Isolation, Identification And Genetic Diversity Of Clostridium Perfringens Of Healthy Broiler Chickens From Commercial Farms

Clostridium perfringens is a common pathogenic bacterium implicated in enteric diseases of animals by production of a variety of toxins. The aim of study is to investigate the epidemiology and genetic diversity of C. perfringens isolated from healthy chickens at commercial farms, to analyze the relationship between the epidemiology and regional difference, and methods for typing its various strains were evaluated. Thirty-four strains of C. perfringens were isolated from 10 different farms of 8 cities in Sichuan province. The gene subtype and genetic diversity of there strains were analyzed by multiplex-PCR technique, Amplified Fragment Length Polymorphism (AFLP), Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) and Repetitive Extragenic Palindromic Polymerase Chain Reaction (REP-PCR). This study is important based on the deficiency of C. perfringens research in China, and it has particular significance in preventing and diagnosing disease of C. perfringens.1 100 cecal and 500 fecal samples of healthy chickens were collected from different commercial farms in Chengdu, Yaan, Yibin et al of Sichuan province. 34 strains of C. perfringens were selected by culture with selective medium. The isolating rate of C. perfringens is 5.7%, which is lower than abroad reports. Isolates were tested for susceptibility to common antimicrobials. All isolates were deemed resistant to benzylpenicillin, gentamicin, vancomycin, et al.2 The isolates were identified and genotyped by multiplex PCR assay for the present ofα,β,εandι. The results showed that a simple and type-specific multiplex PCR could distinguish type A, B, C, D and E of C. perfringens. The 400 bp amplification product of cpa was visualized under UV light after electrophoresis in a 1.5% agarose gel. All isolated C. perfringens belong to type A, this result was different from abroad reports which found alpha toxin positive. The specificity of the PCR is confirmed by the results that only C. perfringens show positive. The main advantages of the method was its specificity and of great value to apply and extend in epidemiologic survey, prevention and diagnosis.3 In order to subtype isolates, the methods AFLP, ERIC-PCR and REP-PCR were compared. Various experimental conditions of AFLP technique were optimized and confirmed. The results of the study were handled with cluster-analysis. The results showed that genetic diversity among these strains was existed. AFLP differentiated 34 strains into 12 different subtypes; ERIC-PCR suggested that 8 genotypes existed, and REP-PCR suggested that 7 genotypes existed, which were found to be equally suitable for subtyping of C. perfringens isolates. At the level of 58 % similarity, all trains collected together, and at the similarity of 75%, 12 AFLP genotypes formed. The subtype of C. perfringen in this study appears to be simple within a single farm, and mixed with other subtypes. The diversity of C perfringens in this study appears to be simple in China. There was close correlation between the epidemiology and regions.