This Article 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.

 Previous Article  |  Next Article 

Clin. Vaccine Immunol. doi:10.1128/CVI.00263-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Development and evaluation of a multi-species real-time RT-PCR assay for interferon-gamma mRNA detection of tuberculosis infection

Canadian Food Inspection Agency, Ottawa Laboratory Fallowfield, Ottawa, Ontario, Canada; Department of Pathobiology, University of Guelph, Guelph, Ontario, Canada; National Animal Disease Center, United States Department of Agriculture, Ames, IA, USA

^* To whom correspondence should be addressed. Email: harringtonnp{at}inspection.gc.ca.

	Abstract

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 many wildlife species. Increasingly, in vitro diagnostic tests including interferon-gamma (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 RT-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 elk and red deer (Cervus elaphus) IFN- mRNA expression 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 superior performance in determining an animal's infection status over either lymphocyte proliferation or IFN- protein ELISA methods. An additional advantage is the ease with which the assay can be modified to reliably quantify the IFN- expression using consensus sequences of closely related species or of other species for which IFN- sequence information is available.