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

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

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.

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.

Test performance of three serological assays for the detection of Mycobacterium bovis infection in common warthogs (Phacochoerus africanus).

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

Modification of the QuantiFERON-TB Gold (In-Tube) assay for the diagnosis of Mycobacterium bovis infection in African buffaloes (Syncerus caffer).

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