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Mycoflora of Commercial Poultry Mixed Feeds
Mycoflora of Commercial Poultry Mixed Feeds MIGUEL A. MORENO ROMO and GUILLERMO SUAREZ FERNANDEZ1 Depanamento de Microbiologic!, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain (Received for publication June 11, 1985)
INTRODUCTION
MATERIALS AND METHODS
Mixed feeds, a basic element in modern husbandry, have not received special mycological attention, in spite of the fact that they are prepared basically with plant raw materials (corn, barley, sorghum, and soya) where fungi are the most important contaminant microorganisms (Lagrandeur and Poisson, 1968; Hesseltine et ai, 1976; Muntanola-Cvetkovic and Borisavljevic, 1979; Clarke and Hill, 1981; McGimpsey and Malone, 1981; El-Kady et ai, 1982). These microorganisms may be present in the final product or originate directly or indirectly from cross contamination. Moreover, actual, mycological studies of food and feedstuffs are significant for determining the distribution of the mycotoxin producing fungi, because these toxic metabolites have been detected in many foods. In this sense, animal feeds are the first stage of one of the chains that may carry mycotoxins to man. Because they can accumulate in animal products, several authors (Van Zytveld et ai, 1970; Mabee and Chipley, 1973; Jacobson and Wiseman, 1974; Trucksess et ai, 1983) have observed aflatoxin accumulation in poultry products (eggs, chicken) when animals received aflatoxins in their diet. We therefore, planned a mycological study of commercial poultry mixed feeds, paying special attention to the mycotoxin producing fungi, which will be reported elsewhere.
Samples. We studied 125 samples of commercial poultry mixed feeds (35 broiler pellets, 31 broiler mashes, 53 layer hen mashes 3 layer pellets, and 3 other poultry mashes) collected from several feed mults and commercial storage in the province of Madrid. Mycological Analysis. Mycological analysis was made by a tenfold dilution method from 10 g of sample homogenized with 90 ml of sterile diluent (Ringer solution plus .01% Tween 80) in a bottom-driver macerator (LoMi, Madrid, Spain) for 1 min. The results were recorded from the 10~2 dilution in two culture media, malt agar (IMISA, Madrid, Spain) and Sabouraud agar (IMISA) both with 100 mg*l _1 of oxytetracycline (Pfizer, Madrid, Spain), incubated at 25 C for 3 to 7 days. Fungi were identified mainly according to von Arx (1981).
'To whom correspondence should be addressed.
RESULTS Tables 1 and 2 show the genera isolated from the samples. The mycoflora of commercial poultry mixed feeds were characterized by an evident predominance of the typical storage fungi (Christensen, 1957). The genera Penicillium, Aspergillus and yeast were present in more than 80% of the samples. In 50% of the samples, we found fungi both field and storage species classified as: Fusarium, Mucor, Cladosporium, and Aureobasidium; next, about 25%, were Circinella and Rhizopus; the remaining genera identified had a lower frequency of isolation and they were designated mycoflora of rare occurrence in mixed feeds.
284
Downloaded from http://ps.oxfordjournals.org/ at UIC Library, Collections Development on May 7, 2015
ABSTRACT Mycological analysis of 125 samples of commercial poultry mixed feeds were performed. Penicillium, Aspergillus, and yeast were present in more than 80% of the samples; Fusarium, Mucor, Cladosporium, and Aureobasidium were in about 50%, of the samples, Circinella and Rbizopus were in about 25%, and the remaining fungi identified had a lower prevalence. Malt agar with oxytetracycline was more useful than Sabouraud agar with oxytetracycline for mycological analysis of poultry mixed feeds. (Key words: mixed feeds, mycoflora, culture media) 1986 Poultry Science 65:284-287
MYCOFLORA IN FEED
285
TABLE 1. Relative frequency (RF) and percentage of isolation (%) of the main genera isolated from mixed feeds Sbt2
AMt 1
Total RF
%
RF
%
RF
%
Penicillium Aspergillus3 Yeast 4 ' 3 Fusarium 5|3 Mucor Cladosporiupt*'3 Aureobasidium3 Circinella Rhizopus Geotricbum
104 101 95 68 68 55 53 34 32 12
83.2 80.8 76 54.4 54.4 44 42.4 27.2 25.6 9.6
94 88 77 65 56 37 43 22 21 10
75.2 70.4 61.6 52 44.8 29.6 34.4 17.6 16.8 8
96 81 76 46 54 38 37 23 21 8
76.8 64.8 60.8 36.8 43.2 30.4 29.6 18.4 16.8 6.4
1
Malt agar with oxytetracycline.
2
Sabouraud agar with oxytetracycline.
3
Sabouraud agar % and total % are significantly different (P<.05).
4
Malt agar with oxytetracycline % and total % are significandy different (P<.05).
s
Malt agar with oxytetracycline % and Sabouraud agar with oxytetracycline % are significantly different (P<.05).
Using the methods of Raper and Fennell (1965), we identified 14 species in the genus Aspergillus: A. flavus, A. parasiticus, A. repens, A. chevalieri, A, amstelodami, A. fumigatus, A. niger, A. ficuum, A. awamori, A. candidus, A. terricola, A. versicolor, A. sydowi, and A.
terreus. Aspergillus flavus were in 63% of the samples. Simultaneous use of two culture media permitted us to analyze their suitability for the study of mycoflora of mixed feeds. In this work, we used the statistical method of com-
TABLE 2. Relative frequency (RF) and percentage of isolation (%) of the remaining genera isolated from mixed feeds Sbt 2
AMt1
Total Genus
RF
%
RF
%
RF
%
Sterile mycelia Acremonium Alternaria Absidia Scopulariopsis Trichoderma Syncephalastrum Amylomyces Chaetopboma Ulocladium Stemphylium Nigrospora Polischema Parapericonia Acremoniella
10 4 3 3 3 2 2 2 2
8 3.2 2.4 2.4 2.4 1.6 1.6 1.6 1.6 .8 .8 .8 .8 .8 .8
4 2 2 2 1 2 1 2
3.2 1.6 1.6 1.6 .8 1.6 .8 1.6
8 3
6.4 2.4 .8 .8 1.6
1 1 1 1 1
.8 .8 .8 .8 .8
1
Malt agar with oxytetracycline.
2
Sabouraud agar with oxytetracycline.
.8 1.6
.8
Downloaded from http://ps.oxfordjournals.org/ at UIC Library, Collections Development on May 7, 2015
Genus
MORENO ROMO AND FERNANDEZ
286
DISCUSSION Penicillium and Aspergillus, the main storage fungi (Christensen, 1957; Jemmali, 1975; Beuchat, 1978) were the genera most frequently isolated by us from the mixed feeds. Similar findings have been reported by other researchers for the mycoflora in raw materials and feeds (Hesseltine et ah, 1976; Moreau, 1978; McGimpsey and Malone, 1981; AbdellFattah et al, 1982; El-Kady et al, 1982). Likewise, genera Fusarium and Cladosporium, as well as the Mucorales, have been reported as highly prevalent by the previously mentioned workers, because these species comprise both field and storage forms. In contrast, our study also found the broad occurrence of yeast, a fact hardly mentioned in other works, although it has been reported by some authors (Ashworth et al, 1965; Lagrandeur and Poisson, 1968; McGimpsey and Malone, 1981). Similarly, Aureobasidium, isolated in 42.4% of our samples, is usually scarcely reported, although Quintanilla (1980) has reported a high occurrence in barley in Spain. The remaining identified genera also occur generally with low frequency, and only Alternaria, which we have isolated in 2.4% of the samples, has consistently been present in other investigations. However, prolonged storage generally reduces the levels of infection by this fungus (Wallace and Sinha, 1975), and our low
results can be explained because Spanish mixed feeds are dependent on international trade. With respect to the culture media used, our results show that oxytetracycline malt agar is more useful than oxytetracycline Sabouraud agar. This is in accord with other workers that have employed this medium and its modifications (malt agar with or without antibiotics, with sodium chloride, with yeast extract, or with rose bengal) for mycological feed analysis (Christensen, 1957; Koburger and Farhat, 1975; Moreau, 1978; Clarke and Hill, 1981; Comi et al., 1981; Abdell-Fattah et al, 1982). Likewise, the mycological characteristics of mixed feeds, frequently containing Mucorales and osmophilic fungi, which are most adapted to substrates with low humidity, suggest that it would be interesting for the analysis of similar feeds to use media with high sugar contents (Christensen, 1957) and with inhibitors that restrict the spread of mold colonies. The mycoflora isolated from poultry mixed feeds pose problems due to the ability of some of them to invade animal tissues (A. fumigatus for example), as well as their capacity to grow on feed, causing spoilage and producing mycotoxins. It is therefore necessary to note the high prevalence of both A. flavus, the main fungus that produces aflatoxins, and, in general, the genera Penicillium, Aspergillus, and Fusarium, which comprise the most important and well-known mycotoxin producing fungi affecting poultry mixed feeds. ACKNOWLEDGMENTS This work was supported in part by a grant of the Spanish Ministry of Education and Science. REFERENCES Abdell-Fattah, H. M., Y. Y. Kamel, S. E. Megalla, and A. H. Hafez, 1982. Aflatoxin and aflatoxicosis. Mycopathologia 77:129-135. von Arx, J. A., 1981. The genera of fungi sporulating in pure culture. 3rd ed. J. Cramer, Vaduz, Liechtenstein. Ashworth, L. J., H. W. Schroeder, and B. C. Langley, 1965. Aflatoxins: environmental factors governing occurrence in Spanish peanuts. Science 148:1228-1229. Beuchat, L. R., 1978. Microbial alterations of grains, legumes and oilseeds. Food Technol. 32:193— 197. Christensen, C. M., 1957. Deterioration of stored grains by fungi. Bot. Rev. 23:108-134. Clarke, J. H., and S. T. Hill, 1981. Mycofloras of moist barley during sealed storage in farm and lab-
Downloaded from http://ps.oxfordjournals.org/ at UIC Library, Collections Development on May 7, 2015
parison of ratios in paired samples (Snedecor and Cochran, 1975) between the percentages of occurrence of genera Penicillium, Aspergillus, yeast, Fusarium, Mucor, Cladosporium, Aureobasidium, Circinella, Rhizopus, and Geotrichum in both media. This method demonstrated that only the percentages of genus Fusarium were significantly different (P<.05). Using the correlation of hierarchy method (Snedecor and Cochran, 1975) to establish a comparison between the arrangements of these 10 genera in decreasing percentage of occurrence, a significant correlation + of .98 was obtained (P<.05). We also determined the differences between the results of each medium and the total results; in oxytetracycline malt agar there was only one difference (Cladosporium), whereas five (Aspergillus, Fusarium, Cladosporium, Aureobasidium, and yeast) in oxytetracycline Sabouraud agar were significant. The hierarchy correlations in both cases were + .99.
MYCOFLORA IN FEED
McGimpsey, H. C , and J. P. Malone, 1981. The fungal flora of stored undried barley grain with special reference to harmful organisms. Rec. Agric. Res. 29:99-102. Moreau, C , 1978. Moisissures des aliments dans une ferme d'elevage porcin. Bull. Soc. Mycol. Fr. 94:359-369. Muntanola-Cvetkovic, M., and J. Borisavljevic, 1979. Mycopopulations in nondried and dried corn grains. Acta Biol. Iugosl. 5:1—21. Ctuintanilla, J. A., 1980. Hongos micotoxigenicos en cebada de la region castellana. A.Y.M.A. 2 1 : 473-484. Raper, K. B., and D. I. Fennell, 1965. The Genus Aspergillus. Williams & Wilkins Co., Baltimore, MD. Snedecor, G. W., and W. G. Cochran, 1975. Metodos estadisticos. 6th ed. C.E.C.S.A., Mexico. Trucksess, M. W., L. Stoloff, K. Young, R. D. Wyatt, and B. L. Miller, 1983. Aflatoxicol and aflatoxins B; and M, in eggs and tissues of laying hens consuming aflatoxin-contaminated feed. Poultry. Sci. 62:2176-2182. Wallace, H.A.H., and R. N. Sinha, 1975. Micoflora of stored grain in international trade. Mycopathologia 57:171-176. Van Zytveld, W. A., D. C. Kelley, and S. M. Dennis, 1970. Aflatoxicosis: the presence of aflatoxins or their metabolites in livers and skeletal muscles of chickens. Poultry Sci. 49:1350-1356.
Downloaded from http://ps.oxfordjournals.org/ at UIC Library, Collections Development on May 7, 2015
oratory silos. Trans. Br. Mycol. Soc. 77:557— 565. Comi, G., C. Marino, and I. Dragoni, 1981. Muffe in mangimi e in tratti intestinali di broilers. Rivisto di Avicoltura 50:43-48. El-Kady, I. A., S. I. Abdel-Hafez, and S. S. El-Maraghy, 1982. Contribution to the fungal flora of cereal grains in Egypt. Mycopathologia 77:103—109. Hesseltine, C. W., O. L. Shotwell, W. F. Kwolek, E. B. Lillehoj, W. K. Jackson, and R. J. Bothast, 1976. Aflatoxin occurrence in 1973 corn at harvest. II. Mycological studies. Mycologia 68:341—353. Jacobson, W. C , and H. G. Wiseman, 1974. The transmission of aflatoxin B t into eggs. Poultry Sci. 53:1743-1745. Jemmali, M., 1975. Les mycotoxines contaminants naturels des produits agricoles. Bull. Anc. Eleves Ec. Fr. Meun 267:131-140. Koburger, J. A., and B. Y. Farhat, 1975. Fungi in foods. VI. A comparison of media to enumerate yeast and molds. J. Milk Food Technol. 38: 466-468. Lagrandeur, G., and J. Poisson, 1968. La microflore du mais: son evolution en fonction des conditions hydriques et thermiques de stockage en atmosphere renouvelee. Ind. Alim. Agric. 85: 775-788. Mabee, M. S., and J. R. Chipley, 1973. Tissue distribution and metabolism of aflatoxin B, 14 C in layer chickens. J. Food Sci. 38:566-570.
287
INTRODUCTION
MATERIALS AND METHODS
Mixed feeds, a basic element in modern husbandry, have not received special mycological attention, in spite of the fact that they are prepared basically with plant raw materials (corn, barley, sorghum, and soya) where fungi are the most important contaminant microorganisms (Lagrandeur and Poisson, 1968; Hesseltine et ai, 1976; Muntanola-Cvetkovic and Borisavljevic, 1979; Clarke and Hill, 1981; McGimpsey and Malone, 1981; El-Kady et ai, 1982). These microorganisms may be present in the final product or originate directly or indirectly from cross contamination. Moreover, actual, mycological studies of food and feedstuffs are significant for determining the distribution of the mycotoxin producing fungi, because these toxic metabolites have been detected in many foods. In this sense, animal feeds are the first stage of one of the chains that may carry mycotoxins to man. Because they can accumulate in animal products, several authors (Van Zytveld et ai, 1970; Mabee and Chipley, 1973; Jacobson and Wiseman, 1974; Trucksess et ai, 1983) have observed aflatoxin accumulation in poultry products (eggs, chicken) when animals received aflatoxins in their diet. We therefore, planned a mycological study of commercial poultry mixed feeds, paying special attention to the mycotoxin producing fungi, which will be reported elsewhere.
Samples. We studied 125 samples of commercial poultry mixed feeds (35 broiler pellets, 31 broiler mashes, 53 layer hen mashes 3 layer pellets, and 3 other poultry mashes) collected from several feed mults and commercial storage in the province of Madrid. Mycological Analysis. Mycological analysis was made by a tenfold dilution method from 10 g of sample homogenized with 90 ml of sterile diluent (Ringer solution plus .01% Tween 80) in a bottom-driver macerator (LoMi, Madrid, Spain) for 1 min. The results were recorded from the 10~2 dilution in two culture media, malt agar (IMISA, Madrid, Spain) and Sabouraud agar (IMISA) both with 100 mg*l _1 of oxytetracycline (Pfizer, Madrid, Spain), incubated at 25 C for 3 to 7 days. Fungi were identified mainly according to von Arx (1981).
'To whom correspondence should be addressed.
RESULTS Tables 1 and 2 show the genera isolated from the samples. The mycoflora of commercial poultry mixed feeds were characterized by an evident predominance of the typical storage fungi (Christensen, 1957). The genera Penicillium, Aspergillus and yeast were present in more than 80% of the samples. In 50% of the samples, we found fungi both field and storage species classified as: Fusarium, Mucor, Cladosporium, and Aureobasidium; next, about 25%, were Circinella and Rhizopus; the remaining genera identified had a lower frequency of isolation and they were designated mycoflora of rare occurrence in mixed feeds.
284
Downloaded from http://ps.oxfordjournals.org/ at UIC Library, Collections Development on May 7, 2015
ABSTRACT Mycological analysis of 125 samples of commercial poultry mixed feeds were performed. Penicillium, Aspergillus, and yeast were present in more than 80% of the samples; Fusarium, Mucor, Cladosporium, and Aureobasidium were in about 50%, of the samples, Circinella and Rbizopus were in about 25%, and the remaining fungi identified had a lower prevalence. Malt agar with oxytetracycline was more useful than Sabouraud agar with oxytetracycline for mycological analysis of poultry mixed feeds. (Key words: mixed feeds, mycoflora, culture media) 1986 Poultry Science 65:284-287
MYCOFLORA IN FEED
285
TABLE 1. Relative frequency (RF) and percentage of isolation (%) of the main genera isolated from mixed feeds Sbt2
AMt 1
Total RF
%
RF
%
RF
%
Penicillium Aspergillus3 Yeast 4 ' 3 Fusarium 5|3 Mucor Cladosporiupt*'3 Aureobasidium3 Circinella Rhizopus Geotricbum
104 101 95 68 68 55 53 34 32 12
83.2 80.8 76 54.4 54.4 44 42.4 27.2 25.6 9.6
94 88 77 65 56 37 43 22 21 10
75.2 70.4 61.6 52 44.8 29.6 34.4 17.6 16.8 8
96 81 76 46 54 38 37 23 21 8
76.8 64.8 60.8 36.8 43.2 30.4 29.6 18.4 16.8 6.4
1
Malt agar with oxytetracycline.
2
Sabouraud agar with oxytetracycline.
3
Sabouraud agar % and total % are significantly different (P<.05).
4
Malt agar with oxytetracycline % and total % are significandy different (P<.05).
s
Malt agar with oxytetracycline % and Sabouraud agar with oxytetracycline % are significantly different (P<.05).
Using the methods of Raper and Fennell (1965), we identified 14 species in the genus Aspergillus: A. flavus, A. parasiticus, A. repens, A. chevalieri, A, amstelodami, A. fumigatus, A. niger, A. ficuum, A. awamori, A. candidus, A. terricola, A. versicolor, A. sydowi, and A.
terreus. Aspergillus flavus were in 63% of the samples. Simultaneous use of two culture media permitted us to analyze their suitability for the study of mycoflora of mixed feeds. In this work, we used the statistical method of com-
TABLE 2. Relative frequency (RF) and percentage of isolation (%) of the remaining genera isolated from mixed feeds Sbt 2
AMt1
Total Genus
RF
%
RF
%
RF
%
Sterile mycelia Acremonium Alternaria Absidia Scopulariopsis Trichoderma Syncephalastrum Amylomyces Chaetopboma Ulocladium Stemphylium Nigrospora Polischema Parapericonia Acremoniella
10 4 3 3 3 2 2 2 2
8 3.2 2.4 2.4 2.4 1.6 1.6 1.6 1.6 .8 .8 .8 .8 .8 .8
4 2 2 2 1 2 1 2
3.2 1.6 1.6 1.6 .8 1.6 .8 1.6
8 3
6.4 2.4 .8 .8 1.6
1 1 1 1 1
.8 .8 .8 .8 .8
1
Malt agar with oxytetracycline.
2
Sabouraud agar with oxytetracycline.
.8 1.6
.8
Downloaded from http://ps.oxfordjournals.org/ at UIC Library, Collections Development on May 7, 2015
Genus
MORENO ROMO AND FERNANDEZ
286
DISCUSSION Penicillium and Aspergillus, the main storage fungi (Christensen, 1957; Jemmali, 1975; Beuchat, 1978) were the genera most frequently isolated by us from the mixed feeds. Similar findings have been reported by other researchers for the mycoflora in raw materials and feeds (Hesseltine et ah, 1976; Moreau, 1978; McGimpsey and Malone, 1981; AbdellFattah et al, 1982; El-Kady et al, 1982). Likewise, genera Fusarium and Cladosporium, as well as the Mucorales, have been reported as highly prevalent by the previously mentioned workers, because these species comprise both field and storage forms. In contrast, our study also found the broad occurrence of yeast, a fact hardly mentioned in other works, although it has been reported by some authors (Ashworth et al, 1965; Lagrandeur and Poisson, 1968; McGimpsey and Malone, 1981). Similarly, Aureobasidium, isolated in 42.4% of our samples, is usually scarcely reported, although Quintanilla (1980) has reported a high occurrence in barley in Spain. The remaining identified genera also occur generally with low frequency, and only Alternaria, which we have isolated in 2.4% of the samples, has consistently been present in other investigations. However, prolonged storage generally reduces the levels of infection by this fungus (Wallace and Sinha, 1975), and our low
results can be explained because Spanish mixed feeds are dependent on international trade. With respect to the culture media used, our results show that oxytetracycline malt agar is more useful than oxytetracycline Sabouraud agar. This is in accord with other workers that have employed this medium and its modifications (malt agar with or without antibiotics, with sodium chloride, with yeast extract, or with rose bengal) for mycological feed analysis (Christensen, 1957; Koburger and Farhat, 1975; Moreau, 1978; Clarke and Hill, 1981; Comi et al., 1981; Abdell-Fattah et al, 1982). Likewise, the mycological characteristics of mixed feeds, frequently containing Mucorales and osmophilic fungi, which are most adapted to substrates with low humidity, suggest that it would be interesting for the analysis of similar feeds to use media with high sugar contents (Christensen, 1957) and with inhibitors that restrict the spread of mold colonies. The mycoflora isolated from poultry mixed feeds pose problems due to the ability of some of them to invade animal tissues (A. fumigatus for example), as well as their capacity to grow on feed, causing spoilage and producing mycotoxins. It is therefore necessary to note the high prevalence of both A. flavus, the main fungus that produces aflatoxins, and, in general, the genera Penicillium, Aspergillus, and Fusarium, which comprise the most important and well-known mycotoxin producing fungi affecting poultry mixed feeds. ACKNOWLEDGMENTS This work was supported in part by a grant of the Spanish Ministry of Education and Science. REFERENCES Abdell-Fattah, H. M., Y. Y. Kamel, S. E. Megalla, and A. H. Hafez, 1982. Aflatoxin and aflatoxicosis. Mycopathologia 77:129-135. von Arx, J. A., 1981. The genera of fungi sporulating in pure culture. 3rd ed. J. Cramer, Vaduz, Liechtenstein. Ashworth, L. J., H. W. Schroeder, and B. C. Langley, 1965. Aflatoxins: environmental factors governing occurrence in Spanish peanuts. Science 148:1228-1229. Beuchat, L. R., 1978. Microbial alterations of grains, legumes and oilseeds. Food Technol. 32:193— 197. Christensen, C. M., 1957. Deterioration of stored grains by fungi. Bot. Rev. 23:108-134. Clarke, J. H., and S. T. Hill, 1981. Mycofloras of moist barley during sealed storage in farm and lab-
Downloaded from http://ps.oxfordjournals.org/ at UIC Library, Collections Development on May 7, 2015
parison of ratios in paired samples (Snedecor and Cochran, 1975) between the percentages of occurrence of genera Penicillium, Aspergillus, yeast, Fusarium, Mucor, Cladosporium, Aureobasidium, Circinella, Rhizopus, and Geotrichum in both media. This method demonstrated that only the percentages of genus Fusarium were significantly different (P<.05). Using the correlation of hierarchy method (Snedecor and Cochran, 1975) to establish a comparison between the arrangements of these 10 genera in decreasing percentage of occurrence, a significant correlation + of .98 was obtained (P<.05). We also determined the differences between the results of each medium and the total results; in oxytetracycline malt agar there was only one difference (Cladosporium), whereas five (Aspergillus, Fusarium, Cladosporium, Aureobasidium, and yeast) in oxytetracycline Sabouraud agar were significant. The hierarchy correlations in both cases were + .99.
MYCOFLORA IN FEED
McGimpsey, H. C , and J. P. Malone, 1981. The fungal flora of stored undried barley grain with special reference to harmful organisms. Rec. Agric. Res. 29:99-102. Moreau, C , 1978. Moisissures des aliments dans une ferme d'elevage porcin. Bull. Soc. Mycol. Fr. 94:359-369. Muntanola-Cvetkovic, M., and J. Borisavljevic, 1979. Mycopopulations in nondried and dried corn grains. Acta Biol. Iugosl. 5:1—21. Ctuintanilla, J. A., 1980. Hongos micotoxigenicos en cebada de la region castellana. A.Y.M.A. 2 1 : 473-484. Raper, K. B., and D. I. Fennell, 1965. The Genus Aspergillus. Williams & Wilkins Co., Baltimore, MD. Snedecor, G. W., and W. G. Cochran, 1975. Metodos estadisticos. 6th ed. C.E.C.S.A., Mexico. Trucksess, M. W., L. Stoloff, K. Young, R. D. Wyatt, and B. L. Miller, 1983. Aflatoxicol and aflatoxins B; and M, in eggs and tissues of laying hens consuming aflatoxin-contaminated feed. Poultry. Sci. 62:2176-2182. Wallace, H.A.H., and R. N. Sinha, 1975. Micoflora of stored grain in international trade. Mycopathologia 57:171-176. Van Zytveld, W. A., D. C. Kelley, and S. M. Dennis, 1970. Aflatoxicosis: the presence of aflatoxins or their metabolites in livers and skeletal muscles of chickens. Poultry Sci. 49:1350-1356.
Downloaded from http://ps.oxfordjournals.org/ at UIC Library, Collections Development on May 7, 2015
oratory silos. Trans. Br. Mycol. Soc. 77:557— 565. Comi, G., C. Marino, and I. Dragoni, 1981. Muffe in mangimi e in tratti intestinali di broilers. Rivisto di Avicoltura 50:43-48. El-Kady, I. A., S. I. Abdel-Hafez, and S. S. El-Maraghy, 1982. Contribution to the fungal flora of cereal grains in Egypt. Mycopathologia 77:103—109. Hesseltine, C. W., O. L. Shotwell, W. F. Kwolek, E. B. Lillehoj, W. K. Jackson, and R. J. Bothast, 1976. Aflatoxin occurrence in 1973 corn at harvest. II. Mycological studies. Mycologia 68:341—353. Jacobson, W. C , and H. G. Wiseman, 1974. The transmission of aflatoxin B t into eggs. Poultry Sci. 53:1743-1745. Jemmali, M., 1975. Les mycotoxines contaminants naturels des produits agricoles. Bull. Anc. Eleves Ec. Fr. Meun 267:131-140. Koburger, J. A., and B. Y. Farhat, 1975. Fungi in foods. VI. A comparison of media to enumerate yeast and molds. J. Milk Food Technol. 38: 466-468. Lagrandeur, G., and J. Poisson, 1968. La microflore du mais: son evolution en fonction des conditions hydriques et thermiques de stockage en atmosphere renouvelee. Ind. Alim. Agric. 85: 775-788. Mabee, M. S., and J. R. Chipley, 1973. Tissue distribution and metabolism of aflatoxin B, 14 C in layer chickens. J. Food Sci. 38:566-570.
287