Acquired transmissibility of sheep-passaged L-type bovine spongiform encephalopathy prion to wild-type mice

L-type bovine spongiform encephalopathy (L-BSE) is an atypical form of BSE that is transmissible to cattle and several lines of prion protein (PrP) transgenic mice, but not to wild-type mice. In this study, we examined the transmissibility of sheep-passaged L-BSE prions to wild-type mice. Disease-associated prion protein (PrP^Sc) was detected in the brain and/or lymphoid tissues during the lifespan of mice that were asymptomatic subclinical carriers, indicating that wild-type mice were susceptible to sheep-passaged L-BSE. The morphological characteristics of the PrP^Sc of sheep-passaged L-BSE included florid plaques that were distributed mainly in the cerebral cortex and hippocampus of subsequent passaged mice. The PrP^Sc glycoform profiles of wild-type mice infected with sheep-passaged L-BSE were similar to those of the original isolate. The data indicate that sheep-passaged L-BSE has an altered host range and acquired transmissibility to wild-type mice.     

Pathological characterization of TgElk mice injected with brain homogenate from elk with chronic wasting disease

Chronic wasting disease (CWD) is classified as a transmissible spongiform encephalopathy or prion disease that affects cervids. CWD has been reported in 15 US states, two Canadian provinces, and in imported elk on several farms in Korea. This study was conducted to examine the molecular biological and pathogenic characteristics of a CWD-associated prion isolated in Korea. The epidemiological origin of this pathogen was also determined. Homozygous TgElk mice were infected with a CWD-affected elk brain pool prepared from the brain of an imported Canadian elk. We measured the incubation time of the pathogen, neuropathological changes by immunohistochemical staining, the pattern(s) of scrapie prion protein (PrP^Sc) deposition, and PrP^Sc protein profiles by Western blotting. We found that TgElk mice infected with brain homogenate from the elk suffering from CWD showed incubation times, vacuolar degeneration, and PrP^Sc accumulation similar to those previously reported in the literature. Our results suggest that homozygous TgElk mice efficiently transmit CWD with short incubation times and that this animal can serve a valuable research model and reliable in vivo diagnostic tool.     

Cellular prion proteins in humans and cattle but not sheep are characterized by a low-solubility phenotype

A feature of transmissible spongiform encephalopathies is the accumulation of infectious prion proteins (PrP^Sc), which are formed by the conversion of physiological prion proteins (PrP^C). As PrP^C, which is modified posttranslationally with various types of glycoproteins, serves as the substrates for PrP^Sc conversion, various PrP^C subtypes may play a role in the formation of PrP^Sc and species-specific transmission; the cattle disease BSE is transmissible naturally to humans, but the sheep disease scrapie is not. To reveal new mechanisms modulating prion conversion, we analyzed the PrP^C profiles by determining the differential PrP^C protein solubilities in the anionic and nonionic detergents N-lauroylsarcosine, N-octyl-β-d-glucopyranoside, CHAPS and deoxycholic acid. We compared the resulting solubility profiles of human PrP^C with the solubility profiles of PrP^C from sheep and cattle. The PrP^C subtypes were differentially soluble. However, non-glycosylated PrP^C from cattle and human was found explicitly in the insoluble fraction, while non-glycosylated ovine PrP^C was detected in the soluble fraction. These findings indicate the existence of low-solubility PrP^C phenotypes in cattle and humans.     

Hepatitis in dogs. An indexed bibliography of journal articles (1994–2004)

Transmissible spongiform encephalopathies (TSEs) or prion diseases are unique disorders that are not caused by infectious micro-organisms (bacteria or fungi), viruses or parasites, but rather seem to be the result of an infectious protein. TSEs are comprised of fatal neurodegenerative disorders affecting both human and animals. Prion diseases cause sponge-like degeneration of neuronal tissue and include (among others) Creutzfeldt–Jacob disease in humans, bovine spongiform encephalopathy (BSE) in cattle and scrapie in sheep. TSEs are characterized by the formation and accumulation of transmissible (infectious) disease-associated protease-resistant prion protein (PrP^Sc), mainly in tissues of the central nervous system. The exact molecular processes behind the conversion of PrP^C into PrP^Sc are not clearly understood. Correlations between prion protein polymorphisms and disease have been found, however in what way these polymorphisms influence the conversion processes remains an enigma; is stabilization or destabilization of the prion protein the basis for a higher conversion propensity? Apart from the disease-associated polymorphisms of the prion protein, the molecular processes underlying conversion are not understood. There are some notions as to which regions of the prion protein are involved in refolding of PrP^C into PrP^Sc and where the most drastic structural changes take place. Direct interactions between PrP^C molecules and/or PrP^Sc are likely at the basis of conversion, however which specific amino acid domains are involved and to what extent these domains contribute to conversion resistance/sensitivity of the prion protein or the species barrier is still unknown.     

Incubation of ovine scrapie with environmental matrix results in biological and biochemical changes of PrPSc over time

Ovine scrapie can be transmitted via environmental reservoirs. A pool of ovine scrapie isolates were incubated on soil for one day or thirteen months and eluted prion was used to challenge tg338 mice transgenic for ovine PrP. After one-day incubation on soil, two PrP^Sc phenotypes were present: G₃₃₈ or Apl₃₃₈ii. Thirteen months later some divergent PrP^Sc phenotypes were seen: a mixture of Apl₃₃₈ii with either G₃₃₈ or P_338, and a completely novel PrP^Sc deposition, designated Cag₃₃₈. The data show that prolonged ageing of scrapie prions within an environmental matrix may result in changes in the dominant PrP^Sc biological/biochemical properties.     

Experimental infection of meadow voles (Microtus pennsylvanicus) with sheep scrapie

Meadow voles (Microtus pennsylvanicus) are permissive to chronic wasting disease (CWD) infection, but their susceptibility to other transmissible spongiform encephalopathies (TSEs) is poorly characterized. In this initial study, we intracerebrally challenged 6 meadow voles with 2 isolates of sheep scrapie. Three meadow voles acquired a TSE after the scrapie challenge and an extended incubation period. The glycoform profile of proteinase K-resistant prion protein (PrP^res) in scrapie-sick voles remained similar to the sheep inocula, but differed from that of voles clinically affected by CWD. Vacuolization patterns and disease-associated prion protein (PrP^Sc) deposition were generally similar in all scrapie-affected voles, except in the hippocampus, where PrP^Sc staining varied markedly among the animals. Our results demonstrate that meadow voles can acquire a TSE after intracerebral scrapie challenge and that this species could therefore prove useful for characterizing scrapie isolates.     

Central nervous system extracellular matrix changes in a transgenic mouse model of bovine spongiform encephalopathy

Bovine spongiform encephalopathy (BSE) is a transmissible spongiform encephalopathy characterised by accumulation of resistant prion protein (PrP^BSE), neuronal loss, spongiosus and glial cell proliferation. In this study, properties of the extracellular matrix (ECM) were investigated in boTg110 transgenic mice over-expressing the bovine cellular prion protein (PrP^c) and infected with BSE. Using immunohistochemistry with Wisteria floribunda agglutinin as a specific marker for perineuronal nets (PNNs) and antibodies against aggrecan and hyaluronic acid binding protein, loss of ECM was correlated with PrP^BSE accumulation and activation of astrocytes and microglia. PrP^BSE accumulation and glial cell activation were detected from the earliest stages of the disease and increased in the terminal stages. Decreases in PNNs, aggrecan and hyaluronic acid were observed only in the terminal stages and correlated with the distribution of PrP^BSE and activated glial cells. This study suggests that the loss of PNNs, aggrecan and hyaluronic acid is a consequence of PrP^BSE accumulation. Degradation of ECM in BSE may be due to secretion of degradative enzymes by activated glial cells.     

朊病毒检测方法综述

传染性海绵状脑病(transmissible spongiform encephalopathies,TSEs)是由朊病毒引起的人和多种哺乳动物以神经退行性变化为主要特征的一种慢性消耗性传染病,也称作朊病毒病;其是由体内正常细胞表面的PrP^C转变成PrP^Sc蛋白所导致。但PrP^Sc主要在脑内表达,在其他组织表达量很低,因此快捷准确的诊断方法对于该病有重要意义。作者重点介绍以朊病毒的致病特点为依据建立的检测方法,便于对该疾病的早期诊断、预防以及食品安全检测提供帮助,保障畜牧业的有序发展以及人类的健康。     

Co-existence of classical scrapie and Nor98 in a sheep from an Italian outbreak

Nor98 is an atypical scrapie strain characterized by a molecular pattern and brain distribution of the pathological prion protein (PrP^Sc) different from classical scrapie. In Italy, 69 atypical cases have been identified so far and all were characterized as Nor98 strain. In this paper we report an unusual case in a sheep which showed immunohistochemical and molecular features of PrP^Sc different from the other atypical cases. The sheep was from an outbreak where the index and the other four cases were affected by classical scrapie. Histopathological, immunohistochemical and Western blot analyses on the brain of the unusual case revealed the simultaneous presence of pathological features characteristic of Nor98 and classical scrapie. Interestingly, the prevalent disease phenotype in the brainstem was classical scrapie-like, while in the cerebral cortex and cerebellum the Nor98 phenotype was dominant. The sub-mandibular lymph node was positive and showed a PrP^Sc molecular pattern referable to classical scrapie. The PrP genotype was AL₁₄₁RQ/AF₁₄₁RQ. Taken together, the occurrence of classical scrapie in the outbreak, the PrP genotype, the involvement of different cellular targets in the brain and the pathological and molecular PrP^Sc features observed suggest that this unusual case may result from the co-existence of Nor98 and classical scrapie.     

General aspects of transmissible spongiform encephalopathies and hypotheses about the agents

Summary

This article reviews the shared characteristics of the group of transmissible spongiform encephalopathies (SEs), both human and animal, and the major theories regarding the nature of the agents involved.

All transmissible SE diseases share two striking characteristics: the degenerative changes including vacuolation in the central nervous system, and the assumption that these disorders are caused by unconventional, transmissible agents. This article examines the major hypotheses that have been postulated about these agents: the virus theory, the virino theory, the prion theory, and the recently proposed ‘unified theory’. Both the virus and the virino hypotheses assume that a small nucleic acid is involved as part of the agent, while the prion hypothesis does not.

The prion model obviates the need for a role of a nucleic acid in the propagation and replication of the agent, but does not explain the existence of strain variation. Nucleic acids in a micro‐organism, as proposed in the virino and the virus hypotheses, could explain this variation. However, to date, no disease‐specific nucleic acids have been identified. The ‘unified’ theory tries to reconcile the essentials of the virino and prion theories.

The article also describes the discovery of the so‐called prion protein (PrP), its isoforms, and the coding host gene, the PrP gene. It goes on to discuss the results of experiments with transgenic animals, indicating that mutations in the PrP gene may play a decisive role in the pathogenesis of at least some SEs. Finally, two different models, both involving the conversion of normal PrP^C into PrP^Sc as part of the pathogenesis of SE, are discussed.     

Sensitivity and specificity of a commercial BSE kit for the detection of ovine scrapie

To examine the sensitivity of a commercially available bovine spongiform encephalopathy (BSE) kit (NippIBL) for the detection of ovine scrapie, 50 scrapie‐positive ovine samples from the UK, and 54 scrapie‐negative ovine samples from Japan were obtain and tested using this kit. The sensitivity and specificity of NippIBL for ovine samples were 96% and 100%, respectively. The detection limit of the abnormal isoform of prion protein (PrP^Sc) of NippIBL was examined using diluted scrapie‐positive samples. The sensitivity of NippIBL to ovine scrapie was 3–10 times superior to that of another commercial BSE diagnosis kit. Thus, the NippIBL kit proved more effective for the detection of ovine scrapie.     

Observations on thermostable subpopulations of the unconventional agents that cause transmissible degenerative encephalopathies

When scrapie agent is exposed to partially inactivating autoclave cycles, the fraction of infectivity that survives remains thermostable during relatively long periods of autoclaving. This resistant subpopulation can also be differentiated from the main population by its prolonged incubation periods in assay animals, compared with control material. Stabilisation of this subpopulation may occur through the smearing and drying of infected tissue that can occur prior to autoclaving, in which the disease-specific form of PrP protein (PrP^Sc) could become rapidly heat-fixed. This may paradoxically be what protects this fraction of PrP^Sc from further inactivation during autoclaving. Data are presented showing that the thermostability acquired by the resistant subpopulation is a stable characteristic; autoclaving for a second time results in very little further loss of infectivity. These observations suggest that inactivation procedures that do not involve rapid and effective fixation of PrP^Sc may be better candidates for dealing effectively with scrapie-like agents.     

Studies on the Putative Interactions between the Organophosphorus Insecticide Phosmet and Recombinant Mouse PrP and its Implication in the BSE Epidemic

It has been suggested that exposure of cattle to the ectoparasiticide Phosmet in the 1980s caused a conformational change in the cellular prion protein (PrP^C) to form the BSE prion (PrP^SC), which initiated the epidemic of bovine spongiform encephalopathy (BSE).Recombinant mouse cellular prion (r[mouse]PrP^C) was exposed to the organophosphorus pesticide Phosmet in vitro and the conformation of the prion before and after exposure was monitored using circular dichroism (CD) spectroscopy, utilizing synchrotron radiation at the Council for the Central Laboratory of the Research Councils (CLRC) facilities at Daresbury, UK. Metabolites of Phosmet, generated in situ by rat microsomes, were investigated in the same way, to determine whether they might initiate the conformational change due to their high chemical reactivity.Our studies showed that exposure of r[mouse]PrP^C to Phosmet or microsomes-generated metabolites of Phosmet did not result in the conformational change in the protein from -helix to -pleated sheet that is characteristic of the PrP^C to PrP^SC conversion and, therefore, Phosmet is very unlikely to have initiated the BSE epidemic by a simple direct mechanism of conformational change in the prion protein.     

Reflections on scrapie and related disorders,with consideration of the possibility of a viral aetiology

The transmissible spongiform encephalopathies of domesticated animals, scrapie in-sheep and bovine spongiform encephalopathy (BSE), and transmissible mink encephalopathy are more than a scientific curiosity; under certain circumstances their impact on commercial activities can be calamitous. Knowledge of their causation and pathogenesis is still rudimentary, but many consider than an unconventional agent, the prion (a brain protein, PrP), that is not associated with nucleic acid is involved in both. Others believe that conventional viruses, which replicate by virtue of their nucleic acid-defined genes, are involved in the causation and progression of the encephalopathies but that technical problems have prevented their identification. Others postulate even more exotic causative agents. While this paper will particularly address the possibility of a viral aetiology for these diseases, it is also emphasized that our knowledge of the state of the immune system in animals with encephalopathy needs broadening. There are remarkable gaps in our knowledge of the histopathology of these diseases, particularly the nature of the characteristic vacuoles. Much further work is needed on the biochemical changes in the brain and the serum, particularly of the latter as it could lead to an additional means of recognizing clinical cases without waiting for the animal to die with subsequent examination of the brain for characteristic lesions and the presence of protease-K-resistant PrP.Abbreviations AI artificial insemination - BSE bovine spongiform encephalopathy - CJD Creutzfeldt-Jakob disease - ET embryo transfer - GSSD Gerstmann-Sträussler-Scheinker disease - HDV hepatitis delta virus - MCF mink cell focus - PK proteinase K - PrP prion protein - PrPSc scrapie prion protein - PrP-C the proteinase-K sensitive homologue in normal brain - SAF scrapie-associated fibrils - TME transmissible mink encephalopathy     

Classical natural ovine scrapie prions detected in practical volumes of blood by lamb and transgenic mouse bioassays

Scrapie is diagnosed antemortem in sheep by detecting misfolded isoforms of prion protein (PrP^Sc) in lymphoid follicles of the rectal mucosa and nictitating membranes. Assay sensitivity is limited if (a) the biopsy is collected early during disease development, (b) an insufficient number of follicles is collected, or (c) peripheral accumulation of PrP^Sc is reduced or delayed. A blood test would be convenient for mass live animal scrapie testing. Currently approved techniques, however, have their own detection limits. Novel detection methods may soon offer a non-animal-based, rapid platform with detection sensitivities that rival the prion bioassay. In anticipation, we sought to determine if diseased animals could be routinely identified with a bioassay using B lymphocytes isolated from blood sample volumes commonly collected for diagnostic purposes in small ruminants. Scrapie transmission was detected in five of six recipient lambs intravenously transfused with B lymphocytes isolated from 5~10 mL of blood from a naturally scrapie-infected sheep. Additionally, scrapie transmission was observed in 18 ovinized transgenic Tg338 mice intracerebrally inoculated with B lymphocytes isolated from 5~10 mL of blood from two naturally scrapie-infected sheep. Based on our findings, we anticipate that these blood sample volumes should be of diagnostic value.     

The Potential of Mesenchymal Stem Cell in Prion Research

Scrapie and bovine spongiform encephalopathy are fatal neurodegenerative diseases caused by the accumulation of a misfolded protein (PrP^res), the pathological form of the cellular prion protein (PrP^C). For the last decades, prion research has greatly progressed, but many questions need to be solved about prion replication mechanisms, cell toxicity, differences in genetic susceptibility, species barrier or the nature of prion strains. These studies can be developed in murine models of transmissible spongiform encephalopathies, although development of cell models for prion replication and sample titration could reduce economic and timing costs and also serve for basic research and treatment testing. Some murine cell lines can replicate scrapie strains previously adapted in mice and very few show the toxic effects of prion accumulation. Brain cell primary cultures can be more accurate models but are difficult to develop in naturally susceptible species like humans or domestic ruminants. Stem cells can be differentiated into neuron‐like cells and be infected by prions. However, the use of embryo stem cells causes ethical problems in humans. Mesenchymal stem cells (MSCs) can be isolated from many adult tissues, including bone marrow, adipose tissue or even peripheral blood. These cells differentiate into neuronal cells, express PrP^C and can be infected by prions in vitro. In addition, in the last years, these cells are being used to develop therapies for many diseases, including neurodegenerative diseases. We review here the use of cell models in prion research with a special interest in the potential use of MSCs.     

Comparison of brain PrPd distribution in ovine BSE and scrapie

Scrapie and bovine spongiform encephalopathy (BSE) are both prion diseases affecting ruminants, and these diseases do not share the same public health concerns. Surveillance of the BSE agent in small ruminants has been a great challenge, and the recent identification of diverse prion diseases in ruminants has led to the development of new methods for strain typing. In our study, using immunohistochemistry (IHC), we assessed the distribution of PrP(d) in the brains of 2 experimentally BSE-infected sheep with the ARQ/ARQ genotype. Distribution of PrP(d) in the brain, from the spinal cord to the frontal cortex, was remarkably similar in the 2 sheep despite different inoculation routes and incubation periods. Comparatively, overall PrP(d) brain distribution, evaluated by IHC, in 19 scrapie cases with the ARQ/ARQ, ARQ/VRQ, and VRQ/VRQ genotypes, in some cases showed similarities to the experimentally BSE-infected sheep. There was no exclusive neuroanatomical site with a characteristic and specific PrP(d) type of accumulation induced by the BSE agent. However, a detailed analysis of the topography, types, and intensity of PrP(d) deposits in the frontal cortex, striatum, piriform cortex, hippocampus, mesencephalon, and cerebellum allowed the BSE-affected sheep group to be distinguished from the 19 scrapie cases analyzed in our study. These results strengthen and emphasize the potential interest of PrP(d) brain mapping to help in identifying prion strains in small ruminants.     

Properties of L-type bovine spongiform encephalopathy in intraspecies passages

The origin and transmission routes of atypical bovine spongiform encephalopathy (BSE) remain unclear. To assess whether the biological and biochemical characteristics of atypical L-type BSE detected in Japanese cattle (BSE/JP24) are conserved during serial passages within a single host, 3 calves were inoculated intracerebrally with a brain homogenate prepared from first-passaged BSE/JP24-affected cattle. Detailed immunohistochemical and neuropathologic analysis of the brains of second-passaged animals, which had developed the disease and survived for an average of 16 months after inoculation, revealed distribution of spongiform changes and disease-associated prion protein (PrP(Sc)) throughout the brain. Although immunolabeled PrP(Sc) obtained from brain tissue was characterized by the presence of PrP plaques and diffuse synaptic granular accumulations, no stellate-type deposits were detected. Western blot analysis suggested no obvious differences in PrP(Sc) molecular mass or glycoform pattern in the brains of first- and second-passaged cattle. These findings suggest failures to identify differences in mean incubation period and biochemical and neuropathologic properties of the BSE/JP24 prion between the first and second passages in cattle.