Assessing the susceptibility of transgenic mice overexpressing deer prion protein to bovine spongiform encephalopathy.

Several transgenic mouse models have been developed which facilitate the transmission of chronic wasting disease (CWD) of cervids and allow prion strain discrimination. The present study was designed to assess the susceptibility of the prototypic mouse line, Tg(CerPrP)1536(+/-), to bovine spongiform encephalopathy (BSE) prions, which have the ability to overcome species barriers. Tg(CerPrP)1536(+/-) mice challenged with red deer-adapted BSE resulted in 90% to 100% attack rates, and BSE from cattle failed to transmit, indicating agent adaptation in the deer.

The interpretation of disease phenotypes to identify TSE strains in mice: characterisation of BSE using PrPSc distribution patterns in the brain.

In individual animals affected by transmissible spongiform encephalopathies, different disease phenotypes can be identified which are attributed to different strains of the agent. In the absence of reliable technology to fully characterise the agent, classification of disease phenotype has been used as a strain typing tool which can be applied in any host. This approach uses standardised data on biological parameters, established for a single host, to allow comparison of different prion sources. Traditionally prion strain characterisation in wild type mice is based on incubation periods and lesion profiles after the stabilisation of the agent into the new host which requires serial passages. Such analysis can take many years, due to prolonged incubation periods. The current study demonstrates that the PrPSc patterns produced by one serial passage in wild type mice of bovine or ovine BSE were consistent, stable and showed minimal and predictable differences from mouse-stabilised reference strains. This biological property makes PrPSc deposition pattern mapping a powerful tool in the identification and definition of TSE strains on primary isolation, making the process of characterisation faster and cheaper than a serial passage protocol. It can be applied to individual mice and therefore it is better suited to identify strain diversity within single inocula in case of co-infections or identify strains in cases where insufficient mice succumb to disease for robust lesion profiles to be constructed. The detailed description presented in this study provides a reference document for identifying BSE in wild type mice.

The interpretation of disease phenotypes to identify TSE strains following murine bioassay: characterisation of classical scrapie.

Mouse bioassay can be readily employed for strain typing of naturally occurring transmissible spongiform encephalopathy cases. Classical scrapie strains have been characterised historically based on the established methodology of assessing incubation period of disease and the distribution of disease-specific vacuolation across the brain following strain stabilisation in a given mouse line. More recent research has shown that additional methods could be used to characterise strains and thereby expand the definition of strain "phenotype". Here we present the phenotypic characteristics of classical scrapie strains isolated from 24 UK ovine field cases through the wild-type mouse bioassay. PrPSc immunohistochemistry (IHC), paraffin embedded tissue blots (PET-blot) and Western blotting approaches were used to determine the neuroanatomical distribution and molecular profile of PrPSc associated with each strain, in conjunction with traditional methodologies. Results revealed three strains isolated through each mouse line, including a previously unidentified strain. Moreover IHC and PET-blot methodologies were effective in characterising the strain-associated types and neuroanatomical locations of PrPSc. The use of Western blotting as a parameter to define classical scrapie strains was limited. These data provide a comprehensive description of classical scrapie strain phenotypes on isolation through the mouse bioassay that can provide a reference for further scrapie strain identification.

Prion-induced toxicity in PrP transgenic Drosophila.

Prion diseases are fatal transmissible neurodegenerative diseases of humans and various vertebrate species. In their natural hosts these conditions are characterised by prolonged incubation times prior to the onset of clinical signs of terminal disease. Accordingly, tractable models of mammalian prion disease are required in order to better understand the mechanisms of prion replication and prion-induced neurotoxicity. Transmission of prion diseases can occur across a species barrier and this is facilitated in recipients transgenic for the same PrP gene as the individual from which the infectious prions are derived. Here we have tested the hypothesis that exogenous ovine prions can induce neurotoxicity in Drosophila melanogaster transgenic for ovine PrP. Drosophila that expressed ovine PrP pan neuronally and inoculated with ovine prions at the larval stage by oral exposure to scrapie-infected sheep brain homogenate showed markedly accelerated locomotor and survival defects. ARQ PrP transgenic Drosophila exposed to scrapie-infected brain homogenate showed a significant and progressive reduction in locomotor activity compared to similar flies exposed to normal sheep brain homogenate. The prion-induced locomotor defect was accompanied by the accumulation of potentially misfolded PrP in the brains of prion-inoculated flies. VRQ PrP transgenic Drosophila, which expressed less ovine PrP than ARQ flies, showed a reduced median survival compared to similar flies exposed to normal sheep brain homogenate. These prion-induced phenotypic effects were PrP-mediated since ovine prions were not toxic in non-PrP transgenic control flies. Our observations provide the basis of an invertebrate model of transmissible mammalian prion disease.

Paraffin-embedded tissue blot as a sensitive method for discrimination between classical scrapie and experimental bovine spongiform encephalopathy in sheep.

The paraffin-embedded tissue (PET) blot was modified for use as a tool to differentiate between classical scrapie and experimental bovine spongiform encephalopathy (BSE) in sheep. Medulla (obex) from 21 cases of classical scrapie and 6 cases of experimental ovine BSE were used to develop the method such that it can be used as a tool to differentiate between BSE and scrapie in the same way that differential immunohistochemistry (IHC) has been used previously. The differential PET blot successfully differentiated between all of the scrapie and ovine BSE cases. Differentiation was permitted more easily with PET blot than by differential IHC, with accurate observations possible at the macroscopic level. At the microscopic level, sensitivity was such that discrimination by the differential PET blot could be made with more confidence than with differential IHC in cases where the immunohistochemical differences were subtle. The differential PET blot makes use of harsh epitope demasking conditions, and, because of the differences in the way prion protein is processed in different prion diseases, it can serve as a new, highly sensitive method to discriminate between classical scrapie and experimental BSE in sheep.

A retrospective immunohistochemical study reveals atypical scrapie has existed in the United Kingdom since at least 1987.

Atypical scrapie is a relatively recent discovery, and it was unknown whether it was a new phenomenon or whether it had existed undetected in the United Kingdom national flock. Before 1998, the routine statutory diagnosis of transmissible spongiform encephalopathy (TSE) in sheep relied on the presence of TSE vacuolation in the brainstem. This method would not have been effective for the detection of atypical scrapie. Currently, immunohistochemistry (IHC) and Western blot are commonly used for the differential diagnosis of classical and atypical scrapie. The IHC pattern of PrPd deposition in atypical scrapie is very different from that in classical scrapie using the same antibody. It is thus possible that because of a lack of suitable diagnostic techniques and awareness of this form of the disease, historic cases of atypical scrapie remain undiagnosed. Immunohistochemistry was performed on selected formalin-fixed, paraffin-embedded (FFPE) blocks of ovine brain from the Veterinary Laboratories Agency archives that were submitted for various reasons, including suspect neurological disorders, between 1980 and 1989. It was found that PrPd deposits in a single case were consistent with atypical scrapie. A method was developed to obtain a PrP genotype from FFPE tissues and was applied to material from this single case, which was shown to be AHQ/AHQ. This animal was a scrapie suspect from 1987, but diagnosis was not confirmed by the available techniques at that time.