The Anemia Caused by Aflatoxin12

The Anemia Caused by Aflatoxin1'2 H S I - T A N G T U N G , F . W . C O O K , R. D . WYATT AND P . B . HAMILTON

Department of Poultry Science and Department of Microbiology, North Carolina State Raleigh, North Carolina 27607

University,

(Received for publication March 10, 1975)

POULTRY SCIENCE 54: 1962-1969, 1975

INTRODUCTION

FLATOXINS are a group of toxic metabolites produced by Aspergillus flavus (Ciegler and Lillehoj, 1968). The aflatoxins are highly carcinogenic to some species such as the rat (Wogan and Newberne, 1967) while they are acutely toxic to other species such as the chicken (Smith and Hamilton, 1970). The response of the hematopoietic system during aflatoxicosis has not been studied extensively. Prior to the discovery of aflatoxin, A. flavus was reported to cause the hemorrhagic anemia syndrome of chickens (Forgacs and Carll, 1962). The hemorrhagic anemia syndrome which results from eating moldy feed was characterized by spontaneous hemorrhages into the musculature and internal organs and by aplastic anemia. Whether

A

1. Paper Number 3720 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, N.C. 2. A preliminary account of part of this work was given at the Mycotoxin Seminar at the 71st Annual Meeting of the American Society for Microbiology, Minneapolis, Minnesota, May 4, 1971.

aflatoxin is involved in the economically important hemorrhagic anemia syndrome has not been investigated directly, but Tung et al. (1970, 1971) found that dietary aflatoxin causes in young chickens increased capillary fragility and increased susceptibility to bruising but not spontaneous hemorrhaging. The specific effects of aflatoxin on the circulating blood cells and the hematopoietic system have been investigated only superficially despite an abundant literature on aflatoxicosis (Ciegler and Lillehoj, 1968). In one of the first descriptions of aflatoxicosis, Wannop (1961) reported an increase of heterophils and monocytes, while lymphocytes and erythrocytes were decreased in a field outbreak of aflatoxicosis in turkeys. Brown and Abrams (1965) observed a slight anemia in ducklings and New Hampshire chicks fed 0.5 p.p.m. of aflatoxin for six weeks but noted that too few animals were used to permit conclusions as to significance. Juskiewicz et al. (1967), on the other hand, did not find any significant changes in erythrocyte and leucocyte counts or in hemoglobin determinations in ducklings fed 0.9 p.p.m. aflatoxin

1962

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ABSTRACT The effects of graded doses of dietary aflatoxin (0, 0.625, 1.25, 2.5, 5.0, and 10.0 (j.g./g.) on hemoglobin, packed blood cell volume, erythrocyte count, leucocyte counts, bone marrow lipid, and bone marrow nucleic acids of chickens were measured. The hemoglobin, packed cell volume, and erythrocyte count were reduced significantly (P < 0.05) to about the same extent by any given dose. Microscopic examination of stained smears of the bone marrow revealed a hyperplastic response including the granulocytic elements. Chemical analyses of the marrow revealed a decreased lipid content and an increased content of ribonucleic acid and deoxyribonucleic acid. Total leucocytes were increased about three fold by aflatoxin (10 (xg./g.). Differential leucocyte counts revealed that the heterophils were increased while the eosinophils were unaffected and the basophils, lymphocytes, and monocytes were decreased. The increase in nucleic acids of the marrow and in circulating leucocytes occurred with doses which inhibit growth rate. These data suggest that aflatoxin causes a hemolytic anemia in chickens, that aflatoxin, by itself, is not involved in the hemorrhagic anemia syndrome of chickens, and that aflatoxin does not cause in chickens a general inhibition of ribonucleic acid and protein synthesis as assumed from studies on rats.

ANEMIA CAUSED BY AFLATOXIN

The objective of our research was to investigate the status of the hematopoietic system during aflatoxicosis in chickens. In particular, the values of various hematologic parameters over a dose-response range were determined to ascertain whether aflatoxin at the doses used would produce the hemorrhagic anemia syndrome of chickens. METHODS Aflatoxicosis was produced experimentally in chickens by adding known amounts of aflatoxin to a commercial diet. Aflatoxin was produced by growing Aspergillus parasiticus NRRL 2999 on rice according to the method of Shotwell et al. (1966) using the flasks described by Smith and Hamilton (1969). The moldy rice was steamed, dried, and ground to a fine powder which was analyzed for aflatoxin content by the colorimetric method of Nabney and Nesbitt (1965) with the adaptation of Wiseman et al. (1967). The analysis was confirmed by the less precise chromatographic method of Pons et al. (1966) which also served the purpose of identification. The commercial feed was analyzed before use to insure freedom from extraneous aflatoxin. Weighed amounts of rice powder were added to the feed so that the concentrations of

aflatoxin were 0, 0.625, 1.25, 2.5, 5.0, and 10 M-g./g. of diet. The chicks were housed under continuous lighting in electrically heated batteries where feed and water were available ad libitum. Each experimental treatment was given to four groups of ten male broiler chicks from hatching until three weeks of age when the experiments were terminated. The experimental design was completely randomized and the statistical analyses were done on group means as outlined in Bruning and Kintz (1968). Blood samples for determination of hemoglobin, packed cell volume, and erythrocyte counts were collected from the wing vein when the birds were 3 weeks old. Hemoglobin was determined by the method of Sunderman et al. (1953) and packed cell volume was determined with a micro-hematocrit tube. The blood was kept from clotting with ethylenediaminetetraacetate. The total erythrocyte and leucocyte counts were made according to Natt and Herrick (1952). The blood for the leucocyte total counts and for the leucocyte differential count was obtained by cardiac puncture. The blood (1.0 ml.) from each bird in a group was pooled and the above mentioned analyses were done on the pooled sample. No clumping of the cellular elements was observed in the pooled sample. The leucocyte differential counts were done using the methods and the differential criteria of Lucas and Jamroz (1961). After the birds were sacrificed at 3 weeks of age, bone marrow from each bird was obtained by scooping the entire marrow from a tibia broken lengthwise and was pooled on a group basis. The marrow then was homogenized with an equal volume of distilled water. The dry weight of 2 ml. of the homogenate was determined and the total lipid content of the marrow was determined gravimetrically on another 2 ml. by extracting with 10 ml. of chloroform: methanol (2:1) using a tissue homogenizer and drying the extract

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for two weeks. Gagne et al. (1968) observed only minimal deviations of hemoglobin and blood cells in pigs fed aflatoxin for four months. Cysewski et al. (1968) observed a decrease in hematocrit values and a transitory increase in leucocyte counts of pigs given doses of aflatoxin lethal within 24 to 72 hours. Harding et al. (1963) observed an increase in both leucocytes and erythrocytes in pigs fed aflatoxin for some weeks. Sisk et al. (1968) found no significant changes in hemoglobin or packed cell volume of pigs given daily oral doses of aflatoxin for three weeks. In a controlled experiment with beef steers, aflatoxin ingestion did not affect blood cell values (Garrett et al., 1968).

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TUNG, COOK, WYATT AND HAMILTON

acid (DNA) content of the marrow was determined by the method of Dische (1955).

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FIG. 1. The effect of graded doses of dietary aflatoxin on hemoglobin concentration in the chicken. Each data point represents the mean of four groups of ten chickens; vertical bars are the standard error of the mean. in a tared weighing dish. Photomicrographs of smears of the marrow were obtained after staining by the method of Cook (1959). Ribonucleic acid (RNA) and deoxyribonucleic 40

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FIG. 2. The effect of graded doses of dietary aflatoxin on packed blood cell volume in the chicken. Each data point represents the mean of four groups of ten chickens; vertical bars are the standard error of the mean.

5.0 AFLAT0XIN(/Jg/g)

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FIG. 3. The effect of graded doses of dietary aflatoxin on the number of circulating erythrocytes in the chicken. Each data point represents the mean of four groups of ten chickens; vertical bars are the standard of the mean.

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Hemoglobin concentrations in birds administered graded doses of dietary aflatoxin are given in Fig. 1. Aflatoxin administration caused an anemia and an analysis of variance showed that aflatoxin at a dose of 1.25 u.g. / g . or above produced a significant (P < 0.05) lowering of the hemoglobin concentration. Fig. 2 is a summary of the effect of dietary aflatoxin on packed blood cell volume. The volume was decreased significantly (P < 0.05) by aflatoxin at dietary doses of 2.5 u-g./g. and above. In Fig. 3 the effect of graded doses of dietary aflatoxin on the number of circulating erythrocytes is given. Total erythrocytes were reduced significantly ( P < 0 . 0 5 ) even by a dose of 0.625 u-g./g. At the higher doses all three hematologic parameters were reduced to about the same extent by any given dose of aflatoxin. Reduced volume and number of erythrocytes and decreased hemoglobin concentra-

ANEMIA CAUSED BY AFLATOXIN

1965

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FIG. 4. The effect of dietary aflatoxin on the bone marrow of the chicken. This photomicrograph was taken of a pooled sample from ten birds receiving 5.0 |xg./g. Note the cluster of the granuloblasts in the center.

FIG. 5. The effect of graded doses of dietary aflatoxin on the lipid content of bone marrow of the chicken. Each point represents the mean of four pooled samples from ten chickens each; vertical bars are the standard error of the mean. S 4.0r

tion are seen in aplastic anemia (Silver, 1970). Because the bone marrow ceases to function and its hematopoietic cells are replaced with fat in aplastic anemia, stained smears of bone marrow were examined microscopically (Fig. 4). The impression was one of increased cytopoesis. Instead of having a decreased number of hematopoietic cells and an increased amount of fat as would be expected if the marrow were aplastic, the marrow from chickens with aflatoxicosis was quite cellular with a proliferation of both erythroid and myeloid cells. There was an increase of granuloblasts in the marrow of birds receiving aflatoxin, and the cells in general appeared immature when compared to the marrow from control birds which exhibited mainly late polychromatic erythrocytes. This visual impression of a proliferative response was confirmed by chemical analysis. Fig. 5 is a summary of the marrow lipid values as a function of dietary aflatoxin. The marrow lipid was decreased significantly (P < 0.05)

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FIG. 6. The effect of graded doses of dietary aflatoxin on the RNA and DNA content of the bone marrow of chickens. Each point represents the mean of four pooled samples from ten chickens each; vertical bars are the standard error of the mean. even by the smallest dose (0.625 p.g. /g.) tried. Fig. 6 shows the effect of graded doses of dietary aflatoxin on the nucleic acid content

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i

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TUNG, COOK, WYATT AND HAMILTON

TABLE 1.—Effect of graded doses of dietary aflatoxin on the total number of circulating leucocytes in the chicken Aflatoxin (p-g-/g-) 0 0.625 1.25 2.50 5.00 10.0

Leucocyte count'

(xlOVmm. 3 ) 17.5 18.0 15.1 17.4 30.5 45.4

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of the bone marrow. Both RNA and DNA content were increased significantly (P x 0.05) at doses of 2.5 p g . / g . and above. Also, there was no differential effect on the two nucleic acids. The increased cytopoiesis of the marrow including the granulocytic or myeloid cells (Fig. 4) suggested that the leucocytes should be circulating in increased numbers if their maturation is not inhibited. Table 1 gives the total leucocyte count as a function of dietary aflatoxin. As expected, the number of circulating leucocytes was increased markedly. The number at the highest dose of aflatoxin (10 p-g./g.) was about three times the number in the control animals. An analysis of variance revealed that doses of 5.0 and 10.0 pg./g. had a significant (P < 0.05) effect. Differential counts of the leucocytes (Table 2) showed that the heterophils were increased while the eosinophils were unaffected and TABLE 2.—Effect Aflatoxin (M-g-/g-) 0 0.625 1.25 2.50 5.00 10.0

DISCUSSION The hemorrhagic anemia syndrome of poultry was reported to be caused by A. flavus and to be characterized by spontaneous hemorrhaging of the musculature and internal organs and by aplastic anemia (Forgacs and Carll, (1962). Tung et al. (1970, 1971), found that aflatoxin caused instead an increased susceptibility to bruising. It now appears that aflatoxin also does not cause aplastic anemia. While there was an anemia (Fig. 1), the bone marrow showed an increased cytopoesis (Fig. 4) and a decreased rather than increased content of lipid (Fig. 5) which are just the opposite of effects noted with aplastic anemia (Keleman, 1969). These findings make it very likely that aflatoxin, by itself, does not cause the hemorrhagic anemia syndrome. However, there remains the possibility that aflatoxin interacts with other toxins to cause the syndrome. Aflatoxin has been reported to interact with rubratoxin (Edwards and Wogan, 1968) which is produced by Penicillium rubrum, another fungus implicated in the hemorrhagic anemia syndrome (Forgacs and Carll, 1962). It is possible that A. flavus might produce another toxin which interacts with aflatoxin or which produces the syndrome itself. This possibility is not unlikely since A. flavus has been reported to produce sever-

of graded doses of dietary aflatoxin on the differential leucocyte counts of the chicken Eosinophils 0.7 a 0.3 0.7 1.0 0.3 1.0

Basophils Heterophils Lymphocytes (Percent of total leucocytes) 42.8 43.5 6.8 45.5 42.0 8.5 8.8 46.8 36.5 55.0 32.8 8.8 74.0 21.3 3.5 12.7 2.0 80.1

Monocytes 6.3 3.8 7.2 2.0 1.0 0.0

"Values are the mean of four pooled samples from ten chickens each at each treatment level of aflatoxin.

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1 The mean total leucocyte count of four pooled samples from ten chickens each is given with the standard error of the mean for each treatment level of aflatoxin. Values with different small letters differ significantly (P < 0.05).

the basophils were reduced. The dramatic increase in the heterophils occurred while the lymphocytes and monocytes were reduced sharply.

ANEMIA CAUSED BY AFLATOXIN

The elevated white blood cell count measured at the higher doses of aflatoxin also suggests a hemolytic anemia since hemolytic anemias generally result in increased leucocyte counts and marrow hyperplasia of the granulocytic cells (Silver, 1970). The heterophils were increased in this study while the nongranular lymphocytes and monocytes were decreased. In addition, aflatoxin caused a decrease in the number of basophils. A similar response of circulating leucoctyes is also found when a physiological stress is applied to chickens (Siegel, 1971). This similarity of the leucocytosis in aflatoxicosis and stress might be expected since aflatoxin interacts in chickens with several different stressors (Hamilton and Harris, 1971). The

leucocytosis measured in these experiments also resembles that seen in field cases of aflatoxicosis in turkeys (Wannop, 1961) except that an increase of monocytes was seen in the turkeys. This discrepancy might be a species difference or the result of the heavy dependence of the identification of avian leucocytes on the staining procedures (Lucas and Jamroz, 1961). The other contradictory reports in the literature about the effects of aflatoxin on the circulating blood elements presumably reflect the species, time, and dose dependence of the effects. The effects we observed in chickens were reproducible and dramatic. The mechanism whereby aflatoxin exerts its effects is thought to be the result of binding to DNA with the subsequent general inhibition of RNA polymerase and protein synthesis (Clifford and Rees, 1967; Wogan and Pong, 1971; Lafarge and Frayssinet, 1970). However, our data have some aspects which are difficult to explain with such a general and nonspecific mechanism. The response of the bone marrow to graded doses of aflatoxin, in particular, suggests that this general mechanism does not apply in chickens. The significant decrease in the lipid content of the marrow with the smallest dose tried, which is without any effect on growth rate in this experimental system (Smith and Hamilton, 1970) but which also decreases serum lipids (Tung et al., 1972), suggests that lipid metabolism is somehow specifically inhibited. The concomitant increase in DNA and RNA content of the marrow and the dramatic increase in circulating leucocytes with doses of aflatoxin that inhibit growth rate show beyond doubt that aflatoxin does not cause a general inhibition of nucleic acid and protein synthesis. Instead, aflatoxin stimulates the marrow to cell growth and division and to the associated synthesis of nucleic acid and protein. Since 90 to 95% of the total lipid synthesis in chickens occurs in the liver (O'Hea and Leveille, 1969), the reduced lipid content of

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al other less potent toxins (Wilson, 1966). At any rate, the elusive hemorrhagic anemia syndrome has not yet been produced in the laboratory with a single chemical entity. In the anemia caused by aflatoxin, the hemoglobin, packed cell volume, and erythrocyte count were decreased generally to about the same extent by most doses (Fig. 1,2, and 3). Such an anemia can be labeled as a normocytic anemia (Silver, 1970). However, the marrow of chicks with aflatoxicosis was hyperplastic rather than aplastic (Fig. 4). The other main class of normocytic anemias are known as hemolytic anemias and are characterized by an increased rate of red blood cell destruction and by marrow hyperplasia. The slight macrocytic response at doses of 0.625 and 1.25 u.g./g. in which there was a significant decrease in erythrocyte count (Fig. 3) without a corresponding decrease in hemoglobin (Fig. 1) or packed cell volume (Fig. 2) supports the possibility that aflatoxin induces a hemolytic anemia in chicks since a slight macroytosis is seen generally in hemolytic anemia (Silver, 1970). The spleen is enlarged in hemolytic anemias (Kelemen, 1969) and the spleen of chickens is almost doubled in size by aflatoxin (Smith and Hamilton, 1970).

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TUNG, COOK, WYATT AND HAMILTON

the marrow may be a reflection of the lipid transport which is impaired in avians during aflatoxicosis (Shank and Wogan, 1966; Tung et al., 1972) instead of a simple replacement of lipid cells by hematopoietic cells as a result of hyperplasia. The hypothesis that aflatoxin exerts its primary effect by binding to DNA and causing inhibition of RNA polymerase can still be retained but it must be modified to allow the specificities observed in the observed that the synthesis of all types of RNA was not inhibited identically in rat liver and proposed that the synthesis of long chain messenger RNA is inhibited

preferentially

over that of short chain messenger RNA. An additional specificity may reside in the fact that aflatoxin is regarded as a hepatotoxin and that it is concentrated and metabolized there (Ciegler and Lillehoj, 1968). This could screen the marrow against inhibitory concentrations of aflatoxin and enable it to respond in a proliferative manner to the physiological stress induced by aflatoxin. However, aflatoxin does inhibit growth at concentrations which result in proliferation of

the

marrow and presumably this growth inhibition is an effect on nucleic acid and protein synthesis although it could be the result of the liver no longer being able to compensate for the specific effects of aflatoxin on it. Resolution of these interesting considerations awaits further investigation.

ACKNOWLEDGMENTS We thank

Sharon

West, Rini

Hardjo-

pranjoto, and Nancy Goodwin for technical assistance. REFERENCES Brown, J. M. M., and L. Abrams, 1965. Biochemical studies on aflatoxicosis. Onderstepoort J. Vet Res. 32: 119-146. Bruning, J. L., and B. L. Kintz, 1968. Computational Handbook of Statistics. Scott Foresman Co., Glenview, 111.

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present study. Lafarge and Frayssinet (1970)

Ciegler, A., and E. B. Lillehoj, 1968. Mycotoxins. Adv. Appl. Microbiol. 10: 155-219. Clifford, J. I., and K. R. Rees, 1967. The action of aflatoxin B, on the rat liver. Biochem. J. 102: 65-75. Cook, F. W., 1959. Staining fixed preparations of chicken blood cells with combination May-Greenwald-Wright-Phloxine B stain. Avian Dis. 3: 272290. Cysewski, S. J., A. C. Pier, G. W. Engstrom, J. L. Richard, R. W. Dougherty and J. R. Thurston, 1968. Clinical pathologic features of acute aflatoxicosis of swine. Am. J. Vet. Res. 29: 1577-1590. Dische, Z., 1955. Color reactions of nucleic acid components, p. 270-284. In: E. Chargaff and J. N. Davidson (ed.), The Nucleic Acids Vol. 1, Academic Press, New York. Edwards, G. S., and G. N. Wogan, 1968. Acute and chronic toxicity of rubratoxin in rats. Fed. Proc. 27: 552. Forgacs, J., and W. T. Carll, 1962. Mycotoxicoses. Adv. Vet. Sci. 7: 273-382. Gagne, W. E., D. L. Dungworth and J. E. Moulton, 1968. Pathologic effects of aflatoxin in pigs. Path. Vet. 5: 370-384. Garrett, W. H., H. Heitman and A. N. Booth, 1968. Aflatoxin toxicity in beef cattle. Proc. Soc. Exp. Biol. Med. 127: 188-190. Hamilton, P. B., and J. R. Harris, 1971. Interactions of aflatoxin with Candida albicans infections and other stresses in the chicken. Poultry Sci. 50: 906-912. Harding, J. D. J., J. T.Done.G. Lewis and R. Allcroft, 1963. Experimental groundnut poisoning in pigs. Res. Vet. Sci. 4: 217-229. Juskiewicz, T., J. Stec, B. Stefaniak, Z. Rakalska and Z. Madjeska, 1967. Biochemical and pathological effects of aflatoxin poisoning in ducklings. Vet. Rec. 81: 297-298. Kelemen, E., 1969. Physiopathology and Therapy of Human Blood Diseases. Pergamon Press, New York. Lafarge, C , and C. Frayssinet, 1970. The reversibility of inhibition of RNA and DNA synthesis induced by aflatoxin in rat liver. A tentative explanation for carcinogenic mechanism. Int. J. Cancer 6: 74-83. Lucas, A. M., and C. Jamroz, 1961. Atlas of Avian Hematology. Agriculture Monograph 25, United States Department of Agriculture, Washington, D.C. Nabney, J., and B. F. Nesbitt, 1965. A spectrophotometric method of determining the aflatoxins. Analyst, 90: 155-160. Natt, M. P., and C. A. Herrick, 1952. A new blood diluent for counting erythrocytes and leucocytes of the chicken. Poultry Sci. 31: 735-738. O'Hea, E. K., and G. A. Leveille, 1969. Lipid bio-

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ANEMIA CAUSED BY AFLATOXIN

G. F. Stevenson and B. C. Copeland, 1953. Symposium on clinical hemoglobinometry. Am. J. Clin. Path. 23: 519-598. Tung, H. T., W. E. Donaldson and P. B. Hamilton, 1970. Effects of aflatoxin on some marker enzymes of lysosomes. Biochem. Biophys. Acta, 222: 665667. Tung, H. T., W. E. Donaldson and P. B. Hamilton, 1972. Altered lipid transport during aflatoxicosis. Toxicol. Appl. Pharmacol. 15: 97-104. Tung, H. T., J. W. Smith and P. B. Hamilton, 1971. Aflatoxicosis and bruising in the chicken. Poultry Sci. 50: 795-800. Wannop, C. C , 1961. The histopathology of turkey " X " disease in Great Britain. Avian Dis. 5: 371-381. Wilson, B. J., 1966. Toxins other than aflatoxins produced by Aspergillus flavus. Bacteriol. Rev. 30: 478-484. Wiseman, H. C., W. C. Jacobson andW. C. Harmeyer, 1967. Note on removal of pigments from chloroform extracts of aflatoxin cultures with copper carbonate. J. Ass. Offic. Agr. Chem. 50: 982-983. Wogan, G. N., and P. M. Newberne, 1967. Dose response characteristics of aflatoxin B, carcinogenesis in the rat. Cancer Res. 27: 2370-2376. Wogan, G. N., and R. S. Pong, 1970. Aflatoxins. Ann. New York Acad. Sci. 174: 623-635.

NEWS AND NOTES (Continued from page 1961) HOFFMANN-LA ROCHE NOTES Dr. Allen A. Kurnick has been promoted to General Manager of the Agriculture, Agrochemical, and Animal Health Departments of the Roche Chemical Division, Hoffmann-La Roche Inc., Nutley, New Jersey. He joined the Technical Services Department of the Chemical Division on the West Coast in 1962, after serving as Head of the Poultry Science Department at the University of Arizona. HUBBARD FARMS NOTES Dr. James A. Ranson, Jr., has been appointed Director of Technical Service, Hubbard Farms, Walpole, New Hampshire. He will be responsible for development and execution of technical service training programs for Hubbard's international franchised distributors. He also will be active in specific areas

of technical and nutritional work in Hubbard Farms' domestic and subsidiary operations. His background in poultry management and nutrition includes more than 10 years experience in poultry flock supervision and management, as well as four years in Honduras and Central America. DELMARVA NOTES At the recent Delmarva Chicken Festival, Col. Harland Sanders, of Kentucky Fried Chicken fame, was presented with a plaque in appreciation of his many contributions to the poultry industry. TEXAS NOTES Dr. Cecil B. Ryan, Poultry Scientist, Texas Agricultural Experiment Station, Associate Professor, Department of Poultry Science, Texas A and M

(Continued on page 1989)

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synthesis and transport in the domestic chick (Gallus domesticus). Comp. Biochem. Physiol. 30: 149-159. Pons, W. A., A. F. Cucullu, L. S. Lee, J. A. Robertson, A. O. Franz and L. A. Goldblatt, 1966. Determination of aflatoxins in agricultural products: Use of aqueous acetone for extraction. J. Ass. Offic. Agr. Chem. 49: 554-562. Shank, R. C , and G. N. Wogan, 1966. Acute effects of aflatoxin B, on liver composition and metabolism in the rat and duckling. Toxicol. Appl. Pharmacol. 9: 468-476. Shotwell, O. L., C. W. Hesseltine, R. D. Stubblefield and W. G. Sorenson, 1966. Production of aflatoxin on rice. Appl. Microbiol. 14: 425-428. Siegel, H. S., 1971. Adrenals, stress, and the environment. World's Poultry Sci. J. 27: 327-349. Silver, R. T., 1970. Morphology of the Blood and Marrow in Clinical Practice. Grune and Stratton, Inc., New York. Sisk, D. B., W. W. Carlton and J. M. Curtin, 1968. Experimental aflatoxicosis in young swine. Am. J. Vet. Res. 29: 1591-1602. Smith, J. W., and P. B. Hamilton, 1970. Aflatoxicosis in the broiler chicken. Poultry Sci. 49: 207-215. Smith, J. W., and P. B. Hamilton, 1969. Technique for the aseptic addition of liquid to flask cultures. Appl. Microbiol. 17: 317. Sunderman, F. W., R. P. MacFate, D. A. McFayden,