The application of subtractive hybridisation to detect intra-species variation in Campylobacter jejuni.
Degree GrantorUniversity of Canterbury
Degree NameMaster of Science
Significant intra-species variation is evident in many pathogenic bacteria. Possession of a specific gene or cluster of genes can lead to the greater pathogenicity of that strain. Campylobacter jejuni is seemingly ubiquitous in the environment and is frequently isolated from many species of birds, cattle, sheep and other animals, as well as water. Molecular typing studies suggest C. jejuni populations are heterogenous, with the emergence of some stable clones. Strains of C. jejuni are naturally competent with a propensity for intraspecies DNA uptake. Virulence determinants and disease mechanisms are poorly understood and research with regards to the pathogenic potential of this organism is contradictory. The primary objective of this study was to determine whether all strains of C. jejuni are equally pathogenic to humans. A PCR-based subtractive hybridisation method was applied to detect differences between two strains of C. jejuni, NCTCll168 (tester) and F38011 (driver). Using this method, a total of eleven DNA fragments were isolated and sequenced. Blast searches revealed all fragments corresponded to the tester strain NCTCl1168. The fragments showed significant amino acid identities with the flagella hook protein (FlgE), an AJG-specific adenine glycosylase and a L-serine dehydratase. Nucleotide sequences corresponding to flgE were further analysed and one base pair substitution was observed between the tester and the driver (F38011). This study demonstrates that the PCR-based subtractive hybridisation method was reproducible and has the potential to identify fragments corresponding to genes with relevance to virulence. However, this method requires optimisation for the efficient isolation of strain differences in C. jejuni. The feasibility of the application of mRNA approaches and RAPD-PCR to detect intra-species variation in C. jejuni were also investigated. Difficulties encountered with mRNA detection excluded further investigation of mRNA-based techniques. RAPD-PCR was not sufficiently reproducible to continue analysis of the significance of differences in RAPD profiles in relation to the pathogenic potential of C. jejuni.