Molecular analysis of cases of Italian sheep scrapie and comparison with cases of bovine spongiform encephalopathy (BSE) and experimental BSE in sheep

Concerns have been raised about the possibility that the bovine spongiform encephalopathy (BSE) agent could have been transmitted to sheep populations via contaminated feedstuffs. The objective of our study was to investigate the suitability of molecular strain typing methods as a surveillance tool for studying scrapie strain variations and for differentiating PrP(Sc) from sheep scrapie, BSE, and sheep BSE. We studied 38 Italian sheep scrapie cases from 13 outbreaks, along with a British scrapie case, an experimental ovine BSE, and 3 BSE cases, by analyzing the glycoform patterns and the apparent molecular masses of the nonglycosylated forms of semipurified, proteinase-treated PrP(Sc). Both criteria were able to clearly differentiate sheep scrapie from BSE and ovine experimental BSE. PrP(Sc) from BSE and sheep BSE showed a higher glycoform ratio and a lower molecular mass of the nonglycosylated form compared to scrapie PrP(Sc). Scrapie cases displayed homogeneous PrP(Sc) features regardless of breed, flock, and geographic origin. The glycoform patterns observed varied with the antibody used, but either a monoclonal antibody (MAb) (F99/97.6.1) or a polyclonal antibody (P7-7) was able to distinguish scrapie from BSE PrP(Sc). While more extensive surveys are needed to further corroborate these findings, our results suggest that large-scale molecular screening of sheep populations for BSE surveillance may be eventually possible.     

Decontamination studies with the agents of bovine spongiform encephalopathy and scrapie

Summary Macerates of bovine brain infected with bovine spongiform encephalopathy (BSE) agent, and rodent brain infected with the 263K or ME7 strains of scrapie agent, were subjected to porous-load autoclaving at temperatures between 134 and 138 °C for 60 min. Bioassay in rodents showed that none of the regimes produced complete inactivation. Homogenates of BSE-infected bovine brain were exposed for 120 min to solutions of sodium hypochlorite or sodium dichloroisocyanurate containing 16,500 ppm available chlorine. There was no detectable survival of infectivity after the hypochlorite treatments but none of the dichloroisocyanurate solutions produced complete inactivation. Homogenates of BSE-infected bovine brain, and rodent brain infected with the 263K and ME7 strains of scrapie agent, were exposed for 120 min to 1M or 2M sodium hydroxide but no procedure produced complete inactivation of all agents tested.     

Clusterin in bovine spongiform encephalopathy (BSE).

Clusterin mRNA, detected in increased quantities in the cervical spinal cord of cattle with bovine spongiform encephalopathy (BSE), was localized mainly in the neuroglia (including astrocytes) of the lateral and ventral areas of white matter. Axonal degeneration was also observed in these areas. The dorsal horns of the spinal cord in which BSE prion protein (PrP(BSE)) was deposited did not exhibit strong clusterin "up-regulation" but showed increased clusterin immunolabelling with a punctate distribution in the neuropil. Labelling of adjacent sections of the grey matter in BSE-affected spinal cord and thalamus demonstrated that the clusterin was deposited in association with extracellular PrP(BSE).     

Human prion diseases and bovine spongiform encephalopathy (BSE)

Prion diseases are transmissible neurodegenerative disorders which affect a range of mammalian species. In humans they can be inherited and sporadic as well as acquired by exposure to human prions. Prions appear to be composed principally of a conformational isomer of host- encoded prion protein and propagate by recruitment of cellular prion protein. Recent evidence argues that prion protein can also encode disease phenotypes by differences in its conformation and glycosylation. Such molecular analysis of prion strains suggests that new variant Creutzfeldt-Jakob disease is caused by BSE exposure. The novel biology of prion propagation may not be unique to these rare degenerative brain diseases.      

Immunohistochemical distinction between preclinical bovine spongiform encephalopathy and scrapie infection in sheep

Sheep are susceptible experimentally to bovine spongiform encephalopathy (BSE), the clinical signs being indistinguishable from those of scrapie. Because of the possibility of natural ovine BSE infection, laboratory tests are needed to distinguish between scrapie and BSE infection. The objectives of this study were to determine whether (1) PrPSc accumulates in biopsy samples of the tonsil or third eyelid, or both, of BSE-infected sheep before the appearance of clinical disease, and (2) such samples from BSE- and scrapie-infected sheep differ in respect of PrPSc accumulations. Homozygous ARQ sheep (n = 10) were dosed orally at 4-5 months of age with a brain homogenate from BSE-infected cattle. Third eyelid and tonsillar biopsy samples were taken at < or = 6 monthly intervals post-infection and examined immunohistochemically for PrPSc. Third eyelid protuberances were difficult to identify, resulting in many unsuitable samples; however, third eyelid samples shown to contain lymphoid follicles were invariably negative for PrPSc. In contrast, tonsillar biopsy samples became positive for PrPSc from 11 to 20 months post-infection. Consistent differences in the morphology of PrPSc granules in tingible body macrophages (TBMs) between BSE- and scrapie-infected sheep were detected with anti-peptide antibodies directed towards amino acids 93-106 of the ovine prion protein: thus, PrPSc appeared as single granules in TBMs of tonsillar sections from BSE-infected sheep, whereas clusters of PrPSc granules were observed within TBMs in the tonsils of scrapie-infected sheep. In contrast, antibodies against epitopes situated N- and C-terminally from the 93-106 region of the ovine prion protein revealed no differences between BSE- and scrapie-infected sheep in terms of PrPSc granules in TBMs.     

Post-mortem immunodiagnosis of scrapie and bovine spongiform encephalopathy

Two polyclonal antisera were raised in rabbits against the scrapie-associated fibril protein (PrP) prepared from sheep and mice which were terminally infected with experimental scrapie. The anti-mouse PrP serum identifies the proteins of scrapie-associated fibrils (SAF) from all the host species studied (mouse, hamster, sheep and goat) and bovine spongiform encephalopathy (BSE) fibrils from cow. The anti-sheep PrP serum displays species restricted immunoreactivity. While it identifies several PrP polypeptides from terminally affected sheep, goat and cow material, only the highest molecular weight band is recognised from hamster and there is no detection of mouse PrP. The use of these antisera in routine laboratory testing at post mortem provides a highly sensitive test for scrapie and BSE and may allow the identification of infected animals prior to the onset of clinical signs.     

Predominant involvement of the cerebellum in guinea pigs infected with bovine spongiform encephalopathy (BSE)

This study reports the experimental transmission of bovine spongiform encephalopathy (BSE) to guinea pigs and describes the cerebellar lesions in these animals. Guinea pigs were inoculated intracerebrally with 10% brain homogenates from BSE-affected cattle. These animals were designated as the first passage. Second and third passages were subsequently performed. All guinea pigs developed infection at each passage. The mean incubation period of the first passage was 370 days post-infection (dpi) and this decreased to 307 dpi and 309 dpi for the second and third passages, respectively. Mild to severe spongiform degeneration and gliosis were observed in the cerebral cortex, thalamus and brainstem. In addition, the affected animals had marked pathological changes in the cerebellum characterized by severe cortical atrophy associated with Bergmann radial gliosis of the molecular layer and reduction in the width of the granular cell layer. Immunohistochemically, intense PrP^Sc deposition and scattered plaque-like deposits were observed in the molecular and granular cell layers. Cerebellar lesions associated with severe atrophy of the cortex have not been reported in animal prion diseases, including in the experimental transmission of PrP^Sc to small rodents. These lesions were similar to the lesions of human kuru or the VV2 variant of sporadic Creutzfeldt–Jakob disease, although typical kuru plaques or florid plaques were not observed in the affected animals.     

Evaluation of a rapid western immunoblotting procedure for the diagnosis of bovine spongiform encephalopathy (BSE) in the UK.

Bovine brain tissue samples from 625 UK cattle, clinically suspected as bovine spongiform encephalopathy (BSE) cases, were used in a blind analysis to assess a rapid Western immunoblotting technique (Prionics Check; Prionics AG, Zurich), which detects bovine disease-specific protease-resistant prion protein (PrP(Sc)). By means of statutory histopathological examination, 599 of the 625 cattle were confirmed as BSE cases by the demonstration of spongiform encephalopathy, the remaining 26 being classified as negative. Duplicate samples from the same animals were also examined by electron microscopy for the presence of abnormal brain fibrils (scrapie-associated fibrils; SAFs). The Prionics technique showed a high sensitivity, particularly when compared with the fibril detection test; the detection rates were 99.3% and 92.0% respectively, with histopathology being used as the "gold standard". The false negative results by the Prionics test were possibly related to the sampling procedure. Analysis of 50 BSE-positive samples revealed similar glycoprofiles, the majority of PrP(Sc)isoforms being di-glycosylated protein. The Prionics test also detected PrP(Sc)in the four brain samples from the 26 histopathologically negative animals, apparently reducing the specificity of the test to 84.6%; however, confirmatory positive results in these samples were obtained by demonstrating SAF or by immunohistochemical examination, or both. It was concluded that the Prionics test detected PrP(Sc)in a small percentage (0.64%) of clinically suspected BSE cases showing no spongiform change. Since January 2000, the Prionics Western blot test has been introduced as one of the statutory tests for the diagnosis of clinically suspected BSE and scrapie cases in the UK. Copyright Harcourt Publishers Ltd.     

Pathogenesis of bovine spongiform encephalopathy in sheep

The pathogenesis of bovine spongiform encephalopathy (BSE) in sheep was studied by immunohistochemical detection of scrapie-associated prion protein (PrP^Sc) in the gastrointestinal, lymphoid and neural tissues following oral inoculation with BSE brain homogenate. First accumulation of PrP^Sc was detected after 6 months in the tonsil and the ileal Peyer’s patches. At 9 months postinfection, PrP^Sc accumulation involved all gut-associated lymphoid tissues and lymph nodes as well as the spleen. At this time point, PrP^Sc accumulation in the peripheral neural tissues was first seen in the enteric nervous system of the caudal jejunum and ileum and in the coeliac-mesenteric ganglion. In the central nervous system, PrP^Sc was first detected in the dorsal motor nucleus of the nervus Vagus in the medulla oblongata and in the intermediolateral column in the spinal cord segments T7–L1. At subsequent time points, PrP^Sc was seen to spread within the lymphoid system to also involve all non-gut-associated lymphoid tissues. In the enteric nervous system, further spread of PrP^Sc involved the neural plexi along the entire gastrointestinal tract and in the CNS the complete neuraxis. These findings indicate a spread of the BSE agent in sheep from the enteric nervous system through parasympathetic and sympathetic nerves to the medulla oblongata and the spinal cord.     

Monitoring and analysis of bovine spongiform encephalopathy (BSE) testing in Denmark using statistical models

The evolution of monitoring and surveillance for bovine spongiform encephalopathy (BSE) from the phase of passive surveillance that began in the United Kingdom in 1988 until the present is described. Currently, surveillance for BSE in Europe consists of mass testing of cattle slaughtered for human consumption and cattle from certain groups considered to be at higher risk of having clinical or detectable BSE. The results of the ongoing BSE testing in Denmark have been analyzed using two statistical approaches: the "classical" frequentist and the Bayesian that is widely used in quantitative risk analysis. The analyses were intended to provide information for decision-makers, the media and the public as well as to provide inputs for future BSE surveillance models. The results to date suggest that the total number of BSE cases that will be found in Denmark in 2001 will not exceed 16.     

Ovine infection with the agents of scrapie (CH1641 isolate) and bovine spongiform encephalopathy: immunochemical similarities can be resolved by immunohistochemistry

Immunochemical ("rapid") tests, which recognize a partly protease-resistant conformer of the prion protein (PrP(res)) are now widely used in Europe for the diagnosis of transmissible spongiform encephalopathies (TSEs). Some of these tests can be used to distinguish natural scrapie from experimental bovine spongiform encephalopathy (BSE) in sheep, on the basis of migration pattern differences of PrP(res) in Western immunoblots. However, PrP(res) from sheep inoculated with CH1641 scrapie gives an immunoblot profile similar to that of sheep inoculated with BSE. Therefore, field scrapie strains similar to CH1641 might be misclassified as ovine BSE in the rapid tests currently employed. This study confirmed that the Western blot similarities (size of the unglycosylated band and distinct reactivity with 6H4 and P4 antibodies) between CH1641 and BSE remained consistent regardless of the PrP genotype of the sheep, but the two infections resulted in accumulation of disease-associated PrP (PrP(d)) that could easily be distinguished by the immunohistochemical "peptide mapping" method. This method, which reveals conformational differences of PrP(d) by the use of a panel of antibodies, indicated that PrP(d) from the CH1641 isolate was truncated further upstream in the N terminus than was PrP(d) from other ovine TSEs, including experimental BSE. In addition, the immunohistochemical "PrP(d) profile method", which defines the phenotype of PrP(d) accumulation in the brain of affected sheep, showed that CH1641 infection leads to much more intra-neuronal and considerably less extracellular PrP(d) than does experimental BSE. The overall results demonstrate that a combined Western blotting and immunohistochemical approach is required to discriminate between different TSE strains in sheep.     

Oral inoculation of sheep with the agent of bovine spongiform encephalopathy (BSE). 1. Onset and distribution of disease-specific PrP accumulation in brain and viscera.

Sixty-three Romney sheep aged 6 months, consisting of three groups (PrP(ARQ/ARQ), PrP(ARQ/ARR), and PrP(ARR/ARR)genotypes) of 21 animals, were infected orally with brain tissue from BSE-infected cattle. Sub-groups of the 21 PrP(ARQ/ARQ) animals were killed, together with uninfected controls 4, 10, 16, 22 or 24-28 (after the development of full clinical disease) months post-inoculation (mpi). One sheep from each of the two groups of four killed at 4 or 10 mpi were shown by immunohistochemical examination to possess disease-specific PrP accumulations in single lymph nodes. At 16 mpi, such accumulations were detected in two of four infected sheep in some viscera and in the spinal cord and brain. At 22 mpi, three of five infected sheep had widespread disease-specific PrP accumulations in all tissues examined, but the remaining two animals gave positive results only in the central nervous system. Clinical disease appeared at 20-28 mpi. Three sheep killed with advanced clinical signs showed widespread PrP accumulation in brain, spinal cord and peripheral tissues. These results confirmed that PrP(ARQ/ARQ) Romney sheep are susceptible to experimental infection with the BSE agent. The different sites at which initial PrP accumulations were detected suggested that the point of entry of infection varied. Once established, however, infection appeared to spread rapidly throughout the lymphoreticular system. The results suggested that in some BSE-infected sheep neuroinvasion occurred in the absence of detectable PrP accumulations in the viscera or peripheral nervous system. In contrast to cattle with BSE, however, most sheep showed disease-specific PrP accumulations in the lymphoreticular system. In this respect, BSE-infected resembled scrapie-infected sheep; it is possible, however, that future research will reveal differences in respect of targeting of cell types within the lymphoreticular and peripheral nervous systems. The PrP(ARQ/ARR)and PrP(ARR/ARR)sheep were also killed in sub-groups at intervals after inoculation. Up to 24 mpi, however, none of these animals showed disease-specific PrP accumulations. Further results will be reported later.     

Discrimination between scrapie and bovine spongiform encephalopathy in sheep by molecular size, immunoreactivity, and glycoprofile of prion protein

A procedure for discrimination between scrapie and bovine spongiform encephalopathy (BSE) in sheep is of importance for establishing whether BSE has entered the sheep population. Since BSE has not yet been found in sheep at the farm level, such discrimination procedures can be developed only with experimental sheep BSE. Two distinctive molecular features of the prion protein (PrP)-molecular size and glycosylation profile-in proteinase K digests of brain stem tissue from sheep were used here; upon Western blotting, these features led to an unequivocal discrimination among natural scrapie, experimental scrapie, and experimental BSE. The higher electrophoretic mobility of PrP in sheep BSE could be best observed after deglycosylation treatment with N-glycosidase F. A simpler method for confirmation of this size difference involved comparison of the ratios for the binding of two monoclonal antibodies: P4 and 66.94b4. Based on epitope mapping studies with P4 and peptides, it appeared that N-terminal amino acid sequence WGQGGSH was intact only in sheep scrapie digests. Another feature typical for PrP in sheep BSE was the large fraction of diglycosylated PrP (70% or more). These data were obtained for a large group of positive sheep, consisting of 7 sheep with experimental BSE infection (genotypes: six ARQ/ARQ and one AHQ/AHQ), 48 sheep naturally infected with scrapie (six different genotypes), and 3 sheep with primary experimental scrapie infection. Routine tests of slaughter material serve well for the initial detection of both BSE and scrapie. With Western blotting as a rapid follow-up test, a 66.94b4/P4 antibody binding ratio above 1.5 is a practical indicator for serious suspicion of BSE infection in sheep.     

Antigen retrieval using sodium hydroxide for prion immunohistochemistry in bovine spongiform encephalopathy and scrapie

Formalin-fixed and paraffin wax-embedded (FFPE) tissue sections are usually used for histopathological and immunohistochemical analyses in prion diseases in animals and man. However, formalin fixation cross-links proteins, reducing disease-associated prion protein (PrP^Sc) immunolabelling. To detect PrP^Sc in animals naturally affected with bovine spongiform encephalopathy (BSE) and scrapie, we applied minimal pretreatment with sodium hydroxide (NaOH). This simple pretreatment, combined with enzymatic digestion using proteinase K (PK), was equally effective in the detection of PrP^Sc in FFPE tissue, and superior in terms of speed, compared with the usual autoclaving method. The most effective results, without any section loss, were obtained with 10 μg/ml PK in phosphate buffered saline containing 0.1% Triton-X at room temperature for 10 min and 150 mM NaOH at 60°C for 10 min. By this simple procedure, PrP^Sc was visualized in the brain of animals with BSE and scrapie using a range of anti-PrP primary antibodies.     

Comparative ultrastructural neuropathology of naturally occurring bovine spongiform encephalopathy and experimentally induced scrapie and Creutzfeldt-Jakob disease.

We report the ultrastructural neuropathology of bovine spongiform encephalopathy (BSE), a recently described slow virus disease first recognized in Friesian/Holstein cattle, and compare it to that of experimental scrapie and Creutzfeldt-Jakob disease. The spongiform change, which was most pronounced in the central grey matter of the midbrain, consisted of membrane-bound vacuoles within neuronal processes, containing curled membrane fragments, secondary chambers and vesicles. Axons and dendrites accumulated whorls of neurofilaments and other subcellular organelles, such as mitochondria and dense bodies, which were entrapped within the filamentous masses. Other neurites accumulated electron-dense bodies, and still others electron-lucent cisterns and branching tubules. Membrane-bound neuronal inclusions, composed of tubules measuring 10 nm in diameter, were found in axonal terminals. Tubulovesicular structures were loosely packed and were occasionally surrounded by a common membrane, a finding previously described only in natural scrapie in sheep. Except for the intraneuronal inclusions, all of the ultrastructural features of BSE resembled those found in scrapie and Creutzfeldt-Jakob disease.     

Bov-tA short interspersed nucleotide element sequences in circulating nucleic acids from sera of cattle with bovine spongiform encephalopathy (BSE) and sera of cattle exposed to BSE

Circulating nucleic acids (CNA) are known to be enriched in repetitive DNA sequences in humans. Here, bovine sera CNA were analyzed to determine if cell stress-related short interspersed nucleotide elements (SINEs) could be detected in sera from cattle associated with bovine spongiform encephalopathy (BSE). Nucleic acids were extracted, amplified, cloned, and sequenced from the sera of protease-resistant prion protein (PrP(res))-positive cattle (n = 2) and sera from BSE-cohort cows (n = 6); 150 out of 163 clones revealed the presence of, on average, an 80-bp sequence from the 3' region of Bov-tA SINE. A PCR protocol was developed that differentially identified SINE-associated CNA in BSE-exposed versus normal cattle. CNA were extracted from a serum vesicular fraction after controlled blood collection and processing procedures. Sera from four confirmed cases of BSE, 137 BSE-exposed cohort animals associated with eight confirmed BSE cases, and 845 healthy, PrP(res)-negative control cows were tested. All four sera from confirmed BSE cases were repeatedly reactive in the assay. BSE-exposed cohorts had a 100-fold higher occurrence of repeatedly reactive individuals per cohort (average = 63%; range = 33% to 91%), compared to healthy controls (average = 0.6%; P < 0.001). This study shows that BSE-confirmed and cohort animals possess a unique profile of SINE-associated serum CNA that can be utilized as a marker that highly correlates to BSE exposure.     

Population-level retrospective study of neurologically expressed disorders in ruminants before the onset of bovine spongiform encephalopathy (BSE) in Belgium, a BSE risk III country

A retrospective epidemiological study (n = 7,875) of neurologically expressed disorders (NED) in ruminants before the onset of the bovine spongiform encephalopathy epidemic (years studied, 1980 to 1997) was carried out in Belgium. The archives of all veterinary laboratories and rabies and transmissible spongiform encephalopathy (TSE) epidemiosurveillance networks were consulted. For all species, a significantly higher number of NED with virological causes (rabies) was reported south of the Sambre-Meuse Valley. During the period 1992 to 1997, for which the data were complete, (i) the predicted annual incidence of NED varied significantly as a function of species and area (higher numbers in areas where rabies was present) but was always above 100 cases per million, and (ii) the mean incidence of suspected TSE cases and, among them, those investigated by histopathological examination varied significantly as a function of species and area. The positive predictive value of a presumptive clinical diagnosis of NED ranged from 0.13 (game) to 0.63 (sheep). Knowledge of the positive predictive value permits the definition of a reference point before certain actions (e.g., awareness and training campaigns) are undertaken. It also shows the usefulness of a systematic necropsy or complementary laboratory tests to establish an etiological diagnosis. TSE analysis of a small, targeted historical sampling (n = 48) permitted the confirmation of one case and uncovered another case of scrapie. The results of the present study help to develop and maintain the quality of the worldwide clinical epidemiological networks for TSE, especially in countries that in the past imported live animals, animal products, and feedstuffs from countries with TSE cases.     

Decision support tools for clinical diagnosis of disease in cows with suspected bovine spongiform encephalopathy

Reporting of clinically suspected cattle is currently the most common method for detecting cases of bovine spongiform encephalopathy (BSE). Improvement of clinical diagnosis and decision-making remains crucial. A comparison of clinical patterns, consisting of 25 signs, was made between all 30 BSE cases, confirmed in Belgium before October 2002, and 272 suspected cases that were subsequently determined to be histologically, immunohistochemically, and scrapie-associated-fiber negative. Seasonality in reporting suspected cases was observed, with more cases being reported during wintertime when animals were kept indoors. The median duration of illness was 30 days. The 10 most relevant signs of BSE were kicking in the milking parlor, hypersensitivity to touch and/or sound, head shyness, panic-stricken response, reluctance to enter in the milking parlor, abnormal ear movement or carriage, increased alertness behavior, reduced milk yield, teeth grinding, and temperament change. Ataxia did not appear to be a specific sign of BSE. A classification and regression tree was constructed by using the following four features: age of the animal, year of birth, number of relevant BSE signs noted, and number of clinical signs, typical for listeriosis, noted. The model had a sensitivity of 100% and a specificity of 85%. This approach allows the use of an interactive decision-support tool, based entirely on odds ratios, a statistic independent of disease prevalence.     

Nuclear DNA fragmentation and immune reactivity in bovine spongiform encephalopathy.

To investigate whether apoptosis contributes to neuronal degeneration in bovine spongiform encephalopathy (BSE), morphological changes consistent with apoptosis were sought and in-situ end labelling (ISEL) was applied, in a series of 20 BSE cases and 10 age-matched normal control cattle. Apoptotic changes were not found in neurons but were occasionally seen in glial cells. Relatively few ISEL-positive neurons were found, but many labelled nuclei were seen in glial cells in certain areas. None of the labelled cells showed morphological features of apoptosis. ISEL(+)cells occurred in areas of spongiform change and other areas of grey matter lacking spongiform change. Some association was found between degree of cellular DNA fragmentation and accumulation of abnormal prion protein (PrP(Sc)). Interestingly, small or moderate numbers of T lymphocytes, not present in the normal central nervous system (CNS), were detected in the CNS parenchyma in most BSE cases. There was a pronounced astrogliosis, but markers of macrophage or microglial activation were only slightly increased. The results indicate that nuclear DNA vulnerability is enhanced in certain neuroanatomical areas in BSE, but evidence that apoptosis plays a role in neuronal loss in BSE was very limited. 1999 Harcourt Publishers Ltd.     

Ultrastructural pathology of axons and myelin in experimental scrapie in hamsters and bovine spongiform encephalopathy in cattle and a comparison with the panencephalopathic type of Creutzfeldt-Jakob disease.

We report the ultrastructural pathology of axons and myelin sheaths in bovine spongiform encephalopathy (BSE) and experimental scrapie in hamsters and compare it with that found in a panencephalopathic model of Creutzfeldt-Jakob disease (CJD). Intramyelinic vacuoles (myelin ballooning), dystrophic axons, phagocytic astrocytes and macrophages were found in all three models but to different degrees, while axons containing numerous cellular processes and concentric cisterns were observed only in experimental scrapie and CJD. We conclude that axonal and myelin pathology is a widespread phenomenon and the differences between panencephalopathic CJD and polioencephalopathic BSE and scrapie are only quantitative.