Widespread PrPSc accumulation in muscles of hamsters orally infected with scrapie

Scrapie, bovine spongiform encephalopathy and chronic wasting disease are orally communicable, transmissible spongiform encephalopathies (TSEs). As zoonotic transmissions of TSE agents may pose a risk to human health, the identification of reservoirs for infectivity in animal tissues and their exclusion from human consumption has become a matter of great importance for consumer protection. In this study, a variety of muscles from hamsters that were orally challenged with scrapie was screened for the presence of a molecular marker for TSE infection, PrP^Sc (the pathological isoform of the prion protein PrP). Sensitive western blotting revealed consistent PrP^Sc accumulation in skeletal muscles from forelimb and hindlimb, head, back and shoulder, and in tongue. Previously, our animal model has provided substantial baseline information about the peripheral routing of infection in naturally occurring and orally acquired ruminant TSEs. Therefore, the findings described here highlight further the necessity to investigate thoroughly whether muscles of TSE-infected sheep, cattle, elk and deer contain infectious agents.     

Detection of PrPres in genetically susceptible fetuses from sheep with natural scrapie

Scrapie is a transmissible spongiform encephalopathy with a wide PrPres dissemination in many non-neural tissues and with high levels of transmissibility within susceptible populations. Mechanisms of transmission are incompletely understood. It is generally assumed that it is horizontally transmitted by direct contact between animals or indirectly through the environment, where scrapie can remain infectious for years. In contrast, in utero vertical transmission has never been demonstrated and has rarely been studied. Recently, the use of the protein misfolding cyclic amplification technique (PMCA) has allowed prion detection in various tissues and excretions in which PrPres levels have been undetectable by traditional assays. The main goal of this study was to detect PrPres in fetal tissues and the amniotic fluid from natural scrapie infected ewes using the PMCA technique. Six fetuses from three infected pregnant ewes in an advanced clinical stage of the disease were included in the study. From each fetus, amniotic fluid, brain, spleen, ileo-cecal valve and retropharyngeal lymph node samples were collected and analyzed using Western blotting and PMCA. Although all samples were negative using Western blotting, PrPres was detected after in vitro amplification. Our results represent the first time the biochemical detection of prions in fetal tissues, suggesting that the in utero transmission of scrapie in natural infected sheep might be possible.     

Accumulation of pathological prion protein PrPSc in the skin of animals with experimental and natural scrapie

Prion infectivity and its molecular marker, the pathological prion protein PrP(Sc), accumulate in the central nervous system and often also in lymphoid tissue of animals or humans affected by transmissible spongiform encephalopathies. Recently, PrP(Sc) was found in tissues previously considered not to be invaded by prions (e.g., skeletal muscles). Here, we address the question of whether prions target the skin and show widespread PrP(Sc) deposition in this organ in hamsters perorally or parenterally challenged with scrapie. In hamsters fed with scrapie, PrP(Sc) was detected before the onset of symptoms, but the bulk of skin-associated PrP(Sc) accumulated in the clinical phase. PrP(Sc) was localized in nerve fibres within the skin but not in keratinocytes, and the deposition of PrP(Sc) in skin showed no dependence from the route of infection and lymphotropic dissemination. The data indicated a neurally mediated centrifugal spread of prions to the skin. Furthermore, in a follow-up study, we examined sheep naturally infected with scrapie and detected PrP(Sc) by Western blotting in skin samples from two out of five animals. Our findings point to the skin as a potential reservoir of prions, which should be further investigated in relation to disease transmission.     

Calpain-dependent endoproteolytic cleavage of PrPSc modulates scrapie prion propagation

Previous studies using post-mortem human brain extracts demonstrated that PrP in Creutzfeldt-Jakob disease (CJD) brains is cleaved by a cellular protease to generate a C-terminal fragment, referred to as C2, which has the same molecular weight as PrP-(27-30), the protease-resistant core of PrP(Sc) (1). The role of this endoproteolytic cleavage of PrP in prion pathogenesis and the identity of the cellular protease responsible for production of the C2 cleavage product has not been explored. To address these issues we have taken a combination of pharmacological and genetic approaches using persistently infected scrapie mouse brain (SMB) cells. We confirm that production of C2 is the predominant cleavage event of PrP(Sc) in the brains of scrapie-infected mice and that SMB cells faithfully recapitulate the diverse intracellular proteolytic processing events of PrP(Sc) and PrP(C) observed in vivo. While increases in intracellular calcium (Ca(2+)) levels in prion-infected cell cultures stimulate the production of the PrP(Sc) cleavage product, pharmacological inhibitors of calpains and overexpression of the endogenous calpain inhibitor, calpastatin, prevent the production of C2. In contrast, inhibitors of lysosomal proteases, caspases, and the proteasome have no effect on C2 production in SMB cells. Calpain inhibition also prevents the accumulation of PrP(Sc) in SMB and persistently infected ScN2A cells, whereas bioassay of inhibitor-treated cell cultures demonstrates that calpain inhibition results in reduced prion titers compared with control-treated cultures assessed in parallel. Our observations suggest that calpain-mediated endoproteolytic cleavage of PrP(Sc) may be an important event in prion propagation.     

Atypical scrapie in sheep from a UK research flock which is free from classical scrapie

Background  

Colic could be accompanied by changes in the morphology and physiology of organs and tissues, such as the intestine. This process might be, at least in part, due to the accumulation of oxidative damage induced by reactive oxygen (ROS) and reactive nitrogen species (RNS), secondary to intestinal ischemia. Glutathione (GSH), being the major intracellular thiol, provides protection against oxidative injury. The aim of this study was to investigate whether ischemia-induced intestinal injury could be related with alterations in GSH metabolism.     

PrPSc in salivary glands of scrapie-affected sheep

The salivary glands of scrapie-affected sheep and healthy controls were investigated for the presence of the pathological prion protein (PrP(Sc)). PrP(Sc) was detected in major (parotid and mandibular) and minor (buccal, labial, and palatine) salivary glands of naturally and experimentally infected sheep. Using Western blotting, the PrP(Sc) concentration in glands was estimated to be 0.02 to 0.005% of that in brain. Immunohistochemistry revealed intracellular depositions of PrP(Sc) in ductal and acinar epithelia and occasional labeling in the lumina of salivary ducts. The presence of PrP(Sc) in salivary glands highlights the possible role of saliva in the horizontal transmission of scrapie.     

Characterization of scrapie agent isolated from sheep in Japan

A pathogenic agent isolated in mice from the brain of a sheep affected by scrapie-like disease was characterized. The incubation period of the disease in the primary transmission from the sheep to mice was longer than in the secondary and the tertiary transmission in the same strain of mice. Progressive dilution of the inoculum caused prolongation of the incubation period. The infectivity of the agent in a 10% brain homogenate persisted, but decreased about 10(3) to 10(4) times after heating at 100 C for 30 min. Histological changes in the diseased mouse brains consisted of vacuolation of the nerve cells and spongiform degeneration in the gray matter of the central nervous system. Fine rod-shaped granulae with a length of 3 to 5 nm were observed within the swollen neuropil, axon, and perivascular astrocytic process. No serum antibodies against available mouse viruses, parainfluenza type 1 virus, lymphocytic choriomeningitis virus, and mouse reovirus type 3, were detected in any mice used in the experiments. These findings demonstrate that the disease of the sheep was the first case of scrapie in Japan.     

Accumulation and dissemination of prion protein in experimental sheep scrapie in the natural host

Background  

Since 1954, there have been in excess of 800 cases of rabies as a result of European Bat Lyssaviruses types 1 and 2 (EBLV-1, EBLV-2) infection, mainly in Serotine and Myotis bats respectively. These viruses have rarely been reported to infect humans and terrestrial mammals, as the only exceptions are sheep in Denmark, a stone marten in Germany and a cat in France. The purpose of this study was to investigate the susceptibility of foxes to EBLVs using silver foxes (Vulpes vulpes) as a model.     

Markedly increased susceptibility to natural sheep scrapie of transgenic mice expressing ovine prp

The susceptibility of sheep to scrapie is known to involve, as a major determinant, the nature of the prion protein (PrP) allele, with the VRQ allele conferring the highest susceptibility to the disease. Transgenic mice expressing in their brains three different ovine PrP(VRQ)-encoding transgenes under an endogenous PrP-deficient genetic background were established. Nine transgenic (tgOv) lines were selected and challenged with two scrapie field isolates derived from VRQ-homozygous affected sheep. All inoculated mice developed neurological signs associated with a transmissible spongiform encephalopathy (TSE) disease and accumulated a protease-resistant form of PrP (PrPres) in their brains. The incubation duration appeared to be inversely related to the PrP steady-state level in the brain, irrespective of the transgene construct. The survival time for animals from the line expressing the highest level of PrP was reduced by at least 1 year compared to those of two groups of conventional mice. With one isolate, the duration of incubation was as short as 2 months, which is comparable to that observed for the rodent TSE models with the briefest survival times. No survival time reduction was observed upon subpassaging of either isolate, suggesting no need for adaptation of the agent to its new host. Overexpression of the transgene was found not to be required for transmission to be accelerated compared to that observed with wild-type mice. Conversely, transgenic mice overexpressing murine PrP were found to be less susceptible than tgOv lines expressing ovine PrP at physiological levels. These data argue that ovine PrP(VRQ) provided a better substrate for sheep prion replication than did mouse PrP. Altogether, these tgOv mice could be an improved model for experimental studies on natural sheep scrapie.     

Mouse-adapted ovine scrapie prion strains are characterized by different conformers of PrPSc

The agent responsible for prion disease may exist in different forms, commonly referred to as strains, with each carrying the specific information that determines its own distinct biological properties, such as incubation period and lesion profile. Biological strain typing of ovine scrapie isolates by serial passage in conventional mice has shown some diversity in ovine prion strains. However, this biological diversity remains poorly supported by biochemical prion strain typing. The protein-only hypothesis predicts that variation between different prion strains in the same host is manifest in different conformations adopted by PrPSc. Here we have investigated the molecular properties of PrPSc associated with two principal Prnp(a) mouse-adapted ovine scrapie strains, namely, RML and ME7, in order to establish biochemical prion strain typing strategies that may subsequently be used to discriminate field cases of mouse-passaged ovine scrapie isolates. We used a conformation-dependent immunoassay and a conformational stability assay, together with Western blot analysis, to demonstrate that RML and ME7 PrPSc proteins show distinct biochemical and physicochemical properties. Although RML and ME7 PrPSc proteins showed similar resistance to proteolytic digestion, they differed in their glycoform profiles and levels of proteinase K (PK)-sensitive and PK-resistant isoforms. In addition, the PK-resistant core (PrP27-30) of ME7 was conformationally more stable following exposure to guanidine hydrochloride or Sarkosyl than was RML PrP27-30. Our data show that mouse-adapted ovine scrapie strains can be discriminated by their distinct conformers of PrPSc, which provides a basis to investigate their diversity at the molecular level.     

The genetics of scrapie in sheep and goats

Scrapie, an invariably fatal disease of sheep and goats, is a transmissible spongiform encephalopathy (TSE). The putative infectious agent is the host-encoded prion protein, PrP. The development of scrapie is closely linked to polymorphisms in the host PrP gene. The pathogenesis of most TSEs involves conversion of normal, cellular PrP into a protease-resistant, pathogenic isoform called PrPSc. The conversion to PrPSc involves change in secondary structure; it is impacts on these structural changes that may link polymorphisms to disease. Within the structured C-terminal part of PrP polymorphisms have been reported at 15 and 10 codons of the sheep and goat PrP genes respectively. Three polymorphisms in sheep are acutely linked to the occurrence of scrapie: A136V, R154H and Q171R/H. These generate five commonly observed alleles: ARQ, ARR, AHQ, ARH and VRQ. ARR and AHQ are associated with resistance; ARQ, ARH and VRQ are associated with susceptibility. There are subtle effects of specific allele pairings (genotypes). Generally, more susceptible genotypes have younger ages at death from scrapie. Different strains of scrapie occur which may attack genotypes differently. Different sheep breeds vary in the assortment of the five alleles that they predominantly encode. The reason for this variation is not known. Furthermore, certain genotypes may be susceptible to scrapie in some breeds and resistant in others. The explanation is not known, but may relate to different scrapie strains circulating in different breeds, or there may be effects of other genes which modulate the effect of PrP.     

Population dynamics of scrapie in a sheep flock.

A detailed analysis of an outbreak of natural scrapie in a flock of Cheviot sheep is described. A total of 137 cases was reported over 13 years among 1307 sheep born into the flock. The epidemiology of scrapie can only be understood with reference to sheep demography, the population genetics of susceptibility to scrapie, pathogenesis during a long incubation period, and the rate of transmission (by both vertical and horizontal routes), all of which interact in complex ways. A mathematical model incorporating these features is described, parameter values and model inputs are derived from available information from the flock and from independent sources, and model outputs are compared with the field data. The model is able to reproduce key features of the outbreak, including its long duration and the ages of cases. The analysis supports earlier work suggesting that many infected sheep do not survive to show clinical signs, that most cases arise through horizontal transmission, and that there is strong selection against susceptible genotypes. However, important aspects of scrapie epidemiology remain poorly understood, including the possible role of carrier genotypes and of an environmental reservoir of infectivity, and the mechanisms maintaining alleles giving susceptibility to scrapie in the sheep population.     

Two different scrapie prions isolated in Japanese sheep flocks

Two different scrapie prion strains with different characteristics were obtained from two sheep naturally infected with scrapie in Japan. In mice transmission, one (Tsukuba-1) showed shorter incubation periods (133+/-2 days) than the other (Tsukuba-2) (288+/-5 days). Spongiform changes and accumulation of an abnormal isoform of prion protein (PrP(Sc)) were observed throughout the brain in Tsukuba-1 inoculated mice, while the lesions and the PrP(Sc) accumulation were localized in the brain stem of mice with Tsukuba-2. Western blot analysis showed that there was no strain-specific glycoform of PrP(Sc) within these two strains. A super-infection experiment revealed that neither strain interfered with the other's propagation.     

Atypical/Nor98 scrapie infectivity in sheep peripheral tissues

Atypical/Nor98 scrapie was first identified in 1998 in Norway. It is now considered as a worldwide disease of small ruminants and currently represents a significant part of the detected transmissible spongiform encephalopathies (TSE) cases in Europe. Atypical/Nor98 scrapie cases were reported in ARR/ARR sheep, which are highly resistant to BSE and other small ruminants TSE agents. The biology and pathogenesis of the Atypical/Nor98 scrapie agent in its natural host is still poorly understood. However, based on the absence of detectable abnormal PrP in peripheral tissues of affected individuals, human and animal exposure risk to this specific TSE agent has been considered low. In this study we demonstrate that infectivity can accumulate, even if no abnormal PrP is detectable, in lymphoid tissues, nerves, and muscles from natural and/or experimental Atypical/Nor98 scrapie cases. Evidence is provided that, in comparison to other TSE agents, samples containing Atypical/Nor98 scrapie infectivity could remain PrP(Sc) negative. This feature will impact detection of Atypical/Nor98 scrapie cases in the field, and highlights the need to review current evaluations of the disease prevalence and potential transmissibility. Finally, an estimate is made of the infectivity loads accumulating in peripheral tissues in both Atypical/Nor98 and classical scrapie cases that currently enter the food chain. The results obtained indicate that dietary exposure risk to small ruminants TSE agents may be higher than commonly believed.