This Article Full Text Full Text (PDF) Other Versions of this Article: CVI.00263-07v1 14/12/1563    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Harrington, N. P. Articles by Prescott, J. F. Search for Related Content PubMed PubMed Citation Articles by Harrington, N. P. Articles by Prescott, J. F.

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.