|JOSHI, DEEPTI - University Of Minnesota|
|HARRIS, BETH - Animal And Plant Health Inspection Service (APHIS)|
|MATHEMA, BARUN - New Jersey Medical School|
|KRIESWIRTH, BARRY - New Jersey Medical School|
|SREEVATSAN, SRINAND - University Of Minnesota|
Submitted to: Journal of Clinical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/9/2012
Publication Date: 12/1/2012
Citation: Joshi, D., Harris, B., Waters, W.R., Thacker, T.C., Mathema, B., Krieswirth, B., Sreevatsan, S. 2012. Single nucleotide polymorphisms in the Mycobacterium bovis genome resolve phylogenetic relationships. Journal of Clinical Microbiology. 50(12):3853-3861.
Interpretive Summary: Despite highly successful eradication efforts in several countries, tuberculosis of cattle remains a serious health concern worldwide. Within the United States, tuberculosis infection of cattle originates from three primary sources: importation of tuberculous cattle from Mexico, spillover of the pathogen from a wildlife reservoir (i.e., White-tailed deer in Michigan), and from farmed deer. In the present study, genomic DNA from various relevant Mycobacterium bovis isolates were compared to determine potential differences. Minor differences were detected. These differences in the genetic material of the various isolates may be exploited to develop improved molecular methods necessary for epidemiology investigations. Present findings will be useful for the development of tools to control bovine tuberculosis.
Technical Abstract: Mycobacterium bovis isolates carry restricted allelic variation yet exhibit a range of disease phenotypes and host preferences. Conventional genotyping methods target small hyper-variable regions of their genome and provide anonymous biallelic information insufficient to develop phylogeny. To resolve phylogeny and establish trait-allele associations we interrogated 75 Mycobacterium bovis and 61 Mycobacterium tuberculosis genomes for single nucleotide polymorphisms (SNPs) using iPLEXTM Massarray (Sequenom Inc., CA) technology. We indexed nucleotide variations in 306 geneic and 44 intergeneic loci among isolates derived from outbreaks in the US between 1991-2010 and isolated from a variety of mammalian hosts. Based on 206 variant SNPs between the Mycobacterium bovis isolates, five major “SNP-cluster groups” were identified. 49 out of the 51 human Mycobacterium tuberculosis isolates were identical at the 350 loci studied. Using SNP-cluster groups, four virulent Mycobacterium bovis strains from human (n=1), cattle (n=2) and deer (n=1) were selected for evaluation of macrophage survival and relative gene expression of six virulence associated genes at two different time points post-infection. The results revealed a variation in macrophage survivability between the strains over a 48-hr. infection cycle. Gene expression profiles for the six virulence associated proteins including those involved in mammalian cell entry (mce4C), antigenic variation and evasion of innate immune response (PE6), lipid biosynthesis and metabolism (fadD9 & INO1), inhibitor of host anti-mycobactericidal compound NOS (speE) and a transmembrane transport protein (mmpL12) revealed large variations. Thus SNP-based analyses sufficiently capture genotypic and phenotypic differences within Mycobacterium bovis.