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-␥)-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 wholeblood 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.Mycobacterium bovis, the etiological agent of bovine tuberculosis (TB), has an extraordinarily broad mammalian host range that includes humans, domestic livestock, and wildlife. Globally, several wildlife species have been implicated in the maintenance and transmission of M. bovis: the badger (Meles meles) in Great Britain and Ireland (1), the brushtail possum (Trichosurus vulpecula) in New Zealand (2), white-tailed deer (Odocoileus virginianus) in Michigan (28), elk (Cervus elaphus) in Manitoba, Canada (17), and buffalo (Syncerus caffer) and kudu (Tragelaphus strepsiceros) in southern Africa (15). These wildlife hosts are difficult to control, and the transmission of M. bovis to domestic livestock may result in regulatory action, movement restrictions, or isolation of infected individuals and may become a significant barrier to the success of national eradication and control programs. Transmission among freeranging and captive wildlife also creates problems in the management of zoological collections; the risk of infection to other valuable animals is increased, directly affecting the value of the collection, and in some cases, the value of tourism or related economic benefits to a country (15).New control str...

High-Level Cellular and Humoral Immune Responses in Guinea Pigs Immunized Intradermally with a Heat-Inactivated Varicella-Zoster Virus Vaccine

“…Importantly, the i.d. delivery of a 1,995-PFU dose of heat-inactivated VZV vaccine induced a significantly higher level of IFN-␥ mRNA in stimulated splenocytes than the same vaccine given s.c. Splenocytes from all animals expressing IFN-␥ mRNA produced IFN-␥ protein with a correlation coefficient similar to that obtained in subjects with active tuberculosis (31) or animals infected with M. bovis (30).…”

“…VZV-specific CMI assays have also determined the IFN-␥ protein production of immune cells, i.e., the VZV responder cell frequency by enzyme-linked immunospot assays, flow cytometry (7,27), or IFN-␥ ELISA of supernatants of ex vivo-stimulated lymphocytes (28). The qRT-PCR assay allows highly sensitive testing of the mRNA expression of selected genes of importance in humans (29) and animals (30). Measurements of the expression of the mRNAs for IFN-␥, granzyme B, and perforin, which are encoded by the important cellular genes controlling CMI, do not appear to have been utilized in evaluations of CMI responses to VZV vaccine candidates administered in various forms and by various routes.…”

Facts and dilemmas in diagnosis of tuberculosis in wildlife

“…Other BTB diagnostic methods, which are more laboratory based methods, include the lymphocyte transformation assay [69,71] and the measurement of production of nitric oxide (NO) or Tumor Necrosis Factor (TNF) α to measure macrophage activity [157]. The production of NO in response to antigen specific stimulation of PBMC of M. bovis infected white-tailed deer gave promising results [158], but additional studies to measure NO responses of more species, or follow-up studies, are lacking.…”

“…Like the skin test, the IFN-γ release assay has time limits because of decreasing CMI responsiveness during progression of disease, as was also shown in deer [71]. Its test result is known to be influenced by the time lapse between collection of samples and their processing [115,116], though samples may be potentiated by addition of IL-12 [117].…”

“…It can also be easily applied to non-related species if IFN-γ sequence information is available [71,124]. The qRT-PCR is a laboratory based technique and compared to other assays in this review, relatively advanced technical equipment is needed.…”