Antemortem detection of Mycobacterium bovis in nasal swabs from African rhinoceros.

Mycobacterium bovis (M. bovis) infection has been identified in black (Diceros bicornis) and white (Ceratotherium simum) rhinoceros populations in Kruger National Park, South Africa. However, it is unknown whether M. bovis infected rhinoceros, like humans and cattle, can shed mycobacteria in respiratory secretions. Limited studies have suggested that rhinoceros with subclinical M. bovis infection may present minimal risk for transmission. However, recent advances that have improved detection of Mycobacterium tuberculosis complex (MTBC) members in paucibacillary samples warranted further investigation of rhinoceros secretions. In this pilot study, nasal swab samples from 75 rhinoceros with defined infection status based on M. bovis antigen-specific interferon gamma release assay (IGRA) results were analysed by GeneXpert MTB/RIF Ultra, BACTEC MGIT and TiKa-MGIT culture. Following culture, speciation was done using targeted PCRs followed by Sanger sequencing for mycobacterial species identification, and a region of difference (RD) 4 PCR. Using these techniques, MTBC was detected in secretions from 14/64 IGRA positive rhinoceros, with viable M. bovis having been isolated in 11 cases, but not in any IGRA negative rhinoceros (n = 11). This finding suggests the possibility that MTBC/M. bovis-infected rhinoceros may be a source of infection for other susceptible animals sharing the environment.

Evaluation of the performance of the IFN-γ release assay in bovine tuberculosis free herds from five European countries.

The diagnostic methods for granting and maintenance of the official tuberculosis-free (OTF) status and for intra-Community movement of cattle are the tuberculin skin tests (single or comparative) and the interferon-γ (IFN-γ) release assay (IGRA). However, until now, IGRAs have been primarily applied in infected farms in parallel to the skin test to maximize the number of infected animals detected. Therefore, an evaluation of the performance of IGRAs in OTF herds to assess whether if their specificity is equal to or higher than that of the skin tests is needed. For this, a panel of 4365 plasma samples coming from 84 OTF herds in six European regions (five countries) was assembled and analysed using two IGRA kits, the ID Screen® Ruminant IFN-g (IDvet) and the Bovigam™ TB Kit (Bovigam). Results were evaluated using different cut-offs, and the impact of herd and animal-level factors on the probability of positivity was assessed using hierarchical Bayesian multivariable logistic regression models. The percentage of reactors ranged from 1.7 to 21.0% (IDvet: S/P ≥ 35%), and 2.1-26.3% (Bovigam: ODbovis-ODPBS ≥ 0.1 and ODbovis-ODavium ≥ 0.1) depending on the region, with Bovigam disclosing more reactors in all regions. The results suggest that specificity of IGRAs can be influenced by the production type, age and region of origin of the animals. Changes in the cut-offs could lead to specificity values above 98-99% in certain OTF populations, but no single cut-off yielding a sufficiently high specificity (equal or higher than that of skin tests) in all populations was identified. Therefore, an exploratory analysis of the baseline IFN-γ reactivity in OTF populations could help to assess the usefulness of this technique when applied for the purpose of maintaining OTF status.

Using whole blood cultures in interferon gamma release assays to detect Mycobacterium tuberculosis complex infection in Asian elephants (Elephas maximus).

Elephants are susceptible to Mycobacterium tuberculosis (M. tb) complex (MTBC) infections. Diagnosis of tuberculosis (TB) in elephants is difficult, and most approaches used for human TB diagnosis are not applicable. An interferon gamma release assay (IGRA) to diagnose TB in Asian elephants (Elephas maximus) using peripheral blood mononuclear cells (PBMCs) has been previously developed. Although the assay is shown to be valid in determining MTBC infection status, the laborious PBMC isolation process makes it difficult to use. In this study, we simplified the method by using whole blood cultures (WC) as the starting material. Using PBMC cultures for IGRA, the MTBC infection status of 15 elephants was first confirmed. Among these animals, one has been previously confirmed for M. tb infection by both TB culture and PCR and the other was confirmed for MTBC infection in this study by droplet digital PCR (ddPCR) method. WC for IGRA consisted of an unstimulated sample, a mitogen stimulated sample, and sample stimulated with recombinant M. tb antigens, ESAT6 and CFP10. Using WC for IGRA in the 15 enrolled elephants, the results showed that 7 out of 15 samples yielded MTBC infection positive status that were completely concordant with those from the results using PBMCs. To test this method, WC for IGRA were applied in another elephant cohort of 9 elephants. The results from this cohort revealed a perfect match between the results from PBMC and WC. Responses to ESAT6 or CFP10 by PBMC and WC were not completely concordant, arguing for the use of at least two M. tb antigens for stimulation. Given the ease of sample handling, smaller blood sample volumes and equivalent efficacy relative to the PBMC approach, using WC for IGRA provides a novel, rapid, and user-friendly TB diagnostic method for determining the MTBC infection in elephants.

Development of an enzyme-linked immunosorbent assay for the diagnosis of feline tuberculosis.

Cases of feline tuberculosis (TB) can be challenging to diagnose. Currently, this is achieved through a combination of mycobacterial culture, polymerase chain reaction (PCR), or interferon-gamma release assay (IGRA); however, these each have limitations. There is limited data regarding the use of humoral immunodiagnostics for TB in cats. Therefore, we sought to develop an enzyme-linked immunosorbent assay (ELISA) to further facilitate the diagnosis of feline TB. A comparative PPD (purified protein derivative) antibody ELISA was optimised for use on serum and plasma, and was tested against samples from 14 cats with culture-confirmed TB and 24 uninfected controls. Selection of an appropriate positive cut-off value based on receiver-operator characteristic curve analysis gave test sensitivity of 64.3 % and specificity of 100 %. When tested on further samples from cats with strongly suspected mycobacteriosis, 32.9 % (23/70) were antibody positive. Notably, positive results were recorded in cats that failed to respond to the IGRA, and in one PCR and IGRA negative cat. No positive responses were identified in cats with non-tuberculous mycobacterial infections, or with non-mycobacterial diseases (n = 12). Therefore, antibody-based diagnostics may be useful adjunctive tests for cases of TB missed by the IGRA, helping protect both feline and, in turn, human health.

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

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

Reduced levels of serum 25(OH)D3 are associated with tuberculosis positive cattle under conditions of high natural exposure to Mycobacterium bovis.

Serum vitamin D (25(OH)D3) concentrations of < 30 ng/mL in cattle are insufficient to induce an adequate immune response against intracellular pathogens, which suggests that the efficacy of the immune response may be highly dependent on the bioavailability of 25(OH)D3. This study shows an overview of both in vitro and in vivo 25(OH)D3-mediated immune modulation amongst dairy cattle naturally exposed to M. bovis. Tuberculin status was confirmed by interferon gamma release assay (IGRA), and natural exposure was demonstrated by polymerase chain reaction (PCR). Tuberculin (-) cattle have a higher serum concentration of 25(OH)D3 (X¯= 87.12 ng/mL) when compared to tuberculin (+) cattle (X¯ = 45.86 ng/mL). Reduced serum 25(OH)D3 levels are associated with the presence of bovine TB, and serum 25(OH)D3 levels of > 80 ng/mL are necessary to counteract infection by M. bovis. Kill assays were performed to evaluate in vitro 25(OH)D3 modulation of intracellular M. bovis growth in bovine macrophages, which showed that reduced serum 25(OH)D3 levels are associated with diminished mycobactericidal capacity in this experimental model. On the other hand, increased 25(OH)D3 in culture media enhances phagocytosis and nitric oxide production, which in turn improves capacity to combat M. bovis.

Tuberculosis detection in nonhuman primates is enhanced by use of testing algorithms that include an interferon-γ release assay.

OBJECTIVE: To develop a testing algorithm that incorporates multiple assays to evaluate host cellular and humoral immunity and antigen detection concerning Mycobacterium tuberculosis complex (MTBC) infection in captive nonhuman primates. ANIMALS: Cohorts of captive-bred and wild-caught macaques from 5 different geographic regions. PROCEDURES: Macaques were tested for MTBC infection by use of a γ interferon tuberculosis (GIFT) assay, an interferon-γ release assay, and other assays. In the first 2 cohorts (n = 15 and 181), initial validation of the GIFT assay was performed by use of experimentally infected and unexposed control macaques. In the next 3 cohorts (n = 59, 42, and 11), results were obtained for opportunistically collected samples from macaques exposed during spontaneous outbreaks. RESULTS: Sensitivity and specificity of the GIFT assay in the control cohorts were 100% and 97%, respectively, and were variable but enhanced by incorporating results from multiple assays in spontaneous outbreaks. CLINICAL RELEVANCE: The detection and management of MTBC infection in captive nonhuman primate populations is an ongoing challenge, especially with animal imports and transfers. Despite standardized practices of initial quarantine with regular intradermal tuberculin skin testing, spontaneous outbreaks continue to be reported. Since infection encompasses a range of disease manifestations over time, a testing algorithm that incorporates multiple assays, such as the GIFT assay, to evaluate host cellular and humoral immunity in addition to agent detection is needed. Testing a combination of samples from controlled studies and spontaneous outbreaks of MTBC infection in nonhuman primates would advance the development and validation of a functional algorithm that incorporates promising tools such as the GIFT assay.

Diagnostic accuracy of the interferon-gamma release assay (IGRA) for cases of feline mycobacteriosis.

The aim of this study was to evaluate the sensitivity and specificity of the interferon-gamma release assay (IGRA) for diagnosing infections with members of the Mycobacterium (M.) tuberculosis-complex (MTBC) and non-tuberculous mycobacteria (NTM) in domestic cats, and to generate defined feline-specific cut-off values using receiver operating characteristic (ROC) curve analysis to improve test performance. Records of 594 cats that had been tested by IGRA were explored to identify individuals that had a culture and/or polymerase chain reaction (PCR)-confirmed case of mycobacterial disease, and those that had a final diagnosis of non-mycobacterial disease. A total of 117 cats - 80 with mycobacterial disease and 37 diagnosed with a condition other than mycobacteriosis - were identified for further detailed analysis. This population was used to estimate test sensitivity and specificity, as well as likelihood ratios for the IGRA to correctly identify a cat with or without mycobacterial disease. Agreement between IGRA results and culture/PCR using current and proposed new cut-off values was also determined. ROC analysis of defined confirmed infected and non-mycobacterial disease control cats allowed an adjustment of current test cut-offs that increased the overall test sensitivity for MTBC infections from 83.1 % (95 % confidence interval [CI]: 71.5-90.5 %) to 90.2 % (95 % CI: 80.2-95.4%), and M. bovis infection from 43 % (95 % CI: 28.2-60.7%) to 68 % (95 % CI: 51.4-82.1%) while maintaining high test specificity (100 % in both cases). Overall agreement between IGRA results and culture/PCR, while recognising that neither culture nor PCR tests have perfect sensitivity, improved from weak (κ = 0.57) to moderate (κ = 0.71) using new proposed IGRA test cut-off values. Application of these results, based upon the statistical analysis of accumulated test data, can improve the diagnostic performance of the feline IGRA, particularly for identifying infections with M. bovis, without compromising specificity.

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.

CYTOKINE-RELEASE ASSAY FOR THE DETECTION OF MYCOBACTERIUM BOVIS INFECTION IN CHEETAH (ACINONYX JUBATUS).

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-1ß), 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 ELISABasic 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.

Modelling the effect of test-and-slaughter strategies to control bovine tuberculosis in endemic high prevalence herds.

Bovine tuberculosis (bTB) prevalence substantially increased over the past two decades with relatively high impact on large dairy herds, raising the concern of regulatory authorities and industry stakeholders, and threatening animal and public health. Lack of resources, together with the economic and social consequences of whole-herd stamping-out, makes depopulation an impractical disease control alternative in these herds. The increase in bTB prevalence was associated with demographic and management changes in the dairy industry in Uruguay, reducing the efficacy of the current control programme (i.e. status quo) based on intradermal serial testing with caudal fold- and comparative-cervical tuberculin test-and-slaughter of reactors (CFT-CCT). Here, we aimed to assess the epidemiological effectiveness of six alternative control scenarios based on test-and-slaughter of positive animals, using mathematical modelling to infer bTB-within-herd dynamics. We simulated six alternative control strategies consisting of testing adult cattle (>1 year) in the herd every 3 months using one test (in vivo or in vitro) or a combination in parallel of two tests (CFT, interferon-gamma release assay-IGRA- or enzyme-linked immunosorbent assay). Results showed no significant differences overall in the time needed to reach bTB eradication (median ranging between 61 and 82 months) or official bovine tuberculosis-free status (two consecutive negative herd tests) between any of the alternative strategies and the status quo (median ranging between 50 and 59 months). However, we demonstrate how alternative strategies can significantly reduce bTB prevalence when applied for restricted periods (6, 12 or 24 months), and in the case of IGRAc (IGRA using peptide-cocktail antigens), without incurring on higher unnecessary slaughter of animals (false positives) than the status quo in the first 6 months of the programme (p-value < .05). Enhanced understanding bTB-within-herd dynamics with the application of different control strategies help to identify optimal strategies to ultimately improve bTB control and bTB eradication from dairies in Uruguay and similar endemic settings.

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

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

Detection of tuberculosis in cynomolgus macaques (Macaca fascicularis) using a supplementary Monkey Interferon Gamma Releasing Assay (mIGRA).

Cynomolgus monkeys (Macaca fascicularis; MF) are commonly used as nonhuman primate models for pharmaceutical product testing. In their habitat range, monkeys have close contact with humans, allowing the possibility of bidirectional transmission of tuberculosis (TB) between the two species. Although the intradermal tuberculin skin test (TST) is used for TB detection in MF, it has limitations. Herein, we established the mIGRA, combining human QuantiFERON-TB Gold-Plus and monkey IFN-γ ELISApro systems, and used it to investigate 39 captive MF who were cage-mates or lived in cages located near a monkey who died from the naturally TB infection. During a 12-month period of study, 14 (36%), 10 (26%), and 8 (21%) monkeys showed TB-positive results using the mIGRA, the TST, and TB culture, respectively. Among the 14 mIGRA-positive monkeys, 8 (57.1%) were TST-positive and 7 (50%) were culture-positive, indicating early TB detection in the latent and active TB stages with the mIGRA. Interestingly, 3 (37.5%) of the TST-negative monkeys were culture-positive. Our study showed that the mIGRA offers many advantages, including high sensitivity and high throughput, and it requires only one on-site visit to the animals. The assay may be used as a supplementary tool for TB screening in MF.

Accuracy of tuberculosis diagnostic tests in small ruminants: A systematic review and meta-analysis.

Tuberculosis (TB) in small ruminants is a neglected disease despite its major impact on goat and sheep production and the global public health. The awareness of the role of small ruminants in the epidemiology of animal TB has increased in the last two decades, however, there is a lack of standardization of procedures and robust quantitative estimates on the accuracy of diagnostic TB tests in the scientific literature. To address this knowledge gap, all the available information regarding the use of ante-mortem diagnostic techniques in small ruminants was collected and summarized through a systematic review process. Furthermore, a random-effects meta-analysis was conducted to separately estimate the sensitivity (Se) and specificity (Sp) of cell-based tests among the retrieved studies in goats. Studies included in the meta-analysis were also evaluated using the Quality Assessment of Diagnostic Accuracy Studies included in systematic reviews adapted for animal diagnostic tests (VETQUADAS). Median pooled Se estimates of the single intradermal tuberculin (SIT) test (ranged from 0.51 to 0.59), the comparative intradermal tuberculin (CIT) test (ranged from 0.30 to 0.50) and the interferon-gamma (IFN-γ) release assay (IGRA) (ranged from 0.66 to 0.72) were lower than that reported previously in cattle, regardless the interpretation criteria and the reporting of MAP infection or vaccination. However, the specificity was adequate for all the tests (ranged from 0.95 to 0.99), except for the SIT test in MAP vaccinated herds (ranged from 0.78 to 0.90). This study provides an overview of the accuracy of diagnostic tests for TB in goats, however, the considerable between-study heterogeneity found hampered the conclusive interpretation of the pooled Se and Sp estimates. Therefore, further studies in small ruminants are necessary to optimize the diagnostic Se, which could help to design effective control strategies, accelerate the eradication of TB in these species and harmonize test procedures.

CONSERVATION CHALLENGES: THE LIMITATIONS OF ANTEMORTEM TUBERCULOSIS TESTING IN CAPTIVE ASIATIC LIONS (PANTHERA LEO PERSICA).

Genetic diversity of captive wild animals can be enhanced by moving those individuals with valuable genes between collections and through introduction of a new pair from a range country. This requires movement of animals, which is inherent with disease risks, such as the introduction of pathogenic Mycobacterium sp. (MTBC) into a zoological collection. Decisions need to be made based on the outcome of perimovement disease screening using an array of tests, the majority of which are unvalidated in the species. A pair of endangered Asiatic lions (Panthera leo persica) imported from India to the United Kingdom were screened for MTBC using the comparative intradermal tuberculosis (TB) test, the feline interferon-γ blood test, and the experimental bacteriophage assay. Reactions on all three tests prompted screening of the three resident Asiatic lions using the same tests, all of which were negative for MTBC. Based on these test results, the decision had to be made to exclude the genetically valuable pair from the current collection. MTBC could not be identified using further tests, including culture and PCR on a bronchoalveolar lavage, on feces, or on postmortem tissues. This case series highlights the usefulness of a control group when interpreting unvalidated test results for detection of MTBC, the value of training big cats for conscious blood sampling, and the practical implications of placing the comparative intradermal TB test in the eyelids, when dealing with a species that requires a general anesthetic for most hands-on interventions.

Detection of native interferon-γ in nyala (Tragelaphus angasii): Towards diagnosing tuberculosis.

Mycobacterium bovis is the main cause of tuberculosis in wildlife. In South Africa, African buffaloes (Syncerus caffer) are a wildlife maintenance host while a number of other species are considered spillover hosts. Nyala (Tragelaphus angasii), a large antelope species from Southern Africa, is frequently traded and can be infected with M. bovis. Interferon gamma (IFN-γ) release assays that detect cell-mediated immune (CMI) responses to M. bovis infection have shown promise in elephants, rhinoceroses and buffaloes. The BOVIGAM® assay is a commercial IFN-γ release assay designed to detect tuberculosis in cattle and has been validated in buffaloes. We tested the suitability of the BOVIGAM® assay to detect native IFN-γ release in nyala. Blood samples collected from 17 nyalas were stimulated with different mitogens and IFN-γ release measured. We found that incubating whole blood with phorbol 12-myristate 13-acetate and calcium ionophore (PMA/CaI) resulted in the highest levels of IFN-y release. Samples stimulated with tuberculin purified protein derivatives of M. bovis (PPDb) and M. avium (PPDa) did not show significant IFN-γ production. An intradermal tuberculin test (IDT) and culture of tissues from 15 of the 17 culled nyala were also performed, which supported the findings of the BOVIGAM® assay, suggesting the potential value of this assay for the diagnosis of tuberculosis in nyala.

An interferon-gamma release assay for the diagnosis of the Mycobacterium bovis infection in white rhinoceros (Ceratotherium simum).

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 ELISAPRO 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.

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

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

Interferon-Gamma test for the detection of Mycobacterium tuberculosis complex infection in Macaca mulatta and other non-human primates.

We have formatted an assay to detect Mycobacterium tuberculosis complex infections of non-human primates. Commercially available reagents were used to elicit a specific immune response that was measured by interferon-gamma release. Initial evaluation using blood samples from Rhesus macaques experimentally infected with M tuberculosis distinguished infected versus uninfected animals.

Simultaneous measurement of antigen-induced CXCL10 and IFN-γ enhances test sensitivity for bovine TB detection in cattle.

Bovine tuberculosis (BTB) is a disease of economic and zoonotic importance caused mainly by Mycobacterium bovis. In addition to the tuberculin skin test, an interferon-gamma (IFN-γ) release assay (IGRA) blood test has been incorporated in the BTB control programs of numerous countries as an ancillary test to the skin test. A potential disadvantage of the IGRA assay is that it relies solely on the measurement of a single readout (i.e. IFN-γ) for the detection of BTB. In this study we have assessed the practical use of CXCL10 as an additional biomarker for the diagnosis of BTB in the setting of the current testing approach alongside IGRA. To do so, we have assessed both IFN-γ and CXCL10 readouts in blood cultures from a variety of different BTB cattle groups stimulated with standard tuberculin reagents and also with more specific defined antigens (ESAT-6, CFP-10 and Rv3615c). When using a tuberculin based whole blood assay, CXCL10 alone could not substitute for IFN-γ as the analyte measured in the test without reducing the sensitivity of detecting BTB animals. However, when used as an additional test readout, CXCL10 identified BTB animals that failed to induce IFN-γ responses. When tested in non-infected animals, the use of the dual biomarker system had the potential to lower overall test specificity, however this could be overcome by raising the cut-off values for CXCL10 test positivity. Taken together, the results demonstrate that in particular settings, measurement of CXCL10 has the potential to complement the current use of IFN-γ in blood assays to maximise the detection of BTB.