Human prion diseases: biology and transmission by blood

Human prion diseases are a group of rare fatal transmissible neurodegenerative disorders that occur in sporadic, acquired and familial forms. In 1996, a new type of human prion disease, variant Creutzfeldt‐Jakob disease (vCJD), was first identified and has subsequently been identified in 10 additional countries. vCJD results from human exposure to the bovine spongiform encephalopathy (BSE) agent, most likely through the consumption of BSE‐contaminated meat products. Unlike other human prion diseases, both infectivity and the disease‐associated form of the prion protein are readily detected in lymphoid tissues in vCJD, suggesting that infectivity may also be present in blood. Three recent cases of apparent iatrogenic vCJD infection by blood transfusion have occurred in the UK, following red blood cell transfusions from asymptomatic donors who subsequently died from vCJD. The first and third cases resulted in a clinical illness identical to vCJD, while the second case was an asymptomatic infection only detected at autopsy. There are no current means of detecting vCJD infection in asymptomatic donors, so continuing surveillance is required in the UK and other countries to monitor the incidence of vCJD and further cases of secondary transmission by blood transfusion.     

Animal prion diseases: the risks to human health

Transmissible spongiform encephalopathies (TSEs) or prion diseases of animals notably include scrapie in small ruminants, chronic wasting disease (CWD) in cervids and classical bovine spongiform encephalopathy (C‐BSE). As the transmission barrier phenomenon naturally limits the propagation of prions from one species to another, and the lack of epidemiological evidence for an association with human prion diseases, the zoonotic potential of these diseases was for a long time considered negligible. However, in 1996, C‐BSE was recognized as the cause of a new human prion disease, variant Creutzfeldt‐Jakob disease (vCJD), which triggered an unprecedented public health crisis in Europe. Large‐scale epidemio‐surveillance programs for scrapie and C‐BSE that were implemented in the EU after the BSE crisis revealed that the distribution and prevalence of prion diseases in the ruminant population had previously been underestimated. They also led to the recognition of new forms of TSEs (named atypical) in cattle and small ruminants and to the recent identification of CWD in Europe. At this stage, the characterization of the strain diversity and zoonotic abilities associated with animal prion diseases remains largely incomplete. However, transmission experiments in nonhuman primates and transgenic mice expressing human PrP clearly indicate that classical scrapie, and certain forms of atypical BSE (L‐BSE) or CWD may have the potential to infect humans. The remaining uncertainties about the origins and relationships between animal prion diseases emphasize the importance of the measures implemented to limit human exposure to these potentially zoonotic agents, and of continued surveillance for both animal and human prion diseases.     

Prionkrankheiten des Menschen

Prion diseases are rare neurodegenerative transmissible fatal diseases affecting humans and mammals. The causative agent is a novel pathogen termed the prion. Unlike classical infectious agents such as bacteria or viruses, prions lack an independent genome and consist largely of an abnormal form of the host-encoded prion protein. Creutzfeldt-Jakob disease (CJD) is the main representative of human prion diseases that may be sporadic in most cases, hereditary, or acquired. Clinical examination yields only a suspected diagnosis with formal criteria for probable or possible. Definite diagnosis relies on autopsy and neuropathology findings. This is also true for the new variant CJD (vCJD), a previously unknown prion disease of humans that is connected with the same strain of prions as found in bovine spongiform encephalopathy (BSE). The autopsy and handling of laboratory material for histopathological examination requires specific precautionary measures and decontamination. For definite histopathological diagnosis of a human prion disease, immunohistochemical detection of the prion protein deposits is the gold standard. Furthermore, molecular and genetic investigations are necessary for classification because a close correlation could be established between distinct CJD phenotypes, codon 129 genotypes of the prion protein gene, and the prion protein type.     

Peripheral tissue involvement in sporadic, iatrogenic, and variant Creutzfeldt-Jakob disease: an immunohistochemical, quantitative, and biochemical study

Human prion diseases are rare fatal neurodegenerative conditions that occur as acquired, familial, or idiopathic disorders. A key event in their pathogenesis is the accumulation of an altered form of the prion protein, termed PrP(Sc), in the central nervous system. A novel acquired human prion disease, variant Creutzfeldt-Jakob disease, is thought to result from oral exposure to the bovine spongiform encephalopathy agent. This disease differs from other human prion diseases in its neurological, neuropathological, and biochemical phenotype. We have used immunohistochemistry and Western blot techniques to analyze the tissue distribution and biochemical properties of PrP(Sc) in peripheral tissues in a unique series of nine cases of variant Creutzfeldt-Jakob disease. We have compared this with the distribution and biochemical forms found in all of the major subtypes of sporadic Creutzfeldt-Jakob disease and in a case of iatrogenic Creutzfeldt-Jakob disease associated with growth hormone therapy. The results show that involvement of the lymphoreticular system is a defining feature of variant Creutzfeldt-Jakob disease, but that the biochemical isoform of PrP(Sc) found is influenced by the cell type in which it accumulates.     

Immunohistochemical features of PrP(d) accumulation in natural and experimental goat transmissible spongiform encephalopathies

Scrapie is a transmissible spongiform encephalopathy (TSE) or prion disease, which naturally affects sheep and goats. Immunohistochemical epitope mapping of abnormal PrP accumulations (PrP(d)) in brain can help in characterizing sheep TSE sources or strains and in identifying potential bovine spongiform encephalopathy (BSE) infections of sheep. Natural and experimental TSE infections of goats were examined to determine whether the epitope mapping approach could also be applied to aid recognition of BSE infection in goats. Goats experimentally infected with the SSBP/1 or CH1641 sheep scrapie strains or with cattle BSE, together with four field cases of natural TSE in goats, were examined immunohistochemically with six different antibodies. CH1641 and SSBP/1 infections in goats, as in sheep, showed PrP(d) accumulations which were mainly intracellular. Some differences in targeting, particularly of Purkinje cells, was evident in inter-species comparisons of CH1641 and SSBP/1. PrP(d) labelling of goat BSE experimental cases showed extensive intracellular and extracellular accumulations, also similar to those in sheep BSE. Intra-neuronal PrP(d) in both goat and sheep BSE was labelled only by antibodies recognizing epitopes located C-terminally of residue His99, whereas in natural sheep TSE sources, and in sheep and goat SSBP/1, PrP(d) was also detected by antibodies to epitopes located between residues Trp93 and His99. Testing of four natural goat TSE samples showed one case in which epitope mapping characteristics and the overall patterns of PrP(d) accumulation was identical with those of experimental goat BSE. The four natural goat scrapie cases examined showed some degree of immunohistochemical phenotype variability, suggesting that multiple strains exist within the relatively small UK goat population.     

Latrogenic Creutzfeldt-Jakob disease with florid plaques

Florid plaques indistinguishable from those found in vCJD were identified at a postmortem examination in the brain of a 58-year-old clinical suspect case of Creutzfeldt-Jakob disease (CJD). Western blotting of brain tissue revealed an unusual prion protein type. Since the patient had received a dura mater graft 20 years prior to death and florid plaques are not only found in new variant CJD, the findings argue in favor of an iatrogenic origin of the disease with the longest incubation time following a dura mater graft reported to date even though he may have been exposed to BSE. The peculiar pathological, clinical and biochemical features may define a new type of human prion disease.     

Detection and Discrimination of Classical and Atypical L-Type Bovine Spongiform Encephalopathy by Real-Time Quaking-Induced Conversion

Statutory surveillance of bovine spongiform encephalopathy (BSE) indicates that cattle are susceptible to both classical BSE (C-BSE) and atypical forms of BSE. Atypical forms of BSE appear to be sporadic and thus may never be eradicated. A major challenge for prion surveillance is the lack of sufficiently practical and sensitive tests for routine BSE detection and strain discrimination. The real-time quaking-induced conversion (RT-QuIC) test, which is based on prion-seeded fibrillization of recombinant prion protein (rPrP^Sen), is known to be highly specific and sensitive for the detection of multiple human and animal prion diseases but not BSE. Here, we tested brain tissue from cattle affected by C-BSE and atypical L-type bovine spongiform encephalopathy (L-type BSE or L-BSE) with the RT-QuIC assay and found that both BSE forms can be detected and distinguished using particular rPrP^Sen substrates. Specifically, L-BSE was detected using multiple rPrP^Sen substrates, while C-BSE was much more selective. This substrate-based approach suggests a diagnostic strategy for specific, sensitive, and rapid detection and discrimination of at least some BSE forms.     

Highly Sensitive Detection of Small Ruminant Bovine Spongiform Encephalopathy within Transmissible Spongiform Encephalopathy Mixes by Serial Protein Misfolding Cyclic Amplification

It is assumed that sheep and goats consumed the same bovine spongiform encephalopathy (BSE)-contaminated meat and bone meal that was fed to cattle and precipitated the BSE epidemic in the United Kingdom that peaked more than 20 years ago. Despite intensive surveillance for cases of BSE within the small ruminant populations of the United Kingdom and European Union, no instances of BSE have been detected in sheep, and in only two instances has BSE been discovered in goats. If BSE is present within the small ruminant populations, it may be at subclinical levels, may manifest as scrapie, or may be masked by coinfection with scrapie. To determine whether BSE is potentially circulating at low levels within the European small ruminant populations, highly sensitive assays that can specifically detect BSE, even within the presence of scrapie prion protein, are required. Here, we present a novel assay based on the specific amplification of BSE PrP^Sc using the serial protein misfolding cyclic amplification assay (sPMCA), which specifically amplified small amounts of ovine and caprine BSE agent which had been mixed into a range of scrapie-positive brain homogenates. We detected the BSE prion protein within a large excess of classical, atypical, and CH1641 scrapie isolates. In a blind trial, this sPMCA-based assay specifically amplified BSE PrP^Sc within brain mixes with 100% specificity and 97% sensitivity when BSE agent was diluted into scrapie-infected brain homogenates at 1% (vol/vol).     

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.     

The paraffin-embedded tissue blot detects PrP(Sc) early in the incubation time in prion diseases

With the appearance of bovine spongiform encephalopathy (BSE) and a new variant of Creutzfeldt-Jakob disease (nvCJD) that seems to be caused by BSE, there is an increased need for improvement of diagnostic techniques and recognition of all variants of prion diseases in humans and animals. Publications on the immunohistochemical identification of PrP(Sc) in the tonsils and appendix in the incubation period of nvCJD indicate that new and more sensitive techniques for the detection of PrP(Sc) in various tissues may be a valuable tool for early diagnosis in prion diseases. We developed a new and sensitive technique to detect PrP(Sc) in formalin-fixed and paraffin-embedded tissue, the paraffin-embedded tissue blot (PET blot), and reinvestigated archival brain material from CJD as well as BSE and scrapie. In addition, C57/Bl6 mice experimentally infected with the ME7 strain were investigated sequentially during the incubation time to compare this new technique with conventional methodologies. The PET blot detects PrP(Sc) in idiopathic (sporadic) and acquired prion diseases, even in cases with equivocal or negative immunohistochemistry, and is more sensitive than the conventional Western blot and histoblot techniques. The PET blot makes possible the detection of PrP(Sc) during the incubation period long before the onset of clinical disease and in prion disease variants with very low levels of PrP(Sc). In mice experimentally infected with the ME7 strain, the PET blot detects PrP(Sc) in the brain 30 days after intracerebral inoculation-145 days before the onset of clinical signs. Its anatomical resolution is superior to that of the histoblot technique. It may therefore be of particular interest in biopsy diagnosis. Thus it complements other tissue-based techniques for the diagnosis of prion diseases in humans and 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.     

The Neuropathology and Epidemiology of Bovine Spongiform Encephalopathy

Bovine spongiform encephalopathy (BSE), defined originally from its characteristic neuropathology, retains a place of particular interest in the scrapie-like or prion disease group, presenting uniquely an example of such diseases occurring as a nationwide food-borne epidemic in Great Britain. Comprehensive monitoring of the epidemic, both pathologically and epidemiologically, has facilitated our present understanding of the disease. BSE presents the classical neuropathological features of the transmissible spongiform encephalopathies. Although particularly similar to natural scrapie of sheep, BSE has, unlike scrapie, a stereotypic lesion profile from which it has been concluded that host and agent factors, including probably the strain of agent, which influence the profile, are constant in this disease. Neuronal loss in BSE may make an important but hitherto inapparent contribution to functional deficits. Preliminary ultrastructural studies have confirmed light microscopic features of brain changes in BSE but have as yet not established significant new findings. Immunohistochemical studies of PrP accumulation reveal distinctive forms and distributions of immunolabelling, confirming features reported previously in experimental models of scrapie, including perineuronal and perineuritic “synapse-like” reactivity. The histopathological diagnosis of BSE, validated on a single section of the medulla for the statutory diagnosis of large numbers of cases, is supplemented where necessary by fibril (SAF) examination which performs similarly to the histological diagnosis in the majority of cases. Epidemiological studies of BSE have supported the pathological findings that there is no detectable variation in susceptibility within the cattle population. The detailed monitoring of the epidemic has revealed the expected effects on the incidence as a result of statutory measures intended to prevent food-borne exposure after July 1988. The main effect has been a reduction in the national incidence during 1993 which has been continued into 1994. Analytical studies have not revealed any means of transmission, other than the food-borne source, capable of maintaining the epidemic in Great Britain. An international comparison of risk factors for the occurrence of BSE indicates that an epidemic of similar magnitude outside the British Isles is unlikely.     

BSE did not cause variant CJD: an alternative cause related to post-industrial environmental contamination.

The new prion diseases that have emerged in the last 15 years are bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (variant CJD). Although initially confined to the UK, these diseases have recently emerged in other European countries. The accepted cause of the human disease is that BSE spread from cattle to humans by the consumption of infected beef. However, the evidence that supports this is very thin. This article describes this evidence and lists a series of hypotheses concerning the cause of both BSE and variant CJD. The final hypothesis is based on recent evidence linking prion diseases to environmental factors including manganese. High environmental availability of manganese is associated with the prevalence of those prion diseases not linked to BSE. Therefore it is quite possible that BSE and variant CJD have emerged as a result of manganese-rich industrial pollution that has only occurred in the last century.     

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.     

Experimental bovine spongiform encephalopathy: detection of PrP(Sc) in the small intestine relative to exposure dose and age

European regulations for the control of bovine spongiform encephalopathy (BSE) decree destruction of the intestines from slaughtered cattle, therefore producers have been obliged to import beef casings from countries with a negligible BSE risk. This study applies immunohistochemical and biochemical approaches to investigate the occurrence and distribution of disease-associated prion protein (PrP(Sc)) in the duodenum, jejunum and ileum of cattle orally exposed to a 1 g or 100 g dose of a titrated BSE brainstem homogenate. Samples were derived from animals at various times post exposure. Lymphoid follicles were counted and the frequency of affected follicles recorded. No PrP(Sc) was detected in the duodenum or jejunum of animals exposed to a 1 g dose or in the duodenum of animals receiving a 100 g dose. PrP(Sc) was detected in the lymphoid tissue of the ileum of 1/98 (1.0%) animals receiving the 1 g dose and in the jejunum and ileum of 8/58 (13.8%) and 45/99 (45.5%), respectively, of animals receiving the 100 g dose. The frequency of PrP(Sc)- positive follicles was less than 1.5% per case and biochemical tests appeared less sensitive than immunohistochemistry. The probability of detecting lymphoid follicles in the ileum declined with age and for the 100 g exposure the proportion of positive follicles increased, while the proportion of positive animals decreased with age. Detection of PrP(Sc) in intestinal neural tissue was rare. The results suggest that the jejunum and duodenum of BSE-infected cattle contain considerably less BSE infectivity than the ileum, irrespective of exposure dose. In animals receiving the low exposure dose, as in most natural cases of BSE, the rarity of PrP(Sc) detection compared with high-dose exposure, suggests a very low BSE risk from food products containing the jejunum and duodenum of cattle slaughtered for human consumption.     

Bovine spongiform encephalopathy

The identification of variant Creutzfeldt-Jakob disease (vCJD) in human strongly reinforced the perception of risks associated with the infectious agent involved in Bovine Spongiform Encephalopathy (BSE). The development of rapid tests for the diagnosis of BSE by the detection of the abnormal prion protein allowed a huge increase in surveillance of the cattle disease. This first revealed a higher prevalence of the infection than previously believed. However, food safety measures, mainly based on the ban of the use of meat and bone meal in ruminants and the elimination of specified risk materials from the food chain, already allowed significant progress in the control of the cattle disease, especially in the United Kingdom. Nevertheless, the diagnosis can still not be obtained in the live animal, while the disease only appears following a several years incubation period. Another major issue is the identification of the BSE agent when it has been transmitted to another species. This question not only arises in veterinary medicine, with the major question of a possible infection of small ruminants by the BSE agent, but also in human in which the existence of other forms of the disease linked to the BSE agent but possibly differing from Creutzfeldt-Jakob disease cannot be excluded.     

Are humans getting 'mad-cow disease' from eating beef,or something else?

Bovine spongiform encephalopathy (BSE) or 'mad-cow disease' is believed to have been caused by the consumption of scrapie-infected sheep matter that had been added to cattle feed. BSE is then believed to have been transmitted to humans by the consumption of infected beef. We have compared the sequences of human and various animal prion proteins with regards to the fragments that could result from gastric digestion. We noted the close similarity of the sequences of human and rodent prion proteins in a peptic fragment that corresponds very closely to one that had been shown by others to be protease resistant and infective. Since rats and mice are known to be susceptible to prion disease, we propose that ingestion of infected rodent parts, possibly droppings, may be a possible mode of transmission of scrapie or BSE to humans.     

Differential diagnosis of infections with the bovine spongiform encephalopathy (BSE) and scrapie agents in sheep.

Scrapie, bovine spongiform encephalopathy (BSE), and variant Creutzfeldt-Jakob disease belong to the group of disorders called transmissible spongiform encephalopathies or prion diseases. The possibility that some sheep may be infected with the BSE agent is of human and animal health concern. Immunohistochemical methods were used to identify specific prion protein (PrP) peptide sequences in specific cell types of the brain and lymphoreticular system (LRS) of sheep with natural scrapie and Suffolk and Romney sheep infected experimentally with the BSE agent. Clinically affected and some pre-clinical cases of BSE infection could be distinguished from scrapie cases by the lesser amount of labelling of PrP containing the 84-102 amino-acid peptide sequences in phagocytic cells of the LRS and brain. Additionally, BSE-infected sheep had higher degrees of intra-neuronal PrP accumulation in the brain, as detected by labelling for a range of PrP peptide sequences. These results suggest that there is strain-dependent processing of PrP in specific cell types within the nervous system and LRS which can be used to distinguish BSE- and scrapie-infected sheep.     

Pathogenesis and prevalence of variant Creutzfeldt-Jakob disease

In the late 1980s and early 1990s, there was widespread exposure of the UK population to bovine spongiform encephalopathy (BSE)-contaminated food products, which has led to over 150 deaths from variant Creutzfeldt-Jakob disease (vCJD). Although the pathogenesis in humans is not fully understood, data from animal models and, to a lesser extent, patients with vCJD suggest that oral exposure to BSE is rapidly followed by accumulation of PrP(res) in gut-associated lymphoid tissue, then, after haematogenous spread, throughout the lymphoreticular system. Spread to the central nervous system may not occur for several years, but blood from individuals in the pre-clinical phase appears to be able to transmit disease. The incidence of vCJD has remained low and is in decline, but it is known from iatrogenic CJD and kuru that human prion disease can have incubation periods of up to 40 years. Cases of vCJD are therefore likely to occur for many more years and alternative phenotypes may develop in individuals with different PRNP genotypes to those seen to date. Studies in transgenic mice have shown that sub-clinical infection is frequent following oral exposure to BSE and a study looking at the accumulation of PrP in anonymized human lymphoid tissue samples found positive cases. There are likely to be a number of asymptomatic 'carriers' of disease within the UK and although it is unclear whether these individuals will develop clinical disease, there is a potential for iatrogenic spread to others. These uncertainties highlight the importance of developing a reliable blood test for vCJD and the continued need for surveillance.     

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.