The Equine Immune Response to Corynebacterium pseudotuberculosis : The Cytokine Response
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Pigeon fever is a deadly affliction affecting horses and has three forms: internal abscesses, external abscesses, and ulcerative lymphangitis. As with leprosy, where TH1 and TH2 immunity determines the outcome of the disease, we hypothesize that Pigeon fever is manifested as external abscesses when a TH1 response is activated while internal abscesses result from a TH2 response. Blood was collected from infected and control horses in the field, lyphocytes/monocytes were extracted and stimulated with a bacterial toxoid and appropriate controls for 48 hours, and cells were harvested. RNA was then extracted and samples were screened for relative concentrations of cytokine RNA using TaqMan qPCR. The cytokine signatures observed reveal which CD4<sup>+</sup> cell populations are present at the time of infection and blood draw. In pilot testing, uninfected horses were tested at varying concentrations with assorted mitogens in an effort to optimize the assay. We achieved optimal cell stimulation and minimal cell death using 4 μg/ml PHA. We also discovered that RNA in our cytokine profile didn?t always increase after stimulation and actually decreased for some cytokines including IFN-γ and IL-10. This is expected because only uninfected control horses were tested initially. There was little to no amplification of the IL-12 p40 RNA in all three experimental trials, which either indicates a problem with stimulation or with the real-time PCR assay. Finally, the small but definite increases in Treg RNA in most infected horses indicates that this third helper T cell population might play an important role in disease outcome.