Identification and molecular characterization of Mycobacterium bovis DNA in GeneXpert® MTB/RIF ultra-positive, culture-negative sputum from a rural community in South Africa.

This study investigated the presence of Mycobacterium bovis (M. bovis) DNA in archived human sputum samples previously collected from residents who reside adjacent to the M. bovis-endemic Hluhluwe-iMfolozi wildlife park, South Africa (SA). Sixty-eight sputum samples were GeneXpert MTB/RIF Ultra-positive for M. tuberculosis complex (MTBC) DNA but culture negative for M. tuberculosis. Amplification and Sanger sequencing of hsp65 and rpoB genes from DNA extracted from stored heat-inactivated sputum samples confirmed the presence of detectable amounts of MTBC from 20 out of the 68 sputum samples. Region of difference PCR, spoligotyping and gyrB long-read amplicon deep sequencing identified M. bovis (n = 10) and M. tuberculosis (n = 7). Notably, M. bovis spoligotypes SB0130 and SB1474 were identified in 4 samples, with SB0130 previously identified in local cattle and wildlife and SB1474 exclusively in African buffaloes in the adjacent park. M. bovis DNA in sputum, from people living near the park, underscores zoonotic transmission potential in SA. Identification of spoligotypes specifically associated with wildlife only and spoligotypes found in livestock as well as wildlife, highlights the complexity of TB epidemiology at wildlife-livestock-human interfaces. These findings support the need for integrated surveillance and control strategies to curb potential spillover and for the consideration of human M. bovis infection in SA patients with positive Ultra results.

Detection of Mycobacterium bovis in nasal swabs from communal goats (Capra hircus) in rural KwaZulu-Natal, South Africa.

Animal tuberculosis, caused by Mycobacterium bovis, presents a significant threat to both livestock industries and public health. Mycobacterium bovis tests rely on detecting antigen specific immune responses, which can be influenced by exposure to non-tuberculous mycobacteria, test technique, and duration and severity of infection. Despite advancements in direct M. bovis detection, mycobacterial culture remains the primary diagnostic standard. Recent efforts have explored culture-independent PCR-based methods for identifying mycobacterial DNA in respiratory samples. This study aimed to detect M. bovis in nasal swabs from goats (Capra hircus) cohabiting with M. bovis-infected cattle in KwaZulu-Natal, South Africa. Nasal swabs were collected from 137 communal goats exposed to M. bovis-positive cattle and 20 goats from a commercial dairy herd without M. bovis history. Swabs were divided into three aliquots for analysis. The first underwent GeneXpert® MTB/RIF Ultra assay (Ultra) screening. DNA from the second underwent mycobacterial genus-specific PCR and Sanger sequencing, while the third underwent mycobacterial culture followed by PCR and sequencing. Deep sequencing identified M. bovis DNA in selected Ultra-positive swabs, confirmed by region-of-difference (RD) PCR. Despite no other evidence of M. bovis infection, viable M. bovis was cultured from three communal goat swabs, confirmed by PCR and sequencing. Deep sequencing of DNA directly from swabs identified M. bovis in the same culture-positive swabs and eight additional communal goats. No M. bovis was found in commercial dairy goats, but various NTM species were detected. This highlights the risk of M. bovis exposure or infection in goats sharing pastures with infected cattle. Rapid Ultra screening shows promise for selecting goats for further M. bovis testing. These techniques may enhance M. bovis detection in paucibacillary samples and serve as valuable research tools.

Advancing animal tuberculosis surveillance using culture-independent long-read whole-genome sequencing.

Animal tuberculosis is a significant infectious disease affecting both livestock and wildlife populations worldwide. Effective disease surveillance and characterization of Mycobacterium bovis (M. bovis) strains are essential for understanding transmission dynamics and implementing control measures. Currently, sequencing of genomic information has relied on culture-based methods, which are time-consuming, resource-demanding, and concerning in terms of biosafety. This study explores the use of culture-independent long-read whole-genome sequencing (WGS) for a better understanding of M. bovis epidemiology in African buffaloes (Syncerus caffer). By comparing two sequencing approaches, we evaluated the efficacy of Illumina WGS performed on culture extracts and culture-independent Oxford Nanopore adaptive sampling (NAS). Our objective was to assess the potential of NAS to detect genomic variants without sample culture. In addition, culture-independent amplicon sequencing, targeting mycobacterial-specific housekeeping and full-length 16S rRNA genes, was applied to investigate the presence of microorganisms, including nontuberculous mycobacteria. The sequencing quality obtained from DNA extracted directly from tissues using NAS is comparable to the sequencing quality of reads generated from culture-derived DNA using both NAS and Illumina technologies. We present a new approach that provides complete and accurate genome sequence reconstruction, culture independently, and using an economically affordable technique.

"Spotting" Mycobacterium bovis infection in leopards (Panthera pardus) - novel application of diagnostic tools.

Background: Mycobacterium bovis (M. bovis) is the causative agent of animal tuberculosis (TB) which poses a threat to many of South Africa's most iconic wildlife species, including leopards (Panthera pardus). Due to limited tests for wildlife, the development of accurate ante-mortem tests for TB diagnosis in African big cat populations is urgently required. The aim of this study was to evaluate currently available immunological assays for their ability to detect M. bovis infection in leopards. Methods: Leopard whole blood (n=19) was stimulated using the QuantiFERON Gold Plus In-Tube System (QFT) to evaluate cytokine gene expression and protein production, along with serological assays. The GeneXpert® MTB/RIF Ultra (GXU®) qPCR assay, mycobacterial culture, and speciation by genomic regions of difference PCR, was used to confirm M. bovis infection in leopards. Results: Mycobacterium bovis infection was confirmed in six leopards and individuals that were tuberculin skin test (TST) negative were used for comparison. The GXU® assay was positive using all available tissue homogenates (n=5) from M. bovis culture positive animals. Mycobacterium bovis culture-confirmed leopards had greater antigen-specific responses, in the QFT interferon gamma release assay, CXCL9 and CXCL10 gene expression assays, compared to TST-negative individuals. One M. bovis culture-confirmed leopard had detectable antibodies using the DPP® Vet TB assay. Conclusion: Preliminary results demonstrated that immunoassays and TST may be potential tools to identify M. bovis-infected leopards. The GXU® assay provided rapid direct detection of infected leopards. Further studies should aim to improve TB diagnosis in wild felids, which will facilitate disease surveillance and screening.

Mycobacterium tuberculosis complex detection in rural goat herds in South Africa using Bayesian latent class analysis.

Animal tuberculosis affects a wide range of domestic and wild animal species, including goats (Capra hircus). In South Africa, Mycobacterium tuberculosis complex (MTBC) testing and surveillance in domestic goats is not widely applied, potentially leading to under recognition of goats as a potential source of M. bovis spread to cattle as well as humans and wildlife. The aim of this study was to estimate diagnostic test performance for four assays and determine whether M. bovis infection was present in goats sharing communal pastures with M. bovis positive cattle in the Umkhanyakude district of Northern Zululand, KwaZulu Natal. In 2019, 137 M. bovis-exposed goats were screened for MTBC infection with four diagnostic tests: the in vivo single intradermal comparative cervical tuberculin test (SICCT), in vitro QuantiFERON®-TB Gold (QFT) bovine interferon-gamma release assay (IGRA), QFT bovine interferon gamma induced protein 10 (IP-10) release assay (IPRA), and nasal swabs tested with the Cepheid GeneXpert® MTB/RIF Ultra (GXU) assay for detection of MTBC DNA. A Bayesian latent class analysis was used to estimate MTBC prevalence and diagnostic test sensitivity and specificity. Among the 137 M. bovis-exposed goats, positive test results were identified in 15/136 (11.0%) goats by the SICCT; 4/128 (3.1%) goats by the IPRA; 2/128 (1.6%) goats by the IGRA; and 26/134 (19.4%) nasal swabs by the GXU. True prevalence was estimated by our model to be 1.1%, suggesting that goats in these communal herds are infected with MTBC at a low level. Estimated posterior means across the four evaluated assays ranged from 62.7% to 80.9% for diagnostic sensitivity and from 82.9% to 97.9% for diagnostic specificity, albeit estimates of the former (diagnostic sensitivity) were dependent on model assumptions. The application of a Bayesian latent class analysis and multiple ante-mortem test results may improve detection of MTBC, especially when prevalence is low. Our results provide a foundation for further investigation to confirm infection in communal goat herds and identify previously unrecognized sources of intra- and inter-species transmission of MTBC.

DETECTION OF ELEPHANT ENDOTHELIOTROPIC HERPESVIRUS (EEHV) IN FREE-RANGING AFRICAN ELEPHANTS (LOXODONTA AFRICANA) IN THE KRUGER NATIONAL PARK, SOUTH AFRICA.

Elephant endotheliotropic herpesvirus (EEHV) infection can cause acute, often fatal, EEHV hemorrhagic disease in free-ranging and human-managed Asian elephants (Elephas maximus) and human-managed African elephants (Loxodonta africana). However, significant knowledge gaps exist pertaining to the presence of EEHV in free-ranging African elephant populations. We retrospectively screened 142 opportunistically collected samples (blood, n=98; bronchoalveolar lavage (BAL) fluid, n=21; trunk wash (TW) fluid, n=23) obtained between 2010 and 2020 from 98 free-ranging African elephants in the Kruger National Park, South Africa, for the presence of different EEHVs, as well as determining the real-time quantitative PCR positivity rate in this population. With the use of validated, previously published DNA extraction and real-time quantitative PCR protocols provided by the National Elephant Herpesvirus Laboratory (Washington, DC, USA), EEHV was detected in nine male African elephants from samples collected in 2011 (n=1), 2013 (n=1), 2018 (n=2), 2019 (n=4), and 2020 (n=1). Viral detection was more common in respiratory compared with blood samples. Six elephants tested positive for EEHV2 subtype (blood, n=2; BAL, n=3; TW, n=2), including one individual that tested positive on matched respiratory samples (BAL and TW). Four elephants tested positive for EEHV3-4-7 (blood, n=1; BAL, n=2; TW, n=1), whereas EEHV6 was not detected in any of the study animals. One elephant tested positive for both EEHV2 and EEHV3-4-7 in the same BAL sample. Even though the levels of viremia varied between 158 and 1,292 viral genome equivalents/mL blood and viral shedding of EEHV2 and EEHV3-4-7 was detected in respiratory samples, no clinical signs were observed in these apparently healthy elephants. These findings are consistent with reports of asymptomatic EEHV infection in human-managed African elephants.

High-Specificity Test Algorithm for Bovine Tuberculosis Diagnosis in African Buffalo (Syncerus caffer) Herds.

Ante-mortem bovine tuberculosis (bTB) tests for buffaloes include the single comparative intradermal tuberculin test (SCITT), interferon-gamma (IFN-γ) release assay (IGRA) and IFN-γ-inducible protein 10 release assay (IPRA). Although parallel test interpretation increases the detection of Mycobacterium bovis (M. bovis)-infected buffaloes, these algorithms may not be suitable for screening buffaloes in historically bTB-free herds. In this study, the specificities of three assays were determined using M. bovis-unexposed herds, historically negative, and a high-specificity diagnostic algorithm was developed. Serial test interpretation (positive on both) using the IGRA and IPRA showed significantly greater specificity (98.3%) than individual (90.4% and 80.9%, respectively) tests or parallel testing (73%). When the SCITT was added, the algorithm had 100% specificity. Since the cytokine assays had imperfect specificity, potential cross-reactivity with nontuberculous mycobacteria (NTM) was investigated. No association was found between NTM presence (in oronasal swab cultures) and positive cytokine assay results. As a proof-of-principle, serial testing was applied to buffaloes (n = 153) in a historically bTB-free herd. Buffaloes positive on a single test (n = 28) were regarded as test-negative. Four buffaloes were positive on IGRA and IPRA, and M. bovis infection was confirmed by culture. These results demonstrate the value of using IGRA and IPRA in series to screen buffalo herds with no previous history of M. bovis infection.

Identification and Characterisation of Nontuberculous Mycobacteria in African Buffaloes (Syncerus caffer), South Africa.

Diagnosis of bovine tuberculosis (bTB) may be confounded by immunological cross-reactivity to Mycobacterium bovis antigens when animals are sensitised by certain nontuberculous mycobacteria (NTMs). Therefore, this study aimed to investigate NTM species diversity in African buffalo (Syncerus caffer) respiratory secretions and tissue samples, using a combination of novel molecular tools. Oronasal swabs were collected opportunistically from 120 immobilised buffaloes in historically bTB-free herds. In addition, bronchoalveolar lavage fluid (BALF; n = 10) and tissue samples (n = 19) were obtained during post-mortem examination. Mycobacterial species were identified directly from oronasal swab samples using the Xpert MTB/RIF Ultra qPCR (14/120 positive) and GenoType CMdirect (104/120 positive). In addition, all samples underwent mycobacterial culture, and PCRs targeting hsp65 and rpoB were performed. Overall, 55 NTM species were identified in 36 mycobacterial culture-positive swab samples with presence of esat-6 or cfp-10 detected in 20 of 36 isolates. The predominant species were M. avium complex and M. komanii. Nontuberculous mycobacteria were also isolated from 6 of 10 culture-positive BALF and 4 of 19 culture-positive tissue samples. Our findings demonstrate that there is a high diversity of NTMs present in buffaloes, and further investigation should determine their role in confounding bTB diagnosis in this species.

Reduced capability of refrigerated white rhinoceros whole blood to produce interferon-gamma upon mitogen stimulation.

Ante-mortem surveillance for Mycobacterium bovis (M. bovis) infection in the Kruger National Park (KNP) rhinoceros population currently relies on results from the QuantiFERON-TB Gold (In-Tube) Plus (QFT)-interferon gamma (IFN-γ) release assay (IGRA). However, same-day processing of rhinoceros blood samples for this test is a logistical challenge. Therefore, a pilot study was performed to compare mitogen-stimulated and unstimulated IFN-γ concentrations in plasma from rhinoceros whole blood processed within 6 h of collection or stored at 4°C for 24 and 48 h prior to incubation in QFT tubes. Replicate samples of heparinized whole blood from seven subadult male white rhinoceros were used. Results showed no change in IFN-γ levels in unstimulated samples, however the relative concentrations of IFN-γ (based on optical density values) in mitogen plasma decreased significantly with increased time blood was stored post-collection and prior to QFT stimulation. These findings support a need for same-day processing of rhinoceros blood samples for QFT-IGRA testing as per the current practice. Further investigation using TB-antigen stimulated samples is warranted to properly assess the impact of blood storage on TB test results in rhinoceros.

Field evaluation of the tuberculin skin test for the detection of Mycobacterium tuberculosis complex infection in communal goats (Capra hircus) in KwaZulu-Natal, South Africa.

In South Africa, animal tuberculosis (TB) control programs predominantly focus on domestic cattle and African buffaloes (Syncerus caffer) despite increasing global reports of tuberculosis in goats (Capra hircus). Left undetected, Mycobacterium tuberculosis complex (MTBC) infected goats may hinder TB eradication efforts in cattle and increase zoonotic risk to humans. Since the publication of animal TB testing guidelines in 2018, prescribing the use of the tuberculin skin test (TST) for goats in South Africa by the Department of Agriculture, Land Reform, and Rural Development (DALRRD), there have been no published reports of any field application of the prescribed test criteria in goat herds. Therefore, this study aimed to evaluate the performance of these DALRRD guidelines using the single intradermal cervical tuberculin test (SICT) and the single intradermal comparative cervical tuberculin test (SICCT). Between October and December 2020, 495 goats from communal pastures of Kwa-Zulu Natal (KZN), where M. bovis infection has been identified in cattle and where cattle and goats cohabitate, were tested using the SICT and SICCT (M. bovis-exposed group). Additionally, 277 goats from a commercial Saanen dairy herd, with no history of M. bovis, were also tested (M. bovis-unexposed group). Estimated apparent prevalence of TST positive goats was determined based on published test interpretation criteria as described by DALRRD. When proportions of test-positive goats were compared between different DALRRD criteria, the ≥ 4 mm cut-off criterion for the SICCT resulted in the lowest proportion of positive results in the presumably uninfected group (1/277 positive in the unexposed group). The apparent prevalence of TB in the exposed group was estimated at 3.0% (95% CI: 1.7-4.9%), which is similar to previous reports of M. bovis prevalence in cattle from this area (6%). The detection of a significantly greater proportion of SICCT positive goats in the M. bovis-exposed group compared to the unexposed group suggests that MTBC infection is present in this population. Further investigations should be undertaken, in conjunction with confirmatory molecular tests, mycobacterial culture, and advanced pathogen sequencing to establish whether MTBC infection in domestic goats is a true under-recognized threat to the eradication of animal TB in South Africa.

Adaptation and Diagnostic Potential of a Commercial Cat Interferon Gamma Release Assay for the Detection of Mycobacterium bovis Infection in African Lions (Panthera leo).

Mycobacterium bovis (M. bovis) infection in wildlife, including lions (Panthera leo), has implications for individual and population health. Tools for the detection of infected lions are needed for diagnosis and disease surveillance. This study aimed to evaluate the Mabtech Cat interferon gamma (IFN-γ) ELISABasic kit for detection of native lion IFN-γ in whole blood samples stimulated using the QuantiFERON® TB Gold Plus (QFT) platform as a potential diagnostic assay. The ELISA was able to detect lion IFN-γ in mitogen-stimulated samples, with good parallelism, linearity, and a working range of 15.6-500 pg/mL. Minimal matrix interference was observed in the recovery of domestic cat rIFN-γ in lion plasma. Both intra- and inter-assay reproducibility had a coefficient of variation less than 10%, while the limit of detection and quantification were 7.8 pg/mL and 31.2 pg/mL, respectively. The diagnostic performance of the QFT Mabtech Cat interferon gamma release assay (IGRA) was determined using mycobacterial antigen-stimulated samples from M. bovis culture-confirmed infected (n = 8) and uninfected (n = 4) lions. A lion-specific cut-off value (33 pg/mL) was calculated, and the sensitivity and specificity were determined to be 87.5% and 100%, respectively. Although additional samples should be tested, the QFT Mabtech Cat IGRA could identify M. bovis-infected African lions.

Culture-Independent PCR Detection and Differentiation of Mycobacteria spp. in Antemortem Respiratory Samples from African Elephants (Loxodonta Africana) and Rhinoceros (Ceratotherium Simum, Diceros Bicornis) in South Africa.

Since certain Mycobacterium tuberculosis complex (MTBC) members, such as M. bovis, are endemic in specific South African wildlife reserves and zoos, cases of clinically important nontuberculous mycobacteria (NTM) in wildlife may be neglected. Additionally, due to the inability of tests to differentiate between the host responses to MTBC and NTM, the diagnosis of MTBC may be confounded by the presence of NTMs. This may hinder control efforts. These constraints highlight the need for enhanced rapid detection and differentiation methods for MTBC and NTM, especially in high MTBC burden areas. We evaluated the use of the GeneXpert MTB/RIF Ultra, the Hain CMdirect V1.0 line probe assay, and novel amplicon sequencing PCRs targeting the mycobacterial rpoB and ku gene targets, directly on antemortem African elephant (n = 26) bronchoalveolar lavage fluid (BALF) (n = 22) and trunk washes (n = 21) and rhinoceros (n = 23) BALF (n = 23), with known MTBC culture-positive and NTM culture-positive results. Our findings suggest that the Ultra is the most sensitive diagnostic test for MTBC DNA detection directly in raw antemortem respiratory specimens and that the rpoB PCR is ideal for Mycobacterium genus DNA detection and species identification through amplicon sequencing.

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.

Improved detection of Mycobacterium tuberculosis and M. bovis in African wildlife samples using cationic peptide decontamination and mycobacterial culture supplementation.

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.

Diagnosis of Mycobacterium bovis infection in free-ranging common hippopotamus (Hippopotamus amphibius).

Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis) infection, is a multi-host disease which negatively affects the wildlife industry, with adverse consequences for conservation, ecotourism, and game/wildlife sales. Although interspecies transmission has been reported between some wildlife hosts, the risk of spread in complex ecosystems is largely unknown. As a controlled disease, tools for accurate detection of M. bovis infection are crucial for effective surveillance and management, especially in wildlife populations. There are, however, limited species-specific diagnostic tests available for wildlife. Hippopotamuses are rarely tested for M. bovis infection, and infection has not previously been confirmed in these species. In this study, blood and tissue samples collected from common hippopotamus (Hippopotamus amphibius) residing in a bTB-endemic area, the Greater Kruger Protected area (GKPA), were retrospectively screened to determine whether there was evidence for interspecies transmission of M. bovis, and identify tools for M. bovis detection in this species. Using the multi-species DPP® VetTB serological assay, a bTB seroprevalence of 8% was found in hippopotamus from GKPA. In addition, the first confirmed case of M. bovis infection in a free-ranging common hippopotamus is reported, based on the isolation in mycobacterial culture, genetic speciation and detection of DNA in tissue samples. Importantly, the M. bovis spoligotype (SB0121) isolated from this common hippopotamus is shared with other M. bovis-infected hosts in GKPA, suggesting interspecies transmission. These results support the hypothesis that M. bovis infection may be under recognized in hippopotamus. Further investigation is needed to determine the risk of interspecies transmission of M. bovis to common hippopotamus in bTB-endemic ecosystems and evaluate serological and other diagnostic tools in this species.

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.

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.

Cell-Mediated Immunological Biomarkers and Their Diagnostic Application in Livestock and Wildlife Infected With Mycobacterium bovis.

Mycobacterium bovis has the largest host range of the Mycobacterium tuberculosis complex and infects domestic animal species, wildlife, and humans. The presence of global wildlife maintenance hosts complicates bovine tuberculosis (bTB) control efforts and further threatens livestock and wildlife-related industries. Thus, it is imperative that early and accurate detection of M. bovis in all affected animal species is achieved. Further, an improved understanding of the complex species-specific host immune responses to M. bovis could enable the development of diagnostic tests that not only identify infected animals but distinguish between infection and active disease. The primary bTB screening standard worldwide remains the tuberculin skin test (TST) that presents several test performance and logistical limitations. Hence additional tests are used, most commonly an interferon-gamma (IFN-γ) release assay (IGRA) that, similar to the TST, measures a cell-mediated immune (CMI) response to M. bovis. There are various cytokines and chemokines, in addition to IFN-γ, involved in the CMI component of host adaptive immunity. Due to the dominance of CMI-based responses to mycobacterial infection, cytokine and chemokine biomarkers have become a focus for diagnostic tests in livestock and wildlife. Therefore, this review describes the current understanding of host immune responses to M. bovis as it pertains to the development of diagnostic tools using CMI-based biomarkers in both gene expression and protein release assays, and their limitations. Although the study of CMI biomarkers has advanced fundamental understanding of the complex host-M. bovis interplay and bTB progression, resulting in development of several promising diagnostic assays, most of this research remains limited to cattle. Considering differences in host susceptibility, transmission and immune responses, and the wide variety of M. bovis-affected animal species, knowledge gaps continue to pose some of the biggest challenges to the improvement of M. bovis and bTB diagnosis.

Mycobacterium bovis Infection in Free-Ranging African Elephants.

Mycobacterium bovis infection in wildlife species occurs worldwide. However, few cases of M. bovis infection in captive elephants have been reported. We describe 2 incidental cases of bovine tuberculosis in free-ranging African elephants (Loxodonta africana) from a tuberculosis-endemic national park in South Africa and the epidemiologic implications of these infections.

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.