The Analysis Of Retail-Fresh-Chicken Polluted In Guang Zhou City By Clostridium Perfringens And Its Pathogenicity Of NetB Strain

Necrotic enteritis is caused by Clostridium perfringens type A or type C isolates and its one of the most important enteric diseases in poultry and is a high cost to the worldwide.Clostridium perfringens can also causes human disease,in the western developed countries, the food poisoning caused by Clostridium perfringens ranked the second among the food poisoning cases, and the human diarrhea contact with Clostridium perfringens,According to foreign research, netB strain of Clostridium perfringens can cause necrotic enteritis.In order to investigate the distribution and pollution of Clostridium perfringens and analyze the potential hazards to people,and its pathogenicity of netB strain.Retail Cold fresh chicken samples were collected randomly from Supermarkets in Guangzhou,Selective media was used to isolate Clostridium perfringens,Biochemical test and iron milk medium test and Specific PCR of Clostridium toxin were used to identify and confirm the Clostridium perfringens gene type and drug sensitivity was used to analysis the drug resistance of Clostridium perfringens and ERIC-PCR was used to subtyping the isolates.A new method reported by Keyburn et al,was used to study the pathogenicity of netB in Clostridium perfringens isolated from chickens necrotic enteritis, intestinal microflora changes of the jejunum and ileum were analysis by denaturing gradient gel electrophoresis(DGGE)to analysis the jejunum and ileum intestinal microflora changes from netB group.This work could probide useful date for the prevention and control the disease of people related with Clostridium perfringens and chicken necrotic enteritis.70 isolates were isolated from 102 chicken samples, the isolates and gentyped by Multiple PCR method refered to Yoo et al. only one band was showed in all the Multi-PCR results and this band amplified was 402 bp by gene sequencing, consistent with Clostridium perfringens alpha toxin gene sequence in Genbank and its also consistent with multiple PCR estimate,all isolates were Clostridium perfringens type A. netB, cpb2(Atypical) and cpb2(Consensus) genes were also detected by PCR, 48 isolates carried cpb2( Atypical) gene, there was no isolate that carried netB and cpb2(consensue) genes.Enterobacterial Repetitive Intergenic Consensus Senquence is an extremely conservative in the animal intestinal pathogenic bacteria and a variety of bacterial genome,to design the primers by ERIC to analysis the subtype of Clostridium perfringens.With the NTSTY software of data by UPGMA cluster analysis, the similarity coefficient of 83%, they were classified as one subtype, 70 strains of Clostridium perfringens were divided into 24 subtypes.The drug sensitivity test was according to the CLSI standard. Results of drug resistance test showed that most of isolates were resistant to clindamycin(75.71%), Enrofloxacin(62.86) Lincomyci(81.43%)、Erythromycin(74.28%)、doxycycline(68.57%)and Oxytetracycline(72.86%)cefalexin(31.58%),most of isolates were sensitive to florfenicol, penicillinand ampicillin. It noteworthy that most of these isolates were resistant to clindamycin(75.71%) and cefalexin(31.43%),which can only be used in human being.whether the Clostridium perfringens of netB strain can cause chicken necrotic enteritis has always been a focus of researchIn the pathogenicity test of Clostridium perfringens netB strains from chicken necrotic enteritis,blank group, there were three groups standared A type group, netB group test results show that only in the netB group necrosis of the intestinal mucosa appeared, and the lesions were mainly concentrated in jejunum and ileum,the blank group were well; standared A type test group, intestinal mucosal became thickening, there were individual congestion and swelling but no intestinal mucosal shedding and necrosis. It means the pathogenicity of netB positive strain of Clostridium perfringens strains from chicken is very strong.There are16 s rRNA gene sequence s in all species of bacteria,and this16 s rRNA gene sequence was highly conservative and specific. A special pair of primers containing GC clamp was designed, and PCR products of DNA of each species of bacterium was unique, so after DGGE, one bands pattern means one species of bacterium,by this way we can analyze changes in the intestinal microflora.Six bird’s intestinal contents of each group were collected respectively before DNA extraction, then PCRs were done,and products were showed by DGGE. the microfloras from jejunum to ileum in netB gene positive group were much more richer than that in the blank control, represented extremely significant difference(P<0.01), that is to say after inoculated with netB positive C. perfringens, the chickens’ microbial diversity were significantly increased in the intestinal from jejunum to ileum.