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Open Access Research article

In vitro amplification of ovine prions from scrapie-infected sheep from Great Britain reveals distinct patterns of propagation

Leigh Thorne1, Thomas Holder1, Andrew Ramsay1, Jane Edwards1, Maged Mohamed Taema2, Otto Windl1, Ben Charles Maddison3, Kevin Christopher Gough2 and Linda Ann Terry14*

Author Affiliations

1 Animal Health Veterinary Laboratories Agency (AHVLA), Woodham Lane, New Haw, Addlestone, Surrey, KT15 3NB, UK

2 School of Veterinary Medicine and Science, The University of Nottingham, Sutton Bonington Campus, College Road, Sutton Bonington, Leicestershire, LE12 5RD, UK

3 ADAS UK, School of Veterinary Medicine and Science, The University of Nottingham, Sutton Bonington Campus, College Road, Sutton Bonington, Leicestershire, LE12 5RD, UK

4 Current address: Department of Immunology and Pathology, CMU, University of Geneva, 1 Rue Michel Servet, Geneva 4, 1211, Switzerland

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BMC Veterinary Research 2012, 8:223  doi:10.1186/1746-6148-8-223

Published: 15 November 2012

Abstract

Background

Protein 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.

Results

PrPSc 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.

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.

Keywords:
Prions; Transmissible spongiform encephalopathy; Scrapie; Sheep; PMCA