Epidemiological Investigation Of Salmonella In Eastern China And Developmentof A Gene Deletion Attenuated Vaccine Of Salmonella Typhimurium

Salmonella is an important zoonotic pathogen,which can not only cause a variety of diseases in livestock and poultry(fowl pullorosis,fowl typhoid,fowl paratyphoid,swine paratyphoid,etc.),but also can lead to human food poisoning through contamination of food.Thus,salmonellosis lead to economically devastating to breeding industries,moreover,it will be potential threat to human health.Recently,antibiotics are wildly used to prevent and treat the salmonellosis.However,the abuse of antibiotics and drug residues seriously affect the development of the breeding industry and the public health.Therefore,it is necessary to develop the vaccine to prevent Salmonella infection.For inactivated vaccine,it is inconvenient,such as multiple subcutaneous inoculation.Moreover,it can not induce cellular immunity andprovide effective local protection.Whereas,attenuated vaccines could simulate cellullar,humoral and mucosal immunity.Therefore,development of attenuated vaccine by genetic engineering technology has attracted much attention.In this study,epidemiological investigation of Salmonella in Eastern China showed that Salmonella was widely distributed and the drug resistance of these isolates was very serious.Then,we constructed the aroA and luxS gene deletion mutant strains of Salmonella typhimurium isolate SAT52,and evaluated the safety and immune protection of this mutant strain in order to provide a vaccine for the prevention and control of salmonellosis.1.The epidemiological investigation of Salmonella in Eastern ChinaTo investigate the prevalence of Salmonella in the farms of Eastern China,285 samples were collected from Shanghai,Jiangsu,Anhui and Zhejiang provinces.Bacteria were isolated and identified by selective medium and PCR.Then,we detected serotype and virulence genes of isolates by PCR,and the antibiotics susceptibility of Salmonella isolates were performed in accordance to the routine methods.Also,the median lethal dose(LD50)of 7 Salmonella isolates were detected.The results showed that 48 isolates were identified as Salmonella,which accounted for 16.84%.Serological detection showed that 64.58%isolates was Salmonella typhimurium,which is the predominant serotype and 8.33%isolates belonged to Salmonella enteritidis.The prevalence of virulence genes detected were over 97%,except the sopE gene,which was present in 25%Salmonella isolates.Animal infection results showed that the LD50 of 7 Salmonella isolates were 1.26×104 CFU,1.26×104 CFU,5.01×104 CFU,5.01×104 CFU,5.01×104 CFU,1.26×105 CFU and 2.00×105 CFU,respectively,indicated that these Salmonella isolates were high virulent to mice.The results of antibiotic sensitivity test indicated that Salmonella isolates were multiple resistance to most antibiotics.More than 50%Salmonella isolates were resistant to Erythromycin,Azithromycin,Clindamycin,Neomycin,Spectinomycin,Tetracyclines,Rifampin and Compound Sulfamethoxazole.These results indicated that Salmonella typhimurium is wildly distributed in the farms of Eastern China.These isolates habor various virulence genes and show multiple resistant to antibiotics.2.Construction and characterization of aroA gene mutant and aroA,luxS double gene mutant strain in Salmonella typhimuriumIn this study,the aroA deletion mutant strain SAT52?aroA and aroA,luxS double gene deletion mutant strain SAT52?aroA-luxS in Salmonella typhimurium strain SAT52 were constructed by the Red recombination system.Then we compared the growth curve,motility,biofilm formation,adhesion and invasion to HeLa cells and virulence of mutant and wild-type strains.The result of growth curve showed that mutant strains SAT52AaroA and SAT52AaroA-luxS showed slightly slower growth than the wild-type strain SAT52 during the logarithmic period,but the difference was not significant.During other growth period,the strains SAT52,SAT52?aroA and SAT52AaroA-luxS showed similar bacterial growth rates.The result showed that mutant strains SAT52AaroA and SAT52?aroA-luxS displayed decreased biofilm formation compared with the wild-type strain SAT52.There was no significant difference between SAT52?aroA and SAT52,while the motility of SAT52?aroA-luxS was decreased than wild-type strain SAT52.Bacterial adherence and invasion assays showed that the adhesion capacity of mutant strain SAT52AaroA was significantly enhanced compared with wild-type strain SAT52,while there was no significant difference for the invasion capacity.The mutant strain SAT52?aroA-luxS showed increased invasion capacity compared to wild-type strain,but no difference was observed for the adhesion capacity.Animal infection results showed that the LD50 of wild-type SAT52,SAT52AaroA and SAT52?aroA-luxS were 3.16×104 CFU,1.78 ×107 CFU and 4.22×107 CFU,respectively.The mutant strain SAT52AaroA-luxS has a 1400-fold and 2.4 times reduction in virulence compared to the wild-type strain SAT52 and mutant strain SAT52AaroA.These results suggested that the aroA and luxS double gene deletion lead to significantly attenuated virulence for Salmonella typhimurium.3.Evaluation of immune protection effect and safety of the attenuated mutant strains of Salmonella typhimuriumThe immune effect and safety of attenuated mutant strain SAT52AaroA-luxS were evaluated using six weeks BALB/c mice.The optimal dose of immunization and period of immune protection of attenuated mutant strain SAT52?aroA-luxS were determined through the infection of wild-type strain SAT52.After immunization,the bacterial loads in the spleen were determined.Moreover,the virulence of mutant strain SAT52AaroA-luxS isolated from mice were determined to evaluate the safety of the gene deletion attenuated vacccine The results showed that attenuated vaccines SAT52AaroA-luxS could provide good immunization protection effect for mice against the infection of Salmonella typhimurium.SAT52AaroA-luxS could sitmulate relatively high antibody titers,and the titer was maintained at a relatively high level after 6 weeks and 8 weeks post-immunization.Moreover,7 weeks after immunization with 1×106 CFU SAT52AaroA-luxS per mice,the immunization protection rate was still 100%.Thus,the optimal imIunization dose was 1×106 CFU per mice.The bacterial loads of SAT52AaroA-luxS were reduced in the spleen than the mutant strain SAT52AaroA,and no bacteria SAT52AaroA-IuxS was determined after 10 days post-immunization.We isolated SAT52AaroA-luxS from mice and then detect it's virulence,which was similar to that of the vaccine,indicating attenuated mutant vaccine is safe.These results indicated that attenuated mutant strain SAT52AaroA-luxS constructed in this study will be a vaccine candidate for the prevention and treatment of Salmonella typhimurium infection.