Construction And Characterization Of Riemerella Anantipestifer 06537/06542 Deleted Mutant Strain

Riemerella anatipestifer infection is an important bacterial disease to farm ducks throughout the world. It has caused considerable economic loss to the duck production.However, there is no report about the significant cross-protection among the serotypes of R. anatipestifer, which leads to the difficulty of vaccination. In most duck farms, it would be difficult to eradicate R. anatipestifer after the occurrence, and repeated infectious episodes could be observed. Therefore, it is important to research the molecular biology,pathology and immunology to completely control R. anatipestifer infection.In this study, through constructing a two-component regulatory system genes06537/06542 deleted mutant of R. anatipestifer and identifying its related biological characteristics, we confirmed that 06537/06542 may be the key virulence factor for R.anatipestifier, which would provide theoretical basis for further study on the molecular pathology of R. anatipestifier and designing of genetically engineered vaccine against R.anatipestifier. The research contents are summarized as follows:1 Construction of 06537/06542 deleted mutant from R. anatipestifer strainPrimers for amplification of homology arms were designed according to06537/06542 genes sequence, and the recombinant plasmid p RE-Leftarm-Spc R-Rightarm(p RE-LSR) was constructed by Overlap PCR. The bacteria after conjugation were cultured on TSA plate with spectinomycin, and the colonies survived were cloned as candidate mutants for further PCR identification.2 Biological characteristics of 06537/06542 deleted mutant from R. anatipestifer strainThe biochemical identification shows that RA-YM 06537/06542 genes mutant could not produce H2 S and use gelatin. And the growth curves indicate that the growth rate of06537/06542 mutant was slower than the wild strain. RA-YM 06537/06542 mutant’s resistant ability of acid and alkali condition decreased compared with the wild strain. The results of drug sensitivity test showed that RA-YM 06537/06542 genes mutant’s resistance of amoxicillin, chloramphenicol, ampicillin, and sulfamethoxazole increased significantly compared to the wild strain while the resistance to tetracycline decreased.The bile and osmotic tolerance of RA-YM 06537/06542 genes mutant decreased.SDS-PAGE results showed that the protein expression of RA-YM 06537/06542 genes mutant was different compared with the wild strain. Transmission electron microscopy results showed that bacteria morphology of RA-YM 06537/06542 genes mutant did not change.3 Pathogenicity of 06537/06542 deleted mutant from R.anatipestifer strainThe consequences of LD50 showed that the virulence of RA-YM 06537/06542 genes mutant was significantly reduced. And the LD50 of the wild strain was 4×107 CFU whist the LD50 of the RA-YM 06537/06542 genes mutant was higher than 1×1010CFU. The determination of the liver, spleen, heart, brain and blood bacteria loading from infection groups showed that various organs bacterium capacity was significantly decreased compared to the wild strain infection group. The pathological HE staining results showed that the capacity to damage the heart, brain, spleen decreased significantly compared to the wild strain, but it still had a certain damage capacity on liver cell. The results above show RA-YM 06537/06542 genes are the important virulence of R. anatipestifer.