African buffaloes ( Syncerus caffer ) are maintenance hosts of Mycobacterium bovis , the causative agent of bovine tuberculosis. They act as reservoirs of this infection for a wide range of wildlife and domestic species, and the detection of infected animals is important to control the geographic spread and transmission of the disease. Interferon gamma (IFN-γ) release assays (IGRAs) utilizing pathogen-derived peptide antigens are highly specific tests of M. bovis infection; however, the diagnostic sensitivities of these assays are suboptimal. We evaluated the diagnostic utility of measuring antigen-dependent interferon gamma-induced protein 10 (IP-10) release as an alternative to measuring IFN-γ levels. M. bovis -exposed buffaloes were tested using the Bovigam PC-EC and Bovigam PC-HP assays and a modified QuantiFERON TB-Gold (mQFT) assay. IP-10 was measured in the harvested plasma and was produced in significantly greater abundance in response to M. bovis antigens in Bovigam-positive than in Bovigam-negative animals. For each assay, using the Bovigam results as a reference, receiver operating characteristic curve analysis was done to determine diagnostically relevant cutoff values for IP-10. Thereafter, mQFT test results derived from measurement of IP-10 and IFN-γ were compared and a larger number of Bovigam-positive animals were detected using IP-10 as a diagnostic marker. Moreover, using IP-10, agreement between the mQFT assay and the Bovigam assays was increased, while the excellent agreement between the Bovigam assays was retained. We conclude that IP-10 is a sensitive marker of antigen recognition and that measurement of this cytokine in antigen-stimulated whole blood might increase the sensitivity of conventional IGRAs in African buffaloes.

The study describes the novel use of the Xpert MTB/RIF Ultra assay for detection of Mycobacterium tuberculosis complex (MTBC) DNA in samples from white rhinoceros (Ceratotherium simum) and African elephants (Loxodonta africana). Culture negative respiratory sample matrices were spiked to determine if the Ultra could detect MTBC DNA in rhinoceros and elephant samples. Rhinoceros bronchial alveolar lavage fluid (BALF) was found to have an inhibitory effect on the Ultra. In this study, the limit of detection (LOD) of M. tuberculosis H37Rv in all spiked animal samples were 2 CFU/ml compared to 15.6 CFU/ml for humans, while the LOD for M. bovis SB0121 was 30 CFU/ml compared to 143.4 CFU/ml for M. bovis BCG in humans. Screening was performed on stored tissue and respiratory samples from known MTBC-infected animals and MTBC DNA was detected in 92% of samples collected from six rhinoceros and two elephants. Conversely, 83% of culture-negative tissue and respiratory samples from uninfected animals tested negative on the Ultra. In conclusion, the Ultra assay appears to be a sensitive and rapid diagnostic test for the detection of MTBC DNA from tissue and respiratory samples collected from African elephants and rhinoceros. Furthermore, the Ultra assay could provide a new tool for the detection of MTBC in various sample types from other wildlife species.

In South Africa, mycobacterial culture is regarded as the gold standard for the detection of Mycobacterium tuberculosis complex (MTBC) infection in wildlife even though it is regarded as “imperfect.” We compared a novel decontamination and mycobacterial culture technique (TiKa) to the conventional mycobacterium growth indicator tube (MGIT) system using known amounts of bacilli and clinical samples from MTBC-infected African buffaloes (Syncerus caffer), white rhinoceros (Ceratotherium simum), and African elephants (Loxodonta africana). Use of the TiKa-KiC decontamination agent on samples spiked with 10,000 to 10 colony forming units (cfu) of M. bovis (SB0121) and M. tuberculosis (H37Rv) had no effect on isolate recovery in culture. In contrast, decontamination with MGIT MycoPrep resulted in no growth of M. bovis samples at concentrations < 1,000 cfu and M. tuberculosis samples < 100 cfu. Subsequently, we used the TiKa system with stored clinical samples (various lymphatic tissues) collected from wildlife and paucibacillary bronchoalveolar lavage fluid, trunk washes, and endotracheal tube washes from 3 species with known MTBC infections. Overall, MTBC recovery by culture was improved significantly (p < 0.01) by using TiKa compared to conventional MGIT, with 54 of 57 positive specimens versus 25 of 57 positive specimens, respectively. The TiKa mycobacterial growth system appears to significantly enhance the recovery of MTBC members from tissue and paucibacillary respiratory samples collected from African buffaloes, African elephants, and white rhinoceros. Moreover, the TiKa system may improve success of MTBC culture from various sample types previously deemed unculturable from other species.

Mycobacterium bovis infection, the cause of bovine tuberculosis (BTB), is endemic in wildlife in the Kruger National Park (KNP), South Africa. In lions, a high infection prevalence and BTB mortalities have been documented in the KNP; however, the ecological consequences of this disease are currently unknown. Sensitive assays for the detection of this infection in this species are therefore required. Blood from M. bovis‐exposed, M. bovis‐unexposed, M. tuberculosis‐exposed and M. bovis‐infected lions was incubated in QuantiFERON^®‐TB Gold (QFT) tubes containing either saline or ESAT‐6/CFP‐10 peptides. Using qPCR, selected reference genes were evaluated for expression stability in these samples and selected target genes were evaluated as markers of antigen‐dependent immune activation. The abundance of monokine induced by gamma interferon (MIG/CXCL9) mRNA, measured in relation to that of YWHAZ, was used as a marker of ESAT‐6/CFP‐10 sensitization. The gene expression assay results were compared between lion groups, and lenient and stringent diagnostic cut‐off values were calculated. This CXCL9 gene expression assay combines a highly specific stimulation platform with a sensitive diagnostic marker that allows for discrimination between M. bovis‐infected and M. bovis‐uninfected lions.