Abstract
BackgroundProtein misfolding cyclic amplification (PMCA) is a method that facilitates the detection of prions from many sources of transmissible spongiform encephalopathy (TSE). Sheep scrapie represents a unique diversity of prion disease agents in a range of susceptible PRNP genotypes. In this study PMCA was assessed on a range of Great Britain (GB) sheep scrapie isolates to determine the applicability to veterinary diagnosis of ovine TSE.ResultsPrPSc amplification by protein misfolding cyclic amplification (PMCA) was assessed as a diagnostic tool for field cases of scrapie. The technique was initially applied to thirty-seven isolates of scrapie from diverse geographical locations around GB, and involved sheep of various breeds and PRNP genotypes. All samples were amplified in either VRQ and/or ARQ PrPC substrate. For PrPSc from sheep with at least one VRQ allele, all samples amplified efficiently in VRQ PrPC but only PrPSc from ARH/VRQ sheep amplified in both substrates. PrPSc from ARQ/ARQ sheep displayed two amplification patterns, one that amplified in both substrates and one that only amplified in ARQ PrPC. These amplification patterns were consistent for a further 14/15 flock/farm mates of these sheep. Furthermore experimental scrapie strains SSBP1, Dawson, CH1641 and MRI were analysed. SSBP1 and Dawson (from VRQ/VRQ sheep) amplified in VRQ but not ARQ substrate. MRI scrapie (from ARQ/ARQ sheep) nor CH1641 did not amplify in ARQ or VRQ substrate; these strains required an enhanced PMCA method incorporating polyadenylic acid (poly(A)) to achieve amplification.ConclusionsPrPsc from 52 classical scrapie GB field isolates amplified in VRQ or ARQ or both substrates and supports the use of PMCA as a rapid assay for the detection of a wide range of ovine classical scrapie infections involving multiple PRNP genotypes and scrapie strains.
Highlights
Protein misfolding cyclic amplification (PMCA) is a method that facilitates the detection of prions from many sources of transmissible spongiform encephalopathy (TSE)
Transmissible spongiform encephalopathies (TSEs) or prion diseases are a group of fatal neurodegenerative disorders which include Creutzfeldt–Jakob disease (CJD) and Kuru in humans, bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep and goats and chronic wasting disease (CWD) in deer and elk
Amplification of classical scrapie field isolates by protein misfolding cyclic amplification (PMCA) Prions derived from brain tissue from sheep infected with classical scrapie of 8 different PRNP genotypes were used to seed PMCA reactions
Summary
Protein misfolding cyclic amplification (PMCA) is a method that facilitates the detection of prions from many sources of transmissible spongiform encephalopathy (TSE). Conclusions: PrPsc from 52 classical scrapie GB field isolates amplified in VRQ or ARQ or both substrates and supports the use of PMCA as a rapid assay for the detection of a wide range of ovine classical scrapie infections involving multiple PRNP genotypes and scrapie strains. PMCA allows propagation of PrPSc in vitro from very small amounts of undetectable seeding material to quantities sufficient for detection by Western blot or plate-based immunoassays This ultra-sensitive method has been previously applied to identify prions in a wide range of tissues and fluids from scrapie-infected sheep (blood, faeces, saliva and milk) where only small amounts of the infectious agent reside [5,6,7,8,9]. Given its unique ability to detect prions in readily accessible tissues and at preclinical stages of the disease it presents a viable method for a live animal, preclinical test for prion disease
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
