Development Of Combined Inactivated Vaccine Against Salmonellosis And Streptococcus Equi Subsp. Zooepidimicus In Donkeys

In recent years,there have been sporadic or endemic infectious diseases in donkey breeding areas in Shandong,Inner Mongolia,Gansu and other provinces and cities in China,such as abortion of pregnant donkeys,endometritis,orchitis of male donkeys,diarrhea of young donkeys caused by Streptococcus equi subsp.zooepidemicus and Salmonella abortus equi.Among them,the susceptibility of primiparous young female donkeys and young foals is the highest,causing significant economic losses to the donkey breeding industry.In order to prevent the abortion of pregnant donkeys caused by Streptococcus.equi subsp.zooepidimicus（SEZ）and Salmonella abortus equi（S.abortus equi）infection,the inactivated vaccine of Salmonella abortus-Streptococcosis was prepared and its immunogenicity was evaluated.The isolates of Salmonella abortus equi and Streptococcus equi subsp.zooepidemicus were isolated and identified from a donkey farm in Shandong Province.The two strains were identified by microbiology,molecular biology and pathogenicity to mice.The two strains were used as vaccine candidates.The medium and culture conditions were optimized.The cultures of the two strains were inactivated with a final concentration of 0.25%formaldehyde solution.The inactivated vaccines and the combined inactivated vaccines of the two antigens were prepared with aluminum hydroxide adjuvant and squalene adjuvant,respectively,and the safety of each inactivated vaccine was tested.BALB/c mice were immunized to detect the antibody level induced by inactivated vaccine and the immune protection against pathogen attack,and the immune protection effects induced by aluminum hydroxide adjuvant and squalene adjuvant were compared.Results:Gram staining,automatic microbial identification system biochemical identification and 16Sr DNA amplification and sequencing identification showed that the two strains were consistent with the characteristics of their respective bacterial strains.After optimization of culture conditions,the best antigen culture conditions were as follows:1∶100inoculation of strains in BHI medium,37℃,180 r/min shaking culture for 12 h;the median lethal dose of S.abortus equi SAL210303 to mice was 1.16×10⁸ CFU,and the median lethal dose of SEZ SZ2006 to mice was 3.07×10⁸ CFU.The results of safety test showed that all kinds of inactivated vaccines prepared with two kinds of adjuvants were safe.The protective rates of BALB/c mice challenged with 5 times LD₅₀ Salmonella abortus equi and Streptococcus equi subsp.zooepidemicus were 62.5%（5/8）and 75.0%（6/8）,respectively,while the protective rates of squalene adjuvant inactivated vaccine against the two pathogens were 100%（8/8）.The experimental results showed that both pathogenic antigens could induce immune protection,and the protective effect of squalene adjuvant inactivated vaccine was better than that of aluminum hydroxide adjuvant inactivated vaccine.The squalene adjuvant inactivated vaccine of Salmonella abortus-Streptococcus zooepidemicus in donkeys was inoculated into young donkeys,non-pregnant female donkeys and pregnant female donkeys.The safety of the prepared squalene adjuvant inactivated vaccine was evaluated by single dose single immunization safety test,super dose single immunization safety test and single dose repeated immunization safety test.The donkeys were immunized with the inactivated vaccine,and the serum was separated after the second immunization.The total antibody level of the immune serum was detected by SPA-ELISA and bacterial agglutination test,and the functional antibody level was detected by serum sterilization test to further evaluate the immunogenicity of the inactivated vaccine.The results showed that the bivalent inactivated vaccine prepared with squalene adjuvant was safe enough for donkeys at different physiological stages.SPA-ELISA results showed that antigen-specific antibodies were produced in the serum of donkeys immunized with conventional doses.The results of bacterial agglutination test and serum bactericidal test showed that the geometric mean of antibody agglutination titer against Salmonella abortus equi（10⁸ CFU/m L）in immunized donkey serum could reach 1∶32,and the serum bactericidal titers of functional antibodies against Salmonella abortus equi and Streptococcus equi subsp.zooepidemicus were 1∶16（bactericidal efficiency 68.99%）and 1∶4（bactericidal efficiency 65.47%）.In this study,the M-like protein of Streptococcus equi subsp.zooepidemicus was successfully expressed by prokaryotic expression system,and the recombinant proteins Me CTA and CTB were purified.Immunization of mice could induce specific antibodies,which laid a foundation for further development of new adjuvant subunit vaccines.The results of this study showed that the immunization of two antigens of Salmonella abortus equi and Streptococcus equi subsp.zooepidemicus did not produce immune interference.The protective effect of squalene formula adjuvant combined with antigen against lethal dose challenge of Salmonella abortus equi and Streptococcus equi subsp.zooepidemicus was stronger than that of aluminum hydroxide adjuvant,and the combined inactivated vaccine had strong protective effect and immunogenicity in mice and donkeys.This study laid a solid foundation for the development of the new vaccine adjuvant-based bivalent vaccine.