Detection of PrP(Sc) in rectal biopsy and necropsy samples from sheep with experimental scrapie

Scrapie diagnosis is based on the demonstration of disease-associated prion protein (PrP(Sc)) in brain or, in the live animal, in readily accessible peripheral lymphoid tissue. Lymphatic tissues present at the rectoanal line were readily obtained from sheep without the need for anaesthesia. The presence of PrP(Sc) in such tissue was investigated in sheep infected orally with scrapie-infected brain material. The methods used consisted of immunohistochemistry and histoblotting on biopsy and post-mortem material. PrP(Sc) was detected in animals with PrP genotypes associated with high susceptibility to scrapie from 10 months after infection, i.e., from about the time of appearance of early clinical signs. In the rectal mucosa, PrP(Sc) was found in lymphoid follicles and in cells scattered in the lamina propria, often near and sometimes in the crypt epithelium. By Western blotting, PrP(Sc) was detected in rectal biopsy samples of sheep with the PrP genotype VRQ/VRQ, after electrophoresis of material equivalent to 8 mg of tissue. This study indicated that rectal biopsy samples should prove useful for the diagnosis of scrapie in sheep.     

Occurrence and distribution of infection-specific PrP in tissues of clinical scrapie cases and cull sheep from scrapie-affected farms in Shetland

The prion protein (PrP) genotypes of all cull sheep originating from four scrapie-affected farms in Shetland in 1998-1999 were determined and a representative sample of the different genotypes was selected for necropsy. Samples of brain and selected viscera were removed from 159 such sheep aged 2-11 years. These samples were examined immunohistochemically and by Western blotting for infection-specific forms of PrP. None of the sheep bearing the following genotypes showed any evidence of PrP accumulation in brain, intestine, selected lymph nodes or the cranial mesenteric ganglia: ARQ/ARQ (n = 41), ARQ/ARH (n = 12), ARH/ARH (n = 2), ARQ/ARR (n = 24), ARR/ARR (n= 2). In five of 71 sheep bearing a single VRQ allele, PrP accumulation was detected immunohistochemically in viscera or brain, or both. These results suggested that only a small proportion of susceptible sheep showed evidence of infection (accumulation of PrP) on the farms studied, and that even sheep of the most susceptible genotype (VRQ/VRQ) did not invariably develop disease in an infected environment. Furthermore, there was no evidence that, in sheep of semi-resistant or fully resistant genotypes, infection could be sequestered within the lymphoreticular system or peripheral nervous system and thereby provide a possible "carrier" source of infection. Rather, the data suggested that some sheep, possibly because they had been exposed to a relatively low infective dose, became infected and accumulated the infective agent over a protracted pre-clinical phase of the disease. Such sheep might be potentially infective for many years. In two VRQ/ARR genotype sheep, PrP was confined to the brain. Infection-specific PrP was also confined to the brain in two of 24 clinical cases of VRQ/ARQ scrapie. Thus, direct neuroinvasion, apparently without a prior phase of replication in the lymphoreticular system, occurred in a proportion of VRQ/ARQ sheep. Possibly it may occur in all sheep of the VRQ/ARR genotype. The factors responsible for direct neuroinvasion are not understood. However, it cannot be attributed to genotype alone.     

Vacuolar lesion profile in sheep scrapie: factors influencing its variation and relationship to disease-specific PrP accumulation

Detailed neuropathological examination for vacuolar lesions was performed on the brains of 42 sheep with clinical signs compatible with scrapie. The sheep were grouped according to their breed (Poll-Dorset, Cheviot, Welsh Mountain, Shetland and Suffolk), their PrP genotype at codons 136, 154 and 171 (VRQ/VRQ, VRQ/ARQ, VRQ/ARR and ARQ/ARQ) and the type of infection (experimental infection with SSBP/1, or natural disease). Twenty-two neuroanatomical sites from seven brain regions were examined for vacuolation in the neuropil and five sites at the level of the obex were examined for intraneuronal vacuolation. In 36 sheep, immunohistochemical examination for disease-specific PrP (PrP(d)) accumulation had also been performed in the same brain regions in an earlier study. The magnitude of total neuropil vacuolation was highest in the naturally affected ARQ/ARQ Suffolk sheep and lowest in the experimentally infected VRQ/VRQ Cheviot sheep and VRQ/ARR Poll-Dorset sheep. The severity of neuropil vacuolation at nine of the 22 neuroanatomical sites examined was used to generate a vacuolar lesion profile, which showed variations between the different sheep groups. These variations could be attributed to both PrP genotype and sheep breed and also possibly to scrapie agent; there was, however, considerable individual variation in lesion profile within sheep groups. All groups showed a similar ratio of neuropil vacuolation to neuronal vacuolation at the level of the obex. Although a positive correlation between neuropil vacuolation and PrP(d) deposition was generally observed, it was low except for the astrocyte-associated pattern of PrP(d) accumulation. The study suggests that vacuolar lesion profiles in sheep are affected by several factors and, by comparison with lesion profiles in mice, are of no more than limited value for discriminating between scrapie strains.     

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.     

Effects of agent strain and host genotype on PrP accumulation in the brain of sheep naturally and experimentally affected with scrapie

Different cellular and neuroanatomical types of disease-specific prion protein (PrP(d)) accumulation in the brain were identified in sheep of different breeds and PrP genotypes exposed to experimental or natural scrapie infection. Immunohistochemical examination of the brains of 43 sheep with clinical signs compatible with scrapie revealed 12 different PrP(d)types, which were subjectively quantified in eight different brain regions. The PrP(d)types were grouped into four PrP(d)patterns, the relative magnitude of which provided the PrP(d)profile of each sheep examined. The analysis of the differences in magnitude and relative proportion of each of these PrP(d)types and patterns indicated (1) an effect of the scrapie strain on the PrP(d)profile, and (2) a possible effect of the host genotype on the magnitude of PrP(d)accumulation in the brain, apparently related to the incubation period. Furthermore, intraneuronal deposition of PrP(d)was the type most closely associated with the development of clinical disease. We conclude that different scrapie strains can be distinguished by PrP immunohistochemical examination of brains of affected animals.     

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.     

Ileal tract and Peyer’s patch innervation in scrapie-free versus scrapie-affected ovines

Ileal Peyer’s patches (PPs) are involved early during sheep scrapie infection. This study qualitatively and semi-quantitatively evaluated ileal tract and PP innervation in 29 Sarda ovines of different age, PrP genotype and scrapie status. A prominent network of fibres was detected within PPs, mainly located in interfollicular lymphoid and stromal components. Intrafollicular fibres were rarely observed, with no apparent differences between scrapie-free and scrapie-affected animals, or among ovines carrying different PrP genotypes. In adult sheep, independent of their scrapie status, nerve fibres could be detected infrequently, close to the follicle-associated epithelium. Fibres were also detected within newly formed follicles and intrafollicular microgranulomas.     

Onset and distribution of tissue prp accumulation in scrapie-affected suffolk sheep as demonstrated by sequential necropsies and tonsillar biopsies.

Tonsillar biopsies (single or multiple) or necropsies, or both, were performed on sheep taken from a Suffolk flock in which frequent cases of scrapie had occurred over a period of several years. Clinically affected sheep of the susceptible PrP(AQ/AQ)genotype had widespread disease-specific PrP accumulation in the central nervous system (CNS), lymphoreticular system and peripheral ganglia. In nine healthy PrP(AQ/AQ)Suffolk sheep between 4 and 7 years of age, PrP could not be demonstrated post mortem in any of the lymphoreticular tissues, or in the peripheral ganglia or CNS. Tonsillar biopsies taken from animals of the resistant PrP(AR/AR)and PrP(AR/AQ)genotypes at age 3, 8, 14, 20 or 26 months did not show PrP accumulation. Disease- specific PrP accumulation in tonsillar biopsies from PrP(AQ/AQ)sheep was not seen in 20 animals aged 3 months, but was found in two of 10 animals at age 8 months and in eight of 10 animals at age 20 months. The numbers of PrP-positive tonsillar biopsies obtained from sheep previously biopsied on more than one occasion was greater than the number of positive tonsils obtained from other susceptible sheep of comparable ages. The earliest disease-specific PrP accumulation seen was in tingible body macrophages within germinal centres and only later was it detected in cells resembling follicular dendritic cells. Fourteen PrP(AQ/AQ)sheep examined post mortem at up to 17 months of age and which had not previously been biopsied or were biopsied only once had no CNS or tonsillar PrP accumulations. Two of these sheep subjected to necropsy at 14 months had PrP accumulation in lymphoreticular tissue, where it was confined to the mesenteric lymph nodes. In susceptible sheep, only low levels of immunohistochemically detectable PrP were present in a minority of follicles from tonsillar biopsies of young lambs, but by 14 months of age widespread PrP accumulation, affecting many or even all follicles, was present. Although clinical cases had widespread PrP accumulations in viscera, susceptible survivors had no such accumulations in tissues of the lymphoreticular system, peripheral nervous system or CNS, suggesting that some animals were not exposed to infection or were exposed to a non-infectious dose. Copyright Harcourt Publishers Ltd.     

Detection of PrPSc in lymphoid tissues of lambs experimentally exposed to the scrapie agent

Histoblotting and immunohistochemistry were used to detect disease-associated prion protein (PrP(Sc)) in lymphoid tissues of lambs of known PrP genotype infected with the scrapie agent by stomach tube at the age of 2 months. The ileal and jejunal Peyer's patches and retropharyngeal and distal jejunal lymph nodes were studied 1 week, 5 weeks, 5 months and 11 months after inoculation. Other lymphoid tissues examined included superficial cervical lymph node, tonsil and spleen. PrP(Sc) was not detected in any tissue of any lamb at 1 week post-inoculation. At 5 weeks, PrP(Sc) was detected in tissues of lambs of susceptible PrP genotypes (AV(136)QQ(171) and VV(136)QQ(171)), but not lambs of other PrP genotypes (AA(136)QQ(171), AA(136)QR(171) and AV(136)QR(171)). PrP(Sc) was present in the germinal centres of tonsils, distal jejunal and retropharyngeal lymph nodes, and spleen. In the nodules of ileal and jejunal Peyer's patches, only occasional solitary cells showed the presence of PrP(Sc). At 5 months post-inoculation, increased accumulations of PrP(Sc) were detected in ileal and jejunal Peyer's patches, as well as in the retropharyngeal and distal jejunal lymph nodes of a single lamb inoculated with the agent from a sheep of the same susceptible PrP genotype. Eleven months after exposure to the scrapie agent, PrP(Sc) was detected in all lymphoid tissues examined from sheep of susceptible PrP genotypes. These studies show that PrP(Sc) was detectable in lymphoid tissues 5 weeks after exposure to the scrapie agent by stomach tube in lambs as young as 3 months of age and indicate that the PrP genotype is a significant factor for the rapid uptake and spread of the agent through lymphoid tissues.     

Prion-induced toxicity in PrP transgenic Drosophila

Prion diseases are fatal transmissible neurodegenerative diseases of humans and various vertebrate species. In their natural hosts these conditions are characterised by prolonged incubation times prior to the onset of clinical signs of terminal disease. Accordingly, tractable models of mammalian prion disease are required in order to better understand the mechanisms of prion replication and prion-induced neurotoxicity. Transmission of prion diseases can occur across a species barrier and this is facilitated in recipients transgenic for the same PrP gene as the individual from which the infectious prions are derived. Here we have tested the hypothesis that exogenous ovine prions can induce neurotoxicity in Drosophila melanogaster transgenic for ovine PrP. Drosophila that expressed ovine PrP pan neuronally and inoculated with ovine prions at the larval stage by oral exposure to scrapie-infected sheep brain homogenate showed markedly accelerated locomotor and survival defects. ARQ PrP transgenic Drosophila exposed to scrapie-infected brain homogenate showed a significant and progressive reduction in locomotor activity compared to similar flies exposed to normal sheep brain homogenate. The prion-induced locomotor defect was accompanied by the accumulation of potentially misfolded PrP in the brains of prion-inoculated flies. VRQ PrP transgenic Drosophila, which expressed less ovine PrP than ARQ flies, showed a reduced median survival compared to similar flies exposed to normal sheep brain homogenate. These prion-induced phenotypic effects were PrP-mediated since ovine prions were not toxic in non-PrP transgenic control flies. Our observations provide the basis of an invertebrate model of transmissible mammalian prion disease.     

Preclinical diagnosis of scrapie by immunohistochemistry of third eyelid lymphoid tissue

Ovine scrapie is a member of the transmissible spongiform encephalopathies (TSEs), a heterogeneous family of fatal neurologic disorders characterized by deposition of an abnormal isoform (prion protein [PrP] PrP-Sc) of a cellular sialoglycoprotein in neural tissue. PrP-Sc is detectable in some lymphoid tissues of infected sheep months or years before development of clinical disease. Detection of PrP-Sc in these tissues is the basis for live-animal testing. In this study, we characterize the performance of a preclinical diagnostic test for ovine scrapie based on a monoclonal antibody (MAb)-based immunohistochemistry assay of nictitating membrane ("third eyelid")-associated lymphoid tissue. The results of third eyelid immunohistochemistry assay agreed with the scrapie status of the sheep for 41 of 42 clinical suspects with confirmed scrapie and 174 of 175 sheep without scrapie. Third eyelid sampling agreed with the scrapie status for 36 of 41 clinically normal sheep positive for PrP-Sc immunostaining of brain tissue, including 27 sheep with positive biopsy specimens that progressed to clinical disease with confirmed scrapie 3 to 20 months after biopsy. The assay used MAb F89/160.1.5, which binds to residues 142 to 145 of ovine PrP. This antibody can be used in combination with MAb F99/97. 6.1, which binds to residues 220 to 225. One or both MAbs in this cocktail recognize PrP sequences conserved in most mammalian species in which natural TSEs have been reported. Immunohistochemistry assay of routinely formalin-fixed lymphoid tissues with a cocktail of pan-specific MAbs is a practical, readily standardized live-animal and preclinical test for ovine scrapie.     

Immunohistochemical detection of prion protein in lymphoid tissues of sheep with natural scrapie.

The scrapie-associated form of the prion protein (PrPSc) accumulates in the brain and lymphoid tissues of sheep with scrapie. In order to assess whether detecting PrPSc in lymphoid tissue could be used as a diagnostic test for scrapie, we studied the localization and distribution of PrPSc in various lymphoid tissues collected at necropsy from 55 sheep with clinical scrapie. Samples collected from the spleen, palatine tonsil, ileum, and five different lymph nodes were immunohistochemically stained for PrPSc. PrPSc was found to be deposited in a reticular pattern in the center of both primary and secondary lymphoid follicles. In addition, granules of PrPSc were seen in the cytoplasm in macrophages associated with the lymphoid follicles. In 54 (98%) of the 55 scrapie-affected sheep, PrPSc was detected in the spleen, retropharyngeal lymph node, mesenteric lymph node, and the palatine tonsil. However, only in the palatine tonsils was PrPSc present in a consistently high percentage of the lymphoid follicles. PrP was not detected in any of the lymphoid tissues of 12 sheep that had no neurohistopathological signs of a scrapie infection. We conclude that the tonsils are the best-suited lymphoid tissue to be biopsied for the detection of PrPSc in the diagnosis of clinical scrapie in living sheep.     

Active surveillance for scrapie by third eyelid biopsy and genetic susceptibility testing of flocks of sheep in Wyoming

Control of scrapie, an ovine transmissible spongiform encephalopathy or prion disorder, has been hampered by the lack of conventional antemortem diagnostic tests. Currently, scrapie is diagnosed by postmortem examination of the brain and lymphoid tissues for PrP(Sc), the protein marker for this group of disorders. For live, asymptomatic sheep, diagnosis using tonsil or third-eyelid lymphoid tissue biopsy and PrP(Sc) assay has been described. To evaluate the feasibility and efficacy of third-eyelid testing for identification of infected flocks and individual infected sheep, 690 sheep from 22 flocks were sampled by third-eyelid lymphoid tissue biopsy and immunohistochemistry. Sheep were further evaluated for relative genetic susceptibility and potential contact exposure to scrapie. Third-eyelid testing yielded suitable samples for 80% of the sheep tested, with a mean of 18.1 lymphoid follicles (germinal centers) per histologic section. Three hundred eleven of the sheep were sampled through passive surveillance programs, in which only sheep with potential contact with an infected sheep at a lambing event were tested, regardless of their scrapie susceptibility genotype. In addition, 141 genetically susceptible sheep with no record of contact with an infected animal at a lambing event were sampled through a targeted active surveillance program. Ten PrP(Sc)-positive sheep were identified through the passive surveillance program, and an additional three PrP(Sc)-positive sheep, including two from flocks with no history of scrapie, were identified through the active surveillance program. All PrP(Sc)-positive sheep had the highly susceptible PrP genotype. Third-eyelid testing is a useful adjunct to flock monitoring programs, slaughter surveillance, and mandatory disease reporting in a comprehensive scrapie eradication and research program.     

Immunophenotype of cells within cervine rectoanal mucosa-associated lymphoid tissue and mesenteric lymph nodes

Rectoanal mucosa-associated lymphoid tissue (RAMALT) is a part of the lymphoid system that can be sampled easily in live animals, especially ruminants. RAMALT biopsy is useful for the diagnosis of transmissible spongiform encephalopathies, including scrapie in sheep and goats and chronic wasting disease (CWD) in cervids. Diagnosis is reliant on detection of abnormal prion protein (PrP(d)), which is associated with lymphoid follicles. For enzyme linked immunosorbent assays (ELISAs) detecting PrP(d) it is necessary to ensure that lymphoid follicles are present in biopsy samples to avoid false-negative results. Monoclonal antibodies known to recognize specific immune cell subsets present in lymphoid tissues of sheep were tested for cross-reactivity with cervine RAMALT and mesenteric lymph nodes (MLNs) preserved in zinc salts fixative. The distribution of cells expressing CD3, CD4, CD79, CD21 and class II molecules of the major histocompatibility complex was determined in these tissues. Cells of each immunophenotype had similar distributions in RAMALT and MLNs and these distributions were similar to those reported previously for sheep and cattle. The identification and validation of cervine lymphoid follicle cell markers (CD79 and CD21) may allow reduction in false-negative results during diagnosis of CWD by ELISA.     

Diagnosis and PrP genotype target of scrapie in clinically healthy sheep of Massese breed in the framework of a scrapie eradication programme

Summary. The application of a selective culling programme in two scrapie affected flocks of Massese breed sheep is described. The genetic susceptibility of this breed and the sensitivity of different diagnostic methods in the pre-clinical diagnosis of scrapie were also investigated. Overall, 2,068 clinically healthy sheep underwent PrP genotyping, providing the basis for selective culling. The prevalence of scrapie infection was investigated in susceptible sheep by two independent diagnostic methods. All the sheep older than 18 months (n = 620) were tested by Prionics^® Check Western rapid test on the obex, with a prevalence of infection of 3.9%. Furthermore, 385 sheep underwent immunohistochemistry (IHC) on retropharyngeal lymph node (RPLN), with a prevalence of infection of 5.2%. Overall, 32 sheep were diagnosed with pre-clinical scrapie. Of these, 31 were positive by Western blot on the spleen, 29 by IHC on the RPLN and tonsil, 28 by IHC on the obex, 24 by rapid test, and only 18 by IHC on the third eyelid. All the scrapie positive sheep were of the ARQ/ARQ, ARQ/AHQ or ARQ/VRQ genotypes. No significant differences in scrapie prevalence were observed among these genotypes. The estimated risk of the three targeted alleles was also similar, suggesting that in this breed the VRQ allele was not at higher risk for scrapie, compared to the ARQ and AHQ alleles.     

Prion protein gene polymorphism and genetic risk evaluation for scrapie in all Turkish native sheep breeds

The aim of this study was to identify the prion protein (PrP) gene polymorphism in a total of 1,110 healthy sheep from 18 Turkish native sheep breeds. There were nine alleles and 22 genotypes observed based on codons 136, 154, and 171 of the PrP gene. The ARQ allele was predominant for all breeds. The most resistant allele to scrapie, ARR, was present in all breeds. The VRQ allele, associated with the highest susceptibility to scrapie, was detected at low frequencies in ?vesi (0.06), K?v?rc?k (0.021), Sak?z (0.010), Karayaka (0.011), Çine Çapar? (0.012), and Güneykaraman (0.017). In general, the ARQ/ARQ genotype was predominant in all breeds. The most resistant genotype to scrapie, ARR/ARR, was found with the frequency lower than 0.180. The most susceptible genotype, VRQ/VRQ, was found in only K?v?rc?k. The TRR and TRH alleles and the genotypes of ARR/TRR, ARR/ARK, and ARH/TRH have been found for the first time in Turkish native sheep breeds. According to these results, all breeds belong to risk group R3 followed by R2. It is propounded that the susceptibility to scrapie increased from eastern to western part of Turkey. Our findings of Turkish native sheep breeds with PrP gene polymorphisms will assist the sheep breeding program for selection of scrapie resistance genotypes to reduce the risk of scrapie.     

High incidence of subclinical infection of lymphoid tissues in scrapie-affected sheep flocks

Prion diseases are characterized by a long incubation period. In scrapie, sheep may incubate and spread the infection for several years before clinical signs evolve. We have previously studied the occurrence of subclincal infection in the brain. Now, we have studied the occurrence of subclinical infection in the brain and several lymphoid tissues in two scrapie-affected Icelandic sheep flocks by immunohistochemistry for PrP^Sc, a molecular marker for infectivity, and correlated this with results of PrP genotyping. At culling, one flock had one confirmed scrapie case, while the other flock had two. Analysis of 106 asymptomatic sheep by immunostaining for PrP^Sc revealed that the incidence of subclinical infection was 58.3% in one flock and 42.5% in the other. PrP^Sc was only detected in lymphoid tissues. The youngest positive sheep were 4 months old. PrP genotyping showed that over 90% of the sheep were of a genotype which is moderately sensitive to infection and may delay neuroinvasion. Our results show that asymptomatic sheep may spread the infection during the long incubation period of several years, which constitutes an important obstacle in the eradication of scrapie. Our findings indicate that contamination of the environment plays an important part in sustaining the infection.