Construction Of Salmonella Enterica Serotype Dublin Sdu189 SpiC,aroA And SopB Deletion Mutants And Their Immunobiological Characteristics

Salmonella enterica serotype Dublin(S.Dublin)is an important zoonotic pathogen with high invasiveness and pathogenicity,which can usually cause S.Dublin infection in cattle and cause serious diseases.Although the use of antibiotics can achieve specific therapeutic effects,it is easy to lead to the form of multi-drug resistant strains and drug residues in meat.Therefore,the development of the safe and effective vaccine is an effective measure to prevent and control S.Dublin infection in cattle.In this study,a virulent S.Dublin strain was screened,and attenuated vaccine candidates were constructed by homologous recombination-mediated by suicide plasmids.The physiological and biochemical characteristics of the five gene deletion strains and their pathogenicity in mice were evaluated.Meanwhile,the immune protective effect of the deletion mutants was preliminarily explored,to provid materials and a basis for the research of live attenuated S.Dublin vaccine.1 Construction and identification of S.Dublin Sdu189 spiC,aroA,sopB mutantsFor constructing spiC,aroA and sopB deleted strain,two DNA fragments which were upstream and downstream of spiC,aroA,sopB gene were amplified from the S.Dublin Sdu189 genome DNA and connected with the suicide plasmid pDM4 respectively.The recombinant plasmids pDM4-spiC,pDM4-aroA,pDM4-sopB were successfully constructed.The recombinant plasmids were transferred into the donor bacterium E.coli x7213 λpir,and then the recombinant plasmids were transferred into S.Dublin by conjugation.Chloramphenicol resistance and sucrose reverse system were used for two screenings;after PCR and sequencing identification,gene deletion strains Sdu189△spiC,Sdu189△aroA,Sdu189△sopB,Sdu189△spiC△aroA and Sdu189△spiC△sopB were successfully constructed.The growth curve and biochemical characteristics of wild strain and deletion strains were identified.Compared with the wild strain,the deletion of spiC,aroA and sopB genes did not significantly change the growth rate,and the deleted strain had good genetic stability.The identification of biochemical characteristics showed that the deletion of aroA gene lost the H2S release ability and also significantly reduced the biofilm formation ability of S.Dublin(P<0.01).2 Evaluation of the safety and protection efficacy of S.dublin as live vaccine candidateUsing mouse macrophage RAW264.7,human colon cancer epithelial cell Caco-2 and bovine macrophage BoMac as cell models,the adhesion,invasion and proliferation abilities of the deletion and wild strains were compared.The results showed that compared with the wild strain,the adhesion and invasion ability of the deletion mutant Sdu189△spiC△aroA to RAW264.7 and Caco-2 were significantly decreased(P<0.001).And the invasion ability of BoMac cells was also significantly lower than that of the wild strain(P<0.001).The LD50 of the Sdu189△spiC(2.22 ×108 CFU),Sdu189△aroA(4.07× 108 CFU)and Sdu189△spiC△aroA(4.07×108 CFU)strains were 586-fold,356-fold and 1073-fold higher than that of parental strain Sdul89(3.79 × 105 CFU)in BALB/c mice.Compared with wild strain,the virulence of the three deletion strains decreased significantly.Mice were immunized by intramuscular injection with the deletion mutants of Sdu189△spiC and Sdu189△spiC△aroA.The bacteria were eradicated at 14,14,9 and 7 days post-challenge in the liver,the spleen,the ileum,and the cecum,respectively.The results of pathological sections showed that compared with the wild strain,the wild group had splenic sinus dilatation,a large number of neutrophils infiltrated,and the number of macrophages increased,the liver and spleen appeared focal necrosis,connective tissue hyperplasia after challenge.However,there was no obvious pathological change in the vaccine group.Experimental results showed that the vaccine candidate strains would not cause functional loss to the organs of mice,and the safety of the vaccine was guaranteed.In this study,qRT-PCR analysis of cytokine expression levels in the spleen showed that both mutants could induce increased IFN-y secretion from 7 to 21 days post-immunization(P<0.001),indicating that the activation of immune cells is beneficial to eliminate S.Dublin and plays a role in eliminating bacteria.On the 7th day post-immunization,the expression of IL-4 reached the highest level and then decreased gradually.The presentation of Sdu189△spiC△aroA was significantly higher than that of Sdu189△spiC(P<0.0001).On the 14th day post-immunization,it caused a high level of the expression of IL-10 and IL-18(P<0.05).Immune protection experiments in mice showed that the vaccine candidate strain could provide good immune protection,and the double deletion strain has a more significant effect on the elimination of wild strain than the single deletion strain.The survival percentages of mice immunized with Sdu189△spiC△aroA reached 92.8%,respectively,after challenge with the parental strain.At the same time,the results of monitoring the weight gain of mice after immunization showed that the immunity of the mutant had no significant effect on the weight gain of mice.Serum IgG levels were determined using an indirect ELISA assay.At 28 days post-immunization,the serum IgG level was significantly higher in the Sdu189△spiC△aroA-immunized group than that in the Sdu189△spiC-immunized mice(P<0.001).The above results indicated that the vaccine candidate strain had good safety and immune protection efficacy.