Evaluation Of The Immunogenicity And Protection Efficacy Of An Constructed Attenuated Salmonella Typhimurium Strain △arcA△fnr△fliC In Mice

Salmonella can cause gastrointestinal diseases m a variety of animals,posing a major threat to aquaculture and public health.With the growing problem of antibiotic resistance,it is necessary to develop efficient new attenuated vaccines.In this study,an attenuated strain of Salmonella Typhimunum △arcA△fnr△fliC was constructed by suicide plasmid-mediated homologous recombination,and its lmmunogemcity and protective effect were evaluated m a mouse model.1 Construction and identification of Salmonella Typhimurium mutant strainIn this study,Salmonella Typhimunum ATCC14028 was used as the base strain,and the regulatory gennes arcA.fnr and flagellin gene fliC were used as target genes,he mutant strains △arcA,△fnr,AfliC,AarcAAfnr and △arcA△fnr△fliC were constructed by suicide plasmid-mediated homologous recombination △fliC,double gene deletion strain△arcA△nr and three gene deletion strain △arc△fnr△fliC.The target gene was successfully deleted by PCR.The growth status and migration ability of each deletion strain were identified by using toluidme blue solid medium,M9 low-salt solid medium and semi-solid LB medium.The results confirmed that the growth of the strains on the corresponding plates was caused by the mutation of arcA and fnr.Limits and fliC mutations resulted m a significant decrease in strain motility,indicating that each deletion strain was successfully constructed.2 Biological characteristics and virulence determination of Salmonella Typhimurium mutantIn this study,the five biological strains were constructed for partial biological characteristics and virulence determination.The results showed that the in vitro growth curves of each mutant strain were similar to those of the wild strain.ArcA,fnry and fliC gene deletions can cause the bacteria to lose their ability to exercise,and their mutants have significantly reduced exercise capacity compared to wild strains.At the same time,the mouse infection test confirmed that the in vivo colonization ability of △fhr,△arcA△fnr and △arcA△fnr△fiiC decreased significantly compared with the wild type strain;at the same time,only the LD50 of △arc△Afnr△fliC was significantly increased 1.3 ×109 CFU is nearly 104 times that of wild strain LD50,and the virulence of other mutants is weak.3 Immunogenicity and protective efficacy of Salmonella Typhimurium△arcA△fnr△fliCIn this study,△arcA△fnr△fliC was orally immunized to mice at 108 CFU and 107 CFU,and immunized twice,at intervals of 28 days,to evaluate the immunogenicity and immunoprotective efficacy of this mutant.At the same time,the tscrp mutant strain was orally administered(109 CFU)and the BSG immunized group.The results showed that compared with the BSG group,the △arcA△fnr△fliC two-dose group and the △crp group produced high levels of serum IgG and fecal IgA antibody responses,and serum IgG2a levels were higher than IgG1.At the same time,the serum bactericidal experiment confirmed that the serum of the strain immunization group could effectively kill Salmonella Typhiimunuumm ATCC14028.Further,after the lethal dose of Salmonella Typhimurium vimlent strain,the bacterial load of the △arcA△fnr△fliC 108CFU dose group,the 107 CFU dose group,and the Acrp group were significantly lower in the Peyer’s knot,liver annd sspleen.In the BSG group,87.5%,75%and complete immune protection were provideedd at the same time.In summary,five strains of Salmonella Typhimunum ATCC 14028 were successfully costruncted in this study.The virulence of △arcA△fnr△fliC was highly attenuated and had certain immunogenicity and high protective power.