Isolation And Characterization Of Type A Clostridium Perfringens And Selecting Of Vaccine Strains

A total of 135 samples of intestine,liver,lung,heart and other organs were collected from 22 sudden death dairy cattle of dairy farms in Inner Mongolia,Shanxi,Sichuan and other places.After the samples were cultured on 10% blood agar plate of sheep,42 plates were found double hemolytic ring.Through typing PCR detection to 106 single colonies(strains)selected from 42 plates,103 strains were Type A Clostridium Perfringens,and 3strains were Type C Clostridium Perfringens.Based on the result,the 103 strains were cultured and determine the virulence(minimal lethal dose in mice),and 30 strains were not higher than 0.1m L.After further screening,only one strain was obtained with virulence(minimum lethal dose in mice)not higher than 0.025 m L and named CNM0708 strain.The screened CNM0708 strain was further identified for culture characteristics,biochemical characteristics,Serotype,molecular biology,virulence,passage stability,pathogenicity for animals,and immunogenicity.The results showed that the strain was gram-positive bacilli,with blunt ends,and the MLD of the toxin in the culture liquid is not higher than 0.025 m L to mice.There were translucent,off-white colonies,and there were ? and ? double hemolytic rings around the colonies,after anaerobic culture for 24 hours.The phenomenon of "violent fermentation" appeared in litmus milk medium culture.The results of biochemical identification were identical with Clostridium perfringens.The serotype identification result is consistent with the serological characteristics of type A Clostridium perfringens.The homology in nucleotide of the 16 S r DNA gene and sequence of ?-toxin of CNM0708 strain were 99.70%and 98.1%-100%,compared with the reference of Clostridium perfringens strains published on Genbank.The culture solution of CNM0708 strain is pathogenic to cattle,and the extracted toxin is pathogenic to cattle,rabbits and mice.Both of them were able to lead to the death of inoculated animals.We prepared seed batches of CNM0708 strain,expanded the culture broth.After inactivation,centrifugation and the supernatant of concentration,we prepared the vaccine and immunized rabbits.The results showed that the neutralizing antibody was 1 in 0.1m L serum after immunization for 21 days.After the second immunization of the cattle for 14 days,5 immunized cattle and 2 control cattle were respectively challenged by the jugular vein with a lethal dose of toxin Type A Clostridium perfringens.The control cattle all died(2/2)and the immunized cattle were all protected(5/5).It was confirmed that the isolated strain which was type A Clostridium perfringens and had good immunogenicity could be used for further development research.