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<p>Bovine Tuberculosis (BTb) is an important zoonosis caused by <i>Mycobacterium bovis</i>. 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 <i>6110</i> (PCR<sub>TD−IS6110</sub>) for the detection of <i>Mycobacterium bovis</i> in dairy milk. To optimize this diagnostic approach, a PCR <sub>BIO/DIG</sub> 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<sub>TD−IS6110</sub> and TSTS, used as composite gold standard. The diagnostic sensitivity and specificity were 97% and 66%, respectively. The analytical sensitivity of the PCR<sub>BIO/DIG</sub>-ELISA was 25 times higher than that of the PCR<sub>TD</sub>-<sub>IS6110</sub>, since 16 milk samples analysed by PCR<sub>BIO/DIG</sub>-ELISA were positive, while all were negative by PCR<sub>TD−IS6110</sub> and 9 were TSTS-negative. Therefore, the developed PCR<sub>BIO/DIG</sub>-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.</p> Graphical Abstract <p></p>

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Strategy for Improving the Diagnostic Efficacy of Bovine Tuberculosis Using a Novel PCRBIO/DIG-ELISA Assay

  • 期刊论文

  • 发表日期：

    2026年5月22日

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  • Silvia Noemi Fabiano,
  • Ana Paula Cislaghi,
  • Ana Maria Canal,
  • Silvina Vanesa Kergaravat,
  • Silvia Raquel Hernández,
  • Adriana Rosa Soutullo

摘要

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 (PCRTD−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 PCRTD−IS6110 and TSTS, used as composite gold standard. The diagnostic sensitivity and specificity were 97% and 66%, respectively. The analytical sensitivity of the PCRBIO/DIG-ELISA was 25 times higher than that of the PCRTD-IS6110, since 16 milk samples analysed by PCRBIO/DIG-ELISA were positive, while all were negative by PCRTD−IS6110 and 9 were TSTS-negative. Therefore, the developed PCRBIO/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.

Graphical Abstract