Characterization and identification of a novel candidate vaccine protein through systematic analysis of extracellular proteins of Erysipelothrix rhusiopathiae.

Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas, is a facultative intracellular Gram-positive bacterium. It has been shown that animals immunized with a filtrate from E. rhusiopathiae cultures are protected against lethal challenge. In this study, we identified and characterized the extracellular proteins of E. rhusiopathiae to search for novel vaccine antigens. A concentrated culture supernatant from the E. rhusiopathiae Fujisawa strain, which has been found to induce protection in mice, was analyzed using two-dimensional electrophoresis. From more than 40 confirmed protein spots, 16 major protein spots were selected and subjected to N-terminal amino acid sequence determination, and 14 protein spots were successfully identified. The identified proteins included housekeeping proteins and other metabolic enzymes. We searched for surface-localized proteins by analyzing the genomes of two E. rhusiopathiae strains: Fujisawa and ATCC 19414. Genome analysis revealed that the ATCC 19414 strain has three putative surface-exposed choline-binding proteins (CBPs): CbpA, CbpB, and CbpC. Each CBP contains a putative choline-binding domain. The CbpC gene is mutated in Fujisawa, becoming a nonfunctional pseudogene. Immunogold electron microscopy confirmed that CbpA and CbpB, as well as the majority of the metabolic enzymes examined, are associated with the cell surface of E. rhusiopathiae Fujisawa. Immunization with recombinant CbpB, but not with other recombinant CBPs or metabolic enzymes, protected mice against lethal challenge. A phagocytosis assay revealed that antiserum against CbpB promoted opsonin-mediated phagocytosis by murine macrophages in vitro. The protective capabilities of CbpB were confirmed in pigs, suggesting that CbpB could be used as a vaccine antigen.

Oral vaccination against mycoplasmal pneumonia of swine using a live Erysipelothrix rhusiopathiae vaccine strain as a vector.

Erysipelothrix rhusiopathiae Koganei 65-0.15 strain, the live swine erysipelas vaccine for subcutaneous injection, has been shown to colonize the tonsils of pigs after oral inoculation. We thus evaluated the possible use of the strain as a vector for oral vaccination against mycoplasmal pneumonia of swine. Recombinant E. rhusiopathiae strains expressing the C-terminal domain of the P97 adhesin of Mycoplasma hyopneumoniae were constructed and examined for vaccine efficacy in mice and pigs. Mice subcutaneously inoculated with the recombinant strains were protected from challenge exposure to a virulent E. rhusiopathiae. Administration of milk replacer containing recombinant E. rhusiopathiae expressing the M. hyopneumoniae protein protected pigs from death after exposure to E. rhusiopathiae and significantly reduced the severity of pneumonic lung lesions caused by infection with M. hyopneumoniae.