Molecular Epidemiological Study Of Clostridium Perfringens Type A Containing ?₂ Toxin Gene From Piglets In Jiangxi Partial Area And Screening Of Lactobacillus Against CpA

Clostridium perfringens?Cp? is considered be one of the most common causes of enteritis in livestock, according to the different types of pathogenic toxins, the Cp can be divided into five different toxin type A, B, C, D, and E, of which, Clostridium perfringens type A and C?CpA and CpC?are major pathogens of swine intestinal diseases. In past years,Clostridial enteritis of piglets was mainly caused by the CpC, but recently the number of piglets enteritis reports caused by the CpA increased a lot, and the studies demonstrated the beta 2-toxin played an important role in the pathogenesis of neonatal enteritis in pigs. Due to the problems of antibiotic resistance we faced a lot difficulty to prevent piglets clostridial enteritis in clinical, and lactobacillus can markedly inhibit the CpC. So the aim of this study was to investigate the epidemiology of CpA containing ?₂ gene?CpA-?₂? in Jiangxi province, analyze the intestinal microflora community diversity of the healthy and diarrheal piglets and screen lactobacillus which can inhibite CpA from healthy piglets for further study. Finally, the in vitro inhibition test of CpA and the cloning and expression of CpA strain of beta 2 toxin gene were carried out to provide reference and materials for the prevention and treatment of CpA.In this study, we detected 255 fecal samples from both healthy and diarrheal piglets by established double and multiplex PCR. Results showed CpA was the only genotype can be detected in 96% of the samples, and all of them were detected ?₂ toxin gene at the same time; 229 strains of Clostridium perfringens were isolated from piglets fecal samples, and all turn up to Clostridium perfringens type A, and the results proved there had no significant difference between healthy and diarrheal piglets.We analyzed intestinal microflora of healthy and diarrhea piglets by DGGE and high-throughput sequencing. The results showed diarrhea significantly affected neonatal intestinal flora diversity, which resulting in reduction of bacterial diversity; Fusobacterium is a dominant microflora in the feces of diarrhea piglets, and had a higher proportion of21.78% compare to the healthy piglets, the proportion of Lactobacillus was lower?0.12%?than the healthy piglets, this results showed Fusobacterium and suckling piglets diarrhea had close connection; Lactobacillus might inhibit the colonization of Fusobacterium in suckling piglets intestinal.We screened the lactobacillus which can inhibite CpA from healthy piglets fecal samples by using MRS medium and resistance assays. Finally, three candidate strains of lactobacillus were selected from 212 strains. of these three strains, two strains were identified as Lactobacillus salivarius and one stains was Lactobacillus plantarum by16 SrDNA sequencing.The results of Antibiotics inhibition test of CpA agaist variety of feed additive antibiotics demonstrated that Enramycin, olaquindox and 15% Bacitracin Zinc had goodantibacterial effects. Then, we cloned ?₂ toxin gene and expressed recombinant ?₂ toxin protein and the size of the recombinant ?₂ toxin protein was 47 kDa.The results of this study showed CpA was correlated with porcine diarrhea. and toxin beta 2 was likely to be involved in the process of piglet enteritis pathology caused by CpA,Lactobacillus might inhibit the colonization of CpA in the intestinal tract of piglets.Enramycin, olaquindox and 15% Bacitracin Zinc can be used to prevent CpA enteritis.Recombinant ?₂ toxin proteins can also be employed to study the roles of ?₂ toxin in the pathogenic process of piglets infected with Clostridium and be the candidate genetic engineered subunit vaccine.