Effects of feed-borne Fusarium mycotoxins on hematology and immunology of turkeys

IMMUNOLOGY, HEALTH, AND DISEASE Effects of Feed-Borne Fusarium Mycotoxins on Hematology and Immunology of Turkeys S. R. Chowdhury,* T. K. Smith,*,1 H. J. Boermans,† and B. Woodward‡ *Department of Animal and Poultry Science, †Department of Biomedical Sciences, and Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada N1G 2W1 with controls. Supplementation of the contaminated diet with GMA prevented these effects on blood cell counts. Biliary IgA concentrations were significantly increased (4.45-fold) when birds were fed contaminated grains compared with controls, but serum IgA concentrations were not affected. Contact hypersensitivity to dinitrochlorobenzene, which is a CD8+ T-cell-mediated delayedtype hypersensitivity response, was decreased (48%) by feed-borne mycotoxins compared with the control. By contrast, the primary and secondary antibody response to sheep red blood cells, a CD4+ T-cell-mediated response, was not affected. It was concluded that chronic consumption of grains naturally contaminated with Fusarium mycotoxins exerts only minor adverse effects on the hematology and some immunological indices of turkeys. Consumption of grains naturally contaminated with Fusarium mycotoxins may, however, increase the susceptibility of turkeys to infectious agents against which CD8+ T cells play a major role in defense.

(Key words: Fusarium mycotoxin, hematology, antibody-mediated immunity, cell-mediated immunity, biliary IgA) 2005 Poultry Science 84:1698–1706

Mycotoxins are fungal secondary metabolites toxic to vertebrates. The co-occurrence of the Fusarium mycotoxins, deoxynivalenol (DON), and zearalenone (ZEN), is common in grains of colder and temperate regions such as Canada, the northern United States, and most of Europe (Scott, 1989). Economic losses due to contamination of feedstuffs have been estimated at more than $1 billion in Canada during the 1990s (Lombaert, 2002) and over $2.5 billion in the United States (Windel, 2000). The economic impact of mycotoxins includes mortality of domestic animals, veterinary care, and reduced production efficiency (Hussein and Brasel, 2001). Immunotoxicity of Fusarium mycotoxins has been demonstrated in numerous species including rodents, poultry,

pigs, and ruminants by using purified toxins or a combination of toxins from naturally contaminated grains. The feeding with diets containing 50 mg of purified DON/ kg depresses mitogen induced lymphocyte proliferation and the antibody response to Newcastle disease vaccine in 3-wk-old broiler chickens (Harvey et al., 1991). Danicke et al. (2002) found a decreased antibody titer against the Newcastle disease virus in laying hens consuming a diet containing 17.6 mg of DON/kg and 1.6 mg of ZEN/kg. The consumption of grains naturally contaminated with Fusarium mycotoxins, however, did not affect the immune competence of starter pigs after 3 wk (Swamy et al., 2003) and did not affect immune competence of broiler chickens after 8 wk (Swamy et al., 2004). Most studies indicate that Fusarium mycotoxins are immunotoxic, and, even though production parameters may not be altered adversely at

2005 Poultry Science Association, Inc. Received for publication March 4, 2005. Accepted for publication July 11, 2005. 1 To whom correspondence should be uoguelph.ca.

Abbreviation Key: CHS = contact hypersensitivity; DNCB = dinitrochlorobenzene; DON = deoxynivalenol; GMA = glucomannan mycotoxin adsorbent; Hb = hemoglobin; HRP = horseradish peroxidase; ZEN = zearalenone.

INTRODUCTION

addressed:

tsmith@

1698

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ABSTRACT Feeding grains naturally-contaminated with Fusarium mycotoxins has been shown to alter the metabolism and performance of turkeys. The objectives of the current experiment were to examine the effects of feeding turkeys with grains naturally contaminated with Fusarium mycotoxins on their hematology and immunological indices (including functions), and the possible protective effect of feeding a polymeric glucomannan mycotoxin adsorbent (GMA). Two hundred twenty-five 1-dold male turkey poults were fed corn, wheat, and soybean meal-based starter (0 to 3 wk), grower (4 to 6 wk), developer (7 to 9 wk), and finisher (10 to 12 wk) diets formulated with uncontaminated grains, contaminated grains, or contaminated grains with 0.2% GMA. The chronic consumption of Fusarium mycotoxins caused minor and transient changes in hematocrit (0.33 L/L) and hemoglobin (106 g/L) concentrations as well as in blood basophil (0.13 × 109/L) and monocyte counts (3.42 × 109/L) compared

FUSARIUM MYCOTOXINS AND HEMATOLOGY AND IMMUNOLOGY OF TURKEYS

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Table 1. Composition of experimental diets (%) Ingredient

Grower (4-6 wk)

Developer (7-9 wk)

Finisher (10-12 wk)

36.50 10.00 37.00 5.00 5.00 2.10 1.00 2.00 0.20 1.00 0.20 ±0.20

41.90 10.00 32.20 5.00 5.00 2.50 0.80 1.20 0.20 1.00 0.20 ±0.20

49.00 10.00 25.00 5.00 5.00 3.00 0.60 1.00 0.20 1.00 0.20 ±0.20

54.50 10.00 17.00 5.00 5.00 5.00 0.60 1.00 0.20 1.00 0.20 ±0.20

2,828 27.90 1.63 0.68 1.58 0.9

2,941 26.00 1.50 0.65 1.34 0.73

3,043 23.10 1.31 0.62 1.21 0.68

3,204 20.00 1.11 0.58 1.20 0.67

90.20 27.32 .38 0.85

90.36 26.49 1.26 0.79

89.72 22.60 1.11 0.77

90.15 20.72 1.07 0.68

1

Either uncontaminated or contaminated. Vitamin-mineral mixture provided per kilogram of diet: vitamin A (all-trans-retinyl palmitate), 8,800 IU; cholecalciferol, 3,300 IU; vitamin E (all-rac-α-tocopheryl acetate), 40 IU; menadione, 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 ␮g; vitamin B12, 12 ␮g; ethoxyquin, 120 mg; manganese, 70 mg; zinc, 70 mg; iron, 60 mg; copper, 10 mg; iodine, 1.0 mg; selenium, 0.3 mg. 3 Polymeric glucomannan mycotoxin adsorbent. 2

low dietary concentrations, alteration in immune function might nevertheless occur (Smith et al., 1990). There are also inconsistent results pertaining to hematological indices of animals fed purified DON or naturally contaminated grain containing DON (Harvey et al., 1991). Moreover, the immunological and hematological effects of long-term feeding of Fusarium mycotoxins have not been well characterized (Sharma, 1993). Information is also lacking regarding the effect of chronic feeding of grains naturally contaminated with Fusarium mycotoxins on hematological and immunological indices and functions in turkeys, which was, therefore, the focus of the current study. A polymeric glucomannan mycotoxin adsorbent (GMA) has been reported to prevent the deleterious effects of mycotoxins on performance and metabolism of broilers (Swamy et al., 2002) and laying hens (Chowdhury and Smith, 2004), which may be attributed to a reduction in intestinal absorption of mycotoxins (Ramos et al., 1996). An additional objective, therefore, was to determine whether any possible adverse effect of mycotoxin-contaminated grains could be prevented by dietary supplementation with GMA.

MATERIALS AND METHODS Birds and Experimental Design Two hundred twenty-five 1-d-old Hybrid male turkey poults2 were individually weighed, wing-banded, and 2

Hybrid Turkeys, Kitchener, ON, Canada.

distributed randomly into groups of 15 poults per floor pen at the Arkell Poultry Research Station of the University of Guelph. Five pens were randomly assigned to each of the 3 diets with each diet fed to 75 birds. Poults were initially maintained at 32°C, and the temperature was gradually lowered by 3°C per week to reach 21°C by the end of wk 4. This temperature was maintained for the duration of the experiment. Turkey poults were fed corn, wheat, and soybean meal-based starter (0 to 3 wk), grower (4 to 6 wk), developer (7 to 9 wk), and finisher (10 to 12 wk) diets formulated with uncontaminated grains, contaminated grains, and contaminated grains with 0.2% GMA (Table 1). Diets were isoenergetic and isonitrogenous, and nutrient concentrations met or exceeded minimal requirements according to the National Research Council (1994). The mycotoxin-contaminated diets were formulated by replacing uncontaminated corn and wheat with corn and wheat naturally contaminated with Fusarium mycotoxins. Feed and water were provided ad libitum. This 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-acetyl-DON, 15-acetylDON, nivalenol, T-2 toxin, iso-T-2 toxin, acetyl-T-2 toxin, HT-2 toxin, T-2 triol, T-2 tetraol, fusarenone-X, diacetoxyscirpenol, scirpentriol, 15-acetoxyscirpentriol, neosolaniol, zearalenone, zearalenol, aflatoxin, and fumonisin

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Corn1 Wheat1 Soybean meal (48%) Pork meal Fish meal Vegetable oil Limestone Dicalcium phosphate Iodized salt Vitamin and mineral mixture2 D,L-Methionine (99%) GMA3 Calculated values ME, kcal/kg Crude protein Lysine Methionine Calcium Available phosphorus Analyzed values DM Crude protein Calcium Total phosphorus

Starter (0-3 wk)

1700

CHOWDHURY ET AL.

were analyzed by gas chromatography and mass spectrometry at the Veterinary Diagnostic Laboratory, North Dakota State University, Fargo, ND (Raymond et al., 2003). Fusaric acid was determined by the HPLC method of Matsui and Watanabe (1988) as modified by Smith and Sousadias (1993) and confirmed by Porter et al. (1995).

Experimental Parameters Measured

Phenotyping of Peripheral Blood Lymphocytes Peripheral blood mononuclear cells were separated from the blood of the same birds used for hematology in each of wk 4, 8, and 12 by using Histopaque-1077.9 One million PBMN cells suspended in 50 ␮L of phosphatebuffered saline were transferred to a 96-well round-bottom plate10 and resuspended with 50 ␮L of the appropriate antibody.11 Cells were double stained with mouse anti-chicken CD4 antibody [CT-4 clone, mouse IgG1K conjugated to fluorescein isothyocyanate, 1 ␮g/106 cells] and mouse anti-chicken CD8 antibody [3-298 clone, mouse IgG2bK conjugated to R-phycoerythrin (R-PE), 0.2 ␮g/106 cells] to identify CD4+ and CD8+ T cells, respectively. B lymphocytes were bound with mouse anti-chicken IgM (␮ heavy chain specific) unlabeled primary antibody [M-

Mycotoxin DON1

15-acetyl-DON

ZEN2

+ GMA

0.2 6.8 7.1

ND3 0.6 0.7

ND 0.4 0.4

+ GMA

0.5 7.2 7.4

ND 0.8 0.7

ND 0.5 ND

+ GMA

0.9 10.7 11.4

ND 1.0 1.1

ND 0.6 0.7

+ GMA

1.7 13.6 14.3

0.2 1.3 1.5

ND 0.7 0.8

Dietary group Starter (0-3 wk) Control Contaminated grains Contaminated grains Grower (4-6 wk) Control Contaminated grains Contaminated grains Developer (7-9 wk) Control Contaminated grains Contaminated grains Finisher (10-12 wk) Control Contaminated grains Contaminated grains

4

1

Deoxynivalenol. Zearalenone. 3 Below the limit of detection. 4 Polymeric glucomman mycotoxin adsorbent. 2

4 clone, mouse IgMK, 1 ␮g/106 cells] and labeled with R-PE-conjugated goat anti-mouse IgM (␮ heavy chain specific, 0.1 ␮g/106 cells) secondary antibody. Appropriate isotype controls11 were included in the analysis. The isotype control was used to determine nonspecific binding of the lymphocyte subset-specific antibodies and to set the cut-off between fluorescence-negative and fluorescence-positive staining. Ten thousand cells were acquired and analyzed using a Becton Dickinson FACScan flow cytometer12 and Cell Quest software.12 For data analysis, a size (FSC) versus internal complexity (SSC) dot plot was generated, and a region was drawn around the small, live cell population containing the lymphocyte population. Because this region may include variable amounts of thrombocytes and red blood cells, the cell population data obtained from the quadrant statistics (2color staining) or histogram statistics (1-color staining) were standardized for the proportion of lymphocytes using the sum of CD4+, CD8+, and IgM+ cells within this region (e.g., CD4−CD8− γg-δ T cells were not accounted). Lymphocyte subset data were then adjusted by calculation to yield an estimate of the percentage of CD4+ lymphocytes, CD8+ lymphocytes, and B lymphocytes. To estimate the concentration of these lymphocyte subsets in the blood circulation, the concentration of lymphocytes in whole blood was multiplied by the proportion of various lymphocyte subsets obtained from the flow cytometric analysis.

3

Damon Division of Exxson Corp., Boston, MA. Diamed Lab Supplies Inc., Mississauga, ON, Canada. 5 Coulter Electronics Inc., Chicago, IL. 6 Bayer Inc., Toronto, ON, Canada. 7 Electron Microscopy Sciences, Hatfield, PA. 8 Unopette, Becton Dickinson, Franklin Lakes, NJ. 9 Sigma Chemical Co., St. Louis, MO. 10 Sarstedt, Inc., Newton, NC. 11 Southern Biotechnology Associates, Birmingham, AL. 12 Becton-Dickinson Immunocytometry System, San Jose, CA. 4

Analysis of IgA in Serum and Bile Two milliliters of blood was collected from the jugular vein of 2 birds per replicate (10 birds per diet) after 12 wk of feeding. Birds were euthanized by cervical dislocation after collection of blood and bile. Serum and bile samples were frozen at −80°C until further analysis. IgA was ana-

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Hematology. Blood was collected into heparinized vials from the jugular veins of 2 birds per pen (10 birds/ diet) at wk 4, 8, and 12. Hematocrit was determined by Micro-Capillary Reader3 after 5-min of centrifugation in a Hettich Haematokrit centrifuge.4 Hemoglobin (Hb) was measured as cyanomethemoglobin using a hemoglobinometer5 after the red blood cells were lysed. Mean corpuscular hemoglobin concentrations (MCHC) were calculated as MCHC = Hb/ hematocrit. Differential white blood cell counts were performed using blood smears stained with modified Wright’s stain using the Hematek Stain Pak.6 One hundred white blood cells were examined per bird using a Nikon microscope with 400× magnification, and heterophils, lymphocytes, monocytes, eosinophils, and basophils were identified. Total heterophil and eosinophils counts were made using a Neubauer Hemacytometer7 and a Nikon microscope set at 100× magnification after staining the cells with phloxine B in aqueous propylene glycol.8 Total counts of lymphocytes, monocytes, and basophils were determined indirectly by calculation from the percentages of cell and total counts of heterophils and eosinophils (Jain, 1986).

Table 2. Mycotoxin concentrations (␮g/g) in experimental diets

FUSARIUM MYCOTOXINS AND HEMATOLOGY AND IMMUNOLOGY OF TURKEYS

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Table 3. Effect of dietary Fusarium mycotoxins on hemoglobin concentration, mean corpuscular hemoglobin concentration and hematocrit1 Diet

Wk 8

115 106 104 3 0.05 NS

96 104 98 3 0.05 NS

121 124 118 3 NS3 NS

333 325 334 4 NS NS

303 312 307 6 NS NS

342 336 342 5 NS NS

0.35 0.33 0.31 0.005 0.05 0.05

0.32 0.34 0.32 0.006 0.05 NS

Wk 12

0.35 0.37 0.34 0.008 NS NS

1

Values are least square means; for each diet and phase n = 5 pens and 2 birds per pen. Polymeric glucomannan mycotoxin adsorbent. 3 P > 0.05. 4 MCHC [mean corpuscular hemoglobin concentration = (hemoglobin/hematocrit)]. 2

lyzed using a sandwich ELISA kit.13 The concentration of affinity purified polyclonal goat anti-chicken IgA (αchain specific) coating antibody was 0.01 mg/mL. The same polyclonal antibody, conjugated with horseradish peroxidase (HRP), was used as the detection antibody at a concentration of 0.01 ␮g/mL. Sample concentrations were determined from a standard curve drawn using graded concentrations of chicken reference serum13 (15.625 to 1,000 ng/mL). Dilutions of antibody, reference serum, and samples were established on the basis of a preliminary study.

Antibody Response to SRBC In wk 8 of the experiment, immediately following the collection of preimmune sera, 2 birds per replicate (10 birds per diet) were given an intrajugular injection of 1 mL of 5% SRBC14 in PBS9 containing NaCl at 120 mmol/ L, KCl at 2.7 mmol/L, and phosphate buffer at 10 mmol/ L, pH 7.4. Primary immune serum from jugular vein blood was collected subsequently at wk 9 and 10. The SRBC were readministered as described above at wk 10, and secondary immune response serum was subsequently collected at wk 11 and 12. Serum samples were frozen at −80°C to until analysis. 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

13

Bethyl Laboratories, Montgomery, TX. Cleveland Scientific, Bath, OH. 15 Fisher Scientific Ltd., Nepean, ON, Canada. 16 Mitutoya Corp., Tokyo, Japan. 14

anti-chicken IgM13 (␮-chain specific, 2 ng/mL) or HRPconjugated goat anti-chicken IgG13 (Fc-fragment specific, 2 ng/mL). 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 wells without serum.

Contact Hypersensitivity Response to Dinitrochlorobenzene This procedure was performed as described previously (Prescott et al., 1982) with minor modifications. At wk 9, 50 ␮L of 90% dimethylsulfoxide15 in deionized water was applied to an area of wing skin previously cleaned with 70% ethyl alcohol in deionized water (2 birds/replicate, 10 birds/diet). Ten minutes later, birds were sensitized by topical application of 50 ␮L of 2.5% dinitrochlorobenzene (DNCB)9 dissolved in acetone. The same procedure was subsequently conducted every 48 h for 6 d. Two week after the first sensitization, the right webfoot of the sensitized birds as well as that of 2 unsensitized birds per replicate (10 birds/diet) was challenged by topical application of 50 ␮L of 1% DNCB dissolved in a 4:1 mixture of acetone and olive oil. Fifty microliters of acetone:olive oil was applied to the left webfoot as an internal control for each bird. The thickness of the webfoot was measured before the challenge as well as 24, 48, and 72 h after the challenge using a constant-tension dial micrometer.16 Three measurements were taken from each webfoot and the averages of 3 measurements were calculated. The percentage of thickness increase was calculated as follows: percentage of thickness increase = [(skin thickness after challenge–skin thickness before challenge)/skin

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Hemoglobin (g/L) Control Contaminated diet Contaminated diet + GMA2 Pooled SE Control vs. contaminated diet Contaminated diet vs. contaminated diet + GMA MCHC (g/L)4 Control Contaminated diet Contaminated diet + GMA Pooled SE Control vs. contaminated diet Contaminated diet vs. contaminated diet + GMA Hematocrit (L/L) Control Contaminated diet Contaminated diet + GMA Pooled SE Control vs. contaminated diet Contaminated diet vs. contaminated diet + GMA

Wk 4

1702

CHOWDHURY ET AL. Table 4. Effect of dietary Fusarium mycotoxins on white blood cell, granulocyte, and monocyte counts (×109/L)1 Diet

Wk 8

Wk 12

diet + GMA

15.41 19.20 14.69 2.70 NS3 NS

30.63 32.34 24.7 2.81 NS NS

29.41 36.24 31.08 4.05 NS NS

diet + GMA

6.39 6.74 5.66 1.44 NS3 NS

10.15 11.94 9.15 1.52 NS NS

12.18 16.36 11.09 2.33 NS NS

diet + GMA

0.06 0.09 0.16 0.10 NS NS

0.23 0.28 0.23 0.12 NS NS

0.19 0.16 0.29 0.08 NS NS

diet + GMA

0.60 1.26 0.71 0.39 NS NS

1.21 1.66 0.96 0.26 NS 0.05

1.36 0.13 1.25 0.30 0.01 0.02

diet + GMA

0.83 1.42 1.44 0.32 NS NS

1.37 3.42 1.54 0.35 0.01 0.01

1.83 2.59 1.36 0.45 NS NS

1

Values are least square means; for each diet and phase n = 5 pens and 2 birds per pen. Differential white blood cell counts were performed to determine the proportion of heterophils, lymphocytes, monocytes, eosinophils, and basophils. The concentrations of heterophils and eosinophils in whole blood were determined using a hemacytometer after staining the cells with phloxine B in aqueous propylene glycol. The concentrations of white blood cells, basophils, and monocytes were then determined by calculation based on the concentrations (cells/ L) of heterophils and eosinophils in whole blood. 2 Polymeric glucomannan mycotoxin adsorbent. 3 P > 0.05.

thickness before challenge] × 100. The contact hypersensitivity (CHS) response resulting from challenge was calculated as the percentage of thickness increase in the right webfoot minus the percentage of thickness increase in the left webfoot. In turn, the contact hypersensitivity (CHS) response due to sensitization was calculated by subtracting the response to challenge exhibited by unsensitized birds from the response exhibited by sensitized birds.

Statistical Analyses The experimental design was a completely randomized design with subsampling (Kuehl, 1994). Data were tested for homogeneity of variance using the Levene test. Data were then subjected to analysis of variance using the general linear model procedure of SAS (SAS Institute, 2000). Preplanned contrasts were used to determine the influence of diet on each index. Statements of statistical significance were based on P < 0.05. For each index as-

sessed, the pen was taken as the statistical unit so that data from individual birds of the same pen were pooled.

RESULTS Mycotoxin Concentrations in Diets Dietary concentrations of DON, 15-acetyl-DON, and ZEN are given in Table 2. Levels of other mycotoxins were below the limits of detection, which were 0.02 mg/ kg for aflatoxin, 2 mg/kg for fumonisin, 0.77 mg/kg for fusaric acid, and 0.2 mg/kg for the remainder of the compounds analyzed.

Hematology Hemoglobin concentrations and hematocrit were reduced compared with controls (P < 0.05) when birds were fed diets containing Fusarium mycotoxins for 4 wk but increased after 8 wk (Table 3). Supplementation of con-

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White blood cell count Control Contaminated diet Contaminated diet + GMA2 Pooled SE Control vs. contaminated diet Contaminated diet vs. contaminated Heterophils Control Contaminated diet Contaminated diet + GMA Pooled SE Control vs. contaminated diet Contaminated diet vs. contaminated Eosinophils Control Contaminated diet Contaminated diet + GMA Pooled SE Control vs. contaminated diet Contaminated diet vs. contaminated Basophils Control Contaminated diet Contaminated diet + GMA Pooled SE Control vs. contaminated diet Contaminated diet vs. contaminated Monocytes Control Contaminated diet Contaminated diet + GMA Pooled SE Control vs. contaminated diet Contaminated diet vs. contaminated

Wk 4

1703

FUSARIUM MYCOTOXINS AND HEMATOLOGY AND IMMUNOLOGY OF TURKEYS 9

1

Table 5. Effect of dietary Fusarium mycotoxins on lymphocyte subset concentration (×10 /L) Diet

Wk 8

Wk 12

1.10 1.59 1.18 0.36 NS3 NS

3.02 2.36 2.06 0.46 NS NS

2.44 1.81 2.76 0.49 NS NS

3.50 4.26 3.34 1.04 NS NS

4.43 3.48 3.13 0.85 NS NS

4.29 4.91 4.93 0.83 NS NS

1.35 1.00 1.05 0.36 NS NS

6.52 3.94 4.22 1.04 NS NS

4.23 4.72 4.92 0.80 NS NS

1 Values are least square means; for each diet and phase n = 5 pens and 2 birds per pen. The concentration of heterophils and eosinophils in whole blood was determined using a hemacytometer after staining the cells with phloxine B in aqueous propylene glycol. The concentration of lymphocytes was determined indirectly by calculation based on the proportions of various leukocytes and the concentrations (cells/L) of heterophils and eosinophils in whole blood. The percentages of CD4+, CD8+, and IgM+ cells were determined by flow cytometry and expressed as CD4+ lymphocytes, CD8+ lymphocytes, and B lymphocytes, respectively, based on the proportions of lymphocytes (estimated as the sum of CD4+, CD8+, and IgM+ cells) in the peripheral blood mononuclear cell population. The concentrations of these lymphocyte subsets were then calculated based on the concentration of lymphocytes in whole blood. 2 Polymeric glucomannan mycotoxin adsorbent. 3 P > 0.05.

taminated diets with GMA further aggravated the adverse effect on HCT after wk 4 compared with the unsupplemented contaminated diet. The mean corpuscular hemoglobin concentrations were not affected by diet.

Peripheral White Blood Cell Counts The total white blood cell count and granulocytes (Table 4) as well as the number of B lymphocytes, CD4+, and CD8+ lymphocytes (Table 5) were not affected by diet. Peripheral blood monocyte counts were increased transiently by consumption of (P < 0.05) the contaminated diet for 8 wk, whereas basophil counts decreased after 12 wk. Supplementation of contaminated diets with GMA prevented these effects on monocyte and basophil counts (cells/L; Table 4).

Serum and Biliary IgA Biliary IgA concentrations were increased (P < 0.05) compared with controls when birds were fed the diet containing contaminated grains (Table 6). Supplementation of the contaminated diet with GMA did not prevent the elevation in biliary IgA (P > 0.05). Serum IgA concentrations, however, were not affected by diet.

Immune Responses to Antigen There was no effect of diet on IgM or IgG primary and secondary antibody titers to SRBC (Table 7). The CHS

response was reduced (P < 0.05) 24 h after challenge when birds fed contaminated grains were compared with controls (Table 8). Supplementation of contaminated diets with GMA did not alter this effect. Moreover, no effect of diet was noticed on skin thickness 48 or 72 h after DNCB challenge.

DISCUSSION Mycotoxins can act as immunosuppressive agents affecting cell-mediated and humoral immune compartments (Bondy and Pestka, 2000). The DON was detected in all control diets, and 15-acetyl-DON was detected in the control diet during the finisher phase, thus indicating that presumably uncontaminated raw materials were, in fact, contaminated, albeit at low levels. Naturally contamTable 6. Effect of dietary Fusarium mycotoxins on biliary and serum IgA concentration (mg/mL)1 Diet

Bile

Serum

Control Contaminated diet Contaminated diet + GMA2 Pooled SE Control vs. contaminated diet Contaminated diet vs. contaminated diet + GMA

3.69 16.41 5.44 4.32 0.05 NS

0.34 0.15 0.24 0.15 NS3 NS

1 Values are least square means; for each diet n = 5 pens and 2 birds per pen. Results shown for wk 12. 2 Polymeric glucomannan mycotoxin adsorbent. 3 P > 0.05.

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B lymphocytes Control Contaminated diet Contaminated diet + GMA2 Pooled SE Control vs. contaminated diet Contaminated diet vs. contaminated diet + GMA CD4+ lymphocytes Control Contaminated diet Contaminated diet + GMA Pooled SE Control vs. contaminated diet Contaminated diet vs. contaminated diet + GMA CD8+ lymphocytes Control Contaminated diet Contaminated diet + GMA Pooled SE Control vs. contaminated diet Contaminated diet vs. contaminated diet + GMA

Wk 4

1704

CHOWDHURY ET AL. Table 7. Effect of dietary Fusarium mycotoxins on serum IgM and IgG titer specific to SRBC antigens1 Primary Diet Serum IgM titer Control Contaminated diet Contaminated diet + GMA2 Pooled SE Control vs. contaminated diet Contaminated diet vs. contaminated diet + GMA Serum IgG titer Control Contaminated diet Contaminated diet + GMA Pooled SE Control vs. contaminated diet Contaminated diet vs. contaminated diet + GMA

Secondary

1st wk

2nd wk

1st wk

2nd wk

9.8 11.3 10.0 0.5 NS3 NS

8.4 8.7 9.0 0.7 NS NS

0.5 9.1 9.6 0.7 NS NS

8.0 7.5 7.8 0.6 NS NS

9.0 11.1 9.9 0.7 NS NS

9.2 10.7 10.3 0.6 NS NS

11.0 9.9 11.6 0.6 NS NS

10.0 10.5 10.5 0.6 NS NS

inated corn and wheat in the current study contain mixtures of mycotoxins (DON at 6.8 to 13.6 mg/kg, 15-acetylDON at 0.6 to 1.3 mg/kg, and ZEN at 0.4 to 0.7 mg/kg) that can exert a greater negative impact on health and productivity of livestock than the sum of their individual effects (Huff et al., 1988). The toxicity of 15-acetyl-DON is similar to that of DON (Eriksen et al., 2004), and, therefore, 15-acetyl-DON concentrations in the current study will likely be additive to the toxicity of DON. The toxic effect of dietary DON may also be enhanced by ZEN at the concentration detected in this investigation (Trenholm et al., 1989). Dietary contents of 3-acetyl-DON, nivalenol, T-2 toxin, iso-T-2 toxin, acetyl-T-2 toxin, HT-2 toxin, T-2 triol, T-2 tetraol, fusarenone-X, diacetoxyscirpenol, scirpentriol, 15-acetoxyscirpentriol, neosolaniol, zearalenol, fusaric acid, aflatoxin, and fumonisin were below the limits of detection. T-2 toxin, aflatoxin, and fumonisin, however, were reported to be potent immunotoxins (Meky et al., 2001). Immunotoxic mechanisms of mycotoxins include direct lymphocyte cytotoxicity (Sharma,

1993), stimulation of apoptosis (Bondy and Pestka, 2000), inhibition of protein synthesis either by inhibition of peptidyl transferase activity (with trichothecenes) or by reaction with DNA and RNA (with aflatoxins) (Corrier, 1991), and alteration of sphingolipid metabolism by fumonisins (Martinova, 1998). Therefore, unidentified mycotoxins and their metabolites in the naturally contaminated grains of this investigation might have contributed additive or synergistic effects to produce the outcomes reported herein. The hematocrit and Hb concentrations have been shown to decrease when chicks are fed a diet naturally contaminated with 18 mg of DON/kg for 9 wk (Harvey et al., 1991). In agreement with this previous study, hematocrit and Hb concentrations decreased in the early stages of the present study of turkeys. It is important to note that even though treatment differences exist, the variation observed in hematocrit and Hb concentrations were within the normal range for turkeys (Jain, 1986). The effect

Table 8. Effect of dietary Fusarium mycotoxins on the delayed-type hypersensitivity response to dinitrochlorobenzene (DNCB)1 Diet

24 h

Control Contaminated diet Contaminated diet + GMA2 Pooled SE Control vs. contaminated diet Contaminated diet vs. contaminated diet + GMA

23 12 21 4 0.05 NS

48 h

72 h

(% increase in webfoot thickness) 7 5 10 2 NS3 NS

3 2 6 1 NS NS

1 Values are least square means; for each diet and time point n = 5 pens and 2 birds per pen. Birds were sensitized by topical application of 2.5% DNCB at every 48 h for 6 d. Two weeks after the first sensitization, the right webfoot of the sensitized birds was challenged by topical application of 1% DNCB. The thickness of the webfoot was measured before the challenge as well as 24, 48, and 72 h after the challenge using a constanttension dial micrometer. The percentage of thickness increase was calculated as follows: percentage of thickness increase = [(skin thickness after challenge–skin thickness before challenge)/skin thickness before challenge] × 100. 2 Polymeric glucomannan mycotoxin adsorbent. 3 P > 0.05.

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1 Values are least square means; for each diet and time point n = 5 pens and 2 birds per pen. Values are reciprocal of base 2 log. 2 Polymeric glucomannan mycotoxin adsorbent. 3 P > 0.05.

FUSARIUM MYCOTOXINS AND HEMATOLOGY AND IMMUNOLOGY OF TURKEYS

The GMA is derived from the cell wall of Saccharomyces cerevisiae. The outer surface of the yeast cell wall is composed of a complex mixture of carbohydrates that contain mannose and a mixture of mannose-proteins, and the inner surface contains glucans and other complex carbohydrates. The cell walls containing polysaccharides, proteins, and lipids exhibit numerous adsorption mechanisms, such as hydrogen bonding and ionic or hydrophobic interaction (Karlovsky, 1999). The GMA has been shown beneficial in in vivo to prevent adverse effects of Fusarium mycotoxins on performance and metabolism of broiler chickens (Swamy et al., 2002), laying hens (Chowdhury and Smith, 2004), pigs (Swamy et al., 2003), and horses (Raymond et al., 2003). In the current study, GMA prevented the adverse effect of Fusarium mycotoxins on basophil and monocyte counts when adverse effects on biliary IgA and CHS response to DNCB were unaltered. The efficacy of GMA in preventing intestinal mycotoxin absorption is not known. The sensitivity of different immunological indices to absorbed mycotoxins differed. Taken together, the current results suggest that longterm consumption of grains naturally contaminated with Fusarium mycotoxins could render turkeys susceptible to infectious diseases where CD8+ T cells play major role. Moreover, the results pertaining to biliary IgA indicate a need for investigation of the impact of Fusarium mycotoxins on the resistance of turkeys to intestinal infections. In the current study, experimental birds were, however, raised under high standards of hygiene. Under commercial conditions, environmental stresses may exaggerate the adverse effects of mycotoxins. Therefore, grains naturally contaminated with Fusarium mycotoxins should be fed to turkeys with caution. The effects observed in this study were minor; therefore, the efficacy of GMA as a preventative agent could not be assessed.

ACKNOWLEDGMENTS Technical supports of Haladi V. L. N. Swamy and Robert Braham are gratefully acknowledged. Financial support for this study was provided by the Ontario Ministry of Agriculture and Food and Alltech Inc., Nicholasville, KY.

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of Fusarium mycotoxins on the hematology of turkeys, therefore, appeared limited. The feeding of DON (5.8 mg/kg of diet) in naturally contaminated grains did not alter peripheral white blood cell counts of pigs, a mycotoxin-sensitive species (Swamy et al., 2003). The results of the present investigation are generally consistent with this finding and extend it to a more resistant species. The transient, minor changes observed in blood monocyte and basophil counts (cells/ L) remain unexplained, and it is also unknown whether these minor effects have any real influence on immunocompetence. Consumption of a diet containing 5.8 mg of DON/ kg from naturally contaminated grains did not alter the primary antibody response to SRBC of starter pigs (Swamy et al., 2003). In agreement with, and extending this previous study, a diet containing comparatively high concentrations of Fusarium mycotoxins from naturally contaminated grains (13.6 mg of DON/kg, 1.3 mg 15acetyl-DON/kg, and 0.7 mg of ZEN/kg) did not affect the primary or secondary antibody response to SRBC in turkeys. Similarly, serum IgA concentrations were not influenced by Fusarium mycotoxins in this species. In contrast to the apparent resistance of humoral competence to Fusarium mycotoxins, the cell-mediated response assessed in this investigation was much depressed in the current study at 24 h. The kinetics of the contact hypersensitivity response was as reported previously in mice (Okazaki et al., 2002) and was affected by consumption of Fusarium mycotoxins. It is noteworthy, however, that the anti-SRBC antibody response and the production of IgA require CD4+ T-cell help (Goldsby et al., 2000), whereas the anti-DNCB response is mediated by CD8+ T cells, as reported for humans and mice (Bour et al., 1995) with CD4+T cells being involved mainly in a suppressive role as reported in lambs (Gunnes et al., 2004). The present results, therefore, suggest that the cell-mediated immunity, where CD8+ T cells play major role, may be more sensitive to dietary mycotoxins than the antibody-mediated immune response. The cell-mediated response to keyhole limpet hemocyanin, a CD4+ T-cell-mediated response (Pestka et al., 1987), was depressed in mice consuming a diet containing 25 mg of purified DON/kg. Antibody response to Newcastle disease virus also a CD4+ T-cell-mediated response decreased in broiler chickens consuming a diet containing 50 mg of purified DON/kg (Harvey et al., 1991) or feeding grains naturally contaminated with 17.6 mg of DON/kg and 1.6 mg of ZEN/kg in laying hens (Danicke et al., 2002). It is unknown whether a lower level resembling natural contamination would affect these responses. Little information exists as to the cellular and molecular alterations responsible for the observed mycotoxin-induced immunosuppression. Trichothecene-induced immunosuppression is most readily explained by the capacity of these compounds to bind to ribosomes and inhibit protein synthesis (Bamburg, 1983). Naturally contaminated grains, however, did not alter the gross weight of lymphoid organs such as the bursa and spleen in turkeys (Chowdhury, 2005).

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