Detection of Mycobacterium tuberculosis complex DNA in oronasal swabs from infected African buffaloes (Syncerus caffer).

Mycobacterium bovis (M. bovis), a member of the Mycobacterium tuberculosis complex (MTBC), is the causative agent of bovine TB (bTB) in animals. Spread occurs through inhalation or ingestion of bacilli transmitted from infected individuals. Early and accurate detection of infected African buffaloes shedding M. bovis is essential for interrupting transmission. In this pilot study, we determined if MTBC DNA could be detected in M. bovis infected buffalo oronasal secretions using a molecular transport media (PrimeStore MTM) with oronasal swabs and a rapid qPCR assay (Xpert MTB/RIF Ultra). Bovine TB test-positive buffaloes were culled, then tissue samples and oronasal swabs collected post-mortem for mycobacterial culture and Ultra testing, respectively. The Ultra detected MTBC DNA in 5/12 swabs from M. bovis culture-confirmed buffaloes. Oronasal swabs from M. bovis negative buffaloes (n = 20) were negative on Ultra, indicating the high specificity of this test. This study showed that MTM can successfully preserve MTBC DNA in oronasal swabs. The proportion of MTBC positive oronasal swabs was higher than expected and suggests that the Ultra may be an additional method for identifying infected buffaloes. Further studies are needed to confirm the utility of the Ultra assay with oronasal swabs as an assay to evaluate possible MTBC shedding in buffaloes.

Optimized interferon-gamma release assays for detection of Mycobacterium bovis infection in African buffaloes (Syncerus caffer).

The African buffalo (Syncerus caffer) is an economically and ecologically important wildlife species in South Africa; it is also a primary wildlife maintenance host of Mycobacterium bovis. Accurate and early detection of M. bovis infection in buffaloes is important for controlling transmission. Assays that detect cell-mediated immune responses to M. bovis in buffaloes have been developed although these often display suboptimal sensitivity or specificity. Therefore, the aim of this study was to evaluate the newly available Mabtech bovine interferon-gamma (IFN-γ) ELISAPRO kit and optimize its use for detection of buffalo IFN-γ in whole blood samples stimulated with the QuantiFERON® TB Gold Plus antigens. Additionally, the test performance of the Mabtech IFN-γ release assay (IGRA) was compared to the currently used Cattletype® IGRA by determining buffalo-specific cut-off values for the two IGRAs and using gold standard-positive (M. bovis culture-confirmed) and M. bovis-unexposed negative cohorts. Validation of the Mabtech ELISA revealed negligible matrix interference and a linear and parallel response for recombinant bovine and native buffalo IFN-γ in the range 1.95-250 pg/mL. Intra- and inter-assay reproducibility produced coefficients of variation <5.5 % and <6.1 %, respectively, with a limit of detection at 3.2 pg/mL. Using receiver operator characteristic curve analyses, buffalo-specific cut-off values were calculated as 8 pg/mL for the Mabtech IGRA and 5 % (signal to positive control ratio) for the Cattletype® IGRA. The sensitivities were 89 % and 83 % for the Mabtech and Cattletype IGRAs with specificities of 94 % and 97 %, respectively. Although the species-specific cut-off values require further evaluation in a relevant test group, the results suggest that the Mabtech IGRA is a promising, sensitive and specific diagnostic tool for M. bovis detection in African buffaloes.

Novel molecular transport medium used in combination with Xpert MTB/RIF ultra provides rapid detection of Mycobacterium bovis in African buffaloes.

Mycobacterium bovis is the causative agent of bovine tuberculosis (bTB) in wildlife. Confirmation of M. bovis infection relies on mycobacterial culture, which is time-consuming. Collection and transportation of infectious material also pose a human health risk. PrimeStore Molecular Transport Medium (MTM) has been shown to effectively inactivate infectious organisms, making it a safe method for handling infectious samples. This study investigated an in-field sampling technique for rapid, safe detection of M. bovis in buffalo tissues. Potentially infected tissues from bTB test-positive buffaloes were swabbed at post-mortem examination and stored in PrimeStore MTM at ambient temperature until Xpert MTB/RIF Ultra testing was performed. Additionally, tissue samples were frozen and transported before homogenisation for culture and Ultra testing. Oral swabs were collected from M. bovis-unexposed buffaloes as a negative control cohort. Mycobacterium tuberculosis complex (MTBC) DNA was detected by Ultra in 13/16 tissue swabs and 9/16 matched tissue homogenates from culture-confirmed M. bovis-positive buffalo tissues. MTBC DNA was not detected in swabs from M. bovis-unexposed animals, showing the potentially high specificity of Ultra with PrimeStore swabs. PrimeStore MTM sample processing, in combination with the Ultra assay, has the potential to provide a safe, rapid post-mortem screening test for M. bovis in buffaloes.

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.

Mycobacterium bovis prevalence affects the performance of a commercial serological assay for bovine tuberculosis in African buffaloes.

The endemic presence of bovine tuberculosis (BTB) in African buffaloes in South Africa has severe consequences for BTB control in domestic cattle, buffalo ranching and wildlife conservation, and poses a potential risk to public health. This study determined the BTB prevalence in free-ranging buffaloes in two game reserves and assessed the influence of the prevalence of mycobacterial infections on the performance of a commercial cattle-specific serological assay for BTB (TB ELISA). Buffaloes (n = 997) were tested with the tuberculin skin test and TB ELISA; a subset (n = 119) was tested longitudinally. Culture, PCR and sequencing were used to confirm infection with M. bovis and/or non-tuberculous mycobacteria (NTM). Prevalence of BTB, but not NTM, influenced the TB ELISA performance. Multiple testing did not increase test confidence. The findings strongly illustrate the need for development of novel assays that can supplement existing assays for a more comprehensive testing scheme for BTB in African buffaloes.

Test Characteristics of Assays to Detect Mycobacterium bovis Infection in High-Prevalence African Buffalo (Syncerus caffer) Herds.

A herd of African buffaloes (Syncerus caffer) was tested for Mycobacterium bovis infection using three cytokine release assays. All animals were subsequently euthanized and mycobacterial culture determined the infection prevalence (52%) and diagnostic characteristics. Sensitivities were lower than previously reported and results provide new insight into the practical utility of these assays.

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.

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.

Parasites of domestic and wild animals in South Africa. XLIX. Ticks (Acari: Ixodidae) infesting white and black rhinoceroses in southern Africa.

The objectives of the study were to determine the species composition of ticks infesting white and black rhinoceroses in southern Africa as well as the conservation status of those tick species that prefer rhinos as hosts. Ticks were collected opportunistically from rhinos that had been immobilised for management purposes, and 447 white rhinoceroses (Ceratotherium simum) and 164 black rhinoceroses (Diceros bicornis) were sampled in South Africa, 61 black rhinos in Namibia, 18 white and 12 black rhinos in Zimbabwe, and 24 black rhinos in Zambia. Nineteen tick species were recovered, of which two species, Amblyomma rhinocerotis and Dermacentor rhinocerinus, prefer rhinos as hosts. A. rhinocerotis was collected only in the northeastern KwaZulu-Natal reserves of South Africa and is endangered, while D. rhinocerinus is present in these reserves as well as in the Kruger National Park and surrounding conservancies. Eight of the tick species collected from the rhinos are ornate, and seven species are regularly collected from cattle. The species present on rhinos in the eastern, moister reserves of South Africa were amongst others Amblyomma hebraeum, A. rhinocerotis, D. rhinocerinus, Rhipicephalus maculatus, Rhipicephalus simus and Rhipicephalus zumpti, while those on rhinos in the Karoo and the drier western regions, including Namibia, were the drought-tolerant species, Hyalomma glabrum, Hyalomma rufipes, Hyalomma truncatum and Rhipicephalus gertrudae. The species composition of ticks on rhinoceroses in Zambia differed markedly from those of the other southern African countries in that Amblyomma sparsum, Amblyomma tholloni and Amblyomma variegatum accounted for the majority of infestations.