ENVIRONMENT, HEALTH, AND BEHAVIOR

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1 ENVIRONMENT, HEALTH, AND BEHAVIOR Effects of Feeding Blends of Grains Naturally Contaminated with Fusarium Mycotoxins on Growth and Immunological Parameters of Broiler Chickens 1 H. V. L. N. Swamy,* T. K. Smith,*,2 N. A. Karrow,* and H. J. Boermans *Department of Animal and Poultry Science, Biomedical Sciences, University of Guelph, Guelph, ON, Canada N1G 2W1 ABSTRACT An experiment was conducted to investigate (d 21 to 42). Efficiency of feed utilization, however, was the effects of feeding grains naturally contaminated with Fusarium mycotoxins on growth and immunological not affected by diet. Production parameters were not significantly affected by the supplementation of GM polymer parameters of broiler chickens. Three hundred sixty, 1- to the contaminated grains. Peripheral blood d-old male broiler chicks were fed 1 of 4 diets containing monocytes decreased linearly in birds fed contaminated grains naturally contaminated with Fusarium mycotoxins grains. The feeding of contaminated diets linearly reduced the B-cell count at the end of the experiment, for 56 d. The diets included (1) control; (2) low level of contaminated grains (5.9 mg/kg deoxynivalenol (DON), whereas the T-cell count on d 28 responded quadratically to the contaminated diets. The feeding of contaminated 19.1 mg/kg fusaric acid (FA), 0.4 mg/kg zearalenone, diets did not significantly alter serum or bile immunoglobulin concentrations, contact hypersensitivity to dini- and 0.3 mg/kg 15-acetyldeoxynivalenol; (3) high level of contaminated grains (9.5 mg/kg DON, 21.4 mg/kg FA, trochlorobenzene, or antibody response to SRBC. 0.7 mg/kg zearalenone, and 0.5 mg/kg 15-acetyldeoxynivalenol); and (4) high level of contaminated grains + 0.2% diet nonspecifically increased white blood cell count and Supplementation with GM polymer in the contaminated polymeric glucomannan mycotoxin adsorbent (GM polymer). lymphocyte count, while preventing mycotoxin-induced Body weight gains and feed consumption of chick- ens fed contaminated grains decreased linearly with the inclusion of contaminated grains during the grower phase decreases in B-cell counts. It was concluded that broiler chickens are susceptible during extended feeding of grains naturally contaminated with Fusarium mycotoxins. (Key words: chicken, deoxynivalenol, fusaric acid, Fusarium mycotoxin, immunotoxicity) 2004 Poultry Science 83: INTRODUCTION Contamination of feed grains with Fusarium mycotoxins has resulted in economic losses worldwide in the animal industries. Losses have been estimated at more than $1 billion in Canada (Lombaert, 2002) and over $2.5 billion in the US (Windel, 2000) from 1990 to It has been reported that broiler chickens have a high tolerance to deoxynivalenol (DON, vomitoxin)-contaminated grains (Hulan and Proudfoot, 1982; Kubena et al., 1997). It has been observed, however, that broiler chicks fed diets containing 16 to 18 mg DON/kg from naturally contaminated wheat had reduced performance, reduced immune function, and changes in hematology and serum chemistry (Huff et al., 1986; Kubena et al., 1988, 1989a; Harvey et al., 1991). In support of the latter findings, 2004 Poultry Science Association, Inc. Received for publication January 31, Accepted for publication November 10, This study was supported in part by the Ontario Ministry of Agriculture and Food and by Alltech Inc., Nicholasville, KY. 2 To whom correspondence should be addressed: tsmith@ uoguelph.ca. feed intake and weight gains of broiler chickens has shown quadratic responses to the prolonged feeding of grains naturally contaminated with Fusarium mycotoxins during the finisher phase (Swamy et al., 2002a). This discrepancy among studies might be due to the co-occurrence of different mycotoxins and DON in contaminated grains. Trichothecene mycotoxins, being potent inhibitors of protein synthesis, can increase disease susceptibility of animals when ingested in sufficient quantity (Bondy and Pestka, 2000). Supplementation of diets with graded levels of purified fusaric acid (FA) has been shown to increase antibody-mediated immune response to SRBC but decreased in vivo cell-mediated immune response to phytohemagglutinin-a in broiler chickens (Chu et al., 1993). Limited information, however, is available on immune alterations in broiler chickens caused by chronic ingestion of mixture of Fusarium mycotoxins from contaminated grains. The identification of specific lympho- Abbreviation Key: CD = cluster designation-determinant; DON = deoxynivalenol; DNCB = 1-chloro-2,3-dinitrobenzene; FA = fusaric acid; GM = glucomannan polymer; HRP = horseradish peroxidase; PBMN cells = peripheral blood mononuclear cells. 533

2 534 SWAMY ET AL. cyte subsets affected by the feeding of Fusarium mycotoxin-contaminated grains might aid us in better understanding the immunotoxicity of Fusarium mycotoxins in poultry. The adverse effects in livestock and poultry associated with ingestion of mycotoxin-contaminated feed can be reduced through the use of physical, chemical, nutritional, or biological approaches. Among the various adsorbents that have been reported to prevent the deleterious effects of mycotoxins, glucomannan polymer (GM polymer) has been more promising given its ability to prevent mycotoxicoses arising from combinations of mycotoxins (Swamy et al. 2002a,b). The current study was conducted, therefore, to investigate the effects of feeding blends of grains naturally contaminated with Fusarium mycotoxins on growth and immune parameters of broiler chickens. The efficacy of GM polymer in preventing these adverse effects was also determined. MATERIALS AND METHODS Experimental Birds and Diets Three hundred sixty, 1-d-old male broiler chicks of a commercial strain 3 were individually weighed and distributed randomly into groups of 30 chicks per floor pen at the Arkell Poultry Research Station of the University of Guelph. Chicks were initially maintained at 31 C; the temperature was gradually lowered by 2 C/wk to reach 21 C by the end of wk 5, and this temperature was maintained for the duration of the experiment. Continuous lighting and water ad libitum were provided throughout the experiment. The birds were vaccinated on farm with one eye drop of Mildvac-Ma5 4 Mass. Type live virus on d 14-posthatching. Three pens (replicates) were assigned to each of the 4 treatments. The control diets (Table 1) were formulated to meet or exceed all nutritional specifications of starter, grower, and finisher broiler chickens (Leeson and Summers, 1997). The mycotoxin-contaminated diets were formulated to the nutrient specifications of the control diet by replacing control diet corn and wheat with the mycotoxin-contaminated corn and wheat. The combinations of control and contaminated corn and wheat are given in Table 2. To test the efficacy of GM polymer (Mycosorb 5 ) as a dietary preventative for Fusarium mycotoxicoses, a diet with a high level of contaminated grains was supplemented with 0.2% GM Dietary crude protein content was analyzed by the method of the Association of Official Analytical Chemists (1980). Representative feed samples were taken at the beginning of the starter, grower, and finisher periods and were 3 Ross Ross, Maple Leaf Poultry, New Hamburg, ON, Canada. 4 Intervet Inc., Millsboro, DE. 5 Alltech Inc., Nicholasville, KY. 6 North Dakota State University, Fargo, ND. 7 Sigma, Oakville, ON, Canada. 8 Southern Biotechnology Associates, Inc., Birmingham, AL. analyzed for nutrient content and Fusarium mycotoxin concentrations. The project was approved by the University of Guelph Animal Care Committee and met the guidelines of the Canadian Council on Animal Care. Analysis of Dietary Mycotoxins Dietary contents of DON, 3-acetyldeoxynivalenol, 15- acetyldeoxynivalenol, nivalenol, T-2 toxin, isot-2 toxin, acetylt-2 toxin, HT-2 toxin, T-2 triol, T-2 tetraol, fusarenon-x, diacetoxyscirpenol, scirpentriol, 15-acetoxyscirpentriol, neosolaniol, zearalenone, and zearalenol were analyzed by gas chromatography-mass spectrometry. 6 The detection limit for these mycotoxins was 0.2 µg/g. Dietary FA content was determined in our laboratory using the HPLC method (0.77 µg/g detection limit) of Matsui and Watanabe (1988) as modified by Smith and Sousadias (1993) and confirmed by Porter et al. (1995). Experimental Parameters Measured Body Weight and Feed Consumption. Chicks were weighed individually, and feed consumption for each pen was measured weekly during the 8-wk experimental period. Cumulative weight gain and feed consumption were calculated along with weekly and cumulative gain:feed. Feed consumption and gain:feed were adjusted for mortalities when appropriate. Organ Weights. On d 56, liver, kidney, bursa of Fabricius, and spleen were excised and weighed. Organ weights were expressed on a relative body weight basis. Differential Leukocyte Counts. At the end of 21, 28, 42, and 56 d, blood was collected into heparinized vials from the jugular vein of 4 chicks per pen. Complete white blood cell counts and differential leukocyte counts were performed manually to test for changes in absolute numbers of leukocytes, lymphocytes, segmented neutrophils, banded neutrophils, monocytes, eosinophils, and basophils. Phenotyping of Peripheral Blood Lymphocytes. Blood (5 ml) was mixed with 10 ml of Dulbecco s PBS and then underlayed with 5 ml of Ficoll Hypaque 7 for density gradient separation of peripheral blood mononuclear cells (PBMN cells). The blood samples were centrifuged at 400 g at room temperature for 30 min. The cell layer at the Ficoll Hypaque interface was transferred, washed 3 times, and suspended in 1 ml of Dulbecco s PBS. The total number of PBMN cells was calculated. Based on trypan blue dye exclusion, cell viability was consistently >95%. Fifty microliters of the suspended PBMN cells ( cells) were transferred to a 96-well round-bottom plate and resuspended with 50 µl of the appropriate antibody. 8 Cells were dual stained with mouse anti-chicken CD4 (cluster designation-determinant 4) monoclonal antibody [conjugated to R-phycoerythrin, 1:25)] and mouse anti-chicken CD3 monoclonal antibody [conjugated to fluorescein isothyocyanate, 1:25], and mouse antichicken CD8 monoclonal antibody (R-phycoerythrin

3 FUSARIUM MYCOTOXINS IN BROILER DIETS 535 TABLE 1. Composition of control diets (%) Ingredient Starter Grower Finisher Corn Wheat Soybean meal (48%) Vegetable oil Limestone Dicalcium phosphate Iodized salt Vitamin and mineral mixture DL-Methionine Coban Calculated content ME, kcal/kg 3,023 3,155 3,200 Crude protein Lysine Methionine Calcium Available phosphorus Analyzed content Crude protein Provided per kilogram of diet: vitamin A (retinyl plamitate), 8,800 IU; cholecalciferol, 3,300 IU; vitamin E (DL-α-tocopheryl acetate), 40 IU; vitamin K 3, 3.3 mg; thiamin, 4.0 mg; riboflavin, 8.0 mg; pantothenic acid, 15.0 mg; niacin, 50 mg; pyridoxine, 3.3 mg; choline, 600 mg; folic acid, 1.0 mg; biotin, 220 µg; vitamin B 12,12µg; ethoxyquin, 120 mg; manganese, 70 mg; zinc, 70 mg; iron, 60 mg; copper, 10 mg; iodine, 1.0 mg; selenium, 0.3 mg. 2 Coccidiostat. conjugated, 1:25) and mouse anti-chicken CD3 monoclonal antibody (conjugated to fluorescein isothyocyanate, 1:25) to label CD3 + CD4 + and CD3 + CD8 + T cells, respectively. B lymphocytes were labeled with R-phycoerythrin conjugated mouse anti-chicken IgM (heavy chain specific) monoclonal antibody (1:25). Appropriate isotype controls were included in the analysis and were subtracted from the sample values. The plate was shaken manually for 5 to 10 s and incubated at 4 C for 30 min in the dark for each step of antibody labeling. The plate was subsequently washed 3 times with Dulbecco s PBS containing 1% bovine serum albumin. Cells were suspended at the end in 500 µl of PBS with BSA and analyzed by flow cytometry using FACScan. 9 Analysis of Ig in Serum and Bile. Blood was collected from the jugular vein of 4 birds per pen on d 21, 42, and 56. After the blood collection on d 56, the 4 birds per pen were humanely euthanized, and bile was collected from the gall bladder. Serum and bile samples were frozen at 80 C until further analysis. Immunoglobulin M, IgG, and IgA were analyzed by Sandwich ELISA according to Swamy et al. (2002b). All antibodies and reference sera used in the assay were purchased from Bethyl Laboratories. 10 Affinity purified coating antibodies included goat anti-chicken IgM (µ-chain specific), goat anti-chicken IgG (Fc-fragment specific), and goat anti-chicken IgA (α-chain specific). Concentrations of IgM, IgG, and IgA in the reference sera were 0.6, 6.0, 9 Becton-Dickinson Immunocytometry Systems, San Jose, CA. 10 Montgomery, TX. 11 Fisher Scientific, Ottawa, Canada. and 0.4 mg/ml, respectively. Horseradish peroxidase (HRP)-conjugated antibodies included 1/60,000 goat anti-chicken IgM-HRP (µ-chain specific), 1/125,000 goat anti-chicken IgG-HRP (Fc-fragment specific), and 1/ 100,000 goat anti-chicken IgA-HRP (α-chain specific). The efficiency of Ig detection in serum and bile was quantified with 50 ng/ml of purified chicken IgG, IgM, or IgA. Detection efficiency was 97% for serum Ig and 65% for IgA in bile samples. Antibody Response to SRBC. On d 21 of the experiment following the collection of pre-immune sera, 12 birds per treatment were injected i.v. with 0.5 ml of 10% SRBC in PBS. Primary immune serum was collected subsequently on d 28 and 35. The SRBC were re-administered as described above on d 35, and subsequently secondary immune serum was collected on d 42, 49, and 56. Serum IgM and IgG antibody titers specific to SRBC were measured according to Temple et al. (1995) with some modifications. Detection antibodies used in the assay were HRP-conjugated goat anti-chicken IgM (µchain specific) and goat anti-chicken IgG (Fc-fragment specific) (Bethyl Laboratories 10 ). The antibody titer was defined as the highest dilution of the test serum whose absorbance was greater than the average absorbance plus 3 standard deviations of 8 serum control wells at an optical density of 405. Titers were expressed as reciprocal of base 2 log values. Contact Hypersensitivity Response to Dinitrochlorobenzene. This procedure was performed according to Prescott et al. (1982) with some modifications. On d 27, 29, and 31 of the experiment, 12 birds per treatment were sensitized by topical application of 50 µl of 90% dimethyl sulfoxide 11 in water followed 10 min later with

4 536 SWAMY ET AL. Mycotoxins 1 (µg/g) TABLE 2. Composition of experimental diets Grains (% of diet) GM 2 Contaminated Contaminated Diet DON 15-DON FA ZEA (%) Corn Wheat corn wheat Starter Control Low High High Grower Control Low High High Finisher Control Low High High Other mycotoxins (T-2 toxin, isot-2 toxin, acetylt-2 toxin, HT-2 toxin, T-2 triol, T-2 tetraol, fusarenon-x, 3- acetyldeoxynivalenol, nivalenol, diacetoxyscirpenol, scirpentriol, 15-acetyl scirpenol, neosolaniol, and zearalenol) measured in the complete feed were below the detection limit of 0.2 mg/kg. 2 GM = glucomannan polymer; DON = deoxynivalenol; 15-DON = 15-acetyl deoxynivalenol; FA = fusaric acid; ZEA = zearalenone. 3 Control = diet with control corn and wheat; Low = diet with low level of contaminated grains; High = diet with high level of contaminated grains; High+ = diet with high level of contaminated grains supplemented with GM an application of 50 µl of 5% 1-chloro-2, 3-dinitrobenzene 12 (DNCB) in ethanol. Three weeks later, the sensitized birds and one unsensitized bird per pen were challenged by application on the right wattle with 50 µl of 0.4% DNCB dissolved in 4:1 acetone:olive oil. Fifty microliters of acetone:olive oil was applied on the left wattle as a control. The thickness of the wattle was measured at 24, 48, and 72 h after the challenge application using a micrometer. 13 The percentage of increase in thickness was calculated using the formula, % = {[(test site thickness at 24, 48, or 72 h test site thickness at 0 h)/test site thickness at 0 h] [(control site thickness at 24, 48, or 72 h control site thickness at 0 h)/control site thickness at 0 h]} 100 (Furesz et al., 1997). Statistical Analyses Data were analyzed by analysis of covariance with initial BW as the covariate (for production parameters) or by ANOVA (for other parameters) using GLM procedure of SAS software (2000) as completely randomized design with subsamples. Repeated measures analysis was applied to study the effects of diets over time on differential leukocyte count, peripheral blood count of lymphocyte subsets, and serum Ig concentrations. Orthogonal polynomial contrasts were used to determine the nature of the response exhibited by different parameters to the dietary inclusion of contaminated grains. The efficacy of a supplemental GM polymer in preventing 12 Sigma, St. Louis, MO Mitutoyo, Mississauga, ON, Canada. Fusarium mycotoxicoses was determined by employing a simple contrast between highly contaminated diets with and without GM Statements of statistical significance were based on P RESULTS Dietary Mycotoxin Concentrations The diets high in contaminated grains with and without GM polymer were formulated to result in the same level of DON. The concentration of DON in the diet supplemented with GM polymer during the grower phase, however, was 18% higher than in the diet containing just the high level of contaminated grains. Dietary FA, 15-acetyl DON, and zearalenone concentrations ranged between 11.9 to 32.6, 0 to 0.6, and 0 to 0.7 mg/kg, respectively (Table 2). Production Parameters Feed consumption and weight gains were not affected (P > 0.05) by the dietary inclusion of mycotoxin-contaminated grains during the starter period. During the grower period, however, feed consumption and weight gains were linearly reduced as the dietary inclusion of contaminated grains increased (P 0.05) (Table 3). Feed intake and weight gains recovered during the finisher period (P > 0.05). Feed efficiency was not affected by diet (P > 0.05) (data not shown). The supplementation of GM polymer to the diets high in contaminated grains did not have any effect on feed consumption, weight gains, or feed efficiency (P > 0.05). Approximately 10%

5 FUSARIUM MYCOTOXINS IN BROILER DIETS 537 TABLE 3. Effect of dietary Fusarium mycotoxins on feed consumption and body weight gain of broiler chickens Feed consumption (g/bird) 2 Body weight gain (g/bird) 3 Diet d d d 0 56 d 0 21 d d d 0 56 d Control 908 2,797 2,544 6, ,678 1,303 3,316 Low 841 2,565 2,437 5, ,522 1,274 3,172 High 923 2,392 2,456 5, ,479 1,319 3,184 High ,472 2,532 5, ,538 1,348 3,278 SEM Linear 4 NS NS NS NS 0.04 NS NS Quadratic 5 NS NS NS NS NS NS NS NS High+ vs. high 6 NS NS NS NS NS NS NS NS 1 Control = diet with control corn and wheat; Low = diet with low level of contaminated grains; High = diet with high level of contaminated grains; High+ = diet with high level of contaminated grains supplemented with 0.2% glucomannan 2 Values are least-square means; n = 3. 3 Values are least-square means; n = 90. 4,5 Linear and quadratic responses to the dietary inclusion of contaminated grains, respectively. 6 Simple contrast comparing diets containing high level of contaminated grains with and without glucomannan 7 P > of the birds died during the finisher phase of the experiment due to heat stress. Organ Weights The weights of liver, kidney, spleen, and bursa of Fabricius, expressed as a percentage of BW, were not altered by the dietary inclusion of contaminated grains (P > 0.05; data not shown). The supplementation of GM polymer to the contaminated diet also had no effect on organ weights (P > 0.05). Differential Leukocyte Count Repeated measures analysis indicated no significant interaction between diet and time effects on differential leukocyte count. The data presented, therefore, are as the means of 4 periods (wk 3, 4, 6, and 8; Table 4). Peripheral blood monocyte numbers were linearly decreased in birds fed contaminated grains (P 0.05). Supplementation of GM polymer to the diet containing high level of contaminated grains increased total white blood cell count and the lymphocyte counts in differential leukocyte count as compared with the unsupplemented highly contaminated diet (P 0.05). Other leukocyte subpopulations were not affected by the dietary treatments (P > 0.05). Phenotype of Lymphocytes Repeated measures analysis indicated interactions between diet and time effects on peripheral blood B- and T-cell percentages (P 0.05) but not on CD4 + and CD8 + T-cell percentages (P > 0.05; Table 5). B- and T-cell counts shown, therefore, were for each period, whereas CD4 + and CD8 + T-cell percentages were the means of 3 periods TABLE 4. Effect of dietary Fusarium mycotoxins on peripheral blood differential leukocyte count (10 9 /L) of broiler chickens 1 Diet 2 WBC 3 Heterophils Lymphocytes Monocytes Eosinophils Basophils Control Low High High SEM Linear 4 NS 7 NS NS 0.01 NS NS Quadratic 5 NS NS NS NS NS NS High+ vs. high NS 0.02 NS NS NS 1 Values are least-square means (n =12). No interaction was observed between diet and time effects. The data, therefore, was pooled over week 3, 4, 6 & 8. 2 Control = diet with control corn and wheat; Low = diet with low level of contaminated grains; High = diet with high level of contaminated grains; High+ = diet with high level of contaminated grains supplemented with glucomannan 3 White blood cells. 4,5 Linear and quadratic responses to the dietary inclusion of contaminated grains, respectively. 6 Simple contrast comparing diets containing high level of contaminated grains with and without glucomannan 7 P > 0.05.

6 538 SWAMY ET AL. TABLE 5. Effect of dietary Fusarium mycotoxins on percentage of lymphocyte subsets in the peripheral blood of broiler chickens 1 B cells 2 T cells 2 CD4 + CD8 + Diet wk 4 wk 6 wk 8 T cells 3,4 T cells 3,5 wk 4 wk 6 wk 8 Control Low High c High SEM Linear 10 NS 13 NS 0.04 NS NS NS NS NS Quadratic 11 NS NS NS NS NS 0.04 NS NS High+ vs. high 12 NS NS 0.04 NS NS NS NS NS 1 Values are least-square means; n =12. 2 There was an interaction between diet and time effects on B- and T-cell counts. The data, therefore, were analyzed separately for wk 4, 6, and 8. 3 No interaction was observed between diet and time effects. The data, therefore, were pooled over wk 4, 6, and 8. 4,5 Cluster of differentiation 4 and 8, respectively, are subsets of T cells. 6 Diet with control corn and wheat. 7 Diet with low level of contaminated grains. 8 Diet with high level of contaminated grains. 9 Diet with high level of contaminated grains supplemented with glucomannan 10,11 Linear and quadratic responses, respectively, to the dietary inclusion of contaminated grains. 12 Simple contrast comparing diets containing high level of contaminated grains with and without glucomannan 13 P > (wk 4, 6, and 8). The feeding of contaminated diets linearly reduced B-cell percentages at the end of the experiment, whereas T-cell percentages at the end of wk 4 responded quadratically to the contaminated diets (P 0.05). Supplementation of GM polymer to the contaminated diet prevented the decrease in B cell count caused by the feeding of a high level of contaminated grains (P 0.05). Serum and Bile Immunoglobulin Concentrations The feeding of contaminated diets to chickens did not cause significant changes in serum Ig concentrations (Table 6). Glucomannan polymer supplementation to the diet containing 24.5% contaminated grains also did not result in any significant changes in serum and bile Ig concentrations. Contact Hypersensitivity to DNCB Negative control chickens, those not sensitized with DNCB, exhibited a 10% increase in the skin thickness 24 h after challenge, but no change in thickness was observed 48 and 72 h after challenge. The chickens fed the control diet and sensitized with DNCB had 97, 31, and 15% increases in skin thickness 24, 48, and 72 h after challenge, respectively (Table 7). No significant diet effect, however, was observed on skin thickness. Antibody Response to SRBC Low levels of cross-reacting IgM and IgG antibodies to SRBC antigen were detectable in pre-immune sera (Tables 8 and 9). Postimmune sera titers were, therefore, statistically analyzed using pre-immune sera titer as covariate. Primary and secondary antibody response to SRBC followed the classical pattern of humoral immune response to foreign antigens. IgM antibody titers to SRBC reached the peak 7 d after the first injection of antigen and then were gradually decreased over time (Table 8). IgG antibody titers to SRBC reached the peak 7 d after the second injection and were maintained until the end of the experiment (Table 9). There was no effect of diet on the specific IgM and IgG antibody titers to SRBC (Tables 8 and 9). DISCUSSION Dietary Mycotoxin Concentrations The diets used in the current experiment were formulated to achieve similar concentrations of mycotoxins as those fed in the previous broiler chicken trial (Swamy et al., 2002a) by incorporating the same level and source of naturally contaminated corn and wheat. The mean concentrations of FA, 15-acetyldeoxynivalenol, and zearalenone in starter, grower, and finisher diets were approximately the same in both the experiments. Average DON concentrations (ppm) over 3 periods were, however, higher in the current experiment than the previous (0.56 vs. 0.13, control diet; 5.93 vs. 4.7, low diet; 9.5 vs. 8.16, high diet; and 10.1 vs. 9.7, high + GM polymer diet). Failure to achieve same concentrations of DON in the 2 experiments may be attributed to lack of uniformity in occurrence of mycotoxins in contaminated corn and wheat (Hamilton, 1978) and sampling limitations (Davis et al., 1980). The current experiment was conducted 2 yr

7 FUSARIUM MYCOTOXINS IN BROILER DIETS 539 TABLE 6. Effect of dietary Fusarium mycotoxins on serum and biliary immunoglobulin concentrations (mg/ml) of broiler chickens 1,2 Diets Serum IgM Serum IgG Serum IgA Biliary IgA Control Low High High SEM Linear 7 NS 10 NS NS NS Quadratic 8 NS NS NS NS High+ vs. high 9 NS NS NS NS 1 Immunoglobulin concentrations in serum and bile were measured using sandwich ELISA. 2 Values are least-square means (n =12). No interaction was observed between diet and time effects. The data, therefore, were pooled over wk 3, 6, and 8. 3 Diet with control corn and wheat. 4 Diet with low level of contaminated grains. 5 Diet with high level of contaminated grains. 6 Diet with high level of contaminated grains supplemented with 0.2% glucomannan 7,8 Linear and quadratic responses to the dietary inclusion of contaminated grains, respectively. 9 Simple contrast comparing diets containing high level of contaminated grains with and without glucomannan 10 P > after the first trial, and fungal growth and mycotoxin production might have continued during the storage of contaminated grains. According to literature reports, broiler chickens should not be adversely affected by exposure to the concentrations of zearalenone detected in the present study (Bacon and Marks, 1976). Feeding of 2 ppm 15-acetyldeoxynivalenol along with 6 ppm DON did not enhance the toxicity of DON to pigs (Rotter et al., 1992). The adverse effects associated with the contaminated diet TABLE 7. Effect of dietary Fusarium mycotoxins on DNCB 1 - induced percent increase in thickness of wattle in broiler chickens 2 24 h 48 h 72 h Diets thickness 3 thickness 3 thickness 3 Control Low High High SEM Linear 8 NS 11 NS NS Quadratic 9 NS NS NS High+ vs. high 10 NS NS NS 1 2,4-Dinitrochlorobenzene is a chemical that induces contact hypersensitivity upon topical application. 2 Values are least-square means after subtracting thickness of unsensitized chickens; n = At 24, 48, and 72 h after the challenge with DNCB, percentage of increase in thickness of skin fold was calculated using the formula, % = {[(test site thickness at 24, 48, or 72 h test site thickness at 0 h)/test site thickness at 0 h] [(control site thickness at 24, 48, or 72 h control site thickness at 0 h)/control site thickness at 0 h]} Diet with control corn and wheat. 5 Diet with low level of contaminated grains. 6 Diet with high level of contaminated grains. 7 Diet with high level of contaminated grains supplemented with glucomannan 8,9 Linear and quadratic responses to the dietary inclusion of contaminated grains, respectively. 10 Simple contrast comparing diets containing high level of contaminated grains with and without glucomannan 11 P > in the current experiment, therefore, were probably due to the combined effects of DON, FA, and any other unidentified Fusarium mycotoxins. Production Parameters Most of the previous experiments related to feeding of DON-contaminated grains to broiler chickens were confined only to the starter period (0 to 21 d), and the current findings during starter period are in agreement with those researchers (Hulan and Proudfoot, 1982; Kubena et al., 1997). Unlike the previous studies, the current and Swamy et al., (2002a) studies were extended over 56 d, which provided a means of studying the chronic toxicity of Fusarium-mycotoxin contaminated grains. In contrast to results in Swamy et al. (2002a), feed intake and weight gains of broiler chickens fed contaminated grains during the grower period were significantly reduced in the current study. In both experiments, strain of birds, management conditions, and nutrient composition of the diets were the same. Broiler chickens in the current study grew, however, faster than those in Swamy et al. (2002a) during the starter period, and this faster growth rate might also have contributed to the growth depression during the grower period (Huff et al., 1986). Growth inhibitory effects of 16 to 18 ppm DON from contaminated wheat in broiler chickens were earlier reported (Huff et al., 1986; Kubena et al., 1988, 1989a). These earlier studies have used >50% wheat in the diet as the source of DON and, therefore, there can be other factors in the wheat that have contributed to the observed toxic effects (Morris et al., 1999). Failure to observe the significant differences in growth parameters during the finisher period of the current experiment might be suggestive of adaptation of birds to mycotoxins over time. Pigs have shown to adapt to Fusarium mycotoxicoses by improving efficiency of nutrient utilization (Rotter et al., 1994). No improvement in nutri-

8 540 SWAMY ET AL. TABLE 8. Effect of dietary Fusarium mycotoxins on anti-srbc IgM titers (reciprocal of log 2 ) of broiler chickens 1,2,3,4 Primary antibody titers Secondary antibody titers Pre-immune Diet titers d 7 d 14 d 21 d 28 d 35 Control Low High High SEM Linear 9 NS 12 NS NS NS NS NS Quadratic 10 NS NS NS NS NS NS High+ vs. high 11 NS NS NS NS NS NS 1 Values are least-square means; n = Chickens were injected with 0.5 ml of 10% SRBC on d 21 of the experiment after collecting preimmune serum (pre-immune titers). Serum was also collected on d 7 and 14 for testing primary antibody titers. On d 14, a booster dose of SRBC was given, and subsequently serum was tested for secondary antibody titers on d 21, 28, and 35 postimmunization. 3 The antibody titer was defined as the highest dilution of the test serum whose absorbance was greater than the average absorbance plus three standard deviations of 8 negative sera control wells at an optical density of Titers on d 7, 14, 21, 28, and 35 were statistically analyzed using pre-immune titers as the covariate. 5 Diet with control corn and wheat. 6 Diet with low level of contaminated grains. 7 Diet with high level of contaminated grains. 8 Diet with high level of contaminated grains supplemented with 0.2% glucomannan 9,10 Linear and quadratic responses to the dietary inclusion of contaminated grains, respectively. 11 Simple contrast comparing diets containing high level of contaminated grains with and without glucomannan 12 P > ent utilization, however, was observed in the current study. Unlike the study by Swamy et al. (2002a), there was no growth stimulatory effect of mycotoxins during the finisher phase of the current study. The reason for this discrepancy, however, is not known. These 2 studies clearly indicated that broiler chickens could be susceptible to extended feeding of contaminated grains containing mixture of mycotoxins. The term DON-contaminated grains has often been used in the literature, but in reality the contaminated grains contain a mixture of Fusarium mycotoxins. This mixture of mycotoxins can vary from region to region, even within a province, and necessitates the thorough analysis of contaminated grains for all the possible mycotoxins. Deoxynivalenol toxicity in animals can vary based on the presence of other mycotoxins in the contaminated grains. Fusaric acid (Smith et al., 1997) and T-2 toxin (Kubena et al., 1989a) have been shown to enhance DON toxicity. The susceptibility of broiler chickens to DON, therefore, might vary depending on which mycotoxins are present in contaminated grains, in addition to DON. Immunotoxicity of Fusarium Mycotoxins In the current study, evaluation of organ weights, serum Ig concentrations, differential leukocyte counts, and enumeration of peripheral blood lymphocyte subpopulations were used as tier I parameters, whereas contact hypersensitivity to DNCB and antibody response to SRBC were tier II parameters (Miller and Meredith, 1999). Organ Weights The effects of feeding Fusarium mycotoxin-contaminated grains on organ weights of broiler chickens are very contradictory. Kubena et al. (1985) reported decreased absolute relative weights of liver in growing chicks fed DON-contaminated grains. No changes in organ weights (liver, kidney, spleen, and bursa of Fabricius) were observed in the study of Kubena et al. (1988). In yet another study, Kubena et al. (1989a) reported increased weight of bursa of Fabricius. All these studies used 16 mg/kg DON from contaminated wheat fed to broiler chickens for 21 d posthatch. The highly variable outcome of these studies suggested that organ weights might not be a good indicator of toxicity of some of the Fusarium mycotoxins. Conversely, time of toxin exposure may be a significant factor as the organ initially swells with toxin exposure followed by shrinkage. Serum and Bile Ig Concentrations The effect of feeding mycotoxin-contaminated grains on serum Ig concentrations in broiler chickens in the current study was in accordance with Swamy et al. (2002a). The effect on bile IgA concentrations, however, was contradictory to Swamy et al. (2002a). This discrepancy might be due to the different immunological assays used in these 2 studies. Ouchterlony double diffusion test, the assay used in Swamy et al. (2002a) is mainly a qualitative tool for determining the presence or absence of antigen

9 FUSARIUM MYCOTOXINS IN BROILER DIETS 541 TABLE 9. Effect of dietary Fusarium mycotoxins on anti-srbc IgG titers (reciprocal of log 2 ) of broiler chickens 1,2,3,4 Primary antibody titers Secondary antibody titers Preimmune Diet titers d 7 d 14 d 21 d 28 d 35 Control Low High High SEM Linear 9 NS 12 NS NS NS NS NS Quadratic 10 NS NS NS NS NS NS High+ vs. high 11 NS NS NS NS NS NS 1 Values are least-square means; n = Chickens were injected with 0.5 ml of 10% SRBC on d 21 of the experiment after collecting pre-immune serum (pre-immune titers). Serum was also collected on d 7 and 14 for testing primary antibody titers. On d 14, a booster dose of SRBC was given, and subsequently serum was tested for secondary antibody titers on d 21, 28, and 35 postimmunization. 3 The antibody titer was defined as the highest dilution of the test serum whose absorbance was greater than the average absorbance plus three standard deviations of 8 negative sera control wells at an optical density of Titers on d 7, 14, 21, 28, and 35 were statistically analyzed using pre-immune titers as a covariate. 5 Diet with control corn and wheat. 6 Diet with low level of contaminated grains. 7 Diet with high level of contaminated grains. 8 Diet with high level of contaminated grains supplemented with 0.2% glucomannan 9,10 Linear and quadratic responses to the dietary inclusion of contaminated grains, respectively. 11 Simple contrast comparing diets containing high level of contaminated grains with and without glucomannan 12 P > or antibody. Few studies, however, have used this assay to quantify bile IgA concentrations by measuring the length of the precipitin formed at the point of antigenantibody equivalence. This assay assumes that the length of the precipitin is directly proportional to the amount of IgA in the bile. The ELISA sandwich assay used in the current study is more sensitive, and the resultant bile IgA concentrations were based on the standard curve derived using known amounts of affinity-purified chicken IgA (Bianchi et al., 1995). Differential Leukocyte Count and Phenotype of Lymphocytes Monocytes originate in the bone marrow and are released into the blood, where they circulate briefly before migrating into the tissues to become the tissue macrophages. Macrophages play an integral role in inflammation and wound healing, cellular and humoral immunity, regulation of immune response, and various normal metabolic and homeostatic functions (Lewis, 1995). The decreased number of peripheral blood monocytes in birds fed contaminated diets in the current study might indicate their reduced synthesis in bone marrow or increased removal from the blood. Altered macrophage function following exposure to contaminated grains remains to be tested. Antigen-reactive circulating B lymphocytes form the humoral branch of the immune system through the synthesis of antigen-specific antibodies. Mycotoxin-induced reduction in peripheral blood B-cell numbers can be considered as a significant finding, only if it correlates with functional assays such as antibody response (Holsapple, 1995). Transient quadratic response of T cell percentages to the feeding of contaminated grains illustrated the possibility that Fusarium mycotoxins, especially trichothecene mycotoxins, can be immunostimulatory or immunosuppressive based on the dose of toxin (Bondy and Pestka, 2000). Pestka et al. (1990) have reported a drastic increase in the number of T cells in both Payer s patches and spleen of mice fed diet with 25 ppm of DON for 12 weeks. Contact Hypersensitivity to DNCB To the best of our knowledge, no other studies to date have reported the effect of feeding Fusarium mycotoxincontaminated grains on contact hypersensitivity of broiler chickens. Blastogenic responses are nonspecific in nature, whereas contact hypersensitivity is an antigen-specific response of T cells. In spite of this difference, lymphocyte blastogenic responses to mitogens have been routinely used in immunotoxicity testing because they provide important information about the capability of T and B lymphocytes to proliferate, which is an essential event for most immune responses (Smialowicz, 1995). Harvey et al. (1991) used in vitro lymphocyte blastogenic responses to phytohemagglutinin (PHA) as a measure of cell-mediated immune response and reported a significant reduction in the stimulation index of splenocytes in 3-wk-old broiler chickens fed 50 ppm of purified DON. Chu et al. (1993) reported that FA reduced in vivo cell-mediated cutaneous response to phytohemaggluti-

10 542 SWAMY ET AL. nin-a in broiler chickens. These 2 earlier studies indicated that unlike in pigs, both DON and FA could reduce lymphocyte proliferation in broiler chickens. This reduced proliferation capability, however, may not be apparent in the contact hypersensitivity response. Reasons might include that (1) Fusarium mycotoxins affect both downregulatory and up-regulatory lymphocytes of contact hypersensitivity and (2) DON and FA levels were considerably lower in the current study than in the aforementioned studies. Antibody Response to SRBC To the best of our knowledge, the current study is the first to adapt this assay, with some modifications for evaluating SRBC-specific serum IgM and IgG titers in broiler chickens. Pre-immune sera in the current study showed detectable amounts of natural antibodies, of both IgM and IgG isotypes, to SRBC. The presence of these antibodies indicated that the highly conserved elements on the SRBC membrane might share common epitopes with self-constituents (Ochsenbein and Zinkernagel, 2000). Reduced antibody titers to SRBC have been observed in mice administered 0.75 or 2.5 mg of DON/kg body weight per day for 5 consecutive wk beginning at 21 d of age (Tryphonas et al., 1984). Harvey et al. (1991) fed 18 mg of DON/kg from contaminated grains to White Leghorn chicks for 18 wk and observed significantly reduced primary Newcastle disease antibody titers at the end of the experiment. In contrast, Chu et al. (1993) reported elevated serum antibody titers against SRBC in broiler chickens fed 35 mg of purified FA/kg. It can be speculated, therefore, that the failure to observe an effect of Fusarium mycotoxins on the primary antibody response to SRBC in the current study might be the outcome of an antagonistic interaction between DON and FA. Effect of GM Polymer Supplementation Nonspecific increases in WBC and lymphocyte counts in birds supplemented with GM polymer might be suggestive of its immunostimulation properties. Glucomannan polymer is composed of outer mannose-proteins and inner glucans. The ability of outer mannan oligosaccharides to prevent colonization of pathogens (Spring et al., 2000) and enhance immune function of animals (Savage et al., 1996) is well documented. Currently it is unclear how exactly the oligosaccharides act to modify the immune system. Although the adsorbent was not effective in preventing mycotoxin-induced growth depression, it was able to prevent the mycotoxin-induced decreases in the peripheral blood B-cell counts. Some beneficial effects of GM polymer in preventing mycotoxin-induced alterations were previously reported (Swamy et al., 2002a,b). 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