Martin Jeffrey

The transmissible spongiform encephalopathies (TSEs) or prion diseases of animals are characterised by CNS spongiform change, gliosis and the accumulation of disease-associated forms of prion protein (PrP^d). Particularly in ruminant prion diseases, a wide range of morphological types of PrP^d depositions are found in association with neurons and glia. When light microscopic patterns of PrP^d accumulations are correlated with sub-cellular structure, intracellular PrP^d co-localises with lysosomes while non-intracellular PrP^d accumulation co-localises with cell membranes and the extracellular space. Intracellular lysosomal PrP^d is N-terminally truncated, but the site at which the PrP^d molecule is cleaved depends on strain and cell type. Different PrP^d cleavage sites are found for different cells infected with the same agent indicating that not all PrP^d conformers code for different prion strains. Non-intracellular PrP^d is full-length and is mainly found on plasma-lemmas of neuronal perikarya and dendrites and glia where it may be associated with scrapie-specific membrane pathology. These membrane changes appear to involve a redirection of the predominant axonal trafficking of normal cellular PrP and an altered endocytosis of PrP^d. PrP^d is poorly excised from membranes, probably due to increased stabilisation on the membrane of PrP^d complexed with other membrane ligands. PrP^d on plasma-lemmas may also be transferred to other cells or released to the extracellular space. It is widely assumed that PrP^d accumulations cause neurodegenerative changes that lead to clinical disease. However, when different animal prion diseases are considered, neurological deficits do not correlate well with any morphological type of PrP^d accumulation or perturbation of PrP^d trafficking. Non-PrP^d-associated neurodegenerative changes in TSEs include vacuolation, tubulovesicular bodies and terminal axonal degeneration. The last of these correlates well with early neurological disease in mice, but such changes are absent from large animal prion disease. Thus, the proximate cause of clinical disease in animal prion disease is uncertain, but may not involve PrP^d.

The cause of the bovine spongiform encephalopathy (BSE) epidemic in the United Kingdom (UK) was the inclusion of contaminated meat and bone meal in the protein rations fed to cattle. Those rations were not restricted to cattle but were also fed to other livestock including farmed and free living deer. Although there are no reported cases to date of natural BSE in European deer, BSE has been shown to be naturally or experimentally transmissible to a wide range of different ungulate species. Moreover, several species of North America's cervids are highly susceptible to chronic wasting disease (CWD), a transmissible spongiform encephalopathy (TSE) that has become endemic. Should BSE infection have been introduced into the UK deer population, the CWD precedent could suggest that there is a danger for spread and maintenance of the disease in both free living and captive UK deer populations. This study compares the immunohistochemical and biochemical characteristics of BSE and CWD in experimentally-infected European red deer (Cervus elpahus elaphus).

Prion diseases are associated with the accumulation of an abnormal form of the host‐coded prion protein (PrP). It is postulated that different tertiary or quaternary structures of infectious PrP provide the information necessary to code for strain properties. We show here that different light microscopic types of abnormal PrP (PrP^d) accumulation found in each of 10 sheep scrapie cases correspond ultrastructurally with abnormal endocytosis, increased endo‐lysosomes, microfolding of plasma membranes, extracellular PrP^d release and intercellular PrP^d transfer of neurons and/or glia. The same accumulation patterns of PrP^d and associated subcellular lesions were present in each of two scrapie strains present, but they were present in different proportions. The observations suggest that different trafficking pathways of PrP^d are influenced by strain and cell type and that a single prion strain causes several PrP^d–protein interactions at the cell membrane. These results imply that strains may contain or result in production of multiple isoforms of PrP^d.

Background: The variability in the clinical or pathological presentation of transmissible spongiform encephalopathies (TSEs) in sheep, such as scrapie and bovine spongiform encephalopathy (BSE), has been attributed to prion protein genotype, strain, breed, clinical duration, dose, route and type of inoculum and the age at infection. The study aimed to describe the clinical signs in sheep infected with the BSE agent throughout its clinical course to determine whether the clinical signs were as variable as described for classical scrapie in sheep. The clinical signs were compared to BSE-negative sheep to assess if disease-specific clinical markers exist.Results: Forty-seven (34%) of 139 sheep, which comprised 123 challenged sheep and 16 undosed controls, were positive for BSE. Affected sheep belonged to five different breeds and three different genotypes (ARQ/ARQ, VRQ/VRQ and AHQ/AHQ). None of the controls or BSE exposed sheep with ARR alleles were positive. Pruritus was present in 41 (87%) BSE positive sheep; the remaining six were judged to be pre-clinically infected. Testing of the response to scratching along the dorsum of a sheep proved to be a good indicator of clinical disease with a test sensitivity of 85% and specificity of 98% and usually coincided with weight loss. Clinical signs that were displayed significantly earlier in BSE positive cases compared to negative cases were behavioural changes, pruritic behaviour, a positive scratch test, alopecia, skin lesions, teeth grinding, tremor, ataxia, loss of weight and loss of body condition. The frequency and severity of each specific clinical sign usually increased with the progression of disease over a period of 16–20 weeks.Conclusion: Our results suggest that BSE in sheep presents with relatively uniform clinical signs, with pruritus of increased severity and abnormalities in behaviour or movement as the disease progressed. Based on the studied sheep, these clinical features appear to be independent of breed, affected genotype, dose, route of inoculation and whether BSE was passed into sheep from cattle or from other sheep, suggesting that the clinical phenotype of BSE is influenced by the TSE strain more than by other factors. The clinical phenotype of BSE in the genotypes and breed studied was indistinguishable from that described for classical scrapie cases.

The epidemic form of Bovine Spongiform Encephalopathy (BSE) is generally considered to have been caused by a single prion strain but at least two strain variants of cattle prion disorders have recently been recognized. An additional neurodegenerative condition, idiopathic brainstem neuronal chromatolysis and hippocampal sclerosis (IBNC), a rare neurological disease of adult cattle, was also recognised in a sub-set of cattle submitted under the BSE Orders in which lesions of BSE were absent. Between the years of 1988 and 1991 IBNC occurred in Scotland with an incidence of 7 cases per 100,000 beef suckler cows over the age of 6 years.

Bovine spongiform encephalopathy (BSE), a member of the transmissible spongiform encephalopathies (TSE), primarily affects cattle. Transmission is via concentrate feed rations contaminated with infected meat and bone meal (MBM). In addition to cattle, other food animal species are susceptible to BSE and also pose a potential threat to human health as consumption of infected meat products is the cause of variant Creutzfeldt-Jakob disease in humans, which is invariably fatal. In the UK, farmed and free ranging deer were almost certainly exposed to BSE infected MBM in proprietary feeds prior to legislation banning its inclusion. Therefore, although BSE has never been diagnosed in any deer species, a possible risk to human health remains via ingestion of cervine products. Chronic wasting disease (CWD), also a TSE, naturally infects several cervid species in North America and is spreading rapidly in both captive and free-ranging populations.

Scrapie is a prion disease or transmissible spongiform encephalopathy (TSE) of sheep, goats and moufflon. As with its human counterparts, pathology consists of vacuolation, gliosis and accumulations of abnormal forms of a host prion protein (PrP^d) in the brain of affected individuals. Immunohistochemical methods can be used to identify both the intracellular truncation sites of PrP^d in different cell types (PrP^d epitope mapping) and the different morphological patterns of accumulation (PrP^d profiling). Differences in the inferred truncation sites of PrP^d are found for different strains of sheep TSEs and for different infected cell types within individual strains. Immunochemical methods of characterizing strains broadly correspond to PrP^d mapping discriminatory results, but distinct PrP^d profiles, which provide strain‐ and source‐specific information on both the cell types which sustain infection (cellular tropisms) and the cellular processing of PrP^d, have no immunoblotting counterparts. The cause of neurological dysfunction in human is commonly considered to be neuronal loss secondary to a direct or indirect effect of the accumulation of PrP^d. However, in sheep scrapie there is no significant neuronal loss, and relationships between different magnitudes, topographical and cytological forms of PrP^d accumulation and clinical signs are not evident. PrP^d accumulation also occurs in lymphoid tissues, for which there is indirect evidence of a pathological effect, in the peripheral nervous system and in other tissues. It is generally assumed that neuroinvasion results from infection of the enteric nervous system neurones subsequent to amplification of infectivity in lymphoid tissues and later spread via sympathetic and parasympathetic pathways. The evidence for this is, however, circumstantial. Accumulation of PrP^d and presence of infectivity in tissues other than the nervous and lymphoreticular systems gives insights on the ways of transmission of infection and on food safety.

To determine the mechanisms of intestinal transport of infection, and early pathogenesis, of sheep scrapie, isolated gut‐loops were inoculated to ensure that significant concentrations of scrapie agent would come into direct contact with the relevant ileal structures (epithelial, lymphoreticular, and nervous). Gut loops were inoculated with a scrapie brain pool homogenate or normal brain or sucrose solution. After surgery, animals were necropsied at time points ranging from 15 min to 1 month and at clinical end point. Inoculum‐associated prion protein (PrP) was detected by immunohistochemistry in villous lacteals and in sub‐mucosal lymphatics from 15 min to 3.5 h post‐challenge. It was also detected in association with dendritic‐like cells in the draining lymph nodes at up to 24 h post‐challenge. Replication of infection, as demonstrated by the accumulation of disease‐associated forms of PrP in Peyer's patches, was detected at 30 days and sheep developed clinical signs of scrapie at 18–22 months post‐challenge. These results indicate discrepancies between the routes of transportation of PrP from the inoculum and sites of de novo‐generated disease‐associated PrP subsequent to scrapie agent replication. When samples of homogenized inoculum were incubated with alimentary tract fluids in vitro, only trace amounts of protease‐resistant PrP could be detected by western blotting, suggesting that the majority of both normal and abnormal PrP within the inoculum is readily digested by alimentary fluids. Copyright © 2006 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

It has previously been reported that disease-associated prion protein (PrP^d) derived from natural scrapie and from sheep infected experimentally with bovine spongiform encephalopathy (BSE) differed in respect of their immunohistochemical and immunoblotting properties. For BSE, however, these initial observations were restricted to orally challenged sheep of the ARQ/ARQ PrP genotype. Here, extended examinations were performed on 28 sheep that developed neurological signs after BSE experimental infection by one of three routes. Intracerebrally infected ARQ/ARQ sheep showed more widespread and abundant accumulations of PrP^d in tissues of the lymphoreticular system (LRS) than VRQ/VRQ animals, whereas no peripheral PrP^d was detected in ARR/ARR sheep. The intensity and dissemination of PrP^d accumulation in LRS tissues were less than those found previously in orally dosed sheep. AHQ/AHQ sheep challenged orally and ARQ/AHQ and ARQ/ARQ animals infected intravenously showed similar LRS-tissue PrP^d distributions and levels to those of ARQ/ARQ sheep infected intracerebrally. The patterns of intra- and extracellular immunoreactivity to different PrP antibodies in brain and LRS tissues and the immunoblotting characteristics of PrP^res from brain samples remained constant, irrespective of the route of inoculation and the PrP genotype, and were the same as described previously for ARQ/ARQ sheep dosed orally with BSE. These results suggest that the intracellular truncation of BSE PrP^d and the proteinase K cleavage site of BSE PrP^res are not altered by PrP genotype or by route of inoculation and that, therefore, screening tests based on these properties can be applied to identify potential sheep BSE cases occurring naturally.