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. Mycobacterium tuberculosis complex (MTBC) members, Mycobacterium bovis (M. bovis) and Mycobacterium tuberculosis (M. tuberculosis), are the cause of bovine tuberculosis (bTB) and human tuberculosis (TB), respectively. These chronic infectious diseases are a significant global health threat for human and animal populations. Since many high TB burden countries are dependent on animal-related industries such as agriculture and tourism, the consequences of infection can be devastating to their economies. Increased opportunities for disease transmission at human-animal interfaces occur as human settlements encroach on agricultural lands or natural habitats. Since TB can cross species barriers, cases of bTB in people in African countries have been reported 1,2 as well as reports of M. tuberculosis infection in domestic cattle in rural areas of Africa as well as captive wildlife and pets 3-6. The risks of MTBC spread to endangered wildlife have been highlighted by the recent discoveries of M. bovis infection in free-ranging white and black rhinoceros' (Ceratotherium simum, Diceros bicornis) as well as the death of an African elephant (Loxodonta africana) bull shown to have M. tuberculosis infection in the Kruger National Park (KNP) 4,5,7. Infection of these animals was not unexpected since KNP is endemic for bTB; however, M. tuberculosis infection in a free-ranging elephant is a novel finding. As controlled veterinary diseases, infections with MTBC have significant consequences for species management, public health, veterinary disease control, and conservation endeavours.

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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.

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-␥) 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-␥ 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-␥ 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. Mycobacterium bovis is the causative agent of bovine tuberculosis (BTB) in a wide range of domestic animals and wildlife (1). BTB in cattle populations is intensively controlled in many countries as it can result in reduced productivity or death of infected animals and poses a serious zoonotic risk. In South Africa, the African buffalo (Syncerus caffer) is a maintenance host of M. bovis, and the early detection of infected animals is important to control the transmission of the pathogen to other wildlife and domestic species and to prevent the geographic spread of this disease by translocation (2, 3).The most sensitive method for diagnosing M. bovis infection is by detection of the host's cell-mediated immune response to pathogen-specific antigens (4). Examples of such tests are the in vivo tuberculin skin test (TST) and in vitro interferon gamma (IFN-␥) release assays (IGRAs). The latter detect the release of interferon gamma in whole blood or from isolated peripheral blood mononuclear cells (PBMCs) in response to M. bovis purified protein derivative (PPD) (5) or to more specific antigens such as the 6-kDa early secreted antigenic target (ESAT-6) and the 10-kDa culture filtrate protein (CFP-10) (6, 7). Recently, IGRAs utilizing the latter antigens, i.e., the modified Quant...