Evaluation of the infectivity present in white blood cells prepared from scrapie infected sheep by four different methods: PMCA, SCA, Cerebellar Organotypic Slice Culture Assay (COSCA) and inoculation into tg338 mice (bioassay).

Five susceptible VRQ/VRQ sheep were orally challenged with 2 g of brain homogenate (106.6 ID50/g IC in tg338 mice) between 6 and 10 months of age. The five VRQ/VRQ sheep respectively died at 198, 193, 198, 194 and 191days post inoculation (dpi). Classical scrapie was confirmed by histopathology (vacuolar change in central nervous system) and detection of abnormal PrP deposit in central nervous system and lymphoid tissues. At different time points whole blood was collected from each donor and aliquots of 108 white blood cells corresponding to 15 mL of plasma were prepared. These cells were tested by in vitro (PMCA), ex vivo (SCA and COSCA) and in vivo (bioassay) approaches in parallel. For PMCA (using tg338 mice brain homogenate as substrate), each sample was run in 5 replicates for 2 successive rounds and the number of positive reactions is presented. For SCA, 4×107 white blood cells were inoculated to ovRK13 cells and abnormal PrP accumulation was assayed 60 days later. For COSCA, 8–10 cerebellar slices prepared from tg338 pups received 106 cells (in 2μL) each and were cultured for 42 days then tested for the presence of abnormal PrP. For bioassay, 108 white blood cells (in 200μL) were inoculated in groups of six tg338 mice. Mice were monitored up to occurrence of TSE compatible clinical sign onset or killed after 250 days post inoculation. All mice were tested for presence of abnormal PrP deposition in brain. Incubation period in mice are presented in days (+/−SD). When less than 3 mice were positive, individual incubation periods are given. Not done: n.d.