THE EFFECT OF CHRONIC FEEDING OF DIACETOXYSCIRPENOL, T-2 TOXIN, AND AFLATOXIN ON PERFORMANCE, HEALTH, AND ANTIBODY PRODUCTION IN CHICKS

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1 2001 Poultry Science Association, Inc. THE EFFECT OF CHRONIC FEEDING OF DIACETOXYSCIRPENOL, T-2 TOXIN, AND AFLATOXIN ON PERFORMANCE, HEALTH, AND ANTIBODY PRODUCTION IN CHICKS D. SKLAN, 1 E. KLIPPER, and A. FRIEDMAN Faculty of Agricultural, Food Environmental Sciences, Hebrew University of Jerusalem, Rehovot, Israel Phone: Fax: sklan@agri.huji.ac.il M. SHELLY Diagnostic Laboratory of Poultry Diseases, Beer-Tuvia, Israel B. MAKOVSKY Matmor Central Feed Mill, MP Evtach, Israel Primary Audience: Poultry Producers, Veterinarians SUMMARY The effects of feeding T-2 toxin, diacetoxyscirpenol (DAS), or aflatoxin B1 at levels up to 1,000 ppb for 5 weeks on performance, health, and immune response of enterally and parenterally immunized chicks were examined. No decreases in growth or feed efficiency were observed when T-2, DAS, or a mixture of these mycotoxins were fed for 35 days. at concentrations above 800 ppb resulted in decreased growth and feed efficiency after 4 weeks. Feeding T-2 and DAS resulted in oral lesions and mild intestinal inflammation, but no other pathological or histopathological lesions. caused enlargement and discoloration of liver and kidneys and mild intestinal inflammation. No effects of T-2, DAS, or aflatoxin B1 were observed on antibody production to antigens administered by enteral or parenteral routes. Key words: Broilers, mycotoxins, growth DESCRIPTION OF PROBLEM The presence of mycotoxins in poultry feeds is of concern as economic losses may ensue because of reduced performance and health. One group of mycotoxins are the tricothecenes, which are produced mainly by Fusarium spp. before harvesting. Tricothecene type A toxins have been most often chacterized by their effects (oral lesions and reduced growth in chickens) after feed J. Appl. Poultry Res. 10:79 85 ing 4 mg/kg feed or higher concentrations for 1 to 3 weeks [1, 2, 3]. Tricothecenes appear to inhibit protein synthesis and thus affect rapidly dividing cells such as those of the oral cavity, gastrointestinal tract, and lymphoid cells [1, 4]. is produced by Aspergillus spp., which often proliferate upon storage. Aflatoxicosis is characterized by acute hepatotoxicity with vacuolation of hepatocytes; lymphocytic depletion of the bursa is the earliest symptom 1 To whom correspondence should be addressed

2 80 JAPR: Research Report TABLE 1. Concentrations of the mycotoxins in the different experiments EXPERIMENT MYCOTOXIN USED CONCENTRATIONS A (ppb) MIXTURE, CONCENTRATIONS (ppb) 1 T-2 0, 110, 530, 1,050 2 DAS B 0, 204, 501, 1,005 T-2, 510; DAS , 102, 222, 552, 1,125, 210; T-2, 105; DAS, , 185, 381, 760 A Determined analytically as described in the Materials and Methods section. B DAS = diacetoxyscirpenol. observed [1]. Several studies have indicated that mycotoxins are immunosuppressive [5]. High aflatoxin doses have been reported to reduce the bursa weight [6, 7] and the secondary immune reponse to injected sheep red blood cells in chicks [7, 8]. In contrast, Weibking et al. [9] reported an enhanced primary immune response to sheep red blood cells when aflatoxin or fumonisim was fed to poults, but Pier [10] reported no change in immunoglobin levels when aflatoxin was fed. Delayed-type hypersensitivity reactions of chicks were depressed by aflatoxin [11], and lymphocytopenia was observed in chicks fed aflatoxin [12]. However, few direct measurements of the effects of mycotoxins on immune function have been carried out. The effects of mycotoxins are related to both dose and time of exposure. Almost all studies have examined the effects of doses of 1,000 ppb or higher when fed for periods of up to 3 weeks [1, 13]. Another possible scenario under practical feeding conditions is exposure to lower mycotoxin doses for a considerable length of time. The objectives of this study were to examine the effects of relatively low, chronic administration of selected mycotoxins on performance and health of chicks and, in addition, to examine their effect on antibody production to enteral and parenteral antigens. MATERIALS AND METHODS Male chicks (Ross) from a commercial hatchery were weighed at hatching, wing-banded, divided into groups of 10 chicks per floor pen, and fed mash diets meeting or exceeding NRC [14] requirements. Mycotoxins were added at various concentrations stated in Table 1 for 35 days. Chicks in two pens were fed each mycotoxin concentration. In succeeding tables, the mycotoxin concentrations are rounded. B 1, DAS, and T-2 toxin were from Sigma Chemical Company [15]. Chicks were vaccinated with Newcastle disease virus (NDV) and infectious bronchitis virus in the hatchery by innoculation with attenuated live virus and again with inactivated NDV vaccine in oil emulsion at 13 days and with a coarse spray of live attenuated NDV vaccine at 17 days. Chicks were orally immunized with bovine serum albumin solution (BSA fraction V) [15] by gently placing a blunt needle above the tongue and slowly dripping the solution into the pharynx, allowing the chick to voluntarily swallow the solution. Chicks were given three doses of 25 mg BSA daily from 14 to 16 days [16]. Blood samples were drawn from the jugular vein at 12, 25, and 32 days, and serum was separated and stored at 70 C until analysis. At 35 days, birds were killed, and post-mortem examinations were performed. Lesion scores were assigned as follows: the most severe lesion observed was assigned a score of four on a fourpoint scale where normal = 0. Samples of liver, small intestines, pancreas, kidney, spleen, and bursa were fixed in a 4% neutral buffered formalin solution and embedded in paraffin. All tissues were examined microscopically on hematoxylineosin-stained, 5-µ sections [17]. Antibodies to NDV and BSA were determined by ELISA [18, 19, 20]. In brief, dilutions (1:50 to 1:3,200) of sera were added to microtiter plates previously coated with NDV (crude vaccine extract) or BSA antigens in carbonate-bicarbonate buffer (ph 9.6) and blocked with skimmilk [21] at 0.5% dilution in PBS. After extensive washings to remove unbound antibodies, plates were blocked again, and bound antibodies were determined using horseradish peroxidase-labeled isotype-specific anti-chicken

3 SKLAN ET AL.: MYCOTOXINS IN CHICKS 81 IgG [22]. The assay was allowed to develop for 5.5 min in the presence of 3,3,5,5 -tetramethylbenzidine [23] (optimal absorbance) and then stopped by stop solution [22]. The assay was read by a Spectra 2 ELISA reader [24] at 450 nm. The results of individual chicks are the average of duplicate measurements and are expressed in absorbance 450 units. Group means ± SD at a serum dilution of 1:800 are presented; serum without antibody activity against the tested antigens was prepared from unimmunized chicks [19]. [25] and T-2 [26] were determined by HPLC, and DAS was determined by GC-MS [27]. All feeds were examined for these three mycotoxins and also screened for the presence of ochratoxin and deoxynivalenol using quantitative test kits [28]. STATISTICAL ANALYSIS Least squares means of results are presented; each bird served as a replicate after analysis of variance using the General Linear Models procedures of SAS. Differences between means were tested using t tests; significance was p < 0.05 unless otherwise stated [29]. RESULTS GROWTH AND FEED EFFICIENCY Growth and feed efficiency are shown in Table 2 for Experiments 1 through 4. Neither DAS nor T-2 toxin nor a mixture of 500 ppb DAS and 500 ppb T-2 influenced performance of the broilers when fed for 35 days (Experiments 1 and 2). Feeding aflatoxin significantly affected performance in both Experiments 3 and 4; growth was reduced after 4 weeks in chicks fed concentrations of 800 ppb aflatoxin or more. Feed efficiency was reduced in parallel at these concentrations in both Experiments 3 and 4. A mixture of DAS, T-2, and aflatoxin also reduced growth and feed efficiency. GROSS PATHOLOGY Oral lesions. Chicks were examined for oral lesions every 3 to 4 days. Of the chicks fed 500 ppb T-2, 90% had oral lesions after 10 to 15 days, and all chicks fed 1,000 ppb T-2 devloped oral lesions after 10 to 15 days; severity was greater with higher intake of T-2. In chicks fed 100 ppb T-2, mild lesions were found in 40% of the birds at 25 and 35 days (Experiment 1). Chicks fed 200 ppb or more DAS also developed oral lesions, which were found after 10 to 15 days and were more severe with increasing intake of DAS. Lesions were observed in 58% of chicks fed 200 ppb DAS and in 92% of chicks fed 500 ppb DAS on Day 20. The severity of oral lesions was maximal between 15 and 20 days and then decreased slightly with time (Experiment 2). Lesion scores at 35 days are shown in Table 3. In Experiment 3, mild oral lesions were observed in some chicks fed aflatoxin; however, as indicated, these lesions were less severe than those in chicks fed T-2 and DAS. INTERNAL LESIONS AT 35 DAYS In chicks fed 1,000 ppb T-2, some mild diffuse intestinal inflammation was observed, and some slight diarrhea was observed in some chicks fed 500 and 1,000 ppb DAS and in chicks fed the T-2/DAS mixture between 13 and 19 days (Table 3). In chicks fed T-2 and DAS, no other lesions were observed. In Experiments 3 and 4, in which chicks were fed aflatoxin B 1, several lesions were apparent at 35 days. In chicks fed 200 ppb aflatoxin or more, hepatic enlargement and discoloration were observed; livers appeared yellowish at the highest aflatoxin intakes and brown at 200 ppb. This effect was more pronounced with increasing aflatoxin concentration in the feed. Some lesser discoloration was noted in kidneys, which again did not respond in a completely linear manner with dose. Mild diffuse intestinal inflammation was observed, which was more severe at the higher aflatoxin intakes (Table 3). HISTOLOGY In Experiment 1, when T-2 was included in the diet, in some of the chicks fed 1,000 ppb T- 2, slight thickening of the wall of the proventriculus was noted, and occasional lymphoid aggregates were found within the tubular glands. No other abnormalities were observed apart from oral lesions in these chicks and in some chicks fed lower concentrations of T-2. In Experiment 2, when DAS was included in the diet, chicks fed 1,000 ppb DAS exhibited mild lymphoid depletion in the spleen and occasional small hepatic lymphocytic perivascular aggre-

4 82 JAPR: Research Report TABLE 2. Body weights and feed efficiency at 35 days in Experiments 1 through 4 A CONCENTRATION OF MYCOTOXIN IN FEED (ppb) EXPERIMENT ,000 1 T-2 BW, kg 1.56 ± ± ± ± 0.12 FE, kg/kg ,000 T-2 + DAS B 2 DAS BW, kg 1.55 ± ± ± ± ± 0.12 FE, kg/kg T-2 + DAS ,100 AFLATOXIN 3 BW, kg 1.72 ± 0.08 a 1.75 ± 0.11 a 1.70 ± 0.10 ab 1.62 ± 0.10 ab 1.55 ± 0.09 b 1.50 ± 0.10 b FE, kg/kg BW, kg 1.58 ± 0.07 a 1.53 ± 0.12 a 1.50 ± 0.11 a 1.26 ± 0.10 b FE, kg/kg A Results are means ± SD of 18 to 20 chicks. B DAS = diacetoxyscirpenol. a,b Values in rows not followed by the same superscript differ significantly. gates. Other abnormalities were not observed apart from the oral lesions in these chicks and in chicks fed lower concentrations of DAS. In Experiments 3 and 4, when aflatoxin was added to the rations, chicks fed 400 ppb aflatoxin or more exhibited occasional hepatic small periportal aggregations of lymphocytes and in the kidneys; some lymphocytic aggregates were observed in the interstitium. In the bursa, some scattered piknotic cells were observed with some mild depletion of lymphoid tissue. All other tissue samples appeared normal. ANTIBODY PRODUCTION Antibodies to NDV administered parenterally and antibodies to BSA administered per os were determined in all chicks at 12, 25, and 32 days. Antibodies to NDV were determined both by heamagglutinin inhibition and by ELISA; no differences in trends were apparent between methods. The heamagglutinin inhibition values ranged between five and seven in 25- and 32-day-old chicks; however, only results from the ELISA are presented. Antibody concentrations to both antigens were highest at 25 days. Antibody concentrations to NDV increased by 30 to 65% between Days 12 and 25 and then decreased by 5 to 10% between 25 and 32 days in all treatments; no difference in trends was observed. Antibodies to BSA decreased by 5 to 15% between 25 and 32 days. No differences in trends were found between treatments; thus, only results of the ELISA at 25 days are presented. Serum antibody concentrations to NDV and BSA from chicks fed increasing concentrations of T-2 toxin are shown in Table 4. No significant effects of this mycotoxin were observed on serum antibody concentrations. Similar results were observed with chicks fed increasing amounts of DAS or a mixture of DAS and T-2 toxin (Table 4), i.e., concentrations of both antibodies were not influenced by the intake of mycotoxins. Table 4 also presents results of Experiments 3 and 4, and it is clear that increasing amounts of aflatoxin

5 SKLAN ET AL.: MYCOTOXINS IN CHICKS 83 or a mixture of aflatoxin, T-2, and DAS did not influence serum antibody concentrations to antigens from either route of challenge. DISCUSSION The overall effects of the three mycotoxins examined in this study on performance were, in general, similar to previous reports in which mycotoxins were fed at higher concentrations for shorter times. Thus, the minimum growth inhibitory response to T-2 in a 3-week feeding period was found at 4 mg/kg [1, 13]. The mycotoxin DAS has been reported to depress growth when fed at 1 to 2 ppm after 3 weeks [1, 2]. In this study, when T-2 and DAS were fed at up to 1,000 ppb for 5 weeks, no decrease in either weight gain or feed efficiency was observed. Oral lesions were observed in chicks fed 200 ppb or higher after about 10 d; these lesions, however, did not TABLE 3. Lesion scores A in Experiments 1 through 4 at 35 days CONCENTRATION OF MYCOTOXIN IN FEED (ppb) EXPERIMENT ,000 1 T-2 Oral lesions 0 ± 0 a 0.3 ± 0.5 a 1.7 ± 0.7 b 1.6 ± 0.6 b Intestines B 0 ± 0 a 0.1 ± 0.3 ab 0.8 ± 0.6 ab 1.0 ± 0.7 b ,000 MIXTURE 2 DAS C Oral lesions 0 ± 0 a 0.5 ± 0.6 a 1.8 ± 0.7 b 2.4 ± 0.6 b 2.2 ± 0.7 b Intestines 0 ± 0 0 ± ± ± ± ,100 MIXTURE 3 Oral lesions 0.1 ± ± ± ± ± ± 0.6 Liver 0 ± 0 a 0.3 ± 0.4 a 0.4 ± 0.5 a 1.6 ± 0.5 b 2.2 ± 0.7 b 0.2 ± 0.6 a Kidney 0 ± 0 a 0.3 ± 0.5 ab 0.5 ± 0.6 ab 1.2 ± 0.6 b 2.8 ± 0.7 c 0.4 ± 0.6 a Intestines 0 ± 0 a 0.2 ± 0.4 ab 0.4 ± 0.5 ab 0.9 ± 0.6 b 1.0 ± 0.7 b 0.5 ± 0.4 ab influence body weight or feed efficiency, and no other histopathological effects were observed at the end of the 5-week feeding period. In most previous studies, it was found that the minimum growth inhibitory concentration of aflatoxin was 2,500 ppb when aflatoxin B 1 was fed to chicks for 3 weeks [13]. However, Doerr et al. [30] reported that 75 ppb (g/g aflatoxin depressed growth in one 7-week feeding experiment; however, in their second experiment, growth was depressed only at 2,700 ppb. In this study, diets containing concentrations of 800 ppb aflatoxin B 1 or more depressed both growth and feed efficiency after 4 weeks of exposure. Thus, these effects occurred at slightly lower concentrations than those generally reported previously [1, 13]. The mixtures of T-2 and DAS examined did not appear to have effects that were greater than either of the individual mycotoxins. 4 Liver 0 ± 0 a 1.1 ± 0.5 b 1.6 ± 0.6 bc 2.3 ± 0.7 c Kidney 0 ± 0 a 1.2 ± 0.5 b 1.1 ± 0.6 b 2.1 ± 0.6 b Intestines 0.1 ± ± ± ± 0.7 A Lesion scores were assigned on a four-point scale, where normal = 0. B Small and large intestines. C DAS = diacetoxyscirpenol. a,b,c Values in rows not followed by the same superscript differ significantly.

6 84 JAPR: Research Report TABLE 4. Effect of dietary concentrations of mycotoxins on antibody responses (absorbance 450 units) to parenteral NDV A and enteral BSA B at 25 days C CONCENTRATION OF MYCOTOXIN IN FEED (ppb) EXPERIMENT ,000 1 T-2 Ab-NDV 1.07 ± ± ± ± 0.05 Ab-BSA 0.93 ± ± ± ± ,000 MIXTURE 2 DAS Ab-NDV 0.61 ± ± ± ± ± 0.10 Ab-BSA 0.40 ± ± ± ± ± ,100 MIXTURE 3 Ab-NDV 1.07 ± ± ± ± ± ± 0.06 Ab-BSA 0.89 ± ± ± ± ± ± Ab-NDV 0.98 ± ± ± ± 0.10 Ab-BSA 0.47 ± ± ± ± 0.11 A NDV = Newcastle disease virus. B BSA = bovine serum albumin. C Results are means ± SD of 18 to 20 chicks. Naïve serum had absorbance 450 units ranging between 0.03 to 0.07 in these experiments. No significant differences were observed between means in rows. Several previous studies have indicated that immune responses are affected by mycotoxins. In this study, we examined systemic antibody production following enteral (BSA) and parenteral (NDV) immunizations as influenced by the three mycotoxins. The enteral immunization procedure used herein allows generation of immune responses against soluble protein antigens [16], thus allowing direct measurement of gut immune competence. To our knowledge, this is the first study to assess effects of mycotoxins on gutoriginating immune responses. The T-2, DAS, and aflatoxin B 1 mycotoxins did not change the response to NDV, which was administered by parenteral routes at the three sampling times. In parallel, the response to BSA, administered enterally, was also not influenced by the three mycotoxins tested. These results indicate that the mycotoxins, at the doses and conditions used in this study, did not impair enteral or parenteral immune competence. These results are different from those of Virdi et al. [7] who showed a low persistence of parenteral antibody response when aflatoxin was fed. However, in that study, the aflatoxin levels used were 10-fold higher than those used in the present report. Qureshi et al. [8] reported depressed antibody production in progeny of chicks exposed in embryo to aflatoxin. However, they suggest that these effects may be due to functional impairment of the immune system occurring during embryonic development. Thus, mycotoxin immunosuppression is probably dose-related and could also be influenced by the administration of mycotoxins at some specific developmental stage, which then causes irreversible damage. Alternatively, at the doses and exposure times used in this study, mycotoxins may not affect antibody production in chicks.

7 SKLAN ET AL.: MYCOTOXINS IN CHICKS 85 CONCLUSIONS AND APPLICATIONS 1. Chronic administration to chicks of 1,000 ppb T-2 or DAS or less for 5 weeks did not appear to affect growth or feed efficiency, despite the presence of oral lesions. 2. depressed body weights and feed efficiency when fed at 800 ppb or higher after 4 weeks. 3. The T-2, DAS, and aflatoxin mycotoxins at the levels fed did not appear to impair antibody production. 1. Leeson, S., G. Diaz, and J.D. Summers, Pages In: Poultry Metabolic Disorders and Mycotoxins. University Books, Guelph, Canada. 2. Ademoyero, A.A. and P.B. Hamilton, Mouth lesions in broiler chickens caused by scirpenol mycotoxins. Poult. Sci. 70: Wyatt, R.D., P.B. Hamilton, and H.R. Burmeister, The effects of T-2 toxin in broiler chickens. Poult. Sci. 52: Ueno, Y., Biochemical mode of action of mycotoxins. Pages In: Mycotoxins in Animal Feeds. J.E. Smith and R.S. Henderson, Eds. CRC Press, Boca Raton, FL. 5. Sharma, R.P., Immunotoxicity of mycotoxins. J. Dairy Sci. 76: Thaxton, J.P., H.T. Tung, and P.B. Hamilton, Immunosuppression in chickens by aflatoxin. Poult. Sci. 53: Virdi, J.S., R.P. Tiwari, M. Saxena, V. Khanna, G. Singh, S.S. Saini, and D.V. Vadehra, Effects of aflatoxin on the immune system of the chick. J. Appl. Toxicol. 9: Qureshi, M.A., J. Brake, P.B. Hamilton, W.M. Hagler, and S. Nesheim, Dietary exposure of broiler breeders to aflatoxin results in immune dysfunction in progeny chicks. Poult. Sci. 77: Weibking, T.S., D.R. Ledoux, A.J. Bermudez, and G.E. Rottinghaus, Individual combined effects of feeding Fusarium monoliforme cultrue material containing known levels of Fumonisin B 1 B 1 in the young turkey poult. Poult. Sci. 73: Pier, A.C., Influence of the mycotoxins on the immune system. Pages In: Mycotoxins in Animal Feeds. J.E. Smith and R.S. Henderson, Eds. CRC Press, Boca Raton, FL. 11. Kadian, S.K., D.R. Monga, and M.C. Goel, Effect of aflatoxin B 1 on the delayed type hypersensitivity phagocytic activity of reticuloendothelial system in chickens. Mycopathol. 104: Ghosh, R.C., H.V.S. Chauhan, and G.J. Jha, Supression of cell mediated immunity by purified aflatoxin B 1 in broiler chicks. Vet. Immunol. Immunopathol. 28: Wyatt, R. D., Poultry. Pages In: Mycotoxins in Animal Feeds. J.E. Smith and R.S. Henderson, Eds. CRC Press, Boca Raton, FL. REFERENCES AND NOTES 14. National Research Council, Nutrient Requirements for Poultry. 9th rev ed. National Academy Press, Washington, DC. 15. Sigma Chemical Company, St. Louis, MO. 16. Klipper, E., D. Sklan, and A. Friedman, Immune responses of chickens to dietary protein antigens: I. Induction of systemic intestinal immune responses following oral administration of soluble proteins in the absence of adjuvant. Vet. Immunol. Immunopathol. 74: Uni, Z., R. Platin, and D. Sklan, Cell proliferation in chicken intestinal epithelium occurs both in the crypt and along the villus. J. Comp. Physiol. B 168: Friedman, A. and D. Sklan, Antigen specific immune response impairment in the chick as influenced by vitamin A. J. Nutr. 119: Sklan, D., D. Melamed, and A. Friedman, The effect of varying levels of vitamin A on immune response in the chick. Poult. Sci. 73: Friedman, A., A. Meidovsky, G. Leitner, and D. Sklan, Decreased resistance and immune response to E. coli infection in chicks with low and high intakes of vitamin A. J. Nutr. 121: Difco Laboratories, Sparks, MD. 22. Bethyl, Montgomery, TX. 23. KPL, Gaithersberg, MD. 24. SLT, Zalsburg, Austria. 25. Official Methods of Analysis, Action th edition. Assoc. Official Analytical Chemists, Arlington, VA. 26. Schmidt, R. and K. Dose, HPLC: A tool for the analysis of T-2 toxin and HT-2 toxin in cereals. J. Analyt. Toxicol. 8: Rood, H.D., Jr., W.B. Buck, and S.P. Swanson, Gas chromatographic screening method for T-2 toxin, diacetoxyscirpenol, deoxynivalenol, and related trichothecenes in feeds. J. Assoc. Official Anal. Chem. 71: Neogen, Co., Lansing, MI. 29. SAS Institute SAS User s Guide. Version 6 Edition. SAS Institute Inc., Cary, NC. 30. Doerr, J.A., W.E. Huff, C.J. Wabek, G.W. Chaloupka, J.D. May, and J.W. Merkley, Effects of low level chronic aflatoxicosis in broiler chickens. Poult. Sci. 62: