Location: Infectious Bacterial Diseases ResearchTitle: Development and Evaluation of a Real-time Reverse Transcription-PCR Assay for Quantification of Gamma Interferon mRNA to Diagnose Tuberculosis in Multiple Animal Species) Author
Submitted to: Clinical and Vaccine Immunology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 10/7/2007
Publication Date: 12/1/2007
Citation: Harrington, N.P., Surujballi, O.P., Waters, W.R., Prescott, J.F. 2007. Development and Evaluation of a Real-time Reverse Transcription-PCR Assay for Quantification of Gamma Interferon mRNA to Diagnose Tuberculosis in Multiple Animal Species. Clinical and Vaccine Immunology. 14(12):1563-1571. Interpretive Summary: Captive deer are included in the Uniform Methods and Regulations for the eradication of bovine tuberculosis within the United States. In the past, tuberculosis has been spread from infected captive deer to cattle and bison. The current approved method for detecting tuberculous deer is the skin test. Improved diagnostic methods are urgently needed as skin testing procedures are not widely accepted by the cervid industry. In this study, three blood-based tests were evaluated for potential use as an initial tuberculosis screening tool with samples from red deer/elk. Results from this study demonstrate the development and potential application of a new blood based test for the detection of tuberculous deer. Further studies with field samples are required for definitive evaluation. These findings are useful for the continued development of improved control strategies for bovine tuberculosis, particularly in countries with significant captive deer industries.
Technical Abstract: Tuberculosis (TB) 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 TB 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. Recently, in vitro diagnostic tests, in particular, interferon-gamma (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 from several TB susceptible target species by quantitative real-time RT-PCR. Oligonucleotide primers and probes were designed and tested for reactivity towards several species of interest with respect to TB infection. The assay was subsequently optimized to quantify elk and deer (Cervus elaphus) IFN-gamma mRNA expression and was evaluated for its ability to detect mycobacterial antigen specific responses of experimentally TB-infected deer. 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 superior performance at determining an animal’s infectious status than either lymphocyte proliferation or IFN-gamma ELISA methods. An additional advantage is the ease with which the assay can be modified to reliably quantify the IFN-gamma expression using consensus sequences of closely related species or of other species for which sequence information is available.