Submitted to: Research Workers in Animal Diseases Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 12/4/2006
Publication Date: 12/4/2006
Citation: Harrington, N.P., Waters, W.R., Surujballi, O.P., Prescott, J.F. 2006. Development of a multispecies real-time RT-PCR whole blood assay to detect interferon-gamma response to mycobacterial antigens and evaluation in Mycobacterium bovis-infected elk (Cervus elaphus)[abstract]. Research Workers in Animal Diseases Conference Proceedings. p. 72. Interpretive Summary:
Technical Abstract: Recent outbreaks of Mycobacterium bovis infection in North American wild and captive Cervidae emphasize their role as potential reservoirs of tuberculosis for cattle, wild animals, and humans. Current diagnostic tests lack sufficient sensitivity and specificity for accurate detection of many infected wildlife and non-traditional domestic species. For humans and cattle, in vitro assays (ELISA or ELISPOT) which measure the production of gamma interferon (IFN-gamma) in response to mycobacterial antigens are more sensitive than other tests for tuberculosis. Unfortunately, these assays do not react with many non-traditional domestic and wildlife species. Furthermore, these protein-based assays are difficult to develop because of a lack of immunological reagents. This report describes the development and evaluation of a simple and rapid multispecies whole blood assay for the quantitative measurement of IFN-gamma mRNA response to M. bovis antigens by real-time reverse transcriptase (RT) polymerase chain reaction (PCR). Oligonucleotide primers and an internal fluorogenic probe were designed to cross-react with multiple species and then optimized and validated for use in cervids. The assay was evaluated to determine its potential usefulness in detecting M. bovis infection using experimentally infected elk and was compared to other cellular assays including a commercial cervid IFN-gamma ELISA (Cervigam(tm)). The IFN-gamma primer/probe set detected IFN-gamma mRNA from a variety of target species and demonstrated high sensitivity and reproducibility for cervids. In response to mycobacterial antigens, the production of IFN-gamma mRNA correlated well with lymphocyte blastogenesis and the production of the IFN-gamma protein as detected by ELISA. Real-time RT-PCR measurement of IFN-gamma mRNA is a simple and rapid assay for the ante-mortem diagnosis of tuberculosis which is particularly relevant for wildlife and non-traditional domestic species for which protein based assays are not available.