Attenuation and immunogenicity of Δcya Δcrp derivatives of Salmonella choleraesuis in pigs

Six different isogenic Δcya Δcrp derivatives of a strain of Salmonella choleraesuis var. kunzendorf-χ3246 virulent for pigs were constructed by transposon-mediated deletion mutagenesis. These strains were evaluated for virulence and ability to elicit a protective immune response in young weaned pigs after oral administration and were compared to a commercially available vaccine which lacks the 50-kb virulence plasmid (vpl^-). These derivatives were Δcya Δcrp vpl⁺, Δcya Δcrp vpl^-, Δcya Δ(crp-cdt) vpl⁺, Δcya Δ(crp-cdt) vpl^-, Δcya Δcrp pmi3834 vpl⁺, and Δcya Δ(crp-cdt) pmi-3834. In experiments to evaluate safety, no significant adverse effects of any of the vaccine constructs were observed, except that two of the strains which carried the virulence plasmid (vpl⁺) caused a small, short-term elevation in maximum temperature compared to pretreatment temperature values. Orally immunized animals, except for those vaccinated with the Δcya Δcrp pmi-3834 vpl⁺ strain or SC-54, developed significant serum antibody responses 21 days postvaccination as measured by enzyme-linked immunosorbent assay. No cell- mediated immune responses to heat-killed S. choleraesuis were noted at the same time point as measured with heat-killed bacteria as antigen in a lymphocyte proliferation assay. In an oral challenge exposure model with a highly virulent heterologous strain of S. choleraesuis, the Δcya Δcrp strains with deletions in pmi were not protective. As measured by morbidity scores, the responses to challenge of the pigs vaccinated with the other four Δcya Δcrp derivatives were significantly better than those of the nonvaccinated, challenged group. With the exception of temperature elevation and slight differences in diarrhea scores postchallenge, none of these strains differed significantly from each other in the other clinical parameters analyzed. While the commercial vaccine was protective by most of the parameters measured, it was not fully protective against challenge with virulent S. choleraesuis as judged by diarrhea scores and temperature elevation. Collectively, these data demonstrate that Δcya Δcrp derivatives, with or without the virulence plasmid but not with deletions in the pmi gene, are candidates for vaccines for protection against salmonellosis in pigs.