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.

Tuberculosis transmission by Mycobacterium bovis in a mixed cattle and goat herd.

A tuberculosis (TB) outbreak caused by Mycobacterium bovis occurred in a mixed herd of three cattle and eighteen goats in Northern Italy in 2005. All the cattle were removed, as opposed to the co-existing goats, who remained in the farm and were not subsequently tested by the official intradermal tuberculin test. At the beginning of May 2006, a 7-day old calf was introduced into the herd from an officially TB-free (OTF) farm. On October 2006, tuberculous lesions were detected at the slaughterhouse in the same animal. The following epidemiological investigation on the herd highlighted a clinical suspicion of TB in one goat out of 35, and visible lesions were found at necropsy in the respiratory and intestinal tracts. Bacteriological culture and molecular tests confirmed the presence of M. bovis in both animals. Spoligotyping and Mycobacterial Interspersed Repetitive Units - Variable Number of Tandem Repeats (MIRU-VNTR) showed the same genomic profile of the previous breakdown occurred in 2005. Since this profile has never been described in Italy, these findings suggest the probable transmission of TB within the farm among cattle and goats. The remaining 34 goats were also tested by single intradermal cervical comparative tuberculin (SICCT) test, Interferon (IFN)-γ assay and ELISA for antibody to M. bovis. The SICCT test and the IFN-γ showed a good concordance with 20 and 19 positive reactors, respectively. By ELISA we found 12Ab-positive animals, seven of which had not been detected by the tests for cell-mediated immunity. Finally, 15 goats were found positive for gross lesions at necropsy. The in vivo tests revealed a total of 27 positive animals out of 35, which highlights the usefulness of the serology in parallel with SICCT and IFN-γ when an advanced stage of infection is suspected. Moreover, our results confirm the necessity for adopting the official tuberculin test on goats co-existing with cattle.