Katy E. Beck

Isolation of Prion with BSE Properties from Farmed Goat

BSE can infect small ruminants and could be misdiagnosed as scrapie.

Ovine PrP genotype is linked with lesion profile and immunohistochemistry patterns after primary transmission of classical scrapie to wild-type mice.

It is currently believed that primary transmission of classical scrapie to wild-type mice is inefficient and characterized by low attack rates and variable incubation periods and lesion profiles. Consequently, strain characterization of classical scrapie in these mice relies on subpassage. The aim of this study was to perform a retrospective analysis of lesion profiles and immunohistochemistry patterns after transmission of a large number of classical scrapie sources to wild-type mice and to investigate trends that might be used to characterize the agent without subpassaging. Scrapie field cases (n = 31) collected from individual farms between 1996 and 1999 were inoculated into RIII, C57BL, and VM mice and profiled using standard methodology and analyzed by immunohistochemistry. Using cluster analysis to resultant lesion profiles produced groups of similar lesion profiles in RIII and C57BL mice. We observed correlations between lesion profile clusters and the ovine prion protein (PrP) genotype. Immunohistochemistry indicated donor-mediated trends in the PrPSc pattern. These results indicate that ovine PrP genotype is a factor that is linked to both the lesion profile and the pattern of PrPSc deposition on primary transmission of classical scrapie to wild-type mice.

Lesion Profiling at Primary Isolation in RIII Mice is Insufficient in Distinguishing BSE from Classical Scrapie

Primary isolation of bovine spongiform encephalopathy (BSE) in RIII mice generates a lesion profile believed to be reproducible and distinct from that produced by classical scrapie. This profile, which is characterized by peaks at gray matter areas 1, 4 and 7 (dorsal medulla, hypothalamus and septal nuclei), is used to diagnose BSE on primary isolation. The aim of this study was to investigate whether the BSE agent could be present in sheep diagnosed with classical scrapie, using lesion profiles in RIII mice as a discriminatory method. Sixty‐two positive scrapie field cases were collected from individual farms between 1996 and 1999 and bioassayed in RIII mice. Fifty‐five of these isolates transmitted successfully to at least one mouse. Of the 31 that produced adequate data to allow lesion profile analysis, 10 showed a consistent profile with peaks at brain areas 1, 4 and 7. All inocula for this subgroup were derived from sheep of genotype ARQ/ARQ. While the 1‐4‐7‐scrapie profile exhibited similarities to BSE in RIII mice at primary isolation, it was distinguishable based on histopathology, immunohistochemistry and cluster analysis. We conclude that caution should be taken to distinguish this profile from BSE and that additional parameters should be considered to reach a final diagnosis.

Use of murine bioassay to resolve ovine transmissible spongiform encephalopathy cases showing a bovine spongiform encephalopathy molecular profile.

Two cases of unusual transmissible spongiform encephalopathy (TSE) were diagnosed on the same farm in ARQ/ARQ PrP sheep showing attributes of both bovine spongiform encephalopathy (BSE) and scrapie. These cases, UK‐1 and UK‐2, were investigated further by transmissions to wild‐type and ovine transgenic mice. Lesion profiles (LP) on primary isolation and subpassage, incubation period (IP) of disease, PrPSc immunohistochemical (IHC) deposition pattern and Western blot profiles were used to characterize the prions causing disease in these sheep. Results showed that both cases were compatible with scrapie. The presence of BSE was contraindicated by the following: LP on primary isolation in RIII and/or MR (modified RIII) mice; IP and LP after serial passage in wild‐type mice; PrPSc deposition pattern in wild‐type mice; and IP and Western blot data in transgenic mice. Furthermore, immunohistochemistry (IHC) revealed that each case generated two distinct PrPSc deposition patterns in both wild‐type and transgenic mice, suggesting that two scrapie strains coexisted in the ovine hosts. Critically, these data confirmed the original differential IHC categorization that these UK‐1 and UK‐2 cases were not compatible with BSE.

Evidence for Co-infection of Ovine Prion Strains in Classical Scrapie Isolates

The diversity of strains of ovine prions within classical scrapie isolates was investigated by transmission studies in wild type mice. To determine the maximum diversity of prion strains present in each ovine scrapie isolate examined, isolates from mice having the shortest and longest incubation times for terminal disease after primary inoculation were passaged serially. Serial passage of ARQ/ARQ scrapie isolates in RIII mice revealed the ME7 prion strain in mice with short incubation times for terminal prion disease and the 87A strain in those mice with long incubation times. Serial passage of VRQ/VRQ scrapie isolates in RIII mice led to emergence of the 221C prion strain in mice with short incubation times and a variant of the 221C strain in those mice with long incubation times. RIII mice with short incubation times had higher levels of total and proteinase K-resistant PrP(Sc) compared with those RIII mice with long incubation times, while mice with long incubation times had large aggregates and plaques of PrP(Sc). ME7 PrP(Sc) differed in stability compared with the 87A prion strain, while PrP(Sc) associated with 221C had similar stability to that of the 221C variant. Serial passage in VM mice led to identification of ME7 and 87V in the same scrapie isolate. The data show that different prion strains can emerge from the same ovine scrapie isolate following serial passage in wild type mice and that the transmission properties of these strains correlate with distinct patterns of PrP(Sc) deposition.

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.

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.

Disease characteristics of bovine spongiform encephalopathy following inoculation into mice via three different routes

Mouse‐adapted transmissible spongiform encephalopathy (TSE) strains are routinely distinguished based on reproducible disease characteristics in a given mouse line following inoculation via a consistent route. We investigated whether different administration routes (oral, intragastric (i.g.) and intracerebral (i.c.)) can alter the disease characteristics in IM mice after serial dilution of a stabilized mouse‐adapted bovine spongiform encephalopathy (BSE) strain (301V). In addition, the infectivity of distal ileum and mesenteric lymph nodes (ln) sampled at three time points (35 days postinoculation (dpi), 70 dpi and terminal disease) after i.g. inoculation of 301V strain was assessed in mice by i.c. challenge. Strain characteristics were assessed according to standard methodology and PrPSc immunohistochemistry deposition patterns. Mean incubation periods were prolonged following oral or i.g. inoculations compared to the i.c. route. Lesion profiles following i.c. challenges were elevated compared to i.g. and oral routes although vacuolation in the dorsal medulla was consistently high irrespective of the route of administration. Nevertheless, the same PrPSc deposition pattern was associated with each route of administration. Distal and mesenteric ln infectivity was detected as early as 35 dpi and displayed consistent lesion profiles and PrPSc deposition patterns. Our data suggest that although 301V retained its properties, some phenotypic parameters were affected by the route of inoculation. We conclude that bioassay data should be interpreted carefully and should be standardized for route of inoculation.

Assessing the Susceptibility of Transgenic Mice Overexpressing Deer Prion Protein to Bovine Spongiform Encephalopathy

ABSTRACT 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.

Strain typing of classical scrapie by transgenic mouse bioassay using protein misfolding cyclic amplification to replace primary passage.

According to traditional murine bioassay methodology, prions must be serially passaged within a new host before a stable phenotype, and therefore a strain, can be assigned. Prions often transmit with difficulty from one species to another; a property termed the transmission barrier. Transgenic mouse lines that over express prion protein (PrP) genes of different species can circumvent the transmission barrier but serial passages may still be required, particularly if unknown strains are encountered. Here we sought to investigate whether protein misfolding cyclic amplification (PMCA), an in-vitro method of PrPSc replication, could be used to replace serial passage of VRQ/VRQ classical scrapie isolates undergoing strain typing in ovine transgenic tg338 mice. Two classical scrapie field isolates that do not readily transmit to wild-type mice underwent bioassay in tg338 mice pre- and post- PMCA and the phenotype of disease in inoculated mice was compared. For one of the sources investigated, the PMCA product gave rise to the same disease phenotypes in tg338 mice as traditional bioassay, as indicated by lesion profile, IHC analysis and Western blot, whilst the second source produced phenotypic characteristics which were not identical with those that arose through traditional bioassay. These data show that differences in the efficiency of PMCA as a strain-typing tool may vary between ovine classical scrapie isolates and therefore suggest that the ability of PMCA to replace serial passage of classical scrapie in tg338 mice may depend on the strain present in the initial source.