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Clinical and Vaccine Immunology, December 2007, p. 1563-1571, Vol. 14, No. 12
1071-412X/07/$08.00+0     doi:10.1128/CVI.00263-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Development and Evaluation of a Real-Time Reverse Transcription-PCR Assay for Quantification of Gamma Interferon mRNA To Diagnose Tuberculosis in Multiple Animal Species

Canadian Food Inspection Agency, Ottawa Laboratory Fallowfield, Ottawa, Ontario, Canada,¹ Department of Pathobiology, University of Guelph, Guelph, Ontario, Canada,² National Animal Disease Center, United States Department of Agriculture, Ames, Iowa³

Received 27 June 2007/ Returned for modification 8 August 2007/ Accepted 7 October 2007

Tuberculosis of free-ranging and captive wildlife, including species implicated in the maintenance and transmission of Mycobacterium bovis, is a difficult disease to diagnose and control. Historically, diagnosis of tuberculosis has relied largely upon assays of cell-mediated immunity (CMI), such as tuberculin skin testing. This approach, however, is problematic or impractical for use with many wildlife species. Increasingly, in vitro diagnostic tests, including gamma interferon (IFN-)-based assays, are replacing or complementing skin testing of cattle and humans. Analogous assays are unavailable for most wildlife because of a lack of species-specific immunological reagents. This report describes the development and validation of a whole-blood assay to quantify antigen-specific IFN- mRNA expression by quantitative real-time reverse transcription-PCR. Oligonucleotide primers and probes were designed and tested for reactivity towards several susceptible species of interest with respect to tuberculosis infection. The assay was subsequently optimized to quantify the IFN- mRNA expression in elk and red deer (Cervus elaphus) and was evaluated for its ability to detect mycobacterial antigen-specific responses of experimentally tuberculosis-infected animals. The assay was a simple, rapid, and sensitive measure of antigen-specific CMI. The IFN- mRNA responses correlated well with IFN- protein production and showed performance in determining an animal's infection status superior to that of either lymphocyte proliferation or IFN- protein enzyme-linked immunosorbent assay methods. An additional advantage is the ease with which the assay can be modified to reliably quantify IFN- expression by using consensus sequences of closely related species or of other species for which IFN- sequence information is available.

Published ahead of print on 17 October 2007.