| This thesis is an investigation of the development of a safe and potentially efficacious attenuated strain of Rhodococcus equi for use in oral immunization of foals. Initial work involved the attempted development of a killed but metabolically active ("KMBA") strain of R. equi through use of two double crossover mutations in the uvrA and uvrB genes. The suicide vector for uvrA, pSuvrA, and the first two steps of a second sucide vector for uvrB were developed. A double crossover mutation in the uvrA gene of R. equi 103+ was not successfully obtained due to the presence of multiple homologous sites for insertion on the chromosome, therefore this approach was abandoned. The second approach involved expression of VapA in a live, virulence-plasmid negative strain ofR. equi (strain 103-), with assessment of virulence and immunogenicity in mice. PCR-amplified fragments of entire vapA gene with and without the upstream virulence-plasmid genes virR, orj5, vapH, orf7 and orf8 (orf4-8) were cloned into a shuttle vector pNBV1. These plasmids, named pAWVapA and pAW48A, were electroporated into strain 103-. The presence of the recombinant vectors in the attenuated strain (103-) and the integrity of the inserted genes were confirmed by antibiotic selection, PCR and DNA sequencing. SDS-PAGE and Western blotting showed that both constructs expressed VapA. The virulence for mice of the two recombinant R. equi was compared to that of wild type R. equi 103+ by intravenous inoculation of mice with the organisms and examination of liver clearance 4 days later. Mice inoculated with R. equi 103-, 103-/pAWVapA and 103-/pNBV1 completely cleared infection. Strain 103-/pAW48A persisted in 47% of the mice, whereas wild-type 103+ persisted in all treatment group mice. No detectable IgG to VapA was noted in any of the mice. R. equi was isolated from the feces of some mice after oral vaccination. No major granulomatous lesions were noted on histopathology in any mice and there was no significant difference in liver bacterial colonization in any of the groups challenged with R. equi 103+, showing that oral immunization of mice does not lead to effective immunization against this organism. |
