Strategy for Improving the Diagnostic Efficacy of Bovine Tuberculosis Using a Novel PCR<sub>BIO/DIG</sub>-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 TD-IS6110 ) for the detection of Mycobacterium bovis in dairy milk. To optimize this diagnostic approach, a PCR BIO/DIG was 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 PCR TD-IS6110 and TSTS, used as composite gold standard. The diagnostic sensitivity and specificity were 97% and 66%, respectively. The analytical sensitivity of the PCR BIO/DIG -ELISA was 25 times higher than that of the PCR TD - IS6110 , since 16 milk samples analysed by PCR BIO/DIG -ELISA were positive, while all were negative by PCR TD-IS6110 and 9 were TSTS-negative. Therefore, the developed PCR BIO/DIG -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.