Molecular epidemiology and biology of small ruminant mycoplasmas

© 2015 Dr. Olusola Martins OlaogunMembers of the genus Mycoplasma are the smallest and simplest self-replicating living cells. Their evolution is characterised by both genome downsizing and horizontal gene transfer. They are among the most important bacterial pathogens of small ruminants worldwide and are associated with a number of chronic illnesses of considerable economic importance. The most costly production disease of the small ruminant dairy industry is contagious agalactia (CA). This multi-aetiological syndrome is found in most countries with significant small ruminant diary industries but is believed to be absent from Australia. To provide insights into the molecular epidemiology and biology of small ruminant mycoplasmas, the studies reported here investigated the molecular epidemiology of mycoplasmas associated with CA in other countries in Victorian populations of sheep and goats, and also investigated the mechanisms involved in the mobilization of mycoplasmal integrative and conjugative elements (MICEs), a class of mobile genetic elements that are thought to be a key driver of mycoplasma evolution. Firstly, sheep and goats from different geographical locations in Victoria were assessed for the presence of CA-associated mycoplasmas and isolates obtained from the study were genotyped using a multilocous sequence typing approach. The findings from the epidemiological survey suggested the persistence of some of the agents associated with CA in Australia, but there was no evidence of the clinical syndrome in the sampled population. Secondly, a multilocus sequence typing (MLST) scheme was developed to characterise M. arginini isolates obtained from the epidemiological survey. The MLST study, which was the first attempt to genotype M. arginini, was able to successfully capture intermediate-level evolutionary relationships among the isolates and revealed a high level of genetic diversity among strains of M. arginini. The scheme offers a promising platform for future molecular epidemiological studies of this ubiquitous mycoplasma species that may improve our understanding of its clinical significance. Thirdly, the machinery involved in the mobilization of the MICE ICEA from Mycoplasma agalactiae was extensively characterized in a mycoplasmal model, as well as in a non-mycoplasma species, Escherichia coli. Using in vitro functional studies, the coding sequence required for the excision and integration of this element, as well as the essential sequences involved in its catalytic actions, were identified and characterized, and the protein encoded by it was also expressed and shown to be functional in a non-mycoplasma organism, E. coli. The findings from this study of MICE biology have generated a foundation for the development of a novel integrase-based molecular tool for genetic manipulation of mycoplasmas