| Clostridium perfringens is one of the most causative strains in the genus Clostridium and it is the main pathogen of gas gangrene and animal necrotizing enterocolitis,gas gangrene,and enterotoxemia.With the rapid development of China’s cattle industry,the strain,especially the type A strain,has become the main cause of sudden death of cattle.In order to understand the genotypes of Clostridium perfringens isolates from cattle sources in Heilongjiang province,a high-performance vaccine was developed.In this study,four types toxins（cpa,cpb,etx,and itx）of Clostridium perfringens isolates and cpb2,two alleles cpb2（atypical cpb2 and consensus cpb2）and enterotoxin（cpe）were detected by PCR;screening for toxin-producing media and development of inactivated trivalent toxoid vaccines.First,thirteen wild bovine strains of Clostridium perfringens were resuscitated and enriched.PCR typing was used to identify the toxin-producing medium and the median lethal dose was measured in mice.As a result,twelve strains of type A and one strain of type D were identified.PCR detection of Beta2 and CPE virulence genes revealed that only type A strains（8/12）carry Beta2 virulence genes and 75%of strains carry atypical Beta2 toxin;CPE was not detected in all strains Virulence genes.The best media for toxin production was meat liver and stomach digestion.The median lethal dose of type A exotoxin was 2-1.5/mL for mice,2-7.17/mL for type C,and 2-2.5/mL for type D.Second,the use of molecular sieve gel chromatography to purify exotoxin proteins as coating antigens.The optimized ELISA reaction conditions were coating concentration of 4μg/mL,the best blocking solution was 5%skim milk,and secondary antibody 1:5000 dilution incubation time is 1 h.The optimal working time of TMB substrate is 15 min.The results showed that the established indirect ELISA method was specific,sensitive,and reproducible.Third,by inactivating the formaldehyde of Clostridium perfringens exotoxin solution and adding aluminum salt adjuvant,A,C,and D-type Clostridium perfringens trivalent toxoid vaccines were prepared,and the mice were vaccinated to determine the safety of the vaccine Immunopotency.The results showed that the vaccine had good safety to mice and stimulated the mice to have a good humoral immune response.The antibody level reached the highest level after the first week of second immunity.After 21 days of the second exemption,the challenge protection experiment was conducted.As a result,the immune protection rate of Clostridium perfringens type A and C exotoxicity challenge group was 100%,and the immunoprotection rate of Clostridium perfringens type D exotoxicity challenge group was 60%,all mice in the control group died.In summary,the isolates of Clostridium perfringens from cattle in Heilongjiang Province were mainly type A,and the detection rate of Beta2 virulence genes in isolates of type A was relatively high.CPE virulence gene not detected.The prepared trivalent toxoid vaccine has a good protective effect against Clostridium perfringens type A and C infections.This study laid a theoretical foundation for the development of Clostridium perfringens toxoid vaccine. |
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