Evidence of mycobacteriaemias and mycobacterial co-infections uncovered in cattle at slaughter using a novel phage-based PhMS-qPCR assay for viable Mycobacterium bovis and Mycobacterium avium subsp. paratuberculosis

Accurate diagnosis of Bovine Tuberculosis (bTB) in cattle is important for success of eradication programmes, but the current reliance on the single intradermal comparative cervical tuberculin (SICCT) test is not achieving disease eradication in all jurisdictions, including Northern Ireland (NI). In this study, a novel Phagomagnetic separation (PhMS)-qPCR assay to rapidly detect viable Mycobacterium bovis in bovine blood samples was evaluated. A total of 149 heart blood samples were collected from cattle at exsanguination point of the slaughter line at a NI abattoir between June and August 2023 - 74 from TB reactor cattle (compulsorily culled within days of a positive SICCT test result) and 75 from routine slaughter cattle (< 30 months). Peripheral blood mononuclear cells (PBMCs) were isolated and lysed to release any mycobacteria present before PhMS-qPCR and culture were performed to detect presence of viable M. bovis. The DNAs obtained were subsequently tested for evidence of viable Mycobacterium avium subsp. paratuberculosis (MAP) also. PhMS-qPCR results indicate that of the TB reactor cattle with conclusive PhMS-qPCR results, 7.5% of bloods tested positive for viable M. bovis only, 41.8% for viable MAP only and 28.4% showed evidence of co-infection (both M. bovis and MAP detected). Of the routine slaughter cattle with conclusive PhMS-qPCR results, 22.4% of bloods tested positive for viable M. bovis only, 19.4% for viable MAP only and 20.9% showed evidence of co-infection. Of the blood samples with conclusive culture results, 19.6% TB reactors and 25.4% routine slaughter cattle were M. bovis culture positive. No agreement was observed between the M. bovis PhMS-qPCR assay and M. bovis culture results (Kappa - 0.028, 95% CI -0.162 to 0.105). Results of this study provide proof-of-concept that the PhMS-qPCR assay is able to detect viable M. bovis in bovine blood, although a different qPCR assay with greater detection sensitivity will need to be identified going forward. A surprisingly high number of M. bovis/MAP co-infections were detected in the blood of NI cattle, which may be contributing to failure of the bTB eradication scheme. The latter warrants further investigation.