Development of a live, attenuated, potential vaccine strain of R. equi expressing vapA and the virR operon, and virulence assessment in the mouse

Pneumonia caused by Rhodococcus equi remains a significant problem in foals. The objective of this study was to develop a safe and efficacious attenuated strain of R. equi for eventual use in oral immunization of foals. The approach involved expression of vapA in a live, virulence plasmid-negative, strain of R. equi (strain 103-). PCR-amplified fragments of the vapA gene, with and without the upstream genes virR, orf5, vapH, orf7 and orf8 (orf4–8), were cloned into a shuttle vector pNBV1. These plasmids, named pAW48A and pAWVapA respectively, 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, and both constructs expressed VapA. The virulence of the two strains was compared to that of wild type R. equi 103+ and negative controls by their intravenous inoculation into mice, followed by examination of liver clearance 4 days later. Mice inoculated with R. equi 103-, 103-/pAWVapA and 103-/pNBV1 completely cleared infection, whereas strain 103-/pAW48A persisted in 47% of mice.