The objectives of this study were to examine whether DON at concentrations in the feed below the EU maximum guidance level predisposes for NE in broilers, and to gain insights in the mechanisms responsible for this interaction. Therefore, the effects of DON on the intestinal epithelial barrier function and on intestinal protein availability for clostridial proliferation were evaluated. Also, the direct effect of DON on in vitro bacterial proliferation, alpha toxin production and netB transcription was studied. Our data demonstrate that the mycotoxin DON is a predisposing factor for the development of NE in broiler chickens. Indeed, contamination of the diet with DON at concentrations below the EU maximum guidance level of 5,000 mg/kg feed, significantly increased the number of chickens affected with NE. The distribution of NE lesions in the present infection study, mainly in duodenum and jejunum, is similar as in a previously described NE infection trial, where coccidiosis was included as predisposing factor. The proximal part of the intestinal tract is the main absorption site for DON. Such sensitive due to their high protein turnover. DON negatively affected the proximal part of the intestinal tract, demonstrated by the significantly reduced villus height in the duodenum. These results are in accordance with those observed by Awad et al., who tested a similar contamination level and duration of exposure of DON. The decreased villus height will compromise the effectiveness of nutrient absorption due to the decreased absorption surface area. Enterocytes must differentiate during their migration along the crypt-villus axis to fully express their digestive functions. The sucrase and maltase activities increase for example towards the villus tip in chicks. As such, the negative impact of DON on the villus height can be associated with an impaired nutrient digestion due to a reduced number of PCI-32765 differentiated epithelial cells. DON also modulates the intestinal paracellular transport leading to an increased passage of macromolecules and bacteria. The intestinal barrier function is maintained by intercellular structures, including tight junctions, adherence junctions and desmosomes. The TEER is considered as an indicator of the epithelial integrity and thus of the organization of tight junctions. In accordance with literature, we demonstrated a reduction of the TEER of the duodenal epithelium after DON exposure. These toxic effects on epithelial cells contribute to an increased protein availability in the intestinal lumen due to leakage of plasma amino acids or proteins into the gut. Consequently, this creates an environment that favors for massive overgrowth of C. perfringens. Indeed, in this study, the total duodenal protein level was increased. This could be caused by malabsorption, a negative effect on nutrient digestion or plasma amino acid or protein leakage in the intestine due to the altered intestinal barrier integrity.