Strategy for Improving the Diagnostic Efficacy of Bovine Tuberculosis Using a Novel PCR-ELISA Assay.

Bovine Tuberculosis (BTb) is an important zoonosis caused by Mycobacterium bovis. In Argentina, the Official National Control Program mandates cattle slaughter when a positive Tuberculin Skin Test Single (TSTS) is observed. Herds that test negative twice a year are considered infection-free; however, many false negatives have been observed in anergic cattle. In our previous study, 16% of herds with TSTS-positive animals were found to be negative by the Polymerase Chain Reaction Touch Down of Insertion Sequence 6110 (PCR) for the detection of Mycobacterium bovis in dairy milk. To optimize this diagnostic approach, a PCRwas developed using primers labelled with biotin (BIO) and digoxigenin (DIG) and coupling the detection of amplified Deoxyribonucleic Acid (DNA) by means of an Enzyme-Linked Immunosorbent Assay (ELISA). The cut-off, the diagnostic and analytical characteristics were determined, considering results from both PCRand TSTS, used as composite gold standard. The diagnostic sensitivity and specificity were 97% and 66%, respectively. The analytical sensitivity of the PCR-ELISA was 25 times higher than that of the PCR-, since 16 milk samples analysed by PCR-ELISA were positive, while all were negative by PCRand 9 were TSTS-negative. Therefore, the developed PCR-ELISA technique would be a useful tool for identifying infected herds, especially those with TSTS-negative animals, as they pose a risk to epidemiological control. Additionally, it could help confirm infection in slaughtered cattle with TSTS-negative results by analysing tissues with lesions compatible with BTb.