AVIAN DISEASES 54:6773, 2010

Morphologic Changes in the Intestine of Broiler Breeder Pullets Fed Diets Naturally Contaminated with Fusarium Mycotoxins With or Without Coccidial Challenge
G. N. Girgis,A J. R. Barta,B M. Brash,C and T. K. SmithAD
Department of Animal and Poultry Science B Department of Pathobiology C Animal Health Laboratory University of Guelph, Ontario, Canada N1G 2W1 Received 29 May 2009; Accepted and published ahead of print 13 October 2009 SUMMARY. The effects of feeding diets containing grains naturally contaminated with Fusarium mycotoxins on intestinal histology were studied in chickens raised to 10 wk of age in the absence or presence of coccidial challenge. Experimental diets included the following: controls, diets containing grains naturally contaminated with Fusarium mycotoxins, and diets containing contaminated grains + 0.2% polymeric glucomannan mycotoxin adsorbent. Contaminated diets contained up to 3.8 mg/g deoxynivalenol (DON), 0.3 mg/g 15-acetyl DON, and 0.2 mg/g zearalenone. An optimized mixture (inducing lesions without mortality) of Eimeria acervulina, Eimeria maxima, and Eimeria tenella was used to challenge birds at 8 wk of age. Intestinal tissues were collected from duodenum, jejunum, and ileum prior to challenge; at the end of the challenge period (7 days postinfection; PI); and at the end of the recovery period (14 days PI). Mean villus height (VH) in the duodenum of birds fed the contaminated diets in the absence of coccidial challenge was significantly lower than that of the controls. Mean VH in the jejunum and ileum of the same birds was significantly higher compared to controls, indicating a compensatory mechanism. Fusarium mycotoxins retarded duodenal recovery from coccidial lesions, as indicated by lower duodenal VH and apparent villus surface area comparing challenged birds fed the contaminated diets to challenged controls of the same age. Increased VH was frequently associated with cryptal hyperplasia and increased numbers of mitotic figures in crypts. It was concluded that diets contaminated with Fusarium mycotoxins below levels that negatively affect performance could alter intestinal morphology and interfere with intestinal recovery from an enteric coccidial infection. gicos en el intestino de pollitas reproductoras pesadas que fueron alimentadas con dietas RESUMEN. Cambios morfolo o de coccidias. contaminadas de manera natural con micotoxinas de hongos Fusarium, con o sin desaf a del intestino por dietas alimenticias que contienen granos contaminados naturalmente Se estudiaron los efectos en la histolog o por con micotoxinas de hongos Fusarium en pollos criados hasta las 10 semanas de edad en la ausencia o en presencia de un desaf an granos naturalmente contaminados coccidias. Las dietas experimentales fueron las siguientes: dieta control, dietas que conten an granos contaminados con micotoxinas mas un adsorbente de micotoxinas tipo con micotoxinas de Fusarium, y dietas que conten an hasta 3.8 mg/g de deoxinivalenol (DON), 0.3 mg/g de 15rico al 0.2%. Las dietas contaminadas conten glucomanano polime a lesiones sin mortalidad) de Eimeria una mezcla optimizada (que induc acetil DON, y 0.2 mg/g de zearalenona. Se utilizo acervulina, Eimeria maxima y Eimeria tenella para desafiar a las aves a las 8 semanas de edad. Se recolectaron tejidos intestinales del o, al final del per odo de desaf o (7 d as postinfeccio odo de n; PI), y al final del per duodeno, yeyuno y el leon antes de desaf as postinfeccio n (14 d n). Se observo que la altura media de las vellosidades (VH) en el duodeno de las aves alimentadas recuperacio o por coccidias fue significativamente menor en comparacio n con de los controles. con dietas contaminadas en la ausencia de desaf n con La altura media de las vellosidades en el yeyuno y en el leon de las mismas aves fue significativamente mayor en comparacio n duodenal de las los controles, indicando un mecanismo compensatorio. Las micotoxinas de Fusarium retardaron la recuperacio n lo indicado por la menor altura de las vellosidades duodenales y la superficie aparente de las lesiones por coccidias, segu vellosidades comparando las aves alimentadas con dietas contaminadas y desafiadas con los controles desafiados de la misma edad. frecuentemente con hiperplasia de las criptas y un incremento en el El aumento en la altura media de las vellosidades se asocio mero de figuras mito ticas en las criptas. Se concluyo que las dietas contaminadas con micotoxinas de Fusarium por debajo de los nu a intestinal e interferir con la recuperacio n por una niveles que afectan negativamente el rendimiento pueden alterar la morfolog n ente rica por coccidias. infestacio Key words: Fusarium mycotoxins, coccidia, intestine, histology, pullets Abbreviations: AVSA 5 apparent villus surface area; CD 5 crypt depth; C : V 5 crypt : villus ratio; DON 5 deoxynivalenol; GMA 5 glucomannan mycotoxin adsorbent; IELs 5 intraepithelial lymphocytes; PI 5 postinfection; SEM 5 standard error of the mean; VH 5 villus height; VW 5 villus width
A

Fungi of the genus Fusarium can produce a large number of mycotoxins in grains used for poultry feed (15). Among these mycotoxins, the trichothecene deoxynivalenol (DON, vomitoxin) and its acetylated derivatives are a major concern in North America as a result of their high incidence and high chemical stability (17,24). Three-acetyl and 15-acetyl DON often co-occur with

Corresponding author. E-mail: tsmith@uoguelph.ca

DON. The acetylated DON is transformed to DON in the gastrointestinal tract before absorption (9). Inhibition of protein synthesis has been described as a major effect of trichothecenes on eukaryotic cells (3,8,19). Repetitive exposure to trichothecenes results in injury to actively dividing cells, such as those present in bone marrow, intestinal mucosa, spleen, and thymus (8). Following ingestion of contaminated feed, the intestinal epithelium could be exposed to high concentrations of mycotoxins (5). Adverse effects of Fusarium mycotoxins on intestinal morphol-

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Table 1.

G. N. Girgis et al.

Composition (%) and energy levels of experimental diets.

Ingredient Starter Grower

Wheat Corn Soybean meal Vegetable fat Wheat shorts Bicalcium phosphate Limestone Vitamin and mineral premixA Salt DL-methionine HCL-lysine Calculated metabolizable energy (Kcal/kg) Analyzed gross energy (Kcal/kg) Control diet ContaminatedB diet Contaminated + GMAC diet

48.2 16 22.6 1.85 6 1.8 1.88 1 0.41 0.16 0.10 2870 3410 3440 3430

52.4 9.2 10.9 3 20 1.5 1.5 1 0.34 0.10 0.06 2870 3470 3530 3520

The current study reports the histopathologic and morphometric findings in the intestinal tract of birds utilized in the previous study. To mimic field situations, birds were raised following a commercial feeding program and were fed diets containing grains that were graded clean or grains that were rejected because of Fusarium mycotoxin contamination. The efficacy of a polymeric glucomannan mycotoxin adsorbent (GMA) derived from yeast cell wall was also evaluated.
MATERIALS AND METHODS Experimental birds. One hundred and twenty one-day-old Ross 308 commercial broiler breeder female chicks were obtained from Aviagen, Inc. (Blairsville, GA). Chicks were not vaccinated in the hatchery against coccidiosis. Chicks were raised in the Central Animal Facility Isolation Unit of the Ontario Veterinary College. Birds were managed as has been prescribed by the Canadian Council on Animal Care with the Animal Utilization Protocols approved by the Animal Care Committee of the University of Guelph (6). Experimental diets. Corn-, wheat-, and soybean mealbased control diets were formulated to meet the standard nutritional specifications for starter and grower broiler breeder chickens (16). The mycotoxincontaminated diets were formulated to the nutrient specifications of the control diets by replacing control corn and wheat with grains naturally contaminated with Fusarium mycotoxins. Contaminated + GMA diets contained 0.2% GMA (MycosorbH, Alltech, Inc., Nicholasville, KY) added at the expense of corn. No coccidiostat was included in any of the experimental diets. The composition of the experimental diets is shown in Table 1. Dietary contents of DON, 3-acetyl DON, 15-acetyl DON, nivalenol, T-2 toxin, acetyl T-2 toxin, iso T-2 toxin, HT-2 toxin, T-2 triol, T-2 tetraol, fusarenone-X, diacetoxyscirpenol, scirpenol, 15-acetoxyscirpenol, neosolaniol, zearalenone, zearalenol, aflatoxin, and fumonisin were analyzed by gas chromatographymass spectrometry, as described by Raymond et al. (18). Limits of detection were 0.02 mg/g for aflatoxin, 2 mg/g for fumonisin, and 0.2 mg/g for the other mycotoxins. Experimental design. One-day-old chicks were randomly divided into three groups of 40 birds each, each of which was fed one of the three experimental diets. Appropriate temperature, humidity, and lighting programs were followed according to the recommendations of the supplier. Birds were fed starter diets ad libitum from day 1 to 3 wk of age and grower diets on a 4/3 skip-a-day feed restriction program from 3 wk to 10 wk of age. At 8 wk of age, eight birds from each group (n 5 24) were randomly selected, euthanatized by cervical dislocation, and immediately sampled as described below. Half of the birds remaining in each group were orally inoculated with a standardized mixture of Eimeria acervulina (Guelph strain; 100,000 sporulated oocysts/bird), Eimeria maxima (U.S. Department of Agriculture strain 68; 30,000 sporulated oocysts/bird), and Eimeria tenella (U.S. Department of Agriculture strain 80; 7500 sporulated oocysts/bird). Oocyst doses were predetermined to cause lesions without mortality in a pilot study. Coccidial challenge and sampling were done 6 hr after feeding. Birds were reared on the floor for 8 wk. Upon oral inoculation with Eimeria

A Provided per kilogram of diet: vitamin A, 8800 IU; vitamin D3, 3300 IU; vitamin E, 40 IU; vitamin K, 3.3 mg; thiamine, 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 mg; vitamin B12, 12 mg; ethoxyquin, 120 mg; manganese, 70 mg; zinc, 70 mg; iron, 60 mg; copper, 10 mg; iodine, 1.0 mg; and selenium, 0.3 mg. B Control clean wheat and corn were replaced by naturally mycotoxin-contaminated wheat and corn. C 0.2% Glucomannan mycotoxin adsorbent (MycosorbH, Alltech, Inc., Nicholasville, KY) added at the expense of corn.

ogy have been reported in broilers (1) and turkey poults (21). There are no reports regarding the effects of Fusarium mycotoxins on the morphology of the intestinal tract of broiler breeder pullets. Feeding programs for broiler breeder pullets differ from those for broilers or turkeys in that they include a feed restriction period during the growing phase. Reports on the effects of Fusarium mycotoxins on the recovery of intestinal epithelium following coccidial infection are also lacking. We observed immunomodulation in broiler breeder pullets infected with coccidia and fed diets naturally contaminated with Fusarium mycotoxins. Total serum immunoglobulin A and immunoglobulin G concentrations in challenged birds fed the contaminated diet were higher than those of controls at the end of the challenge period. In contrast to challenged controls, the prechallenge percentage of blood CD8+ cells that decreased at the end of the challenge period was not restored at the end of the recovery period in challenged birds fed the contaminated diet. Interferon-c gene expression in cecal tonsils was up-regulated in challenged birds fed the contaminated diet at the end of the challenge period (11). Body weight gains, however, were not significantly affected by diet.

Table 2. Mycotoxin concentrations (mg/g) detected in the experimental diets.

Experimental diets Control Mycotoxin Starter Grower Starter Contaminated Grower Starter Contaminated + GMA Grower

DON 15-acetyl DON Zearalenone

1.2 NDA ND

0.9 ND ND

2.5 ND ND

3.8 0.3 0.2

2.4 ND ND

3.2 0.3 0.2

A ND 5 not detected. Limits of detection were 0.02 mg/g for aflatoxin, 2 mg/g for fumonisin, and 0.2 mg/g for the other mycotoxins. Dietary contents of nivalenol, T-2 toxin, acetyl T-2 toxin, iso T-2 toxin, HT-2 toxin, T-2 triol, T-2 tetraol, fusarenone-X, diacetoxyscirpenol, scirpenol, 15acetoxyscirpenol, neosolaniol, zearalenol, aflatoxin, and fumonisin were below the detectable levels.

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Fig. 1. Effects of diet and coccidial challenge on villus height, villus width, and crypt depth in different intestinal regions. Bars represent mean 6 standard error of the mean (SEM). White bars represent birds fed the control diet; gray bars represent birds fed the contaminated diet; and black bars represent birds fed the contaminated + GMA diet. Birds were challenged with coccidia at 8 wk of age, and day 14 postinfection was considered the end of the recovery period. Different letters in each group of bars indicate significant differences. Asterisk indicates significant difference relative to nonchallenged age-matched birds fed the same diet (P # 0.05). and until the end of the experiment at 10 wk, all birds were assigned in cages (two birds/cage) with a wire-mesh floor in order to prevent fecaloral reinfection. At day 7 postinfection (PI; the end of the infection cycle), eight birds in each group (n 5 48) were randomly selected, euthanatized by cervical dislocation, and sampled. At 14 days PI (the end of the recovery period), the remaining birds (n 5 48) were similarly euthanatized and sampled. Sample collection and processing. Intestinal segments were collected from freshly killed birds from the midpoint of the descending loop of the duodenum, 5 cm proximal to the Meckel diverticulum (jejunum) and 10 cm proximal to the ileo-cecal junction (ileum). Intestinal segments were opened longitudinally, flushed with 0.9% NaCl, and immediately fixed in freshly prepared 10% neutral buffered formalin for 48 hr at room temperature. Tissues were trimmed parallel to the longitudinal axis of the intestine, dehydrated, cleared, and paraffin embedded. Longitudinal sections (5 mm in thickness) were cut from each sample, placed on glass slides, and processed for hematoxylin and eosin staining (14).

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Fig. 2. Effects of diet and coccidial challenge on crypt : villus ratio and apparent villus surface area in different intestinal regions. Bars represent mean 6 standard error of the mean (SEM). White bars represent birds fed the control diet; gray bars represent birds fed the contaminated diet; and black bars represent birds fed the contaminated + GMA diet. Birds were challenged with coccidia at 8 wk of age, and day 14 postinfection was considered the end of the recovery period. Different letters in each group of bars indicate significant differences. Asterisk indicates significant difference relative to nonchallenged age-matched birds fed the same diet (P # 0.05). Morphometric measurements. Fifteen intact, axially sectioned, welloriented villus-crypt units were randomly selected from sections in each sample. Morphometric analyses relative to each villus-crypt unit were performed using a computer-aided light microscope imager and OpenLabH software (Improvision, Inc., Lexington, MA). The parameters measured in each intestinal segment included villus height (VH), villus width (VW), crypt depth (CD), crypt : villus ratio (C : V), and apparent villus surface area (AVSA). VH was measured from the tip of the villus to the top of the crypt. VW was expressed as the average of apical width (at upper one third of the villus) and basal width (at lower one third of the villus). CD was measured from the base of the crypt upward to the region of transition between the crypt and the villus. AVSA was calculated by multiplying VH by the average VW. Samples collected from challenged birds at 9 wk of age (the end of the infection cycle) were not analyzed morphometrically because of the widespread mucosal destruction caused by developing coccidia. Histopathologic scoring. Tissue sections were scored for epithelial atrophy, cryptal dilatation, necrosis, and hyperplasia. Examination of the cellularity of villus-crypt units included scoring of intraepithelial lymphocytes (IELs) and total cell population in the lamina propria as well as categorizing of cell infiltrates as mononuclear cells, granulocytes, and lymphoid nodules. Scoring was done on 15 villus-crypt units in each section and included three different sections per tissue sample. Description of each parameter was digitalized for statistical analysis. The parameters were, therefore, subjectively scored using a 0-to-3-point scale with 0.5-unit increments. Normal tissues were scored 0, while the scores 1, 2, and 3 indicated mild, moderate, and marked increase in cellularity, respectively. All tissues were scored blind by a single operator throughout the study. Mitotic figures were identified according to Van Diest et al. (23) based on the absence of nuclear membrane, which indicates that the cells have passed prophase. The presence of clear hairy extensions of the nuclear material of condensed chromosomes either clotted (beginning of metaphase), in a plane (metaphase/anaphase), or separated (telophase) was also observed. Mitotic figures were counted from the crypt base to the crypt-villus junction in three different sections for each bird, and these three values were used to calculate the mean for one bird. Fifteen crypts were examined in each section. Statistical analysis. Data were analyzed by ANOVA using a PROC GLM model of SAS software (20). Tukey test was used to compare least square means among treatments. Comparisons were considered significant at P # 0.05.

RESULTS

Dietary mycotoxin concentrations. DON was the predominant Fusarium mycotoxin in experimental diets. Contaminated diet and contaminated + GMA diet contained 15-acetyl DON and zearalenone

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Fig. 3. Examples of the changes in the duodenum of birds fed the contaminated diets. Compared to duodenum of birds fed the control diet (a), duodenum of birds fed the contaminated diet had increased intraepithelial lymphocyte population (b and c), increased cell infiltration in the lamina propria (arrows) (c), and cryptal hyperplasia with increased numbers of mitotic figures (d). Hematoxylin and eosin, 403.

in addition to DON. Nivalenol, T-2 toxin, acetyl T-2 toxin, iso T-2 toxin, HT-2 toxin, T-2 triol, T-2 tetraol, fusarenone-X, diacetoxyscirpenol, scirpenol, 15-acetoxyscirpenol, neosolaniol, zearalenol, aflatoxin, and fumonisin were not detected in any of the experimental diets. The detected mycotoxin levels in the experimental diets are shown in Table 2. Morphometric analysis of the duodenum. VH in the duodenum of birds fed either the contaminated diet or the contaminated + GMA diet was significantly lower than that of the controls at each sampling point (Fig. 1). VW, CD, and C : V in the same birds were higher than those in the controls (Figs. 1, 2). AVSA was significantly lower in birds fed the contaminated diet compared to birds fed the control diet before coccidial challenge and upon recovery from infection. Birds recovering from infection and fed the contaminated diet or the contaminated + GMA diet had VH, CD, and AVSA values that were lower than those in nonchallenged age matches of the same age fed the same diet. Morphometric analysis of the jejunum. VH in the jejunum of nonchallenged birds fed either the contaminated diet or the contaminated + GMA diet was significantly higher than VH in the controls (Fig. 1). VW and CD in the jejunum of nonchallenged birds fed the contaminated diet were significantly greater than those of the controls at 8 wk of age (before challenge).

In birds recovering from coccidial challenge, all morphologic parameters of the jejunum were not significantly different when comparing birds fed the contaminated diet and those fed the control diet. Birds recovering from coccidial challenge and fed the contaminated + GMA diet had significantly greater VW, CD, C : V, and AVSA values when compared with recovering birds fed either the contaminated or control diet (Figs. 1, 2). Morphometric analysis of the ileum. VH in the ileum of nonchallenged birds fed either the contaminated diet or the contaminated + GMA diet was significantly higher than VH of the controls before challenge, but the differences were not significant thereafter. In birds recovering from coccidia infection and fed the contaminated diet or contaminated + GMA diet, VW was significantly greater than that in controls. CD in the ileum of nonchallenged birds fed the contaminated diet was significantly lower than that of controls. Histopathologic findings. IELs in the duodenum of birds fed the contaminated diet were significantly higher than those of controls at all sampling points in challenged or nonchallenged birds. There was a significant increase in cellularity (mainly mononuclear cells) of the lamina propria of duodenal villi, duodenal crypts, and jejunal crypts in the absence of infection in birds fed the contaminated diets compared to the controls (Fig. 3). No differences in lamina propria

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cellularity were observed upon challenge. Birds fed the contaminated + GMA diet showed increased cryptal hyperplasia associated with increased numbers of mitotic figures in the duodenum and jejunum in the absence of challenge (Fig. 4). Increased numbers of mitotic figures were also observed in the duodenum and jejunum of birds fed the contaminated diet before challenge (Fig. 4).
DISCUSSION

The intestine of chickens possesses an inherent ability to maintain regional differences in mucosal structure, especially with respect to VH, even though birds lack the distinct delineations of these regions that are clearly evident in mammals (12). Absorptive villus surface area is related to VH, and, therefore, VH and AVSA are considered suitable indicators for assessing intestinal function (25). The increase of VH in the jejunum and ileum of nonchallenged birds fed the contaminated diets in the current study may represent compensation to the reduced surface area of the duodenal villi resulting from their reduced heights. Such compensatory changes also included increased VW in the duodenum and jejunum. The small intestine has surplus absorptive capacity, and the decrease in the absorptive surface in the proximal small intestine due to the feeding of contaminated diets could have been compensated for by displacing absorption to more distal intestinal sites (21). The lack of effect of contaminated diets on body weight gain, as observed earlier (11), may be related to these changes. The overall compensatory capacity might have been so high that performance was not impaired, despite the apparent alterations of gut structure. Broilers fed contaminated wheat containing 5 mg/ kg DON for 21 days have been reported (2) to have shorter and thinner villi in the duodenum, and poults fed T-2 toxin or diacetoxyscirpenol at a rate of up to 1 mg/kg have been reported (21) to have shorter and thinner villi of the jejunum. Growth was not depressed in either case, which is in agreement with the current findings. Mature cells derived from the intestinal stem cells migrate upward from the crypt toward the tip of the villus, gradually differentiating as they reach the tip (4). The observed increase of CD in the duodenum and jejunum of birds fed the contaminated diets was associated with hyperplasia of the crypt epithelium. Increased numbers of mitotic figures were frequently observed in such crypts. This may be associated with a greater need for increasing intestinal absorptive surface (10). The differences in C : V among different regions of the intestine may reflect differences in the distance migrated by enterocytes relative to the total mucosal depth and differences in cell migration rates (10,12). The feeding of Fusarium mycotoxincontaminated diets appeared to impair recovery of duodenal villi from coccidial lesions. This was indicated by reduced duodenal VH and AVSA in challenged birds fed the contaminated diets compared to controls of the same age. In the jejunum of birds recovering from coccidial infection, VW, CD, C : V, and AVSA were significantly greater in birds recovering from coccidial infection and fed GMA-supplemented diets compared to birds fed other diets. Villus growth promotion has been shown to be an effect of feeding yeast cell wallderived glucomannan to broilers (27). Yeast extract containing b-glucan and mannan-oligosaccharide has been reported to accelerate gastrointestinal maturation in turkey poults, in which VH, CD, and surface area of the ileum were enhanced in a dose-dependent manner (22). Apart from its ability to ameliorate the toxic effects of mycotoxins, the observed hyperplasia and increased mitotic figures in the duodenum and jejunum of birds fed diets supplemented with GMA indicate a direct mitogenic effect of this yeast cell wall product.

Fig. 4. Effects of diet and coccidial challenge on the number of mitotic figures in crypts. Bars represent mean 6 standard error of the mean (SEM). White bars represent birds fed the control diet; gray bars represent birds fed the contaminated diet; and black bars represent birds fed the contaminated + GMA diet. Birds were challenged with coccidia at 8 wk of age, and day 14 postinfection was considered the end of the recovery period. Different letters in each group of bars indicate significant differences. Asterisk indicates significant difference relative to nonchallenged age-matched birds fed the same diet (P # 0.05).

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Increased IELs and cellular infiltrations in the lamina propria of the duodenum and jejunum of nonchallenged birds fed the contaminated diet could possibly be attributed to the direct irritating effects of Fusarium mycotoxins on intestinal mucosa. Such an increased cellularity was not, however, associated with changes in immune parameters in the absence of infection, as investigated earlier (11). Repeated stimulation of immune cells can limit their function (13). DON was the major mycotoxin contaminant detected in the experimental diets in the present study. DON has been the most frequently detected trichothecene contaminant of Canadian-grown grains. DON has persistently occurred in Ontario grains from 1979 to 1995, with an annual incidence of up to 100% (7). The recommended maximum level of DON in Canadian grains used in poultry diets (not exceeding 50% of the total diet) is 5 mg/kg. The level of DON in the experimental diets in the current study is, therefore, of practical relevance to the field. It has been reported (26) that broiler breeders can tolerate higher levels of Fusarium mycotoxins than did those used in the current study without apparent adverse effects on performance. The feeding of grains naturally contaminated with Fusarium mycotoxins in the current study represents chronic exposure of growing broiler breeders to relatively low concentrations of mycotoxins rather than an acute toxin challenge. The current findings indicate that diets containing grains naturally contaminated with Fusarium mycotoxins below levels that negatively affect performance could alter small intestinal morphology in broiler breeder pullets. Fusarium mycotoxins also appeared to interfere with intestinal recovery from coccidial infection. Some histologic alterations caused by Fusarium mycotoxins were modulated by dietary GMA.
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ACKNOWLEDGMENTS Financial support for this study was provided by the Ontario Ministry of Agriculture, Food, and Rural Affairs (OMAFRA) and by Alltech, Inc. (Nicholasville, KY). We also thank Aviagen, Inc. (Blairsville, GA) for donating the experimental birds.