Evaluation Of Safety And Immunogenicity Of Attenuated SsrAB Gene Deletion In Salmonella Enteritidis

Salmonella enteritidis is a facultative intracellular parasite that can cause Salmonella in humans and various animals.It is mainly characterized by gastroenteritis and sepsis.It is one of the major pathogens of human food poisoning.Salmonella enteritidis plays a pathogenic role mainly through Salmonella Pathogenicity Island?SPI?.The virulence gene ssr AB,as a two-component regulatory system on SPI-2,plays an important role in Salmonella enteritidis colonization in host cells.Immunization is the most effective method for the prevention of salmonellosis.Compared to inactivated vaccines,attenuated live vaccines have a better effect in preventing SE colonization and systemic infection in the intestine.Compared with live vaccines weakened by traditional methods,Salmonella genetically engineered attenuated live vaccines not only can more effectively stimulate the host's humoral and cellular immunity,but also have the advantages of stable mutation characteristics and less occurrence of virulence.This study was based on the successful construction of an attenuated ssr AB gene deletion strain of Salmonella enteritidis?G9?ssr AB?that was successfully constructed using genetic engineering technology in the previous period,and further research on the evaluation of biosafety,immunogenicity,and immunoprotective capacity was conducted.Therefore,it provides a scientific basis for the application of the attenuated deletion gene strain in the immune prevention of enteritis Salmonellosis/infection.Using the constructed attenuated strain of G9?ssr AB In of S.enteritidis,The following studies were carried out in this paper by the experimental animal model of Kunming mice.First,different doses of G9?ssr AB were inoculated by oral inoculation and serum Ig G antibodies were measured by indirect ELISA.The optimal immune dose of G9?ssr AB was determined in combination with the survival rate.Secondly,G9?ssr AB was serially passaged in LB liquid medium to the 30th generation.The in vitro genetic stability test was performed every five generations.The corresponding sequences were amplified by PCR and verified by sequencing.Third,the liver,spleen,and small intestine of mice inoculated with G9?ssr AB and its parental strain G9 were aseptically sampled,weighed,ground,homogenized,and serially diluted,and inoculated into Salmonella chromogenic medium by pour plate method to count colonies.The colonization clearance efficiency of G9?ssr AB and G9was measured.Fourth,indirect ELISA was used to determine the levels of Ig G in peripheral blood and Ig A antibodies in intestinal mucosa of mice immunized with G9?ssr AB,and the levels of five cytokines?IL-4,IL-10,IFN-?,TNF-?,MCP-1?.The spleen lymphocyte proliferation index was measured by MTT assay,and the percentage of CD4⁺and CD8⁺T cell subsets in peripheral blood was determined by flow cytometry.Fifth,the immunoprotective ability of G9?ssr AB and the cross-protective force of G9?ssr AB were determined by immune attack bacteria tests.After immune attack bacteria tests,the liver,spleen,and small intestine of mice were made into histopathological sections to observe pathological changes.Comprehensive evaluation of G9?ssr AB's safety,immunogenicity and immune protection.The results showed that the optimal immunization dose for oral immunization with G9?ssr AB was 1.0×10⁷ CFU;G9?ssr AB could be stably inherited for 30 generations in vitro;The G9?ssr AB colonized in vivo was substantially eliminated after 12 days of inoculation,and G9?ssr AB was cleared more quickly in mice than in the parent strain G9.The mice were immunized with 1.0×10⁷ CFU of G9?ssr AB and boosted with the same dose two weeks later.The Ig G antibody titers respectively were 1:800,1:1600,1:12800,1:51200 in immunization once a week,immunized once every two weeks,immunization twice a week,immunization twice every two weeks.Compared with parental strain G9,G9?ssr AB induced 5 levels of cytokines produced by the body?IL-4,IL-10,IFN-?,TNF-?,MCP-1?,intestinal mucosal secretory Ig A antibody levels,spleen lymphocyte proliferation index,percentage of CD4⁺and CD8⁺T cell subsets were no significant difference?P>0.05?,and there was a significant difference compared with the control group.The G9 attack bacteria with 10 times LD₅₀.The protection rate of attack bacteria was 90%.After the attacking bacteria,there was no obvious pathological changes in the liver,spleen,and small intestine of the mice.The Salmonella typhimurium standard strain CMCC50115attack bacteria with 10 times LD₅₀,and the protection rate of attack bacteria was 60%.The results showed that:G9?ssr AB meets the safety requirements of SE attenuated vaccine strains,and the strain has good immunogenicity and immune protection,and has the necessary conditions to become SE attenuated vaccine strains.