Genetic Diversity Of Clostridium Perfringens Isolated From Healthy Broiler Chickens At Commercial Farms

In order to fully know the occurrence of Clostridium perfrigens in chicken from poultry farms in Sichuan province,this study was carried out to isolate Clostridium perfringens from chickens and to detected the gene encoding theα-toxin,β-toxin,ε-toxin andι-toxin by a multiplex polymerase chain reaction (Multi-PCR).Our objective was to characterize the population diversity of Clostridium perfringens colonizing healthy birds.In order to subtype Clostridium perfringens isolates,the two methods ERIC-PCR and REP-PCR were compared on Clostridium perfringens type A isolates.A probiotic is defined as a live microbial feed supplement that beneficially affects the host animal by improving its intestinal microbial balance. Few studies have been performed on the protective effects of probiotic strains against C.perfringens in chickens.The aim of this study was to obtain lots of probiotic which can inhibit C.perfringens in vitro.150 samples were collected from four different region including Yaan,Minshan Shuangliu,Deyang.Eight strains of C.perfringens were isolated from fecal specimens and intestines.In this study,five medium[sheep blood agar added 1%glucose,tryptose sulfite cycloserine(TSC) agar,tryptone sulfite neomycin(TSN),sulfite polymyxin sulfadiazine(SPS) agar,brain heart infusion-egg yolk agar medium added kanamycin(CW)]were compared.Eligibility of a medium for a specific microorganism is based on both the selectivity of the antibiotic(s) used and the growth-supporting capabilities of the agar for the organism of choice.The medium utilized D-cycloserine as the selective agents to give a high degree of selectivity and specificity for C.perfrigens.Additionally the growth of C.perfringens was well.After overnight incubation,C.perfringens produced black colonies and an opaque zone around it due to lecithinase activity.The obviously characteristic easily distinguished C.perfringens from the other strains.It was concluded that TSC was the most favorable medium for the isolation of C.perfringens from fecal and intestinal contents.Isolated strains were inoculated into iron milk medium(IMM) and made stormy fermentation.The isolates were identified and genotyped by Multi-PCR.A simple and type-specific multiplex PCR could distinguish type A,B,C,D and E of C.perfringens. The 402 bp amplification product of cpa was visualized under UV light after electrophoresis in a 2%agarose gel.The results showed that all isolated C.perfringens belong to type A.The specific of the PCR is higher only C.perfringens show positive, other 7 strains(E,coli 08,Salmonella 339,Bacillus cereus SA39,Bacillus aeruginosus,Staphylococcus aureus) all showed negative.To study the pathogenesis and epidemiology of disease caused by C.perfringens, methods for typing its various strains need to be evaluated.In order to subtype isolates, the two methods ERIC-PCR and REP-PCR were compared.All 8 isolates were typeable(100%typeability) by repetitive-element PCR.Isolates were classified into five subgroups or clades by ERIC-PCR and three subgroups or clades by REP-PCR. This subtyping method was highly reproducible and discriminatory.To test the efficacy of probiotics in inhibiting C.perfringens,9 Lactobacillus strains(D11,D22,E31,E41,E42,F61,G31,G52,H51)and 8 Bafillus(ND1,ND2,ND3,ND4,ND5,ND6,JS01,Z2) were evaluate in vitro.6 strains,E41,G31,H51,ND2,ND3,Z2 were screened from the 17 bacteria strains by bacteriostatic test.