Construction And Biological Features Of Rough Salmonella Choleraesuis And Salmonella Typhimurium (?)rfaJ Strain

Food safety is one of important social issues,pork is the main animal food,but pigs and their pork are susceptible to Salmonella infection to threaten food safety and human health.Salmonella Choleraesuis is adaptable to swine,which is easy to cause 2-4-months-old pigs to paratyphoid disease,this strain is very serious in the pig industry.Salmonella Typhimurium is the most common serotype,with a wide range of animal hosts including pigs.As one of the food borne pathogens,it is the main cause of human food poisoning.Practice has proved that the vaccine is also one of the effective measures to prevent and control Salmonella.Salmonella Choleraesuis attenuated vaccine C500 strain has been used in China for over 40 years,vaccination can prevent Salmonella infection to animals and its products contamination,reduce the Salmonella to infect humans through the food chain,block Salmonella to spread between humans and animals,to prevent the occurrence of zoonoses by Salmonella.But the currently vaccine can not offer simply and rapid differentiating infected from vaccinated individuals(DIVA)based on the slide agglutination test.So the rfaJ-deficient strain of Salmonella Choleraesuis was constructed,which is dedicated to distinguishing between immunized animals and naturally infected animals.At the same time,the rfaJ-deficient strain of Salmonella Typhimurium D6 was constructed.Preliminary evaluation of their biological characteristics,virulence and immunoprotection were made.The feasibility of rfaJ gene was explored in DIVA vaccine design and diagnosis in the study.1.Construction and biological characteristics of Salmonella Choleraesuis C500?rfaJThe C500?rfaJ strain of Salmonella Choleraesuis was constructed by using ?-red homologous recombination method.The results of PCR and sequencing showed that the rfaJ gene was deleted from the genomic DNA,and the complementary strain C500?rfaJ::rfaJ was constructed.The growth speed of C500?rfaJ did not change significantly comparing to that of parent strain of C500.The self-agglutination precipitate occurred in the process of liquid culture.Comparing to the parent strain,the LPS structure of C500?rfaJ strain was no longer complete due to the absence of O antigen,and C500?rfaJ was shown to be rough type,not like smooth type of C500.The mice serum induced by C500?rfaJ was not agglutinated with the parent strain C500,which could distinguish the animal to be infected or immunized.After C500?rfaJ infection of porcine jejunum epithelial cells of IPEC-J2,the adhesion and invasion to cells of C500?rfaJ strain decreased significantly(P<0.01),its proliferation in the cell is no significant changes.The RNA expressed level of cytokines of TNF-a,IL-4 and IL-6 in IPEC-J2 cells infected by the C500?rfaJ was significantly decreased comparing to that of the parent strain C500.2.Construction and biological characteristics of Salmonella Typhimurium D6?rfaJSalmonella Typhimurium D6?rfaJ was constructed.Its biological characteristics were almost same with Salmonella Choleraesuis C500?rfaJ.The growth speed of D6ArfaJ strain is not different significantly with that of the parent strain D6,and the self-agglutination of the D6?rfaJ strain occurred during the liquid culture.LPS structure of D6ArfaJ is no longer complete due to O antigen deficiency,the D6ArfaJ was rough type,and the mice serum induced by D6ArfaJ did not agglutinated with parent strain D6.Some biological characteristics of D6ArfaJ changed and its swarming motility decreased.In the cell assay,the adhesion and invasion ability of D6ArfaJ to IPEC-J2 cell decreased significantly,the proliferation ability was no change significantly,but after 8 hours of infection,the proliferation ability of D6ArfaJ exceeded that of D6 in the IPEC-J2 cells.D6ArfaJ could provide 80%of the immunoprotection efficiency challenged against the highvirulent strain SL1344 at two weeks after the second immunization by oral administration of 6-weeks BALB/c mice,and the antibody in serum reached a high level at 14 days.After oral infection of mice,the load of D6?rfaJ was less and was cleaned faster than that of D6 in the organs and feces,and the organs in the mice infected by the D6?rfaJ was no obvious lesions comparing to that of D6.In summary,D6?rfaJ had not only safety,but also good immuneprotection,which could lay an important foundation for DIVA vaccine.