Evaluation Of Immune Efficacy And Safety Of Genetic Engineering Attenuated Strains For Salmonella Enteritidis

Salmonella is an important zoonotic pathogen with a wide range of animal hosts.So far,there are more than 2700 serotypes,of which the main serotypes are Salmonella enteritidis?SE?and Salmonella typhimurium?ST?.The acute gastroenteritis caused by SE?food poisoning?has increased in all countries of the world,and has become an important issue in international public health.At present,drug control is often used clinically to reduce the risk of Salmonella,which have caused a series of problems such as bacterial resistance and drug-residue.Immunization against Salmonellosis is an ideal choice due to the emergence of multi-resistant Salmonella on the basis of food safety and public health safety.Compared with inactivated vaccine and subunit vaccine,Salmonella gene engineering attenuated live vaccine can stimulate the humoral immunity and cellular immunity of the host more effectively.Studies have shown that Salmonella Pathogenicity Island?SPI?is a specific region of the virulence related genes on the chromosomes,which are related to the pathogenicity,and the most important virulence genes are located on SPI-1 and SPI-2.Among them,ssr AB is located on SPI-2,and hil A and hil D are located on SPI-1.In this study,4 strains of SE[G9??hil A??G9??hil D??G9??ssr ABhil A?and G9??ssr ABhil Ahil D?]were successfully constructed by genetic engineering technology in the early.The evaluation of immunogenicity,immune protection and biological safety should be further carried out to provide a scientific basis for the application of the gene deletion attenuated strains in the immunization and infection.This study takes four strains of SE[G9??hil A??G9??hil D??G9??ssr ABhil A?and G9??ssr ABhil Ahil D?]from pet dog as the research objects.Kunming mice were used as experimental animal models.First,different doses of strains were immunized by intragastric administration.According to the mortality rate of the immunized mice and the specific antibody Ig G titer,the optimal immunity dose of the four strains were determined.Secondly,through immunizing mice with the optimal immunity dose,Specific antibody Ig G titer and intestinal mucosal antibody Ig A titer and Levels of IFN-?,IL-4,IFN-?,TNF-?and MCP-1 in serum were detected by indirect ELISA.Determination of CD4⁺/CD3⁺and CD8⁺/CD3⁺T cell subsets by flow cytometry and spleen lymphocyte proliferation index was measured by MTT assay.The challenge tests measured the immunity protection and cross-immunity protection as well as histopathological changes.Finally,the colonization amount of the tested strains in mice was determined by a pour plate method;the PCR stability and gene sequencing were used to determine the genetic stability of the tested strains.The results showed that the best immunization dose of four strains should be 5×10⁷CFU.The mice were intragastrically administered at 5×10⁷CFU/0.2 m L/dose and boosted at the same dose 2 weeks later.During the 4-week experimental period,G9??hil D?induced the highest serum antibody Ig G titer,which was higher than that of G9,followed by G9??ssr ABhil Ahil D?,G9??ssr ABhil A?,G9??hil A?,significantly higher than the control groups?P<0.05?.The highest titer of intestinal mucosal Ig A antibody was G9??hil D?and G9??ssr ABhil Ahil D?,higher than G9??ssr ABhil A?,G9,and G9??hil A??P<0.05?.The levels of immune-related cytokines?IL-4,IL-10,IFN-?,TNF-?,MCP-1?of four strains were not significantly different with G9?P>0.05?.The ratio of CD4⁺/CD3⁺and CD8⁺/CD3⁺in T cells stimulated of four strains showed an upward trend,in which G9??ssr ABhil A?and G9??ssr ABhil Ahil D?were not significantly different from G9,and G9??ssr ABhil A?and G9??ssr ABhil Ahil D?were significantly higher than G9??hil D?and G9??hil A??P<0.05?.G9??ssr ABhil Ahil D?induced significant differences in spleen lymphocyte proliferation index with G9?P<0.05?,and were higher than G9??hil A?,G9??hil D?and G9??ssr ABhil A??P<0.05?.The protective immunity of mice infected with the four strains against the G9challenge of 10LD₅₀ was 80%to 100%.The liver,spleen and small intestine villus of mice were relatively intact,with no obvious pathological changes,and the provided protection against challenge of 10LD₅₀SE was 50%to 80%.The tested strains can be rapidly cleared in the liver,spleen,and small intestine of mice,and the strains that were colonized in vivo can be substantially eliminated 12 days after inoculation,and can be stably inherited after30 passages in vitro.The resluts showed that G9??hil A?,G9??hil D?,G9??ssr ABhil A?and G9??ssr ABhil Ahil D?have good immunogenicity,immune protection,cross-protective force,and biosafety requirements for the development of strains for enteropathogenic Salmonellosis infection.The utility model has the application prospect in preparing genetically engineered attenuated live vaccine of SE.