The Genomic Diversity and Molecular Epidemiology of Mycobacterium avium subsp. paratuberculosis in Canadian Dairy Cattle

Abstract

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative bacterium of Johne’s disease, a chronic enteritis in ruminants. Extensive collaborations with Johne’s disease researchers from across Canada provided an opportunity to build a collection of MAP isolates representing all provinces. Difficulties in interpreting the minimal genetic diversity between isolates have previously limited the power of traditional molecular epidemiological approaches in determining transmission patterns. However, the use of whole genome sequencing (WGS) now permits the diversity and relationship of MAP isolates to be assessed in much greater detail. The focus of this thesis was to investigate MAP genetic diversity and molecular epidemiology using molecular tools and phylogenetic analyses based primarily on WGS. In the following chapters the phylogeography and evolutionary dynamics of MAP in Canadian dairy cattle, explored through a combination of complementary approaches, will be presented. In Chapter 2, matrix-assisted laser desorption ionization-time of flight mass spectrometry was applied to interrogate a discriminatory short sequence repeat locus in the MAP genome, improving the discrimination of long mononucleotide repeats. In Chapter 3, WGS was used to determine the reliability of variable number tandem repeat typing (VNTR) to appropriately assess epidemiologically related and unrelated isolates. The use of WGS clarified the level of diversity and relationship between isolates and revealed that VNTR typing does not always reflect extensive within-herd MAP diversity. In Chapter 4, several thousand single nucleotide polymorphisms (SNPs) were used to infer the phylogeny of Canadian isolates in the context of the known global diversity, identifying a dominant clade that includes more than 85% of the Canadian isolates. The phylogeography of MAP at a national and provincial level was further explored, in which considerable overlap of MAP types between different provinces and within a single province was found. In Chapter 5, the impact of SNPs on the microevolution of the Canadian MAP population was investigated, identifying specific genes with an overrepresentation of nonsynonymous mutations and observing selection over time in multiple subtypes. Lastly, in Chapter 6 a novel PCR assay is described that targeted phylogenetically informative SNPs to further identify the relative importance of MAP subtypes in over 600 Canadian isolates.