Duck infectious serositis results in significant economic losses to the farming industry,and the pathogen that causes the disease has been isolated and identified as Riemerella anatipestifer(R.anatipestifer).When RA infects a duck,the first bacterial structure encountered by the immune system is the capsular polysaccharide(CPS)that exists in the outermost layer of the bacteria.CPS is widely used for serotyping.Currently,21 RA serotypes have been reported,but the polysaccharide antigens that determine RA serotyping are unknown,and cross-protection is among them,which leads to poor prevention of R.anatipestifer infections with commercial vaccines.Previous studies in our laboratory have predicted that the RA CH-2 CPS synthesis gene cluster.The genes G148_RS04400 and G148_RS04405 in this gene cluster are jointly involved in the synthesis of the bacillosamine(D-Qui NAc4Nac).D-Qui NAc4 Nac is a monosaccharide that forms CPS.We used this as a starting point to explore the relationship between CPS and RA serotyping.In this study,the gene G148_RS04400 and G148_RS04405 were deleted by natural transformation method.The results of real-time q PCR showed successful gene deletion without affecting of downstream gene expression.We constructed complement strains of the corresponding genes by using conjugation transfer.Capsule staining showed that the surface capsule of the two deletion strains were missing whereas the parent strain and the two complement strains had capsule on the surface.Transmission electron microscopy results confirmed these findings,indicating that the deletion of gene G148_RS04400 and G148_RS04405 resulted in capsule defects.Extraction and visualization of CPS from the parent strain RA CH-2 and the deletion strains showed that only Alcian blue staining allows the visualization of CPS,but not silver staining.The deletion strain still extracts CPS,indicating that the synthesis of CPS continues inside the bacterium.Extraction and visualization the lipopolysaccharide(LPS)showed that the LPS of RA CH-2 lacked the O antigen long chain.The slide agglutination test showed that both deletion strains of these two genes were able to agglutinate with serum types 1-13.The agar-gel precipitin test indicates that the binding of LPS to serum was not specific while the binding of CPS to serum was specific.Mice immunized with RA CH-2 CPS produced specific serum,indicating that the extracted CPS was also immunogenic.The agar-gel precipitin test of this serum with CPS further demonstrated the specificity of the binding of the CPS to the serum.In summary,this study demonstrates that the genes G148_RS04400 and G148_RS04405 are involved in CPS synthesis,and that their deletion results in the disappearance of capsule from the surface of RA,leading to a change in the serotype of the strain.CPS is the specific antigen that determines the serotyping of RA,as shown by the extraction of CPS and LPS for agar-gel precipitin test. |