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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., (© 2024. The Author(s).)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Pongma et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., Competing Interests: Declarations of Competing Interest None., (Crown Copyright © 2022. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Conflict of interest 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 Elsevier B.V. All rights reserved.)

Serum vitamin D (25(OH)D 3 ) 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)D 3 . 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)D 3 (X¯= 87.12 ng/mL) when compared to tuberculin (+) cattle (X¯ = 45.86 ng/mL). Reduced serum 25(OH)D 3 levels are associated with the presence of bovine TB, and serum 25(OH)D 3 levels of > 80 ng/mL are necessary to counteract infection by M. bovis. Kill assays were performed to evaluate in vitro 25(OH)D 3 modulation of intracellular M. bovis growth in bovine macrophages, which showed that reduced serum 25(OH)D 3 levels are associated with diminished mycobactericidal capacity in this experimental model. On the other hand, increased 25(OH)D 3 in culture media enhances phagocytosis and nitric oxide production, which in turn improves capacity to combat M. bovis., (Copyright © 2022. Published by Elsevier Ltd.)

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.

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.

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., (Crown Copyright © 2021. Published by Elsevier B.V. All rights reserved.)

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.)

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) ELISA PRO 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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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 ELISA pro 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.

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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.

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.

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.)

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., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

In South Africa, the largest proportion of the African wild dog ( Lycaon pictus ) population resides in regions where buffaloes have a high prevalence of Mycobacterium bovis , the causative agent of bovine tuberculosis (bTB). Recent reports of deaths of wild dogs associated with bTB have raised concerns regarding the threat this disease might pose for this species. In order to understand the potential impact of the disease in wild dogs, diagnostic tools are required to identify infected individuals. The interferon gamma (IFN-γ) release assay (IGRA) is commonly used for tuberculosis (TB) screening of humans, cattle, and other species, and the aim of this study was to develop an IGRA for wild dogs to detect immune sensitization. Blood was collected from immobilized wild dogs from the Ann van Dyk Cheetah Centre (AvDCC; n =9) and Kruger National Park (KNP; n =31). Heparinized whole blood was incubated overnight in QuantiFERON ® -TB Gold (QFT) blood collection tubes and with selected mitogens, after which the plasma fraction was harvested. Three canine IFN-γ enzymelinked immunosorbent assays (ELISAs) were compared for detection of wild dog IFN-γ in plasma and the R&D Quantikine canine IFN-γ ELISA was selected for measurement of M. bovis -specific IFN-γ release in plasma samples. An IGRA result was calculated as the concentration in plasma derived from the QFT TB Antigen tubes minus that in the QFT Nil tube. An IGRA cut-off value was calculated using the IGRA results of M. bovis- unexposed individuals from AvDCC. Using this cut-off value, 74% (23/31) of M. bovis -exposed KNP wild dogs were IGRA positive, indicating immune sensitization to TB antigens in these animals. Three M. bovis culture-positive wild dogs from KNP had IFN-γ concentrations between 758 and 1,445 pg/mL, supporting this interpretation. This warrants further investigation into the prevalence of M. bovis infection in the KNP population.

Mycobacterium bovis can cause tuberculosis (TB) in social mammals including lions, cattle and man, but canine infections are considered rare. In 2016/17 we investigated a M. bovis TB outbreak in a pack of approximately 180 Foxhounds within the bovine TB Edge Area of England. We employed a combination of immunological tests including an interferon gamma release assay (IGRA) and a serological assay (DPP VetTB, Chembio). Test-positive hounds were euthanased and subjected to post-mortem examination (PME). Overall 164 hounds were tested; 97 (59%) responded positively to at least one test. Eighty-five (52%) dogs responded to M. bovis antigens by IGRA while only 21 (12.9%) had detectable serological responses. At PME three hounds (3.1%) had visible lesions (VL) due to M. bovis infection, later confirmed by culture. Samples from 24 non-VL hounds were cultured and M. bovis infection was confirmed in a further three hounds (11%). This study is the first investigation and report of an outbreak of M. bovis TB in a canine species. We establish that, in principle, diagnostic tests used for identifying infected individuals of other species can effectively be used in the dog. Further work is urgently needed to establish the sensitivity and specificity of the testing approach used in this study for future clinical application., (© 2018 The Authors. Transboundary and Emerging Diseases Published by Blackwell Verlag GmbH.)

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.)

Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) is a devastating and terminal disease in non-human primates (NHPs). Regular TB screenings using the intradermal tuberculin test (TST) have been the mainstay of TB surveillance and control in NHPs. Historically, Aotus monkeys have been considered less susceptible to TB than other NHPs. Here we present the diagnosis and epidemiology of a TB outbreak at The Gorgas Memorial Institute Aotus colony in Panama, and the results of two cross-sectional randomized TB screening studies, using antibody (Ab) and IFN-gamma release assay testing., Results: Epidemiological and spatial analysis confirmed that the outbreak was the result of a continuing intermittent exposure, with human to monkey transmission as the most likely source. During the outbreak that lasted five months (January-June 2015), Mycobacterium kansassi and MTB were isolated from lung caseous granulomas in 1/7 and 3/7 TB suspicious animals respectively. Furthermore, MTB was detected by qRT-PCR in formalin fixed lung and liver granulomas in 2/7 and 1/6 monkeys respectively, suggesting an aerosol route of infection. Likewise, a random sample that included 63 / 313 adult (>2 year-old) monkeys, screened for latent TB with the Primagam® IFN-gamma release assay, between March-May, 2016, were all non-reactors; indicating that the outbreak was self-limiting and the colony was likely free or latent TB infection. Control measures included, quarantine, disinfection and TST screening of all personnel. In conclusion, this study demonstrates that Aotus are highly susceptible to TB, therefore, TB prevention measures should be strictly enforced in Aotus monkey colonies., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The diagnosis of tuberculosis (TB) in goats is based mainly on the single and comparative intradermal tuberculin (SIT and CIT) tests and, exceptionally, on the interferon-gamma (IFN-γ) assay, however they are not perfect in terms of sensitivity and specificity. Nevertheless, various serological assays that provide a potential cost-effective approach for the control of TB are also available or under development, and a variety of results have been reported regarding the ability of these tests to detect infected animals, particularly in the early stages of infection. In the present study, SIT/CIT and IFN-γ tests and three different serological assays were evaluated during two consecutive herd testing events in a recently infected caprine herd (n = 447) with a high prevalence of infection in order to evaluate their performance and provide field data with which to improve the TB control programs in this species. The proportion of infected animals that tested positive among all the infected goats (T+/I+ value) in the last herd testing event ranged from 26.2% (IC95%; 19.3-34.5) to 85.7% (IC95%; 78.5-90.7) using cell-based diagnostic tests. The SIT/SCIT tests detected more infected goats than the IFN-γ test, regardless of the interpretation criteria. The T+/I+ value of serology was 83.2 (IC95%; 75.2-89), although it increased significantly (P < 0.05) when using samples collected 15 days after the intradermal test (100%, IC95%; 97-100). In general, a parallel interpretation of intradermal tests with serology maximized the detection of infected goats. These results demonstrate that serological tests are valuable diagnostic tools to maximize the detection of TB infected goats, even in recent outbreaks, accelerating the eradication process., (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.)

The interferon-gamma (IFN-γ) test measures cell mediated immune response (CMI) during the early stages of tuberculosis infection. Although Bovine Tuberculosis (BT) spread in feral pigs is widely documented in literature, the effectiveness of IFN-γ in this species has been only recently reported. One of the major obstacle of this assay is that whole blood samples should be stimulated with purified protein derivative (PPD) cocktail within 8 h from the blood sampling. This study set up a defined broth culture in which lymphocytes, the cell population predominantly responsible for IFN-γ production, are maintained in a steady-state and their vitality is preserved. The IFN-γ production measured from the samples added with the maintenance medium and stored at 4 °C was similar to the enzyme-linked immunosorbent assay (ELISA) optical density values obtained from the same assay performed within 8 h from sampling., (Copyright © 2017. Published by Elsevier Ltd.)

Background: Red deer (Cervus elaphus) is regarded as an epidemiologically relevant host for Mycobacterium bovis (M. bovis) and closely related members of the Mycobacterium tuberculosis complex that cause animal tuberculosis (TB). The standard antemortem screening test for the detection of TB in deer is the intradermal tuberculin skin test, but the detection of interferon-gamma (IFNγ) produced by white blood cells exposed to M. bovis antigens can be used as an alternative or supplemental assay in most TB eradication/control programs. This study aims to develop an in-house sandwich ELISA for deer IFNγ, based on the cross-reactivity of the antibodies to both cervid and bovine IFNγ, and to evaluate the potential of this assay to detect M. bovis-infected red deer in response to the in vitro stimulation of whole-blood cells with bovine purified protein derivative (bPPD), p22 protein complex derived from bPPD or using the specific tuberculous mycobacterial proteins ESAT-6/CFP-10, Rv3615c and Rv3020c. The positive control stimulant used in this study was pokeweed mitogen, which resulted in a consistent induction of IFNγ in samples from red deer, thus allowing the interpretation of the assay., Results: The percentage of animals correctly classified by this technique as M. bovis non-infected was 100%. The detection of infected animals as positive was high and ranged widely depending upon the antigen and the cut-off value applied, as well as the time after infection. Our findings indicate that this protocol may serve as a reliable assay for the antemortem diagnosis of TB from the initial stage of M. bovis-infection, and may also be adequately sensitive., Conclusions: The suggested optimal antigens and cut-off are bPPD, p22 and the combination of ESAT-6/CFP-10 and Rv3020c with a 0.05 Δ optical density, which yielded a up to 100% correct classification of TB positive and negatve red deer under our experimental conditions. This technique will aid in TB testing of farmed and translocated deer. Future studies should evaluate the ability of this IFNγ assay to detect specific responses under field conditions.

The diagnosis and control of Mycobacterium bovis infection (bovine tuberculosis: TB) continues to present huge challenges to the British cattle industry. A clearer understanding of the magnitude and duration of immune response to M. bovis infection in the European badger (Meles meles) - a wildlife maintenance host - may assist with the future development of diagnostic tests, and vaccination and disease management strategies. Here, we analyse 5280 diagnostic test results from 550 live wild badgers from a naturally-infected population to investigate whether one diagnostic test (a gamma interferon release [IFNγ] assay, n = 550 tests) could be used to predict future positive results on two other tests for the same disease (a serological test [n = 2342 tests] and mycobacterial culture [n = 2388 tests]) and hence act as an indicator of likely bacterial excretion or disease progression. Badgers with the highest IFNγ optical density (OD) values were most likely to subsequently test positive on both serological and culture tests, and this effect was detectable for up to 24 months after the IFNγ test. Furthermore, the higher the original IFNγ OD value, the greater the chance that a badger would subsequently test positive using serology. Relationships between IFNγ titres and mycobacterial culture results from different types of clinical sample suggest that the route of infection may affect the magnitude of immune response in badgers. These findings identify further value in the IFNγ test as a useful research tool, as it may help us to target studies at animals and groups that are most likely to succumb to more progressive disease.

Caprine tuberculosis is a major health problem for goats, and an important zoonosis. Eradication programmes using the comparative skin test are being developed to aid in decreasing the prevalence of infection. However, persistent tuberculosis infections are found in herds subjected to eradication programmes. In the present study a commercial IFN-γ release assay and an experimental ELISA based on MPB70, were evaluated as potential ancillary tests to detect infection. The relationship between the three techniques (skin test, IFN-γ release and ELISA) and histopathological lesions was analyzed in 162 goats from herds with persistent tuberculosis infection. The presence of related pathogens (paratuberculosis and pseudotuberculosis) was also studied. The IFN-γ release assay and the ELISA had a higher sensitivity than the comparative skin test (65.3% and 66.3% vs 44.5%) using as a gold standard a combination of histopathological analysis and isolation. Used in combination, ELISA and the skin test detected 89.1% of goats with tuberculosis while a combination of IFN-γ assay and skin test detected 78.2%. The types of macroscopic and microscopic lesions reflected the results of diagnostic tests., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In the current study, the results of an intradermal tuberculin test and a gamma interferon (IFN-γ) release assay were compared. IFN-γ release assay is based on the detection of IFN-γ production after in vitro stimulation with Mycobacterium avium subsp. avium-specific antigen for the discrimination of pigs naturally infected with M. avium subsp. hominissuis. Fifty-five clinically healthy pigs were used in the study. Three of these were proven by culture and real-time quantitative polymerase chain reaction methods to be infected with M. avium subsp. hominissuis (2 animals) and Mycobacterium xenopi (1 animal). No animals were positive by the tuberculin test. Both M. avium subsp. hominissuis-positive pigs were evaluated as positive by the IFN-γ release assay. Bacteriologically negative and M. xenopi-positive pigs were unresponsive in the IFN-γ release assay, indicating the specificity of the method. The results suggest that the IFN-γ release assay has a higher sensitivity than the tuberculin test and that the assay can be used for diagnosis of M. avium infections in live, naturally infected pigs.