Search for healthy carriers of scrapie: an assessment of subclinical infection of sheep in an Icelandic scrapie flock by three diagnostic methods and correlation with PrP genotypes

Summary.  Subclinical infection in scrapie of sheep, characterized by a long incubation period, may be of importance for the spread of the disease. We screened brain samples from all 65 sheep in a scrapie-affected flock for subclinical infection and correlated with results of PrP genotyping, which is of relevance for the epidemiology and the question, whether by breeding for resistant genotypes one would be breeding for healthy carriers. The sensitivity of three methods was compared, i.e. histopathological examination for vacuoles (HP), immunohistochemical staining (IHC) and Western blotting (WB) for PrP^Sc. Five sheep showed definite clinical signs and histological scrapie lesions, and signs of infection were detected in 25 of 60 asymptomatic sheep, by HP and/or IHC and WB. The IHC was slightly more sensitive than HP and WB. Sheep with subclinical infection were, with one exception, either homo- or heterozygotes for 136-V, as were four of the five sheep with clinical scrapie. The incidence of the VRQ allelic variant in the flock was unusually high compared to the Icelandic sheep population probably contributing to the high prevalence of both clinical and subclinical infection in the flock. Neither sheep with definite scrapie nor detectable subclinical infection, were of the resistant AHQ genotype, indicating that Icelandic AHQ sheep are not healthy carriers of scrapie infection. Received September 7, 2001 Accepted December 13, 2001     

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.     

PrPSc deposition in nervous tissues without lymphoid tissue involvement is frequently found in ARQ/ARQ Sarda breed sheep preclinically affected with natural scrapie

Summary. The pathogenesis of natural scrapie in Sarda breed sheep was investigated in 1050 asymptomatic and 49 sick sheep from scrapie-affected flocks. Central and peripheral nervous system, along with lymphoreticular system (LRS) tissues, were subjected to immunohistochemistry (IHC) and Western-blotting (WB) for detection of pathological isoform of the prion protein (PrP^Sc). A total of 69 of the 1050 clinically healthy sheep were found to be infected with scrapie, with PrP^Sc being detected in both the central nervous system (CNS) and enteric nervous system (ENS) plexuses of 60 of the sheep, while IHC and WB yielded evidence of (PrP^Sc) deposition only in lymphoid tissues of the remaining 9 clinically healthy sheep. PrP^Sc was also detected in the CNS, as well as in ENS plexuses from all of the 49 clinically affected sheep. Nevertheless, 18 of the 69 clinically healthy animals (26%, 17 ARQ/ARQ and 1 ARQ/AHQ sheep), along with 3 ARQ/ARQ sheep (6%) of the clinically affected group, showed no IHC or WB evidence of PrP^Sc in lymphoid tissues, but PrP^Sc could be still detected in their CNS and ENS plexuses. The study demonstrates dual CNS and ENS PrP^Sc deposition in Sarda sheep with scrapie, in spite of an apparent lack of lymphoid tissue involvement in a number of cases.     

The association between PrP and infectivity in scrapie and BSE infected mouse brain

Summary The structure of the scrapie agent remains unknown. However, scrapie infectivity tends to co-sediment with an infection specific fraction of the glycoprotein PrP (PrP^Sc) under conditions which solubilise the normal form of this protein (PrP^c); accordingly, PrP has been proposed as a candidate component of the agent. To investigate this further we have been examining a new scrapie-related murine model in conjunction with established scrapie models. A bovine spongiform encephalopathy (BSE) derived murine model has short incubation periods, high infectivity titre and low amounts of PrP deposited in the brain. A membrane fraction from scrapie/BSE infected brain is solubilised with Sarkosyl at pH9.0. Most PrP is also solubilised. In models of the disease with little deposition of PrP in the brain, this solubilisation step is particularly effective in reducing the amounts of PrP sedimented from brain extracts. Gradient centrifugation of the sedimented fraction shows further separation of infectivity and the residual PrP. It is concluded that at least some PrP^Sc in the brain need not be associated directly with infectious agent but is deposited in brain solely as a pathological product of infection. However, a residual sedimentable fraction contains PrP which may be a component of the agent.     

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.     

Evaluation of Infective Property of Recombinant Prion Protein Amyloids in Cultured Cells Overexpressing Cellular Prion Protein

Misfolded isoform of prion protein (PrP), termed scrapie PrP (PrP^Sc), tends to aggregate into various fibril forms. Previously, we reported various conditions that affect aggregation of recombinant PrP into amyloids. Because amyloidogenesis of PrP is closely associated with transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease in humans, we investigated infectivity of recombinant PrP amyloids generated in vitro. Using cultured cell lines which overexpress cellular PrP of different species, we measured the level of de novo synthesized PrP^Sc in cells inoculated with recombinant mouse PrP amyloids. While PrP-overexpressing cells were susceptible to mouse-adapted scrapie prions used as the positive control, demonstrating the species barrier effect, infection with amyloids made of truncated recombinant PrP (PrP[89-230]) failed to form and propagate PrP^Sc even in the cells that express mouse cellular PrP. This suggests that infectivity of PrP amyloids generated in vitro is different from that of natural prions. Recombinant PrP (89-230) amyloids tested in the current study retain no or a minute level, if any, of prion infectivity.

Graphical Abstract

相似文献   

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.     

Improvement of PrPSc-detection in mouse spleen early at the preclinical stage of scrapie with collagenase-completed tissue homogenization and Sarkosyl-NaCl extraction of PrPSc

Summary Scrapie in sheep has recently become again a target of control measures and eradication programs. Crucial for the effectiveness of these measures is the detection of infected sheep during the long and potentially hazardous incubation period. However, routine-diagnosis is mostly limited to clinical examinations when disease becomes apparent, and to postmortem investigations. Through the detection of the scrapie-specific isoform of the prion protein (PrP^Sc) by Western blot in the spleen and lymph nodes from scrapie-infected mice and sheep, we have shown previously that diagnosis during the preclinical stage is possible. We introduce here an improved method for the diagnosis of mouse scrapie shortly after infection. Through a homogenization procedure that includes a collagenase digestion step, and through extraction and salting-out of PrP^Sc by Sarkosyl and NaCl, respectively, we were able to detect PrP^Sc in spleen tissue of intraperitoneally infected mice seven days postinfection. Moreover, the new protocol makes sample-handling easier and reduces the hands-on time. We also successfully enriched PrP^Sc from spleen tissue through immobilized metal affinity chromatography (IMAC); however, for the diagnosis at the earliest stage of infection, extraction of PrP^Sc by Sarkosyl and NaCl was more effective.     

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.     

Alteration of cell responses to PrPSc in prolonged cell culture and its effect on transmission of PrPSc to neural cells

The mechanisms and processes of the uptake, intracellular trafficking and intercellular spread of PrP^Sc and its transfer to neural cells are not clearly defined. The involvement of immune, intestinal, mast or peripheral neural cells in this process also remains unclear. The role of these cell types in the accumulation and transfer of PrP^Sc to neural cells was investigated following short and prolonged exposure to the Chandler and Obihiro strains of scrapie PrP^Sc for up to 28 days. Eight cell lines of murine immune, neural, intestinal and fibroblast cell types were tested. After transient degradation phases, certain immune, intestinal and neural cells accumulated PrP^Sc for up to 28 days postinfection. When co-cultured with N2a-3/EGFP neuroblastoma cells for 4 days followed by several passages, the immune, intestinal and the neural cell lines were able to transfer infection to neural cells. Our results suggest that some of these cell types may have a role in PrP^Sc accumulation and intercellular spread of PrP^Sc infection to neural cells in vivo.     

Detection of PrPSc in sheep at the preclinical stage of scrapie and its significance for diagnosis of insidious infection

Summary A field sheep insidiously infected with natural scrapie was diagnosed at the preclinical stage through detection of the core fragment of Prp^Sc (PrP^core) in peripheral lymph nodes by the biopsy method. Three out of 32 euthanized healthy sheep were found positive for PrP^core in the spleen and lymph nodes. Mice that were inoculated with spleen homogenate of one of these sheep showed clinical signs of scrapie and were positive for PrP^core in their brain samples. These results suggest that the detection of PrP^core is significant for diagnosis in the preclinical phase of scrapie.     

Uptake and Degradation of Protease-Sensitive and -Resistant Forms of Abnormal Human Prion Protein Aggregates by Human Astrocytes

Sporadic Creutzfeldt-Jakob disease is the most common of the human prion diseases, a group of rare, transmissible, and fatal neurologic diseases associated with the accumulation of an abnormal form (PrP^Sc) of the host prion protein. In sporadic Creutzfeldt-Jakob disease, disease-associated PrP^Sc is present not only as an aggregated, protease-resistant form but also as an aggregated protease-sensitive form (sPrP^Sc). Although evidence suggests that sPrP^Sc may play a role in prion pathogenesis, little is known about how it interacts with cells during prion infection. Here, we show that protease-sensitive abnormal PrP aggregates derived from patients with sporadic Creutzfeldt-Jakob disease are taken up and degraded by immortalized human astrocytes similarly to abnormal PrP aggregates that are resistant to proteases. Our data suggest that relative proteinase K resistance does not significantly influence the astrocyte''s ability to degrade PrP^Sc. Furthermore, the cell does not appear to distinguish between sPrP^Sc and protease-resistant PrP^Sc, suggesting that sPrP^Sc could contribute to prion infection.Prion diseases, or transmissible spongiform encephalopathies, are rare fatal neurologic disorders of mammals that include Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy in cattle, and scrapie in sheep. Prion diseases are characterized by the conversion of normal prion protein (PrP^C) into a disease-associated and aggregated isoform (PrP^Sc), which is thought to be the main component of the infectious agent or prion (reviewed in Priola and Vorberg¹). PrP^C is a glycoprotein that contains two N-linked glycosylation sites^2,3 and is bound to the plasma membrane via a glycosyl-phosphatidyl-inositol anchor.⁴ Although PrP^C is detergent soluble and fully susceptible to proteolytic degradation, PrP^Sc has an increased detergent insolubility and partial resistance to proteinase K (PK).⁵ The presence of amino-terminally truncated, PK-resistant core fragments of PrP^Sc (rPrP^Sc) after limited proteolysis is considered the most reliable diagnostic marker for prion infection,^6,7 and biochemical profiles of rPrP^Sc based on molecular mass and/or the degree of glycosylation are used to help differentiate distinct prion disease phenotypes in humans.^8–11In recent years, alternative approaches for analyzing PrP^Sc that do not rely on the enzymatic removal of PrP^C have indicated that not all forms of PrP^Sc are necessarily resistant to proteolytic treatment. The conformation-dependent immunoassay, which uses conformational differences between the N termini of PrP^C and PrP^Sc, has found that often a majority of PrP^Sc present in prion-affected brains is susceptible to proteolytic degradation.^12–15 In sporadic CJD (sCJD), this PK-sensitive species of aggregated PrP^Sc, termed sPrP^Sc, was found in some cases to account for up to 90% of the total PrP^Sc.^13,16 Careful analysis of the size distribution of PrP^Sc has also found that sPrP^Sc forms much smaller aggregates than rPrP^Sc.^17,18 Thus, sPrP^Sc appears to represent a population of PrP^Sc aggregates which tends to be both smaller and more protease sensitive than rPrP^Sc.Evidence suggests that, like rPrP^Sc, sPrP^Sc has seeding activity and can convert PrP^C to protease resistance.^18,19 It has also been associated with prion infectivity²⁰ and may influence the incubation time of prion disease.²¹ Although these data suggest that PK-sensitive disease-associated PrP aggregates may be actively involved in prion pathogenesis, no studies have been performed to determine how this population of PrP aggregates might interact with cells and influence prion infection. In this study, we have looked at the uptake and degradation of PK-sensitive and PK-resistant disease-associated PrP aggregates in an established human astrocyte cell line. Our results indicate that, despite their biochemical differences, PK-sensitive PrP aggregates are taken up and degraded similarly to PK-resistant PrP aggregates, suggesting that relative PK resistance does not significantly influence the cell''s ability to degrade PrP^Sc. Thus, the astrocyte does not appear to distinguish between sPrP^Sc and rPrP^Sc aggregates, suggesting that sPrP^Sc could be involved in prion pathogenesis.     

Protease-resistant prion protein in brain and lymphoid organs of sheep within a naturally scrapie-infected flock

The hallmark of transmissible spongiform encephalopathies (TSE), such as scrapie in sheep, is the accumulation in tissues of an insoluble and protease resistant form (PrPres) of the cellular prion protein. In this study, we evaluated whether the diversity in both the clinical pattern and the PrP genotypes of scrapied sheep from the same flock was connected with different levels and/or glycoform patterns of the PrPres in the brain and lymphoid organs of the animals. Whereas the PrPres levels in spleen, lymph nodes and tonsils from sheep of different PrP genotypes and clinical status appeared comparable, they were highly variable in brain, particularly in the brain stem and the cerebellum. PrPres was only detected in sheep bearing at least one VRQ allele, including three asymptomatic sheep and the highest PrPres load was found in the cerebellum of VRQ/VRQ animals. All together, levels of PrPres in brain did not necessarily correlate with the severity of the clinical disease but might depend on the PrP genotype of the animals. Different brain regions from a given sheep displayed a similar glycopattern of PrPres, whereas the apparent molecular sizes of the unglycosylated and diglycosylated forms of the protein differed between brain and lymphoid tissues. We did not find any notifiable differences in the glycopattern of PrPres in brain from sheep of different PrP genotypes or different clinical status and this PrPres glycotype was also similar to that found in brain from four cattle BSE.     

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).     

Prion protein allele A136 H154Q171 is associated with high susceptibility to scrapie in purebred and crossbred German Merinoland sheep

Summary. Prion protein (PrP) genotypes were determined in eight sheep that have been tested positive for atypical scrapie from purebred or crossbred Merinoland sheep flocks in Germany and compared with the PrP genotypes of their flock mates. Two restriction fragment length polymorphism (RFLP) analyses were developed to determine all PRNP haplotypes occurring by variations at codons 136, 154 and 171. At least one copy of the A₁₃₆H₁₅₄Q₁₇₁ (AHQ) allele was found in all scrapie-positive sheep while the frequency of AHQ varied from over 23% to less than 3% in the whole flocks. There was a significant association between PrP genotype and a positive scrapie diagnosis over all flocks, suggesting a high scrapie susceptibility of PrP genotypes including the AHQ allele, at least in sheep of Merinoland type. These results argue that sheep with the AHQ allele are not generally less susceptible to scrapie and support the hypothesis that the influence of this allele on scrapie susceptibility may vary from flock to flock depending on genetic and/or epidemiological factors. This has to be considered when strategies for the eradication of scrapie in sheep are based on PrP genotypes.     

PrP octarepeats region determined the interaction with caveolin-1 and phosphorylation of caveolin-1 and Fyn

Caveolin-1 is one of the major constituents of caveolae. Both Cav-1 and PrP are plasma membrane proteins, which show active capacities for molecular interactions with many other proteins or agents, including themselves. Using yeast two-hybrid system and immunoprecipitation, we reconfirmed the molecular interaction between human Cav-1 and PrP. With co-immunoprecipitation tests, PrP^C–Cav-1 and PrP^Sc–Cav-1 complexes were identified in the brain homogenates of normal and scrapie agent 263K-infected hamsters, respectively. Transient expression of wild-type PrP (PrP-PG5) in HEK293 cells did not change the situation of Cav-1 and subsequent signal transduction pathways, while cross-linking of the expressed PrP with specific antibody induced remarkable colocalization of PrP and Cav-1 on the plasma membrane and significant increases of phosphorylated Cav-1 and phosphorylated Fyn. With deleted and inserted PrP mutants within octarepeat region, we observed obvious octarepeat-associated phenomena, including lower binding capacity with Cav-1 in vitro, unable to co-localize with Cav-1 in the cells and to induce up-regulation of p-Cav-1 and p-Fyn when removal of octarepeats in the context of full-length PrP. Moreover, we found that treatment on HEK293 cells with fibrous form of recombinant PrP protein led to up-regulating the levels of p-Cav-1 and p-Fyn. Our data here provide strong evidence that octarepeats of PrP are critical for the interaction between PrP and Cav-1. Significant alterations in the cultured cells, either the distributions of PrP and Cav-1 morphologically or the up-regulations of p-Cav-1 and p-Fyn, induced by antibody-mediated cross-linking or fibrous forms of PrP may suggest a possible internalization process of PrP^Sc.     

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.     

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.     

Cellular uptake of the prion protein fragment PrP106-126 in vitro

The aetiological agent of prion disease is proposed to be an aberrant isoform of the cell surface glycoprotein known as the prion protein (PrP^c). This pathological isoform (PrP^Sc) is abnormally deposited in the extracellular space of diseased CNS. Neurodegeneration in these disease has been shown to be associated with accumulation of PrP^Sc in affected tissue. To investigate the possible uptake mechanisms that may be required for PrP^Sc-induced neurodegeneration we studied the cellular trafficking of the neurotoxic fragment, PrP106-126. We were able to detect, by fluorescence microscopy, PrP106-126 inclusions in murine neurones, astrocytes and microglia in vitro. These inclusions were abundant after 24 hour exposure and still present 48h post-exposure. Shorter exposure times yielded only occasional cells with inclusions. Large extracellular aggregates of PrP106-126 could also be detected, which appeared in a time dependent manner. The appearance of inclusions or aggregates was not dependent on PrP^c expression as determined by exposure of peptides from PrP-null mice. Using transmission electron microscopy and gold particle detection, positively labelled osmiophilic inclusions of peptide could be detected in the cytoplasm of exposed cells. These results demonstrate that cultured cells are capable of sequestering PrP106-126 and may indicate uptake pathways for PrP^Sc in various cell types. Toxicity of PrP106-126 may thus be mediated via a sequestration pathway that is not effective for this peptide in PrP-null cells.