Optimisation of the lion (Panthera leo) specific interferon gamma assay for detection of tuberculosis in lions in South Africa

Abstract

Mycobacterium bovis is the causative agent of bovine tuberculosis (BTB) which has a diverse host range. The maintenance host of BTB in South Africa is the African buffalo (Syncerus caffer). It is believed that lions get infected by feeding on infected buffalo or through wounds. The spread of the disease amongst lions has raised concern regarding the future of the animals and the impact on tourism in the country. Diagnoses of tuberculosis in free ranging wildlife is often dependent on post-mortem samples due to logistical challenges, the use of the lion specific interferon gamma release assay as an antemortem test offers a simpler methodology to testing live animals. The aim was to optimise an already developed assay by Maas et al.,2012 and to harmonise it with the Rhinoceros specific interferon gamma assay developed by Morar-Leather et al 2007. Optimisation of the interferon gamma specific ELISA included: determination of optimal concentrations for the capture and detection monoclonal antibodies; optimal concentrations for the conjugate and evaluation of alternative blocking agents. Different mitogens and incubation times were evaluated for the stimulation of whole blood as positive control in the assay. The optimum concentration for coating the plates with the capture monoclonal antibody was 2 g/ml. An optimum dilution of 1:5000 was selected for both the biotinylated detection monoclonal antibody and the streptavidin horseradish peroxidase conjugate. The assay was optimised using recombinant lion interferon gamma and the lower detection limit was calculated to be 109 pg/ml. Phosphate buffered saline with 1% bovine serum albumin was found to be Chapter 1 © University of South Africa iii a suitable blocking agent. Native interferon gamma was detected in whole blood samples from 5 lions and a 24 hour incubation time with PMA and ionomycin was selected as the optimal mitogen positive control. This assay system demonstrated good potential as an ante mortem test for the diagnosis of tuberculosis in lions. In conclusion, the assay can detect IFN- from supernatants harvested from whole blood cultures stimulated with specific antigens and mitogens