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Immunosuppression in Chickens by Aflatoxin1,2
Immunosuppression in Chickens by Aflatoxin1'2 J. P . THAXTON, H . T . TUNG AND P . B . HAMILTON
Department of Poultry Science and Department of Microbiology, North Carolina State Raleigh, North Carolina 27607
University,
(Received for publication July 9, 1973)
affected similarly. When this hypothesis was tested directly, HE aflatoxins are a group of closely aflatoxicosis and S. gallinarum infections related toxic metabolites produced in were found to exert their effects on body feedstuffs by Aspergillus flavus (Ciegler and weight and mortality independently and withLillehoj, 1968). While aflatoxin in chickens out interaction (Smith et al., 1969). On the generally causes an acute toxicosis characother hand, Hamilton and Harris (1971) found terized on a practical basis by lowered growth an interaction on body weights and crop rates and inefficient conversion of feed (Carweights of chickens consuming dietary aflanaghan et al., 1966; Gardiner and Oldroyd, 1965; and Smith and Hamilton, 1970), the toxin and infected with Candida albicans. possibility of interaction of aflatoxicosis with In another laboratory Pier and Heddleston other diseases has been studied since the (1970) found that dietary aflatoxin consumed disease was discovered. One of the original by turkey poults and young chickens during reports of aflatoxicosis (Siller and Ostler, or after immunization with Pasteurella mul1961) described the isolation of Salmonella tocida interfered with the development of from turkeys during a field outbreak of the resistance to a lethal challenge with the disease. Brown and Abrams (1965) consis- pathogenic organism; however, these authors tently isolated Salmonella from ducklings and did not find any aflatoxin induced alterations chickens with typical aflatoxicosis. They also in -/-globulin levels or in levels of agglutinins measured a hypoproteinemia which included against P. multocida. Contrariwise, Pier et low levels of globulins and suggested that al. (1971) reported that aflatoxin did not aflatoxicosis made birds more susceptible to impair acquired resistance to Newcastle disSalmonella. Abrams (1965) hypothesized that ease virus. Richard et al. (1973) in an interestother bacterial and viral diseases would be ing study found no interaction in turkey poults between aflatoxin and A. fumigatus on mortality, histopathologic lesions and growth 1. Paper No. 3617 of the Journal Series of the North rate; although, the two factors in combination Carolina State University Agricultural Experiment resulted in the occurrence of precipitating Station, Raleigh, North Carolina. antibodies against A. fumigatus. INTRODUCTION
T
2. A preliminary report of part of this paper was presented at the 60th Annual Meeting of the Poultry Science Association, Fayetteville, Arkansas, 1971.
The object of the present study was to investigate the effect of aflatoxin on immuni-
721
Downloaded from http://ps.oxfordjournals.org/ at New York University on October 12, 2014
ABSTRACT Aflatoxin, a potent natural contaminant in feedstuffs, suppressed hemagglutinin formation in chickens. The primary immune response was reduced in a dose-related fashion when aflatoxin was incorporated into the diet at levels as low as 0.625 n.g. per gram. The relative sizes of the bursa of Fabricius and the thymus, the primary initiators of immunity in the chicken, were reduced by 30% and 55%, respectively, when chickens ate a diet containing 10 u.g. of aflatoxin per gram of feed. The state of immunosuppression can account for the carcinogenecity of aflatoxin as well as the enhanced susceptibility to some infectious agents found during aflatoxicosis. POULTRY SCIENCE 53: 721-725, 1974
722
J. P. THAXTON, H. T. TUNG AND P. B. HAMILTON
ty in a system uncomplicated by pathogenesis. Our report describes the suppression by aflatoxin of the development of specific antibodies and the regression of the thymus and bursa of Fabricius. MATERIALS AND METHODS
Immunoassay. The birds were immunized with sheep red blood cells (SRBC) at 24 days of age by injecting intravenously 1 ml. of a 7% suspension of SRBC in 0.15 M NaCl. Blood was collected by venipuncture immediately prior to SRBC injection and additional bleedings were made at three day intervals for 15 days following SRBC injections. The blood samples were incubated for at least two hours at 37° C. and then refrigerated overnight at 10° C. before the serum was collected. The complement fractions of the serum were inactivated by heating in a water bath at 56° C. for 30 min. The hemagglutinins
Statistical A nalyses. The group means were submitted to an analysis of variance in which the F-ratio was calculated. If a significant F-ratio were obtained, the least significant differences among treatments was calculated. The details are outlined by Bruning and Kintz (1968). Organ Size. The thymus and bursa of Fabricius were excised from three-week old birds which had been fed graded concentrations of dietary aflatoxin from hatching. The glands were blotted and weighed, and the size of the glands was expressed relative to the body weight of the birds from which they were excised.
10.0
3 6 9 "9 12 TIMEtdays after antigen injection)
FIG. 1. Effect of dietary aflatoxin on the hemagglutinin response in chickens. The birds were fed graded doses of aflatoxin from hatching until termination of the experiment. The birds were immunized at three weeks of age with 1 ml of a 7% suspension of SRBC in 0.15 M NaCl and then were bled at three day intervals for the next 15 days. Each data point represents the mean of four groups of ten birds. Symbols: 0, controls; t , 0.625 ixg./g.; A, 1.25 ^g./g.; A 5.0 (xg./g.; and • , 10.0 (xg./g. of dietary aflatoxin.
Downloaded from http://ps.oxfordjournals.org/ at New York University on October 12, 2014
Animal Husbandry and Diet Preparation. The commercial male broiler chicks (Vantress x Arbor Acres) used in this study were housed in electrically-heated cages where feed and water were available ad libitum. Aflatoxicosis was induced by incorporating known amounts of aflatoxin in a broiler-starter diet obtained commercially. Aflatoxin was produced by growing A. flavus NRRL 2999 on rice according to the method of Shotwell et al. (1966). The moldy rice was steamed to kill the fungus, dried, and ground to a fine powder. The aflatoxin content of the powder was determined by the colorimetric method of Nabney and Nesbitt (1965) with the modification of Wiseman et al. (1967). This analysis was confirmed by the less precise chromatographic method of Pons et al. (1966). Weighed amounts of the powder were mixed into the diet which was analyzed and found not to contain aflatoxin. Each concentration of aflatoxin (0,0.625,1.25, 2.5, 5.0, and 10 u,g./g.) was given to four groups of ten birds.
(HA) produced in response to SRBC were determined by a microtitration procedure (Sever, 1962).
723
IMMUNOSUPPRESSION BY AFLATOXIN
RESULTS
Organ Size. The relative sizes of both the bursa of Fabricius and thymus were reduced by dietary aflatoxin (Table 2). The bursa decreased by approximately one-fourth at the higher doses of aflatoxin while the thymus regressed to about one-half of normal size. DISCUSSION Our results indicate that dietary aflatoxin is a potent immunosuppressant in the young chicken and that the extent of suppression
TABLE 1.—Effect on hemagglutinin titers of aflatoxin fed only during the immune response Time (days after antigen injection) Aflatoxin (|xg./g.)
0
3
6
9
12
0 0.625 1.25 2.50 5.00 10.0
0.77a 0.49= 0.59s 0.67a 0.55" 0.40"
4.73 a 3.31 b 3.66" 4.17a 2.84° 2.71 c
5.95a 4.82b 4.48" 4.57" 4.36b 3.46c
4.05 a 3.54a 3.30b 3.70" 3.40b 3.68"
3.54a 2.94a 3.04a 2.86b 2.50" 2.75b
15 3.36a 3.14a 3.56a 3.11 a 3.26" 2.99a
abc Mean titers (log2) of four groups of ten birds. Means in a column accompanied by different superscripts differ significantly (P s 0.05).
TABLE 2.- -Effect of dietary aflatoxin on the relative weights of the bursa of Fabricius and thymus in
chickens Relative organ weights" Aflatoxin (M-8-/g-) 0 0.625 1.25 2.50 5.00 10.0
Bursa of fabricius (g. /100 g. body weight) 0.36 ± 0.01" 0.37 ± 0.02b 0.32 ± 0.02 c 0.32 ± 0.01 c 0.28 ± 0.01 d 0.27 ± 0.02d
Thymus (g./100 g. body weight) 0.79 ± 0.06b 0.75 ± 0.10b 0.77 ± 0.03 b 0.66 ± 0.05c 0.45 ± 0.01 d 0.34 ± 0.04*
"Each value represents the mean ± standard error for four groups ten birds each. b.c.a.e Means in a column accompanied by different superscripts differ significantly (P : 0.05).
Downloaded from http://ps.oxfordjournals.org/ at New York University on October 12, 2014
Antibody Levels. Fig. 1 shows the HA response in birds fed graded levels of aflatoxin from hatching until the experiment was terminated. The HA responses were depressed significantly (P < 0.05) by all concentrations of aflatoxin at 3, 6, and 9 days after SRBC injection. The degree of immunosuppression was directly related to the concentration of dietary aflatoxin on days 3 and 6. At 6 days after SRBC injection the primary response reached a maximum and the antibody levels decreased thereafter except in the birds fed 5.0 or 10 \xg. of aflatoxin per g. of diet. The antibody levels in birds on the higher concentrations of aflatoxin did not reach their maxima until 12 days after SRBC injection when the antibody levels at these concentrations of aflatoxin exceeded those of the control birds. At 15 days these antibody levels
had decreased from their maxima but they still exceeded the control values. Initiating the feeding of aflatoxin at the same time that the antigen was injected gave similar though more variable results (Table 1) except that the increase of antibody levels above control values at 12 and 15 days was not obtained with the higher concentrations of aflatoxin.
724
J. P. THAXTON, H. T. TUNG AND P. B. HAMILTON
produce antibodies is dependent on the bursa and thymus, the regression of these two organs by aflatoxin would be expected to result in impaired immunological performance. A similar mechanism is postulated for the effects of actinomycin D, a carcinogenic antibiotic, on the immune system (Kaufman, 1971). Our data indicate that aflatoxin is a potent immunosuppressant in young chickens being able to suppress the primary HA response and to concomitantly reduce the relative size of the tissues responsible for initiating immunological competence. Immunosuppression induced by aflatoxin would appear to be an excellent model for the study of humoral as well as tissue related immune responses. An animal experiencing immunological deficiencies is expected to be more susceptible to carcinogens (Burnet, 1970) and the immunosuppressing property of aflatoxin may account, at least in part, for its strong carcinogenecity. Certainly this hypothesis should be entertained as well as the hypothesis based on deletions and malignant transformations as a result of binding to DNA (Lafarge and Frayssinet, 1970). The interference of aflatoxin with immune responses should result in an enhanced susceptibility of animals with aflatoxicosis to infectious agents. However, when this hypothesis has been tested with actual diseases, both positive and negative results have been obtained as outlined in the introduction to this paper. A possible explanation for the conflicting literature lies in the finding (Fig. 1 and Table 1) that the occurrence and degree of immunosuppression is dose and time related. Perhaps interactions between aflatoxin and infectious agents would be more readily demonstrated if the proper dose and time were selected for the test. The ability of small amounts of an environmental contaminant to cause such disruptive effects emphasizes anew the importance of multivariant studies on factors affecting animal and public health.
Downloaded from http://ps.oxfordjournals.org/ at New York University on October 12, 2014
of HA response is related to the dose of aflatoxin as well as being dependent on the duration of treatment. Several alternative explanations for the immunosuppressive ability of aflatoxin exist. 1. Aflatoxin has been demonstrated to inhibit RNA polymerase in vivo and subsequently to limit protein synthesis (LaFarge and Frayssinet, 1970). Immunosuppression by aflatoxin then would be the result of inhibition of the synthesis of specific immunoglobulins. 2. An enhanced degradation of antibodies would account for our results. Aflatoxin causes a rapid and dramatic increase in the specific activity of lysosomal enzymes in skeletal muscle and liver of chickens (Tung et al., 1970) and a dose-related decrease in tissue strength and integrity (Tung et al., 1971). Since lysosomes and their hydrolytic enzymes are involved in the extracellular and intracellular digestion of macromolecules (DeDuve and Wattiaux, 1966), aflatoxin would be an immunosuppressant by virtue of its ability to stimulate lysosomal degradation of immunoglobulins. 3. Inhibition of the processing of antigen would also explain our results. Aflatoxin inhibits the reticuloendothelial system in a dose-related fashion (Michael et al., 1973). These phagocytic cells possibly are involved in the processing of antigen (Karnovsky, 1962). 4. Inhibition of specific immunological tissues is an equally attractive alternative. The efferent limb of the immunological system in chickens is dependent on the bursa of Fabricius for initiating humorally-related antibodies (Glick, 1970) and on the thymus for initiating cellularly-related antibodies (Cooper et al., 1965). Modification of the cellular integrity of these tissues by chemical or physical agents results in immunosuppression (Glick, 1967). It should be noted that the regression of the bursa and the suppression of the HA responses occurred with concentrations of aflatoxin that do not inhibit growth (Smith and Hamilton, 1970). Since the potential of lymphoid tissue to
IMMUNOSUPPRESSION BY AFLATOXIN
REFERENCES
Pier, A. C , and K. L. Heddleston, 1970. The effect of aflatoxin on immunity in turkeys. I. Impairment of actively acquired resistance to bacterial challenge. Avian Dis. 14: 797-809. Pier, A. C , K. L. Heddleston, W. A. Boney and P. D. Lukert, 1971. The effect of aflatoxin on immunity. 19th Congresso Mundial de Medicina Veterinaria y Zootechnia, 1: 216-219. Pons, W. A., A.F.Cucullu, L. S.Lee, J. A. Robertson, A. D. Franz and L. A. Goldblatt, 1966. Determination of aflatoxin in agricultural products: Use of aqueous acetone for extraction. J. Assoc. Off. Agr. Chem. 49: 554-562. Richard, J. L., A. C. Pier, S. J. Cysewski and C. K. Graham, 1973. Effect of aflatoxin and aspergillosis on turkey poults. Avian Dis. 17: 111-121. Sever, J. L., 1962. Application of a microtechnique to viral serological investigations. J. Immunol. 88: 320-329. Shotwell, O. L., C. W. Hesseltine, R. D. Stubblefield and W. G. Sorenson, 1966. Production of aflatoxin on rice. Appl. Microbiol. 14: 425-428. Siller, W. G., and D. C. Ostler, 1961. The histopathology of enterohepatic syndrome of turkey poults. Vet. Rec. 73: 134-138. Smith, J. W., and P. B. Hamilton, 1970. Aflatoxicosis in the broiler chicken. Poultry Sci. 49: 207-215. Smith, J. W., W. R. Prince and P. B. Hamilton, 1969. Relationship of aflatoxicosis to Salmonella gallinarum infections of chickens. Appl. Microbiol. 18: 946-947. Tung, H. T., W. E. Donaldson and P. B. Hamilton, 1970. Effect of aflatoxin on some marker enzymes of lysosomes. Biochim. Biophys. Acta, 222: 665-667. Tung, H. T„ J. W. Smith and P. B. Hamilton, 1971. Aflatoxicosis and bruising in the chicken. Poultry Sci. 50: 795-800. Wiseman,H.G., W. C. JacobsonandW. C. Harmeyer, 1967. Note on removal of pigments from chloroform extracts of aflatoxin cultures with copper carbonate. J. Assoc. Off. Agr. Chem. 50: 982-983.
ACKNOWLEDGEMENTS
This investigation was supported in part by the Biomedical Science Support Grant RR07071 from the National Institutes of Health. We thank Linda Travis and Sharon West for technical assistance.
JANUARY 27-30. SOUTHEASTERN-INTERNATIONAL POULTRY TRADE SHOW, ATLANTA CIVIC CENTER, ATLANTA, GEORGIA.
Downloaded from http://ps.oxfordjournals.org/ at New York University on October 12, 2014
Abrams, L., 1965. Mycotoxins in veterinary medicine. South African Med. J. 58: 767-771. 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, and Co., Glen view, 111. Burnet, F. M., 1970. A concept of immunological surveillence. Prog. Exp. Tumor Res. 13: 1-27. Carnaghan, R. B. A., G. Lewis, D. S. P. Patterson and R. Allcroft, 1966. Biochemical and pathological aspects of groundnut poisoning in chickens. Pathol. Vet. 3: 601-605. Ciegler, A., and E. B. Lillehoj, 1968. Mycotoxins. Appl. Microbiol. 10: 155-219. Cooper, M. D., R. D. A. Peterson, M. A. South and R. A. Good, 1965. The functions of the thymus system and bursa system in the chicken. J. Exp. Med. 123: 75-123. DeDuve, C , and R. Wattiaux, 1966. Functions of lysosomes. Ann. Rev. Physiol. 28: 435-492. Gardiner, M. R., and B. Oldroyd, 1965. Avian aflatoxicosis. Australian Vet. J. 41: 272-276. Glick, B., 1967. Antibody and gland studies in cortisone and ACTH-injected birds. J. Immunol. 98: 1076-1084. Glick, B., 1970. The bursa of Fabricius as a central issue. Bioscience, 20: 10-13. Hamilton, P. B., and J. R. Harris, 1971. Interaction of aflatoxicosis with Candida albicans infections and other stresses in chickens. Poultry Sci. 50: 906-912. Karnovsky.M. L., 1962. Metabolic basis of phagocytic activity. Physiol. Rev. 42: 143-165. Kaufman, S. J., 1971. Selective inhibition of cells in the immune response by actinomycin D. J. Immunol. 106: 781-785. 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. Michael, G. Y., J. P. Thaxton and P. B. Hamilton, 1973. Inpairment of the reticuloendothelial systems of chickens during aflatoxicosis. Poultry Sci. 52: 1206-1207. Nabney, J., and B. F. Nesbitt, 1965. A spectrophotometric method of determining the aflatoxins. Analyst, 90: 155-160.
725
Department of Poultry Science and Department of Microbiology, North Carolina State Raleigh, North Carolina 27607
University,
(Received for publication July 9, 1973)
affected similarly. When this hypothesis was tested directly, HE aflatoxins are a group of closely aflatoxicosis and S. gallinarum infections related toxic metabolites produced in were found to exert their effects on body feedstuffs by Aspergillus flavus (Ciegler and weight and mortality independently and withLillehoj, 1968). While aflatoxin in chickens out interaction (Smith et al., 1969). On the generally causes an acute toxicosis characother hand, Hamilton and Harris (1971) found terized on a practical basis by lowered growth an interaction on body weights and crop rates and inefficient conversion of feed (Carweights of chickens consuming dietary aflanaghan et al., 1966; Gardiner and Oldroyd, 1965; and Smith and Hamilton, 1970), the toxin and infected with Candida albicans. possibility of interaction of aflatoxicosis with In another laboratory Pier and Heddleston other diseases has been studied since the (1970) found that dietary aflatoxin consumed disease was discovered. One of the original by turkey poults and young chickens during reports of aflatoxicosis (Siller and Ostler, or after immunization with Pasteurella mul1961) described the isolation of Salmonella tocida interfered with the development of from turkeys during a field outbreak of the resistance to a lethal challenge with the disease. Brown and Abrams (1965) consis- pathogenic organism; however, these authors tently isolated Salmonella from ducklings and did not find any aflatoxin induced alterations chickens with typical aflatoxicosis. They also in -/-globulin levels or in levels of agglutinins measured a hypoproteinemia which included against P. multocida. Contrariwise, Pier et low levels of globulins and suggested that al. (1971) reported that aflatoxin did not aflatoxicosis made birds more susceptible to impair acquired resistance to Newcastle disSalmonella. Abrams (1965) hypothesized that ease virus. Richard et al. (1973) in an interestother bacterial and viral diseases would be ing study found no interaction in turkey poults between aflatoxin and A. fumigatus on mortality, histopathologic lesions and growth 1. Paper No. 3617 of the Journal Series of the North rate; although, the two factors in combination Carolina State University Agricultural Experiment resulted in the occurrence of precipitating Station, Raleigh, North Carolina. antibodies against A. fumigatus. INTRODUCTION
T
2. A preliminary report of part of this paper was presented at the 60th Annual Meeting of the Poultry Science Association, Fayetteville, Arkansas, 1971.
The object of the present study was to investigate the effect of aflatoxin on immuni-
721
Downloaded from http://ps.oxfordjournals.org/ at New York University on October 12, 2014
ABSTRACT Aflatoxin, a potent natural contaminant in feedstuffs, suppressed hemagglutinin formation in chickens. The primary immune response was reduced in a dose-related fashion when aflatoxin was incorporated into the diet at levels as low as 0.625 n.g. per gram. The relative sizes of the bursa of Fabricius and the thymus, the primary initiators of immunity in the chicken, were reduced by 30% and 55%, respectively, when chickens ate a diet containing 10 u.g. of aflatoxin per gram of feed. The state of immunosuppression can account for the carcinogenecity of aflatoxin as well as the enhanced susceptibility to some infectious agents found during aflatoxicosis. POULTRY SCIENCE 53: 721-725, 1974
722
J. P. THAXTON, H. T. TUNG AND P. B. HAMILTON
ty in a system uncomplicated by pathogenesis. Our report describes the suppression by aflatoxin of the development of specific antibodies and the regression of the thymus and bursa of Fabricius. MATERIALS AND METHODS
Immunoassay. The birds were immunized with sheep red blood cells (SRBC) at 24 days of age by injecting intravenously 1 ml. of a 7% suspension of SRBC in 0.15 M NaCl. Blood was collected by venipuncture immediately prior to SRBC injection and additional bleedings were made at three day intervals for 15 days following SRBC injections. The blood samples were incubated for at least two hours at 37° C. and then refrigerated overnight at 10° C. before the serum was collected. The complement fractions of the serum were inactivated by heating in a water bath at 56° C. for 30 min. The hemagglutinins
Statistical A nalyses. The group means were submitted to an analysis of variance in which the F-ratio was calculated. If a significant F-ratio were obtained, the least significant differences among treatments was calculated. The details are outlined by Bruning and Kintz (1968). Organ Size. The thymus and bursa of Fabricius were excised from three-week old birds which had been fed graded concentrations of dietary aflatoxin from hatching. The glands were blotted and weighed, and the size of the glands was expressed relative to the body weight of the birds from which they were excised.
10.0
3 6 9 "9 12 TIMEtdays after antigen injection)
FIG. 1. Effect of dietary aflatoxin on the hemagglutinin response in chickens. The birds were fed graded doses of aflatoxin from hatching until termination of the experiment. The birds were immunized at three weeks of age with 1 ml of a 7% suspension of SRBC in 0.15 M NaCl and then were bled at three day intervals for the next 15 days. Each data point represents the mean of four groups of ten birds. Symbols: 0, controls; t , 0.625 ixg./g.; A, 1.25 ^g./g.; A 5.0 (xg./g.; and • , 10.0 (xg./g. of dietary aflatoxin.
Downloaded from http://ps.oxfordjournals.org/ at New York University on October 12, 2014
Animal Husbandry and Diet Preparation. The commercial male broiler chicks (Vantress x Arbor Acres) used in this study were housed in electrically-heated cages where feed and water were available ad libitum. Aflatoxicosis was induced by incorporating known amounts of aflatoxin in a broiler-starter diet obtained commercially. Aflatoxin was produced by growing A. flavus NRRL 2999 on rice according to the method of Shotwell et al. (1966). The moldy rice was steamed to kill the fungus, dried, and ground to a fine powder. The aflatoxin content of the powder was determined by the colorimetric method of Nabney and Nesbitt (1965) with the modification of Wiseman et al. (1967). This analysis was confirmed by the less precise chromatographic method of Pons et al. (1966). Weighed amounts of the powder were mixed into the diet which was analyzed and found not to contain aflatoxin. Each concentration of aflatoxin (0,0.625,1.25, 2.5, 5.0, and 10 u,g./g.) was given to four groups of ten birds.
(HA) produced in response to SRBC were determined by a microtitration procedure (Sever, 1962).
723
IMMUNOSUPPRESSION BY AFLATOXIN
RESULTS
Organ Size. The relative sizes of both the bursa of Fabricius and thymus were reduced by dietary aflatoxin (Table 2). The bursa decreased by approximately one-fourth at the higher doses of aflatoxin while the thymus regressed to about one-half of normal size. DISCUSSION Our results indicate that dietary aflatoxin is a potent immunosuppressant in the young chicken and that the extent of suppression
TABLE 1.—Effect on hemagglutinin titers of aflatoxin fed only during the immune response Time (days after antigen injection) Aflatoxin (|xg./g.)
0
3
6
9
12
0 0.625 1.25 2.50 5.00 10.0
0.77a 0.49= 0.59s 0.67a 0.55" 0.40"
4.73 a 3.31 b 3.66" 4.17a 2.84° 2.71 c
5.95a 4.82b 4.48" 4.57" 4.36b 3.46c
4.05 a 3.54a 3.30b 3.70" 3.40b 3.68"
3.54a 2.94a 3.04a 2.86b 2.50" 2.75b
15 3.36a 3.14a 3.56a 3.11 a 3.26" 2.99a
abc Mean titers (log2) of four groups of ten birds. Means in a column accompanied by different superscripts differ significantly (P s 0.05).
TABLE 2.- -Effect of dietary aflatoxin on the relative weights of the bursa of Fabricius and thymus in
chickens Relative organ weights" Aflatoxin (M-8-/g-) 0 0.625 1.25 2.50 5.00 10.0
Bursa of fabricius (g. /100 g. body weight) 0.36 ± 0.01" 0.37 ± 0.02b 0.32 ± 0.02 c 0.32 ± 0.01 c 0.28 ± 0.01 d 0.27 ± 0.02d
Thymus (g./100 g. body weight) 0.79 ± 0.06b 0.75 ± 0.10b 0.77 ± 0.03 b 0.66 ± 0.05c 0.45 ± 0.01 d 0.34 ± 0.04*
"Each value represents the mean ± standard error for four groups ten birds each. b.c.a.e Means in a column accompanied by different superscripts differ significantly (P : 0.05).
Downloaded from http://ps.oxfordjournals.org/ at New York University on October 12, 2014
Antibody Levels. Fig. 1 shows the HA response in birds fed graded levels of aflatoxin from hatching until the experiment was terminated. The HA responses were depressed significantly (P < 0.05) by all concentrations of aflatoxin at 3, 6, and 9 days after SRBC injection. The degree of immunosuppression was directly related to the concentration of dietary aflatoxin on days 3 and 6. At 6 days after SRBC injection the primary response reached a maximum and the antibody levels decreased thereafter except in the birds fed 5.0 or 10 \xg. of aflatoxin per g. of diet. The antibody levels in birds on the higher concentrations of aflatoxin did not reach their maxima until 12 days after SRBC injection when the antibody levels at these concentrations of aflatoxin exceeded those of the control birds. At 15 days these antibody levels
had decreased from their maxima but they still exceeded the control values. Initiating the feeding of aflatoxin at the same time that the antigen was injected gave similar though more variable results (Table 1) except that the increase of antibody levels above control values at 12 and 15 days was not obtained with the higher concentrations of aflatoxin.
724
J. P. THAXTON, H. T. TUNG AND P. B. HAMILTON
produce antibodies is dependent on the bursa and thymus, the regression of these two organs by aflatoxin would be expected to result in impaired immunological performance. A similar mechanism is postulated for the effects of actinomycin D, a carcinogenic antibiotic, on the immune system (Kaufman, 1971). Our data indicate that aflatoxin is a potent immunosuppressant in young chickens being able to suppress the primary HA response and to concomitantly reduce the relative size of the tissues responsible for initiating immunological competence. Immunosuppression induced by aflatoxin would appear to be an excellent model for the study of humoral as well as tissue related immune responses. An animal experiencing immunological deficiencies is expected to be more susceptible to carcinogens (Burnet, 1970) and the immunosuppressing property of aflatoxin may account, at least in part, for its strong carcinogenecity. Certainly this hypothesis should be entertained as well as the hypothesis based on deletions and malignant transformations as a result of binding to DNA (Lafarge and Frayssinet, 1970). The interference of aflatoxin with immune responses should result in an enhanced susceptibility of animals with aflatoxicosis to infectious agents. However, when this hypothesis has been tested with actual diseases, both positive and negative results have been obtained as outlined in the introduction to this paper. A possible explanation for the conflicting literature lies in the finding (Fig. 1 and Table 1) that the occurrence and degree of immunosuppression is dose and time related. Perhaps interactions between aflatoxin and infectious agents would be more readily demonstrated if the proper dose and time were selected for the test. The ability of small amounts of an environmental contaminant to cause such disruptive effects emphasizes anew the importance of multivariant studies on factors affecting animal and public health.
Downloaded from http://ps.oxfordjournals.org/ at New York University on October 12, 2014
of HA response is related to the dose of aflatoxin as well as being dependent on the duration of treatment. Several alternative explanations for the immunosuppressive ability of aflatoxin exist. 1. Aflatoxin has been demonstrated to inhibit RNA polymerase in vivo and subsequently to limit protein synthesis (LaFarge and Frayssinet, 1970). Immunosuppression by aflatoxin then would be the result of inhibition of the synthesis of specific immunoglobulins. 2. An enhanced degradation of antibodies would account for our results. Aflatoxin causes a rapid and dramatic increase in the specific activity of lysosomal enzymes in skeletal muscle and liver of chickens (Tung et al., 1970) and a dose-related decrease in tissue strength and integrity (Tung et al., 1971). Since lysosomes and their hydrolytic enzymes are involved in the extracellular and intracellular digestion of macromolecules (DeDuve and Wattiaux, 1966), aflatoxin would be an immunosuppressant by virtue of its ability to stimulate lysosomal degradation of immunoglobulins. 3. Inhibition of the processing of antigen would also explain our results. Aflatoxin inhibits the reticuloendothelial system in a dose-related fashion (Michael et al., 1973). These phagocytic cells possibly are involved in the processing of antigen (Karnovsky, 1962). 4. Inhibition of specific immunological tissues is an equally attractive alternative. The efferent limb of the immunological system in chickens is dependent on the bursa of Fabricius for initiating humorally-related antibodies (Glick, 1970) and on the thymus for initiating cellularly-related antibodies (Cooper et al., 1965). Modification of the cellular integrity of these tissues by chemical or physical agents results in immunosuppression (Glick, 1967). It should be noted that the regression of the bursa and the suppression of the HA responses occurred with concentrations of aflatoxin that do not inhibit growth (Smith and Hamilton, 1970). Since the potential of lymphoid tissue to
IMMUNOSUPPRESSION BY AFLATOXIN
REFERENCES
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ACKNOWLEDGEMENTS
This investigation was supported in part by the Biomedical Science Support Grant RR07071 from the National Institutes of Health. We thank Linda Travis and Sharon West for technical assistance.
JANUARY 27-30. SOUTHEASTERN-INTERNATIONAL POULTRY TRADE SHOW, ATLANTA CIVIC CENTER, ATLANTA, GEORGIA.
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