultra-high-efficiency air filter (6). While packs were being processed, the airflow was set at a rate of 1 ac/ min. The hood was purged for at least 2 min at a rate of 3 ac/min between processings of each pack. After the packs were opened, seven of the sponges were placed in each of two screw-cap serum bottles containing 100 ml of Trypticase soy broth (TSB, BBL) and incubated aerobically at 37 C. The remaining six sponges were placed into another serum bottle containing 100 ml of TSB and incubated under anaerobic conditions in a Brewer jar at 37 C. All cultures were incubated for 21 days before being considered negative for viable microorganisms. An estimate of the amount of viable surface contamination that may have collected on the outside of the packs was made by using stainless-steel strips [1 by 2 inches (2.54 by 5.08 cm)]. Using these strips, two evaluations were made of open shelves versus closed cabinets in hospital 2: one was carried out in the cold months (series 2 and 3), and the other was carried out during the hot months (series 5 and 6). Strips were placed on a stainless-steel tray wrapped in doublethick aluminum foil and sterilized in a hot-air oven at 150 C for 3 hr. The trays with strips were transported to the hospital with the test packs. The trays of strips were placed on the same shelves with the packs and opened. Five randomly selected strips were collected for microbial assay when each set of packs was picked up for examination. Each strip was aseptically placed into a sterile 4-oz specimen jar and returned to the laboratory. Upon return to the laboratory, 50 ml of TSB was aseptically added to each jar, and the jars were vigorously shaken for 5 min on a wrist-action shaker. Just before assay, each jar also was handshaken 50 times. Sets of two pour plates each for aerobic and for anaerobic incubation were prepared by using 5-ml sample amounts mixed with 10 to 15 ml of Trypticase soy agar (TSA). The remaining sample in the jars was then heat-shocked for 15 min at 80 C, and four additional pour plates were prepared, as described above, again for aerobic and anaerobic incubation. Aerobic incubation was carried out in a waterjacketed incubator at 37 C, and anaerobic incubation was carried out in Brewer jars at 37 C. Colonies on the plates were enumerated after 48 hr and after 7 days of aerobic incubation and after 7 days of anaerobic incubation. Concurrent with these studies, single-wrap (two layers) muslin packs prepared and sterilized as previously described were sealed in sterile 3-mil polyethylene bags (0.5 by 18 inch). Eight packs were stored in each hospital CSSD on shelves that collected the most dust. Two packs were examined at 1, 3, 6, and 9 months for assay as previously described. In an attempt to determine depth of penetration of contamination, six single-wrap (two layers) muslin packs containing nine stacks of 15 sponges per stack, sterilized as previously described, were placed on open shelves with other sterile supplies in the CSSD of hospital no. 2 for a period of 5 weeks. The first nine sponges were placed in separate 2-oz screw-cap bottles containing 25 ml of TSB. These specimens were incubated aerobically at 37 C for 21 days before being considered negative for viable microorganisms. RESULTS Microbial contamination was determined from single-wrap (two layers) muslin packs, doublewrap (each two layers) muslin packs, and singlewrap (single layer) two-way crepe paper packs. Table 1 shows the first time in days at which each type of pack was found contaminated in the openshelf study in both hospitals; contamination occurred as early as 3 days after initiation of storage with single-wrap muslin and in 21 and 28 days (first noted at 28 days) with double-wrap muslin or single-wrap two-way crepe paper. Results from the closed-cabinet studies (series 3 and 6) are shown in Table 2. Contamination first became apparent at 14 days for single-wrap muslin and at 56 days for double-wrap muslin, and no contamination was found for at least 63 days with two-way crepe paper. Series 1, 2, and 3 were done during cold months, and series 4, 5, and 6 were done during hot months. Series 1 and 4 were done in hospital no. 1; 2, 3, 5, and 6 were done in hospital no. 2. The time interval in which contamination first became apparent in single-wrap muslin packs was shorter in hot months than in c3ld months; however, the double-wrap muslin and single-wrap two-way crepe did not appear to follow this pattern. Averages of weekly average indoor temperatures in the CSSD did not vary significantly between the hot (25 C) and the cold (25.5 C) months and only slightly between hospital 1 (27.2 C) and hospital 2 (24.4 C). The CSSD in hospital 1 was more humid in the summer and less humid in the winter than the CSSD of hospital 2. Averages of weekly average indoor relative humidities were about 35% in the studies during the cold months and about 48% in the studies during the hot months. Table 3 shows the total microbial counts from stainless-steel strips exposed in two evaluations of open shelves versus closed cabinets in hospital 2. The microbial counts were calculated on the basis of an area measuring 80 square inches, equal to the surface area of the packs used in the study. The doors on the closed cabinets were opened (and closed) an average of 28 times per day during the investigation. The results of these evaluations showed that only about one-tenth as much viable microbial contamination settled onto horizontal surfaces in the closed cabinets as on the open shelves. About 50 % of the settled microorganisms were aerobic and not heat-shocked, about 20% were anaerobic and unshocked, over 10% were molds, and less than 10% each were aerobic and anaerobic, heat-shocked organisms. bNo packs or strips processed. Table 4 shows the types and frequencies of organisms isolated from contaminated packs. About half of the organisms isolated were grampositive rods; the next most frequently isolated organisms were staphylococci, Aspergillus spp., and Micrococcus spp. The single-wrap (two layers) muslin packs sealed in 3-mil polyethylene bags remained sterile throughout the 9-month study. Examination of the six single-wrap muslin packs used to study depth of microbial contamination after 5 weeks of open-shelf storage showed penetration down to the fifth sponge in one pack. Two to seven of the nine surface sponges per pack examined were found to be contaminated. A total of 195 control packs were utilized throughout the study. Only two (1.0%), both single-wrap (two layers) muslin, were found to be contaminated. No growth was found in any of the ampoules of resistant spores placed in the packs to check the effectiveness of sterilization. DISCUSSION Many wrapping materials are commercially available to hospital CSSD today, but almost no data are available on how well these materials maintain sterility. Basically, bacteria penetrate muslin draping material much more rapidlf than they penetrate water-repellent paper drape fabrics (7). It has been shown that bacteria penetrate a surgeon's intact gown more readily and in greater numbers during surgery requiring unusual physical effort than during simple procedures (3). The same may be true of the more porous materials used to wrap sterile supplies. That is, the less the materials are handled and moved, the less likely it is for contamination to occur. It has been suggested that a change in atmospheric conditions may cause a breathing effect in packs and thus contribute to penetration of microorganisms (14). In the present study, neither the temperature nor the relative humidity varied greatly, and there were few rapid changes in either of these variables; thus, the frequency of contamination could not be shown to be directly related to atmospheric conditions. Gradual increases in settled contamination on the outside of packs together with handling or vibration and possibly atmospheric changes were responsible for ultimate contamination of packs. This study utilized the entire contents of the sterile package as the assay system. Using this system, single-wrap (two layers) muslin packages become contaminated as early as 3 days, and double-wrap (each two layers) muslin and single-wrap (single layer) two-way crepe paper maintained sterility for at least 3 weeks (contamination was first found at 4 weeks) stored on open shelves. Packs stored in closed cabinets remained sterile for at least twice as long as those held on open shelves; however, even in closed cabinets, the time of sterile storage for single-wrap muslin (less than 14 days) was too short to be practical. In an effort to show a visual comparison of a single thickness of muslin and a single thickness of two-way crepe paper, a photograph at approximately 40 X magnification was made of each. Figure 1 shows the muslin; there is a visible opening through the material at almost every thread junction. Figure 2 shows the two-way crepe paper; in this figure, three dark areas near the center indicate very thin areas in the paper fiber. These photographs help demonstrate the possibility of rapid recontamination of sterile objects wrapped in a single wrap of muslin. The results of the study on the amount of microbial contamination settling on stainless-steel strips corroborate the findings of Fitzwater (9); only about one-tenth as many viable microorganisms settled in the closed cabinets as on the open shelves. Thus, the results of our studies clearly show the advantages of closed-cabinet over openshelf storage of sterile packs. Of course, cabinet doors must be kept closed except for replacement or removal of contents. Although a single thickness of two-way crepe paper was observed to maintain sterility as long as double-wrap muslin, it is not recommended that any sterile pack be wrapped with only a single thickness of material. With a single wrapper, the possibilities for contamination are greatly increased via contamination from the outside surface of the pack (16). Impervious plastic wrappers have been observed to maintain sterility for as long as 18 months (13). The 9-month period of sterile storage obtained from this study of materials sealed in polyethylene bags at least in part confirms these earlier observations. With proper rotation of stocks, no material should be held for as long as 9 months. All sterile materials in pervious wrappers should be handled as little as possible and then only with extreme care and caution. Closed cabinets offer more protection than open shelves, and single wrappers are not recommended. LrITRATURE CITED

v3-fos

2020-12-10T09:04:16.972Z

1971-03-01T00:00:00.000Z

237235019

s2

Classification of Streptomyces Spore Surfaces into Five Groups Streptomyces spores surfaces have been classified into five groups, smooth, warty, spiny, hairy, and rugose, by examination of carbon replicas of spores with the transmission electron microscope and by direct examination of spores with the scanning electron microscope. Kriss et al. (12) were probably the first to do electron microscopy of streptomycete spores. Their finding of smooth spores was followed by those of Carvajal (3) and Bringman (2), who also found smooth spores on the cultures they studied. Flaig et al. (10) described thorny or spiny spores from certain streptomycete species. Kuster (13) confirmed the presence of spiny spores on certain species and also found hairy spores and warty spores on other species. Baldacci and Grein (1) observed smooth, spiny, and hairy spores but failed to mention warty spores. Enghusen (8) suggested that the warty surface of spores was an artefact of preparation. Flaig and Kutzner (11), Ettlinger et al. (9), Preobrazhenskaya et al. (15), and Tresner et al. (17) found and discussed four spore surface types: smooth, warty, spiny, and hairy. Lechevalier and Tikhonenko (14) studied the formation of spines during spore maturation. The concept of smooth, warty, spiny, and hairy spore surfaces was sufficiently established that Cross and Maclver (4) and Shirling and Gottlieb (16) listed this as one of the criteria to be used in characterizing species. These studies were based on the direct observation by electron microscopy of whole streptomycete spores. Instead of spore surfaces these workers were looking at silhouettes of spores. The size and density of whole spores prevent penetration of the electron beam with sufficient strength to reveal surface structure. Information about the spore surface obtained by studying the silhouette is at best conjecture and may be misleading. In 1961, the authors began to use preshadowed carbon replicas in studying streptomycete spore surfaces. This technique provides more information about gross characteristics of the spore surface and permits study of the fine detail of the spore coat. We have examined more than 500 cultures by this method. Our findings (5,6) show that, in addition to the four recognized spore surface types, a fifth type should be designated, namely "rugose." This paper presents pictures of the five spore surface types as revealed by transmission electron microscopy (TEM) of whole spores and preshadowed carbon replicas, scanning electron microscopy (SEM) of intact cultures, and phasecontrast microscopy of intact cultures. Characteristics of the fifth spore surface type will be discussed. MATERIALS AND METHODS Based on previous information, cultures examined in this study were chosen to represent the four established types and the proposed new type. These cultures and the types they represent are: smooth, Streptomyces albus, ATCC 3004; warty, S. steffisburgensis, NRRL 3193; spiny, S. echiniatus, NRRL 2587; hairy, S. pactum, NRRL 2939; rugose, S. hygroscopicus, (Jensen) Waksman, CBS. Conditions of culture and growth have been described previously (5, 7). Whole spore mounts and preshadowed carbon replicas were prepared for TEM examination by the procedures previously described (5). Cultures for examination by SEM were prepared by the method of Williams and Davies (18) as described previously (7). Cover slips with adherent culture were cemented to stubs, coated with a thin film of evaporated aluminum, and examined with a Stereoscan SEM (Cambridge Scientific Instruments, Ltd.) in the laboratories of Alpha Research and Development, Inc., Blue Island, 111. Extra cover slips with adherent growth were placed, growth side up, on glass slides and examined by phase-contrast microscopy. Appropriate, representative photographs were made by each of the three methods and are used to substantiate our designation of a fifth spore surface type. carbon replicas of spores by TEM. The phasecontrast pictures, designated part A, in the figures show the maximum information that can be obtained by this method with its relatively poor resolution and shallow depth of field. In compari-son, the low-magnification SEM pictures, designated section B, show much more concerning spatial arrangement, attachment of spores to mycelium, and development of spores. At higher magnification ( Looking at Fig. 2D and 5D, one sees spore chains with poorly defined segmentation and many short protrusions. One could easily classify both of these cultures as the warty spore type based on these two pictures alone. Figures Figure 5B shows what are apparently two types of spores. These are only found by SEM. We believe that the light-colored spirals of unseg-.-y, -:-. RESULTS -y @ 't4- mented, rugose-surfaced spore chains are those which develop as aerial spores with no direct contact with the agar surface. The darker, segmented, smooth spores are those which have developed while in direct contact with the moist agar surface. This latter spore configuration is never found on replicas or whole spore mounts by TEM, since such spores would be difficult to pick up on a filmed grid or a glass disc. On the basis of our previously reported obser->. STREPTOMYCES SI vations (5-7) plus the pictures presented here, we believe that in addition to the smooth, warty, spiny, and hairy spore surface types a fifth type should be recognized. Because of the nature of the surface we have designated this the rugose type. LITERATURE CITED

v3-fos

2018-04-03T02:05:56.416Z

1971-03-01T00:00:00.000Z

32348509

s2

Fungi isolated from flue-cured tobacco inoculated in the field with storage fungi. Flue-cured tobacco inoculated in the field with A. amstelodami, A. flavus, A. ochraceus, A. repens, A. ruber, and a species of Penicillium was rarely invaded by these fungi. Regardless of inoculum, the predominant fungi reisolated from green tissue were species of Alternaria and Cladosporium. After curing, A. repens, A. niger, and species of Alternaria and a species of Penicillium were the most commonly isolated fungi. The fungus used as inoculum was not the predominant fungus reisolated from green or cured tissue. Conditions during handling and storage prior to marketing probably determine when storage fungi become associated with the leaf and which species becomes predominant. Several species of Aspergillus and Penicillium are commonly isolated from marketed, damaged, and nondamaged (4) flue-cured tobacco (Nicotiana tabacum L.) but are rarely isolated from green tobacco leaves immediately after flue-curing (5). The purpose of this study was to determine whether selected storage fungi could infect living tobacco leaves in the field and whether the fungi used to inoculate the growing plant survived flue-curing and were the predominant fungi isolated from the cured leaf after the usual handling of the tobacco in preparation for marketing. Flue-cured tobacco variety 'Coker 319' was grown under normal field conditions at the Oxford Tobacco Research Station, Oxford, N.C., in the summer of 1967. The plants were topped and suckered, and the lower six leaves on each plant were harvested before the test began. Test plots contained 10 plants per row, and each plot was separated from other plots by nontreated rows of the same variety. For each treatment, upper leaf surfaces on 10 test plants in each plot were sprayed with the spore suspension until run-off occurred on each leaf. Plants in two plots were sprayed with the Tween-80 solution for controls. Plants were sprayed on 12 August 1967, and mature leaves were harvested on 16, 23, and 30 August and 6 September 1967. Immediately after harvest, about half of the leaves from each treatment were placed in an ice chest and transported to the laboratory in Raleigh; the remainder was flue-cured in the usual manner (3). In the laboratory, 50 discs (9 mm) from each treatment were cut from the green leaves, surface disinfected for 30 sec in 1% NaOCl, rinsed with sterile distilled water, and cultured on Czapek solution-agar (Difco) with 6% NaCl. Culture dishes were observed for microorganisms after 5, 9, and 12 days of incubation at room temperature. The fungi growing from the discs were identified, whenever possible, in the original petri dish culture. When this was not possible, the fungi were subcultured and stored for later identification. Species of Aspergillus were identified by the method of Raper and Fennell (1). From the other portion of the harvested leaves that had been flue-cured, 50 discs (9 mm) from each treatment were cut, washed in running tap water for 30 min, and cultured on Czapek solution-agar (Difco) with 6% NaCl. These samples were not surface disinfected because earlier studies had demonstrated that treatment with 1% NaOCl eliminated fungi from cured leaf tissue. The petri dishes were observed and fungi were identified as previously described. Results (Table 1) demonstrate that species of report. The inability to reisolate these species Aspergillus and Penicillium sprayed on tobacco supports the generally accepted theory that their leaves in the field are rarely reisolated from green primary nutritional role is saprophytic. The tissue by using the procedures described in this results also show that the species of storage fungi TABLE 1. Fungi isolated from 9-mm discs of tobacco before (BC) and after (AC) curing, inoculated in the field with five species of Aspergillus and a species of Penicillium, and harvested at four intervals, based on culturing 50 discs of tobacco for both samples used to inoculate tobacco plants in the field are not necessarily the fungi most frequently isolated after flue-curing and handling. After curing, A. repens grew from 0 to 98% of the discs and was absent in only one sample; A. niger Van Tiegham grew from 0 to 68% of the discs and was absent in two samples; and a species of Penicillium grew from 0 to 100% of the discs and was absent in three samples. Invasion by these three fungi was not associated with field infection. In addition to species of Aspergillus and Penicillium, other fungi frequently were isolated from green and cured tobacco. A species of Alternaria (probably A. tenuis) grew from 94 to 100 % r of the green-leaf discs and from 6 to 100%c of cured-tobacco discs. A species of Cladosporium was the second most common fungus growing from green tissue, but curing reduced its frequency of isolation to 0 to 14%xand in nine samples it was eliminated. This work indicates that storage fungi rarely infect tobacco in the field and supports a similar conclusion of Tuite and Christensen (2) regarding storage fungi on wheat. The study also indicates that exposure to specific storage fungi in the field has little effect in determining which storage fungi become associated with a particular tobacco sample. The technical assistance of Virginia Perez and field plot supervision of Billy N. Ayscue are gratefully acknowledged.

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2020-12-10T09:04:11.832Z

1971-03-01T00:00:00.000Z

237232215

s2

Analysis of Clostridium botulinum Toxigenic Types A, B, and E for Fatty and Carbohydrate Content Lyophilized, 48-hr log-phase vegetative cells were extracted with chloroform-methanol (2:1, v/v) and ethanol-ether (3:1, v/v) and then saponified with methanolic KOH. Gas-liquid chromatography of the methyl esters of extractable fatty acids revealed distinctive “pattern profiles” of Clostridium botulinum toxigenic types “A,” “B,” and “E.” C. perfringens type “A” and Escherichia coli strain “B” were also studied in a similar manner and were found to give pattern profiles which were distinct even from those obtained for the C. botulinum microorganisms. Amino sugar content of the five microorganisms was determined by using a Beckman amino acid analyzer. The molar ratio of glucosamine to that of galactosamine was found to be of further assistance in distinguishing the individual microorganisms. There has been increased interest in the bacterial lipid and other chemical constituents of the genus Clostridium, both from a physiological standpoint and as a means of species differentiation (6,17,22,24,27). Several approaches to this species differentiation have been explored. Henis et al. (10) used gas chromatographic analysis of bacterial metabolic products to provide a means for rapid detection and differentiation of several different microorganisms. Moore (20) also utilized gas chromatography for the study of the genus Clostridium and demonstrated that the patterns obtained allowed species differentiation. Fugate and Hansen (Bacteriol. Proc., 1969, p. 46-47) reported preliminary results of an investigation in the use of lipid and carbohydrate patterns obtained by gas-liquid chromatography (GLC) of whole cell extracts to distinguish toxigenic C. botulinum strains. Kimble et al. (13) also reported a detailed study of the fatty acid composition in whole-cell extracts of C. botulinum and suggested that variances in composition served as a means of differentiating the chiefly proteolytic C. botulinum types A and B from the nonproteolytic types E and F. It is well established that carbohydrates confer antigenic specificity to many bacterial cell components (2,15). Thus, it would be of interest to determine whether C. botulinum vegetative cells contain carbohydrate moieties in the easily 1 A preliminary report of this investigation was presented before the 69th Meeting of The American Society for Microbiology, Miami Beach, Fla., 4-9 May, 1969. extractable lipid portion of their cell surface. Since members of this genus are frequently involved in food poisoning outbreaks, it would be advantageous to characterize the lipid and carbohydrate content of selected, single strains of C. botulinum as a basis for a rapid, preliminary means of identification. Moore (20) has pointed out that gas chromatography allows simple and rapid identification of species through characteristic compounds, although such analyses do not replace necessary morphological and biochemical studies. The primary purpose of this investigation was to quantitatively characterize the lipid and carbohydrate content of whole cells of three strains of toxigenic C. botulinum. The three strains selected for extensive study (type E. Beluga; type B, 169B; type A, 62A) were found to be representative of several strains which were investigated during this study. Since this represents an investigation of single strains and since there are a large number of strains which were not included in this study, it may be necessary in the future to substitute other single strains for these clostridia so that the strains chosen are representative of the majority of the strains in each toxigenic type. It is good to remember that these strains constitute a diverse group of microorganisms (9,21). Cultural conditions. Clostridium cultures were grown in 15-liter quantities in the Trypticase-peptoneglucose broth of Schmidt, Nank, and Lechowich (25). A 2.0% (of total volume) inoculum of a 12-hr culture was used when the cells were grown in large quantities. Vegetative cultures were incubated for 36and 48-hr periods at 30 C. The cells were harvested by centrifugation and washed four times in 0.85% (w/v) NaCl and lyophilized in a VirTis lyophilizer before extraction procedures. Cultures of E. coli B were grown and harvested as reported by Brian and Gardner (3) for the detection of cyclopropane fatty acids in bacterial lipids. Cultures were grown in 25 ml of Trypticase Soy Broth (TSB) and 3% agar at 35 C for 18 hr. These 18-hr cultures were then used as the inoculum for 6 liters of TSB with 3% agar. Cultures were incubated for 24 hr at 35 C. Cells were harvested by centrifugation and washed four times with 0.85% (w/v) NaCl. These cells were lyophilized. GLC. All GLC analyses were performed with a Barber-Colman model 5000 equipped with flame ionization detection and having a flow rate of 40 ml of hydrogen per min and 150 ml of air per min. Polar column number 1. An all-glass column (180 cm by 3 mm) with 8% LAC-728 on 1% phosphoric acid-precoated 100/120 mesh Anakrom AB was used. Nitrogen flow rate was 40 ml/min at 16 psi. The column was temperature-programmed during analysis from 100 to 250 C at 5 degrees per min. Injector temperature was 250 C, and detector temperature was set at 260 C. Nonpolar column number 2. An all-glass column (180 cm by 3 mm) with 8% SE-33 on 100/110 mesh Gas-Chrom Q was used. Nitrogen flow and temperature conditions were the same as described for column number 1. Hexose column number 3. An all-glass column (90 cm by 3 mm) with 4% XE-60 and 3% neopentylglycol-adipate on 100/110 mesh Gas-Chrom Q was used. Nitrogen flow rate was 50 ml/min at 18 psi. The column was temperature-programed from 100 to 250 C at 10 degrees per min. Injector temperature was 250 C, and detector temperature was 260 C. Analytical methods: lipids. One gram of lyophilized cells was extracted with chloroform-methanol (2:1, v/v) and then with ethanol-ether (3:1, v/v) as shown in Fig. 1. The combined lipid extracts (number 1 and number 2) were concentrated under a stream of nitrogen. The combined extracts were washed by the procedure of Folch et al. (7) to begin lipid characterization. The upper phase lipids were made to a 10.0-ml volume A 5.0-ml portion of each sample was saponified with 5.0 ml of 15% KOH in methanolic aqueous solution (1:1, v/v) for 1 hr at 80 C. The remaining sample was chromatographed on diethylaminoethyl cellulose by the method of Creech (5) and Rouser et al. (23) to remove nonlipid contaminants. Thin-layer chromatography of the lipid mixture was accomplished by using the techniques of Mangold (18). Nonsaponifiable material was extracted three times with 5-, 3-, and 2-ml volumes of mixed ethers (ligroin diethyl ether, 1: 1, v/v) successively, and the combined extract was stored at 0 C for hexose analysis. The extracted residue of cells was adjusted to pH 2.0 with 6 N HCI, and the saponified acids were extracted with the mixed ethers three times by using 5, 3, and 2 ml successively. The remaining aqueous portion was stored at 0 C for hexose analysis. A sample of the extracted fatty acid was esterified with methanolic BC1, as reported by Metcalf and Schmitz (19) and determined by GLC analysis on a polar column. The fatty acid methyl esters were then hydrogenated immediately for 20 min in 5 ml of chloroform-methanol (2:1, v/v) with 100 mg of 5% platinum on charcoal and chromatographed on the same column. The fatty acid esters were brominated as described by Brian and Gardner (3) and chromatographed on the same column. A second sample of the extracted fatty acids was treated similarly but chromatographed on a nonpolar column; as a result, six chromatograms were available for each microorganism studied. With both columns, quantitative results agreed within 5% of stated composi-VOL. 21,1971 tion for the National Institutes of Health fatty acid standards (Applied Science Laboratories Inc., State College, Pa., reference 11). Fatty acids were identified by the method suggested by Kimble et al. (13). Analytical methods: hexoses. Hexose content was determined by the method of Kim et al. (12). In most experiments, 10 mg of extract (scheme B-9 and 10, Fig. 1) was hydrolyzed in 7.5 ml of 0.25 N sulfuric acid plus 500 mg of Dowex 50-X12, 200 to 400 mesh (H+ form) ion-exchange resin in evacuated sealed ampoules at 100 C for 24 hr. The hexoses, in a portion of the hydrolysate, were converted to the alditol derivatives by the procedure of Kim et al. (12). Thin-layer chromatography of a second portion of the hydrolysate was performed on glass plates coated with Adsorbosil-3 (Applied Science Laboratories, Inc.) in 0.1 N boric acid at a thickness of 0.5 mm. A mixture of butanol-acetone-water (40:50:10) was used as the developer. Silver nitrate spray followed by NaOH spray and heating at 100 C for 10 min was used for visualization. Ninhydrin in acetone was employed to locate the amino sugars. Analytical methods: amino sugars. A 5-mg amount of dried lipid solvent extracts (scheme B-5, 9, Fig. 1) was hydrolyzed in 6 N HCI for 18 hr at 110 C. The hydrolysates were examined with an amino acid analyzer (model 120 C; Beckman Instruments, Inc., Palo Alto, Calif.) for amino sugars after being extracted with diethyl ether which removed noncovalently bound fatty acids. The analyzer was fitted with a 20-cm column packed with PA-35 spherical resin and maintained at 55 C. The buffer system was 0.35 N sodium citrate at pH 5.26, and a flow rate of 70 ml/hr was maintained. A 10-mg amount of extracted whole cells (scheme A-1, Fig. 1) was hydrolyzed in 2 ml of 6 N HCl under reduced pressure at 110 C. After 18 hr, the sealed ampoules were broken and the filtered hydrolysates were examined for amino sugar content by using the same conditions. The amino acid analyzer was calibrated with amino acid standard calibration mixture type 1 (Beckman Instruments, Inc.) and with glucosamine and galactosamine standards prepared to a concentration of 0.125 ,umole per 0.1 ml. Analytical methods: amino acids. Amino acid content of the whole cell was determined after the whole cell mass had been through the lipid extraction procedures. A 10-mg sample (scheme A-1) was hydrolyzed in 6 N HCI for 24 hr at 110 C. Hydrochloric acid was removed by repeated evaporation to dryness from water solution. These hydrolysates were then freed from noncovalently bound fatty acids by extraction with diethyl ether, redried, and dissolved in sodium citrate buffer (pH 2.2) for analysis. The amino acid analyzer operating conditions were the same as previously described. RESULTS The total lipid extracted accounted for 3.3, 3.0, 3.2, and 4.0% of the dry weight of C. botulinum vegetative cell types A, B, E and C. perfringens, respectively, with percentages based on 48-hr vegetative cells cultured as described above. A comparison of the different fatty acid "pattern profiles" obtained from the microorganisms studied is shown in Fig. 2 by Brian and Gardner (3), to facilitate analysis of the fatty acid data. The lipid portions of C. botulinum types A, B, and E were found to contain essentially the same fatty acids but in different concentrations. Tetradecanoic (C: 14) and hexadecanoic (C:16) acids were found to be the predominant fatty acids in the whole cell extracts of types A, B, and E. Together they make up over 50% of the total fatty acids. Table 1 lists the fatty acids identified in the five microorganisms studied. The results agreed favorably with those reported by Kimble et al. (13). Figure 2 also shows that a distinctive pattern profile is obtained from C. perfringens, which is different in that relatively large percentages of dodecanoic (C:12) and tetradecanoic (C:14) were found. Smaller percentages of hexadecanoic (16.6%) and octadecanoic acids (1.3%) were also present, which agrees with results reported by Moss and Lewis (22). The lipids of C. botulinum types A, B, and E are essentially the same in their fatty acid composition. The quantitative distribution of the various fatty acids were found to differ, but only with the additional information supplied by the amino acid analyzer may they be distinguished easily. However, the limited number of strains examined does not rule out the possibility that the differences observed between types are actually differences between individual strains. (13) does substantiate preliminary findings that a similarity does exist between strains of the same toxigenic type. The chromatograms of C. botulinum type A after methylation, hydrogenation, and bromination are shown in Fig. 3. Cyclopropane fatty acids containing 17, 19, and 23 carbon atoms were demonstrated to be present in each of the C. botulinum types studied. E. coli was used as a standard of reference in detecting the cyclopropane fatty acids, since E. coli is a ready source of these acids, especially for GLC identification procedures (3). E. coli was found to have 37.3% hexadecanoic acid (C:16), with 12.0% cyclopropane methylene-hexadecanoic acid (C: 17) and 2.8% cyclopropane methylene-octadecanoic (C:19) acid. No hexose was detected by GLC of the alditol acetate derivatives from any of the lipid extracts of C. botulinum vegetative cells. C. perfringens, on the other hand, contains mannose, which distinguishes it quite readily from the C. botulinwn types studied. After hydrolysis, lipid solvent extracts from E. coli were found to contain glucose, fucose, galactose, and rhamnose. The results obtained in the separation of the amino sugars in the Beckman amino acid analyzer (model 120C) are shown in Table 2. The molar ratio of galactosamine to glucosamine appears to be quite useful in distinguishing the microorganisms studied in that it varied from 1:2 in C. botulinum type A, 2:3 in type B, and 4:1 in type E. In contrast, the ratio for C. perfringens was 1:8 and 2:1 for E. coli B. The amino acid residues detected after whole cell hydrolysis failed to reveal significant differences between the Clostridium species studied. DISCUSSION The lack of carbohydrate in the lipid of the Clostridium species studied may be one of the major factors in explaining the findings of Lynt et al. (16). They reported that somatic antigens were common to all C. botulinum strains studied. This may well be due to the lipid composition and corresponding low antigenic specificity of this class of chemical constituents. Solomon et al. (26) expressed the belief that serological classification of the clostridia would require that the entire antigenic makeup, including the fiagellar, somatic, and spore antigens of the organisms, be considered. An alternative approach to the identification of clostridia by purely serological techniques could be by GLC. Abel et al. (1) suggested that differences in chemical composition of microorganisms be detected by gas chromatography and used for their classification, since gas chromatography offers the necessary degree of sensitivity, rapidity, and selectivity required. The need for more rapid bacterial species identification, especially with genera associated with food-borne illness, is quite apparent. Several novel methods, including immunological and enzymatic, have been proposed (14). The present investigation suggests that principles of immunochemistry and gas chromatography can also be combined and utilized in this respect. There are several possible approaches to the problem of rapid identification of toxigenic Clostridium species. The present report demonstrates that analysis of the whole cell is preferable to analysis of sonically disrupted and enzyme-cleansed cell walls when rapidity is desired. The use of whole cells in preference to disrupted cells was investigated originally since whole cells gave promise of more rapid analysis. The unique pattern profiles obtained through the use of GLC analysis of fatty acids coupled with the amino sugar ratios for the microorganisms studied appears to offer a rapid means of differentiation. This investigation is being continued through a study of additional strains to determine whether the strains reported are as representative for C. botulinum as those investigated for C. pertringens by Moss and Lewis (22).

v3-fos

2020-12-10T09:04:11.117Z

1971-04-01T00:00:00.000Z

237231176

s2

Identification of Mycoplasmatales: Characterization Procedures A large collection of avian Mycoplasma cultures was used in studies to improve and develop biological and biochemical characterization techniques. Differential patterns among 11 avian serotypes were shown by carbohydrate fermentation, tetrazolium- and methylene blue-reduction reactions, breakdown of arginine, and the formation of film on egg yolk-agar. Some cultures fermented as many as 14 carbohydrates. Polyhydric alcohols and pentoses were among the compounds fermented. An improved procedure for determining methylene blue reduction by Mycoplasma was developed. These simple, rapid procedures are reproducible and should be useful in grouping Mycoplasma isolates prior to definitive identification by serological or other means. Studies of Mycoplasma have been stimulated by their identification as pathogens of man, cattle, sheep, goats, swine, rodents, and fowl (17). Specific identification of highly pathogenic Mycoplasma species is relatively easy. Often the pathogen is limited to one species. However, proving the identity of most Mycoplasma, especially those of low or undetermined virulence, is more difficult. Because increasing numbers of Mycoplasma are being found in all animal species (19) and in plants (16), the need for their identification is becoming more evident. A recent review (10) listed 35 named species in the genus Mycoplasma. It pointed out that some doubt is cast on the concept that these organisms are highly host-specific. Both factors complicate identification of the organisms and indicate the need to improve identification techniques (10)(11)(12). Very few biochemical reactions have been routinely used in identification of Mycoplasma (10). The exacting requirements for their growth render most standard bacteriological procedures of little value. A complement of biochemical reactions, like those used in bacterial identification (7), is needed. Approaches to identification have been primarily serological (19,20,23). Serological screening of new isolates against all other recognized species is too cumbersome (10). However, preliminary grouping of new isolates I Submitted by the senior author to Cornell University, Ithaca, N.Y., in partial fulfillment of the requirements for the Ph.D. Degree. 2 Other growth media were prepared as follows (M. M. Sabry, Ph.D. Thesis, Cornell University, Ithaca, N.Y., 1968). For basal medium, a slurry was prepared from 500 g of frozen fat-free rabbit muscle (Pel-Freeze Biologicals, Inc., Rogers, Ark.) and 1 liter of distilled water. The slurry was held overnight at 5 C; it was then heated with stirring for 30 min in a boilingwater bath. The suspension was filtered sequentially through four layers of cheesecloth, coarse filter paper, and fine filter paper. To the filtrate was added 10 g of peptone (Difco), 5 g of sodium chloride, and 10 ml of 10% sodium hydroxide. The suspension was autoclaved and passed through coarse and fine paper filters. Thallium acetate was added to a final concentration 0.1%, and the pH was adjusted to 7.8. The basal medium was filtered to sterilize, held at 37 C overnight, and stored at 5 C. Agar medium (RYEP) was prepared as follows. A solution of 2 g of agar (Difco) was added to 34 ml of distilled water. The complete medium was made by mixing 100 ml of basal medium with the agar solution at 50 C. Test media. (i) Carbohydrate fermentation broth was BS with 1% carbohydrate, 0.002% phenol red with pH adjusted to 7.6 to 7.8. Carbohydrates were prepared as 10% stock solutions and filtered to sterilize, except glycerol which was added directly to the basal medium. All 12 carbohydrates selected for extensive testing were freely soluble except salacin which dissolved with gentle heating. (ii) The methylene blue-reduction medium was biphasic with liquid over a small agar slant. The slant was made by adding 0.12 ml of a 1% stock solution of methylene blue to 100 ml of HIA. Approximately 0.25 ml was allowed to solidify as a slanted butt in a glass tube. The overlay solution was 2 ml of BS medium with 0.024 ml of 1% methylene blue. (iii) For tetrazolium reduction, a 1% stock solution of 2,3,5-triphenyl tetrazolium chloride (General Biochemicals, Chagrin Falls, Ohio) was prepared in distilled water. It was added to both BS and RYE media to final concentrations of 0.01%. It was also incorporated in viande foie medium and E medium as described elsewhere (1,12). Cultures were tested in all four media with tetrazolium. (iv) Arginine medium (11) was BS medium with 1% L-arginine monohydrochloride and 0.002% phenol red. The pH was adjusted to 7.0. (v) Film formation on egg yolkagar (12) was on plates prepared from HIA with 5% swine serum and 10% concentrated egg yolk emulsion (Oxoid Ltd., London, distributed by Colab Lab., Inc., Chicago Heights, Ill.). (vi) Resazurin-reduction medium (15) was prepared by adding 0.004% resazurin (Allied Chemical Corp., New York, N.Y.) to BS medium. Test procedures. All test media were inoculated with 3to 4-day-old broth cultures. Carbohydrates, arginine, and tetrazolium were inoculated with 0.1 ml; methylene blue-reduction medium and resazurin were inoculated with 0.2 ml; egg yolk-agar was streaked with 0.01 ml for maximum spread. Inoculated carbohydrate media were held 10 days and other liquid media for 5 days at 37 C. Egg yolk-agar plates were read during 6 days of incubation at 37 C. Interpretation of tests. (i) A change in color from red to yellow was evidence of carbohydrate fermentation with subsequent acid formation. Visual readings were confirmed at intervals with a pH-meter. (ii) Methylene blue reduction was interpreted as positive when the bluish-green medium became tan or colorless. Although decolorization in the liquid was often transitory, decolorization in the agar butt tended to persist. (iii) When tetrazolium was reduced, the colorless medium became pink or red with or without precipitated tetrazolium salts in the tube. When reduction was rapid, the red sometimes faded to purple. (iv) Breakdown of arginine was indicated by a change of the phenol red from pale reddish-orange to a deep purplish-red. This indicated an alkaline shift from a starting pH of 7.0. (v) Film formation on egg yolkagar was a crystalline film overlayering the colonies. When viewed at an angle to the light, the film had a faint purplish sheen. The film was usually too dense to be properly viewed with a microscope. When the plate was gently flooded with water, the film easily detached and floated to the surface. In this manner, film formation could be distinguished from dense colony growth. RESULTS Carbohydrate fermentation. Forty-five carbohydrates or related compounds (Table 1) were tested with avian Mycoplasma. After extensive preliminary tests, certain carbohydrates were valueless in differential tests for one of these reasons: (i) all fermenting cultures fermented them, (ii) no cultures fermented them, (iii) the carbohydrates became spontaneously acidic, or (iv) variable results were obtained in repeated tests with the same cultures or with different members of a single Mycoplasma serotype ( Table 1). As a control, the fermentation medium without carbohydrate was inoculated. In control medium, no cultures caused the phenol red to change to any shade of yellow. Fermenting cultures generally reduced the pH slightly in control medium, whereas nonfermenting strains usually caused a slight pH increase. Uninoculated carbohydrate-containing controls, incubated with every test, were usually satisfactory, but three carbohydrates were discarded because of spontaneous acid formation (Table 1). Table 2 lists fermentation results for 11 avian Mycoplasma serotypes and unclassified cultures from ducks with the 10 most useful differential carbohydrates. Glycerol and alpha-methyl glucoside appeared potentially useful, but only limited data were obtained. The four serotypes B, E, H, and L did not ferment any carbohydrates. Differential patterns for the fermenting (Table 1) which were not of differential value. Avian Mycoplasma serotype C-0 was unique in fermenting dextrose and saccharose ( Table 2). No other avian serotypes consistently fermented saccharose. About one-half of the cultures in this group fermented either mannose or levulose. Cultures in avian Mycoplasma serotype D-P usually fermented dextrose but were more variable in ability to ferment saccharose. The only other avian Mycoplasma group tested in adequate numbers was I, J, K, N, Q, R. These cultures ( (Table 3). Avian serotype B reduced methylene blue but not tetrazolium; M. anatis reduced tetrazolium but not methylene blue. Resazurin was reduced by representatives of all avian Mycoplasma serotypes except F and H. Resazurin-reduction studies were done with fewer cultures than used for other reactions, so results were not tabulated. Arginine dihydrolase. Cultures of five avian serotypes (Table 3) split arginine. Four of these, B, E, H, and L, were nonfermenting serotypes. The other serotype positive for the arginine dihydrolase reaction was the complex I, J, K, N, Q, R group which caused reduction reactions and carbohydrate fermentations. Film on egg yolk-agar. Four of 11 avian Mycoplasma serotypes caused film formation on egg yolk-agar (Table 3). Serotypes positive in this reaction (B, E, L, and M. anatis) did not react identically in any other characteristic. DISCUSSION The use of dextrose fermentation alone has allowed the grouping of Mycoplasma into fermenting and nonfermenting types (2,9,14,25). The value of carbohydrates other than dextrose has been questioned since patterns for all fermenting species have been markedly similar (10,19,25). It was generally concluded (17) that most fermenting strains produced acid from dextrose, fructose, mannose, maltose, starch, and glycogen but that some avian strains fermented sucrose and galactose. No Mycoplasma serotypes were reported to ferment lactose, pentoses, or polyhydric alcohols. In developing differential patterns, certain carbohydrates were discarded after preliminary tests with representatives of avian serotypes (Table 1). It was nevertheless concluded that no carbohydrate should be considered useless for differentiating Mycoplasma until it has been tested with representatives from all known serotypes and with unclassified cultures. Hundreds of cultures were tested before any were found to ferment xylose or galactose. In fermentation studies by other workers (8,26), different procedures and several basal media were used. Both groups regarded a pH change to 6.5, determined either by color or a pH-meter, as a positive reaction. Most cultures in the present study changed the pH of the carbohydrate test medium to 6.5 or lower, but a drop to pH 6.9 was sufficient to change the color of the medium to yellow and was considered positive. This study and other reports (8,26) indicate that avian Mycoplasma serotype A ferments dextrose, mannose, levulose, and the seven other carbohydrates (Table 1) which we regarded to have no differential value. Only one of the latter group, trehalose, was reported (26) not to be fermented by serotype A. Avian Mycoplasma serotypes B, E, H, and L did not ferment any of the carbohydrates tested (Table 2), which agrees with previous reports (8,26). The fermentation pattern of the complex avian serotype I, J, K, N, Q, R was of great interest. These cultures consistently fermented more carbohydrates than any other serotype. Some members of the group were capable of fermenting dextrose, mannose, levulose, mannitol, sorbitol, cellobiose, salicin (Table 2), and all seven carbohydrates which had no differential value (Table 1). No other avian Mycoplasma serotype fermented sorbitol or mannitol. Thus, the fermentation of sorbitol or mannitol immediately marked the isolate as a possible member of the I, J, K, N, Q, R group. Fermentation of different carbohydrates within the I, J, K, N, Q, R group did not correlate to other differential biochemical reactions or to antigenic differences (T. L. Barber and J. Fabricant, Avian Dis., in press). In similar work with cultures from cattle, 8 of 13 bovine Mycoplasma serotypes (J. M. Al-Aubaidi, Ph.D. Thesis, Cornell University, Ithaca, N. Y., 1969) fermented dextrose and other carbohydrates. One bovine serotype fermented sorbitol and three fermented xylose. Differential patterns were obtained for most serotypes. Reduction of tetrazolium compounds by Mycoplasma was reported (22) and later used in classification studies (11,12,24). This study confirmed reports that certain avian Mycoplasma serotypes consistently reduce tetrazolium and that it may be used as a differential characteristic (Table 3). A shortcoming of the test is that more than one medium must be used to demonstrate the phenomenon consistently (11). Our method for demonstrating methylene blue reduction is an improvement over more cumbersome procedures (5,14,18). This technique is rapid, simple, and reproducible. It is an excellent differential reaction because 5 of 11 avian serotypes tested were positive (Table 3). These five serotypes (A, B, L, M. laidlawii, and the complex I, J, K, N, Q, R group) were not grouped together on any other single characteristic. Serotypes reducing methylene blue did not necessarily reduce tetrazolium, and the reverse was also true. Both fermenting and nonfermenting serotypes were positive in reduction reactions. It was also found that 7 of 13 bovine Mycoplasma serotypes were positive for methylene blue reduction by this technique. These seven serotypes, all of which reduced tetrazolium, included both fermenting and nonfermenting organisms (J. M. Al-Aubaidi, Ph.D. Thesis, Cornell University, Ithaca, N.Y., 1969). The reduction of resazurin was of little differential value but was found useful in the metabolic inhibition serological procedure with cultures which were negative in fermentation, arginine dihydrolase, and tetrazolium reduction (T. L. Barber, Ph.D. Thesis, Cornell University, Ithaca, N.Y., 1969). The ability of Mycoplasma to split arginine was first noted in contaminated cell cultures (21). The arginine dihydrolase pathway and its value for identification and classification of these microorganisms were subsequently reported (3). The present study found this reaction to be reliable and consistent in agreement with other reports (2,6,11,13). The need for additional standard test procedures for identification of Mycoplasma still exists. Results from different laboratories cannot be accurately compared until similar media and procedures are used. Techniques reported in this study are useful in preliminary grouping of avian Mycoplasma. Modification may be necessary before they are applied to Mycoplasma isolated from other species.

v3-fos

2018-04-03T06:07:48.039Z

1971-06-01T00:00:00.000Z

26050205

s2

Rapid Infrared Determination of the Potency of Chlorinated Bactericides A rapid infrared reflectance method for evaluating the germicidal potency of synthetic materials containing various amounts of two chlorinated bactericides was developed. The dimeric product 2,2'-methylenebis (4,6-dichlorophenol) exhibited a characteristic C=C skeletal inplane stretching infrared absorption band at 1,640 cm7l. The monomeric 2,4-dichlorophenol precursor showed a character- istic absorption band at 1,579 cm7l. These characteristic infrared absorptions may be used for analysis of the potency of the manufactured chlorinated bactericide. For a series of samples known to vary in dimer content, the micrograms per milliliter required for a 100% bacterial kill is first determined by a standard American Petroleum Institute method. Then the area ratio of the infrared absorption bands characteristic ofthe chlorinated bactericides is measured for each sample and plotted versus the microgram per milliliter required for 100% bacterial kill. The potency of subsequent samples is simply and rapidly determined by measuring this ratio from the infrared absorption curve and calculating micrograms per milliliter required for 100% kill from the calibration curve. Analysis time is approximately 1 hr com-pared to biocidal tests in current use requiring approximately a 1-month incu-bation period. A rapid infrared reflectance method for evaluating the germicidal potency of synthetic materials containing various amounts of two chlorinated bactericides was developed. The dimeric product 2,2'-methylenebis (4,6-dichlorophenol) exhibited a characteristic C=C skeletal inplane stretching infrared absorption band at 1,640 cm7l. The monomeric 2,4-dichlorophenol precursor showed a characteristic absorption band at 1,579 cm7l. These characteristic infrared absorptions may be used for analysis of the potency of the manufactured chlorinated bactericide. For a series of samples known to vary in dimer content, the micrograms per milliliter required for a 100% bacterial kill is first determined by a standard American Petroleum Institute method. Then the area ratio of the infrared absorption bands characteristic of the chlorinated bactericides is measured for each sample and plotted versus the microgram per milliliter required for 100% bacterial kill. The potency of subsequent samples is simply and rapidly determined by measuring this ratio from the infrared absorption curve and calculating micrograms per milliliter required for 100% kill from the calibration curve. Analysis time is approximately 1 hr compared to biocidal tests in current use requiring approximately a 1-month incubation period. The Baroid Division of the National Lead Co. has developed a method for the preparation of the bactericide 2,2'-methylenebis (4,6-dichlorophenol) from 2,4-dichlorophenol. Bacterial potency of experimental preparations was measured by a typical test for potency in which the concentration in micrograms per milliliter required for a 100% kill of a standard bacterial culture is used. In the American Petroleum Institute (API) method (1), a sulfate-reducer medium is inoculated with an actively growing culture of a standard sulfate-reducing bacterium, which is added to various concentrations of the chemical to be tested and incubated. Growth is indicated by a blackening of the medium, whereas cultures with no growth remain clear. The API method requires 1 month of incubation, which delays observing the effect of variations in the synthesis conditions on the yield of the reaction. Although pure samples of the dimer were not evaluated, it has been estimated that the dimer is approximately seven times as potent as the monomer precursor. The higher the dimer content, the higher the potency of the product and the lower the amount required for germicidal action. Therefore, the measure of potency is directly related to dimer content or yield of the synthesis reaction. Since the biocidal test is time-consuming, it was highly desirable to develop a rapid method for measuring potency of synthetic bactericide for production control. Before the successful application of the infrared method, various approaches were investigated without success. These included ultraviolet and visible spectrophotometry; gas, thin-layer, and liquid chromatography; and nuclear magnetic resonance spectroscopy. Since the product is not entirely soluble in many transparent solvents used in spectroscopy or chromatography, we chose the multiple internal reflection technique for obtaining infrared spectra. MATERIALS AND METHODS Apparatus. A Perkin-Elmer infrared spectrophotometer (model 621) with a frustrated multiple internal reflectance accessory and a KRS-5 internal reflecting crystal was used. Any infrared spectrophotometer capable of resolving the two peaks of interest (1,640 and 1,579 cm7l) would be satisfactory. An analytical balance for weighing traced peak areas or a planimeter for measuring peak areas was at about 105 C and thoroughly mixed while fluid. The sample is then allowed to cool until it assumes a pasty form and is buttered onto a KRS-5 (thallium bromidethallium iodide salt) internal reflecting crystal. The crystal is placed in the reflectance accessory, and the infrared spectrum is obtained by using a quantitative schedule. Samples are removed from the crystal by washing with isopropanol. The areas under the analytical peaks (1,640 cm-' for the dimer and 1,579 cm-' for the monomer) are measured by tracing and weighing or by planimetry. A single base line is drawn from 1,685 to 1,540 cm-' for both peaks. Since the peaks are not fully resolved, it is necessary to compromise somewhat in defining the specific boundary for each peak. Figure 1 illustrates a typical area determination. The work described in this paper is based on area determination by the cutting and weighing technique. A calibration curve is obtained, by using a series of samples of various bactericidal potencies previously determined by the biocidal test by plotting peak area ratio versus micrograms per milliliter required for 100% kill. Figure 2 shows the calibration curve obtained by the authors. Samples are handled in exactly the same manner as the standards, and the ratios obtained are applied to the previously plotted calibration curve to estimate biocidal potency. RESULTS AND DISCUSSION The multiple reflection infrared method can be used in the rapid estimation of biocidal potency of binary mixtures of the chlorinated phenols. Although the method is not designed to replace entirely the biocidal tests, it affords a rapid means of control for estimation of biocidal potency of products in production. Once a calibration curve has been prepared, with samples whose potency has been determined by biocidal test, an infrared analysis may be completed in approximately 1 hr. The analysis of a group of samples may be completed in a relatively short time by heating in a thermostated oven, mixing to obtain a soft homogeneous melt, and running the infrared spectra over the 1,800 to 1,500 cm-l range in succession. LITERATURE

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2019-03-20T13:04:50.067Z

1971-05-01T00:00:00.000Z

237232470

s2

Dry-Heat Resistance of Bacterial Spores Recovered from Mariner-Mars 1969 Spacecraft The dry-heat resistances of 70 bacterial spore isolates recovered from Mariner-Mars 1969 spacecraft were determined and expressed as D values (decimal reduction times). Fifty per cent of the spore isolates had D values of 60 min or less at 125 C. Of organisms with D values greater than 60 min, four were selected for a study of the effect of sporulation medium and suspension menstruum on dry-heat resistance. Both sporulation medium and suspension menstruum were found to affect significantly the dry-heat resistance of the bacterial spores tested. It is imperative that an understanding of the dry-heat resistance of microorganisms on spacecraft that require sterilization, i.e., landers for an extraterrestrial life-detection mission, be acquired (6). An important step toward obtaining this necessary knowledge for the determination of a sterilization cycle for such a spacecraft was made by the prelaunch recovery of spore isolates from the Mariner-Mars 1969 flybys. During the Mariner-Mars 1969 Microbiological Moni-toring Program at Cape Kennedy, Fla., 70 spore isolates were recovered from the craft and their dry-heat resistance was tested. In addition, samples of environmental fallout were subjected to dry-heat testing. This program served to further the knowledge of the dry-heat resistance of microorganisms accumulating on ffight hardware during assembly. A similar program is planned for the Mariner-Mars 1971 mission. A major difficulty in defining the dry-heat resistance of microbial populations in spacecraft assembly areas rests in the inability to work with a natural population because of the low population densities present (8). Hence, a study of the heat resistance of organisms from such areas requires that the organisms be cultured to produce sufficient numbers for testing. It has been observed that the D value [a D value (or decimal reduction time) is the duration of exposure at a given temperature necessary to reduce a microbial population by 90%0] for a microorganism may vary according to the microorganism's inherent thermal resistance and environmental influences acting on that resistance (7). Of these environmental influences, the chemi-I Present address: Resources Planning and Control Corporation, El Segundo, Calif. cal milieu of the organism prior to dry-heat treatment is of importance in determining its dry-heat resistance (1). Therefore, a separate study was undertaken to define the effect of certain sporulation media and storage menstrua on the dry-heat resistance of bacterial spores isolated from Mariner 1969. MATERIALS AND METHODS Spore recovery and selection. Bacteria were collected from Mariner 1969 by the swab-rinse method, as defined by NASA (5). The samples were heatshocked at 80 C for 15 min prior to quadrant streaking on Trypticase Soy Agar (TSA; BBL). Colonies were removed from the TSA plates and, from these, 70 organisms capable of sporulation in a synthetic sporulation medium (SSM; 4), were selected for dry-heat resistance testing (phase I). Four isolates capable of satisfying the criteria of D values at 125 C (D125 c) in excess of 60 min when sporulated on SSM and of 95% or greater sporulation in both SSM and TAM sporulation agar (Difco; supplemented with 80 ,g of CaCd2 per mg and 20 ,ug of MgSO4 per mg) were chosen for further study (phase II). Culture and sporulation technique. Isolated colonies were removed from TSA streak plates, added to Trypticase Soy Broth (TSB; BBL), and incubated at 37 C until visible turbidity occurred. For phase I testing, 2 ml of the TSB suspension was inoculated into a flask containing 250 ml of SSM. (Phase II included inoculation onto a TAM agar plate.) The flask (or plate) was then incubated at 37 C until spores constituted 95% or more of the cells (as determined by microscopic examination of a stained preparation) at which time the spores were harvested. (For the TAM-grown isolates, harvesting consisted of washing the spores from the agar surface with sterile distilled water. The SSM spore cultures were harvested by centrifugation.) The suspension was centrifuged (10,400 X g for 15 min), resuspended, and washed eight times in sterile distilled water. After the final washing, the spore pellet was resuspended and divided equally to form both 95% ethanol and sterile distilled water suspensions which were then stored at 4 This facility provided temperature and humidity control by circulating air through a system of heating and cooling coils. The system used a water spray and cooling coil to assure that air leaving the cooling coil was at saturation. The air then underwent a reheat function to provide air at the specific temperature and humidity conditions required for the room. After the equilibra- tion period, the coupons were placed on trays in a dryheat oven and exposed to 125 C (+0.5) for designated time intervals. The oven was a mechanical convection oven modified by installing removable sliding trays into the door (Fig. 1). To minimize temperature fluctuations in the oven, the trays were individually removed and replaced without opening the door. Thermocouples were mounted at 23 oven locations (with an additional thermocouple exposed to ambient conditions), and the leads were connected to a 24-point recorder for continuous temperature monitoring during an experiment. After a 10-min temperature come-up time, coupons inoculated with TAM-grown spores were removed and assayed at 30-min intervals, whereas those with SSM-grown spores were removed at 1-hr intervals. This variation in assay intervals was established because of the differences in heat resistance that resulted from sporulation on the two different media. The Mariner 1969 Microbiological Monitoring Program included a study to determine the heat resistance of the microbial burden collected on stainlesssteel strips exposed in the Assembly Operations (AO) Building and the Explosive Safe Facility (ESF), at the Air Force Eastern Test Range (AFETR), Cape Kennedy. Upon collection, the strips were exposed to given temperatures (125 or 115 C) for designated intervals, at which time groups of eight were removed from the dry-heat oven and subjected to a microbiological assay. Assay procedure and data handling. After a designated heat exposure of the spore isolate, the three coupons were removed from the dry-heat oven tray with sterile forceps and placed individually into three flasks each of which contained 20 ml of sterile 0.1% peptone water. The flasks, partially immersed in an aqueous solution of 0.1 % Tween-80, were then treated in an ultrasonic bath for 12 min at 25 kHz. After this treatment, 10-fold serial dilutions were made in sterile 0.1% peptone water. Dilutions were then plated in triplicate by the pour-plate method by using TSA. a D125c value is duration of exposure at 125 C necessary to reduce a microbial population by 90%. b 0, no significant effect on dry-heat resistance; +, significant effect on dry-heat resistance (P < 0.01). Plates were incubated for 48 hr at 32 C, and the dilutions yielding 30 to 300 colony-forming units per plate were counted and recorded. The resulting data were subjected to a computerized linear regression analysis which generated a survivor curve and calculated the reciprocal of the slope of the curve (or D value) and the 95% confidence limits about the D value. An analysis of variance and Duncan multiple-range tests were performed on phase II data (2). Strips collected in the AO and ESF areas were aseptically placed into a flask containing 50 ml of sterile TSB. All flasks were incubated at 32 C for 48 hr and examined for growth. Results were recorded as growth or no growth. Isolate identification. Identification of the phase II isolates was performed as described by Bergey's Manual (7th ed.). Figure 2 shows a cumulative relative frequency distribution of D125 c values (expressed in minutes) for the Mariner 1969 isolates sporulated in SSM and suspended in 95% ethanol prior to heat testing. The graph gives a measure of the likelihood that bacterial spores accumulating on spacecraft surfaces under Mariner 1969 assembly conditions would not exceed a specified D value. For example, 20 to 30% of the spores accumulating on the spacecraft were found to have Di2zc values of 30 min or less; 49 to 57%, 1 hr or less; and 86 to 90%, 3 hr or less. In general, the survivor curves for those spores with D125C values greater than 180 min were not satisfactorily explained by linear regression analysis; i.e., the resulting R (SQ) terms were quite low. [R (SQ) refers to the measure of the proportion of total variation about the mean explained by linear regression.] Identification of the four phase II isolates (see above) indicated that isolate number 1 was Bacillus cereus, whereas isolates 2, 3, and 4 were classified as B. licheniformis. Identification was not pursued to the subspecies level; however, because of differences in heat resistance of isolates 2, 3, and 4, it was believed that more than one variety of B. licheniformis was tested. Four D125c determinations were made for each phase II isolate test condition (a total of 64 heat-resistance tests). An analysis of variance was performed on the data to determine the significance of the sources of variation present in the experiment (Table 1). No effect on D125 c due to replication was noted, thus indicating the ability to repeat significantly D value estimates for an organism handled in a specified way. Differences in strain, sporulation medium, and suspension menstruum were all seen to affect D values. A strain-sporulation medium interaction that affected heat resistance was also noted; i.e., for all four organisms tested a significantly higher D125 c value was observed when the sporulation was performed with SSM rather than TAM (P < 0.05; Table 2). Table 3 summarizes the media-menstruum effects on the four Mariner isolates. The far right-hand column of this table indicates a significantly higher D125 c value for the ethanol versus the water-suspended spores; however, this observation did not hold for all strainmedia combinations. For example, the ethanol and water suspended-TAM cultured isolates, with the exception of isolate number 4, had a similar D125 c- Table 4 shows the results of determinations of thermal extinction points of environmental fallout strips from the AFETR. No growth was observed from the assay of strips exposed to 125 C for 90 min. However, growth was still present after 60 min of exposure, indicating The U.S. Public Health Service at Phoenix, Ariz., has classified and dry-heat tested 103 Mariner 1969 bacterial spore isolates (9). Spores were cultured by using TAM agar and found to have substantially lower heat resistance than the 70 SSM-cultured spores discussed in the present study. Such findings would be in agreement with the sporulation medium effects on heat resistance reported above. Work by the Phoenix group (M. Favero, personal communication) suggests that spores harvested from SSM retain a dry-heat resistance more closely approximating their natural dry-heat resistance, i.e., resistance before culturing, than do spores cultured on TAM. RESULTS AND DISCUSSION The present study of the dry-heat resistance of bacterial spores recovered from the surfaces of flight craft provides some of the information necessary for the formulation of a dry-heat sterilization cycle for unmanned spacecraft. In addition to a knowledge of the dry-heat resistance of microorganisms on such a craft, a thorough understanding of other factors must be acquired, e.g., the level of microbial contamination and the temperature profile of the craft (3).

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2020-12-10T09:02:51.962Z

1971-10-01T00:00:00.000Z

237231150

s2

Diacetyl and Acetoin Production by Lactobacillus casei Agitation of broth cultures of Lactobacillus casei retarded cellular dry weight accumulation but enhanced production of both diacetyl and acetoin. Addition of pyruvate overcame this retardation, but addition of sulfhydryl-protecting reagents did not. Both pyruvate and citrate enhanced accumulated dry weight of L. casei incubated without agitation, but only pyruvate increased diacetyl accumulation. Both actively dividing cells and cells suspended in buffer converted pyruvate to diacetyl and acetoin. Maximum production of diacetyl and acetoin occurred during the late logarithmic or early stationary phases. Cells isolated from pyruvate- or citrate-containing cultures showed the greatest ability to convert pyruvate to diacetyl and acetoin. The optimum pH for diacetyl and acetoin formation by whole cells was in the range of 4.5 to 5.5. The presence of citrate or acetate enhanced diacetyl and acetoin formation by L. casei cells in buffer suspension. Lactobacillus casei, an organism utilized in several food fermentations, produces diacetyl as one of its more important volatile constituents (1,11,15). Since diacetyl is important in the flavor of certain fermented foods, production and utilization of this compound can play a significant role in the development of flavor. We reported previously that citrate stimulates the growth of L. casei (4) and that citrate and pyruvate increase the levels of diacetyl reductase in this organism (3). During these studies, pyruvate and citrate were found to have a profound influence on diacetyl production (5). Thus the present investigation was initiated to elucidate the nature of certain chemical and environmental factors which can influence diacetyl and acetoin production by actively growing cultures and by isolated whole cells of L. casei. MATERIALS AND METHODS Organism and culturing conditions. L. casei 393, obtained from the American Type Culture Collection, was used throughout this study. The organism was propagated routinely by culturing in Elliker broth (Difco) for 36 to 48 hr at 30 C. To determine the effects of citrate or pyruvate, given amounts of sodium citrate or sodium pyruvate were dissolved in deionized water and added to Elliker broth, which was then sterilized by autoclaving for 15 min at 121 C. Acetaldehyde and sodium acetate solutions were sterilized by autoclaving (2), and solu-tions of glutathione, 2-mercaptoethanol, and dithiothreitol were sterilized by membrane filtration (0.45-,m pore size; Millipore Corp., Bedford, Mass.) before addition to sterile Elliker broth. All cultures were incubated at 30 C. Aerobic conditions were maintained by agitating cultures in a gyratory shaker (model G-25, New Brunswick Scientific Co., New Brunswick, N.J.) which was operated at 90 strokes/ min unless specified otherwise. Static conditions refer to undisturbed incubation. Cell growth was measured by determining absorbance at 660 nm and converting to dry weight of cells as described previously (4). Diacetyl and acetoin assay. Diacetyl levels were determined by the specific colorimetric method of Pack et al. (13). The total diacetyl plus acetoin content of reaction mixtures was determined as acetoin by the method of Hill et al. (7). This method also measures 2, 3-butanediol, but gas-chromatographic analysis of reaction mixtures and cultures by a slight modification (5) of the technique of Rogosa and Love (14) revealed the absence of 2, 3-butanediol in all cases. Diacetyl and acetoin production by whole cells. Cells were recovered from Elliker broth by centrifugation at 15,000 X g for 30 min at 2 C. The cells were washed once with 0.1 M potassium phosphate buffer (pH 7.0) and resuspended in phosphate buffer. The ability of isolated cells to produce diacetyl and acetoin was determined in the pyruvate assay mixture described by Speckman and Collins (17). This mixture contained 100 MAmoles of sodium pyruvate, 0.02 pAmole of thiamine pyrophosphate, 4.5 ,moles of magnesium sulfate, 0.1 ml of cell suspension, and 2.0 ml of 0.1 M phosphate buffer (pH 4.5) in a final volume of 3.0 ml. These mixtures were incubated at 30 C. The effect of pH on the reaction was determined by using a series of 0.1 M phosphate buffers to yield final pH values ranging from 4.5 to 7.0. Preparation of cell-free extracts. Buffer suspensions of washed cells were cooled in ice and sonically oscillated for 15 min with a Branson Sonicator (model B110) operated at maximum amplitude. Cell debris was removed by centrifugation at 15,000 X g for 20 min at 2 C, and the cell-free supernatant fluid was recovered. The ability of this supernatant fluid to produce diacetyl and acetoin was determined in the pyruvate assay mixture described previously. RESULTS AND DISCUSSION L. casei 393, when cultured in milk medium, utilized acetaldehyde and produced small amounts of ethanol and diacetyl (10,11). Addition of citrate to the medium decreased the levels of diacetyl which accumulated by increasing the diacetyl reductase activity of the cells (3,11). We have found pyruvate to be a precursor for the formation of diacetyl and acetoin by L. casei and that the presence of pyruvate in the culture medium enhanced diacetyl reductase activity, thus increasing the rate of diacetyl utilization by the test organism (3,5). Pyruvate, in levels of 16 h-6 PYR -260 CPM medium, increased dry weight of L. casei incubated without shaking (Fig. 1). Stimulation was first evident after 18 hr, and the higher cellular dry weight of treated cultures persisted through 48 hr of incubation. Agitation of cultures at either 90 or 260 strokes/min caused a significant decrease in accumulated dry weight of L. casei. Addition of pyruvate overcame this effect ( Fig. 1). At 90 strokes/min, cultures containing pyruvate displayed the same cellular dry weight as unagitated controls. At 260 strokes/min, pyruvate restored the accumulated cell weight to nearly the same level as that of the unagitated controls. We had previously demonstrated that inclusion of citrate into the medium significantly stimulated the accumulated dry weight of L. casei under agitated and unagitated conditions (4). This was confirmed in the present study and it was observed that, in the same molar concentrations, pyruvate was more effective than citrate in stimulating accumulation of L. casei cells. The inhibition induced by agitation appeared to be related directly to increased contact with oxygen. Agitated cultures held in flasks tightly stoppered with rubber-lined screw caps showed much higher accumulations of cellular dry weights than agitated cultures contained in flasks closed with cotton plugs. In both cases, however, accumulation was still retarded relative to unagi- (5), the sulfhydryl-protecting reagents glutathione, 2-mercaptoethanol, and dithiothreitol were tested for their effect on accumulation of cells in agitated cultures. None of these reagents, when added in levels ranging from 2 to 20 ,umoles per ml of medium, had any demonstrable effect on L. casei. Thus, factors other than oxidation of enzyme sulfhydryl groups appear to be responsible for retardation of Lactobacillus growth under aerobic conditions. Gilliland and Speck (6) observed no growth enhancement when catalase was added to cultures of lactic streptococci and lactobacilli. Citrate, pyruvate, and acetaldehyde were tested for their effect on diacetyl and acetoin production by the test organism (Fig. 2). Agitation resulted in increased diacetyl accumulation with all compounds tested when compared to unagitated controls. Agitation also increased diacetyl accumulation by the test organism in the absence of additives. By far the greatest increase in diacetyl and acetoin accumulation occurred in agitated cultures containing pyruvate. Pyruvate also stimulated diacetyl and acetoin accumulation under still conditions, but the increase was much greater with agitation and the levels of diacetyl accounted for a much larger percentage of the total diacetyl plus acetoin levels under these conditions. Addition of combinations of pyruvate and citrate decreased the production of diacetyl and acetoin relative to the amount produced when pyruvate alone was present. Acetaldehyde had little effect when added either alone or together with pyruvate. Based on a constant dry weight of cells, there was a much greater accumulation of diacetyl plus acetoin in agitated cultures compared to still controls (Fig. 3). Addition of either pyruvate or citrate to the broth medium resulted in both increased rate of synthesis and accumulation of total diacetyl and acetoin; pyruvate had a greater effect in this regard. With combinations of pyruvate and citrate, production of diacetyl plus acetoin was retarded relative to production in broths containing pyruvate alone. In nearly all cases, maximal levels of diacetyl plus acetoin were attained after 18 hr, but maximal cell populations were not reached until 24 to 36 hr. These observations indicate that pyruvate enhances both growth and diacetyl and acetoin formation by L. casei. Diacetyl and acetoin contents of cultures increased throughout the first 16 hr of incubation, indicating that these compounds arise as a result of the metabolism of cells during logarithmic and early stationary phases. Further, these results strongly imply that L. casei converts pyruvate to diacetyl and acetoin. We have observed previously that cell-free extracts of L. casei convert pyruvate to a-acetolactate, which is decarboxylated subsequently to diacetyl and acetoin (5). Isolated washed L. casei cells suspended in buffer actively converted pyruvate to diacetyl and acetoin (Fig. 4). Resting cells, isolated at different intervals during the growth curve, yielded diacetyl plus acetoin production patterns nearly identical to those observed with actively growing cells in broth medium, with maximal production of these compounds being attained with cells harvested during the late logarithmic or early stationary phases (cf. Fig. 3 and 4). Cells harvested from broths containing pyruvate or citrate produced more diacetyl plus acetoin than did cells from control broths at all stages examined. In contrast to the situation with actively growing cultures, where agitation enhanced diacetyl production, cells isolated from agitated cultures produced less total diacetyl plus acetoin Effect ofpH on diacetyl plus acetoin formation by isolated cells and cell-free extracts. Levels of diacetyl and acetoin, determined after incubation for 2 hr in the standard assay mixture, are expressed as acetoin. Pyruvate and citrate refer to cells harvested from media containing 16 ,umoles per ml of the respective acid. APPL. MICROBIOL. than did cells isolated from cultures incubated without agitation (Fig. 4). This indicates that the enhanced diacetyl and acetoin formation in agitated cultures resulted from something other than increased enzymatic ability to produce these compounds. Increased production of diacetyl and acetoin appears to be due to increased conversion of a diacetyl and acetoin precursor. One known precursor of both diacetyl and acetoin, a-acetolactate, readily undergoes nonenzymatic decarboxylation to yield both acetoin and diacetyl (5, 8,9,16,17), and increased oxygen content greatly stimulates this conversion (8). An alternative explanation is that aerobic conditions induce the cells to direct more pyruvate to oxidative breakdown reactions, such as diacetyl and acetoin formation, without increasing the enzymatic ability of cells to produce diacetyl and acetoin. Although citrate did not enhance significantly diacetyl formation when added to broth cultures, resting cells isolated from citrate-containing broth showed an increased ability to produce diacetyl and acetoin from pyruvate. The optimum pH for diacetyl and acetoin formation from pyruvate was in the range of 4.5 to 5.5 for control, citrate-, and pyruvate-cultured resting cells (Fig. 5). However, the pH optimum for citrate-cultured cells was in the range of 4.5 to 5.0, whereas the range for control and pyruvatecultured cells was between 5.0 and 5.5. In contrast, the optimum pH for diacetyl and acetoin formation in cell-free extracts from all cell types was 5.5 (Fig. 5). This indicated that the optimum pH for diacetyl and acetoin formation by whole cells reflected something other than the influence of pH on the enzymatic reaction. At the lower pH values, the charge on both the cells and on pyruvate would be neutralized, which may have facilitated the accumulation of pyruvate. Citrate can reduce the ionic charge of cells (12), and it was assumed to enhance the formation of diacetyl and acetoin by isolated whole cells by reducing cellular charge and thus facilitating pyruvate uptake. Addition of citrate to pyruvate-containing buffer suspensions of whole cells indicated that this was the case (Fig. 6). Although citrate itself was not converted to diacetyl and acetoin in this system, it stimulated diacetyl and acetoin formation from pyruvate. In concentrations ranging from 4 to 16 mM, citrate stimulated diacetyl formation by cells isolated from control, citrate, and pyruvate broths. In contrast, the presence of citrate inhibited diacetyl formation by cell-free extracts, indicating that it stimulated uptake of a precursor and not an enzymatic reaction (Fig. 7). Acetate, another compound which in itself was not converted to diacetyl or acetoin, stimulated formation of these compounds by whole cells and inhibited their formation in cell-free extracts (Fig. 7). This again indicated stimulation of uptake of a precursor. Results presented herein revealed that pyruvate, as well as citrate, enhanced production of diacetyl in L. casei cultures, with pyruvate being much more effective in this regard. Aeration of cultures by agitation markedly enhanced diacetyl accumulation. Addition or omission of pyruvate or citrate and the control of culturing conditions would appear to be powerful factors to consider for the control of diacetyl formation in fermentations involving L. casei. Furthermore, these results point to the fallability of extending results obtained with cell-free systems to the situation which exists in organisms in cultures. From a practical standpoint, our results point to the importance of considering the ionic charge on cells when attempting to alter the metabolic activity of microorganisms, particularly with regard to diacetyl and acetoin formation.

v3-fos