Monoclonal Antibody Preparation, Mutants Screening And Characterization Of Riemerella Anatipestifer Lipopolysaccharide

Riemerella anatipestifer（RA） infection causes high mortality for ducks and results in major economic losses in the duck industry. In this study, we successfully prepared anti-RA LPS（lipopolysaccharide） monoclonal antibodies 7H1 and 8A9 which were used for screening LPS mutants from the RA random transposon mutant library. We identified two LPS mutants, in which the gene M949₁556 or M949₁603 was deleted. Biological characterization showed the both mutants were attenuated in animal experiments. Furthermore, the cross-protection against RA serotypes 1 、 2 and 10 were investigated. This study mainly contained three parts as described blow.The objective of this study was to prepare monoclonal antibodies（MAbs） against Riemerella anatipestifer（RA） lipopolysaccharide（LPS）. BALB/c mice were immunized with inactivated RA CH3 cells for 3 times and spleen cells were fused with murine myeloma SP2/0 cells. Hybridomas secreting specific MAbs were screened using indirect ELISA, using purified CH3 LPS（0.5 μg/100 μL/well） as the coating antigen. After sub-cloned for 3 times, two hybridoma cell lines（7H1 and 8A9） stably producing anti-LPS MAbs were identified. The subtype of both MAbs was identified as IgG1, and the antibody titers were determined to be 1:12800 and 1:6400 in 7H1 and 8A9 ascetic fluids, respectively, by ELISA assay. By slide agglutination and suspended immuno-fluorescence assays, both MAbs were determined to be reacted with R. anatipestifer serotype 1 strain WJ4, but not with serotype 2 strain NJ-3 and serotype 10 strain HXb2. The development of the MAbs paved a path for further study of structure analysis and pathogenesis of R. anatipestifer LPS.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, M949₁556 gene inactivated mutant strain CH3ΔM949₁556 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 CH3ΔM949₁556 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 CH3ΔM949₁556 infected ducks were significantly decreased, compared with those of CH3-infected ducks. In addition, CH3ΔM949₁556 presented significant higher susceptibility to complement-dependent killing than CH3 did in vitro. Furthermore, CH3ΔM949₁556 showed increased bacterial adhesion and invasion capacities to Vero cells. Immunization with CH3ΔM949₁556-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 CH3ΔM949₁556 could provide a broad cross-protection among R. anatipestifer serotypes 1, 2 and 10 strains. Our results demonstrated that the M949₁556 gene plays a role on the bacterial antigenicity and pathogenicity of R. anatipestifer.Riemerella anatipestifer infection causes high mortality for ducks that results in major economic losses in the duck industry. In this study, we identified a mutant strain RA-M1 by Tn4351 transposon mutagenesis, in which the M949₁603 gene encoding glycosyl transferase was inactivated. Distribution analysis of the M949₁603 gene revealed that this gene is specifically existed in R. anatipestifer serotype 1 strains. The mutant strain presented no reactivity to the anti-LPS MAb in an indirect ELISA. SDS-PAGE followed by western blotting demonstrated that the lipopolysaccharide（LPS） of the mutant RA-M1 had a deficiency in ladder-like binding pattern to rabbit antiserum against R. anatipestifer serotype 1 strain CH3, indicating that the O-antigen structure of mutant RA-M1 was changed. The mutant strain RA-M1 showed significant attenuated virulence in ducks and higher sensitivity to normal duck serum, compared with its parent strain CH3. Furthermore, the cross-protection of mutant RA-M1 among serotype 1, 2 and 10 was evaluated by inactivated RA-M1 vaccine. Ducks that received two immunizations with RA-M1-inactivated vaccine were 100% protected from challenge with R. anatipestifer serotype 1 strain WJ4, serotype 2 strain Yb2 and serotype 10 strain HXb2. No changes were observed in the liver, spleen, or brain samples from the protected ducks during autopsy and histological examination. Furthermore, the ducks with two immunizations generated high antibody titers of 1:12,800 against the three serotypes of strains. The vaccine significantly enhanced the production of interleukin 2（IL-2） and IL-4 after two immunizations. These results suggested that the type-specific M949₁603 gene is associated with the O-antigen biosynthesis and mutant RA-M1 could be used as a novel cross-protection vaccine candidate to protect ducks against R. anatipestifer infection.