Construction and vaccine potential of acapsular mutants of Erysipelothrix rhusiopathiae:: Use of excision of Tn916 to inactivate a target gene

We previously showed that acapsular transposon Tn916 mutants of Erysipelothrix rhusiopathiae are avirulent for mice. In this study, we constructed nonreverting acapsular mutants and examined the vaccine potential of the mutants in mice. A representative acapsular transposon mutant, 33H6, was plated on selective agar containing autoclaved chlortetracycline and quinaldic acid, and two tetracycline-sensitive mutants were obtained. Sequence analysis of chromosomal regions of the mutants in which Tn916 had flanked revealed that Tn916 had spontaneously excised from the region and that the six new nucleotides, which were presumably inserted with Tn916 into 33H6 chromosome, substituted for those present at the insertion site. The mutants were confirmed to be devoid of capsular antigen by Western immunoblotting and were nonvirulent for mice (subcutaneous 50% lethal dose [LD50], > 10(9) CFU). The safety and efficacy of acapsular mutants for live vaccines was further studied by using one mutant strain, named YS-1, The YS-1 bacteria were cleared from the skin sites of inoculation, livers, and spleens of the inoculated mice by 7 days after subcutaneous (s.c.) inoculation. Mice immunized s.c. with doses ranging from 2 x 10(4) to 2 x 10(8) CFU of strain YS-1 were completely protected against challenge with 100 LD50 of the homologous, highly virulent strain Fujisawa-SmR 21 days postimmunization, and protective immunity conferred by immunization with 2 x 10(8) CFU of the strain lasted for as long as the 3 months of the observation period. In passive immunization experiments, sera collected from mice immunized,vith strain YS-1 at days 14 and 21 postimmunization provided protection against challenge with Fujisawa-SmR, whereas sera collected at days 4 and 7 did not. Furthermore, specific spleen cell responses to E. rhusiopathiae antigens were observed in mice immunized with strain YS-1, and cross-protection against the antigenically heterologous bacterium Listeria monocytogenes was observed at 7 days after immunization in the mice, suggesting that cell-mediated immunity had been induced, These results suggest that E. rhusiopathiae YS-1 may be a suitable choice for further studies of vaccine efficacy in swine.