Immunogenicity And Evaluation Of Safety Of Attenuated HilD Gene Deletion In Salmonella Typhimurium

Salmonella is a non-host-specific or panophilic zoonotic pathogen,which mainly causes intestinal diseases in animals and foodborne diseases in humans.There are more than 2700 serotypes of the bacteria,among which S.typhimurium is one of the serotypes with the highest isolation rate in all countries in the world.At present,antibiotics are often used to control the harm of Salmonella in clinic,but the drug resistance of Salmonella is becoming more and more serious due to long-term unreasonable use.Vaccination has become an effective method for the prevention and control of salmonellosis/infection.Genetically engineered live attenuated vaccine is also a live attenuated vaccine,which is made of partially knockout or mutation of pathogenic genes related to the virulence of pathogenic microorganisms to screen out attenuated strains with lost virulence or reduced virulence and can maintain the original antigenicity.Salmonella mainly causes disease through the volatilization of virulence island(Salmonella Pathogenicity Island,SPI).Hil D has the function of regulating genes on SPI-1 and SPI-2,which is the core of the virulence gene regulatory system of Salmonella.In this study,genetic engineering homologous recombination technique was used to delete and supplement the hilD gene of the parent strain S.T(pc DNA3.1),and the gene deletion strain S.T(?hilD+pc DNA3.1)and supplement strain S.T(?hilD+pc DNA3.1-hilD)were obtained respectively.The immune efficacy and safety evaluation of hilD gene deletion strain of Salmonella typhimurium were studied.The aim is to provide scientific basis and technical reserve for the prevention and control of salmonellosis/infection.Inthisstudy,S.T(pc DNA3.1),S.T(?hilD+pc DNA3.1)and S.T(?hilD+pc DNA3.1-hilD)were used as test strains and Kunming mice as experimental animal models.The following experiments were carried out:(1)different doses of tested strains were inoculated by intragastric administration and intraperitoneal injection,and the immunization procedure was determined according to the mortality of immunized mice and the titer of serum Ig G antibody.(2)the mice were inoculated with the best immune dose and route and immunized with the same dose and route two weeks later.The serum and intestinal tract of mice were collected for four consecutive weeks after the first immunization.The levels of serum Ig G and intestinal mucosal lg A antibody were detected by indirect ELISA method,and the levels of serum related cytokines(IL-4,IL-10,IFN-?,TNF-?,MCP-1)were detected by double antibody sandwich ELISA method.Serum bacteriostatic test was used to detect the bacteriostatic ability,Wright Giemsa staining was used to detect the phagocytic ability of leukocytes,flow cytometry was used to determine the percentage of CD4+and CD8+T lymphocyte subsets,and MTT method was used to determine the splenic lymphocyte proliferation index.(3)the immune protection rate and cross-immune protection rate of mice were determined by intraperitoneal challenge test,and the pathological sections of liver,spleen and small intestine were made,and the pathological changes were observed.(4)routine bacterial isolation and cultivation methods,PCR technology were used to identify fecal bacteria,indirect ELISA method was used to detect serum Ig G antibody,and to evaluate the horizontal transmission ability of the tested strains;coating plate method was used to determine the colonization and clearance efficiency of the tested strains in vivo;30 generations of the tested strains were passed on continuously in mice,and every 5 generations were identified and sequenced by PCR method to verify the genetic stability in vivo.The results showed that:(1)the immunization procedure was determined as intraperitoneal injection of 5×10⁵CFU/0.2m L/each,and the immunity was enhanced with the same dose and route two weeks later.(2)during the four-week immune period,the serum Ig G antibody titer,intestinal mucosal Ig A antibody level and related cytokine levels(IL-4,IL-10,IFN-?,TNF-?,MCP-1)of S.T(?hilD+pc DNA3.1)were the highest,and S.T(?hilD+pc DNA3.1)had the strongest bacteriostatic activity,followed by S.T(pc DNA3.1)and S.T(?hilD+pc DNA3.1-hilD).After enhanced immunization,S.T(?hilD+pc DNA3.1)leukocytes had the strongest ability of phagocytosis of bacteria(P<0.05),and S.T(?hilD+pc DNA3.1)stimulated mice to produce the highest percentage of CD4+/CD3+and CD8+/CD3+T cells,but there was no significant difference between S.T(?hilD+pc DNA3.1)and S.T(pc DNA3.1)(P>0.05).The proliferation index of spleen lymphocytes induced by S.T(?hilD+pc DNA3.1)was the highest and was significantly different from that of other immune groups(P<0.05).(3)the protective rate of mice immunized with S.T(?hilD+pc DNA3.1)against 10LD₅₀S.T was the highest(100%),and the cross protection rate against 10LD₅₀Salmonella enteritis was the highest(60%).The villi of liver,spleen and small intestine of mice in S.T(?hilD+pc DNA3.1)group were intact and the degree of tissue damage was the lowest.(4)S.T(?hilD+pc DNA3.1)did not show horizontal transmission ability and was safe,it could basically eliminate the tested strains colonized in vivo within 12 days after inoculation,and could be stably inherited in mice for 30 generations.The results showed that S.T(?hilD+pc DNA3.1)could induce good humoral immunity,cellular immunity and mucosal immunity in mice,and provide good immune protection and cross immunity after challenge,which met the biological safety requirements of the development of live attenuated vaccine strain against salmonellosis/infection,and had the application prospect in the preparation of genetic engineering live attenuated Salmonella typhimurium vaccine.