Construction Of Multigene Deletion Strains Of Salmonella Typhimurium And Analysis On Their Pathogenicity

Salmonella is an aggressive and facultative intracellular pathogen with the ability to colonize a variety of host organisms.Salmonella typhimurium is a common and important zoonotic pathogen with a wide host spectrum.Ingestion of food and drinking water contaminated by this bacteria can cause gastrointestinal diseases in humans and animals.In recent years,with the development of rabbit breeding,salmonella is more common in rabbits.The pathogens are mainly Salmonella typhimurium and Salmonella enteritidis.Rabbits of different breeds and ages can be infected.After Salmonella typhimurium infects rabbits,it can cause miscarriage and death of female rabbits and diarrhea of young rabbits,but also pollute fur products and meat products of rabbits.The occurrence and prevalence of salmonellosis in rabbits not only bring huge economic losses to the rabbit industry,but also serious public health and safety problems.In the world,Salmonella typhimurium is a serious threat to human health and causes more than millions of infections every year.Studies have shown that the pathogenicity of Salmonella is closely related to its virulence factors.The reason why Salmonella can survive,reproduce and spread in the host is mainly because there are a large number of virulence related genes and their interaction to cause the host infection.The research on Salmonella disease and prevention and treatment has been attracting people's attention.How to better prevent and control Salmonella from livestock and poultry and protect human health has become a hot topic of international public health.It is very important to develop a more stable attenuated Salmonella vaccine by constructing single or multiple virulence gene deletions of Salmonella,and research and prevention of Salmonella infection have important social benefits for medicine,veterinary public health and food safety.This study is based on three rabbit farms in Yangling,Baoji and Wugong,Shaanxi Province,with the female rabbits are abortion,death,diarrhea as a prominent clinical symptoms.The liver,spleen and feces of the dead rabbits with hepatosplenomegaly and intestinal air were collected,and five suspected Salmonella strains were obtained by bacterial isolation and culture methods.The Salmonella typhimurium were isolated from suspected strains by screening,biochemical test,molecular biological identification and animal pathogenicity test.We used CRISPR/Cas9 gene editing technology to construct knockout vectors of virulence genes Sop B and Ssr B of Salmonella typhimurium,and successfully constructed mutant strains?Sop B,?Ssr B and?Sop B-Ssr B gene deletion of Salmonella typhimurium,then conducted pathogenicity tests in mice.This study provided the basis for the establishment of precise editing technology of CRISPR/Cas9 gene in Salmonella typhimurium,and explored the feasibility of?Sop B,?Ssr B and?Sop B-Ssr B gene deletion mutants of Salmonella typhimurium as attenuated live vaccines or vectors,and provided reference for the development of attenuated Salmonella vaccine.Results of this study are as follows:1.The suspected Salmonella strains obtained from three rabbit farms were medium-sized,round,transparent or translucent,smooth and slightly raised colonies on Mac Conkey agar plates;they were round on SS agar plates with a small black dot in the center or almost all black,smooth,moist,and neatly edged colonies.The results of biochemical tests and specific PCR tests confirmed that there were three isolated strains of Salmonella typhimurium(Y3,B2,W1).The results of drug susceptibility test showed that the isolated strains were almost resistant to penicillin,vancomycin,tetracycline,erythromycin,etc.,and resistant to tobramycin,chloramphenicol,kanamycin,etc.Both gentamicin and gentamicin are sensitive,and the resistance of Salmonella strains isolated from the three rabbit farms is still different.The test of the isolated strains infecting young rabbits can cause clinical symptoms such as rabbit diarrhea and malaise.The dead rabbits were re-isolated by autopsy and Salmonella typhimurium was identified by PCR.Stool samples collected from three rabbit farms were detected by PCR and the contamination rate of Salmonella typhimurium was between 30%and 47%.2.Using CRISPR/Cas9-mediated homologous recombination technology,through continuous screening of the optimal sg RNA,a recombination knockout vector containing Cas9,sg RNA and homologous arm sequences based on the virulence genes Sop B and Ssr B of Salmonella typhimurium was established to form a A more effective gene editing.The recombinant vector was transformed into Salmonella typhimurium by electrotransformation,and the Salmonella typhimurium gene deletion mutant strains?Sop B,?Ssr B and?Sop B-Ssr B were successfully constructed and screened.3.In the biological characteristics and virulence tests of the wild and deleted Salmonella typhimurium strains?Sop B,?Ssr B and?Sop B-Ssr B,the results showed that compared with the wild strains,the deleted strains?Sop B and?Sop B-Ssr B grew faster compared with the wild strain,the growth rate and motility of the deleted strain?Ssr B were relatively slow.In the mouse pathogenicity test,the LD₅₀ of the three deletion strains was 10 to 42 times higher than that of the wild strain,it indicates that the virulence of the deletion strain was reduced.The double gene deletion strain?Sop B-Ssr B was compared with the two single deletion strains?Sop B and?Ssr B's virulence weakening effect is not obvious.These results suggest that the pollution rate of salmonella in rabbit farms is relatively serious.Although the breeding and management measures and prevention and control of rabbit farms are in place,there are still loopholes.The three strains of Salmonella typhimurium gene deletion constructed in this study showed weakened virulence,which could reduce the pathogenicity to mice,in order to provide a reference for in-depth study of Salmonella typhimurium attenuated vaccines or as vaccine vectors.The attenuation effect of double gene deletion strain was not as expected as that of the single gene deletion strain,so further tests and strict screening of virulence candidate genes are needed.