Duodenal morphology and immune responses of broiler chickens fed low doses of deoxynivalenol

Morphometry and flow cytometry for intraepithelial lymphocyte phenotyping were used to determine the changes in duodenal mucosae after administration of low doses of deoxynivalenol in chickens. Moreover, functions of phagocytes and immunocompetent cells in peripheral blood were evaluated by flow cytometry. In total, sixty chickens of Ross hybrid broilers 308 were used in this experiment. Two experimental groups of 20 birds were continually fed for 14 days a diet containing deoxynivalenol at a dose of 1 and 3 mg·kg-1; 20 birds of the control group were fed uncontaminated diet. Morphometry showed only tendency to decrease the height of villi and surface area of duodenal mucosae in chickens fed the diet supplemented with 3 mg·kg-1 deoxynivalenol. Phenotyping of intraepithelial lymphocytes showed a decrease of CD45+ (P < 0.034) in duodenum of birds fed diets supplemented with deoxynivalenol. Examination of white blood cells showed a decrease of monocytes (P < 0.020) in chickens fed 3 mg·kg-1 of deoxynivalenol. Both experimental groups revealed higher metabolic burst of peripheral blood heterophils (P < 0.001). Phenotyping of immunocompetent cells showed an increase (P < 0.003) of CD3+ and a decrease (P < 0.001) of MHC II+ cells in peripheral blood of chickens fed with 3 mg·kg-1 dose of deoxynivalenol. The experimental feeding of chickens with deoxynivalenol resulted in immunomodulation of immunocompetent cells in duodenum and blood with mild atrophy of intestinal villi, mainly after the feeding of the dose of 3 mg·kg-1. We proved that even low doses of deoxynivalenol can cause changes in haemathological, immunological and morphological profiles already during two weeks, and lead to the activation of compensatoryadaptive mechanisms with unfavourable impact on health and performance of birds. Intestine, immunity, vomitoxin, morphometry, toxicity, poultry Deoxynivalenol (DON, vomitoxin) is mycotoxin produced by Fusarium graminearum and is included into B type trichothecenes which can cause serious problems of animals and poultry when consumed via contaminated cereal grains. Consumption of lower amounts of fungal toxins may result in impaired immunity and decreased resistance to infection diseases (Oswald et al. 2005). Mycotoxins may act on all types of immune cells and on different levels of the immune response to produce their adverse effects. Numerous studies conducted on host resistance, antibody responses, and cell mediated immunity have revealed that trichothecenes stimulate or suppress the immune function depending on the dose, exposure frequency, and timing of functional immune assay (Pestka et al. 2004). It is very likely that mycotoxins have their greatest effect on the mucosal lymphoid tissue (particularly gut and bronchial) before they are absorbed and subsequently metabolized (Oswald et al. 2005). The avian gut associated lymphoid tissue (GALT) comprises a diverse set of cell subsets, distinct from that of systemic tissues but includes representatives of each of the major cell populations found on other sites. Overall the gut is populated with heterophils, macrophages, dendtritic cells, natural killer cell, and B and T lymphocytes, although the proportions of each cell type differ widely according to site and age (Brown et al. 2008). Intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) have ACTA VET. BRNO 2013, 82: 337–342; doi:10.2754/avb201382030337 Address for correspondence: Doc. MVDr. Viera Revajová, PhD. Department of Pathological Anatomy and Pathological Physiology University of Veterinary Medicine and Pharmacy in Košice Komenského 73, Košice 041 81, Slovak Republic Phone: +421 915 984 708 Fax: +421 55 67 11 674 E-mail: revajova@uvlf.sk http://actavet.vfu.cz/ fundamental importance in host prevention after antigenic stimulation in the intestinal tract (Davison et al. 2008). Enterocytes as part of the integral mucosal immune system are multi-functional epithelial cells that play an important role in the organization and function of the enteric immune system. Studies using the mouse model have shown the importance of enterocyte-expressed chemokines in the recruitment and retention of IEL cell populations (Onai et al. 2002). Diets contaminated with low DON that induce a negative impact on health and performance could affect small intestinal morphology in broilers by diffusion of mycotoxins. Awad et al. (2006) observed that feeding DON for 21 days to broiler chickens at a concentration of up to 5 mg·kg-1 of diet influenced the weight of the small intestine as well as intestinal histology, especially the duodenum, as evidenced by shorter and thinner villi. Changes in the villi may influence also the immunocompetent cells of the intestine. For that reason, the aim of the present study was to examine the effects of low doses of DON in naturally contaminated maize on duodenal morphology and immune responses of broilers. Materials and Methods Animals, housing and diets One-day-old sixty chickens of Ross hybrid broilers 308 were randomly divided into 3 groups of 20 birds. The broilers were reared in large pens with wood shavings and had free access to water and feed. The experiment was carried out in accordance with established standards for use of animals. Local ethics and scientific authorities approved the Ro-2518/05-211/c protocol. Chickens of all groups were fed the commercial diet HYD-02 for 2 weeks, and during the following 2 weeks broilers of DON groups were fed diets contaminated with different doses of DON mycotoxin. Commercial diet of the control group was naturally contaminated with 0.2 mg·kg-1 DON. Diets of the second and third groups were experimentally contaminated with 1 mg·kg-1 and 3 mg·kg-1 DON, respectively. The final mycotoxin contents in the diets for each group of birds are shown in Table 1. Contaminated batches of maize were obtained by their cultivation with Fusarium graminearum for four weeks at the Slovak Agriculture University in Nitra (Labuda et al. 2003). To provide stable dietary contents of mycotoxins throughout the whole experimental period, the chickens were fed only one type of diet, HYD-02. The composition of this diet is given in Table 2. At the age of 4 weeks, 6 randomly chosen chickens from each group were anaesthetized with intraperitoneal injection of xylazine and ketamine (Rometar 2% and Narkamon 5%, Spofa, Czech Republic) at doses of 0.6 and 0.7 ml·kg-1 body weight, respectively. After laparotomy, blood was collected into heparinized tubes by intracardial punction and immediately used for counting of leukocytes, and flow cytometry analysis of granulocytes and lymphocytes. Duodenal samples were taken into Hanks solution for flow cytometry, as well as into 10% formaline for histology and morphometry. Mycotoxin analysis Mycotoxins in maize were detected using gas chromatography-mass spectrophotometry (GC-MS) method (Raymond et al. 2003). Mycotoxin contents in the basal diet (the part of HYD-02 diet before addition of 40% portion of control or contaminated maize) were analyzed using NOACK kits for enzyme-linked immunosorbent assay with spectrophotometric evaluation. 338 Table 1. Content of mycotoxins in complete diets for control and experimental groups of chickens. DON deoxynivalenol, ZEA zearalenone, 15-ADON 15-acetyldeoxynivalenol Groups of birds Mycotoxins (mg·kg-1 of complete feed) DON ZEA 15-ADON Total aflatoxins Control group 0.2 0.03 0 0.002 Group fed 1 mg DON 1.0 0.06 0.07 0.002 Group fed 3 mg DON 3.0 0.15 0.24 0.002 Table 2. Composition of diet HYD-02 fed to broilers during the experiment. Metabolizable energy (12.75 MJ·kg-1 of complete feed), CP crude protein analysed by Kjelahl method (210.6 g·kg-1 of complete feed) Components g·kg-1 Wheat ground, 10.5% of CP 260

Deoxynivalenol (DON, vomitoxin) is mycotoxin produced by Fusarium graminearum and is included into B type trichothecenes which can cause serious problems of animals and poultry when consumed via contaminated cereal grains.Consumption of lower amounts of fungal toxins may result in impaired immunity and decreased resistance to infection diseases (Oswald et al. 2005).
Mycotoxins may act on all types of immune cells and on different levels of the immune response to produce their adverse effects.Numerous studies conducted on host resistance, antibody responses, and cell mediated immunity have revealed that trichothecenes stimulate or suppress the immune function depending on the dose, exposure frequency, and timing of functional immune assay (Pestka et al. 2004).It is very likely that mycotoxins have their greatest effect on the mucosal lymphoid tissue (particularly gut and bronchial) before they are absorbed and subsequently metabolized (Oswald et al. 2005).
The avian gut associated lymphoid tissue (GALT) comprises a diverse set of cell subsets, distinct from that of systemic tissues but includes representatives of each of the major cell populations found on other sites.Overall the gut is populated with heterophils, macrophages, dendtritic cells, natural killer cell, and B and T lymphocytes, although the proportions of each cell type differ widely according to site and age (Brown et al. 2008).Intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) have fundamental importance in host prevention after antigenic stimulation in the intestinal tract (Davison et al. 2008).
Enterocytes as part of the integral mucosal immune system are multi-functional epithelial cells that play an important role in the organization and function of the enteric immune system.Studies using the mouse model have shown the importance of enterocyte-expressed chemokines in the recruitment and retention of IEL cell populations (Onai et al. 2002).
Diets contaminated with low DON that induce a negative impact on health and performance could affect small intestinal morphology in broilers by diffusion of mycotoxins.Awad et al. (2006) observed that feeding DON for 21 days to broiler chickens at a concentration of up to 5 mg•kg -1 of diet influenced the weight of the small intestine as well as intestinal histology, especially the duodenum, as evidenced by shorter and thinner villi.Changes in the villi may influence also the immunocompetent cells of the intestine.
For that reason, the aim of the present study was to examine the effects of low doses of DON in naturally contaminated maize on duodenal morphology and immune responses of broilers.

Animals, housing and diets
One-day-old sixty chickens of Ross hybrid broilers 308 were randomly divided into 3 groups of 20 birds.The broilers were reared in large pens with wood shavings and had free access to water and feed.The experiment was carried out in accordance with established standards for use of animals.Local ethics and scientific authorities approved the Ro-2518/05-211/c protocol.
Chickens of all groups were fed the commercial diet HYD-02 for 2 weeks, and during the following 2 weeks broilers of DON groups were fed diets contaminated with different doses of DON mycotoxin.Commercial diet of the control group was naturally contaminated with 0.2 mg•kg -1 DON.Diets of the second and third groups were experimentally contaminated with 1 mg•kg -1 and 3 mg•kg -1 DON, respectively.The final mycotoxin contents in the diets for each group of birds are shown in Table 1.Contaminated batches of maize were obtained by their cultivation with Fusarium graminearum for four weeks at the Slovak Agriculture University in Nitra (Labuda et al. 2003).To provide stable dietary contents of mycotoxins throughout the whole experimental period, the chickens were fed only one type of diet, HYD-02.The composition of this diet is given in Table 2.At the age of 4 weeks, 6 randomly chosen chickens from each group were anaesthetized with intraperitoneal injection of xylazine and ketamine (Rometar 2% and Narkamon 5%, Spofa, Czech Republic) at doses of 0.6 and 0.7 ml•kg -1 body weight, respectively.After laparotomy, blood was collected into heparinized tubes by intracardial punction and immediately used for counting of leukocytes, and flow cytometry analysis of granulocytes and lymphocytes.Duodenal samples were taken into Hanks solution for flow cytometry, as well as into 10% formaline for histology and morphometry.

Mycotoxin analysis
Mycotoxins in maize were detected using gas chromatography-mass spectrophotometry (GC-MS) method (Raymond et al. 2003).Mycotoxin contents in the basal diet (the part of HYD-02 diet before addition of 40% portion of control or contaminated maize) were analyzed using NOACK kits for enzyme-linked immunosorbent assay with spectrophotometric evaluation.12.5 Premix HYD-02 (vitamins and minerals)

White blood cell counting
Routine laboratory method using haemocytometer and Hemacolor staining (Merck, Germany) were used for evaluation of total count of leukocytes and their differentiation on blood smears.Absolute numbers (total numbers) of different white blood cell counting (WBC) were counted as follows: total leukocyte count/100 counted cells × relative % of differential cell count.

Phagocytosis and oxidative burst assay
The functions of polymorphonuclear cells were measured by flow cytometry in whole heparinized blood (heparin 10-20 U•ml -1 in PBS, Zentiva, Czech Republic).A commercial Phagotest and Bursttest kits (ORPEGEN ® Pharma, Germany) were used for examination of phagocytosis and metabolic activity by the manual instructions.

Flow cytometry
Duodenal intraepithelial lymphocytes were isolated by modification some methods and published in detail by Levkut et al. (2009).Mononuclear cells from blood and intestine were separated over Histopaque-1077 gradient sedimentation (Sigma, Germany).Indirect and direct immunofluorescence methods of single staining cells were used.Labelled and unlabelled primary mouse anti-chicken monoclonal antibodies (Serotec, GB, and Southern Biotechnology Associates, Inc., Birmingham, USA) were used (Table 3).Polyclonal goat antimouse FITC-conjugated immunoglobulins F(ab') 2 fragment (Dako, Denmark) at a working dilution 1:50 with phosphate-buffered saline and 0.1% natrium azide (PBS+NaN 3 ) was used for staining lymphocytes in indirect immunofluorescence.
For each cell suspension (1.10 6 lymphocytes in PBS), cell population acquisition and analysis was carried out based on 10,000 cells using FACScan flow cytometer and Cell Quest programme (Becton Dickinson, Germany).For each marker, the relative percentage of fluorescent positive cells was recorded and absolute subpopulation's lymphocyte counts in peripheral blood were computed as follows: absolute lymphocyte counts/100 × relative % subpopulation's lymphocytes.

Histology and morphometry of duodenum
Routine histological method with haematoxylin-eosin staining was used.Photos of histological sections were taken by Nikon LABOPHOT-2 with camera adapter (DS Camera Control Unit DS -U2) with × 4 magnification.The height and surface area of villi were measured by NIS-Elements programme.The height of villi was measured from the base to the apex.

Statistical analysis
Statistical analysis was done using one-way analysis of variance (ANOVA) with post hoc Tukey multiple comparison test.Differences between the mean values for the groups of chickens were considered significant when P < 0.05.

Results
Determination of peripheral blood heterophils demonstrated their increased (P < 0.002) frequency in birds fed with the 3 mg•kg -1 DON dose than in chickens fed with the 1 mg•kg -1 DON dose.On the contrary, values of monocytes were lower (P < 0.02) in chickens fed with the 3 mg•kg -1 DON dose than in control.Metabolic burst of heterophils was higher (P < 0.001) in birds fed with 1 mg•kg -1 and 3 mg•kg -1 DON doses compared to control (Table 4).
Density of lymphocyte subpopulations in peripheral blood revealed higher frequency of CD3+ cells in birds fed with 3 mg•kg -1 DON dose than in 1 mg•kg -1 DON dose (P < 0.003) and in control (P < 0.003).Numbers of CD4+ and CD8+ cells were higher (P < 0.003, P < 0.016, respectively) in birds fed with 3 mg•kg -1 DON dose than in chickens fed with 1 mg•kg -1 DON dose.However, density of MHC II+ cells was lower (P < 0.001) in birds fed with 3 mg•kg -1 DON dose than in control (Table 5).
Evaluation of intraepithelial lymphocytes showed lower (P < 0.034) density of CD45+ cells in both experimental groups fed with DON than in control chickens, but with tendency in increase of CD8+ cells (Table 6).
During the experiment, chickens did not reveal clinical signs.Similarly, no gross and histological lesions were found in the intestine of birds fed the diet naturally contaminated with deoxynivalenol.Duodenal morphology demonstrated only tendency to decrease the height of villi and surface area of villi in birds fed with 3 mg•kg -1 DON dose compared to 1 mg•kg -1 and control (Table 7).

Table 1 .
Content of mycotoxins in complete diets for control and experimental groups of chickens.

Table 2 .
Composition of diet HYD-02 fed to broilers during the experiment.

Table 3 .
Primary mouse anti-chicken monoclonal antibodies used in the experiment.

Table 4 .
Number of peripheral white blood cells (G•l -1 = 10 9 •l -1 ) and functions of phagocytes in broilers fed diets contaminated with deoxynivalenol.DON -deoxynivalenol.Data are presented as means ± SD (n = 6), different letters within the same row mark significant differences (P < 0.05).

Table 6 .
Relative percentage of duodenal intraepithelial lymphocytes of broilers fed diets contaminated with deoxynivalenol.DON -deoxynivalenol.Data are presented as means ± SD (n = 6), different letters within the same row mark significant differences (P < 0.05).