Development of a cytokine gene expression assay for the relative quantification of the African elephant (Loxodonta africana) cell-mediated immune responses.

Immunological assays are the basis for many diagnostic tests for infectious diseases in animals and humans. Application in wildlife species, including the African elephant (Loxodonta africana), is limited however due to lack of information on immune responses. Since many immunoassays require both identified biomarkers of immune activation as well as species-specific reagents, it is crucial to have knowledge of basic immunological responses in the species of interest. Cytokine gene expression assays (GEAs) used to measure specific immune responses in wildlife have frequently shown that targeted biomarkers are often species-specific. Therefore, the aim of this study was to identify elephant-specific cytokine biomarkers to detect immune activation and to develop a GEA, using pokeweed mitogen stimulated whole blood from African elephants. This assay will provide the foundation for the development of future cytokine GEAs that can be used to detect antigen specific immune responses and potentially lead to various diagnostic tests for this species.

The VetMAX™ M. tuberculosis complex PCR kit detects MTBC DNA in antemortem and postmortem samples from white rhinoceros (Ceratotherium simum), African elephants (Loxodonta africana) and African buffaloes (Syncerus caffer).

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.

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

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

MYCOBACTERIUM BOVIS IN FREE-RANGING LIONS (PANTHERA LEO) - EVALUATION OF SEROLOGICAL AND TUBERCULIN SKIN TESTS FOR DETECTION OF INFECTION AND DISEASE.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis infection, causes morbidity and mortality in free-ranging lions in bTB-endemic areas of South Africa. However, the only currently used diagnostic test is the tuberculin skin test (TST). This test is logistically challenging to perform because it requires immobilization of lions twice in a 72-hr period. Blood-based diagnostic tests, such as serological assays, have been previously reported for M. bovis detection in lion populations, and have the advantage of only requiring a single immobilization. In addition, serological assays can be used for retrospective testing. Therefore, the aim of this study was to test free-ranging lions with the STAT-PAKt (Chembio Diagnostics Systems, Medford, NY 11763, USA) and DPPt VetTB (Chembio Diagnostics Systems) serological assays and compare those results with the tuberculin skin test. The serological assays were also used to determine prevalence in bTB-endemic and uninfected lion populations. The results showed that the serological assays could distinguish between M. bovis culture-positive and -negative lions. In addition, antigen-specific humoral responses were present in lions that had clinical signs of bTB disease or were shedding M. bovis antemortem. Although the seroprevalence of M. bovis infection in Kruger National Park lions was similar to that obtained from antemortem mycobacterial culture (4.8 and 3.3%, respectively), it was less than that estimated by the TST (72%). These findings support the hypothesis that assays based on cell-mediated immune responses are more sensitive than serology is in detecting M. bovis infection in lions. However, serological assays can have a role in bTB disease detection in lions and are especially useful for retrospective studies.

Conservation of White Rhinoceroses Threatened by Bovine Tuberculosis, South Africa, 2016-2017.

During 2016-2017, when Kruger National Park, South Africa, was under quarantine to limit bovine tuberculosis spread, we examined 35 white and 5 black rhinoceroses for infection. We found 6 infected white rhinoceroses during times of nutritional stress. Further research on Mycobacterium bovis pathogenesis in white rhinoceroses is needed.

A pilot study evaluating the utility of commercially available antibodies for flow cytometric analysis of Panthera species lymphocytes.

BACKGROUND: The immune response against tuberculosis in lions is still poorly defined and our understanding is hampered by the lack of lion specific reagents. The process for producing antibodies against a specific antigen is laborious and not available to many research laboratories. As the search for antibody cross-reactivity is an important strategy for immunological studies in veterinary medicine, we have investigated the use of commercially available antibodies to characterize T cell subsets in African lions (Panthera leo). RESULTS: Commercially available antibodies were screened and investigated the influence of two different sample processing methods, as well as the effect of time delay on cell surface marker expression on lion lymphocytes. Using commercially available antibodies, we were able to identify CD4+, CD5+, CD8+, CD14+, CD25+, CD44+ and CD45+ T lymphocytes in samples obtained by density gradient centrifugation as well as red cell lysis of lion whole blood. Two distinct lymphocyte populations, which differed in size and phenotype, were observed in the samples processed by density gradient centrifugation. CONCLUSION: Commercially available antibodies are able to differentiate between T lymphocyte subsets including immune effector cells in African lion whole blood, and possibly give insight into unique specie phenotypes.

Measuring antigen-specific responses in Mycobacterium bovis-infected warthogs (Phacochoerus africanus) using the intradermal tuberculin test.

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.

Mycobacterium bovis in a Free-Ranging Black Rhinoceros, Kruger National Park, South Africa, 2016.

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.

Multilaboratory Evaluation of a Novel Lateral Flow Immunochromatographic Assay for Confirming Isolation of Mycobacterium bovis from Veterinary Diagnostic Specimens.

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.

Development and evaluation of a diagnostic cytokine-release assay for Mycobacterium suricattae infection in meerkats (Suricata suricatta).

BACKGROUND: Sensitive diagnostic tools are necessary for the detection of Mycobacterium suricattae infection in meerkats (Suricata suricatta) in order to more clearly understand the epidemiology of tuberculosis and the ecological consequences of the disease in this species. We therefore aimed to develop a cytokine release assay to measure antigen-specific cell-mediated immune responses of meerkats. RESULTS: Enzyme-linked immunosorbent assays (ELISAs) were evaluated for the detection of interferon-gamma (IFN-γ) and IFN-γ inducible protein 10 (IP-10) in meerkat plasma. An IP-10 ELISA was selected to measure the release of this cytokine in whole blood in response to Bovigam® PC-HP Stimulating Antigen, a commercial peptide pool of M. bovis antigens. Using this protocol, captive meerkats with no known M. suricattae exposure (n = 10) were tested and results were used to define a diagnostic cut off value (mean plus 2 standard deviations). This IP-10 release assay (IPRA) was then evaluated in free-living meerkats with known M. suricattae exposure, categorized as having either a low, moderate or high risk of infection with this pathogen. In each category, respectively, 24.7%, 27.3% and 82.4% of animals tested IPRA-positive. The odds of an animal testing positive was 14.0 times greater for animals with a high risk of M. suricattae infection compared to animals with a low risk. CONCLUSION: These results support the use of this assay as a measure of M. suricattae exposure in meerkat populations. Ongoing longitudinal studies aim to evaluate the value of the IPRA as a diagnostic test of M. suricattae infection in individual animals.

Zoonotic Mycobacterium bovis-induced tuberculosis in humans.

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.

Novel cause of tuberculosis in meerkats, South Africa.

The organism that causes tuberculosis in meerkats (Suricata suricatta) has been poorly characterized. Our genetic analysis showed it to be a novel member of the Mycobacterium tuberculosis complex and closely related to the dassie bacillus. We have named this epidemiologically and genetically unique strain M. suricattae.

Spontaneous latency in a rabbit model of pulmonary tuberculosis.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is an exquisitely adapted human pathogen capable of surviving for decades in the lungs of immune-competent individuals in the absence of disease. The World Health Organization estimates that 2 billion people have latent TB infection (LTBI), defined by a positive immunological response to Mtb antigens, with no clinical signs of disease. A better understanding of host and pathogen determinants of LTBI and subsequent reactivation would benefit TB control efforts. Animal models of LTBI have been hampered generally by an inability to achieve complete bacillary clearance. Herein, we have characterized a rabbit model of LTBI in which, similar to most humans, complete clearance of pulmonary Mtb infection and pathological characteristics occurs spontaneously. The evidence that Mtb-CDC1551-infected rabbits achieve LTBI, rather than sterilization, is based on the ability of the bacilli to be reactivated after immune suppression. These rabbits showed early activation of T cells and macrophages and an early peak in the TNFα level, which decreased in association with clearance of bacilli from the lungs. In the absence of sustained tumor necrosis factor-α production, no necrosis was seen in the evolving lung granulomas. In addition, bacillary control was associated with down-regulation of several metalloprotease genes and an absence of lung fibrosis. This model will be used to characterize molecular markers of protective immunity and reactivation.

Whole genome sequencing improves the discrimination between Mycobacterium bovis strains on the southern border of Kruger National Park, South Africa.

Background: Mycobacterium bovis forms part of the Mycobacterium tuberculosis complex and has an extensive host range and zoonotic potential. Various genotyping methods (e.g., spoligotyping) have been used to describe the molecular epidemiology of M. bovis. Advances in whole genome sequencing (WGS) have increased resolution to enable detection of genomic variants to the level of single nucleotide polymorphisms. This is especially relevant to One Health research on tuberculosis which benefits by being able to use WGS to identify epidemiologically linked cases, especially recent transmission. The use of WGS in molecular epidemiology has been extensively used in humans and cattle but is limited in wildlife. This approach appears to overcome the limitations of conventional genotyping methods due to lack of genetic diversity in M. bovis. Methods: This pilot study investigated the spoligotype and WGS of M. bovis isolates (n = 7) from wildlife in Marloth Park (MP) and compared these with WGS data from other South African M. bovis isolates. In addition, the greater resolution of WGS was used to explore the phylogenetic relatedness of M. bovis isolates in neighbouring wildlife populations. Results: The phylogenetic analyses showed the closest relatives to the seven isolates from MP were isolates from wildlife in Kruger National Park (KNP), which shares a border with MP. However, WGS data indicated that the KNP and MP isolates formed two distinct clades, even though they had similar spoligotypes and identical in silico genetic regions of difference profiles. Conclusions: Mycobacterium bovis isolates from MP were hypothesized to be directly linked to KNP wildlife, based on spoligotyping. However, WGS indicated more complex epidemiology. The presence of two distinct clades which were genetically distinct (SNP distance of 19-47) and suggested multiple transmission events. Therefore, WGS provided new insight into the molecular epidemiology of the M. bovis isolates from MP and their relationship to isolates from KNP. This approach will facilitate greater understanding of M. bovis transmission at wildlife-livestock-human interfaces and advances One Health research on tuberculosis, especially across different host species.

Survey of infections transmissible between baboons and humans, Cape Town, South Africa.

Baboons on South Africa's Cape Peninsula come in frequent contact with humans. To determine potential health risks for both species, we screened 27 baboons from 5 troops for 10 infections. Most (56%) baboons had antibodies reactive or cross-reactive to human viruses. Spatial overlap between these species poses low but potential health risks.

Comparison of interferon gamma release assay and CXCL9 gene expression assay for the detection of Mycobacterium bovis infection in African lions (Panthera leo).

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.

Cytokine biomarker discovery in the white rhinoceros (Ceratotherium simum).

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 RT2 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 RT2 profiler PCR array as a useful tool for screening biomarkers for the diagnosis of M. bovis infection in rhinoceros.

Combining Analytical Approaches and Multiple Sources of Information to Improve Interpretation of Diagnostic Test Results for Tuberculosis in Wild Meerkats.

Diagnostic tests are used to classify individual animals' infection statuses. However, validating test performance in wild animals without gold standard tests is extremely challenging, and the issue is further complicated in chronic conditions where measured immune parameters vary over time. Here, we demonstrate the value of combining evidence from different diagnostic approaches to aid interpretation in the absence of gold standards, large sample sizes, and controlled environments. Over a two-year period, we sampled 268 free-living meerkats (Suricata suricatta) longitudinally for Mycobacterium suricattae (a causative agent of tuberculosis), using three ante-mortem diagnostic tests based on mycobacterial culture, and antigen-specific humoral and cell-mediated immune responses, interpreting results both independently and in combination. Post-mortem cultures confirmed M. suricattae infection in 22 animals, which had prior ante-mortem information, 59% (13/22) of which were test-positive on a parallel test interpretation (PTI) of the three ante-mortem diagnostic assays (95% confidence interval: 37-79%). A similar ability to detect infection, 65.7% (95% credible interval: 42.7-84.7%), was estimated using a Bayesian approach to examine PTI. Strong evidence was found for a near doubling of the hazard of death (Hazard Ratio 1.75, CI: 1.14-2.67, p = 0.01), associated with a positive PTI result, thus demonstrating that these test results are related to disease outcomes. For individual tests, small sample sizes led to wide confidence intervals, but replication of conclusions, using different methods, increased our confidence in these results. This study demonstrates that combining multiple methodologies to evaluate diagnostic tests in free-ranging wildlife populations can be a useful approach for exploiting such valuable datasets.

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.

Optimisation of the tuberculin skin test for detection of Mycobacterium bovis in African buffaloes (Syncerus caffer).

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 SICTT72h 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; SICTT72h), and specificities of 86 % (95 % CI 80-90; SITT), 96 % (95 % CI 92-98; SICTT72h) 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.