Characterization of the global bovine microRNAome of peripheral blood mononuclear cells isolated from Mycobacterium bovis exposed cattle

Accurate diagnostics are urgently needed for bovine TB in cattle – an economically devastating disease posing a re-emerging threat to veterinary and public health worldwide. MicroRNAs, post-transcriptional regulators of gene expression, are involved in a wide range of biological processes and immunological pathways, and have emerged as promising diagnostic biomarkers for numerous diseases. In human TB, microRNAs have been widely studied, but not much is currently known about their role in bovine TB. The aim of this study was to investigate the diagnostic potential of microRNAs in bTB through comprehensive analysis of their expression profiles in disparate states of M. bovis (BCG) exposure. We used a state-of-the-art RNA sequencing approach to characterize the global microRNAome of peripheral blood mononuclear cells isolated from cattle that were either unvaccinated, BCG-vaccinated, unprotected or protected. A total of 468 microRNAs were detected across all samples, none of which were uniquely expressed in any group. Significant differential expression between groups was observed for 3 microRNAs (bta-miR-6122-3p, bta-miR-3533 and bta-miR29b) in various comparisons. Subsequent target analysis of bta-miR-29b, a candidate biomarker of disease in human tuberculosis, revealed that several genes (ACVR2A, PIK3R1, TBX21, TGFBR1 and TGFBR2) involved in a number of relevant T-cell and immune signaling pathways, were amongst the predicted targets. Overall, this study provides evidence that microRNAs could be a promising novel biomarker for bovine TB diagnostics. Overall design: PBMC were isolated from whole blood of cattle in previous study (doi: 10.3389/fimmu.2020.588180). The present study compared two groups of animals: unvaccinated controls and vaccinated with live M. bovis BCG (Danish SSI 1331; 0.5 mL 4.6 x 10^6). Animals were vaccinated at T=0 and challenged endobronchially with virulent M. bovis (AF2122/97; 10^4 CFU) after 9 weeks. Samples used in this study were taken 8 weeks after vaccination (week 8) and 8 weeks after challenge (week 17). Samples were selected on the basis of presence (VL) or absence (NVL) of lesions at PM. Animals were subsequently grouped according to their immunological and infection status (“status”): controls (unvaccinated, prior to challenge), vaccinated (BCG-vaccinated, prior to challenge), unprotected (unvaccinated, post-challenge, VL) and protected (BCG-vaccinated, post-challenge, NVL).