Rapid recontamination of a farm building occurs after attempted prion removal.

The transmissible spongiform encephalopathy scrapie of sheep/goats and chronic wasting disease of cervids are associated with environmental reservoirs of infectivity. Preventing environmental prions acting as a source of infectivity to healthy animals is of major concern to farms that have had outbreaks of scrapie and also to the health management of wild and farmed cervids. Here, an efficient scrapie decontamination protocol was applied to a farm with high levels of environmental contamination with the scrapie agent. Post-decontamination, no prion material was detected within samples taken from the farm buildings as determined using a sensitive in vitro replication assay (sPMCA). A bioassay consisting of 25 newborn lambs of highly susceptible prion protein genotype VRQ/VRQ introduced into this decontaminated barn was carried out in addition to sampling and analysis of dust samples that were collected during the bioassay. Twenty-four of the animals examined by immunohistochemical analysis of lymphatic tissues were scrapie-positive during the bioassay, samples of dust collected within the barn were positive by month 3. The data illustrates the difficulty in decontaminating farm buildings from scrapie, and demonstrates the likely contribution of farm dust to the recontamination of these environments to levels that are capable of causing disease.

Expression of four S. pneumoniae type 2 polysaccharide biosynthetic enzymes utilising the endogenous Kex2 protease activity in tobacco.

In order to express multisubunit proteins, or to manipulate metabolic pathways in plants it is essential to be able to efficiently express multiple proteins within the same plant cell. To increase the efficiency of multi-protein expression, we demonstrate the use of the Golgi localized Kex2 protease activity in tobacco to process a large polyprotein precursor consisting of four individual protein domains into its individual protein constituents. Four genes encoding enzymes involved in the biosynthesis of S. pneumoniae type 2 polysaccharide were assembled into a single expression cassette as a large polyprotein driven by a single cauliflower mosaic virus (CaMV) 35S promoter. Each of the individual protein domains were separated by three sequential Kex2 protease digestion sites. At the N-terminus a Pr1b signal peptide was incorporated for efficient targeting of the polyprotein to the apoplast. Each individual protein domain was tagged with its own immuno-tag. The construct was used for the transformation of Nicotiana tabacum and stable lines were selected. All four processed proteins could be immunologically detected in protein extracts using Western blotting indicating correct expression and Kex2 processing. Utilisation of the Kex2 protease system represents an efficient way of expressing multiple proteins in the same plant. This method simplifies the transformation procedures, and presents a method for expression of multiple proteins within the same plant.

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

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.