Assessment of classical scrapie infectivity in sheep embryos

Naturally scrapie infected VRQ/VRQ (n = 47) and ARR/ARR control (n = 11) Romanov sheep were super-ovulated and mated with matching genotype rams.Embryos were collected in dams 6 to 7 days after the oestrus and embryos were selected according to guidelines from the International Embryo Technology Society.A total of 267 transferable and 149 non-transferable embryos were collected in the VRQ/VRQ dams and 55 transferable and 23 non-transferable embryos were collected in ARR/ARR controls.The presence of prion seeding activity in non-transferable and transferable embryos from each dam was tested by Protein Misfolding Cyclic Amplification.After four amplification rounds, none of the reactions seeded with embryos displayed detectable levels of abnormal PrP.In contrast, Protein Misfolding Cyclic Amplification reactions seeded with a 10 -8 diluted 10% brainstem homogenate from a VRQ/VRQ infected dam were found to be PrP res positive.Among the 267 transferable VRQ/VRQ embryos, 204 embryos collected from 19 different VRQ/VRQ infected dams were inoculated to ovine PrP transgenic mice (tg338 mice) by intracerebral route.Nineteen embryos from two ARR/ARR dams were inoculated as controls.No clinical signs indicative of Transmissible Spongiform Encephalopathy and no PrP Sc accumulation were observed in any of the tg338 mice inoculated with embryos.Within the limit of the experiment (intrinsic sensitivity of the bioassay and Protein Misfolding Cyclic Amplification) these results indicate that the residual risk of the presence of detectable infectivity or positive seeding activity in transferable embryos from other VRQ/VRQ sheep that would be infected by the Langlade scrapie agent is lower than 1.79% and 1.37%, respectively (upper bound of the exact binomial 95% confidence interval).