title: Development and evaluation of a real-time reverse transcription-PCR assay for quantification of gamma interferon mRNA to diagnose tuberculosis in multiple animal species.
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 use with many wildlife species. Increasingly, in vitro diagnostic tests, including gamma interferon (IFN-gamma)-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-gamma 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-gamma 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-gamma mRNA responses correlated well with IFN-gamma protein production and showed performance in determining an animal's infection status superior to that of either lymphocyte proliferation or IFN-gamma protein enzyme-linked immunosorbent assay methods. An additional advantage is the ease with which the assay can be modified to reliably quantify IFN-gamma expression by using consensus sequences of closely related species or of other species for which IFN-gamma sequence information is available.
authors.affiliation: Ottawa Laboratory Fallowfield, Canadian Food Inspection Agency, 3851 Fallowfield Road, Ottawa, Ontario K2H 8P9, Canada. harringtonnp@inspection.gc.ca
authors.forename: Noel P
authors.initials: NP
authors.lastname: Harrington
authors.forename: Om P
authors.initials: OP
authors.lastname: Surujballi
authors.forename: W Ray
authors.initials: WR
authors.lastname: Waters
authors.forename: John F
authors.initials: JF
authors.lastname: Prescott
mesh_terms: D000818:Animals; D000835:Animals, Wild; D000866:Antelopes; D016164:Bison; D002020:Buffaloes; D002161:Camelids, New World; D002417:Cattle; D002478:Cells, Cultured; D017931:DNA Primers / Q000737:chemistry; D015342:DNA Probes / Q000737:chemistry; D018076:DNA, Complementary / Q000096:biosynthesis; D003670:Deer; D004797:Enzyme-Linked Immunosorbent Assay; D006041:Goats; D007371:Interferon-gamma / Q000032:analysis* / Q000276:immunology; D007700:Kinetics; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000276:immunology; D045969:Mustelidae; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000032:analysis* / Q000378:metabolism; D012053:Reindeer; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity; D012756:Sheep; D013997:Time Factors; D048330:Trichosurus; D014376:Tuberculosis / Q000097:blood / Q000175:diagnosis* / Q000276:immunology / Q000662:veterinary*
chemical_list: D017931:DNA Primers; D015342:DNA Probes; D018076:DNA, Complementary; D012333:RNA, Messenger; D012313:RNA; D007371:Interferon-gamma