Development and evaluation of two multi-antigen serological assays for the diagnosis of bovine tuberculosis in cattle

There is currently an increased interest in the use of serological approaches in combination with traditional cell-mediated immunity-based techniques to improve the detection of tuberculosis (TB)-infected animals. In the present study, we developed and validated two different serological TB-detection assays using four antigens, MPB70, MPB83, ESAT6 and CFP10, and the tuberculin PPDb. A conventional multi-antigen TB-ELISA method and a novel TB multiplex test, based on Luminex technology, were developed to detect antibodies to multiple antigen targets. The performance levels of the two tests were evaluated and compared using selected panels of samples having known TB states. The TB-ELISA test (containing five antigens, including PPDb) had a sensitivity (Se) of 74.2% and a specificity (Sp) of 94.9%, while the TB-Luminex test had higher Se (79.0%) and Sp (99.1%) rates even when only one reactive antigen was used to classify the test as positive. If a more restrictive criterion, requiring two positive antigens to classify the test as positive, was used, then the TB-ELISA's Sp rate increased to 99.8% but the Se decreased to 61.3%, while the TB-Luminex test's Sp rate increased to 100% but the Se decreased to 51.2%. TB-ELISA and TB-Luminex were applied to a panel of 257 sera collected from bTB-positive herds, as determined by a post-mortem inspection. They showed good performance levels, identifying 49 (80.3%) and 48 (78.7%), respectively, of 61 samples that had tested positive by the intradermal tuberculin (IDT) test and/or interferon-gamma assay. In addition, TB-ELISA and TB-Luminex were able to identify 60 and 42 samples as positive, respectively, out of the 196 samples that tested negative to IDT and interferon-gamma at the time of serum collection. Subsequent IDT tests performed after 1–2 months, confirmed the positivity of 18 samples, indicating the strategic value of having two serological assays to detect TB-infected herds that were not reactive to initial IDT testing, thereby allowing for the rapid control of outbreaks and eradication of the disease.