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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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Dr. KL is affiliated with Chembio Diagnostic Systems, Inc., the manufacturer of the DPP® Vet TB for Elephants serological assay., (Copyright © 2023 Gumbo, Goosen, Buss, de Klerk-Lorist, Lyashchenko, Warren, van Helden, Miller and Kerr.)

Mycobacterium bovis ( M. bovis ) infection has been identified in both domestic and wild animals and may threaten the conservation of vulnerable species including African lions ( Panthera leo ). There is a need to develop accurate ante-mortem tools for detection of M. bovis infection in African big cat populations for wildlife management and disease surveillance. The aim of this study was to compare the performances of two immunological assays, the QuantiFERON ® -TB Gold Plus (QFT) Mabtech Cat interferon gamma release assay (IGRA) and QFT CXCL9 gene expression assay (GEA), which have both shown diagnostic potential for M. bovis detection in African lions. Lion whole blood (n=47), stimulated using the QFT platform, was used for measuring antigen-specific CXCL9 expression and IFN-γ production and to assign M. bovis infection status. A subset (n=12) of mycobacterial culture-confirmed M. bovis infected and uninfected African lions was used to compare the agreement between the immunological diagnostic assays. There was no statistical difference between the proportions of test positive African lions tested by the QFT Mabtech Cat IGRA compared to the QFT CXCL9 GEA. There was also a moderate association between immunological diagnostic assays when numerical results were compared. The majority of lions had the same diagnostic outcome using the paired assays. Although the QFT Mabtech Cat IGRA provides a more standardized, commercially available, and cost-effective test compared to QFT CXCL9 GEA, using both assays to categorize M. bovis infection status in lions will increase confidence in results., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gumbo, Sylvester, Parsons, Buss, Warren, van Helden, Miller and Kerr.)

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., (© 2021 Wiley-VCH GmbH.)

The lack of species-specific assays for the diagnosis of infectious diseases, such as bovine tuberculosis, poses a threat to the management of wildlife populations, especially for vulnerable species such as cheetah ( Acinonyx jubatus ). The aim of this study was to identify and develop a cell-mediated immunological cytokine-release assay that could distinguish between Mycobacterium bovis -infected and uninfected cheetahs using commercially available feline cytokine ELISA and domestic cat ( Felis catus ) recombinant proteins. Antibodies against domestic cat cytokines, tumour necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and interferon gamma (IFN-γ), were screened for cross-reactivity with plasma cytokines from cheetah whole blood stimulated using QuantiFERON ® -TB Gold Plus (QFT) tubes. Evidence of cytokine production in response to QFT mitogen stimulation was observed in all four ELISA assays. However only the Mabtech Cat IFN-γ ELISA Basic kit could distinguish between M. bovis -infected ( n = 1) and uninfected ( n = 1) cheetahs and was therefore selected for further evaluation. A preliminary cheetah specific cutoff value (11 pg/ml) for detecting M. bovis infection using the Mabtech Cat IFN-γ release assay was calculated using a M. bovis uninfected cheetah cohort. Although this study only included one confirmed M. bovis culture-positive and one M. bovis culture-negative cheetah, the Mabtech Cat IFN-γ release assay demonstrated its potential for diagnostic application in this species.

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., (© 2021 Wiley-VCH GmbH.)

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.

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.

Effective screening methods are critical for preventing the spread of bovine tuberculosis (bTB) among livestock and wildlife species. The tuberculin skin test (TST) remains the primary test for bTB globally, although performance is suboptimal. African buffaloes (Syncerus caffer) are a maintenance host of Mycobacterium bovis in South Africa, tested using the single intradermal tuberculin test (SITT) or comparative test (SICTT). The interpretation of these tests has been based on cattle thresholds due to the lack of species-specific cut-off values for African buffaloes. Therefore, the aims of this study were to calculate buffalo-specific thresholds for different TST criteria (SITT, SICTT, and SICTT 72h that calculates the differential change at 72 h only) and compare performance using these cut-off values. The results confirm that 3 mm best discriminates M. bovis-infected from unexposed control buffaloes with sensitivities of 69 % (95 % CI 60-78; SITT and SICTT) and 76 % (95 % CI 65-83; SICTT 72h ), and specificities of 86 % (95 % CI 80-90; SITT), 96 % (95 % CI 92-98; SICTT 72h ) and 97 % (95 % CI 93-99; SICTT), respectively. A comparison between TST criteria using buffalo-specific thresholds demonstrates that the comparative TST performs better than the SITT, although sensitivity remains suboptimal. Therefore, further research and the addition of ancillary tests, such as cytokine release assays, are necessary to improve M. bovis detection in African buffaloes., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis) infection, disrupts conservation programs of threatened species such as the white rhinoceros (Ceratotherium simum). Interferon gamma release assays have been developed for the diagnosis of M. bovis infection in rhinoceros, however, the discovery of additional diagnostic biomarkers might improve the accuracy of case detection. The aim of this pilot study was therefore to evaluate a novel unbiased approach to candidate biomarker discovery and preliminary validation. Whole blood samples from twelve white rhinoceros were incubated in Nil and TB antigen tubes of the QuantiFERON® TB Gold (In-Tube) system after which RNA was extracted and reverse transcribed. Using the equine RT 2 profiler PCR array, relative gene expression analysis of samples from two immune sensitized rhinoceros identified CCL4, CCL8, IL23A, LTA, NODAL, TNF, CSF3, CXCL10 and GPI as upregulated in response to antigen stimulation. Novel gene expression assays (GEAs) were designed for selected candidates, i.e. CCL4, CXCL10 and IFNG, and analysis of QFT-processed samples showed the CXCL10 GEA could distinguish between five M. bovis-infected and five uninfected rhinoceros. These findings confirm the value of the equine RT 2 profiler PCR array as a useful tool for screening biomarkers for the diagnosis of M. bovis infection in rhinoceros., (Copyright © 2020 Elsevier B.V. All rights reserved.)

As a recognized Mycobacterium bovis maintenance host, the African buffalo (Syncerus caffer) poses transmission risks to livestock, humans and other wildlife. Early detection of M. bovis infection is critical for limiting its spread. Currently, tests detecting cell-mediated immune responses are used for diagnosis in buffaloes. However, these may have suboptimal sensitivity or specificity, depending on the blood stimulation method. Recent evidence suggests that assays using combinations of host cytokine biomarkers may increase diagnostic performance. Therefore, this study aimed to investigate the application of a MILLIPLEX ® bovine cytokine/chemokine multiplex assay to identify candidate biomarkers of M. bovis infection in buffaloes. Whole blood from twelve culture-confirmed M. bovis-infected buffaloes, stimulated with the QuantiFERON ® TB Gold Plus in-tube system, was tested using the MILLIPLEX ® platform. Results indicated binding of bovine antibodies to fifteen buffalo cytokine/chemokine targets. Moreover, there was a significant difference in concentrations between unstimulated and TB antigen-stimulated buffalo samples for seven cytokines/chemokines included in the kit. Although these preliminary results require further investigation in larger sample sets and a comparison between M. bovis-infected and uninfected cohorts, the utility of the MILLIPLEX ® platform in a novel species was demonstrated, in addition to identifying potential African buffalo cytokines for future research., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Bovine tuberculosis and tuberculosis are chronic infectious diseases caused by the Mycobacterium tuberculosis complex members, Mycobacterium bovis and Mycobacterium tuberculosis, respectively. Infection with M. bovis and M. tuberculosis have significant implications for wildlife species management, public health, veterinary disease control, and conservation endeavours., Results: Here we describe the first use of the VetMAX™ Mycobacterium tuberculosis complex (MTBC) DNA quantitative real-time polymerase chain reaction (qPCR) detection kit for African wildlife samples. DNA was extracted from tissues harvested from 48 African buffaloes and MTBC DNA was detected (test-positive) in all 26 M. bovis culture-confirmed animals with an additional 12 PCR-positive results in culture-negative buffaloes (originating from an exposed population). Of six MTBC-infected African rhinoceros tested, MTBC DNA was detected in antemortem and postmortem samples from five animals. The PCR was also able to detect MTBC DNA in samples from two African elephants confirmed to have M. bovis and M. tuberculosis infections (one each). Culture-confirmed uninfected rhinoceros and elephants' samples tested negative in the PCR assay., Conclusions: These results suggest this new detection kit is a sensitive screening test for the detection of MTBC-infected African buffaloes, African elephants and white rhinoceros.

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.

Mycobacterium bovis infection has been described in many wildlife species across Africa. However, diagnostic tests are lacking for many of these, including warthogs (Phacochoerus africanus). Most literature on suids has focused on using serological tools, with few studies investigating the use of cell-mediated immune response (CMI) assays. A recent study showed that warthogs develop measurable CMI responses, which suggests that cytokine gene expression assays (GEAs) may be valuable for detecting M. bovis-infection, as shown in numerous African wildlife species. Therefore, the aim of the study was to develop GEAs capable of distinguishing between M. bovis-infected and uninfected warthogs. Whole blood was stimulated using the QuantiFERON-TB Gold (In-Tube) system, using ESAT-6 and CFP-10 peptides, before determining the relative gene expression of five reference (B2M, H3F3A, LDHA, PPIA and YWHAZ) and five target (CXCL9, CXCL10, CXCL11, IFNG and TNFA) genes through qPCR. The reference gene H3F3A was the most stably expressed, while all target genes were significantly upregulated in M. bovis-infected warthogs with the greatest upregulation observed for CXCL10. Consequently, the CXCL10 GEA shows promise as an ante-mortem diagnostic tool for the detection of M. bovis-infected warthogs.

Mycobacterium bovis (M. bovis), the cause of bovine tuberculosis, is endemic in Kruger National Park (KNP), South Africa. The risk of spread of M. bovis infection currently prevents translocation of white rhinoceros (Ceratotherium simum) from this population. Therefore, accurate assays are necessary for screening this threatened species. Interferon gamma (IFN-γ) release assays (IGRA) are commonly used for tuberculosis diagnosis in humans and other wildlife species. Hence, the aim of this study was to develop an IGRA for M. bovis detection in white rhinoceros. Heparinized whole blood was collected from immobilized white rhinoceros in KNP (n = 131) and incubated overnight in QuantiFERON®-TB Gold (QFT) blood collection tubes, after which the plasma was harvested following centrifugation. Tissue samples for mycobacterial culture were available from a subset of 21 rhinoceros. The concentration of IFN-γ in plasma samples was measured using the Mabtech equine IFN-γ ELISA PRO kit. An IGRA result was calculated as the difference in IFN-γ concentrations in the QFT Nil and TB antigen tubes. Using test results for the white rhinoceros with known infection status, a diagnostic cut-off value was calculated as 21 pg/ml. Additionally, cut-off values for IFN-γ concentrations for plasma from QFT Nil and QFT Mitogen tubes were calculated to increase confidence in IGRA result interpretation. The combination of the QFT stimulation platform and Mabtech equine IFN-γ ELISA is a promising diagnostic test to distinguish between of M. bovis-infected and -uninfected white rhinoceros., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Integrating biological processes across scales remains a central challenge in disease ecology. Genetic variation drives differences in host immune responses, which, along with environmental factors, generates temporal and spatial infection patterns in natural populations that epidemiologists seek to predict and control. However, genetics and immunology are typically studied in model systems, whereas population-level patterns of infection status and susceptibility are uniquely observable in nature. Despite obvious causal connections, organizational scales from genes to host outcomes to population patterns are rarely linked explicitly. Here we identify two loci near genes involved in macrophage (phagocyte) activation and pathogen degradation that additively increase risk of bovine tuberculosis infection by up to ninefold in wild African buffalo. Furthermore, we observe genotype-specific variation in IL-12 production indicative of variation in macrophage activation. Here, we provide measurable differences in infection resistance at multiple scales by characterizing the genetic and inflammatory variation driving patterns of infection in a wild mammal.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis , is endemic in Kruger National Park, South Africa, home to the largest population of white rhinoceros ( Ceratotherium simum ) in the world. In 2016, the first cases of naturally occurring bTB were reported in white rhinoceros; however, there is a lack of understanding of infection and disease process in this species. Prevention and control of transmission depends on the availability of accurate tools to detect M. bovis infection. Interferon gamma (IFN-γ) assays are a reliable detection method for TB in other animal species, and studies have indicated that these tests can be used in white rhinoceros. We sought to screen and optimize a commercial IFN-γ enzyme-linked immunosorbent assay (ELISA) to detect endogenous white rhinoceros IFN-γ in mitogen-stimulated whole blood as a basis for developing a test for M. bovis infection. Optimizations included identifying ELISA antibodies and determining the effect of sample matrix, ELISA plate incubation temperature, ELISA linearity, assay reproducibility, and the assay's limit of quantification. The optimized assay employed an equine IFN-γ antibody pair that was used to create a commercial ELISA kit. This ELISA had a linear response to recombinant equine and endogenous rhinoceros IFN-γ (range: 7.8-125 pg/mL). When incubated at 37°C, the ELISA was highly reproducible, with an optimal recovery and a low limit of quantification, indicating that the Mabtech equine IFN-γ ELISA PRO kit is a robust assay for measuring white rhinoceros IFN-γ.

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.

Background: Bovine tuberculosis (bTB) caused by Mycobacterium bovis has previously been diagnosed in warthogs and infection can be highly prevalent (> 30%) in endemic areas. Thus, warthogs could potentially be an important species to consider as sentinels for disease surveillance. However, disease surveillance is dependent on availability of accurate diagnostic assays and only a few diagnostic tests have been investigated for warthogs. Furthermore, the tests that have been used in this species require laboratory equipment and trained personnel to obtain results. Therefore, this study investigated the use of the intradermal tuberculin test (ITT) to screen warthogs for bTB, which can be done with minimal equipment and under field conditions by most veterinarians and other qualified professionals. Changes in skin fold thickness measurements at the bovine purified protein derivative (PPD) administration site, between 0 and 72 h, were compared with differential changes between the bovine and avian PPD sites, for 34 warthogs, to evaluate the performance when different interpretation criteria for the ITT was used., Results: Using an increase of 1.8 mm or more at the bovine PPD site as a cut-off for positive responders, 69% of 16 M. bovis culture-positive warthogs had a positive test result, with 100% of the 18 culture-negative warthogs considered as test negative. When a differential of 1.2 mm or more in skin fold thickness at the bovine PPD compared to the avian PPD site was used as a cut-off for the comparative ITT, 81% of culture-positive warthogs were considered as test positive, with 100% of culture-negative warthogs considered as test negative., Conclusion: The findings in this study suggest that the ITT is a promising tool to use when screening warthogs for M. bovis infection.

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Bovine tuberculosis (bTB) is endemic in several areas of South Africa and has been reported in multiple species, including common warthogs (Phacochoerus africanus). Limited diagnostic tests and disease control programs exist for African wildlife. Thus, there is a need to develop techniques for bTB detection in species such as warthogs to assess their role in disease maintenance and spread in multi-host ecosystems. In this study, we obtained blood samples from warthogs in bTB endemic areas to investigate biomarkers for detection of Mycobacterium bovis infection. Warthog whole blood was incubated in QuantiFERON ® TB Gold In-Tube tubes and pathogen specific release of interferon gamma (IFN-γ) and interferon gamma induced protein 10 (IP-10) was measured by a sandwich enzyme-linked immunosorbent assay. Although we were unable to measure IFN-γ, we could successfully measure IP-10. The IP-10 assay was able to distinguish between M. bovis-infected and M. bovis-culture negative warthogs, within bTB endemic areas, with an assay specific sensitivity of 68% and specificity of 84%. Of the 88 M. bovis-exposed warthogs screened, 42% were IP-10 test positive. These results indicate warthogs develop a measurable cell-mediated immune response after antigen stimulation of whole blood, which can distinguish between M. bovis-infected and M. bovis-culture negative animals. Thus, the IP-10 assay shows promise as an ante-mortem test to diagnose bTB in warthogs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

African buffaloes (Syncerus caffer) are wildlife maintenance hosts of Mycobacterium bovis, the cause of bovine tuberculosis. Consequently, M. bovis infected buffaloes pose a transmission risk for cattle and other wildlife species. Previously, a modification to the Qiagen QuantiFERON ® -TB Gold (QFT) system, using QFT tubes and an in-house bovine interferon-gamma (IFN-γ) ELISA, was evaluated for the detection of M. bovis infection in buffaloes. Subsequently, Qiagen has developed a commercially available cattletype ® IFN-gamma ELISA for the detection of antigen-specific IFN-γ release in ruminants. The aim of this study was to investigate the use of QFT tubes and the cattletype ® IFN-gamma ELISA, in a cattletype IFN-γ release assay (IGRA), to detect M. bovis infection in African buffaloes. The test agreements between the cattletype IGRA, single comparative intradermal skin test (SCITT) and Bovigam ® 1G IGRA in two M. bovis-exposed buffalo populations (n = 134 and n = 92) were calculated and κ coefficients ranged from 0.65 (95% CI 0.48-0.82) to 0.86 (95% CI 0.72-0.99). Increasing the QFT incubation time in one M. bovis-exposed buffalo cohort (n = 92), from 20 to 40 h, had no effect on the cattletype IGRA test results. Inter-assay and intra-assay reproducibility determination for the cattletype IGRA produced coefficient of variations (CV) <9.1% and <1.7%, respectively. A total of 21/21 known M. bovis-unexposed buffaloes tested negative in the cattletype IGRA. Moreover, the cattletype IGRA test result values were significantly greater for 13 M. bovis culture-positive buffaloes compared with 14 M. bovis-exposed culture-negative (P < .01) and 21 M. bovis-unexposed (P < .001) buffaloes, respectively. These findings suggest that the combination of QFT tubes and the cattletype ® IFN-gamma ELISA is a promising new diagnostic assay for the detection of M. bovis infection in African buffaloes. However, further research is needed to evaluate the sensitivity and specificity of the assay in larger African buffalo populations., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Bovine tuberculosis is a zoonotic disease with largely unknown impact in Africa, with risk factors such as HIV and direct contact with animals or consumption of Mycobacterium bovis infected animal products. In order to understand and quantify this risk and design intervention strategies, good epidemiological studies are needed. Such studies can include molecular typing of M. bovis isolates. The aim of this study was to apply these tools to provide novel information concerning the distribution of bovine tuberculosis in cattle in Mozambique and thereby provide relevant information to guide policy development and strategies to contain the disease in livestock, and reduce the risk associated with transmission to humans. A collection of 178 M. bovis isolates was obtained from cattle in Mozambique. Using spoligotyping and regions of difference analysis, we classified the isolates into clonal complexes, thus reporting the first characterisation of M. bovis strains in this region. Data from MIRU-VNTR typing was used to compare isolates from a number of African countries, revealing a deeply geographically structured diversity of M. bovis. Eastern Africa appears to show high diversity, suggesting deep evolution in that region. The diversity of M. bovis in Africa does not seem to be a function of recent importation of animals, but is probably maintained within each particular region by constant reinfection from reservoir animals. Understanding the transmission routes of M. bovis in Mozambique and elsewhere is essential in order to focus public health and veterinary resources to contain bovine tuberculosis.

A novel lateral flow immunochromatographic device (LFD) was evaluated in several veterinary diagnostic laboratories. It was confirmed to be specific for Mycobacterium bovis and M. caprae cells. The performance of the novel LFD was assessed relative to the confirmatory tests routinely applied after culture (spoligotyping or quantitative PCR [qPCR]) in each laboratory; liquid (MGIT or BacT/Alert) and/or solid (Stonebrink, Coletsos, or Lowenstein-Jensen) cultures were tested. In comparison to spoligotyping of acid-fast-positive MGIT cultures, percent agreement between positive LFD and spoligotyping results was excellent in two United Kingdom laboratories (97.7 to 100%) but lower in the Spanish context (76%), where spoligotyping was applied to MGIT cultures previously confirmed to be positive for M. tuberculosis complex (MTBC) by qPCR. Certain spoligotypes of M. bovis and M. caprae were not detected by the LFD in Spanish MGIT cultures. Compared to qPCR confirmation, the agreement between positive LFD and qPCR results was 42.3% and 50% for BacT/Alert and MGIT liquid cultures, respectively, and for solid cultures, it ranged from 11.1 to 89.2%, depending on the solid medium employed (Coletsos, 11.1%; Lowenstein-Jensen, 55.6%; Stonebrinks, 89.2%). Correlation between the novel LFD and BD MGIT TBc Identification test results was excellent when 190 MGIT cultures were tested ( r = 0.9791; P < 0.0001), with the added benefit that M. bovis was differentiated from another MTBC species in one MGIT culture by the novel LFD. This multilaboratory evaluation demonstrated the novel LFD's potential utility as a rapid test to confirm isolation of M. bovis and M. caprae from veterinary specimens following culture., (Copyright © 2017 American Society for Microbiology.)

Tuberculosis caused by Mycobacterium bovis is endemic in the African buffalo (Syncerus caffer) population in the Kruger National Park and other conservation areas in South Africa. The disease has been diagnosed in a total of 21 free ranging or semi-free ranging wildlife species in the country with highly variable presentations in terms of clinical signs as well as severity and distribution of tuberculous lesions. Most species are spillover or dead-end hosts without significant role in the epidemiology of the disease. White rhinoceroses (Ceratotherium simum) are translocated from the Kruger National Park in substantial numbers every year and a clear understanding of their risk to manifest overt tuberculosis disease and to serve as source of infection to other species is required. We report the findings of experimental infection of three white rhinoceroses with a moderately low dose of a virulent field isolate of Mycobacterium bovis. None of the animals developed clinical signs or disseminated disease. The susceptibility of the white rhinoceros to bovine tuberculosis was confirmed by successful experimental infection based on the ante mortem isolation of M. bovis from the respiratory tract of one rhinoceros, the presence of acid-fast organisms and necrotizing granulomatous lesions in the tracheobronchial lymph nodes and the detection of M. bovis genetic material by PCR in the lungs of two animals.

Mycobacterium bovis infects multiple wildlife species and domesticated cattle across South Africa, and negatively impacts on livestock trade and movement of wildlife for conservation purposes. M. bovis infection was first reported in the Kruger National Park (KNP) in South Africa during the 1990s, and has since spread to infect numerous animal host species throughout the park and across South Africa. Whole genome sequencing data of 17 M. bovis isolates were analyzed to investigate the genomic diversity among M. bovis isolates causing disease in different animal host species from various locations in South Africa. M. bovis strains analyzed in this study are geographic rather than host species-specific. The clonal expansion of M. bovis in the KNP highlights the effect of an introduction of a transmissible infectious disease leading to a rising epidemic in wildlife, and emphasizes the importance of disease control and movement restriction of species that serve as disease reservoirs. In conclusion, the point source introduction of a single M. bovis strain type in the KNP ecosystem lead to an M. bovis outbreak in this area that affects various host species and poses an infection risk in neighboring rural communities where HIV prevalence is high., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Mycobacterium bovis, the causative agent of bovine tuberculosis (BTB), is endemic in the Kruger National Park (KNP), South Africa. African lions ( Panthera leo ) are susceptible to BTB, but the impact of the disease on lion populations is unknown. In this study, we used a novel gene expression assay for chemokine (C-X-C motif) ligand 9 (CXCL9) to measure the prevalence of M. bovis infection in 70 free-ranging lions that were opportunistically sampled in the southern and central regions of the KNP. In the southern region of the KNP, the apparent prevalence of M. bovis infection was 54% (95% confidence interval [CI]=36.9-70.5%), compared with 33% (95% CI=18.0-51.8%) in the central region, an important difference (P=0.08). Prevalence of M. bovis infection in lions showed similar patterns to estimated BTB prevalence in African buffaloes ( Syncerus caffer ) in the same areas. Investigation of other risk factors showed a trend for older lions, males, or lions with concurrent feline immunodeficiency virus infection to have a higher M. bovis prevalence. Our findings demonstrate that the CXCL9 gene expression assay is a useful tool for the determination of M. bovis status in free-ranging lions and identifies important epidemiologic trends for future studies.

In 2016, an emaciated black rhinoceros (Diceros bicornis) was found in Kruger National Park, South Africa. An interferon-γ response was detected against mycobacterial antigens, and lung tissue was positive for Mycobacterium bovis. This case highlights the risk that tuberculosis presents to rhinoceros in M. bovis-endemic areas.

Bovine tuberculosis (bTB) was first diagnosed in the Kruger National Park (KNP) in 1990. Research has since focused on the maintenance host, the African buffalo ( Syncerus caffer ) and clinically affected lion ( Panthera leo ). However, little is known about the role of small predators in tuberculosis epidemiology. During 2011-12, we screened banded mongooses ( Mungos mungo ) in the bTB high-prevalence zone of the KNP for Mycobacterium tuberculosis complex members. Fecal swabs, tracheal swabs, and tracheal lavages of 76 banded mongooses caught in cage traps within a 2-km radius of Skukuza Rest Camp were submitted for Mycobacterium culture, isolation, and species identification. Lesions and lymph node samples collected from 12 animals at postmortem examination were submitted for culture and histopathology. In lung and lymph nodes of two banded mongooses, well demarcated, irregularly margined, gray-yellow nodules of up to 5 mm diameter were identified with either central necrosis or calcification, characterized on histopathology as caseating necrosis with epithelioid macrophages or necrogranuloma with calcified centre. No acid fast bacteria were identified with Ziehl-Neelsen stain. We isolated Mycobacterium bovis from lung, lymph node, and liver samples, as well as from tracheal lavages and tracheal swab from the same two banded mongooses. Blood samples were positive by ElephantTB STAT-PAK® Assay for 12 and Enferplex™ TB Assay for five animals. Only the two banded mongooses positive on pathology and M. bovis culture were positive on both serologic assays. We provide evidence of bTB infection in banded mongooses in the KNP, demonstrate their ability to shed M. bovis , and propose a possible antemortem diagnostic algorithm. Our findings open the discussion around possible sources of infection and their significance at the human/wildlife interface in and around Skukuza.

Sporadic cases of bovine tuberculosis (bTB) have been reported in warthogs in Southern Africa and confirmed through mycobacterial culture. However, there are no validated ante-mortem tests currently available for bTB in warthogs. In this study, we evaluated the use of three serological assays for the detection of Mycobacterium bovis infection in warthogs; an indirect enzyme-linked immunosorbent assay (ELISA) using bovine purified protein derivative (PPD b ) as a capture antigen (indirect PPD ELISA), as well as two commercial assays, the TB ELISA-VK ® and DPP ® VetTB Assay. Test performance of these assays was compared using sera from 35 warthogs of known Mycobacterium bovis infection status. All three assays were able to distinguish M. bovis-infected from uninfected individuals with high sensitivity (Se) and specificity (Sp) (indirect PPD ELISA Se: 88%, Sp: 89%; TB ELISA-VK ® 88%, 79%; DPP ® VetTB Assay 75%, 89%, respectively). The assays performed very similarly and the ELISA assays showed the greatest agreement (κ=0.89). These results indicate that M. bovis-infected warthogs develop measurable pathogen-specific humoral responses which can be used to distinguish them from uninfected animals. Therefore, serological assays have value as ante-mortem bTB diagnostic tests in warthogs., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The most widely used ante-mortem diagnostic tests for tuberculosis in cattle are the tuberculin skin test and the interferon-gamma (IFN-γ) release assay, both of which measure cell-mediated immune responses to Mycobacterium bovis infection. However, limitations in the performance of these tests results in a failure to identify all infected animals. In attempting to increase the range of diagnostic tests for tuberculosis, measurement of the cytokine IP-10 in antigen-stimulated blood has previously been shown to improve the detection of M. tuberculosis and M. bovis infection, in humans and African buffaloes (Syncerus caffer), respectively. In the present study, 60 cattle were identified by the single intradermal comparative tuberculin test as tuberculosis reactors (n = 24) or non-reactors (n = 36) and the release of IFN-γ and IP-10 in antigen-stimulated whole blood from these animals was measured using bovine specific ELISAs. There was a strong correlation between IP-10 and IFN-γ production in these samples. Moreover, measurement of the differential release of IP-10 in response to stimulation with M. bovis purified protein derivative (PPD) and M. avium PPD distinguished between reactor and non-reactor cattle with a sensitivity of 100% (95% CI, 86%-100%) and a specificity of 97% (95% CI, 85%-100%). These results suggest that IP-10 might prove valuable as a diagnostic biomarker of M. bovis infection in cattle.

We recently reported on our success to generate deletion mutants of the genes encoding glutamate dehydrogenase (GDH) and glutamine oxoglutarate aminotransferase (GOGAT) in M. bovis BCG, despite their in vitro essentiality in M. tuberculosis. We could use these mutants to delineate the roles of GDH and GOGAT in mycobacterial nitrogen metabolism by using M. bovis BCG as a model for M. tuberculosis specifically. Here, we extended our investigation towards the involvement of GDH and GOGAT in other aspects of M. bovis BCG physiology, including the use of glutamate as a carbon source and resistance to known phagosomal stresses, as well as in survival inside macrophages. We find that gdh is indispensable for the utilization of glutamate as a major carbon source, in low pH environments and when challenged with nitric oxide. On the other hand, the gltBD mutant had increased viability under low pH conditions and was unaffected by a challenge with nitric oxide. Strikingly, GDH was required to sustain M. bovis BCG during infection of both murine RAW 264.7 and bone-marrow derived and macrophages, while GOGAT was not. We conclude that the catabolism of glutamate in slow growing mycobacteria may be a crucial function during infection of macrophage cells and demonstrate a novel requirement for M. bovis BCG GDH in the protection against acidic and nitrosative stress. These results provide strong clues on the role of GDH in intracellular survival of M. tuberculosis, in which the essentiality of the gdh gene complicates knock out studies making the study of the role of this enzyme in pathogenesis difficult.

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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Mycobacterium bovis has global public-health and socio-economic significance and can infect a wide range of species including the lion (Panthera leo) resulting in tuberculosis. Lions are classified as vulnerable under the IUCN Red List of Threatened Species and have experienced a 30% population decline in the past two decades. However, no attempt has been made to collate and critically evaluate the available knowledge of M. bovis infections in lions and potential effects on population. In this review we set out to redress this. Arguments suggesting that ingestion of infected prey animals are the main route of infection for lions have not been scientifically proven and research is needed into other possible sources and routes of infection. The paucity of knowledge on host susceptibility, transmission directions and therefore host status, manifestation of pathology, and epidemiology of the disease in lions also needs to be addressed. Advances have been made in diagnosing the presence of M. bovis in lions. However, these diagnostic tests are unable to differentiate between exposure, presence of infection, or stage of disease. Furthermore, there are contradictory reports on the effects of M. bovis on lion populations with more data needed on disease dynamics versus the lion population's reproductive dynamics. Knowledge on disease effects on the lion reproduction and how additional stressors such as drought or co-morbidities may interact with tuberculosis is also lacking. Filling these knowledge gaps will contribute to the understanding of mycobacterial infections and disease in captive and wild lions and assist in lion conservation endeavours., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Diagnosis of tuberculosis in wildlife often relies on postmortem samples because of logistical challenges and lack of field-friendly techniques for live animal testing. Confirmation of infection through detection of infectious organisms is essential for studying the pathogenesis and epidemiology of disease. We describe the application of a technique to obtain respiratory samples from free-ranging living lions to facilitate detection of viable Mycobacterium bovis under field conditions. We identified M. bovis by mycobacterial culture and PCR in tracheobronchial lavage samples from 8/134 (6.0%) lions tested in Kruger National Park, South Africa. This confirms the respiratory shedding of viable M. bovis in living lions. The implications of these results are that infected lions have the potential to transmit this disease and serve as maintenance hosts.

We evaluated commercially available bovine enzyme linked immunosorbent assays (ELISA) and a human IP-10 ELISA to measure IP-10, MIG, MCP-1, MCP-2, MCP-3 and IL1-RA in buffalo plasma in order to identify sensitive markers of the immune response to Mycobacterium bovis-specific peptides. Additionally, we found that all coding mRNA sequences of these cytokines showed very high homology with their homologues in domestic cattle (97-99%) as did the derived amino acid sequences (97-99%). This high sequence homology between cattle and buffaloes supports the use of bovine ELISAs for the detection these cytokines in buffaloes. MCP-1 concentration showed a positive correlation with that of IFN-γ (p=0.0077) and appears to occur in far greater abundance in buffaloes when compared to humans. Using a bovine IP-10 ELISA, levels of this cytokine were found to be significantly increased in antigen-stimulated blood samples from M. bovis test positive buffaloes (p<0.0001) and IP-10 was detected in far greater abundance than IFN-γ. Measurement of IP-10 with this ELISA may prove to be a sensitive marker of M. bovis infection in African buffaloes., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We assessed the use of Mycobacterium bovis-specific peptides for the diagnosis of tuberculosis in African buffaloes (Syncerus caffer) by evaluating the agreement between the single intradermal comparative tuberculin test (SICTT), the Bovigam(®) EC (BEC) assay, the Bovigam(®) HP (BHP) assay and two assays utilizing the QuantiFERON(®) TB-Gold (in tube) system employing 20 h (mQFT20 assay) and 30 h (mQFT30 assay) whole blood incubation periods. Of 84 buffaloes, 45% were SICTT-positive, 48% were BEC-positive, 50% were BHP-positive, 37% were mQFT20-positive and 43% were mQFT30-positive. Agreement between the BEC and BHP Bovigam(®) assays was high (κ=0.86, 95% CI 0.75-0.97) and these detected the most test-positive animals suggesting that they were the most sensitive assays. Interferon-gamma release was significantly greater in buffaloes that were test-positive for all tests than in animals with discordant but positive Bovigam(®) results. Agreement between the mQFT assays was equally high (κ=0.88, 95% CI 0.77-0.98); however, all buffaloes with discordant mQFT results (n=6) were mQFT30-positive/mQFT20-negative, including three confirmed M. bovis-infected animals, suggesting that the mQFT30 assay is the more sensitive of the two. Agreements between the two Bovigam(®) and two mQFT assays were moderate, suggesting that in its current format the mQFT assay is less sensitive than either the BEC or the BHP assays., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: The global epidemiology of parasitic helminths and mycobacterial infections display extensive geographical overlap, especially in the rural and urban communities of developing countries. We investigated whether co-infection with the gastrointestinal tract-restricted helminth, Trichuris muris, and the intracellular bacterium, Mycobacterium bovis (M. bovis) BCG, would alter host immune responses to, or the pathological effect of, either infection., Results: We demonstrate that both pathogens are capable of negatively affecting local and systemic immune responses towards each other by modifying cytokine phenotypes and by inducing general immune suppression. T. muris infection influenced non-specific and pathogen-specific immunity to M. bovis BCG by down-regulating pulmonary TH1 and Treg responses and inducing systemic TH2 responses. However, co-infection did not alter mycobacterial multiplication or dissemination and host pulmonary histopathology remained unaffected compared to BCG-only infected mice. Interestingly, prior M. bovis BCG infection significantly delayed helminth clearance and increased intestinal crypt cell proliferation in BALB/c mice. This was accompanied by a significant reduction in systemic helminth-specific TH1 and TH2 cytokine responses and significantly reduced local TH1 and TH2 responses in comparison to T. muris-only infected mice., Conclusion: Our data demonstrate that co-infection with pathogens inducing opposing immune phenotypes, can have differential effects on compartmentalized host immune protection to either pathogen. In spite of local and systemic decreases in TH1 and increases in TH2 responses co-infected mice clear M. bovis BCG at the same rate as BCG only infected animals, whereas prior mycobacterial infection initiates prolonged worm infestation in parallel to decreased pathogen-specific TH2 cytokine production.

Recent evidence suggests that the regulation of intracellular glutamate levels could play an important role in the ability of pathogenic slow-growing mycobacteria to grow in vivo. However, little is known about the in vitro requirement for the enzymes which catalyse glutamate production and degradation in the slow-growing mycobacteria, namely; glutamine oxoglutarate aminotransferase (GOGAT) and glutamate dehydrogenase (GDH), respectively. We report that allelic replacement of the Mycobacterium bovis BCG gltBD-operon encoding for the large (gltB) and small (gltD) subunits of GOGAT with a hygromycin resistance cassette resulted in glutamate auxotrophy and that deletion of the GDH encoding-gene (gdh) led to a marked growth deficiency in the presence of L-glutamate as a sole nitrogen source as well as reduction in growth when cultured in an excess of L-asparagine.

Background: Tumor necrosis factor (TNF) is a key immune regulator of tuberculosis resistance, as exemplified by the highly increased risk of tuberculosis disease among individuals receiving TNF-blocker therapy., Methods: We determined the extent of TNF production after stimulation with BCG or BCG plus interferon gamma (IFN-γ) using a whole blood assay in 392 children belonging to 135 nuclear families from an area hyperendemic for tuberculosis in South Africa. We conducted classical univariate and bivariate genome-wide linkage analysis of TNF production using the data from both stimulation protocols by means of an extension of the maximum-likelihood-binomial method for quantitative trait loci to multivariate analysis., Results: Stimulation of whole blood by either BCG or BCG plus IFN-γ resulted in a range of TNF release across subjects. Extent of TNF production following both stimulation protocols was highly correlated (r = 0.81). We failed to identify genetic linkage of TNF release when considering each stimulus separately. However, using a multivariate approach, we detected a major pleiotropic locus (P < 10(-5)) on chromosome region 11p15, termed TNF locus 1 (TNF1), that controlled TNF production after stimulation by both BCG alone and BCG plus IFN-γ., Conclusions: The TNF1 locus was mapped in the vicinity of the TST1 locus, previously identified in the same family sample, that controls tuberculin skin test (TST) negativity per se, that is, T-cell-independent resistance to Mycobacterium tuberculosis infection. This suggested that there is a connection between TST negativity per se and TNF production.

We aimed to estimate the global occurrence of zoonotic tuberculosis (TB) caused by Mycobacterium bovis or M. caprae infections in humans by performing a multilingual, systematic review and analysis of relevant scientific literature of the last 2 decades. Although information from many parts of the world was not available, data from 61 countries suggested a low global disease incidence. In regions outside Africa included in this study, overall median proportions of zoonotic TB of ≤1.4% in connection with overall TB incidence rates ≤71/100,000 population/year suggested low incidence rates. For countries of Africa included in the study, we multiplied the observed median proportion of zoonotic TB cases of 2.8% with the continental average overall TB incidence rate of 264/100,000 population/year, which resulted in a crude estimate of 7 zoonotic TB cases/100,000 population/year. These generally low incidence rates notwithstanding, available data indicated substantial consequences of this disease for some population groups and settings.

Bovine tuberculosis (TB), caused by Mycobacterium bovis, has become established in Kruger National Park, South Africa, in the cape buffalo (Syncerus caffer) population and in other species. TB in prey species has resulted in infection and morbidity in the resident lion (Panthera leo) prides. The only validated live animal test currently available for lions is the intradermal tuberculin test. Because this test requires capture twice, 72 hr apart, of free-ranging lions to read results, it is logistically difficult to administer in a large ecosystem. Therefore, development of a rapid animal-side screening assay would be ideal in providing information for wildlife managers, veterinarians, and researchers working with free-living lion prides. This study reports preliminary descriptive results from an ongoing project evaluating two serologic tests for M. bovis (ElephantTB Stat-Pak and dual path platform VetTB). Disease status was determined by postmortem culture and presence of pathologic lesions in 14 free-ranging lions. Seropositivity was found to be associated with M. bovis infection. Extended field studies are underway to validate these rapid animal-side immunoassays for antemortem screening tests for TB in lions.

Tuberculosis pathology was studied on 19 African buffalo (Syncerus caffer) from a herd in the Hluhluwe-iMfolozi Park in South Africa. The animals tested positive with the comparative intradermal tuberculin test and were euthanized during a test-and-cull operation to decrease prevalence of bovine tuberculosis (bTB) in the park. The lymph nodes and lungs were examined grossly for presence of tuberculous lesions, which were scored on a 0-5 scale for macroscopic changes. The gross lesions were examined histologically and classified into grade I, II, III, or IV according to a grading system used for bTB lesions in domestic cattle. Macroscopic lesions were limited to the retropharyngeal, bronchial, and mediastinal lymph nodes and the lungs. The most frequently affected lymph nodes were the bronchial (in 16 animals) and mediastinal (in 11 animals). All four grades of microscopic lesions were observed, grade II lesions were the most frequent. Mycobacterium bovis was detected by PCR in 8 out of 19 animals, and acid-fast bacilli were seen in 7 out of 19 animals, together both techniques identified mycobacteria in 5 out of 19 animals. Lesions were paucibacillary, as acid-fast bacilli were only rarely observed. The absence of lesions in the mesenteric lymph nodes and the high frequency of lesions in respiratory tract associated lymph nodes suggest that the main route of M. bovis infection in African buffalo is by inhalation.

We have identified a globally important clonal complex of Mycobacterium bovis by deletion analysis of over one thousand strains from over 30 countries. We initially show that over 99% of the strains of M. bovis, the cause of bovine tuberculosis, isolated from cattle in the Republic of Ireland and the UK are closely related and are members of a single clonal complex marked by the deletion of chromosomal region RDEu1 and we named this clonal complex European 1 (Eu1). Eu1 strains were present at less than 14% of French, Portuguese and Spanish isolates of M. bovis but are rare in other mainland European countries and Iran. However, strains of the Eu1 clonal complex were found at high frequency in former trading partners of the UK (USA, South Africa, New Zealand, Australia and Canada). The Americas, with the exception of Brazil, are dominated by the Eu1 clonal complex which was at high frequency in Argentina, Chile, Ecuador and Mexico as well as North America. Eu1 was rare or absent in the African countries surveyed except South Africa. A small sample of strains from Taiwan were non-Eu1 but, surprisingly, isolates from Korea and Kazakhstan were members of the Eu1 clonal complex. The simplest explanation for much of the current distribution of the Eu1 clonal complex is that it was spread in infected cattle, such as Herefords, from the UK to former trading partners, although there is evidence of secondary dispersion since. This is the first identification of a globally dispersed clonal complex M. bovis and indicates that much of the current global distribution of this important veterinary pathogen has resulted from relatively recent International trade in cattle., (Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.)

African buffaloes (Syncerus caffer) are the most significant wildlife maintenance hosts of Mycobacterium bovis, the causative organism of bovine tuberculosis (BTB). Current diagnostic tests for the detection of M. bovis infection in free-ranging buffaloes have numerous limitations and we wished to evaluate a modification to a human TB assay, the QuantiFERON-TB Gold (In-Tube) assay (QFT), as a practical diagnostic test for BTB in buffaloes. One hundred and seventy-five buffaloes were tested using the single intradermal comparative tuberculin test (SICTT) and a modified QFT (mQFT). An appropriate cut-off point for the mQFT was derived from SICTT results using receiver operator characteristic curve analysis. Twenty-six SICTT-positive buffaloes were killed and subjected to necropsy, and selected tissues were processed for mycobacterial culture and speciation. An optimal cut-off point for the mQFT was calculated as 66pg/ml. The assay correctly detected 39/40 SICTT-positive buffaloes and 129/134 TST-negative buffaloes and M. bovis was cultured from 21/26 slaughtered SICTT/mQFT-positive animals. The mQFT shows promise as a practical test for M. bovis infection in buffaloes and shows a sensitivity and specificity at least similar to that of the TST., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We have identified a clonal complex of Mycobacterium bovis isolated at high frequency from cattle in Uganda, Burundi, Tanzania, and Ethiopia. We have named this related group of M. bovis strains the African 2 (Af2) clonal complex of M. bovis. Af2 strains are defined by a specific chromosomal deletion (RDAf2) and can be identified by the absence of spacers 3 to 7 in their spoligotype patterns. Deletion analysis of M. bovis isolates from Algeria, Mali, Chad, Nigeria, Cameroon, South Africa, and Mozambique did not identify any strains of the Af2 clonal complex, suggesting that this clonal complex of M. bovis is localized in East Africa. The specific spoligotype pattern of the Af2 clonal complex was rarely identified among isolates from outside Africa, and the few isolates that were found and tested were intact at the RDAf2 locus. We conclude that the Af2 clonal complex is localized to cattle in East Africa. We found that strains of the Af2 clonal complex of M. bovis have, in general, four or more copies of the insertion sequence IS6110, in contrast to the majority of M. bovis strains isolated from cattle, which are thought to carry only one or a few copies.

Most individuals latently infected with Mycobacterium tuberculosis (M.tb) contain the infection by a balance of effector and regulatory immune responses. This balance can be influenced by steroid hormones such as glucocorticoids. The widely used contraceptive medroxyprogesterone acetate (MPA) possesses glucocorticoid activity. We investigated the effect of this hormone on immune responses to BCG in household contacts of active TB patients. Multiplex bead array analysis revealed that MPA demonstrated both glucocorticoid and progestogenic properties at saturating and pharmacological concentrations in peripheral blood mononuclear cells (PBMCs) and suppressed antigen specific cytokine production. Furthermore we showed that PBMCs from women using MPA produced significantly lower levels of IL-1α, IL-12p40, IL-10, IL-13 and G-CSF in response to BCG which corresponded with lower numbers of circulating monocytes observed in these women. Our research study is the first to show that MPA impacts on infections outside the genital tract due to a systemic effect on immune function. Therefore MPA use could alter susceptibility to TB, TB disease severity as well as change the efficacy of new BCG-based vaccines, especially prime-boost vaccine strategies which may be administered to adult or adolescent women in the future.

Mycobacterium bovis is a pathogen of significant importance in livestock and a wide range of wild animal species worldwide. It is also known to cause tuberculosis disease in humans, a fact which has raised renewed concerns regarding the zoonotic risk for humans, especially those living at the animal-human interface. This review consolidates recent reports in the literature mainly on animal and zoonotic tuberculosis with an emphasis on evolution, epidemiology, treatment and diagnosis. The information presented reveals the fundamental differences in the complexity and level at which the disease affects the economy, ecosystem and human population of regions where animal tuberculosis control is achieved and regions where little or no control is implemented. In conclusion the review suggests that bovine tuberculosis has essentially been reduced to a disease of economic importance in the developed world, while low-income countries are facing a multifaceted impact which potentially affects the health of livestock, humans and ecosystems and which is likely to increase in the presence of debilitating diseases such as HIV/AIDS and other factors which negatively affect human livelihoods., (Copyright 2009 Elsevier B.V. All rights reserved.)