Predisposing effect of the mycotoxins deoxynivalenol and fumonisins on necrotic enteritis in broiler chickens

Mycotoxins are secondary fungal metabolites, which frequently contaminate feed and food raw materials. The Fusarium mycotoxins deoxynivalenol (DON) and fumonisins (FBs) are the most frequently detected mycotoxins on a worldwide basis. The majority of feed samples however comply with European Union (EU) maximum guidance levels. The EU maximum guidance level for poultry feed is set at 5 mg DON/kg feed and 20 mg fumonisin B1 (FB1) + fumonisin B2 (FB2)/kg feed. Although these low to moderate contamination levels do not result in obvious clinical symptoms of intoxication, they may impair intestinal health, immune function and/or pathogen fitness, resulting in altered host pathogen interactions and thus a different outcome of eventual infections. Necrotic enteritis (NE) is one of the economically most important enteric diseases in poultry production. NE in broiler chickens may arise when changes in the gut microbial homeostasis allow colonization of the virulent Clostridium perfringens strains expressing the NetB toxin gene. The presence of predisposing factors such as coccidiosis and fishmeal is required in the pathogenesis of NE, allowing the proliferation of C. perfringens by either providing nutrients and/or favorable ecological niches. The general aim of this thesis was to examine whether DON and FBs, at concentrations in the feed approaching EU maximum guidance levels, predispose for NE in broiler chickens; and to gain insights in the mechanisms responsible for this interaction. Chapter 1 describes an experimental C. perfringens infection study in order to evaluate the predisposing effect of DON exposure to the development of NE in broiler chickens. After C. perfringens challenge, DON significantly increased the percentage of birds developing subclinical NE compared to the control group (47±3.0% vs. 20±2.6%). A negative impact of DON was demonstrated on intestinal morphology and the intrinsic component of the intestinal barrier, which is composed of the epithelial cell layer and the tight junctions that interconnect these epithelial cells. Consequently, this intestinal damage led to an increased luminal protein content by leakage of plasma proteins or altered absorption of dietary proteins, which stimulate clostridial proliferation and subsequently the development of NE. In Chapter 2 the impact of feeding a FBs contaminated diet on the intestinal morphology and microbiota composition was studied. The uptake of FBs contaminated feed also induced a negative impact on the intrinsic component of the intestinal barrier, and on the intestinal microbiota as well. A reduced abundance of the immunomodulating bacteria Candidatus Savagella and Lactobacillus spp. such as L. johnsonii, and an increased number of C. perfringens were observed in the ileum of birds fed a FBs contaminated diet. Subsequently, an experimental C. perfringens infection study was performed investigating the predisposing effect of FBs exposure to the development of NE in broiler chickens. After C. perfringens challenge, a significant higher percentage of birds developed subclinical NE in the group fed a FBs contaminated diet compared to the control group (45±2.2% vs. 30±5.5%). In Chapter 3, the impact of DON and FBs on the intestinal barrier was further examined for selected functional and structural extrinsic components of the intestinal barrier, to gain insights in the associated consequences for intestinal colonization and nutrient availability for C. perfringens. In the first part (Chapter 3.1), a negative impact of feeding broiler chickens a DON and/or FBs contaminated diet on intestinal mucus layer integrity was observed and induction of oxidative stress in intestinal epithelial cells. The last study (Chapter 3.2) investigated whether DON-induced intestinal damage influences the intestinal absorption of FBs, which may lead to an altered systemic exposure and increased toxic effects of the latter mycotoxin in broiler chickens. Although, no differences in toxicokinetic parameters of FB1 could be demonstrated between chickens fed a DON contaminated diet compared to birds fed a control diet. This doctoral thesis demonstrates for the first time that feeding a DON or FBs contaminated diet is a predisposing factor for the development of C. perfringens induced NE in broiler chickens. This coincides with negative effects on selected components of the intrinsic and extrinsic intestinal barrier of the chicken host, i.e. villus height, tight junctions, mucus, oxidative stress, and microbiota homeostasis. Consequently, exposure to DON and/or FBs at concentrations approaching the EU maximum guidance level in feed provides both nutrients and a favorable ecological niche for clostridial proliferation. Therefore, this doctoral thesis demonstrates that in addition to the best-known predisposing factors, DON and/or FBs contaminated feed should be included as major risk factor for the development of C. perfringens induced NE in broiler chickens.