Construction And Characterization Of A SpaA Gene Knock-out Mutant Of Erysipelothrix Rhuriopathiae C43065

To develop a recombinant subunit vaccine against swine erysipelas, we evaluatedthe protective effect of surface protective antigen A (rSpaA) and its N-terminalprotective domain (SpaA-N) and C-terimnal reapeat region (SpaA-C) of Erysipelothrixrhusiopathiae strain C43065. The spaA、spaA-N and spaA-C were expresed in E. coliBL21and the recombinant proteins of rSpaA, rSpaA-N and rSpaA-C were purified byNi-IDA SefinoseTMResin affinity chromatography, respevtively. Six groups of fivemice each were subcutaneously inoculated three times with50μg or100μg proteins ofrSpaA, rSpaA-N and rSpaA-C N at two week intervals. Five mice of each group werechallenged with2.74×104CFU of virulent strain C43065two weeks after the thirdinoculation. As the results,100%protections of mice were obtained by vaccination withrSpaA, rSpaA-N. Western blot results indicated that the rSpaA, rSpaA-N and rSpaA-Cwere recognized specifically by an antiserum against the native SpaA of strain C43065.These results indicated that the rSpaA-N might be a useful vaccine candidate antigen forswine erysipelas.To investigate the rSpaA in pathogenicity of Erysipelothrix rhusiopathiae, a spaAdeleted mutant was constructed by homologous recombination. The results of PCR,RT-PCR and Western blot demonstrated that the spaA deleted mutant (C43065spaA)was successfully constructed. Experiments were conducted to compare the differencesof biological characteristics such as growth rate, serum sensitivity, antiphagocyticproperty, adhesion ability and virulence between the C43065spaA and its parentalstrain C43065, as well as the complemented strain C43065C. The C43065spaA wassensitive to the bactericidal action of normal swine serum, whereas the C43065andC43065C were both resistant. In the presence of normal rabbit serum, the C43065spaAwere readily taken up by mice peritoneal macrophages, but C43065and C43065C weresignificantly resistant to phagocytosis. In the presence of anti-SpaA rabbit serum, theC43065, C43065C and C43065spaA were efficiently phagocytosed. The adhesionassay shows that the number of C43065spaA adhered to mouse embryonic fibroblastcells or to chicken embryonic fibroblast cells was significantly lower than that ofC43065and C43065C, and the C43065spaA was relatively attenuated in mice byintraperitoneal injection. These results demonstrated that the SpaA is a major virulencefactor in the pathogenesis for swine erysipelas.