The Identification, Isolation and Partial Characterisation of VapA-encoding virulence plasmids in Rhodococcus equi found in New Zealand.

This thesis describes the identification, isolation and partial characterisation of VapA-encoded virulence plasmids present in Rhodococcus equi (R. equi) found in foals in New Zealand. R. equi infection associated with the presence of virulence plasmids causes severe pyogranulomatous pneumonia in foals and, if left untreated, it can be lethal. Foals are thought to be infected when they ingest or breathe in bacteria present in soil, dust and faecal particles after which the bacteria multiply inside macrophages and cause pneumonia. Studies have shown that virulence is associated with the presence of the vapA gene in a plasmid that encodes a VapA protein. Twelve different types of VapA-encoding virulence plasmids have been described so far. Epidemiological studies of America, Australia, Korea, Japan and some parts of Europe have revealed that more than one subtype can exist in a farm or even a country. The aim of the study was to investigate if more than one subtype of VapA-encoded plasmids exists in a farm in New Zealand. Nasal swabs for the analysis were obtained from the Auckland Veterinary Centre in Papakura. Based on colony morphology and 16S primers, successful identification of R. equi was possible, VapA primers assisted with distinguishing bacteria that carried VapA-encoded plasmids. Since success with plasmid isolations was modest, characterization of plasmids was conducted by sequence analysis of an approximately 1-kb variable region present in VapA-encoded plasmids. Sequence analysis and restriction digestion patterns revealed two distinct patterns of sequences from the variable region which were divided into two groups: PI and PII. Results indicated that PI sequences were significantly similar to published 85-kb type I plasmids and must also be from 85-kb type I plasmids but more detailed analysis of other regions is required to confirm the results. PII sequences on the other hand were distinctly different from PI sequences and displayed poor matches with published plasmid sequences. This is the first attempt at characterising VapA-encoded virulence plasmids from R. equi isolates in New Zealand. This work will contribute towards increasing our knowledge regarding the unique characteristics of the VapA-encoding plasmids, which could define transmission characteristics, possible sources of infection in disease outbreaks and vaccine development