Three-colour flow cytometric detection of PrP in ovine leukocytes.

PrP(c) (cellular prion protein, CD230) expression by subpopulations of lymphoid cells has been widely investigated in a variety of species, possibly because of the possible link between transmissible spongiform encephalopathies (TSE) transmission and blood transfusion. However, the role of the immune cells in the transmission of the disease is still unclear. Here we describe the optimisation and standardisation of a three-colour staining procedure to detect PrP in association with phenotypic and activation markers in ovine immune cells. We demonstrate a reproducible, flexible and sensitive method and that the combination of isotype-specific antibodies and Fab fragments is feasible. To our knowledge, this is the first report of such labelling of ovine cells. Using this method, we were able to detect differences in levels of PrP expression between blood and lymph node cells of the same animal, and considerable variability between animals. Moreover, we were able to explore possible associations between PrP expression and cellular activation and to identify cell subsets with different labelling patterns. We are currently employing this approach to evaluate variations in immunological parameters during experimental infection in sheep.

CD21 B cell populations are altered following subcutaneous scrapie inoculation in sheep.

In order to gain a better understanding of the pathogenesis of scrapie in sheep an experimental model was developed to characterise immune system cells in the minutes following inoculation with scrapie-brain homogenate. Four 1-year-old susceptible (ARQ/ARQ) sheep were inoculated via the subcutaneous route at four different peripheral lymph node (LNs) drainage sites, at specific time points, prior to euthanasia of the sheep. The LNs were removed post-mortem at 30, 90, 180 and 300min after inoculation. Flow cytometric triple-labelling was carried out on the LN cells and indicated that inoculation of scrapie-brain homogenate adjacent to a lymph node may delay or even inhibit the number of host CD21(+) B cells expressed within the first 5h. Immunohistochemistry was used to attempt detection of the abnormal form of prion protein (PrP(sc)) in draining LNs adjacent to inoculation sites, with negative results at those time points.