Transmissible spongiform encephalopathy diagnosis using PrPsc immunohistochemistry on fixed but previously frozen brain samples.

The histological diagnosis of transmissible spongiform encephalopathies (TSEs), such as scrapie and bovine spongiform encephalopathy (BSE), relies on identification in the brain of spongiosis, gliosis, and neuron loss without inflammatory lesions. Because of its sensitivity, immunohistochemistry of abnormal prion protein (PrPsc) is of great help in this diagnosis and can be used on its own or complementary to the biochemical detection of PrPsc. However, in some cases no formalin-fixed material is available, rendering its use as a complementary method impossible. For that purpose, we studied the possibility of detecting PrPsc immunohistochemically in fixed brain samples that had been previously frozen and used for Western blotting analysis. We compared freshly and fixed-frozen brain samples originating from the same sheep, either affected or unaffected with scrapie. We also studied fixed-frozen brain samples from scrapie-affected goats and from cows showing BSE. We showed that in all the species tested, despite damage to the histological structures, PrPsc was still detectable in the fixed-frozen brain sections without unspecific background staining. Notwithstanding the limited number of cases thus far analyzed, we have already demonstrated the possibility of using PrPsc immunohistochemistry on fixed-frozen brain samples with very good efficacy, thus rendering possible its use for diagnostic purposes in TSEs.     

Amplified immunohistochemical detection of PrPsc in animal transmissible spongiform encephalopathies using streptomycin.

Due to its sensitivity, immunohistochemistry (IHC) of abnormal prion protein (PrPsc) is used to study experimental and natural cases of transmissible spongiform encephalopathies (TSEs) such as Creutzfeldt-Jakob disease in humans or scrapie and bovine spongiform encephalopathy (BSE) in animals. The limits of detection are particularly critical when PrPsc IHC is used for diagnostic purposes. In this article, we describe for the first time the use of streptomycin sulfate in IHC, providing a novel original and easy way to amplify specifically PrPsc immunohistochemical detection in natural cases of BSE and scrapie, as well as in experimental TSEs in mice models using two different PrP antibodies.     

Neuropathological characterisation of French bovine spongiform encephalopathy cases

Bovine spongiform encephalopathy (BSE) in cattle is a neurodegenerative disease belonging to the transmissible spongiform encephalopathies, a group of diseases including sheep scrapie and human Creutzfeldt-Jakob disease. The pathological characteristics of BSE are vacuolation, mild gliosis, little neuronal degeneration without inflammatory process and abnormal prion protein (PrPsc) accumulation. The aim of this study was to define precisely the neuropathology of BSE in French cases by assessing the distributions of vacuolar lesions and PrPsc within cattle brains. We showed that vacuolation and PrPsc accumulation varied from one structure to the other, and most often coexisted. These distributions were in accordance with British and Portuguese data previously published. Seven types of PrPsc immunolabelling were described based on morphology and localisation. Besides mild gliosis mainly associated with vacuolation, we observed a very slight neuronal apoptosis. In addition, we saw a moderate vimentin labelling colocalised with vacuolation, a discrete ubiquitin staining and no Tau protein staining. This study provides precise histopathological data that will be completed with a quantitative study on more than 100 obex samples of French BSE cases.     

An alternative pretreatment procedure in animal transmissible spongiform encephalopathies diagnosis using PrPsc immunohistochemistry.

Because of its sensitivity, immunohistochemistry (IHC) of abnormal prion protein (PrPsc) is used more often in the diagnosis of transmissible spongiform encephalopathies (TSEs), such as scrapie and bovine spongiform encephalopathy (BSE). PrPsc IHC requires a combination of pretreatments (chemical, heating, and enzymatic). The method of application may depend on the anti-prion antibody considered. If these pretreatments are efficient for diagnostic purpose, it may, however, be interesting to use an alternative method to efficiently detect PrPsc IHC immunohistochemically using chemical pretreatments solely. Here we describe such pretreatments reporting the difficulty (section adhesion) but also the potential advantages of such methods (easy, quick, inexpensive, and amplifying effect).     

Diversity in neuroanatomical distribution of abnormal prion protein in atypical scrapie

Scrapie is a transmissible spongiform encephalopathy (TSE) in sheep and goats. In recent years, atypical scrapie cases were identified that differed from classical scrapie in the molecular characteristics of the disease-associated pathological prion protein (PrP(sc)). In this study, we analyze the molecular and neuropathological phenotype of nine Swiss TSE cases in sheep and goats. One sheep was identified as classical scrapie, whereas six sheep, as well as two goats, were classified as atypical scrapie. The latter revealed a uniform electrophoretic mobility pattern of the proteinase K-resistant core fragment of PrP(sc) distinct from classical scrapie regardless of the genotype, the species, and the neuroanatomical structure. Remarkably different types of neuroanatomical PrP(sc) distribution were observed in atypical scrapie cases by both western immunoblotting and immunohistochemistry. Our findings indicate that the biodiversity in atypical scrapie is larger than expected and thus impacts on current sampling and testing strategies in small ruminant TSE surveillance.     

Prion protein alleles showing a protective effect on the susceptibility of sheep to scrapie and bovine spongiform encephalopathy

The susceptibility of sheep to classical scrapie and bovine spongiform encephalopathy (BSE) is mainly influenced by prion protein (PrP) polymorphisms A136V, R154H, and Q171R, with the ARR allele associated with significantly decreased susceptibility. Here we report the protective effect of the amino acid substitution M137T, I142K, or N176K on the ARQ allele in sheep experimentally challenged with either scrapie or BSE. Such observations suggest the existence of additional PrP alleles that significantly decrease the susceptibility of sheep to transmissible spongiform encephalopathies, which may have important implications for disease eradication strategies.     

Scrapie strain transmission studies in ovine PrP transgenic mice reveal dissimilar susceptibility

The Tg(OvPrP4) mouse line, expressing the sheep prion protein, is a sensitive model crucial for the identification of the bovine spongiform encephalopathy agent possibly present in natural sheep spongiform encephalopathies. It was also previously demonstrated as susceptible to infection with natural scrapie isolates from sheep harbouring various genotypes. The performance of this new transgenic mouse line in scrapie strain characterization was further assessed by intracranial inoculation of five groups of Tg(OvPrP4) mice with brain homogenate of the wild type mouse-adapted scrapie strains, C506M3, 22A, 79A, 87V, or Chandler. The Tg(OvPrP4) mice were susceptible to the scrapie agent transmitted using mouse-adapted scrapie strains but not equivalently. Strains 87V and Chandler were most readily transmissible followed by 79A and C506M3. Strain 22A was the least transmissible. Clinical signs, survival data, spongiosis, and PrP^sc distribution were also reported. These various data demonstrate the possibility of distinguishing between scrapie strains. Our findings are discussed with regard to agent strain and host factors and already demonstrate the dissimilar susceptibilities of Tg(OvPrP4) mice to the different murine strains studied, thus, reinforcing their potential use in strain typing studies.     

PrP(c) mRNA, but not PrP(Sc) is found in the salivary glands of scrapie-infected sheep

Transmission studies in transmissible spongiform encephalopathies (TSEs) have become increasingly important due to the possible transmission of bovine spongiform encephalopathy to humans resulting in new variant Creutzfeldt-Jacob disease. The horizontal transmission of scrapie, a TSE of sheep, is poorly understood. Possible sources of horizontal transmission are the submandibular and parotid salivary glands. TSEs like natural sheep scrapie are characterized by the conversion of a normal protease sensitive prion protein, PrP(c), to an abnormal protease resistant prion protein, PrP(Sc). Since the presence of PrP(Sc) is an indicator of disease, the salivary glands of scrapie-infected sheep were examined for the presence of PrP(Sc). Although PrP(c) mRNA was detected in the salivary glands, PrP(Sc) was not found in the salivary glands of scrapie-infected sheep. These data suggest that the salivary glands are unlikely sources of horizontal transmission of natural sheep scrapie.     

Clinical Examination Protocol to Detect Atypical and Classical Scrapie in Sheep

The diagnosis of scrapie, a transmissible spongiform encephalopathy (TSEs) of sheep and goats, is currently based on the detection of disease-associated prion protein by post mortem tests. Unless a random sample of the sheep or goat population is actively monitored for scrapie, identification of scrapie cases relies on the reporting of clinical suspects, which is dependent on the individual''s familiarization with the disease and ability to recognize clinical signs associated with scrapie. Scrapie may not be considered in the differential diagnosis of neurological diseases in small ruminants, particularly in countries with low scrapie prevalence, or not recognized if it presents as nonpruritic form like atypical scrapie. To aid in the identification of clinical suspects, a short examination protocol is presented to assess the display of specific clinical signs associated with pruritic and nonpruritic forms of TSEs in sheep, which could also be applied to goats. This includes assessment of behavior, vision (by testing of the menace response), pruritus (by testing the response to scratching), and movement (with and without blindfolding). This may lead to a more detailed neurologic examination of reporting animals as scrapie suspects. It could also be used in experimental TSE studies of sheep or goats to evaluate disease progression or to identify clinical end-point.     

Subclinical prion infection

Prion diseases are transmissible neurodegenerative disorders that include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle and Creutzfeldt–Jakob disease (CJD) in humans. The principal component of the infectious agent responsible for these diseases appears to be an abnormal isoform of the host-encoded prion protein (PrP), designated PrP^Sc. Prion diseases are transmissible to the same or different mammalian species by inoculation with, or dietary exposure to, infected tissues. Although scrapie in sheep has been recognized for over 200 years, it is the recent epidemic of BSE that has centred much public and scientific attention on these neurodegenerative diseases. The occurrence of variant CJD in humans and the experimental confirmation that it is caused by the same prion strain as BSE has highlighted the need for intensive study into the pathogenesis of these diseases and new diagnostic and therapeutic approaches. The existence and implications of subclinical forms of prion disease are discussed.     

The spread of prions through the body in naturally acquired transmissible spongiform encephalopathies

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases that are caused by unconventional pathogens and affect the central nervous system of animals and humans. Several different forms of these diseases result from natural infection (i.e. exposure to transmissible spongiform encephalopathy agents or prions, present in the natural environment of the respective host). This holds true also for scrapie in sheep, bovine spongiform encephalopathy in cattle, chronic wasting disease in elk and deer, or variant Creutzfeldt-Jakob disease in humans, all of which are assumed to originate predominantly from peroral prion infection. This article intends to provide an overview of the current state of knowledge on the spread of scrapie, chronic wasting disease, bovine spongiform encephalopathy and variant Creutzfeldt-Jakob disease agents through the body in naturally affected hosts, and in model animals experimentally challenged via the alimentary tract. Special attention is given to the tissue components and spreading pathways involved in the key stages of prion routing through the body, such as intestinal uptake, neuroinvasion of nerves and the central nervous system, and centrifugal spread from the brain and spinal cord to peripheral sites (e.g. sensory ganglia or muscles). The elucidation of the pathways and mechanisms by which prions invade a host and spread through the organism can contribute to efficient infection control strategies and the improvement of transmissible spongiform encephalopathy diagnostics. It may also help to identify prophylactic or therapeutic approaches that would impede naturally acquired transmissible spongiform encephalopathy infections.     

Increased susceptibility of human-PrP transgenic mice to bovine spongiform encephalopathy infection following passage in sheep

The risk of the transmission of ruminant transmissible spongiform encephalopathy (TSE) to humans was thought to be low due to the lack of association between sheep scrapie and the incidence of human TSE. However, a single TSE agent strain has been shown to cause both bovine spongiform encephalopathy (BSE) and human vCJD, indicating that some ruminant TSEs are transmissible to humans. While the transmission of cattle BSE to humans in transgenic mouse models has been inefficient, indicating the presence of a significant transmission barrier between cattle and humans, BSE has been transmitted to a number of other species. Here, we aimed to further investigate the human transmission barrier following the passage of BSE in a sheep. Following inoculation with cattle BSE, gene-targeted transgenic mice expressing human PrP showed no clinical or pathological signs of TSE disease. However, following inoculation with an isolate of BSE that had been passaged through a sheep, TSE-associated vacuolation and proteinase K-resistant PrP deposition were observed in mice homozygous for the codon 129-methionine PRNP gene. This observation may be due to higher titers of the BSE agent in sheep or an increased susceptibility of humans to BSE prions following passage through a sheep. However, these data confirm that, contrary to previous predictions, it is possible that a sheep prion is transmissible to humans and that BSE from other species is a public health risk.     

Slow viral infections of animals: experimental models for human disease

Sigurdsson's three criterions for a slow viral infection are quoted and discussed and the background of his work briefly described. A fourth criterion for a slow viral infection, is suggested, infection of the hosts lymphoid tissues, which is a common feature of slow infections that have been observed. The general properties of the maedi-visna virus, the diseases and the immune response it causes are discussed. Sheep and goats are susceptible to natural maedi-visna infection. In the goat an identical retrovirus causes arthritis. Arthritis has not been found in maedi-visna infected sheep. Two subacute spongiform encephalopathies of animals are shortly reviewed, scrapie in sheep and goats and transmissible mink encephalopathy (TME). General properties of the very unusual scrapie agent are mentioned briefly. The third type of a slow viral infection is mentioned, Aleutian mink disease, an immunopathological disorder of certain minks caused by a selective infection of lymphoid cells.     

Phenotyping of protein-prion (PrPsc)-accumulating cells in lymphoid and neural tissues of naturally scrapie-affected sheep by double-labeling immunohistochemistry.

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases characterized by amyloid deposition of protein-prion (PrPsc), the pathogenic isoform of the host cellular protein PrPc, in the immune and central nervous systems. In the absence of definitive data on the nature of the infectious agent, PrPsc immunohistochemistry (IHC) constitutes one of the main methodologies for pathogenesis studies of these diseases. In situ PrPsc immunolabeling requires formalin fixation and paraffin embedding of tissues, followed by post-embedding antigen retrieval steps such as formic acid and hydrated autoclaving treatments. These procedures result in poor cellular antigen preservation, precluding the phenotyping of cells involved in scrapie pathogenesis. Until now, PrPsc-positive cell phenotyping relied mainly on morphological criteria. To identify these cells under the PrPsc IHC conditions, a new, rapid, and highly sensitive PrPsc double-labeling technique was developed, using a panel of screened antibodies that allow specific labeling of most of the cell subsets and structures using paraffin-embedded lymphoid and neural tissues from sheep, leading to an accurate identification of ovine PrPsc-accumulating cells. This technique constitutes a useful tool for IHC investigation of scrapie pathogenesis and may be applicable to the study of other ovine infectious diseases.     

Molecular analysis of the protease-resistant prion protein in scrapie and bovine spongiform encephalopathy transmitted to ovine transgenic and wild-type mice

The existence of different strains of infectious agents involved in scrapie, a transmissible spongiform encephalopathy (TSE) of sheep and goats, remains poorly explained. These strains can, however, be differentiated by characteristics of the disease in mice and also by the molecular features of the protease-resistant prion protein (PrP(res)) that accumulates into the infected tissues. For further analysis, we first transmitted the disease from brain samples of TSE-infected sheep to ovine transgenic [Tg(OvPrP4)] and to wild-type (C57BL/6) mice. We show that, as in sheep, molecular differences of PrP(res) detected by Western blotting can differentiate, in both ovine transgenic and wild-type mice, infection by the bovine spongiform encephalopathy (BSE) agent from most scrapie sources. Similarities of an experimental scrapie isolate (CH1641) with BSE were also likewise found following transmission in ovine transgenic mice. Secondly, we transmitted the disease to ovine transgenic mice by inoculation of brain samples of wild-type mice infected with different experimental scrapie strains (C506M3, 87V, 79A, and Chandler) or with BSE. Features of these strains in ovine transgenic mice were reminiscent of those previously described for wild-type mice, by both ratios and by molecular masses of the different PrP(res) glycoforms. Moreover, these studies revealed the diversity of scrapie strains and their differences with BSE according to labeling by a monoclonal antibody (P4). These data, in an experimental model expressing the prion protein of the host of natural scrapie, further suggest a genuine diversity of TSE infectious agents and emphasize its linkage to the molecular features of the abnormal prion protein.     

Lack of Prion Accumulation in Lymphoid Tissues of PRNP ARQ/ARR Sheep Intracranially Inoculated with the Agent of Scrapie

Sheep scrapie is a transmissible spongiform encephalopathy that can be transmitted horizontally. The prion protein gene (PRNP) profoundly influences the susceptibility of sheep to the scrapie agent and the tissue levels and distribution of PrP^Sc in affected sheep. The purpose of this study was to compare the survival time and PrP^Sc tissue distribution in sheep with highly resistant and highly susceptible PRNP genotypes after intracranial inoculation of the agent of scrapie. Five sheep each of genotype VRQ/VRQ, VRQ/ARR or ARQ/ARR were inoculated. Sheep were euthanized when clinical signs of scrapie became severe. Clinical signs, microscopic lesions, and western blot profiles were uniform across genotypes and consistent with manifestations of classical scrapie. Mean survival time differences were associated with the 171 polymorphic site with VRQ/VRQ sheep surviving 18 months, whereas VRQ/ARR and ARQ/ARR sheep survived 60 and 56 months, respectively. Labeling of PrP^Sc by immunohistochemistry revealed similar accumulations in central nervous system tissues regardless of host genotype. Immunoreactivity for PrP^Sc in lymphoid tissue was consistently abundant in VRQ/VRQ, present but confined to tonsil or retropharyngeal lymph node in 4/5 VRQ/ARR, and totally absent in ARQ/ARR sheep. The results of this study demonstrate the susceptibility of sheep with the ARQ/ARR genotype to scrapie by the intracranial inoculation route with PrP^Sc accumulation in CNS tissues, but prolonged incubation times and lack of PrP^Sc in lymphoid tissue.     

Structural and functional analysis of the HSP90AA1 gene: distribution of polymorphisms among sheep with different responses to scrapie

Scrapie is a transmissible spongiform encephalopathy in sheep and goats. Susceptibility to this neurodegenerative disease is mainly controlled by point mutations at the PRNP locus. Other genes, apart from PRNP, have been reported to modulate resistance/susceptibility to scrapie. On the basis of several studies in Alzheimer and different transmissible spongiform encephalopathy models, HSP90AA1 was chosen as a putative positional and functional candidate gene that might be involved in the polygenic variance mentioned above. In the present work, the ovine HSP90AA1 gene including the promoter and other regulatory regions has been isolated and characterized. Several sequence polymorphisms have also been identified. FISH-mapping localized the HSP90AA1 gene on ovine chromosome OAR19q24dist, which was confirmed by linkage analysis. This chromosome region has been shown to include a quantitative trait loci (QTL) for scrapie incubation period in sheep. Expression analyses were carried out in spleen and cerebellum samples. No differences in the expression of the HSP90AA1 gene were found in any of these tissues (p > 0.05) between control and infected animal samples. Nevertheless, association analyses revealed that several polymorphisms in the 5′ and 3′ regions of the HSP90AA1 gene were differentially distributed among animals with different responses to scrapie infection. Thus, results presented here support the hypothesis that HSP90AA1 could be a positional and functional candidate gene modulating the response to scrapie in sheep. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Prion Interaction with the 37-kDa/67-kDa Laminin Receptor on Enterocytes as a Cellular Model for Intestinal Uptake of Prions

Enterocytes, a major cell population of the intestinal epithelium, represent one possible barrier to the entry of prions after oral exposure. We established a cell culture system employing enterocytes from different species to study alimentary prion interaction with the 37-kDa/67-kDa laminin receptor LRP/LR. Human, bovine, porcine, ovine, and cervid enterocytes were cocultured with brain homogenates from cervid, sheep, and cattle suffering from chronic wasting disease (CWD), scrapie, and bovine spongiform encephalopathy (BSE), respectively. PrP^CWD, ovine PrP^Sc, and PrP^BSE all colocalized with LRP/LR on human enterocytes. PrP^CWD failed to colocalize with LRP/LR on bovine, porcine, and ovine enterocytes. Ovine PrP^Sc colocalized with the receptor on bovine enterocytes, but failed to colocalize with LRP/LR on cervid and porcine enterocytes. PrP^BSE failed to colocalize with the receptor on cervid and ovine enterocytes. These data suggest possible oral transmissibility of CWD and sheep scrapie to humans and may confirm the oral transmissibility of BSE to humans, resulting in zoonotic variant Creutzfeldt-Jakob disease. CWD might not be transmissible to cattle, pigs, and sheep. Sheep scrapie might have caused BSE, but may not cause transmissible spongiform encephalopathy in cervids and pigs. BSE may not be transmissible to cervids. Our data recommend the enterocyte model system for further investigations of the intestinal pathophysiology of alimentary prion infections.