The M949_1556 gene plays a role on the bacterial antigenicity and pathogenicity of Riemerella anatipestifer

- M949_1556 gene inactivated RA mutant CH3ΔM949_1556 was identified.
- The mutant lost reactivity to the anti-LPS MAb in an indirect ELISA.
- The virulence of the mutant was attenuated more than 50 times.
- The mutant showed much higher susceptibility to complement-dependent killing.
- The mutant provided a broad cross-protection among RA serotypes 1, 2 and 10 strains.

Riemerella anatipestifer is one of the most economically important pathogens of farm ducks worldwide. However, the molecular mechanisms regarding its antigenicity and pathogenicity are poorly understood. We previously constructed a library of random Tn4351 transposon mutants using R. anatipestifer strain CH3. In this study, M949_1556 gene inactivated mutant strain CH3ΔM949_1556 was identified by screening of the library using monoclonal antibody against R. anatipestifer serotype 1 lipopolysaccharide (LPS) (anti-LPS MAb) followed by sequence analysis. The mutant strain presented no reactivity to the anti-LPS MAb in an indirect ELISA. Animal studies showed that the median lethal dose (LD50) of CH3ΔM949_1556 was >1010 colony forming units (CFU), which was attenuated more than 50 times, compared with that of wild-type strain CH3 (LD50 = 2 × 108 CFU). The bacterial loads in the blood of CH3ΔM949_1556 infected ducks were significantly decreased, compared with those of CH3-infected ducks. In addition, CH3ΔM949_1556 presented significant, higher susceptibility to complement-dependent killing than CH3 did in vitro. Furthermore, CH3ΔM949_1556 showed increased bacterial adhesion and invasion capacities to Vero cells. Immunization with CH3ΔM949_1556-inactived vaccine was effective in protecting the ducks from challenge with R. anatipestifer serotype 1 strain WJ4, serotype 2 strain Yb2 and serotype 10 strain HXb2, suggesting that the mutant strain CH3ΔM949_1556 could provide a broad cross-protection among R. anatipestifer serotypes 1, 2 and 10 strains. Our results demonstrated that the M949_1556 gene plays a role on the bacterial antigenicity and pathogenicity of R. anatipestifer.