Evaluation of Mycobacterial Load Assay for rapid molecular identification of Mycobacterium bovis in bovine tissues

Mammalian tuberculosis (TB) in cattle, caused mostly by Mycobacterium bovis, has a significant impact on veterinary public health, animal welfare, trade, and farming economies. Diagnosis and laboratory confirmation are crucial for disease surveillance and effective control strategies in animals. The relatively low sensitivity of current ante- and postmortem tests for TB often leads to undetected infections in animals. In the absence of a “gold standard”, bacteriological culture of M. bovis is considered the reference standard for confirmation, but it has a long turnaround time and relatively low sensitivity. In reference laboratories there has been a move towards the more rapid turnaround PCR-based testing, with equivalent sensitivity to culture. Although current molecular diagnostic techniques are based on the detection of M. bovis DNA, 16S rRNA offers detection with high sensitivity while also serving as a marker of bacterial viability.  In this pilot study, we evaluated the ability of the mycobacterial load assay (MBLA), a PCR-based method quantifying viable Mycobacterium tuberculosis complex organisms, to rapidly detect M. bovis 16S rRNA directly from culture-positive bovine tissue samples. We assessed the efficacy of MBLA to detect M. bovis in comparison to the reference method, bacteriological culture. We also evaluated the association of MBLA-determined bacterial load to tissue weight, presence or absence of visible lesions, lesion scores, and M. bovis molecular types.  MBLA detected M. bovis in 89.7% of culture positive tissue samples. Quantification was possible in 72.9% of positive samples, with bacterial loads ranging from 3.11x102 CFU/g to 1.09x106 CFU/g.  No significant associations were found between bacterial load and tissue weight, lesion status, lesion scores, or molecular types.  This study is the first to utilize MBLA to detect and quantify M. bovis 16S rRNA bacterial loads directly from infected bovine tissues. MBLA demonstrated promising potential for rapid, robust and reliable quantitative detection of M. bovis in infected bovine tissues. Further research is needed to evaluate its applicability to detect M. bovis in other cattle sample types, environmental samples and tissues from other host species.