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

Abstract

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.

“Spotting” Mycobacterium bovis infection in leopards (Panthera pardus) – novel application of diagnostic tools

“…This study demonstrated that existing assays, previously evaluated for TB diagnosis in African lions ( 12 , 16 , 18 , 31 ) and cheetahs ( 13 ), showed potential for detecting M. bovis -infected leopards. The GXU ® was able to detect MTBC DNA in all five culture positive leopards that were tested, providing rapid results in a significantly shorter time than mycobacterial culture.…”

“…Medians and inter-quartile ranges are indicated by horizontal bars. The lion assay cut-off value (33 pg/ml) is shown as a dotted line on the y-axis ( 18 ). There was a statistically significant difference between the test results (p = 0.0001).…”

“…DY1796), and the Mabtech Cat IFN-γ ELISA Basic kit (catalogue no. 3122-1H-20; Mabtech AB, Nacka Strand, SE-131 28, Sweden) were evaluated to determine if native leopard cytokines could be detected in stimulated whole blood plasma as previously shown in cheetah ( 13 ) and lions ( 18 ). A four-parameter logistic (4PL) regression analysis was performed using GraphPad Prism 7 for Windows (version 7.04, GraphPad Software, Inc., San Diego, CA 92108, USA; www.graphpad.com ).…”

“…The differences between unstimulated and mitogen responses for the three cytokine release assays were evaluated using a one-tailed paired Student t-test. The infection status of leopards based on QFT Mabtech Cat IGRA results was determined using the previously calculated African lion cut-off value (33 pg/ml) ( 18 ).…”

“…Previous studies have shown the upregulation of the C-X-C motif ligand 9 ( CXCL9 ) gene to be a sensitive diagnostic biomarker for M. bovis infection in African lions and a single cheetah ( 16 , 17 ). Cytokine release assays (CRA) have also shown diagnostic potential in cheetahs and African lions ( 13 , 18 ). Therefore, the aim of this study was to evaluate immunological assays, previously validated in African lions and cheetahs, to identify potential TB diagnostic tests for leopards.…”