Lymphocyte subpopulations in blood and duodenal epithelium of broilers fed diets contaminated with deoxynivalenol and zearalenone

Abstract

The effects of two dietary levels of Fusarium mycotoxins deoxynivalenol (DON) and zearalenone (ZEA) on subpopulations of peripheral blood and duodenal intraepithelial lymphocytes in Ross 308 hybrid broiler chickens of both sexes were examined. From the day of hatching to the age of two weeks three groups of chickens were fed the same uncontaminated control diet. After 14 days, broilers in two of the groups were switched to otherwise identical experimental diets containing DON and ZEA. The diet of Group 2 contained DON and ZEA, each at a level of 3.4 mg/kg and the Group 3 diet contained DON and ZEA at 8.2 and 8.3 mg/kg, respectively. Contaminated diets were prepared in the laboratory using maize cultivated with Fusarium graminearum. Group 1 (control) continued with intake of uncontaminated diet for entire experiment. At the age of four weeks samples of blood and duodenal tissue were collected. Intake of both contaminated diets reduced blood phagocytic activity (P<0.001) whereas numbers of various types of peripheral white blood cells were not affected. Among different blood lymphocyte populations investigated, only the counts of CD3⁺ cells were lowered by diet with higher mycotoxin levels (P=0.021). In duodenal epithelium, the large increase in cells expressing MHC II was observed due to intake of both diets contaminated with lower and higher mycotoxins contents (P<0.001) while the IgA⁺ cell subpopulation was decreased (P=0.018). The other intraepithelial lymphocyte subpopulations in duodenum examined (CD3⁺, CD4⁺, CD8⁺ and CD44⁺) were not influenced by intake of contaminated diets (P>0.05). Results suggest that intake of diets contaminated with DON and ZEA at medium levels may affect some blood and intraepithelial duodenal lymphocyte subpopulations and compromise blood phagocytic activity in broilers.

Introduction

Mycotoxins are secondary metabolites of mould that may contaminate food and feed materials. Consumption of mycotoxins may result in impaired immunity and decreased resistance to infectious diseases (Li et al., 2005, Awad et al., 2008).

According to chemical structure, deoxynivalenol (DON) is a member of trichothecene mycotoxin group and belongs to most commonly detected mycotoxins in feedstuffs produced in EU countries. The mode of toxic action of DON is inhibition of protein synthesis, thus affecting rapidly dividing cells, such as those of the gastrointestinal tract and the immune system. DON is absorbed through the intestinal epithelium by simple diffusion and inhibits intestinal cell proliferation (Sergent et al., 2006).

Numerous studies demonstrated trichothecene effects as immunostimulatory or immunosuppressive depending on dose, exposure frequency and timing of functional immune assay (Bondy and Pestka, 2000, Bouhet and Oswald, 2005). High trichothecene exposure injures actively dividing tissues including bone marrow, lymph nodes, spleen, thymus and intestinal mucosa and leads to reduction in circulating blood leukocytes (Pestka et al., 2004). In contrast, low trichothecene exposure may elevate serum IgA levels due to enhanced premature differentiation of IgA secreting cells in Peyer's patches (Pestka, 2007, Pinton et al., 2008). DON was shown as the agent altering claudin expression and thus reducing the barrier function of the intestinal epithelium in pigs (Pinton et al., 2009). In poultry, DON disrupts gut glucose and amino acid transport and reduces the height and width of villi in the duodenum (Awad et al., 2006, Awad et al., 2008). Poultry is known to be relatively resistant to dietary DON in comparison to pigs but more sensitive than ruminants. Diet contaminated with DON at 10 mg/kg had no effect on the growth performance of broilers (Awad et al., 2004). A lowest observed adverse effect level (LOAEL) 9 mg DON/kg feed was suggested for poultry (Eriksen and Pettersson, 2004). Finding of median lethal oral dose (LD₅₀) of 140 mg DON/kg b.w. in 1-day-old broiler chickens also indicates a relative insensitivity of broilers to DON toxicity (Huff et al., 1981).

Zearalenone (ZEA) is a worldwide contaminant of cereals and grains, including maize and soybean. Despite its non-steroidal structure, ZEA activates estrogen receptors resulting in functional and morphological alteration in reproductive organs (Dänicke et al., 2003, Fink-Gremmels and Malekinejad, 2007). The immune system is a potential target for estrogenic endocrine disruptors considering that its cells express estrogen receptors (Igarashi et al., 2001). Contrary to other species, poultry appear to be fairly resistant to ZEA effects. No adverse effects on zootechnical performance of broilers and laying hens were observed at dietary ZEA level up to 800 mg/kg (Chi et al., 1980, Allen et al., 1981).

In practice the food and feed commodities are frequently contaminated with more than one mycotoxin (Speijers and Speijers, 2004); therefore the experimental approach using diets significantly contaminated with both DON and ZEA may yield new information. It is well known that some Fusarium toxins can exert additive and/or synergistic effects. In this study we investigated the effects of feed contaminated with lower and higher DON and ZEA levels on poultry lymphocyte subpopulations by flow cytometry examination of peripheral blood and duodenum epithelium.