Shedding of Mycobacterium bovis in respiratory secretions of free-ranging wild dogs (Lycaon pictus): Implications for intraspecies transmission.

It has recently been discovered that Mycobacterium bovis (M. bovis) causes disease in the endangered African wild dog (Lycaon pictus) in areas endemic for bovine tuberculosis (bTB), including the Kruger National Park (KNP). However, information on M. bovis infection dynamics within this species is limited and requires investigation as M. bovis can cause conservation consequences due to movement restrictions, crucial for genetic management. This study had two aims: firstly, to investigate mycobacterial shedding in free-ranging wild dogs from KNP by culturing oropharyngeal swab (OS) and bronchoalveolar lavage (BAL) samples. Secondly, to determine the relationship between ante-mortem culture and interferon gamma release assay (IGRA) results as well as agreement between OS culture and BAL culture results. Mycobacterial culture revealed that 6 of 173 (3.5%) OS samples and 1 of 32 (3.1%) BAL samples (from 7 different wild dogs) were M. bovis culture positive, suggesting that wild dogs can shed M. bovis through respiratory secretions. However, the possibility of contamination by ingestion of infected prey cannot be excluded in wild dogs with positive OS culture results. Furthermore, the test outcomes between IGRA and culture (OS and BAL) differed substantially. Samples from 172 wild dogs were available for IGRA screening and 134 had positive results (detectable M. bovis immune sensitization). Seven wild dogs had culture-positive results, which included one additional wild dog that did not have an IGRA result (total 173 wild dogs). Out of these 7 M. bovis culture-positive wild dogs, 3 were IGRA positive initially, however, after repeat sampling and testing, 5 out of 7 were IGRA positive. These findings suggest that intraspecies transmission of M. bovis may be possible among wild dogs. Although the risk of intraspecies transmission is currently unknown, this knowledge is important for assessing the risk of M. bovis transmission from infected wild dogs to uninfected populations during translocations.

Review of Diagnostic Tests for Detection of Mycobacterium bovis Infection in South African Wildlife.

Wildlife tuberculosis is a major economic and conservation concern globally. Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis), is the most common form of wildlife tuberculosis. In South Africa, to date, M. bovis infection has been detected in 24 mammalian wildlife species. The identification of M. bovis infection in wildlife species is essential to limit the spread and to control the disease in these populations, sympatric wildlife species and neighboring livestock. The detection of M. bovis-infected individuals is challenging as only severely diseased animals show clinical disease manifestations and diagnostic tools to identify infection are limited. The emergence of novel reagents and technologies to identify M. bovis infection in wildlife species are instrumental in improving the diagnosis and control of bTB. This review provides an update on the diagnostic tools to detect M. bovis infection in South African wildlife but may be a useful guide for other wildlife species.

Impact of Mycobacterium bovis-induced pathology on interpretation of QuantiFERON®-TB Gold assay results in African buffaloes (Syncerus caffer).

The cytokine interferon gamma-inducible protein 10 (IP-10) is a sensitive biomarker of Mycobacterium bovis (M. bovis) infection in African buffaloes (Syncerus caffer). However, elevated levels of IP-10 in QuantiFERON®-TB Gold (QFT) unstimulated whole blood compromises the utility of this biomarker. In this study, IP-10 and interferon gamma (IFN-γ) concentrations in whole blood samples from M. bovis culture-confirmed buffaloes with varying degrees of pathological changes (n = 72) and uninfected controls (n = 70) were measured in the IP-10 release assay (IPRA) and IFN-γ release assay (IGRA), respectively. Findings suggest that concentrations of both cytokines in QFT Nil tubes were higher in infected buffaloes with macroscopic pathological changes consistent with bovine tuberculosis compared to uninfected controls, and IGRA values increased with more severe pathological changes in infected buffaloes (p < 0.05). Finally, in culture-confirmed buffaloes with IPRA-negative and IGRA-positive test results, most animals were also those with the most advanced pathology. We conclude that IP-10 and IFN-γ concentrations measured in QFT Nil tubes may provide insight into the presence of M. bovis pathology in infected buffaloes. Furthermore, this study highlights the value in evaluating cytokine production in both antigen-stimulated and unstimulated samples when interpreting cytokine release assay results.

Parallel measurement of IFN-γ and IP-10 in QuantiFERON®-TB Gold (QFT) plasma improves the detection of Mycobacterium bovis infection in African buffaloes (Syncerus caffer).

The QuantiFERON®-TB Gold (QFT) stimulation platform for cytokine release is a novel approach for diagnosis of bovine tuberculosis in wildlife species. Plasma interferon gamma (IFN-γ) is routinely measured to detect immune sensitization to Mycobacterium bovis. However, the cytokine interferon gamma-inducible protein 10 (IP-10) has been proposed as an alternative, more sensitive, diagnostic biomarker. In this study, we investigated the use of the QFT system with measurement of IFN-γ and IP-10 in parallel to identify M. bovis-infected African buffaloes. The test results of either biomarker in a cohort of M. bovis-unexposed buffaloes (n = 70) led to calculation of 100% test specificity. Furthermore, in cohorts of M. bovis culture-positive (n = 51) and M. bovis-suspect (n = 22) buffaloes, the IP-10 test results were positive in a greater number of animals than the number based on the IFN-γ test results. Most notably, when the biomarkers were measured in parallel, the tests identified all M. bovis culture-positive buffaloes, a result neither the single comparative intradermal tuberculin test (SCITT) nor Bovigam® IFN-γ release assay (IGRA) achieved, individually or in parallel. These findings demonstrate the diagnostic potential of this blood-based assay to identify M. bovis-infected African buffaloes and a strategy to maximise the detection of infected animals while maintaining diagnostic specificity and simplifying test procedures.

Mycobacterium bovis Infection in African Wild Dogs, Kruger National Park, South Africa.

We screened African wild dogs (Lycaon pictus) in Kruger National Park, South Africa, for Mycobacterium bovis infection using an interferon-gamma release assay. We detected M. bovis sensitization in 20 of 21 packs; overall apparent infection prevalence was 83%. These animals experience high infection pressure, which may affect long-term survival and conservation strategies.

AN INTERFERON GAMMA RELEASE ASSAY FOR THE DETECTION OF IMMUNE SENSITIZATION TO MYCOBACTERIUM BOVIS IN AFRICAN WILD DOGS (LYCAON PICTUS).

In South Africa, the largest proportion of the African wild dog (Lycaon pictus) population resides in regions where buffaloes have a high prevalence of Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB). Recent reports of deaths of wild dogs associated with bTB have raised concerns regarding the threat this disease might pose for this species. In order to understand the potential impact of the disease in wild dogs, diagnostic tools are required to identify infected individuals. The interferon gamma (IFN-γ) release assay (IGRA) is commonly used for tuberculosis (TB) screening of humans, cattle, and other species, and the aim of this study was to develop an IGRA for wild dogs to detect immune sensitization. Blood was collected from immobilized wild dogs from the Ann van Dyk Cheetah Centre (AvDCC; n=9) and Kruger National Park (KNP; n=31). Heparinized whole blood was incubated overnight in QuantiFERON®-TB Gold (QFT) blood collection tubes and with selected mitogens, after which the plasma fraction was harvested. Three canine IFN-γ enzymelinked immunosorbent assays (ELISAs) were compared for detection of wild dog IFN-γ in plasma and the R&D Quantikine canine IFN-γ ELISA was selected for measurement of M. bovis-specific IFN-γ release in plasma samples. An IGRA result was calculated as the concentration in plasma derived from the QFT TB Antigen tubes minus that in the QFT Nil tube. An IGRA cut-off value was calculated using the IGRA results of M. bovis-unexposed individuals from AvDCC. Using this cut-off value, 74% (23/31) of M. bovis-exposed KNP wild dogs were IGRA positive, indicating immune sensitization to TB antigens in these animals. Three M. bovis culture-positive wild dogs from KNP had IFN-γ concentrations between 758 and 1,445 pg/mL, supporting this interpretation. This warrants further investigation into the prevalence of M. bovis infection in the KNP population.

Parallel testing increases detection of Mycobacterium bovis-infected African buffaloes (Syncerus caffer).

The diagnosis of Mycobacterium bovis (M. bovis) infection in African buffaloes (Syncerus caffer) relies on detection of the cell-mediated immune response to M. bovis antigens using the single comparative intradermal tuberculin test (SCITT) or interferon gamma release assays (IGRAs). The aim of the present study was to determine whether parallel testing with the SCITT and an IGRA increases the number of M. bovis-infected buffaloes detected by these assays. Culture-confirmed animals (n = 71) tested during routine bovine tuberculosis (bTB) control programmes in Hluhluwe iMfolozi Park and Madikwe Game Reserve in South Africa, were used in this study. Results from 35 buffaloes tested using the SCITT and three Bovigam® IGRAs (cohort A) and 36 buffaloes tested using the SCITT, standard Bovigam® IGRA and Qiagen Cattletype IGRA (cohort B) were analysed. The parallel use of the SCITT with selected IGRAs was able to identify all animals in both cohorts. These findings are in agreement with cattle studies supporting the use of the SCITT and IGRAs in parallel to identify the greatest number of M. bovis-infected animals. The suggested parallel testing algorithm should be strategically applied to maximize detection of M. bovis infection in bTB-positive buffalo herds.