Characterization Of Riemerella Anantipestifer DPP IV Deletion Strain And Construction Of AroA Deletion Strain

Riemerella anatipestifersis which was caused by Riemerella anatipestifer is one of the most important contagious diseases confronting the duck industry throughout the world. The infected duck characterized septic leaking. Infected ducks have systemic serosal inflammatory exudate. It accounts for significant economic losses to the duck industry throughout the world. Presently,21serotypes of R.anafipestifer have been identified and no significant cross-protection was reported. It lead to the difficulty of using vaccines to immunize duck against infection. R.anatipestifer infection has been significant losses in many duck farms. So, we urgently need a safe and effective vaccine to control it. Therefore, emphasis on the study of molecular biology, pathopoiesis and immunologic mechanism, which play a very important role in control and prevent the disease.In this study, RA-YM strain was used as the parent strain. The recombination suicide plasmids were designated, using Homologous recombination, we obtained aroA deleted mutant from R.anatipestifer strain RA-YM. The mutant will be helpful for the further study of aroA as a virulence factor. The research contents are summarized as follows:1Biological characteristic of RA-YM DPP Ⅳ deletedBiochemical assays of DPP IV mutant was the same as those of RA-YM, The growth curve in TSB showed that the DPP IV mutant grew more slowly than RA-YM. The LD50value of DPP IV mutant was determined to be approximately1.0×108CFU, The LD50value of RA-YM was determined to be approximately3.6×106CFU. The virulence of DPP IV mutant in duck reduced about28times than parent strain. Adhesion and invasion assay showed that the capacity for adherence and invasion of DPP IV mutant to Vero cells was decreased than parent strain. It therefore appears that DPP IV is a virulence factor of Ranatipestifer. This laid a foundation for the molecular pathopoiesis mechanism of DPP Ⅳ gene.2Construction of aroA deleted mutant from R.anatipestifer strain RA-YMAccoding to the sequence of aroA, the homologous arm gene were respectively amplified from RA-YM genome, added to the spectinomycin resistance gene, subcloned into suicide plasmid pRE112. The recombination suicide plasmids were designated as pRE-LSR. The aroA deleted mutant was constrcted first. The E.coli donor strain X7213transformed with the suicide plasmid pRE-LSR was conjugated with the recipient strain, the R.anatipestifer strain RA-YM. After transconjugation, spectinomycin-resistant trans conjugants in which the whole plasmid had been incorporated into the recipient chromosome were analyzed by PCR. This laid a foundation for the molecular pathopoiesis mechanism of R.anatipestifer aroA gene.