Detection Of Virulence Factors Of Clostridium Perfringens From Broiler Chickens And Effect On Ileal Tissue And Flora Of Chickens Exposured To Clostridium Perfringens CVCC2030

Necrotic enteritis in broiler chickens is associated with Clostridium perfringens type A, carrying the NetB toxin. Clostridium perfringens type A is also a member of the normal intestinal microbiota of broilers.To investigate the occurrence of the NetB toxin gene in Chinese Clostridium perfringens strains,75 isolates of Clostridium perfringens were examined by PCR. Then detecting two causative agents (collagenolytic enzymes activity and Intra-species growth-inhibition)of Clostridium perfringens strains to screen strains which were used for animal experments.Through doing three animal experments to find the optimal Clostridium perfringens strains and the optimal dose of coccidian and Clostridium perfringens, then observe the effect on the lengh of ileal villus, ileal histopathological change and ileal microbial population structure.The present study investigated the disease producing capability of NetB negative strains recovered from chickens with necrotic enteritis.And this study have plombed a research blank.1.Isolation and identification of Clostridium perfringens from chickens93 samples of chickens with enteritis or diarrhea were collected from different commercial farms in ChengDu and Yaan Sichuan province.19 isolates were selected by blood agar(with 1% glucose), CW agar, TSC agar. The isolating rate was 20.4%.2.Detection of causative agent of Clostridium perfringens75 isolates were examined for the presence of the NetB gene follow the protocol described by literature.And the result shows the detection rate is 0.The results of collagenolytic enzymes activity and Intra-species growth-inhibition shows that different origin of Clostridium perfringens isolates have similar collagenolytic enzymes activity,but have different capability of intra-species growth-inhibition.3.animal experimentIn animal experiment one, comparing the effect on ileum tissue of broiler chickens exposured to Clostridium perfringens CVCC2030 or FR and comparing the effect on ileum tissue between using Clostridium perfringens alone or combination with coccidian. The lengh of villus intestinalis of ileum and histopathological change of ileum reflect degree of necrosis of ileum.To find the change regularity of Clostridium perfringens by detecting viable count after the challenge. The results showed that inoculating coccidian can increase the number of Clostridium perfringens in ileum and can enhance the damage effect of Clostridium perfringens to ileum. And Clostridium perfringens combine with coccidian can get better pathopoiesis result. After challenge, the number of Clostridium perfringens in ileum increased by time, and reach peak value at 23d, then reduced.In animal experiment two, comparing the effect on ileum tissue of broiler chickens by using coccidian and Clostridium perfringens CVCC2030 with different dosage. The results showed that coccidian and Clostridium perfrengens CVCC2030 with different dosage caused damage of ileum have no differnce in the lengh of villus intestinalis and histopathological change.In animal experiment three, all birds of treatment group were inoculated with coccidian(1000,000 oocysts per mL; 1 mL per bird) at 15d, then subsequently inoculated on days 17,18,19 and 20 with Clostridium perfringens CVCC2030 type A((1-2)×108 CFU/mL/bird; 1 time per day). All biuds in control group were inoculated with equal volume physiologic saline at 15d, then subsequently inoculated on days 17,18,19 and 20 with equal volume sterile cooked meat medium. The results of the lengh of ileal villus, ileal histopathological change and ileal microbial population showed that Coccidian and Clostridium perfringens CVCC2030 just can induce ileal inflammatory reaction and focal damage, and cannot induce severe tissue necrosis.They also can reduce the number of Bacillus acidi lactici, Bacillus bifidus, enterococcus, bacteroid, total anaerobes in ileum, and can increase the number of Escherichia coli, total aerobic in ileum, then induce alteration of ileal flora.