The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis

Abstract

In this study, the Riemerella anatipestifer mutant strain RA1062 was obtained by screening a random Tn4351 transposon mutant library. The mutant strain was unreactive with the anti-CH3 lipopolysaccharide monoclonal antibody, as demonstrated with an enzyme-linked immunosorbent assay, and its M949_RS01035 gene was inactivated. When cultured in trypticase soy broth, the late stage growth of the mutant RA1062 was significantly decreased. The mutant RA1062 was stained with crystal violet and presented a rough lipopolysaccharide phenotype, which differed from that of the wild-type strain CH3, suggesting that deletion of the M949_RS01035 gene resulted in defective lipopolysaccharide. Silver staining and Western blot analyses further confirmed that the RA1062 lipopolysaccharide had a deficiency in ladder-like binding pattern, as compared to lipopolysaccharide of the wild-type CH3 strain. In addition, the mutant RA1062 showed a higher susceptibility to complement-dependent killing, increased bacterial adhesion and invasion capacities to Vero cells, decreased blood bacterial loads, and attenuated virulence in infected ducks, when compared to the wild-type strain CH3. Moreover, RNA-Seq and real-time polymerase chain reaction analyses indicated that two genes were up-regulated and two were down-regulated in the mutant RA1062 genome. Furthermore, an animal protection experiment showed that immunization of ducks with inactivated RA1062 bacterin conferred effective cross-protection against challenge with the virulent R. anatipestifer serotypes 1, 2, and 10. This study presents evidence that the M949_RS01035 gene is involved in bacterial phenotype, virulence, and gene regulation in R. anatipestifer. The mutant strain RA1062 could be used as a cross-protective vaccine candidate.Electronic supplementary materialThe online version of this article (10.1186/s13567-018-0589-8) contains supplementary material, which is available to authorized users.

Genetic loci of theR. anatipestiferserotype discovered by Pan-GWAS and its application for the development of a multiplex PCR serotyping method

“…Similarly, Zou et al mutated the M949_1603 gene in the above gene cluster in R. anatipestifer CH-3 and found that the LPS of the mutant strain lacks the O-antigen chain(Zou et al, 2015). Two other studies reported similar results, that is, the expression of M949_RS07580 was significantly downregulated in the CH-3 mutant strain, which lacks O-antigen repeat units(Dou et al, 2017; Dou et al, 2018). M949_RS07580 is also located in the O-antigen gene cluster.…”

Identification and Pathogenicity Analysis of the Pathogen Causing Spotted Spleen in Muscovy Duck

“…The LD 50 of CZG-1 was 35.122 CFU/ml. In Dou et al results about R. anatipestifer , the LD 50 of serotype 1 CH3 strain was 7.50 × 10 7 CFU/ml ( 30 ). Another serotype 1 R. anatipestifer YL4 strain has an LD 50 of about 4.74 × 10 6 CFU/ml ( 31 ).…”

Immunogenicity of live phoP gene deletion strain of Riemerella anatipestifer serotype 1

“…After immunization of young ducks using drinking water, aerosol, or subcutaneous inoculation, analysis of serum titers using ELISA demonstrated that the attenuated vaccine induced high levels of immune antibodies that persisted for a long time compared to inoculation with the inactivated vaccine. Lymphocyte transfer tests also indicated that the attenuated vaccine induced a strong cellular immune response ( Dou et al, 2018 ). Compared with the above study, The R. anatipestifer YMΔ phoP strain was constructed by homologous recombination, which mainly has a protective effect on ducks infected with serotype 1.…”