Optimisation of the lion (Panthera leo) specific interferon gamma assay for detection of tuberculosis in lions in South Africa
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Authors
Khumalo, Nozipho Lindiwe
Issue Date
2017-02
Type
Dissertation
Language
en
Keywords
Mycobacterium bovis , Interferon gamma , Bovine tuberculosis , ELISA , Lion , Optimisation , Recombinant lion
Alternative Title
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
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© 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