We describe the performance of cell-based and antibody blood tests for the antemortem diagnosis of tuberculosis (TB) in South American camelids (SAC). The sensitivity and specificity of the gamma interferon (IFN-␥) release assay, two lateral flow rapid antibody tests (Stat-Pak and Dual Path Platform [DPP]), and two enzyme-linked immunosorbent assay (ELISA)-based antibody tests (Idexx and Enferplex) were determined using diseased alpacas from Mycobacterium bovis culture-confirmed breakdown herds and TB-free alpacas from geographical areas with no history of bovine TB, respectively. Our results show that while the sensitivities of the IFN-␥ and antibody tests were similar (range of 57.7% to 66.7%), the specificity of the IFN-␥ test (89.1%) was lower than those of any of the antibody tests (range of 96.4% to 97.4%). This lower specificity of the IFN-␥ test was at least in part due to undisclosed Mycobacterium microti infection in the TB-free cohort, which stimulates a positive purified protein derivative (PPD) response. The sensitivity of infection detection could be increased by combining two antibody tests, but even the use of all four antibody tests failed to detect all diseased alpacas. These antibody-negative alpacas were IFN-␥ positive. We found that the maximum sensitivity could be achieved only by the combination of the IFN-␥ test with two antibody tests in a "test package," although this resulted in decreased specificity. The data from this evaluation of tests with defined sensitivity and specificity provide potential options for antemortem screening of SAC for TB in herd breakdown situations and could also find application in movement testing and tracing investigations.

Bacillus Calmette-Guérin (BCG), an attenuated strain of Mycobacterium bovis (M. bovis), is the lead candidate vaccine for control of bovine tuberculosis (TB) in cattle. However, BCG vaccination sensitises cattle to bovine tuberculin, thus compromising the use of the current bovine TB surveillance tests. To address this, we have developed a diagnostic skin test that is not compromised by BCG vaccination and is able to detect BCG vaccinated animals that subsequently develop bovine TB following exposure to M. bovis. Building on previous work using ‘in house’ formulated protein cocktail reagents, we herein present test performance data for a single fusion protein (DST-F) containing the mycobacterial antigens ESAT-6, CFP-10 and Rv3615c formulated as a ‘ready to use’ reagent by a commercial manufacturer. Our results demonstrate that, unlike tuberculin reagents, a diagnostic skin test using DST-F maintained high specificity in BCG vaccinated animals. Furthermore, the DST-F skin test demonstrated a high relative sensitivity in identifying M. bovis infected animals, including those where BCG vaccination failed to prevent bovine TB pathology following experimental exposure to M. bovis. The DST-F is currently undergoing field trials in Great Britain to support its licensure and commercialisation.

In the absence of biomarkers of protective immunity, newly developed vaccines against bovine tuberculosis need to be evaluated in virulent Mycobacterium bovis challenge experiments, which require the use of expensive and highly in demand Biological Safety Level 3 (BSL3) animal facilities. The recently developed bovine BCG challenge model offers a cheaper and faster way to test new vaccine candidates and additionally reduces the severity of the challenge compared to virulent M. bovis challenge in line with the remits of the NC3Rs. In this work we sought to establish the sensitivity of the BCG challenge model by testing a prime boost vaccine regimen that previously increased protection over BCG alone against M. bovis challenge. All animals, except the control group, were vaccinated subcutaneously with BCG Danish, and half of those were then boosted with a recombinant adenoviral vector expressing Antigen 85A, Ad85A. All animals were challenged with BCG Tokyo into the prescapular lymph node and the bacterial load within the lymph nodes was established. All vaccinated animals, independent of the vaccination regimen, cleared BCG significantly faster from the lymph node than control animals, suggesting a protective effect. There was however, no difference between the BCG and the BCG-Ad85A regimens. Additionally, we analysed humoral and cellular immune responses taken prior to challenge for possible predictors of protection. Cultured ELISpot identified significantly higher IFN-ɣ responses in protected vaccinated animals, relative to controls, but not in unprotected vaccinated animals. Furthermore, a trend for protected animals to produce more IFN-ɣ by quantitative PCR and intracellular staining was observed. Thus, this model can also be an attractive alternative to M. bovis challenge models for the discovery of protective biomarkers.