The mycotoxin deoxynivalenol predisposes for the development of Clostridium perfringens-induced necrotic enteritis in broiler chickens

Abstract

Both mycotoxin contamination of feed and Clostridium perfringens-induced necrotic enteritis have an increasing global economic impact on poultry production. Especially the Fusarium mycotoxin deoxynivalenol (DON) is a common feed contaminant. This study aimed at examining the predisposing effect of DON on the development of necrotic enteritis in broiler chickens. An experimental Clostridium perfringens infection study revealed that DON, at a contamination level of 3,000 to 4,000 µg/kg feed, increased the percentage of birds with subclinical necrotic enteritis from 20±2.6% to 47±3.0% (P<0.001). DON significantly reduced the transepithelial electrical resistance in duodenal segments (P<0.001) and decreased duodenal villus height (P = 0.014) indicating intestinal barrier disruption and intestinal epithelial damage, respectively. This may lead to an increased permeability of the intestinal epithelium and decreased absorption of dietary proteins. Protein analysis of duodenal content indeed showed that DON contamination resulted in a significant increase in total protein concentration (P = 0.023). Furthermore, DON had no effect on in vitro growth, alpha toxin production and netB toxin transcription of Clostridium perfringens. In conclusion, feed contamination with DON at concentrations below the European maximum guidance level of 5,000 µg/kg feed, is a predisposing factor for the development of necrotic enteritis in broilers. These results are associated with a negative effect of DON on the intestinal barrier function and increased intestinal protein availability, which may stimulate growth and toxin production of Clostridium perfringens.

Figure 1. Lesion scores of individual broiler chickens challenged with C. perfringens.

Chickens were fed either a control or DON-contaminated diet and subsequently challenged with C. perfringens strain 56. The solid bars represent the average lesion score in each group. Error bars represent SEM. Intestinal lesions in the small intestine (duodenum to ileum) were scored as previously described ; 0 no gross lesions; 2 small focal necrosis or ulceration (one to five foci); 3 focal necrosis or ulceration (six to 15 foci); 4 focal necrosis or ulceration (16 or more foci); 5 patches of necrosis 2 to 3 cm long; 6 diffuse necrosis typical field cases. The score 1 used for congested intestinal mucosa was not applied here because of difficulties in scoring this characteristic objectively, and due to the lack of scientific documentation of an association between “congested intestinal mucosa” and necrotic enteritis. Birds with lesion scores of 2 or more were classified as NE positive.

Figure 2. Protein concentration in intestinal content is significantly increased in duodenum of chickens fed a DON-contaminated diet.

Percentage crude protein per dry matter of the intestinal content was determined by the Kjeldahl method. Results are presented as the mean protein level of 27 samples per group per intestinal segment. Error bars represent SD. ^(*) significantly different (P<0.05) within one intestinal segment.

Figure 3. No impact of DON on in vitro growth of C. perfringens.

C. perfringens strains 6 (a) and 56 (b) were grown in TGY broth medium containing 0, 0.2, 2 or 20 µg DON/mL. Samples were taken at 0, 2, 3, 4, 5, 6, 7, 8 and 24 h after inoculation with an overnight culture of C. perfringens. The number of colony forming units (cfu) per mL was determined by bacterial plating of 10-fold dilutions. Results are presented as the mean cfu/mL. There is no significant difference between the different test conditions.

Figure 4. NetB toxin transcription is not influenced by DON.

Transcription level of netB toxin was analysed by qRT-PCR of C. perfringens strain 56 RNA samples collected from in vitro culture material in the mid (after 3 h incubation) and late logarithmic (after 6 h incubation) growth phase. C. perfringens strain 56 was grown in absence or presence (0.2, 2, 20 µg/mL) of DON. The results for the netB gene transcription were normalized to the rpoA gene transcription. Results are presented as the mean value of three biological replicates. Error bars represent SD. There is no significant difference between the different test conditions.

Figure 5. Deoxynivalenol predisposes for C. perfringens induced necrotic enteritis.

DON decreased villus height and reduced transepithelial electrical resistance (1), leading to a decreased absorption and digestion of dietary nutrients; and an increased intestinal barrier permeability, respectively. Taken together with an increased intestinal protein level, these results suggest an impaired nutrient uptake (2) and leakage of plasma amino acids (3) into the intestinal lumen, providing the necessary growth substrate for C. pefringens proliferation (4). Proliferation of virulent (netB positive) C. perfringens induces necrotic enteritis (5).