Isolation And Identification Of Pseudotuberculosis Corynebacterium From Sheep And Preliminary Study On Immune Self-made Inactivated Vaccine

Objective:To master the isolation and identification of corynebacterium pseudotuberculosis from sheep,to understand its pathogenicity,to explore the immune effect of inactivated vaccine,and to lay a foundation for further study on the pathogenic mechanism and prevention and control of this disease.Methods:（1）Pathogen isolation and identification:the diseased materials of goats suspected to be diseased in a sheep farm in Guizhou were inoculated into ordinary agar,blood-nourishing AGAR and LB liquid medium to observe the growth morphological characteristics of the medium.Primers were designed based on the 16Sr RNA sequence of the suspected bacteria,a single colony was selected for PCR amplification,and p MD18-T vector was connected to transform DH5 receptor cells.Then the 16Sr RNA sequence of corynebacterium pseudotuberculosis was sequenced.The strains were identified and named by homology comparison of the sequences.Mice were injected subcutaneously and intraperitoneally to observe the morbidity and mortality of mice.（2）The preliminary study on the immune effect of inactivated vaccine of named strain:the OD600 of named strain was diluted between 0.06-0.09 and coated with enzyme labeling plate.After overnight coating at 4℃,it was sealed with defatted milk powder,diluted serum to be tested was added,incubated at 37℃for 1h,followed by enzymatic-labeled secondary antibody,incubated at 37℃for 1h,and then TMB was added for color development.Then,the reaction was stopped with dilute H₂SO₄,and an indirect ELISA method was set up to detect the named strain.After shaking the named strain overnight,the inactivated vaccine was inactivated with formaldehyde and ISA206VG adjuvant was added to prepare the inactivated vaccine.Ten Balb/c mice were immunized on day 0 and day 14,respectively,and subcutaneously inoculated with bacterial solution for challenge on day 14 after the second exemption.At the same time,non-immune control group and the healthy control group were set.After challenge,the incidence or infection of mice was observed.Results:（1）The bacterial strain was isolated and named corynebacterium GZ2015.The16Sr RNA sequence of the strain was successfully obtained and cloned into p MD18-T vector with a sequence length of 1504 bp.After 6h of intraperitoneal injection,mice developed disease successively,and all mice died within 24h.After subcutaneous injection of mice for 12h,the abscess appeared subcutaneous at the injection site,and 2d later,the abscess site was broken,with yellow and white toothpaste-like pus flowing out.（2）Preliminary study on the immune effect of the inactivated vaccine of corynebacterium pseudomycosis GZ2015 from sheep:after overnight inclusion of the enzyme labeled plate of corynebacterium pseudomycosis GZ2015,the optimized serum dilution to be tested was1:400,and the optimal dilution of the enzyme labeled secondary antibody was 1:500,moreover an indirect ELISA method was established.Serum of immunized mice continued to grow.The incidence of the challenge group was 80%（8/10）,while the incidence of the immune group was only 20%（2/10）.Conclusions:（1）intraperitoneal injection of corynebacterium pseudotuberculosis GZ2015can kill 100%of mice,and subcutaneous injection can produce abscesses.In addition,the bacteria have a strong pathogenicity to mice.（2）A method of indirect ELISA for detecting corynebacterium pseudotuberculosis antibodies were established,which can be utilized to detecting serum antibodies to the pathogen after vaccine immunization.（3）The inactivated vaccine of corynebacterium pseudotuberculosis GZ2015 can induce the body to produce better immune protection.