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.     

The major species specific epitope in prion proteins of ruminants

Summary The species specific nature of an antigenic determinant previously discovered in the scrapie form of prion protein (PrP^D) from cattle, sheep and mice, was further investigated in normal prion protein (PrP^C) from these and other species. This was carried out with eight different anti-peptide sera raised in rabbits against various synthetic peptides representing segments of the amino acid (aa) sequence 101–122 of ovine, bovine, murine and hamster PrP. Anti-peptide serum against a peptide representing aa 107–122 of ovine PrP showed almost specific reaction and crossreacted in immunoblot with caprine and human PrP only. Antisera to the corresponding bovine sequence stained bovine and porcine PrP and to a minor extent PrP of goat, man, cat, and mink, while antiserum to the murine aa sequence reacted with rodent and monkey PrP only. In contrast, antiserum to the corresponding hamster sequence displayed a broader reactivity pattern, just like the four other anti-peptide sera to various ovine and bovine sequences. Antisera were also tested for reactivity with the pathogenic isoforms of PrP of sheep, cow, hamster and mouse and showed generally similar reactivity patterns as by using PrP^C. In conclusion, the region close to the actual or putative proteinase K cleavage sites of PrP seems to exhibit high structural variability among mammalian species.     

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.     

Preparation and characterization of antibodies against mouse prion protein (PrP) peptides.

Antisera were raised in rabbits against three peptides, representing amino acid sequences 150 to 159, 165 to 174, and 213 to 226 of mouse prion (PrP), which were synthesized by using a multiple antigenic peptide (MAP) system. The reactivities of these sera to PrP were examined by an enzyme-linked immunosorbent assay (ELISA), Western immunoblotting (WB), and immunohistochemical procedures. The results of both ELISA and WB showed that antisera to peptide sequence 150 to 159 (Ab150-159) did not react with purified mouse PrP. On the other hand, sera to the sequence 165 to 174 (Ab165-174) reacted weakly with purified mouse PrP, as detected by WB but not by ELISA. However, antiserum to peptide sequence 213 to 226 (Ab213-226) reacted strongly with mouse, Syrian hamster, and sheep PrP by WB and with mouse PrP as shown by the results of ELISA. Moreover, Ab213-226 clearly detected PrP immunohistochemically in mouse, Syrian hamster, and sheep brains affected with scrapie as well as in the brain of a cow with bovine spongiform encephalopathy. From these data, we conclude that rabbit antiserum against the MAP representing amino acid sequence 213 to 226 of mouse PrP is useful as a diagnostic tool for prion disease of animals.     

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.     

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.     

The ultrastructural diversity of scrapie-associated fibrils isolated from experimental scrapie and Creutzfeldt-Jakob disease.

Several different samples of scrapie-associated fibrils (SAF) were extracted in identical fashion from the brains of golden Syrian hamsters infected with the 263K strain of scrapie agent and NIH Swiss mice infected with the Fujisaki strain of Creutzfeldt-Jakob disease (CJD) agent. Based on a total of over 500 measurements in individual fibrils in different extracts, hamster fibrils were more abundant, thicker and had better defined substructure than mouse fibrils. Hamster protofibrils were usually either twisted helically or in parallel arrays, whereas mouse protofibrils were often twisted, occasionally parallel, or could not be morphologically defined. Thus, SAF preparations from scrapie-affected hamsters can be ultrastructurally distinguished from those of CJD-affected mice, an observation that presumably reflects differences in their respective host-encoded amyloid protein subunits.     

Similar Signature of the Prion Protein in Natural Sheep Scrapie and Bovine Spongiform Encephalopathy-Linked Diseases

It has been suggested that specific molecular features could characterize the protease-resistant prion protein (PrP res) detected in animal species as well as in humans infected by the infectious agent strain that causes bovine spongiform encephalopathy (BSE). Studies of glycoform patterns in such diseases in French cattle and cheetahs, as well as in mice infected by isolates from both species, revealed this characteristic molecular signature. Similar studies of 42 French isolates of natural scrapie, from 21 different flocks in different regions of France, however, showed levels of the three glycoforms comparable to those found in BSE-linked diseases. Moreover, the apparent molecular size of the unglycosylated form was also indistinguishable among all different sheep isolates, as well as isolates from BSE in cattle. Overall results suggest that scrapie cases with features similar to those of BSE could be found more frequently in sheep than previously described.     

All major prion types recognised by a multiplex immunofluorometric assay for disease screening and confirmation in sheep

Prion diseases or transmissible spongiform encephalopathies (TSEs) in small ruminants are presented in many forms: classical scrapie, Nor98/atypical scrapie, CH1641 scrapie and bovine spongiform encephalopathy (BSE). We previously described a multiplex immunofluorometric assay (mIFMA), based on a bead array flow cytometry technology, which provided, in a single assay, discrimination between BSE (in cattle and sheep) and classical scrapie (Tang et al., 2010). In this study, we extended the mlFMA to differentiate classical scrapie, atypical scrapie, BSE (experimentally infected sheep and naturally infected cattle) and CH1641 (both experimental and natural CH1641-like infections in sheep). Three capture antibodies were used, two distinct PrP N-terminus specific antibodies 12B2 and 9A2, and a PrP core specific antibody 94B4. All three antibodies were shown to bind classical scrapie PrP(res) strongly, whereas in Nor98/atypical scrapie PrP(res) only 12B2 and 9A2 binding was observed. PrP(res) binding of 12B2 was low for both BSE and CH1641, as expected. Furthermore, analysis of serially diluted samples indicated that the assay provided a similar level of sensitivity for atypical scrapie as that found using a well established commercial test. Unexpectedly, 9A2 binding to CH1641 PrP(res) was reduced by 2.1 fold both for experimental CH1641 and CH1641-like scrapie when compared with BSE, suggesting that major cleavage of the N-terminus occurs further towards the C-terminus in CH1641 than in BSE. The ratios of 12B2/94B4 and 9A2/94B4 were similar between experimental CH1641 and CH1641-like cases, although two CH1641-like subjects displayed slightly elevated ratios of both 12B2/94B4 and 9A2/94B4. To verify this finding for PrP(res), mass spectrometry based quantification was used to determine the absolute abundance of the peptides associated with all three antibody binding regions. There was a 2.2 fold reduction of peptides containing the 9A2 epitope for experimental CH1641 PrP(res) in comparison to BSE PrP(res). Observation of reduced PrP(res) may serve as a new marker for CH1641. This mIFMA may thus provide the basis for simplified TSE diagnosis with capability for simultaneous screening and differential diagnosis.     

A novel epitope for the specific detection of exogenous prion proteins in transgenic mice and transfected murine cell lines

Prion diseases are closely linked to the conversion of host-encoded cellular prion protein (PrPC) into its pathological isoform (PrPSc). PrP conversion experiments in scrapie infected tissue culture cells, transgenic mice, and cell-free systems usually require unique epitopes and corresponding monoclonal antibodies (MAbs) for the immunological discrimination of exogenously introduced and endogenous PrP compounds (e.g., MAb 3F4, which is directed to an epitope on hamster and human but not on murine PrP). In the current work, we characterize a novel MAb designated L42 that reacts to PrP of a variety of species, including cattle, sheep, goat, dog, human, cat, mink, rabbit, and guinea pig, but does not bind to mouse, hamster, and rat PrP. Therefore, MAb L42 may allow future in vitro conversion and transgenic studies on PrPs of the former species. The MAb L42 epitope on PrPC includes a tyrosine residue at position 144, whereas mouse, rat, and hamster PrPs incorporate tryptophane at this site. To verify this observation, we generated PrP expression vectors coding for authentic or mutated murine PrPCs (i.e., codon 144 encoding tyrosine instead of tryptophan). After transfection into neuroblastoma cells, MAb L42 did not react with immunoblotted wild-type murine PrPC, whereas L42 epitope-tagged murine PrPC was strongly recognized. Immunoblot and fluorescence-activated cell sorting data revealed that tagged PrPC was correctly posttranslationally processed and translocated to the cell surface.     

Characterisation of antisera raised against species-specific peptide sequences from scrapie-associated fibril protein and their application for post-mortem immunodiagnosis of spongiform encephalopathies

Summary Transmissible spongiform encephalopathies (TSE), such as scrapie or Creutzfeldt-Jakob disease (CJD), are fatal neurodegenerative diseases of the central nervous system caused by a yet unidentified virus. They are accompanied by a brain specific amyloidosis, during which a host coded protein irreversibly aggregates to form the scrapie-associated fibrils. The diagnosis of TSE relies on histopathological detection of spongiform lesions, on electron microscopical detection of fibrils, or on the immunological detection of SAF protein, which is the most specific diagnostic marker. In order to improve the diagnosis of TSE, we developed a protocol for rapid tissue fractionation and enrichment of SAF protein which subsequently allows the specific detection of SAF protein by western blotting and immunodetection. Using some new antisera raised against synthetic peptides with sequences specific for the hamster, sheep, cattle and human SAF protein, several samples can be diagnosed for TSE within 24 hours, starting with only 10–100 mg of brain tissue from different species.     

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.     

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.     

Distinct proteinase K-resistant prion protein fragment in goats with no signs of disease in a classical scrapie outbreak

Considerable efforts have been directed toward the identification of small-ruminant prion diseases, i.e., classical and atypical scrapie as well as bovine spongiform encephalopathy (BSE). Here we report the in-depth molecular analysis of the proteinase K-resistant prion protein core fragment (PrP(res)) in a highly scrapie-affected goat flock in Greece. The PrP(res) profile by Western immunoblotting in most animals was that of classical scrapie in sheep. However, in a series of clinically healthy goats we identified a unique C- and N-terminally truncated PrP(res) fragment, which is akin but not identical to that observed for atypical scrapie. These findings reveal novel aspects of the nature and diversity of the molecular PrP(res) phenotypes in goats and suggest that these animals display a previously unrecognized prion protein disorder.     

Use of a new immunoassay to measure PrP Sc levels in scrapie-infected sheep brains reveals PrP genotype-specific differences

The diagnosis of prion diseases, such as scrapie and BSE, has traditionally relied upon the identification of the disease-associated form of the prion protein, PrP(Sc), based on its resistance to digestion by proteinase K (PK). A more recent development is the conformation-dependent immunoassay (CDI), which distinguishes between PrP Sc and normal PrP (PrP C) based on their differing solubility in guanidine hydrochloride rather than resistance or sensitivity to PK. We have developed a CDI-formatted sandwich immunoassay for the measurement of PrP Sc in sheep brain, which discriminates between clinically affected scrapie cases (natural or experimental) and uninfected controls of the same PrP genotype. Using this method, we have shown for the first time that, in sheep, the PrP genotype has a significant influence on the amount of PrP Sc deposited in the brains of animals experimentally infected with scrapie.     

Isolation and purification of scrapie-associated fibrils and prion protein from scrapie-infected hamster brain

We report the purification of prion protein (PrP) 27-30 and scrapie-associated fibrils (SAF) from hamsters infected with the 263K strain of scrapie. SDS-PAGE of fractions purified from scrapie-infected brains revealed several bands at approximately 28.5 kDa, 23.9 kDa and 14.3 kDa and, in one set of preparations, a protein of Mr 26 kDa was found in both scrapie-infected and sham-inoculated animals. The specificity of PrPs was confirmed by Western blotting. Ultrastructural analysis of fractions from scrapie-infected brains revealed numerous fibrils measuring approximately 20 nm in diameter and 100 to 200 nm in length. The substructure of these fibrils consisted of protofilaments which were usually straight and rarely helically arranged. We conclude that the electron microscopical appearance of SAF depends much on the purification scheme. The PrP27-30 as well as proteins of lower Mr are easily detectable in scrapie-infected brains. The detection of protein of a Mr 26 kDa in both scrapie-infected and sham-inoculated animals suggests that this form of PrP may exist in equilibrium with PrP33-35c.     

Early and late pathogenesis of natural scrapie infection in sheep

The pathogenesis of scrapie infection was studied in sheep carrying the PrP(VRQ)/PrP(VRQ) genotype, which is associated with a high susceptibility for natural scrapie. The sheep were killed at sequential time points during a scrapie infection covering both the early and late stages of scrapie pathogenesis. Various lymphoid and neural tissues were collected and immunohistochemically examined for the presence of the scrapie-associated prion protein PrP(Sc), a marker for scrapie infectivity The first stage of scrapie infection consisted of invasion of the palatine tonsil and Peyer's patches of the caudal jejunum and ileum, the so-called gut-associated lymphoid tissues (GALT). At the same time, PrP(Sc) was detected in the medial retropharyngeal lymph nodes draining the palatine tonsil and the mesenteric lymph nodes draining the jejunal and ileal Peyer's patches. From these initial sites of scrapie replication, the scrapie agent disseminated to other non-GALT-related lymphoid tissues. Neuroinvasion started in the enteric nervous system followed by retrograde spread of the scrapie agent via efferent parasympathetic and sympathetic nerve fibres innervating the gut, to the dorsal motor nucleus of the vagus in the medulla oblongata and the intermediolateral column of the thoracic spinal cord segments T8-T10, respectively.     

Atypical scrapie cases in Germany and France are identified by discrepant reaction patterns in BSE rapid tests

The intensified surveillance of scrapie in small ruminants in the European Union (EU) has resulted in a substantial increase of the number of diagnosed cases. Four rapid tests which have passed the EU evaluation for BSE testing of cattle are also recommended currently and used for the testing of small ruminants by the EU authorities. These tests include an indirect ELISA (cELISA), a colorimetric sandwich ELISA (sELISA I), a chemiluminescent sandwich ELISA (sELISA II), and a Western blot (WB). To this point, the majority of samples have been screened by using either sELISA I (predominantly in Germany) or WB (predominantly in France). In this study, it is shown that a number of the German and French scrapie cases show inconsistent results using rapid and confirmatory test methods. Forty-eight German sheep, 209 French sheep and 19 French goat transmissible spongiform encephalopathy (TSE) cases were tested. All cases were recognised by the sELISA I and either one of the confirmatory methods (scrapie-associated fibrils (SAF)-immunoblot or immunohistochemistry). Surprisingly, three rapid tests failed to detect a significant number of scrapie cases (29 in France and 24 in Germany). The possible reasons for these inconsistent reaction patterns of scrapie cases are discussed. Similar discrepancies have not been observed during rapid testing of cattle for BSE, the disease for which all diagnostic methods applied have been evaluated.     

Ubiquitin conjugate immunoreactivity in the brains of scrapie infected mice

Sections of brain from normal mice or clinically-ill mice infected with either the 87V or the ME7 strains of sheep scrapie were immunostained to show the localization of ubiquitin-protein conjugates or a specific marker of disease, the scrapie-associated fibril protein (PrP). In both scrapie models immunoreactive ubiquitin-protein conjugates were seen in thread-like structures found throughout the neuropil, in inclusion bodies within vacuolated neurones, and in areas surrounding anti-PrP positive amyloid plaques. The PrP protein was visualized in diffuse deposits in highly vacuolated parts of the scrapie-affected brain, and focally in amyloid plaques, microglia and neuronal processes. The ubiquitin-protein conjugate staining of scrapie amyloid plaques is very similar to that seen in the plaques of Alzheimer's disease. The ubiquitinated intraneuronal inclusion bodies seen in scrapie resemble the granulovacuolar lesions also seen in Alzheimer's disease, but appear much larger and possibly correspond to material in giant autophagic vacuoles. We suggest that these inclusions may be the result of ubiquitinated abnormal proteins being directed to the lysosomal system, and that scrapie and Alzheimer's disease share at least some common processes of neurodegeneration.     

Scrapie-associated prion protein in the gastrointestinal tract of sheep with natural scrapie.

The scrapie-associated prion protein (PrPSc), which is closely associated with scrapie infectivity, accumulates in the brain and lymphoid tissues of sheep with natural scrapie. The most probable portal of entry of the scrapie agent in sheep is the alimentary tract; little attention, however, has been paid to the gastro-intestinal tract in scrapie research. In this study, we examined the presence and distribution of PrPSc within the gastro-intestinal tract of sheep with natural scrapie and scrapie-negative sheep. It was found that PrPSc accumulated in the enteric nervous system (ENS) of all scrapie-infected sheep but not in scrapie-negative sheep. The distribution of PrPSc within the ENS was then studied along the entire gastro-intestinal tract in seven scrapie-infected sheep carrying various PrP genotypes. In sheep with the highest genetically determined susceptibility to scrapie, PrPSc was detected in the ENS from the oesophagus to the rectum. In sheep with a lower genetic susceptibility to scrapie, PrPSc was present in the ENS of the forestomachs, small intestine and large intestine but not in the oesophagus. In a scrapie-negative sheep with a PrP genotype associated with scrapie resistance, no PrPSc was seen in the ENS at any site along the gastro-intestinal tract. The presence of PrPSc within the ENS of scrapie-infected sheep indicates a possible role of the ENS in the pathogenesis of natural scrapie as a portal of entry to the central nervous system.