Transmissible spongiform encephalopathy strain-associated diversity of N-terminal proteinase K cleavage sites of PrP(Sc) from scrapie-infected and bovine spongiform encephalopathy-infected mice.

Assessment of the different conformational states of the abnormal prion protein (PrP(Sc)) in the CNS provides an established basis for distinguishing transmissible spongiform encephalopathy (TSE) strains. PrP(Sc) conformers are variably resistant to N-terminal proteinase K (PK) digestion, and analysis of the consensus products (PrP(res)) by immunoassay enables effective, but relatively low-resolution differentiation. Determination of the precise N-terminal amino acid profile (N-TAAP) of PrP(res) presents a potential high-resolution means of TSE-strain typing, and thus of differential disease diagnosis. This approach was evaluated using individual mice affected by model scrapie (22A, ME7, 87V and 79A) and bovine spongiform encephalopathy (BSE) (301V) strains. Nano liquid chromatography-mass spectrometry (LC-MS) was used to determine PrP(res) N-terminal tryptic digestion products. Four major N-terminal tryptic peptides were generated from all mouse TSE strains investigated, corresponding with predominant N-termination of PrP(res) at G(81), G(85), G(89) and G(91). Both the mass spectrometric abundance of the individual peptides and the ratios of pairs of these peptides were evaluated as markers of conformation in relation to their potential for strain discrimination. The yield of peptides was significantly greater for BSE than scrapie strains and the relative quantities of particular peptide pairs differed between strains. Thus, whereas peptide G(91)-K(105) was a dominant peptide from 301V, this was not the case for other strains and, significantly, the ratio of peptides G(91)-K(105):G(89)-K(105) was substantially higher for BSE-infected compared with scrapie-infected mice. These data support the potential of the N-TAAP approach for high-resolution TSE strain typing and differential diagnosis.     

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.     

Rapid PrP(Sc) detection in lymphoid tissue and application to scrapie surveillance of fallen stock in Japan: variable PrP(Sc) accumulation in palatal tonsil in natural scrapie

Rapid western blot (WB) procedure for an abnormal isoform of prion protein (PrP(Sc) ) detection in lymphoid tissues was established and has been applied to the surveillance of fallen stock. In this program, brain and palatal tonsil were examined by WB and three cases of sheep scrapie were detected. While one clinically scrapie-infected sheep harbored PrP(Sc) in the brain and palatal tonsil, the two sheep in the pre-clinical stage harbored PrP(Sc) in the brain, but not in the palatal tonsil. This study shows that PrP(Sc) accumulation in palatal tonsil is variable in natural scrapie, even among genetically susceptible sheep.     

Surveillance and simulation of bovine spongiform encephalopathy and scrapie in small ruminants in Switzerland

Background  

After bovine spongiform encephalopathy (BSE) emerged in European cattle livestock in 1986 a fundamental question was whether the agent established also in the small ruminants' population. In Switzerland transmissible spongiform encephalopathies (TSEs) in small ruminants have been monitored since 1990. While in the most recent TSE cases a BSE infection could be excluded, for historical cases techniques to discriminate scrapie from BSE had not been available at the time of diagnosis and thus their status remained unclear. We herein applied state-of-the-art techniques to retrospectively classify these animals and to re-analyze the affected flocks for secondary cases. These results were the basis for models, simulating the course of TSEs over a period of 70 years. The aim was to come to a statistically based overall assessment of the TSE situation in the domestic small ruminant population in Switzerland.     

Exposure of sheep scrapie brain homogenate to rumen-simulating conditions does not result in a reduction of PrP(Sc) levels

AIMS: Experiments were designed to evaluate the potential of rumen-simulating conditions to reduce PrP(Sc) levels. METHODS AND RESULTS: Scrapie-positive brain material was incubated under rumen-simulating conditions. Time points were taken over a 24-h period and PrP(Sc) levels were analysed by Western blot. No loss of PrP(Sc) was observed over a 24-h time period. CONCLUSIONS: Our results indicate that a fully developed rumen fermentation does not provide significant protection against prion infection via the oral route. Developmental changes including senescence of immune system function or other developmental changes in the gastrointestinal tract are potential mechanisms by which relative bovine spongiform encephalopathy (BSE) susceptibility might vary with age. SIGNIFICANCE AND IMPACT OF THE STUDY: Epidemiology of the BSE outbreak in the United Kingdom indicates that younger animals were at higher risk of infection. The rumen undergoes pronounced developmental changes early in life, coinciding with the introduction of fibre into the diet. The timeframe of highest risk of infection overlaps the time in life prior to full rumen development. This work indicates that a fully developed rumen does not provide significant protection against prion infection via the oral route of infection. This result implicates other developmental changes that are responsible for the age-dependent susceptibility of cattle to BSE.     

Discriminating scrapie and bovine spongiform encephalopathy isolates by infrared spectroscopy of pathological prion protein

For the surveillance of transmissible spongiform encephalopathies (TSEs) in animals and humans, the discrimination of different TSE strains causing scrapie, BSE, or Creutzfeldt-Jakob disease constitutes a substantial challenge. We addressed this problem by Fourier transform-infrared (FT-IR) spectroscopy of pathological prion protein PrP27-30. Different isolates of hamster-adapted scrapie (263K, 22A-H, and ME7-H) and BSE (BSE-H) were passaged in Syrian hamsters. Two of these agents, 22A-H and ME7-H, caused TSEs with indistinguishable clinical symptoms, neuropathological changes, and electrophoretic mobilities and glycosylation patterns of PrP27-30. However, FT-IR spectroscopy revealed that PrP27-30 of all four isolates featured different characteristics in the secondary structure, allowing a clear distinction between the passaged TSE agents. FT-IR analysis showed that phenotypic information is mirrored in beta-sheet and other secondary structure elements of PrP27-30, also in cases where immunobiochemical typing failed to detect structural differences. If the findings of this study hold true for nonexperimental TSEs in animals and humans, FT-IR characterization of PrP27-30 may provide a versatile tool for molecular strain typing without antibodies and without restrictions to specific TSEs or mammalian species.     

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.     

Immunohistochemical comparison of anti-prion protein (PrP) antibodies in the CNS of mice infected with scrapie.

One of the pathological changes characteristic of the transmissible spongiform encephalopathies (TSEs) is the accumulation of disease-specific PrP (PrP(sc)). Immunolabeling of PrP(sc) was compared using a panel of monoclonal and polyclonal antibodies. To determine the effects of tissue fixation on immunostaining, we performed a supplementary investigation reviewing the fixatives formol saline and periodate-lysine-paraformaldehyde (PLP). The main target sites of the antibodies were similar. However the monoclonal antibodies (MAbs) 6H4, 7A12 and 8H4 revealed targeted PrP(sc) labeling with no background labeling. Although 7A12 and 8H4 did not detect early PrP deposition, we propose that during the later stages of disease 7A12 and 8H4 can be used with equal effectiveness in place of 6H4. Tissues taken during the early stages of disease that had been fixed in PLP displayed more PrP immunolabeling than tissues that had undergone formol fixation. PLP fixation on 6H4-immunostained tissue revealed interweaving granular linear PrP deposits in the hippocampus. This labeling was not observed in tissue that had undergone formol fixation, suggesting that PLP fixation might enhance the sensitivity of the immunohistochemical (IHC) detection of PrP. In the two scrapie mouse models studied here, PLP fixation and immunolabeling with the anti-PrP antibody 6H4 gave superior results.     

Differences in proteinase K resistance and neuronal deposition of abnormal prion proteins characterize bovine spongiform encephalopathy (BSE) and scrapie strains.

Prion diseases are associated with the accumulation of an abnormal isoform of host-encoded prion protein (PrP(Sc)). A number of prion strains can be distinguished by "glycotyping" analysis of the respective deposited PrP(Sc) compound. In this study, the long-term proteinase K resistance, the molecular mass, and the localization of PrP(Sc) deposits derived from conventional and transgenic mice inoculated with 11 different BSE and scrapie strains or isolates were examined. Differences were found in the long-term proteinase K resistance (50 microg/ml at 37 degrees C) of PrP(Sc). For example, scrapie strain Chandler or PrP(Sc) derived from field BSE isolates were destroyed after 6 hr of exposure, whereas PrP(Sc) of strains 87V and ME7 and of the Hessen1 isolate were extremely resistant to proteolytic cleavage. Nonglycosylated, proteinase K-treated PrP(Sc) of BSE isolates and of scrapie strain 87V exhibited a 1-2 kD lower molecular mass than PrP(Sc) derived from all other scrapie strains and isolates. With the exception of strain 87V, PrP(Sc) was generally deposited in the cerebrum, cerebellum, and brain stem of different mouse lines at comparable levels. Long-term proteinase resistance, molecular mass, and the analysis of PrP(Sc) deposition therefore provide useful criteria in discriminating prion strains and isolates (e.g., BSE and 87V) that are otherwise indistinguishable by the PrP(Sc) "glycotyping" technique.     

Comparative molecular analysis of the abnormal prion protein in field scrapie cases and experimental bovine spongiform encephalopathy in sheep by use of Western blotting and immunohistochemical methods

Since the appearance of bovine spongiform encephalopathy (BSE) in cattle and its linkage with the human variant of Creutzfeldt-Jakob disease, the possible spread of this agent to sheep flocks has been of concern as a potential new source of contamination. Molecular analysis of the protease cleavage of the abnormal prion protein (PrP), by Western blotting (PrP(res)) or by immunohistochemical methods (PrP(d)), has shown some potential to distinguish BSE and scrapie in sheep. Using a newly developed enzyme-linked immunosorbent assay, we identified 18 infected sheep in which PrP(res) showed an increased sensitivity to proteinase K digestion. When analyzed by Western blotting, two of them showed a low molecular mass of unglycosylated PrP(res) as found in BSE-infected sheep, in contrast to other naturally infected sheep. A decrease of the labeling by P4 monoclonal antibody, which recognizes an epitope close to the protease cleavage site, was also found by Western blotting in the former two samples, but this was less marked than in BSE-infected sheep. These two samples, and all of the other natural scrapie cases studied, were clearly distinguishable from those from sheep inoculated with the BSE agent from either French or British cattle by immunohistochemical analysis of PrP(d) labeling in the brain and lymphoid tissues. Final characterization of the strain involved in these samples will require analysis of the features of the disease following infection of mice, but our data already emphasize the need to use the different available methods to define the molecular properties of abnormal PrP and its possible similarities with the BSE agent.     

Detection of prion protein particles in blood plasma of scrapie infected sheep

Prion diseases are transmissible neurodegenerative diseases affecting humans and animals. The agent of the disease is the prion consisting mainly, if not solely, of a misfolded and aggregated isoform of the host-encoded prion protein (PrP). Transmission of prions can occur naturally but also accidentally, e.g. by blood transfusion, which has raised serious concerns about blood product safety and emphasized the need for a reliable diagnostic test. In this report we present a method based on surface-FIDA (fluorescence intensity distribution analysis), that exploits the high state of molecular aggregation of PrP as an unequivocal diagnostic marker of the disease, and show that it can detect infection in blood. To prepare PrP aggregates from blood plasma we introduced a detergent and lipase treatment to separate PrP from blood lipophilic components. Prion protein aggregates were subsequently precipitated by phosphotungstic acid, immobilized on a glass surface by covalently bound capture antibodies, and finally labeled with fluorescent antibody probes. Individual PrP aggregates were visualized by laser scanning microscopy where signal intensity was proportional to aggregate size. After signal processing to remove the background from low fluorescence particles, fluorescence intensities of all remaining PrP particles were summed. We detected PrP aggregates in plasma samples from six out of ten scrapie-positive sheep with no false positives from uninfected sheep. Applying simultaneous intensity and size discrimination, ten out of ten samples from scrapie sheep could be differentiated from uninfected sheep. The implications for ante mortem diagnosis of prion diseases are discussed.     

Relationship between clinical signs and postmortem test status in cattle experimentally infected with the bovine spongiform encephalopathy agent

Background  

Various clinical protocols have been developed to aid in the clinical diagnosis of classical bovine spongiform encephalopathy (BSE), which is confirmed by postmortem examinations based on vacuolation and accumulation of disease-associated prion protein (PrP^d) in the brain. The present study investigated the occurrence and progression of sixty selected clinical signs and behaviour combinations in 513 experimentally exposed cattle subsequently categorised postmortem as confirmed or unconfirmed BSE cases. Appropriate undosed or saline inoculated controls were examined similarly and the data analysed to explore the possible occurrence of BSE-specific clinical expression in animals unconfirmed by postmortem examinations.     

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.     

Molecular analysis of the abnormal prion protein during coinfection of mice by bovine spongiform encephalopathy and a scrapie agent

Molecular features of the proteinase K-resistant prion protein (PrP res) may discriminate among prion strains, and a specific signature could be found during infection by the infectious agent causing bovine spongiform encephalopathy (BSE). To investigate the molecular basis of BSE adaptation and selection, we established a model of coinfection of mice by both BSE and a sheep scrapie strain (C506M3). We now show that the PrP res features in these mice, characterized by glycoform ratios and electrophoretic mobilities, may be undistinguishable from those found in mice infected with scrapie only, including when mice were inoculated by both strains at the same time and by the same intracerebral inoculation route. Western blot analysis using different antibodies against sequences near the putative N-terminal end of PrP res also demonstrated differences in the main proteinase K cleavage sites between mice showing either the BSE or scrapie PrP res profile. These results, which may be linked to higher levels of PrP res associated with infection by scrapie, were similar following a challenge by a higher dose of the BSE agent during coinfection by both strains intracerebrally. Whereas PrP res extraction methods used allowed us to distinguish type 1 and type 2 PrP res, differing, like BSE and scrapie, by their electrophoretic mobilities, in the same brain region of some patients with Creutzfeldt-Jakob disease, analysis of in vitro mixtures of BSE and scrapie brain homogenates did not allow us to distinguish BSE and scrapie PrP res. These results suggest that the BSE agent, the origin of which remains unknown so far but which may have arisen from a sheep scrapie agent, may be hidden by a scrapie strain during attempts to identify it by molecular studies and following transmission of the disease in mice.     

Proteolytic inactivation of the bovine spongiform encephalopathy agent

Thermostable proteases have been investigated for their ability to provide a novel biological solution to decontamination of prion agents responsible for transmissible spongiform encephalopathies (TSEs). Proteases were identified that digested total mouse brain homogenate (MBH) protein from uninfected mice. These proteases were then evaluated for digestion of BSE (301V) infectious MBH over a range of pH and temperatures, screened for loss of anti-prion antibody 6H4 immunoreactivity and protease-treated infectious MBH assessed in mouse bioassay using VM mice. Despite a number of proteases eliminating all 6H4-immunoreactive material, only the subtilisin-enzyme Properase showed a significant extension in incubation period in mouse bioassays following a 30-min incubation at 60 degrees C and pH 12. These results demonstrate the potential of the method to provide a practical solution to the problems of TSE contamination of surgical instruments and highlight the inadequacy of using Western blot for assessment of decontamination/inactivation of TSE agents.     

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.     

PrP N-terminal domain triggers PrP(Sc)-like aggregation of Dpl

Transmissible spongiform encephalopathies are fatal neurodegenerative disorders thought to be transmitted by self-perpetuating conformational conversion of a neuronal membrane glycoprotein (PrP^C, for “cellular prion protein”) into an abnormal state (PrP^Sc, for “scrapie prion protein”). Doppel (Dpl) is a protein that shares significant biochemical and structural homology with PrP^C. In contrast to its homologue PrP^C, Dpl is unable to participate in prion disease progression or to achieve an abnormal PrP^Sc-like state. We have constructed a chimeric mouse protein, composed of the N-terminal domain of PrP^C (residues 23-125) and the C-terminal part of Dpl (residues 58-157). This chimeric protein displays PrP-like biochemical and structural features; when incubated in presence of NaCl, the α-helical monomer forms soluble β-sheet-rich oligomers which acquire partial resistance to pepsin proteolysis in vitro, as do PrP oligomers. Moreover, the presence of aggregates akin to protofibrils is observed in soluble oligomeric species by electron microscopy.     

Infectivity in skeletal muscle of cattle with atypical bovine spongiform encephalopathy

The amyloidotic form of bovine spongiform encephalopathy (BSE) termed BASE is caused by a prion strain whose biological properties differ from those of typical BSE, resulting in a clinically and pathologically distinct phenotype. Whether peripheral tissues of BASE-affected cattle contain infectivity is unknown. This is a critical issue since the BASE prion is readily transmissible to a variety of hosts including primates, suggesting that humans may be susceptible. We carried out bioassays in transgenic mice overexpressing bovine PrP (Tgbov XV) and found infectivity in a variety of skeletal muscles from cattle with natural and experimental BASE. Noteworthy, all BASE muscles used for inoculation transmitted disease, although the attack rate differed between experimental and natural cases (～70% versus ～10%, respectively). This difference was likely related to different prion titers, possibly due to different stages of disease in the two conditions, i.e. terminal stage in experimental BASE and pre-symptomatic stage in natural BASE. The neuropathological phenotype and PrP(res) type were consistent in all affected mice and matched those of Tgbov XV mice infected with brain homogenate from natural BASE. The immunohistochemical analysis of skeletal muscles from cattle with natural and experimental BASE showed the presence of abnormal prion protein deposits within muscle fibers. Conversely, Tgbov XV mice challenged with lymphoid tissue and kidney from natural and experimental BASE did not develop disease. The novel information on the neuromuscular tropism of the BASE strain, efficiently overcoming species barriers, underlines the relevance of maintaining an active surveillance.