Scrapie Pathogenesis Research Articles

Evidence of p75 Neurotrophin Receptor Involvement in the Central Nervous System Pathogenesis of Classical Scrapie in Sheep and a Transgenic Mouse Model.

Neurotrophins constitute a group of growth factor that exerts important functions in the nervous system of vertebrates. They act through two classes of transmembrane receptors: tyrosine-kinase receptors and the p75 neurotrophin receptor (p75NTR). The activation of p75NTR can favor cell survival or apoptosis depending on diverse factors. Several studies evidenced a link between p75NTR and the pathogenesis of prion diseases. In this study, we investigated the distribution of several neurotrophins and their receptors, including p75NTR, in the brain of naturally scrapie-affected sheep and experimentally infected ovinized transgenic mice and its correlation with other markers of prion disease. No evident changes in infected mice or sheep were observed regarding neurotrophins and their receptors except for the immunohistochemistry against p75NTR. Infected mice showed higher abundance of p75NTR immunostained cells than their non-infected counterparts. The astrocytic labeling correlated with other neuropathological alterations of prion disease. Confocal microscopy demonstrated the co-localization of p75NTR and the astrocytic marker GFAP, suggesting an involvement of astrocytes in p75NTR-mediated neurodegeneration. In contrast, p75NTR staining in sheep lacked astrocytic labeling. However, digital image analyses revealed increased labeling intensities in preclinical sheep compared with non-infected and terminal sheep in several brain nuclei. This suggests that this receptor is overexpressed in early stages of prion-related neurodegeneration in sheep. Our results confirm a role of p75NTR in the pathogenesis of classical ovine scrapie in both the natural host and in an experimental transgenic mouse model.

Neuroimmune Response in Natural Preclinical Scrapie after Dexamethasone Treatment.

A recently published report on chronic dexamethasone treatment for natural scrapie supported the hypothesis of the potential failure of astroglia in the advanced stage of disease. Herein, we aimed to extend the aforementioned study on the effect of this anti-inflammatory therapy to the initial phase of scrapie, with the aim of elucidating the natural neuroinflammatory process occurring in this neurodegenerative disorder. The administration of this glucocorticoid resulted in an outstanding reduction in vacuolation and aberrant protein deposition (nearly null), and an increase in glial activation. Furthermore, evident suppression of IL-1R and IL-6 and the exacerbation of IL-1α, IL-2R, IL-10R and IFNγR were also demonstrated. Consequently, the early stage of the disease is characterized by an intact neuroglial response similar to that of healthy individuals attempting to re-establish homeostasis. A complex network of neuroinflammatory markers is involved from the very early stages of this prion disease, which probably becomes impaired in the more advanced stages. The in vivo animal model used herein provides essential observations on the pathogenesis of natural scrapie, as well as the possibility of establishing neuroglia as potential target cells for anti-inflammatory therapy.

Pathogenesis, detection, and control of scrapie in sheep.

In sheep, scrapie is a fatal neurologic disease that is caused by a misfolded protein called a prion (designated PrPSc). The normal cellular prion protein (PrPC) is encoded by an endogenous gene, PRNP, that is present in high concentrations within the CNS. Although a broad range of functions has been described for PrPC, its entire range of functions has yet to be fully elucidated. Accumulation of PrPSc results in neurodegeneration. The PRNP gene has several naturally occurring polymorphisms, and there is a strong correlation between scrapie susceptibility and PRNP genotype. The cornerstone of scrapie eradication programs is the selection of scrapie-resistant genotypes to eliminate classical scrapie. Transmission of classical scrapie in sheep occurs during the prenatal and periparturient periods when lambs are highly susceptible. Initially, the scrapie agent is disseminated throughout the lymphoid system and into the CNS. Shedding of the scrapie agent occurs before the onset of clinical signs. In contrast to classical scrapie, atypical scrapie is believed to be a spontaneous disease that occurs in isolated instances in older animals within a flock. The agent that causes atypical scrapie is not considered to be naturally transmissible. Transmission of the scrapie agent to species other than sheep, including deer, has been experimentally demonstrated as has the transmission of nonscrapie prion agents to sheep. The purpose of this review is to outline the current methods for diagnosing scrapie in sheep and the techniques used for studying the pathogenesis and host range of the scrapie agent. Also discussed is the US scrapie eradication program including recent updates.

Potential scrapie-associated polymorphisms of the prion protein gene (PRNP) in Korean native black goats

Small ruminants, including sheep and goats are natural hosts of scrapie, and the progression of scrapie pathogenesis is strongly influenced by polymorphisms in the prion protein gene (PRNP). Although Korean native goats have been consumed as meat and health food, the evaluation of the susceptibility to scrapie in these goats has not been performed thus far. Therefore, we investigated the genotype and allele frequencies of PRNP polymorphisms in 211 Korean native goats and compared them with those in scrapie-affected animals from previous studies. We found a total of 12 single nucleotide polymorphisms (SNPs) including 10 nonsynonymous and 2 synonymous SNPs in Korean native goats. Significant differences in allele frequencies of PRNP codons 143 and 146 were found between scrapie-affected goats and Korean native goats (p < 0.01). By contrast, in PRNP codons 168, 211 and 222, there were no significant differences in the genotype and allele frequencies between scrapie-affected animals and Korean native goats. To evaluate structural changes caused by nonsynonymous SNPs, PolyPhen-2, PROVEAN and AMYCO analyses were performed. PolyPhen-2 predicted “possibly damaging” for W102G and R154H, “probably damaging” for G127S. AMYCO predicted relatively low for amyloid propensity of prion protein in Korean native black goats. This is the first study to evaluate the scrapie sensitivity and the first in silico evaluation of nonsynonymous SNPs in Korean native black goats.

Relative Impact of Complement Receptors CD21/35 (Cr2/1) on Scrapie Pathogenesis in Mice.

Complement receptors 1 and 2 (CR1/2 or CD35/CD21) recognize complement-opsonized antigens to initiate innate and adaptive immunity, respectively. CD35 stimulates phagocytosis on macrophages and antigen presentation on follicular dendritic cells (FDCs). CD21 helps activate B cells as part of the B cell coreceptor with CD19 and CD81. Differential splicing of transcripts from the mouse Cr2 gene generates isoforms with both shared and unique complement binding capacities and cell-type expression. In mouse models, genetic depletion of Cr2 causes either a delay or complete prevention of prion disease, but the relative importance of CD35 versus CD21 in promoting prion disease remains unknown. Here we show that both isoforms act as high-affinity cell surface prion receptors. However, mice lacking CD21 succumbed to terminal prion disease significantly later than mice lacking CD35 or wild-type and hemizygous mice. CD21-deficient mice contained fewer splenic prions than CD35 knockout mice early after infection that contributed to delayed prion neuroinvasion and terminal disease, despite forming follicular networks closer to proximal nerves. While we observed no difference in B cell networks, PrPC expression, or number of follicles, CD21-deficient mice formed more fragmented, less organized follicular networks with fewer Mfge8-positive FDCs and/or tingible body macrophages (TBMφs) than wild-type or CD35-deficient mice. In toto, these data demonstrate a more prominent role for CD21 for proper follicular development and organization leading to more efficient lymphoid prion replication and expedited prion disease than in mice expressing the CD35 isoform. IMPORTANCE Mammalian prion diseases are caused by prions, unique infectious agents composed primarily, if not solely, of a pathologic, misfolded form of a normal host protein, the cellular prion protein (PrPC). Prions replicate without a genetic blueprint, but rather contact PrPC and coerce it to misfold into more prions, which cause neurodegeneration akin to other protein-misfolding diseases like Alzheimer's disease. A single gene produces two alternatively spliced mRNA transcripts that encode mouse complement receptors CD21/35, which promote efficient prion replication in the lymphoid system and eventual movement to the brain. Here we show that CD21/35 are high-affinity prion receptors, but mice expressing only CD21 die from prion disease sooner than CD35-expressing mice, which contain less prions early after infection and exhibit delayed terminal disease, likely due to their less organized splenic follicles. Thus, CD21 appears to be more important for defining splenic architecture that influences prion pathogenesis.

Precision in the design of an experimental study deflects the significance of proteinase-activated receptor 2 expression in scrapie-inoculated mice.

Proteinase-activated receptor 2 (PAR2) is suspected to modulate the pathogenesis of various neurodegenerative conditions. We previously described delayed onset of clinical symptoms and prolonged survival of PAR2-deficient mice after intracerebral inoculation with prions. Here we report the results from a refined blinded study that aimed to investigate the effects of PAR2 deletion on scrapie pathogenesis after peripheral infection. This study failed to confirm that PAR2 deficiency impacts on the length of the incubation period, with PAR2-/- and PAR2+/+ littermates developing scrapie at the same time. To clarify the discrepancy between the two observations, we repeated the intracerebral inoculation study while utilizing our refined protocol, which aimed to limit possible sources of experimental bias. The study again failed to confirm the significant effect of PAR2 expression on the course of prion infection. Our report emphasizes and discusses the importance of unbiased experimental design and the selection of proper genetic controls when using genetically altered animal models for prion pathogenesis studies.

Transgenic over-expression of mammalian heparanase delays prion disease onset and progression

Cellular heparan sulfate (HS) has a dual role in scrapie pathogenesis; it is required for PrPSc (scrapie prion protein) formation and facilitates infection of cells, mediating cellular uptake of prions. We examined the involvement of heparanase, a mammalian endoglycosidase degrading HS, in scrapie infection. In cultured cells, heparanase treatment or over-expression resulted in a profound decrease in PrPSc. Moreover, disease onset and progression were dramatically delayed in scrapie infected transgenic mice over-expressing heparanase. Together, our results provide direct in vivo evidence for the involvement of intact HS in the pathogenesis of prion disease and the protective role of heparanase both in terms of susceptibility to infection and disease progression.

Prion infection of mouse brain reveals multiple new upregulated genes involved in neuroinflammation or signal transduction.

Gliosis is often a preclinical pathological finding in neurodegenerative diseases, including prion diseases, but the mechanisms facilitating gliosis and neuronal damage in these diseases are not understood. To expand our knowledge of the neuroinflammatory response in prion diseases, we assessed the expression of key genes and proteins involved in the inflammatory response and signal transduction in mouse brain at various times after scrapie infection. In brains of scrapie-infected mice at pre- and postclinical stages, we identified 15 previously unreported differentially expressed genes related to inflammation or activation of the STAT signal transduction pathway. Levels for the majority of differentially expressed genes increased with time postinfection. In quantitative immunoblotting experiments of STAT proteins, STAT1α, phosphorylated-STAT1α (pSTAT1α), and pSTAT3 were increased between 94 and 131 days postinfection (p.i.) in brains of mice infected with strain 22L. Furthermore, a select group of STAT-associated genes was increased preclinically during scrapie infection, suggesting early activation of the STAT signal transduction pathway. Comparison of inflammatory markers between mice infected with scrapie strains 22L and RML indicated that the inflammatory responses and gene expression profiles in the brains were strikingly similar, even though these scrapie strains infect different brain regions. The endogenous interleukin-1 receptor antagonist (IL-1Ra), an inflammatory marker, was newly identified as increasing preclinically in our model and therefore might influence scrapie pathogenesis in vivo. However, in IL-1Ra-deficient or overexpressor transgenic mice inoculated with scrapie, neither loss nor overexpression of IL-1Ra demonstrated any observable effect on gliosis, protease-resistant prion protein (PrPres) formation, disease tempo, pathology, or expression of the inflammatory genes analyzed. Prion infection leads to PrPres deposition, gliosis, and neuroinflammation in the central nervous system before signs of clinical illness. Using a scrapie mouse model of prion disease to assess various time points postinoculation, we identified 15 unreported genes that were increased in the brains of scrapie-infected mice and were associated with inflammation and/or JAK-STAT activation. Comparison of mice infected with two scrapie strains (22L and RML), which have dissimilar neuropathologies, indicated that the inflammatory responses and gene expression profiles in the brains were similar. Genes that increased prior to clinical signs might be involved in controlling scrapie infection or in facilitating damage to host tissues. We tested the possible role of the endogenous IL-1Ra, which was increased at 70 days p.i. In scrapie-infected mice deficient in or overexpressing IL-1Ra, there was no observable effect on gliosis, PrPres formation, disease tempo, pathology, or expression of inflammatory genes analyzed.

Gene expression profiling of mesenteric lymph nodes from sheep with natural scrapie

BackgroundPrion diseases are characterized by the accumulation of the pathogenic PrPSc protein, mainly in the brain and the lymphoreticular system. Although prions multiply/accumulate in the lymph nodes without any detectable pathology, transcriptional changes in this tissue may reflect biological processes that contribute to the molecular pathogenesis of prion diseases. Little is known about the molecular processes that occur in the lymphoreticular system in early and late stages of prion disease. We performed a microarray-based study to identify genes that are differentially expressed at different disease stages in the mesenteric lymph node of sheep naturally infected with scrapie. Oligo DNA microarrays were used to identify gene-expression profiles in the early/middle (preclinical) and late (clinical) stages of the disease.ResultsIn the clinical stage of the disease, we detected 105 genes that were differentially expressed (≥2-fold change in expression). Of these, 43 were upregulated and 62 downregulated as compared with age-matched negative controls. Fewer genes (50) were differentially expressed in the preclinical stage of the disease. Gene Ontology enrichment analysis revealed that the differentially expressed genes were largely associated with the following terms: glycoprotein, extracellular region, disulfide bond, cell cycle and extracellular matrix. Moreover, some of the annotated genes could be grouped into 3 specific signaling pathways: focal adhesion, PPAR signaling and ECM-receptor interaction. We discuss the relationship between the observed gene expression profiles and PrPSc deposition and the potential involvement in the pathogenesis of scrapie of 7 specific differentially expressed genes whose expression levels were confirmed by real time-PCR.ConclusionsThe present findings identify new genes that may be involved in the pathogenesis of natural scrapie infection in the lymphoreticular system, and confirm previous reports describing scrapie-induced alterations in the expression of genes involved in protein misfolding, angiogenesis and the oxidative stress response. Further studies will be necessary to determine the role of these genes in prion replication, dissemination and in the response of the organism to this disease.

Identical Pathogenesis and Neuropathological Phenotype of Scrapie in Valine, Arginine, Glutamine/Valine, Arginine, Glutamine Sheep Infected Experimentally by the Oral and Conjunctival Routes

The pathogenesis of scrapie in sheep after natural or oral exposure to the infectious agent generally involves the early accumulation of disease-associated prion protein (PrP(d)) in the lymphoreticular system (LRS). This phase is followed by neuroinvasion, for which two routes, ascending neural and haematogenous, have been postulated. The present study reports the use of immunohistochemistry to track the tissue progression of PrP(d) deposition in sheep of a single, highly scrapie-susceptible PrP genotype administered by the oral or conjunctival routes. Regardless of the route of infection, the earliest detection of PrP(d) was in gut- and pharynx-associated LRS tissues. Subsequently, the brain became PrP(d) positive simultaneously with other LRS tissues, but before the spinal cord and peripheral nervous tissues of the enteric, parasympathetic and sympathetic systems. The sites of initial PrP(d) accumulation in the brain were the dorsal motor nucleus of the vagus and the hypothalamus and their related circumventricular organs (the area postrema and the median eminence, respectively). These were the same for both routes of infection. Rapid progression to clinical disease was observed in sheep infected orally or conjunctivally, with definite signs of scrapie recorded at around 6 and 8 months after infection, respectively. Longer incubation periods in sheep infected by the conjunctival route were probably due to them receiving a lower dose than those infected orally. Irrespective of the route of infection, clinically affected sheep showed the same pathological phenotype (PrP(d) profile) and PrP(d) distribution throughout the brain. The identical peripheral and central pathogenesis observed in sheep of both groups suggests early dissemination of the infectious agent in the bloodstream and a common neuroinvasion pathway. The late involvement of the enteric and autonomic nervous system supports a haematogenous route of infection to the brain.

Three-dimensional reconstruction of the pharyngeal tonsil innervation pattern in sheep

The pharyngeal tonsil has recently been identified as a new participant in airborne contamination by the ovine scrapie agent. In the context of scrapie pathogenesis, we conducted a three-dimensional reconstruction of the innervation pattern in the lymphoid compartments of this tonsil. This model confirmed that very few nerve fibres penetrated the lymphoid follicles and suggested that the nerve fibre distribution in the interfollicular and subepithelial areas is more suitable with neuro-invasion through direct contact between these nerve fibres and prion-transporting cells prior to or after prion amplification in the germinal centre of the pharyngeal tonsil lymphoid follicles.

Pathogenesis of scrapie in ARQ/ARQ sheep after subcutaneous infection: Effect of lymphadenectomy and immune cell subset changes in relation to prion protein accumulation

It is well established that the infectious agent of scrapie can replicate in the lymphoreticular system (LRS). However, the effects of removal of LRS target tissues on the pathogenesis of the infection and the accumulation of disease-associated prion protein (PrPd) in LRS tissues on specific immune cell subsets are poorly understood aspects. To address these questions 16 ARQ/ARQ sheep were subcutaneously inoculated in the drainage area of the prefemoral lymph node with brain homogenate derived from Suffolk sheep naturally infected with scrapie. Fourteen sheep were then subjected to either early (14–17 days post-inoculation [dpi]) or late (175–201 dpi) lymphadenectomy and culled at preclinical or clinical stages of infection. Neither late nor even early lymphadenectomy prevented infection or had any effect on the accumulation of PrPd in the LRS or CNS suggesting a rapid organic dissemination of the infectious agent after inoculation. Lymph nodes from eight scrapie inoculated sheep selected on the basis of the amount of PrPd in their LRS tissues (negative, low or high) were examined for six different immune cell markers. The PrPd negative lymph nodes from two sheep with no evidence of scrapie infection showed lower numbers of cluster of determination (CD) 21 positive cells than PrPd positive nodes, irrespective of their location (hind leg or head). However, quantitative differences in the expression of this marker were not detected when comparing lymph nodes with low and high levels of PrPd accumulation, suggesting that proliferation of CD21 positive cells is related to scrapie infection, but not directly linked to the magnitude of PrPd accumulation. An additional observation of the study was that sheep that were methionin–threonine at codon 112 of the prion protein gene showed lower attack rates than methionine homozygotes (67% and 100%, respectively) and also generally lower levels of PrPd accumulation in the LRS and brain and increased survival times, suggesting an influence of such polymorphism in the susceptibility to scrapie.

The Pathogenesis and Disease Phenotype of Scrapie is not Affected by the Dose or Route of Inoculation

The Pathogenesis and Disease Phenotype of Scrapie is not Affected by the Dose or Route of Inoculation

Medulla oblongata transcriptome changes during presymptomatic natural scrapie and their association with prion-related lesions

BackgroundThe pathogenesis of natural scrapie and other prion diseases is still poorly understood. Determining the variations in the transcriptome in the early phases of the disease might clarify some of the molecular mechanisms of the prion-induced pathology and allow for the development of new biomarkers for diagnosis and therapy. This study is the first to focus on the identification of genes regulated during the preclinical phases of natural scrapie in the ovine medulla oblongata (MO) and the association of these genes with prion deposition, astrocytosis and spongiosis.ResultsA custom microarray platform revealed that 86 significant probes had expression changes greater than 2-fold. From these probes, we identified 32 genes with known function; the highest number of regulated genes was included in the phosphoprotein-encoding group. Genes encoding extracellular marker proteins and those involved in the immune response and apoptosis were also differentially expressed. In addition, we investigated the relationship between the gene expression profiles and the appearance of the main scrapie-associated brain lesions. Quantitative Real-time PCR was used to validate the expression of some of the regulated genes, thus showing the reliability of the microarray hybridization technology.ConclusionsGenes involved in protein and metal binding and oxidoreductase activity were associated with prion deposition. The expression of glial fibrillary acidic protein (GFAP) was associated with changes in the expression of genes encoding proteins with oxidoreductase and phosphatase activity, and the expression of spongiosis was related to genes encoding extracellular matrix components or transmembrane transporters. This is the first genome-wide expression study performed in naturally infected sheep with preclinical scrapie. As in previous studies, our findings confirm the close relationship between scrapie and other neurodegenerative diseases.

The Oral Secretion of Infectious Scrapie Prions Occurs in Preclinical Sheep with a Range of PRNP Genotypes

Preclinical sheep with the highly scrapie-susceptible VRQ/VRQ PRNP genotype secrete prions from the oral cavity. In order to further understand the significance of orally available prions, buccal swabs were taken from sheep with a range of PRNP genotypes and analyzed by serial protein misfolding cyclic amplification (sPMCA). Prions were detected in buccal swabs from scrapie-exposed sheep of genotypes linked to high (VRQ/VRQ and ARQ/VRQ) and low (ARR/VRQ and AHQ/VRQ) lymphoreticular system involvement in scrapie pathogenesis. For both groups, the level of prion detection was significantly higher than that for scrapie-resistant ARR/ARR sheep which were kept in the same farm environment and acted as sentinel controls for prions derived from the environment which might contaminate the oral cavity. In addition, sheep with no exposure to the scrapie agent did not contain any measurable prions within the oral cavity. Furthermore, prions were detected in sheep over a wide age range representing various stages of preclinical disease. These data demonstrate that orally available scrapie prions may be a common feature in sheep incubating scrapie, regardless of the PRNP genotype and any associated high-level accumulation of PrP(Sc) within lymphoreticular tissues. PrP(Sc) was present in buccal swabs from a large proportion of sheep with PRNP genotypes associated with relatively low disease penetrance, indicating that subclinical scrapie infection is likely to be a common occurrence. The significance of positive sPMCA reactions was confirmed by the transmission of infectivity in buccal swab extracts to Tg338 mice, illustrating the likely importance of orally available prions in the horizontal transmission of scrapie.

Transcriptional profiling of peripheral lymphoid tissue reveals genes and networks linked to SSBP/1 scrapie pathology in sheep

Transmissible spongiform encephalopathies (TSEs) are slow and progressive neurodegenerative diseases of humans and animals. The major target organ for all TSEs is the brain but some TSE agents are associated with prior accumulation within the peripheral lymphoid system. Many studies have examined the effects of scrapie infection on the expression of central nervous system (CNS) genes, but this study examines the progression of scrapie pathology in the peripheral lymphoid system and how scrapie infection affects the transcriptome of the lymph nodes and spleen. Infection of sheep with SSBP/1 scrapie resulted in PrP Sc deposition in the draining prescapular lymph node (PSLN) by 25 days post infection (dpi) in VRQ/VRQ genotype sheep and 75 dpi in tonsils and spleen. Progression of PrP Sc deposition in VRQ/ARR animals was 25 dpi later in the PSLN and 250 dpi later in spleen. Microarray analysis of 75 dpi tissues from VRQ/VRQ sheep identified 52 genes in PSLN and 37 genes in spleen cells that showed significant difference ( P ≤ 0.05) between scrapie-infected and mock-infected animals. Transcriptional pathway analysis highlighted immunological disease, cell death and neurological disease as the biological pathways associated with scrapie pathogenesis in the peripheral lymphoid system. PrP Sc accumulation of lymphoid tissue resulted in the repression of genes linked to inflammation and oxidative stress, and the up-regulation of genes related to apoptosis.

Gene Expression Profiling and Association with Prion-Related Lesions in the Medulla Oblongata of Symptomatic Natural Scrapie Animals

The pathogenesis of natural scrapie and other prion diseases remains unclear. Examining transcriptome variations in infected versus control animals may highlight new genes potentially involved in some of the molecular mechanisms of prion-induced pathology. The aim of this work was to identify disease-associated alterations in the gene expression profiles of the caudal medulla oblongata (MO) in sheep presenting the symptomatic phase of natural scrapie. The gene expression patterns in the MO from 7 sheep that had been naturally infected with scrapie were compared with 6 controls using a Central Veterinary Institute (CVI) custom designed 4×44K microarray. The microarray consisted of a probe set on the previously sequenced ovine tissue library by CVI and was supplemented with all of the Ovis aries transcripts that are currently publicly available. Over 350 probe sets displayed greater than 2-fold changes in expression. We identified 148 genes from these probes, many of which encode proteins that are involved in the immune response, ion transport, cell adhesion, and transcription. Our results confirm previously published gene expression changes that were observed in murine models with induced scrapie. Moreover, we have identified new genes that exhibit differential expression in scrapie and could be involved in prion neuropathology. Finally, we have investigated the relationship between gene expression profiles and the appearance of the main scrapie-related lesions, including prion protein deposition, gliosis and spongiosis. In this context, the potential impacts of these gene expression changes in the MO on scrapie development are discussed.

Overexpression of Shadoo protein in transgenic mice does not impact the pathogenesis of scrapie

Shadoo is a glycoprotein expressed in the adult brain that is an interacting protein of prion protein; however, its function remains to be determined. To elucidate its role in prion pathogenesis, we generated transgenic mice overexpressing wild-type (wt) Shadoo driven by the murine PrP promoter. Expression of the murine Sprn transgene significantly increased brain Shadoo protein levels in all three mouse lines generated. Following infection with mouse-adapted scrapie strain 22L, all transgenic mice tested exhibited characteristics of scrapie disease. Importantly, there was no correlation between the expression level or incubation time of Shadoo with disease phenotype. We therefore conclude that Shadoo has little or no influence on the outcome of transmissible spongiform encephalopathy (TSE) disease in transgenic mice.

Features of follicular dendritic cells in ovine pharyngeal tonsil: An in vivo and in vitro study in the context of scrapie pathogenesis

Although the alimentary tract has been suggested as the most likely portal of entry in natural scrapie, a growing amount of data indicates that the respiratory system and more specifically the pharyngeal tonsils serve as a natural portal of entry for scrapie. This study describes for the first time the broad cell populations in the lymphoid compartment of pharyngeal tonsils and more specifically inside the lymphoid follicles where the scrapie agent accumulates during the period of latency. Follicular dendritic cells (FDCs), stromal cells located in the light zone of the germinal centre of lymphoid follicles, seem to be the principal causal factor in the accumulation of the infectious agent in transmissible spongiform encephalopathy (TSE) diseases. Knowing that efficient lymphoreticular prion propagation requires PrPc expression, we analysed the expression of PrPc with the mouse monoclonal antibody Pri 909 both in situ and on FDC-cluster-enriched cell suspensions. In situ, a positive staining was observed in the germinal centre of pharyngeal lymph follicles. The germinal centre labelling was due to the presence of a follicular dendritic network as revealed after immunogold staining of isolated FDC clusters. Our results suggest that the pharyngeal lymphoreticular system and more specifically PrPc expressing follicular dendritic cells could serve as a prion “reservoir” during the latency phase, thus playing a key role during the scrapie lymphoinvasion.

Cartilaginous Metaplasia in the Sclera of Suffolk Sheep

Scleral cartilaginous metaplasia was detected by routine histologic examination of globes from 5 Suffolk sheep from a scrapie pathogenesis study. The extent of the metaplasia varied among the sheep but was always posterior to the tapetal fundus. The matrix surrounding chondrocytes stained intensely with alcian blue and was immunopositive for type II collagen. Retrospective evaluation of additional eyes from Suffolk and Cheviot sheep used in various scrapie pathogenesis studies at the authors' facility revealed similar histologic changes in 40% and 12.7% of eyes examined, respectively. The clinical significance of this previously unreported finding is unknown.