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DON: a predisposing factor for necrotic enteritis

It has been recently discovered that the Fusarium mycotoxin deoxynivalenol (DON) is a predisposing factor for necrotic enteritis (NE) in broiler chickens. This is because this mycotoxin - even at low levels - damages the intestinal wall of the chicken’s gut.

By Gunther Antonissen, Ghent University, Belgium

Necrotic enteritis leads globally to important production losses, increased feed consumption and mortality rates, and a reduced welfare of broiler chickens. Necrotic enteritis is caused by Clostridium perfringens, a bacterium which is ubiquitously present in the environment, in feed and in the gastrointestinal tract of animals and humans. It may present as acute clinical disease or pass subclinical. The acute clinical form of the disease is characterised by a sudden increase in flock mortality, often without premonitory signs. Nowadays, the subclinical form is becoming more prevalent, and is mainly characterised by intestinal mucosal damage without clinical signs or mortality. This leads to a decreased digestion and absorption of nutrients, a reduced weight gain and an impaired feed conversion rate. Notwithstanding the role of C. perfringens in poultry production losses, the mere presence of virulent strains in the intestinal tract of broilers does not always lead to the development of NE. Predisposing factors including dietary, husbandry and immune factors, are required to induce the disease.

Effect of DON in broilers
The mycotoxin deoxynivalenol (DON) is one of the most common contaminants in poultry feed worldwide. DON is produced by Fusarium (F.) fungi among others F. graminearum and F. culmorum. Recent data on global mycotoxin occurrence showed that 59% of 5,819 samples of animal feed tested positive for the presence of DON. The European maximum guidance level for poultry feed is set at 5,000 µg/kg feed. It has been suggested that concentrations higher than 5,000 µg/kg feed are necessary to negatively influence the growth performance of broilers. As a consequence of the negative effect of DON on the intestine, feeding DON-contaminated diets can lead to greater susceptibility to enteric infections. It was hitherto unknown, whether the intestinal epithelial damage caused by contamination levels of DON below 5,000 µg/kg in feed, may act as an additional predisposing factor in broiler necrotic enteritis.

To induce experimentally necrotic enteritis, chickens are infected with C. perfringens at the age of three weeks. To investigate the effect of a DON-contaminated feed on the broiler susceptibility for necrotic enteritis, such an experimental C. perfringens infection trial was conducted.  Chicks were divided into 4 experimental groups, one group was experimentally infected with C. perfringens and received a control diet. A second group was experimentally infected with C. perfringens and received a DON contaminated diet, while a third group was fed a DON contaminated feed but did not receive C. perfringens.

A fourth group was a negative control (no C. perfringens and control feed). The DON contamination level in the different batches of contaminated feed varied between 2,884 ± 800 µg/kg and 4,384 ± 1,300 µg/kg feed. Feeding a DON contaminated diet resulted in more chickens with necrotic enteritis, i.e. 20 ± 2.6% of the chickens in the group inoculated with C. perfringens and fed a control diet had necrotic enteritis lesions, while in the group inoculated with C. perfringens and fed a DON-contaminated diet 47 ± 3.0% of the broilers had necrotic enteritis (P<0.001) (>Figure 1).  No animals with lesions were detected in the groups without bacterial challenge.

Reduced villi length
The small intestine is the major part of the gastrointestinal tract for digestion and absorption of nutrients. The intestinal epithelium, which consists of an organised structure of intestinal epithelial cells, covers the intestine. Following ingestion of DON-contaminated feed, these intestinal epithelial cells are exposed to high concentrations of this mycotoxin. DON has a negative impact on the morphology of the proximal part of the intestinal tract, demonstrated in our study by the significantly reduced villus height in the duodenum. The average duodenal villus height was 2,175 ± 26.8 µm and 2,010 ± 52.9 µm, respectively, of chickens fed a control feed or a DON-contaminated feed. The decreased villus height will compromise the effectiveness of nutrient absorption due to the decreased absorption surface area. DON also modulates the transport of molecules across the intestinal epithelial layer. The intestinal barrier function is maintained by intercellular structures such as tight junctions. The trans-epithelial electrical resistance (TEER) is considered as an indicator of the epithelial integrity and thus of the organisation of tight junctions. We demonstrated a reduction of the TEER of the duodenal epithelium after DON exposure, respectively 369.8 ± 5.47 Ω.cm² in DON-fed birds compared with 392.2 ± 4.72 Ω.cm² in control birds.

Environment for bacteria overgrowth
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. In order to proliferate and cause necrotic enteritis, C. perfringens needs proteins. The total protein concentration in duodenal intestinal content was significantly higher in chickens fed the DON contaminated diet (P=0.023)(Figure 2). 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. Consequently, this creates an environment that favours for massive overgrowth of C. perfringens, and subsequently necrotic enteritis.

Conclusion
In conclusion, our results indicate that the intake of DON contaminated feed at contamination levels below the EU maximum guidance level, is a predisposing factor for the development of necrotic enteritis in broiler chickens due to the negative influence on the intestinal epithelium, and to an increased intestinal nutrient availability for bacterial proliferation. We showed that DON has a cytotoxic effect on enterocytes, leading to an altered intestinal barrier function, resulting in an increased permeability of the intestinal wall. Additionally, the shortened villus height could lead to a decreased absorption of dietary nutrients, resulting in an increased protein concentration in the intestinal lumen. These mechanisms lead to an increased protein content in the intestinal lumen, which is available for clostridial proliferation resulting in the development of necrotic enteritis.

[Source: Managing mycotoxins, 2014]

References are available on request.
This article is based on the original paper: Antonissen G., Van Immerseel F., Pasmans F., Ducatelle R., Haesebrouck F., Timbermont L., Verlinden M., Janssens G.P.J., Eeckhaut V., Eeckhout M., De Saeger S., Hessenberger S., Martel A., Croubels S.
The Mycotoxin Deoxynivalenol predisposes for the development of Clostridium perfringens-induced Necrotic Enteritis in Broiler Chickens. PlosONE, 2014.

Gunther Antonissen

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