Molecular epidemiology, clonality and virulence of Dichelobacter nodosus, the agent of ovine footrot

Dichelobacter nodosus, an anaerobic bacterium, is the major transmissible agent of ovine footrot. The disease expresses as a virulent or benign lesion in the hoof. Virulence is related to the production of serine proteases, particularly a thermostable protease. Isolates of D. nodosus are characterised according to the type of protease produced (either heat-stable or heat-labile) and the electrophoretogram (zymogram) of the protease. This study reports on the use of the DNA-based typing techniques Pulsed-Field Gel Electrophoresis (PFGE) and Infrequent-Restriction-Site-PCR (IRS-PCR) to investigate the molecular epidemiology of D. nodosus, including a consideration of the relationship between genetic type, zymogram patterns and whole cell protein profiles. The aim of the project was to obtain a better understanding of D. nodosus strain diversity and dissemination in Australia and its relationship to virulence within the population. The overall intention was to use this information to assist in the long-term control of virulent footrot. Field isolates of D. nodosus from Western Australia (n = 735), New South Wales (n = 16), Victoria (n = 24) and South Australia (n = 21) were obtained and analysed. Both typing techniques that were used offered good differentiation between isolates for epidemiological purposes, and the results were in general agreement. PFGE provided slightly better discrimination between isolates, with 214 PFGE types (181 from Western Australia) compared to 94 IrsT types (77 from Western Australia). Within this diverse range of molecular types clonality was observed - with clones being defined as clusters of isolates having closely related PFGE types. The strains were categorised as genetically diverse, genetically similar or identified as the same strain. This diversity of genetic types was found overall, within flocks of sheep on a farm and within a single hoof where, on a number of occasions, multiple molecular types and zymogram types were found colonising a single hoof. One isolate that was experimentally inoculated into a flock of sheep produced six different genetic types when tested 12 months after the initial infection. This indicates that D. nodosus undergoes rapid genetic change, which means that follow-up epidemiological investigation of disease outbreaks and trace-backs need to be done as soon after infection as possible. The genetic differences appeared to be due to large insertions or deletions of DNA. Amongst sheep on some properties, isolates that had a different protease expression and virulence expression were found to have the same molecular type. Investigation of these isolates by SDS-PAGE showed that they also had the same whole cell protein profiles. Isolates from the same clonal groups also had the same protein profile, whereas genetically diverse isolates had different protein profiles. The lack of protein differences between isolates of the same molecular type, or within a clonal group, suggests that the differences in protease thermostability may be due to conformational changes in the protein, rather than to overall detectable genetic change and/or expression of different proteins. These results demonstrate that PFGE typing can be useful in predicting likely phenotypic expression of whole cell proteins. Further work is required to elucidate differences between virulent and benign strains of D. nodosus.