Strategy to improve the diagnostic efficacy of Bovine Tuberculosis by PCRBIO/DIG-ELISA

Bovine Tuberculosis (BTb) is an important zoonosis caused by Mycobacterium bovis (M. bovis) . In Argentina, the Official National Control Program mandates cattle slaughter when a positive Tuberculin Skin Test (TST) is observed. Herds that test negative twice a year are considered infection-free; however, unfortunately, many false negatives have been observed in anergic cattle. In our previous study, 16% of herds with TST-positive animals were found to be negative by the Polymerase Chain Reaction Touch-Down Insertion Sequence (PCR _TD-IS6110 ) for detecting M. bovis in dairy milk. To optimise this diagnostic approach, a PCR _{BIO/DIG (Biotin/Digoxigenin)} -ELISA (Enzyme-Linked Immunosorbent Assay) was developed using primers labelled with digoxigenin and biotin and detecting the amplified DNA by ELISA. The experimental parameters were optimised through statistical experimental design. The cut-off and the diagnostic and analytical characteristics were determined, considering results from both PCR and TST. Ultimately, DNA from 25 milk samples and 16 bovine tissue samples were analysed. 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 ; however, 16 negative milk samples by PCR _TD-IS6110 were positive by PCR _BIO/DIG -ELISA, 9 TST-negative. Therefore, PCR _BIO/DIG -ELISA can be proposed as a useful tool for identifying infected herds, especially those with TST-negative animals, as they pose a risk for epidemiological control. Additionally, it could help confirm infection in slaughtered cattle with TST-negative results by analysing tissues with or without lesions compatible with BTb. It is the first to publish a PCR _BIO/DIG -ELISA for the M. bovis detection in raw milk and tissue.