Quantitation of cytokine mRNA by real-time RT-PCR during a vaccination trial in a rabbit model of fascioliasis

Use of the rabbit as disease model has long been hampered by a lack of immunological assays specific to this species. In the present study we developed a SYBR Green-based, real-time RT-PCR protocol to quantitate cytokine mRNA in freshly harvested rabbit peripheral mononuclear cells. The method was validated in the course of a vaccination trial in which animals vaccinated with the recombinant antigen FhSAP2 were challenged with Fasciola hepatica metacercariae. Changes in the levels of rabbit interleukin (IL)-2, IL-4, IL-6, IL-10, tumor necrosis factor-alpha (TNFα), and interferon-gamma (IFNγ) mRNA were determined. Messenger RNA from the universally expressed housekeeping gene GAPDH was used as an amplification control and allowed for correction of variations in the efficiencies of RNA extraction and reverse transcription. Rabbits vaccinated with FhSAP2 showed an 83.3% reduction in liver fluke burden after challenge infection when compared to non-vaccinated controls. All cytokine mRNAs were found at detectable levels; however, the levels of IFNγ, TNFα, IL-2 and IL-10 were significantly higher in the vaccinated group compared to the non-vaccinated group. These results suggest that protection conferred by FhSAP2 protein could be associated with a mixed Th1/Th2 immune response in which Th1 cytokines are dominant. The real-time RT-PCR method described herein can be a useful tool for monitoring changes in basic immune functions in the rabbit model of fascioliasis and may also aid in studies of human diseases for which the rabbit is an important experimental model.