The Research Of Diagnosing Clostridium Perfringens Disease In Rabbits And Biological Characteristics Of Isolated Strains

Clostridium perfringens has been widely existed in soil,intestines of humans and animals,which can cause various diseases.Clostridium perfringens disease in rabbits is an acute,lethal diarrhea infectious disease caused by type A Clostridium perfringe ns.Clostridium perfringens disease is acute onset and high mortality,which causes hu ge economic losses to livestock countries.This study was conducted to diagnose the suspected sick rabbits from two scaled rabbit farms in Linyi and Xintai,Shandong Province in November 2016 and November 2017.The suspected sick rabbits were detected by bacterial separation and identification,DAS-ELISA and immunohistochemistry,which was used for the comprehensive diagnosis of Clostridium perfringens disease.The serotype identification and drug resistance analysis were performed on pathogens isolated from diseased rabbits,and the best toxin production time and the ability of toxin production were detected by using lecithin hydrolysis assay and DAS-ELISA method,screening out strains with good toxin production effects,in order to provide reference for the diagnosis and prevention of Clostridium perfringens disease.Clinical symptoms observation and necropsy results showed that the clinical features and the pathological changes of suspected sick rabbits were consistent with the rabbits which are infected with Clostridium perfringens.By means of bacterial separation and identification,a total of 9 isolates were obtained,which the positive isolation rate in Linyi and Xintai rabbit farm was 66.7% and 83.3%,respectively.By using the multiple PCR identification,all of the isolates were identified as Clostridium perfringens type A.DAS-ELISA was used to detect the level of toxins in cecal contents of rabbits with suspected Clostridium perfringens.The positive rates of Linyi and Xintai rabbit farm were 83.3%.The liver,kidney and stomach of sick rabbits were detected useing immunohistochemical method,as a positive criterion,?toxin should be detected in one organ at least,the positive rate in Linyi and Xintai rabbit farm was 83.3% and 100%,respectively.Antimicrobial susceptibility test results showed that the isolates of sick rabbits fromLinyi rabbit farm were resistant to gentamicin and tetracycline,the drug resistance rate reached 100%,and the isolates were sensitive to penicillin,ampicillin,erythromycin,norfloxacin,ciprofloxacin,lincomycin,and bacitracin;The isolates of sick rabbits from Xintai rabbit farm were resistant to gentamicin,tetracycline,penicillin,ampicillin,erythromycin and bacitracin,the resistance rates were 100%,60%,60%,60%,60% and 20%,respectively;The isolates of sick rabbits from Xintai rabbit farm were sensitive to norfloxacin,ciprofloxacin,and lincomycin.There was a significant difference in the drug resistance between isolates of the two rabbit farms.In this study,three genotypes of CTX-M,SHV and TEM were used for detecting the ?-lactam resistance genes.CTX-M gene was detected from only one isolates,and no SHV and TEM genotypes were detected.The DAS-ELISA method and lecithin hydrolysis assay were used to detect the ability of isolates in producing toxin at different culture times.The results showed that the time of toxin production should not exceed 10 h,and it is better to control it between 6-10 h.The antigenic content and enzyme activity of the alpha toxin of Clostridium perfringens in different isolates were at a relatively stable level during that period.The isolated strain T12 had a good proliferation ability and stable toxin production ability,which was suitable for the preparation of vaccines.