Analysis Of IFN-γ And IL-4 MRNA Expression In Red Deer Infected With Mycobacterium Bovis By Real-time PCR

Object: This study was to elucidate the immune mechanisms operative in red deer infected with Mycobacterium bovis (M. bovis) and establish the feasibility of using TaqMan Real-time PCR to determine the expression levels of cytokines mRNA in red deer.Methods: Ten red deer, which had been bred from stags resistant to M. bovis, and ten red deer bred from susceptible animals were used in this study. Half of the resistant and half of the susceptible groups of animals, were vaccinated with live bacille Calmette-Guerin (BCG). All of animals had been challenged with virulent M. bovis eight weeks after vaccination. Peripheral blood mononuclear cells (PBMC) were collected four weeks before and six weeks after challenged with virulent M. bovis. Cytokines mRNA expression levels were measured following stimulation of cells with Pokeweed Mitogen (PWM). Interferon-y (IFN-y) and interleukin-4 (IL-4) mRNA expression in PBMC was quantified with TaqMan Real-time PCR using an ABI PRISM 7700 Sequence Detector System (Perkin-Elmer Applied Biosystems).Results: The difference of expression levels of IFN- Y and IL-4 mRNA in PBMC from red deer was statistically significant pre and post challenge with virulent M. bovis. This study had established the feasibility of using TaqMan Real-time PCR to examine the expression levels of cytokines mRNA in PBMC from deer.Conclusions: The complex cellular activation patterns can be demonstrated in red deer infected with M. bovis. Such a response is compatible with mixed Thl,Th2 cell activation. Real-time PCR should be the method of choice for anyexperiments requiring sensitive, specific and reproducible quantification of mRNA.