Comparative Identification Of Clostridium Perfringens By Three Methods(SDS-PAGE Et Al.) And The Elementary Study Of The Toxoid Bacterin

The Sudden Death Syndrome caused by Clostridium perfringens came on rapid, so it is necessary to estimating the toxin types of Clostridium perfringens prevalent in the epidemic disease region exactly and timely. This study described SDS-PAGE method particularly and used it to detect 20 airborne Clostridium perfringens samples from cow stable and 57 Clostridium perfringens samples that separated from Shandong province, and compared the results of different detecting methods, the results showed that the accordance was 95.0% between ELISA and SDS-PAGE,100% between Muti-PCR and SDS-PAGE. At the same times, a great deal of exotoxins were distilled and inactivated to produce the toxoid vaccine. And then discussed the pathogenicity of the toxins produced by Clostridium perfringens.This research consists of five parts.PartⅠ: Isolation and Identification of Clostridium perfringens of samples. Samples isolated from the epidemic disease region in Shandong province were detected by aerobic and anaerobic culture, form and API-20A biochemical test. The study result showed 57 Clostridium perfringens strains were isolated from 298 samples.PartⅡ: The amelioration of (NH4)2SO4 depositing method in the purification of the toxins produced by Clostridium perfringens. This research used (NH4)2SO4 of different saturation to salt out the toxins produced by Clostridium perfringens.,and make electrophoresis to the crude toxin. After get the strip and content of the toxins, the results showed that (NH4)2SO4 of 60% saturation could effectively deposited the toxins.PartⅢ: Using SDS-PAGE to detect the toxin types of Clostridium perfringens.The NTCC Type B 6121 Clostridium perfringens standard strains,20 airborne Clostridium perfringens samples from cow stable and 57 strains Clostridium perfringens isolated in Part 1 were gotten rid of the bacterial body and the crude toxin were twice salted out by (NH4)2SO4 of 60% and 40% saturation. The deposition were disdolved and dialysed, then treated with SephadexG–200. The chromatography liquid which had toxin activity was gathered and concentrated . By this way , the purified toxin of Clostridium perfringens was gained. The detecting results of 20 airborne Clostridium perfringens samples from cow stable were 17 of type A, 2 of type C and 1 of type D;the detecting results of 57 strains Clostridium perfringens isolated in Part 1 were all of type A.PartⅣ: Research on ELISA and Multi-PCR methods to detect the toxin typeof Clostridium perfringens. This research used ELISA,Multi-PCR and SDS-PAGE to detect 20 airborne Clostridium perfringens samples from cow stable and 57 Clostridium perfringens samples that separated from Shandong province, and compared the results of different detecting methods, the results showed that the accordance was 95.0% between ELISA and SDS-PAGE,100% between Muti-PCR and SDS-PAGE. The results showed that the difference existed between ELISA method and SDS-PAGE method and the coherence between multiplex-PCR method and SDS-PAGE method.PartⅤ:Research on pathogenicity of Clostridium perfringens exotoxin and the preparation of the toxoid vaccine. The standard strain and the isolated strain of Clostridium perfringens type A were inoculated to the nutrient broth and cultured in 40℃anaerobic environment, then some culture liquid was transformed into the Gordon broth, shaking cultured for 6~7 hour in 43℃anaerobic environment. The cultured liquid was centigraded with high speed and low temperature, and then they were filtered in order to remove the bacterium bodies . We got the Clostridium perfringens exotoxin. The LD50 for the little white mouse was 0.00316(standard strain) and 0.001 (isolated strain). At different time(8h,16h,32h,64h, 128h), the toxin was inactivated with formaldehyde. After inactivation, 5% Al(OH)3 was put in the toxoid in certain proportion, then we got the Clostridium perfringens type A toxoid vaccine. The vaccine was checked for its security, immune dose. The results showed that this vaccine was very safe and had high immunity effect. The studies on pathogenicity consisted of lethal test, putrescence test, pathogenicity test, measurement of LD50 and pathology histology examining.