Toxigenic Fungi from Poultry Feed and Litter

STARVATION AND PLASMA LIPOPROTEINS

there may be changes in the a-lipoproteins which are the result of the anorexia rather than the progress of the disease state. On the positive side, the changes observed were sensitive to changes in feed intake and so may be useful in the evaluation of dietary factors in relation to feed intake on different diets and as a measure of anorexia attendant to disease states.

REFERENCES Bide, R. W., and C. le Q. Darcel, 1969. Changes in the phosphorus in the plasma fractions in avian erythroblastosis. Poultry Sci. 48: 795-798. Bird, H. R., H. J. Almquist, D. R. Clandinin, W. W. Cravens, F. W. Hill and J. McGinnis. Nutrient Requirements of Poultry. 5th Ed. Publication 1345 of Nat. Acad. Sci., National Research Council, Wash., D.C., p. 4, 20. Darcel, C. le Q., 1967. Reduction in the concentration of phosphatidyl serine in the plasma of birds with avian erythroblastosis. Nature, 215: 647-648. Darcel, C. le Q., R. W. Bide and M. Merriman, 1968. Properties of turpentine relative to the effects on fowl plasma in vitro. I. The simulation of "leukemic" changes in normal plasma. Can. J. Biochem. 46: 503-508. Gardiner, E., 1962. The relationship between dietary phosphorus level and the level of plasma inorganic phosphorus in chicks. Poultry Sci. 4 1 : 1156-1163. Merriman, M., and C. le Q. Darcel, 1964. Confirmation of plasma protein changes in avian erythroblastosis. Can. J. Biochem. 42: 293-297.

Toxigenic Fungi from Poultry Feed and Litter JOSEPH LOVETT U. S. Department of Health, Education, and Welfare, Public Health Service, Food and Drug Administration, Division of Microbiology, Cincinnati, Ohio 45226 (Received for publication June 11, 1971) ABSTRACT Fungi isolated from feed and litter of two Ohio poultry farms were screened for toxin production. Fourteen-day-slant cultures were used to inoculate neopeptone dextrose, Czapek-Dox, and Mycological broth media. Four-day chick embryos were inoculated with 0.2 ml. of culture filtrate via the air cell. Embryo death at 9 days was used as the toxicity indicator. Those fungi found toxigenic in one or more media were Aspergillus chevalieri (one), A. fumigatus (one), A. terreus (two), Pmicillium cyclopium (five), P. patulum (two), and one each Fusarium and Scopulariopsis sp. Of those isolates screened 13% were found toxigenic. POULTRY SCIENCE 51:

INTRODUCTION

M

YCOTOXINS in foodstuffs are recognized as a public health problem of considerable importance. Fungal toxins in feed and foods have become a major research area since the 1961 discovery of the carcinogenicity of the aflatoxins. Hundreds

309-313,

1972

of species of more than a dozen fungal genera are known to be toxigenic. Poultry mycotoxicosis results from ingestion by poultry of toxic fungal metabolites in feed, litter, and water into which feed is spilled (Forgacs et al., 1962; Forgacs, 1966; Abrams, 1965). Some fungi

Downloaded from http://ps.oxfordjournals.org/ by guest on June 9, 2015

ACKNOWLEDGMENTS The author wishes to thank Mr. H. Klassen, Mr. S. McGee and Mrs. Norma Dow for their technical assistance in this work. Thanks are also due to Dr. E. Gardiner of Canada Agriculture Research Station, Lethbridge and Dr. S. Magwood for their help with the manuscript. The photographic work was done by Mr. N. Kloppenborg and staff of the Photograph Section, Canada Agriculture Research Station, Lethbridge.

309

J. LOVETT

310

EXPERIMENTAL PROCEDURE

Cultures stored on neopeptone-dextrose (ND) agar at 4°C. were transferred to

fresh ND agar slants, grown at 25°C, and used to inoculate broth of the following composition: neopeptone, 5 g.; dextrose, 10 g.; and distilled water, 1000 ml. (pH 6.3 after autoclaving). Aluminum foil closed 300-ml. erlenmeyer flasks were filled to a depth of 1.5 cm. (90 ml.) with the medium and autoclaved at 121°C. for 15 minutes. Spores washed from 14-day-old slants with sterile 0.005% Triton X-100 in distilled water were used to inoculate the sterile medium. Inoculated flasks were shaken vigorously for 10 seconds and incubated statically for 10 days at 25°C. At the end of this period, each culture was filtered through a 0.45-micron filter and stored in a screw-cap tube at 4°C. until bioassay. Embryonated chicken eggs (Burnside et al., 1957; Piatt et al., 1962; and Verrett et al., 1964) were used as the bioassay system. Babcock-300 eggs were incubated at 65% relative humidity and 38°C. for 4 days. Viability was determined by candling. Eggs with abnormally placed air cells were discarded. Viable embryos were inoculated with 0.2 ml. filtered culture broth into the air cell via a small drilled hole. Inoculated eggs were closed with plastic tape and incubated 2 hours air-cell-up to allow absorption of the inoculum before returning to the regular horizontal position. After 5 days post-inoculation incubation, the eggs were again candled and read for death or life. Aspergillus flavus, NRRL 3145, was used throughout the screening as a positive control. Uninoculated ND broth was used as a negative control. In order to check the embryo bioassay system for non-specific deaths, eggs were inoculated with ND broth adjusted to pH 3.0, 6.3, and 10.0 with HC1 or NaOH and to temperatures of 4.0°C. and 25°C. at pH 6.3. Aspergillus flavus NRRL 3145 and 2999, culture broths were used as positive controls. The 25°C. broth served as a negative

Downloaded from http://ps.oxfordjournals.org/ by guest on June 9, 2015

found capable of producing poultry toxicosis in litter and feed are Aspergillus clavatus, A. flavus, A. fumigatus, A. glaucus, Paecilomyces varioti, Penicillium citrinum, P. purpurogenum, P. rubrum, an unidentified Penicillium sp. and Alternaria and Fusarium species (Forgacs, 1966; Abrams, 1965). The recent proposals to use poultry litter as a nitrogen source for chickens (Wehunt et al., 1960), cows (Noland et al., 1955; Southwell et al., 1958; Camp, 1959; Ray et al, 1964, 1965; Carmody, 1964; Brugmann et al., 1964; Fontenot et al., 1964; and Drake et al., 1965), hogs (Camp, 1959), and sheep (Noland et al., 1955; Fontenot et al., 1964; Bhattacharya et al., 1965, 1966), making up to as much as onethird of the total diet, makes the potential mycotoxin-producing fungi in litter and feed significant. In a previous paper, sampling and analysis of feed and litter from one house on each of two Southern Ohio farms raising pullets for laying stock, were described (Lovett et al., 1971). The mycoflora was enumerated and identified to genus. Fungal densities of feeds varied from 7.0 X 102 to 3.2 X 105 per gram. Twelve mold genera were indentified from feeds with Penicillium, Aspergillus, Fusarium, and Mucor dominating. Seventeen genera of fungi were isolated from litter with Penicillium, Scopulariopsis, and Candida dominating. This paper reports the results of screening of some of the isolates from the above study for toxigenicity in a synthetic medium. Isolates found toxigenic in the laboratory were classified to species and lyophilized for further study. No attempt was made to determine their toxigenicity in natural substrates during this study.

311

FEED AND LITTER FUNGI

Of the approximately 500 cultures isolated from poultry feed and litter, 103 TABLE 1.—Non-specific responses in the chick embryo assay Test conditions

Embryo response*

ND broth (0.2 ml./egg) pH6.3 pH3.0 pH 10.0

1/25 2/25 0/25

ND broth at pH 6.3 (0.2 ml./egg) 4°C 25°C

1/25 0/25

ND broth cultures of NRRL 2999 NRRL 3145

24/25 25/25

Drilled but uninoculated controls

1/25

Undrilled controls

2/25

* Numbers represent deaths/total eggs inoculated per group.

Genus Aspergillus Candida Cephalosporium Cladosporium Fusarium Geotrichum Monilia Mucor Oidium Oospora Penicillium Scopulariopsis Total

Number Screened

Number Toxic

Per cent Toxic

8 3 3 4 11 1 4 7 2 5 39 16

4 0 0 0 1 0 0 0 0 0 7 1

50 0 0 0 9 0 0 0 0

103

13

13

6

were screened for toxigenicity in ND broth. They represented those isolates from the last two weeks of sampling. Before actual screening began, the chick embryo assay system was checked for nonspecific deaths using those test conditions described above and listed in Table 1. Deaths recorded after 5 days post-inoculation incubation showed no significant difference between any solution tested and negative controls, except for the Aspergillus fiavus, NRRL 2999 and 3145, cultures. Using a table of binomial probabilities (Dixon et al., 1957), it can be seen that when background (non-specific) deaths are 10% or less (p = 0.1) and five eggs per culture are used (n = 5), the probability that as many as three observed deaths will be due to a non-specific response is 0.0085 or less than 1 chance in 100. All screening data reported were obtained using five eggs per culture and accepting three or more deaths as indicative of culture toxicity. Negative controls were never less than 25 eggs. Table 2 presents the toxigenicity screening data by genus. The Aspergillus isolates yielded the largest percent positives of any genus screened (50%). Penicillium was the only other genus yielding greater than 10% toxicity. Of the 103 isolates screened for

Downloaded from http://ps.oxfordjournals.org/ by guest on June 9, 2015

RESULTS AND DISCUSSION

TABLE 2.—Toxigenicity screening data

OOO

control as did drilled but uninoculated and undrilled eggs. All isolates found toxic during the initial screening were inoculated into ND broth, Czapek-Dox broth, and Mycological broth by the method previously described and assayed for toxicity after incubation for 10 days at 2S°C. The confirmatory assay procedure was the chick embryo method previously described. Those fungal isolates expressing toxigenicity in one or more of the confirmatory media were transferred to ND agar, cultured for spore production, and lyophilized in double strength milk. Aspergillus and PenicilUum cultures were tranferred to Czapek solution agar and Malt extract agar for groupings according to colony, gross and microscopic characteristics. One or more representative of each group was sent to Miss Dorothy I. Fennell, American Type Culture Collection for identification to species.

312

J . LOVETT TABLE 3.—Toxigenic fungi from poultry Litter and feeds Identification

Aspergillus chevalieri Aspergillus fumigatus Aspergillus terreus Penicillium cyclopium Pencillium patulum Fusarium sp. Scopulariopsis sp.

^™f„

Source

1 1 2 5 2 1 1

Feed Litter Feed Feed Feed Feed Litter

REFERENCES Abrams, L., 1965. Mycotoxins in veterinary medicine. South African Med. J. 39: 767-771. Bhattacharya, A. N., and J. P. Fontenot, 1965. Utilization of different levels of poultry litter

Downloaded from http://ps.oxfordjournals.org/ by guest on June 9, 2015

toxigenicity, 13 were positive for an overall 13%. Those fungal species found toxigenic are shown in Table 3. Feed contributed all but 2 of the 13 toxigenic fungal cultures found. This is not surprising since Christensen et al. (1968) found 54% of the isolates from feeds they tested to produce death in experimental animals when fed as natural substrate cultures. Although their isolates were primarily from feeds suspected of involvement in mycotoxicosis, their results and those reported here are indicative of the common occurrence of toxitenic fungi in nature. The importance of feed as a source of toxigenic fungi makes it imperative that feeds and feed handling equipment be kept dry to prevent fungal growth and toxin formation. Routine cleaning of watering devices into which feed may be carried on birds' beaks is a precaution against mycotoxicosis which should not be overlooked. An investigation of the physical and chemical conditions necessary for mycotoxin production under poultry house conditions would be worthwhile. The recent proposals to use poultry house litter as livestock feed makes research into the production and stability of mycotoxins in poultry litter warranted.

nitrogen by sheep. J. Animal Sci. 24: 1174-1178. Bhattacharya, A. N., and J. P. Fontenot, 1966. Protein and energy value of peanut hull and wood shaving poultry litters. J. Animal Sci. 25: 367-371. Brugmann, H. H., H. C. Dickey, B. E. Plummer and B. R. Poultron, 1964. Nutritive value of poultry litter. J. Animal Sci. 23 : 869. Burnside, J. E., W. L. Sippel, J. Forgacs, W. T. Carll, M. B. Atwood and E. R. Doll, 1957. A disease of swine and cattle caused by eating moldy corn. II. Experimental production with pure cultures of molds. Am. J. Vet. Res. 18: 817-824. Camp, A. A., 1959 .Broiler-house litter as livestock feed. Texas Agri. Prog. 5: 17. Carmody, R., 1964. Chicken litter cow feed. Farm Quarterly. 19: 52, 92, 94. Christensen, C. M., G. H. Nelson, C. J . Mirocha and F. Bates, 1968. Toxicity to experimental animals of 943 isolates of fungi. Cancer Res. 28: 2293-2295. Dixon, W. J., and F. J. Massey, 1957. Introduction to Statistical Analysis. McGraw-Hill Book Co., Inc., New York. 488 pp. Drake, C. L., W. H. McClure and J. P. Fontenot, 1965. Broiler litter as feed for ruminants. Livestock Res. Prog. Report, 1964-65. Virginia Agricultural Experiment Station, Blacksburg, Va. Fontenot, J. P., C. L. Drake, W. H. McClure, F. S. McClaugherty, A. N. Bhattacharya, R. F. Kelly and G. W. Litten, 1964. The value of poultry litter as a feed for ruminants. Livestock Res. Prog. Report, 1963-64. Virginia Agricultural Experiment Station, Blacksburg, Va. Fontenot, J. P., F. S. McClaugherty and W. H. McClure, 1965. The value of urea in wintering rations for weanling beef calves. Livestock Res. Prog. Report, 1964-65. Virginia Agricultural Experiment Station, Blacksburg, Va. Forgacs, J., 1966. Mycoses and mycotoxicosis in poultry. Part II. Feedstuffs, 38: 26, 30, 71. Lovett, J., J. W. Messer and R. B. Read, Jr., 1971. The microflora of Southern Ohio poultry itter. Poultry Sci. 50: 746-751. Noland, P. R., B. F Ford and M. L. Ray. 1955. The use of ground chicken litter as a source of nitrogen for gestating-lactating cows and fattening steers. J. Animal Sci. 14: 860-865. Piatt, B. S., R. J. C. Stewart and S. R. Gupta, 1962. The chick embryo as a test organism for toxic substances in food. Proc. Nutr. Soc. 21. Ray, M. L., and R. D. Child, 1964. He's doing well on broiler house litter. Feed Bag, 40: 46.

313

FEED AND LITTER FUNGI Ray, M. L., and R. D. Child, 1965. Chicken litter as a supplement in wintering beef cows and calves on pasture. Arkansas Farm Res. 14: 5. Southwell, B. L., O. M. Hale and W. C. McCormick. 1958. Poultry house litter as a protein supplement in steer fattening rations. Mimeograph Series N. S. 55. Georgia Agricultural Experiment Station, Athens, Ga.

Verrett, J., J. Marliac and J. McLaughlin, Jr. 1964. Use of the chicken embryo in the assay of anatoxin toxicity. J. Assoc. Off. Agri. Chem. 47: 1003-1006. Wehunt, K. E., H. L. Fuller and H. M. Edwards, Jr., 1960. The nutritional value of hydrolyzed poutry manure for broiler chicks. Poultry Sci. 39: 1057-1063.

J. A. ASMAR, P. L. PELLETT, NUR HARIRI AND M. D. HARIRI Department of Animal Science and Food Technology & Nutrition, Faculty of Agricultural Sciences, American University of Beirut, Beirut, Lebanon (Received for publication June 21, 1971)

ABSTRACT Fertile Single Comb White Leghorn chicken eggs were incubated in an automatic incubator. Embryo weights, dry matter and protein content were determined at 6, 12 and 18 days of incubation. Embryos of the foregoing ages were homogenized and their tissue fluids electrophoresed in acrylamide gel. Amino acid composition was determined at 6 and 18 days. Embryo weight, total solids and protein content increased exponentially but at differing rates. Amino acids were incorporated at exponential rates which differed for different amino acids. With advancing incubation time soluble proteins increased in heterogeneity with new fractions appearing at the anodic end of the spectrum. POULTRY SCIENCE 5 1 : 313-320,

NDER suitable incubation conditions the chicken embryo grows within three weeks from the few blastocystic cells in the freshly laid egg to the large and composite population of differentiated cells and intercellular substances making up the tissues and organs of the fully formed chick. This fast sequence of morphogenetic events is made possible by rapidly occurring changes in metabolic processes and chemical composition affecting egg constituents and embryonic tissues at once. Various aspects of the chemistry of chicken embryo development have retained the attention of a number of investigators (Needham, 1931, 1950; Williams et al., 1954; Rupe and Farmer, 1955; Kaminski and Durieux,

U

1972

1956; Flickinger, 1957; Walter and Mahler, 1958; Romanoff, 1960; Fitzsimmons and Waibel, 1968). Here, as in other systems, the rapidly occurring morphogenic and chemical changes are genetically controlled. This is made possible by the synthesis of the appropriate amounts and quality of proteins mediating phenotypic expressions as directed by genome and affected by environment. Determining total amino acid and protein composition of the embryo in the course of incubation would be of interest to the experimenter studying responses of the developing embryo to genetic, chemical, nutritional or microbial treatments. The usual complexity of the required analyses, however,

Downloaded from http://ps.oxfordjournals.org/ by guest on June 9, 2015

Quantitative and Qualitative Protein Changes in the Developing Single Comb White Leghorn Chicken Embryo