Assessment ofMycobacterium tuberculosisOmpATb as a Novel Antigen for the Diagnosis of Bovine Tuberculosis

Abstract

In the search for better tools to control bovine tuberculosis, the development of diagnostic tests with improved specificity and sensitivity has a high priority. We chose to search for novel immunodiagnostic reagents. In this study, Rv0899 (outer membrane protein A of Mycobacterium tuberculosis [OmpATb]) was evaluated as a stimulation antigen in a gamma interferon (IFN-␥) release assay to diagnose bovine tuberculosis. OmpATb induced IFN-␥ responses in cattle experimentally infected with M. bovis as early and as persistently as ESAT-6 and CFP-10, the current lead diagnostic antigens. In naturally infected cattle, OmpATb stimulated IFN-␥ production in 22 of 26 animals (85%). Importantly, OmpATb detected a portion of M. bovis-infected cattle which did not respond to ESAT-6 and CFP-10 (five of six cattle). The combined diagnostic sensitivity of OmpATb, ESAT-6, and CFP-10 for a preselected group consisting of naturally infected cattle with an overrepresentation of ESAT-6/CFP-10 nonresponders was 96% (25 of 26 animals). The specificity of OmpATb for uninfected cattle was 100% (27 cattle were tested; 12 of them gave false-positive results with tuberculins). In summary, our results indicate that OmpATb has the potential to enhance the sensitivity of previously described diagnostic tests based on ESAT-6 and CFP-10 and that the combined use of OmpATb, ESAT-6, CFP-10, and other proteins may achieve at least equal sensitivity to that obtained with purified protein derivative, but at a higher specificity. Further studies evaluating the diagnostic performance of OmpATb in combination with other proteins are ongoing.Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB), is also responsible for a proportion of human TB cases. Thus, infection of cattle with M. bovis constitutes both a human health hazard and an animal welfare problem, with economic implications in terms of trade restrictions, productivity losses, and massive annual expenditure on bTB eradication programs. The control of bTB is based mainly on a policy of test and slaughter. The persistence of this zoonotic disease combined with the loss of trade and the exponential costs for control justify a need not only for more sensitive but also for more specific diagnostic assays. The occurrence of false-positive results can be attributed, at least in part, to the fact that immune responses to purified protein derivative (PPD) are present not only in animals with TB but also in animals exposed to environmental mycobacteria (reviewed in reference 29). PPDs are prepared by precipitation from heat-killed cultures of mycobacteria and are poorly defined, complex antigens containing many proteins, some of which are shared by different mycobacterial species or even other bacteria. Coinfection of cattle with M. bovis and M. avium subsp. paratuberculosis has been reported to coincide not only with increased numbers of false-positive results in PPD-based diagnostic assays but also with an increased frequency of false-negative results due to a general depression of ce...

Bovine Tuberculosis: A Review of Current and Emerging Diagnostic Techniques in View of their Relevance for Disease Control and Eradication

“…Therefore, a wider range of antigens in addition to ESAT‐6 and CFP‐10 is needed for improving the sensitivity of specific antigen‐based IFN‐γ assays. In recent studies, promising antigen combinations for the IFN‐γ assay resulting in improved sensitivity and specificity have been described (Cockle et al., 2006; Sidders et al., 2008; Schiller et al., 2009b). Importantly, Rv3615c and OmpATb were found to detect cattle with confirmed bTB but not responding to E/C.…”

“…Changes required for the IFN‐γ assay to overcome its current disadvantages and to allow its use as individual animal test comprise improvements of specificity, overall test accuracy and ease of use, in addition to cost reductions. New antigen combinations have been found which complement the current lead diagnostic antigens ESAT‐6 and CFP‐10 and result in superior sensitivity, specificity, and predictive values compared to PPD‐based tests (Sidders et al., 2008; Schiller et al., 2009b; Vordermeier et al., 2009). Interestingly, recent data, using relatively small sample numbers that need to be confirmed in larger trials, indicate that even ESAT‐6 and CFP‐10 alone (without further antigens) could result in superior performance of the IFN‐γ assay compared to that of CCT.…”

Isolation of mycobacteria from clinical samples collected in the United States from 2004 to 2011

“…Several antigens have shown considerable promise in existing and emerging diagnostic assays (e.g. MPB70, MPB83, ESAT6, CFP10, and OmpTb) [9-12]. However, cross-reactivity with other mycobacterial species [13,14] and variation in the immune response from animal to animal [15,16] continues to hamper improved diagnostic specificity [3].…”

Use of a multiplex enzyme-linked immunosorbent assay to detect a subpopulation of Mycobacterium bovis–infected animals deemed negative or inconclusive by the single intradermal comparative tuberculin skin test

“…No single antigen has, thus far, been identified that elicits a response in all animals during different stages of infection. As reported in a 2009 review, 15 many infected animals are missed with assays based on the use of a single antigen. Attempts to increase the sensitivity of the assay using multiple antigens showed this approach has the potential of increasing the ability to detect more infected animals.…”