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.

FIG. 1.

Typical IFN-γ real-time RT-PCR amplification curves from multiple target species. Real-time PCR was performed on cDNA obtained from mitogen-stimulated PBMCs of the following species: bovine (Bos taurus), ovine (Ovis aries), caprine (Capra hircus), bison (Bison bison), red deer (Cervus elaphus elaphus), elk (Cervus elaphus manitobensis), white-tailed deer (WTD) (Odocoileus virginianus), reindeer (Rangifer tarandus), and llama (Llama glama). The amplification curves showed positive reactions for all tested animals with the exception of llamas. The y axis indicates the absolute emission intensity (delta RFU indicates relative fluorescence units subtracted by the background fluorescence signal); the x axis shows the number of PCR cycles. NTC, no template control.

FIG. 2.

qRT-PCR analysis of Cervus elaphus IFN-γ gene expression kinetics from PBMCs stimulated with tuberculin (PPD-bovis). Isolated PBMCs from six M. bovis-infected deer were stimulated with PPD-bovis (10 μg/ml), and RNA was extracted at 0, 2, 4, 8, 16, 24, 48, 72, and 96 h for analysis by qRT-PCR. The results are expressed as box plots of mRNA expression relative to nonstimulated cells after normalization against the β2M housekeeping gene. Shown are the 25% to 75% response ranges (top and bottom lines of boxes) and minima and maxima (whiskers). Asterisks indicate responses by M. bovis-infected deer that differ (P < 0.01) from the respective responses of noninfected control deer.

FIG. 3.

Lymphocyte proliferation (A), IFN-γ protein (B), and IFN-γ mRNA (C) responses to PPD-bovis. Infected cervids (n = 10) were inoculated by intratonsillar instillation of 1.5 × 10³ CFU of M. bovis, whereas control deer (n = 5) received no inoculation. Heparinized blood samples were obtained at the indicated time points, and isolated PBMCs (lymphoproliferation) or whole-blood cells (IFN-γ assays) were stimulated with mycobacterial antigen (PPD-bovis) or received no stimulation. The LPA sample was incubated for 5 days, and the blood cultures were incubated for 16 h and 48 h for qRT-PCR and ELISA, respectively. Shown are the medians (solid lines in boxes), 25 to 75% response ranges (top and bottom lines of boxes), and minima and maxima (whiskers). CCT indicates skin testing performed at 184 days p.i. Asterisks indicate responses by M. bovis-infected deer that differ (P < 0.05) from the respective responses of noninfected control deer. Double asterisks indicate that responses of M. bovis-infected deer post-CCT were greater (P < 0.05) than the respective responses of noninfected control deer and greater than the immediate pre-CCT response (171 days p.i.).

FIG. 4.

Immunoassay classification of cervids experimentally infected with M. bovis. Heparinized blood samples were obtained at the indicated time points, and isolated PBMCs (lymphoproliferation) or whole-blood cells (IFN-γ assays) were stimulated with mycobacterial antigen (PPD-bovis) or received no stimulation. The LPA sample was incubated for 5 days, and the blood cultures were incubated for 16 h and 48 h for qRT-PCR and ELISA, respectively. The CMI-based assays were performed at 11 time points over the course of a 7-month infection study. CCT indicates skin testing performed at 184 days p.i. Results are presented as the percentage of animals classified as M. bovis reactive by criteria outlined in Materials and Methods.