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 Erysip elothrix rhusiopathiae are avirulent for mice. In this study, we const ructed nonreverting acapsular mutants and examined the vaccine potenti al of the mutants in mice. A representative acapsular transposon mutan t, 33H6, was plated on selective agar containing autoclaved chlortetra cycline and quinaldic acid, and two tetracycline-sensitive mutants wer e 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 presumab ly inserted with Tn916 into 33H6 chromosome, substituted for those pre sent at the insertion site. The mutants were confirmed to be devoid of capsular antigen by Western immunoblotting and were nonvirulent for m ice (subcutaneous 50% lethal dose [LD50], > 10(9) CFU). The safety and efficacy of acapsular mutants for live vaccines was further studied b y using one mutant strain, named YS-1, The YS-1 bacteria were cleared from the skin sites of inoculation, livers, and spleens of the inocula ted mice by 7 days after subcutaneous (s.c.) inoculation. Mice immuniz ed s.c. with doses ranging from 2 x 10(4) to 2 x 10(8) CFU of strain Y S-1 were completely protected against challenge with 100 LD50 of the h omologous, highly virulent strain Fujisawa-SmR 21 days postimmunizatio n, and protective immunity conferred by immunization with 2 x 10(8) CF U 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 collecte d at days 4 and 7 did not. Furthermore, specific spleen cell responses to E. rhusiopathiae antigens were observed in mice immunized with str ain YS-1, and cross-protection against the antigenically heterologous bacterium Listeria monocytogenes was observed at 7 days after immuniza tion in the mice, suggesting that cell-mediated immunity had been indu ced, These results suggest that E. rhusiopathiae YS-1 may be a suitabl e choice for further studies of vaccine efficacy in swine.