Toll-like receptor (TLR)6 and TLR1 differentiation in gene expression studies of Johne's disease

Johne's disease (JD) is a mycobacterial infection of the gut affecting ruminants and other species caused by Mycobacterium avium subspecies paratuberculosis (MAP). The role of toll-like receptors (TLR) in the pathogenesis of JD has been previously identified at the level of gene expression. Gene expression studies using reverse transcriptase (RT)-PCR are widely used and powerful, but the results obtained from such studies are dependent on the specificity of the assay. Here we describe an assay designed to detect ovine TLR6 in blood and tissues from sheep. Discrimination between TLR1 and TLR6 at the level of gene expression was challenging due to extensive tracts of homology and identity within the two sequences. Both TLR1 and 6 can form heterodimers with TLR2 in order to bind the ligands of microbial pathogens. The expression of TLR6 was increased in the ileum and jejunum of sheep infected with MAP, with a trend towards TLR6 upregulation in peripheral blood cells in response to exposure to MAP. A likely role for TLR6/TLR2 heterodimers in the pathogenesis of JD was identified. TLR6 may be a potential marker of exposure and could aid in the development of a gene signature for sheep resistant to MAP infection.