NR ASAQ

AU anonym

TI TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES

QU Research Report des Institut for Animal Health 1994

IA http://www.iah.bbsrc.ac.uk/institut/public/3wtse.htm (nicht mehr vorhanden)

PT Research Report

VT identification of candidate molecular species
The nature of the scrapie agent remains ill-defined but the hypothetical requirement for an independent informational molecule (presumably nucleic acid) remains. Accordingly, a search is continuing for candidate scrapie-specific nucleic acids. After purifying scrapie infectivity at high yield from infected brain, nucleic acids are extracted under standard conditions. As an alternative, minimal conditions to remove proteins and release nucleic acids for analysis have also been used.
Nucleic acid extracts have been analysed on silver stained polyacrylamide gels where distinct patterns of bands <300 nucleotides have been identified in extracts from control and scrapie- infected brain. Similar patterns were observed for extracts from mouse brains infected with three different strains of scrapie. The nucleic acid fragments are resistant to RNAase digestion prior to detergent extraction (ie when the extract is still infectious), but all purified fragments of nucleic acid are susceptible to RNAase but not DNAase.
chemical characterisation
The conversion of the normal form of PrP protein (PrPc) to its protease-resistant isoform (PrPsc) is a key process in the development of scrapie and related diseases. PrPsc is enriched in preparations of infectious particles and, either by itself or in association with other molecules, is regarded by many as the infectious agent. The structure of PrPsc (and infectivity) is resistant to heat and chemicals which destroy most other micro-organisms and this stability was a critical factor in the transmission of a spongiform encephalopathy to cattle in the early 1980s which initiated the epidemic of BSE. To understand this stability and its relationship to infectivity, it is necessary to understand the molecular detail of the formation of PrPsc.
Using a TX-114/N-lauryl sarcosinate extraction protocol, it was found that rodent brain PrPc (in contrast to PrPc in cultured cells and cell lines) is extremely heterogeneous. This raised the possibility that not all of the forms of PrPc in brain might act as a precursor for PrPsc. To test this hypothesis, the levels of different fractions of protease-sensitive PrP in three rodent scrapie models - the ME7 and 87V strains of scrapie in Sinc p7 mice and the 263K strain of scrapie in Syrian hamsters - have been investigated. In all models, a significant rise (4-fold) was detected in the level of protease-sensitive PrP associated with a phospholipid- rich, cholesterol-rich fraction of brain membrane during the development of disease. This protease-sensitive PrP (PrP*) may either be an intermediate in the conversion of PrPc to PrPsc or a protease-sensitive component of the 'pre-amyloid.'
If the scrapie phenotype is encoded by a unique conformation of the PrPsc oligomer then this may be 'mutated' by the use of denaturants. In experiments using denaturants to modify PrPsc followed by titration in different strains of mice, both biphasic and non-parallel dose- response curves (incubation time vs PrPsc injected) have been observed. In some cases, the relative incubation time in different strains of mice (one of the criteria for strain typing) appears to depend on dose. The basis of these anomalies is under investigation.
Attempts to re-constitute infectivity from PrPsc dissolved in 6M-guanidinium chloride solutions (which completely abolish the protease-resistance of PrPsc) by dilution techniques have been unsuccessful. If one molecule in 107 had re-folded to the putative 'infectious' conformation, this event would have been detected by the hamster bioassay.
strain variation in scrapie and related agents
The characterisation of isolates of spongiform encephalopathies from a range of natural and experimental sources, by transmission to mice, has continued. Transmissions of scrapie-like disease from kudu, nyala and cats have already given closely similar results to BSE, suggesting a common infection source. This also shows that the donor species has little effect on disease characteristics in mice. In contrast, the transmission characteristics of natural sheep scrapie, chronic wasting disease of mule deer, TME and cow-passaged TME differ from those of BSE, suggesting that different strains of agent are involved.
Transmissions from two 'negative line' Cheviot sheep, intracerebrally or orally infected with BSE, and from a goat, intracerebrally infected with BSE, have given similar results to cattle BSE. However, two further sources, a 'positive line' Cheviot and a goat, both orally infected with BSE, are unlike BSE when transmitted to mice. Either the donor animals were infected with natural scrapie or passage of BSE through these individuals had selected a new strain.
effects of mutagens
An attempt to mutate the 87A scrapie by exposing mice to gamma-irradiation 100-300 days post- infection is continuing. The possibility that nucleic acid repair occurs over the long bioassays of scrapie, following irradiation, is being tested using SCID mice injected with irradiated (21 kGy) or unirradiated agent, as these mice are reported to carry a DNA repair defect.
The possibility that scrapie disease involves the co-replication of agents has been tested. Intracerebral injection of mice with mixed (1+2, 3+4, and 1+2+3+4) 10-7 diluted ME7 or 139A, in which Karber endpoints were 10-6.14 (ME7) and 10-5.9 (139A) failed to produce disease after over 685 days of incubation.
cell culture-based assay for scrapie agent
The conditions for growth, transfection and selection of stably transformed PC12 cells have been thoroughly examined. Suitable protocols have been developed and clonally purified PC12 cell lines transfected with PrP gene constructs have been produced. The inability to repeat the published scrapie infection of PC12 cells appeared to be inexplicable. Repeated attempts using a highly sensitive immunoassay failed to observe the conversion of PrPc to PrPsc. Unpublished information from the authors of the papers has revealed that scrapie infection is absolutely dependent on the passage history of the PC12 cells. Work is now being repeated with new PC12 cells; subtle differences in growth characteristics have already been observed.
No rat PrP-specific antibody reagents are available to distinguish between host (rat PC-12) and exogenous PrP (murine) in the transfected PC-12 cell lines. An E coli gene expression construct has been made and sequenced and will be used to produce sufficient rat recPrP protein for use as an antigen. Mouse anti-rat PrP polyclonal, and ultimately monoclonal, antibodies will then be made.
recombinant prp protein
N-terminal sequence analysis has revealed that the recPrP protein expressed from mammalian cells has the predicted mature N-terminus. No evidence of substantial raggedness was observed. Additional analysis of the protein was performed by electrospray mass spectrometry. This revealed that, unlike recPrP material from E coli, there was a degree of micro-heterogeneity, despite favourable polyacrylamide gel analysis. Some heterogeneity appeared to be due to a partial loss of mass consistent with loss of the C-terminal amino acid residue (loss of serine at the C-terminus in several other proteins has been observed).
Other components found in the sample indicated that appreciable oxidation had occurred since multiple bands differing by + 16 Da were observed in the sample. This oxidation could be a consequence of biosynthesis, purification or sample preparation. Modifications to the protocols used in these three areas are being evaluated.
scrapie-associated protein prp
Soluble recPrP protein has been produced and secreted by mammalian cells and good yields of protein have been purified for structural studies. A micellular concentration of detergent has been found to be necessary to prevent self-aggregation and to maintain the solubility of the protein, especially when it is concentrated (>0.1 mg/ml). The use of sub-micellular levels of detergent with other solubilisation agents (glycerol, DMSO, sucrose) was found not to be an effective alternative.
Concentration of soluble recPrP protein to 1 mg/ml has been achieved by ultrafiltration. This material was observed to be a tight double band by SDS-PAGE analysis and free of detectable breakdown by sensitive Western blot assay. Preliminary sitting drop crystallisation experiments gave encouraging results. Under favourable conditions small crystals (~0.1 mm length) were obtained which were absent in the control drops.
Higher concentrations of protein (10-20 mg/ml) are required to obtain high quality large crystals for detailed structural analysis. The achievement of >1 mg/ml soluble recPrP protein concentrations by ultrafiltration is precluded by the requirement for detergent micelles. Extensive investigation of the physical properties of a wide range of biological detergents has identified an alternative detergent with the appropriate characteristics for protein solubilisation but possessing a reduced micellular diameter. In addition a diversity of ultrafiltration devices are being evaluated for use under a range of buffer and temperature regimes.
The X-ray crystallography studies are in collaboration with University of Oxford.
control of prp messenger rna expression
This project is designed to investigate the potential of the PrP gene 3'-untranslated region (UTR) to control expression at the transcriptional or translational level. Interest in this region of the PrP gene results from the sheep genetic studies in which an EcoRI restriction fragment length polymorphism has been associated with incidence of both experimental and natural scrapie and from the study of PrP mRNA in the sheep where a second short PrP mRNA is expressed in peripheral tissues. The peripheral mRNA is shorter than the full length mRNA in the 3'-UTR.
Reporter gene constructs have been made containing various sections of the PrP 3'-UTR downstream of the CAT gene. Two constructs contain the entire PrP 3'-UTR, one in reverse orientation. Other constructs contain the 3'-UTR with sections deleted. A control construct contains only the CAT gene with no 3'-UTR. In in vitro cell-free systems, expression of CAT protein has not been markedly affected by any of the PrP 3'-UTR regions. The constructs all express mRNA containing both CAT sequences and the appropriate 3'-UTR.
5'- and 3'-non-coding regions of prp messenger rna
PrP gene promoter and exon 1 sequences from NPU Cheviot sheep have been isolated to enable the use of RT-PCR for the detection and characterisation of sheep PrP mRNA. These sequences differed in only a few nucleotides between different PrP alleles.
Sheep mRNA from various tissues and developmental stages has been analysed in RNA protection assays. It was shown, for the first time, that sheep with heterozygous PrP genotype (valine or alanine at codon 136) produce mRNA from both PrP alleles at about the same level. These experiments also revealed new sequence differences in the 3'-untranslated mRNA between these PrP alleles.
intercellular trafficking of scrapie infection in vitro
In the persistently scrapie-infected cell line SMB the production of PrPsc from its precursor protein PrPc is sensitive to the action of sulphated compounds, notably glycosaminoglycans (GAGs) and the dye Congo Red. Addition of soluble GAG or Congo Red to growing cultures of these cells irreversibly inhibits the conversion of the cell-surface PrP to the disease-specific isotype.
Elevated levels of insulin cause an increase in the cellular production of polysulphated GAG giving rise to higher than normal levels of soluble proteoglycan (PG), but without substantially elevating the levels of cell-surface associated free-GAG or PG. Insulin does not affect PrPsc synthesis. beta-D-xyloside also elevates the production of sulphated polyanions but this is released from the cell as soluble GAG or resides as free-GAG in the extracellular matrix. Unlike insulin, beta-D-xyloside does inhibit PrPsc synthesis, most likely by increasing the amount of soluble polysulphates.
Another compound which interferes with PrPsc production in vivo and, importantly, delays the onset of clinical TSE is the antibiotic amphotericin B, but as this has no effect on PrPsc production in neuroblastoma-TSE cell models, defining its mode of action has been impossible by this route. However, it has now been shown that amphotericin B is a potent inhibitor of PrPsc synthesis in the SMB cell model and its mode of action in these cells is being studied. This points to fundamental differences between the only two persistent TSE culture models, neuroblastoma and SMB, which has direct relevance to modelling the TSE disease process in vivo.
in vitro cell systems for replication
Development of the SMB cell line, a persistently scrapie-infected line derived from a scrapie- infected mouse, has been continued for the studies on PrP synthesis, degradation and its conversion to the disease-specific isoform PrPsc. SMB sub-clones have been derived which have lost the ability to produce PrPsc and these have proved to be suitable as uninfected controls, a previously missing link. Retroviral vectors are being developed for introducing PrP coding sequences into SMB cells, allowing the analysis of the fates of co-expressed PrP proteins. The introduction of different species, eg cow, sheep, hamster and human PrP proteins, will allow the confirmation of certain species-barrier effects, whether or not PrP on its own dictates the tropism of the TSE agents. It is also intended to introduce mouse PrP sequences with structural mutations and to study the interaction of these mutant proteins with the PrPc and PrPsc fractions.
expression of prp gene during infection
Despite the marked changes in amount and distribution of PrP protein in scrapie-affected mouse brain compared with controls, no differences were seen in the localisation or amount of PrP mRNA. This suggests that a breakdown in post-transcriptional control mechanisms in scrapie-affected animals may lead to abnormal deposition of PrP protein in the brain.
In scrapie-affected sheep, no differences were seen in levels of PrP mRNA in brain or uterine tissues.
expression of prp in lymphoid tissues
The expression of PrP on the surface of ruminant leukocytes has been studied using recently produced mAbs. Two mAbs raised against recombinant ovine PrP (AF6 and FH11) and two raised against recombinant bovine PrP (BG4 and KG9) were tested; KG9 is specific for the bovine molecule whereas the others recognise PrP from sheep and cattle. All four mAbs detected low levels of PrP on blood leukocytes and on cells obtained from lymph nodes, spleen and thymus. Control chicken leukocytes were not labelled with the mAb. PrP was expressed on all cell lineages (lymphocytes, monocytes and granulocytes). Binding of the mAb was sensitive to enzymatic treatment of the cells with PI-PLC indicating the GPI membrane anchorage expected for native PrP. In contrast to previously reported findings with human lymphocytes, no increase in expression of PrP was observed following activation of sheep lymphocytes with the mitogen conA. Peripheral blood leukocytes from sheep with clinical scrapie showed no detectable increase in cell surface expression of PrP when compared with healthy animals.
mice with prp gene alterations
A double replacement gene targeting strategy has been developed. The first step replaces the PrP coding exon with a selectable marker - the HPRT minigene. This replacement was carried out in HPRT-deficient HM-1 cells, selection was for HPRT expression in HAT medium. Enrichment of gene targeted clones was achieved by use of a negative selectable marker, the Herpes simplex thymidine kinase gene. The clones were assayed for gene targeting by PCR analysis. One in five of the clones contained the targeted allele.
In the second targeting step the HPRT minigene was itself replaced by the PrP coding region containing the Pro(101) to Leu mutation. Selection for the second step involved 6- thioguanine selection against HPRT expression. Targeted cell lines containing one wild-type allele and a second PrP allele with the Pro(101) to Leu mutation were identified by PCR and DdeI digestion.
Chimaeric mice have been produced with the targeted cells. Germ line transmission of the targeted allele has been obtained, and a colony of Pro(101) to Leu mice is currently being established.
molecular pathology of scrapie
Analysis of the transcribed sequences of murine PrP and its relationship to scrapie incubation periods has revealed two polymorphisms in the 3'-untranslated region of the gene. These are the only sequence differences found in the non-translated portion of the PrP mRNA. The Sincs7 mice SV, L129/Ola and NZW all have adenosine at positions 444 and 1010 of the 3'- UTR. The Sincp7 mice VM and I/LnJ have guanosine at these positions. These changes appear to be linked to those at codons 108 and 189 of the PrP protein which are thought to be responsible for Sinc differences. RIII and VL mice which both have short incubation periods and contain the 108 and 189 polymorphisms associated with Sincs7 mice both contain a guanosine for the 3'-UTR polymorphism. They also contain an extra polymorphism in their coding region at codon 188. This is silent with no change in the amino acid. RIII mice do show atypical properties with regards to scrapie incubation periods, most significantly when inoculated with the BSE agent.
peripheral pathogenesis and pathology of scrapie
An immunocytochemical study on the distribution of PrP in a range of peripheral organs in scrapie infected and non-infected mice has been completed, using organs from PrP-null mice as controls. Non-isotopic in situ hybridisation techniques are being used to investigate the expression of the PrP gene in the relevant cell types.
pathology in the central nervous system
The study of neuronal loss in the dorsal lateral geniculate nucleus following intraocular infection with ME7 has been extended to demonstrate the sequence of events preceding neuronal degeneration. Morphometric techniques are also being used to examine neuronal loss in the hippocampus. The nature of this degeneration is being studied using light, electron and confocal microscopy. Examination of Golgi-stained sections has revealed a loss of dendritic spines, and attempts are being made to quantify this. Initial confocal microscopy studies of lucifer yellow-filled neurons indicate that this is the method of choice for future studies of dendritic spine loss and other structural changes such as vacuolation. There is increasing evidence from electron microscopic studies that the mechanism of neuronal loss is apoptotic.
Studies on the immuno-localisation of PrP at the ultrastructural level are being extended to a wider range of experimental models and to earlier times in the incubation period. The aim is to correlate PrP accumulation with the structural changes preceding neuronal loss.
In collaboration with Free University, Amsterdam, an immuno-histochemical study of amyloid 'chaperone' molecules in scrapie has been started. These molecules are commonly associated with amyloid deposits of different types and are thought to participate in the aggregation of precursor proteins into fibrillar amyloid.
scrapie disease in prp-deficient mice
A mouse line with an inactive PrP gene has been produced by gene targeting. In animals heterozygous for this mutation, PrP mRNA is reduced by approximately 50% throughout the brain, compared with wild-type mice. The steady-state level of PrPc is also significantly reduced in heterozygotes compared to wild-type mice. PrP mRNA and protein are not detected in brains of mice homozygous for the mutation. Wild-type mice and mice heterozygous and homozygous for the mutation with ME7 strain of scrapie have been infected.
A gene dosage effect can be seen in time of disease onset and period over which the disease symptoms develop. In heterozygotes disease onset occurs around 220 days and terminal stages are reached by 280 days. In wild-type mice disease onset occurs around 130 days and the terminal stages by 160 days. The PrP-/- mice are resistant to disease up to 475 days. PrP deposition in heterozygous mice starts in the same brain area as wild-type mice and can be detected as early as 50 days. The pattern of PrP deposition in the brain of heterozygotes follows an identical course to that observed in wild type mice and by terminal stages of disease the amount deposited is equivalent to wild-type mice. Vacuolation is detected later than PrP deposition and distribution and degree in the terminal stages of disease is similar in wild-type and heterozygous mice. These results show that signs of disease, vacuolation and PrP deposition are dependent on available PrPc in a rate-dependent manner.
scrapie-associated fibrils and polymeric prp
The most highly purified infectious fractions (InfF) from scrapie-infected tissues contain an abundance of polymeric PrPsc but also various amounts of non-PrP polypeptides, glycosaminoglycan, lipid and nucleic acid fragments. Denaturation of InfF leads to the depolymerisation of the aggregates and controlled renaturation allows the repolymerisation of these complexes to occur without the loss of infectivity. Tests are being carried out on whether the stoichiometry of the components alters during the cycle of denaturation and renaturation and also, by fractionation of the components of the complexes under denaturing conditions before allowing renaturation, whether the removal of particular components alters the structure of the infectious titre of the InfF complex.
antigenic structure of prp isoforms
The sequence of PrP is highly conserved between mammalian species. This limits the production of mAbs recognising PrP to those epitopes that differ between species. To overcome this limitation, attempts are being made to produce monoclonal reagents recognising PrP from immunised chickens. Since no established procedures are available to make mAbs from chickens, bacteriophage display technology is being developed to achieve this.
Several bacteriophage antibodies have been isolated from the naive bursal repertoire by selection with immobilised recombinant bovine PrP. Problems with non-specific binding and limited recognition by many of these antibodies has now been overcome with improved preparations of recombinant PrP. Further libraries have been produced from chickens immunised with PrP and new antibodies from these libraries recognising PrP have been isolated. These are currently being studied and will be used in 'chain shuffling' techniques to recover combining sites closer to those used in the high affinity in-vivo immune response.
targeting and cellular pathology
The early development of PrP-related pathology is being investigated immunohistochemically in three combinations of scrapie strain and mouse genotype with contrasting patterns of pathology (87V/ Sincp7, ME7/ Sincs7, 79A/ Sincs7). After an intracerebral scrapie injection, abnormal PrP accumulation is detectable about a quarter of the way through the incubation period, long before the appearance of vacuolar changes. After intraocular infection, PrP pathology is first seen in the visual projection areas relatively later, at about half-way through the incubation period with all three scrapie strains.
Double immunostaining for PrP and GFAP in the intraocular models has revealed differences between scrapie strains in the astrocytic response. With 79A astrocytes become "reactive" very quickly after the first appearance of PrP pathology. With ME7 (and probably also 87V), more PrP accumulates before the astrocytes become active. Attempts are being made to relate the earliest pathology to the terminals of the retinal ganglion cell axons, using fibre tracing techniques.
BSE transmission in sheep
The likely origin of BSE in cattle is considered to have been scrapie-contamination of meat and bone meal in compounded feedstuffs. For this reason, it was decided to transmit BSE into genetically defined sheep and goats, to compare transmission properties with those of a natural scrapie isolate and other scrapie sources.
Whilst these experiments are necessarily of long duration, it is already apparent that when BSE is passaged in sheep the strain characteristics, as defined by subsequent mouse passage, do not differ greatly from those of primary BSE. Another feature of this work is the relative ease of transmission of BSE in sheep by oral challenge. This was unexpected, largely because of earlier studies in mice which showed this route to be relatively inefficient. However, the transmission results do not support significant differences between the BSE and natural scrapie isolates.
non-lymphoid cells in scrapie pathogenesis
Only high doses of ME7 scrapie produce disease in SCID mice by peripheral routes, probably by agent reaching the CNS via peripheral nerves. Agent replication does not occur in SCID spleen unless its lymphoid deficit and follicular dendritic cell (FDC) immaturity have been reversed with normal (CB20) mouse bone marrow. Reconstitution with hamster bone marrow has permitted ME7 but not 263K (hamster agent) replication in SCID spleen. A putative FDC dysfunction in aged mice has not resulted in a reduction in ME7 replication in spleens of 2- year-old mice.
Analysis of splenic B- and T-lymphocytes in ME7- (scrapie) or in 301V- (BSE) infected and control mice showed no differences.
Bioassay of dendritic (not follicular) cells and unstained cells showed no infectivity when sorted from ME7-scrapie spleen.
neuronal function in scrapie-infected mice
The electrophysiological and pathological changes in thalamic and hippocampal neurones in scrapie infected mice have been correlated. In thalamic neurones these changes appear to be subtle until the last stages of the incubation period. In the hippocampal model the Schaffer collateral-evoked field epsp and associated population spike undergo a gradual increase in threshold and reduction in amplitude as the disease progresses. The field potential disappears by two-thirds of the way through the incubation period. However, individual neurones recorded intracellularly are still able to generate an epsp-ipsp sequence. Intracellular synaptic responses are greatly attenuated and have an increased threshold for activation. The dendrites of CA1 pyramidal cells are normally encrusted with dendritic spines. Intracellular dye injections, in parallel with Golgi studies, suggest a gradual loss of spines and subsequent axonal sprouting. Ultrastructural localisation of PrP on dye-filled neurons from which recordings have been made is in progress in collaboration with Dr M Jeffrey (CVL Lasswade). LTP testing of PrP null mice has also been undertaken.
glia and inflammatory mediators
The nature of the microglial response in scrapie-affected mice showing signs of disease has been characterised and demonstrated that microglia show increased immunoreactivity of leukocyte common antigen, type 3 complement receptor, F4/80 reactivity and greater endocytic and lysosomal activity. This indicates that the microglial response represents a restricted form of inflammation in the scrapie-infected brain. The presence of the inflammatory cytokines IL-1, IL-6 and tumour necrosis factor- alpha was therefore investigated. Immunoreactive glia were detected for all of these and also PGE2, PGF2 alpha and lipocortin-1. Both the above studies were extended into time-course studies to examine the progression and temporal relationship of cytokine immunoreactivity to PrP accumulations, astrocytosis, microglial activation and vacuolation.
In addition, an initial study to determine whether the observed microglial response is derived entirely from intrinsic "resting" microglia, or whether an influx of monocytes/macrophages contributes to the overall microglial response, was completed. The proportion of microglia derived from recruited monocytes varied between individual animals. It was considered, therefore, that it is unlikely that the recruitment of monocytes is a pivotal event in the development of early pathological changes in scrapie.
cerebral metabolism
This project is a collaboration with the University of London. Magnetic resonance imaging (MRI) and spectroscopy (MRS) allow non-invasive monitoring of the internal structure and biochemistry of living organisms. Scrapie-infected rodents provide convenient models for study in the development of MRS/MRI monitoring of the spongiform encephalopathies. Recently, magnetic resonance (MR) imaging has been used in combination with gadolinium- diethylenetriaminepenta-acetic acid (Gd-DTPA) enhancement to investigate the integrity of the blood-brain barrier in the 263K-hamster model of scrapie during the clinical phase of disease. The post-Gd-DTPA images of the infected hamster brain showed marked enhancement, an effect not seen in control animals. These results suggest that function of the blood-brain barrier is severely disrupted in clinically-affected animals and future work aims to map the time course of this lesion.
identification of BSE infection
Previous work in the IAH has demonstrated the usefulness of mice, usually of defined genotypes, in identifying and strain typing the BSE agent from bovine brain. This project was therefore designed to identify whether or not BSE agent was demonstrable in tissues of BSE-affected cattle.
BSE agent has not been isolated from any non-central nervous tissue from BSE-affected cattle. It has been isolated by mouse assay only in spinal cord and brain.
The failure to identify BSE agent in peripheral tissues, particularly lymphoid tissue, needs to be pursued as it shows a major difference from scrapie and other homologous diseases where agent is present in peripheral organs. This also poses questions about the neuropathogenesis of the disease, in particular the routing of infection to the central nervous system in the absence of infection elsewhere.
an antemortem test for BSE and scrapie
In scrapie and BSE some of the host-encoded protein PrP population exhibits altered properties, forming a sedimentable fraction which is partially resistant to proteolytic degradation. Specific detection of this fraction in extracts from peripheral organs would provide the basis for a diagnostic test.
Essentially pure chicken egg-yolk IgY, with greatly improved signal-to-noise ratios in blot analyses, has been prepared from three hens each immunised with recombinant PrP protein (recPrP). IgY fractions of extremely high purity, specificity and titre have also been produced from eggs from the same birds by affinity chromatography on immobilised recPrP.
The purified IgY reagents show high titre and high specificity of binding to PrP when examined by immunoblotting and are now thought to be the most appropriate reagents available for diagnostic detection of PrPsc.
PrP has been clearly detected in sheep and cow brain homogenates but it is less distinguishable in spleen or lymph node homogenates. To assess their utility in the diagnosis of scrapie they have been used to determine whether PrPsc could be detected in a panel of control and scrapie-infected sheep brain extracts. PrPsc was detected in all scrapie-infected and none of the control brains. PrPsc was also detected in cow brain extracts from animals infected with BSE. PrPsc has been detected in spleen extracts from all natural scrapie cases examined.
Developments in the production and processing of recPrP protein continue. Notable improvements in the solubility range of the recPrP have been made by refolding the protein from chaotropic solutions. This has enabled simplified coating procedures for the preparation of ELISA plates. This new method should ensure that a greater range of epitopes are exposed on the solid substrate and therefore should now enable the selection of mAbs with different reactivities. The design of quantitative tests which also rely on the binding of recPrP to solid supports will also be greatly facilitated by this development.
strain typing of BSE isolates
The primary purpose of this project is to explore the epidemiological links between BSE in cattle and scrapie in sheep, by comparing the characteristics of the strains of pathogen isolated from each species. Specific questions being asked include: Did BSE originate from sheep scrapie? Has there been any selection of new strains of BSE in cattle in the course of the epidemic? Has BSE been transmitted back to sheep in feed? What is the relationship between BSE in Switzerland and BSE in the UK?
Eight transmissions to mice have been completed, from separate BSE cases collected in the UK between 1987 and 1990. These sources gave remarkably similar patterns of incubation periods and neuropathology in a standard panel of mouse strains, indicating that the same major agent strain was present in each source. Transmissions from one further UK source and two from Switzerland are in progress.
Transmissions have been set up from five sheep with natural scrapie. Two of these were from flocks where a food-borne source of infection was suspected, thus allowing the possibility that BSE has spread to sheep to be tested. Three of the experiments are complete and two are still in progress. As in previous natural scrapie transmissions, the results have varied between sources. Three so far (the 'non food-borne' sources), have given definite positive transmissions, but none has resembled BSE in the patterns of incubation periods or pathology. One of the possible food-borne sources has given no definite clinical disease in mice, up to 750 days after injection. This suggests that different strains of agent were present in the sheep and cattle sources.
Further serial passages in different mouse genotypes are in progress from one of the sheep sources (a Greyface) and three BSE sources. The same two mouse-passaged strains have been isolated from each BSE source and these differ from all previously characterised scrapie strains. This strain, designated 301V, was isolated in VM mice and has the shortest incubation period ever found for scrapie-like disease in mice. The strain isolated from Greyface scrapie in C57BL or RIII mice closely resembles ME7. This strain has been isolated from the majority of the 20 natural scrapie sources studied in the past. Cloning passages have been set up from one BSE source and the Greyface scrapie source, to attempt to isolate 'wild-type' strains.
The results have been compared with transmissions of TSEs from a range of other sources. Transmissions from a kudu, nyala and three cats have given incubation periods and pathology in mice which are closely similar to those in BSE transmissions, suggesting a common source of infection. These results also show that, amongst these species, donor species has little effect on disease characteristics in mice.
Experimental passage of BSE through sheep, goats and pig has also had little effect on the transmission characteristics to mice.
publications 1994
Black,C.J.; MacLeod,N.; Scott,J. (1994) - An electrophysiological study of scrapie-infected dorsal lateral geniculate neurons in vitro - Annals of the New York Academy of Sciences, 724: 355-357
Bostock,C.J. (1994) - Molecular genetics and strain characteristics of BSE - Livestock Production Science, 38: 35-39
Bruce,M. (1994) - Bovine spongiform encephalopathy: experimental studies in the United Kingdom - Office International des Epizooties Ad Hoc Group on BSE, Paris, 29
Bruce,M.; Chree,A.; McConnell,I.; Foster,J.; Pearson,G.; Fraser,H. (1994) - Transmission of bovine spongiform encephalopathy and scrapie to mice: strain variation and the species barrier - Philosophical Transactions of the Royal Society, London, B343: 405-411
Bruce,M.E.; McBride,P.A.; Jeffrey,M.; Scott,J.R. (1994) - PrP in pathology and pathogenesis in scrapie-infected mice - Molecular Neurobiology, 8: 105-112
Farquhar,CF.; Dornan,J.; Somerville,R.A.; Tunstall,A.M.; Hope,J. (1994) - Effect of Sinc genotype, agent, isolate and route of infection on the accumulation of proteinase-resistant PrP in noncentral nervous system tissues during the development of murine scrapie - Journal of General Virology, 75: 495-504
Foster,J.D.; Hope,J.; McConnell,I.; Bruce,M.; Fraser,H. (1994) - Transmission of bovine spongiform encephalopathy to sheep, goats, and mice. - Annals of the New York Academy of Sciences, 724: 300-303
Fraser,H.; Brown,K. (1994) - Peripheral pathogenesis of scrapie in normal and immunocompromised mice - Animal Technology, 45: 21-23
Fraser,H.; Bruce,M.; Foster,J.D.; Fraser,J.R. (1994) - Biology and pathogenesis of scrapie- like disease. - Proceedings and Abstracts, 17th Nordic Veterinary Congress, Reykjavik, 1: 117
Fraser,H.; Pearson,G.R.; McConnell,I.; Bruce,M.E.; Wyatt,J.M.; Gruffydd-Jones,T.J. (1994) - Transmission of feline spongiform encephalopathy to mice - Veterinary Record, 134: 449
Fraser,H.; Waterston,C.; Parker,A.; Hope,J.; Bruce,M. (1994) - High doses of ultraviolet. (200,000 J/m2) or ionizing. (20 kGy) radiation neither mutate nor inactivate scrapie agent - Abstracts, Neuropathology and Applied Neurobiology, 20: P510
Goldmann,W.; Hunter,N.; Smith,G.; Foster,J.; Hope,J. (1994) - PrP genotype and agent effects in scrapie: change in allelic interaction with different isolates of agent in sheep, a natural host of scrapie - Journal of General Virology, 75: 989-995
Goldmann,W.; Hunter,N.; Smith,G.; Foster,J.; Hope,J. (1994) - PrP genotypes and the Sip gene in Cheviot sheep form the basis for scrapie strain typing in sheep - Annals of the New York Academy of Sciences, 724: 296-299
Hope,J. (1994) - The nature of the scrapie agent: the evolution of the virino - Annals of the New York Academy of Sciences, 724: 282-289
Hope,J. (1994) - Expression of polyubiquitin and heat-shock protein 70 genes increases in the later stages of disease progression in scrapie-infected mouse brain - Journal of Neurochemistry, 62: 1870-1877
Hope,J.; Chong,A. (1994) - Scrapie, Creutzfeldt-Jakob disease and bovine spongiform encephalopathy: the key role of a nerve membrane protein. (PrP) - Biochemical Society Transactions, 22: 159-163
Hope,J.; Jeffrey,M.; Goodsir,C.M.; Chong,A. (1994) - Observation of possible intermediates in the conversion of PrPc to scrapie amyloid PrPsc - Neurobiology of Aging Supplement, 1: 151
Hunter,N.; Goldmann,W.; Smith,G.; Hope,J. (1994) - The association of a codon 136 PrP gene variant with the occurrence of natural scrapie - Archives of Virology, 134: 171-177
Hunter,N.; Goldmann,W.; Smith,G.; Hope,J. (1994) - Frequencies of PrP gene variants in healthy cattle and cattle with BSE in Scotland. - Veterinary Record, 135: 400-403
Hunter,N.; Manson,J.C.; Charleson,F.C.; Hope,J. (1994) - Comparison of expression patterns of PrP MRNA in the developing sheep and mouse - Annals of the New York Academy of Sciences, 724: 353-354
Jeffrey,M.; Goodsir,C.M.; Bruce,M.E.; McBride,P.A.; Fowler,N.; Scott,J.R. (1994) - Murine scrapie-infected neurons in vivo release excess prion protein into the extracellular space - Neuroscience Letters, 174: 39-42
Jeffrey,M.; Goodsir,C.M.; Bruce,M.E.; McBride,P.A.; Scott,J.R. (1994) - Infection-specific prion protein. (PrP) accumulates on neuronal plasmalemma in scrapie-infected mice - Annals of the New York Academy of Sciences, 724: 327-330
Jeffrey,M.; Goodsir,C.M.; Bruce,M.; McBride,P.A.; Scott,J.R.; Halliday,W.G. (1994) - Correlative light and electron microscopy studies of PrP localisation in 87V scrapie - Brain Research, 656: 329-343
Kenward,N.; Hope,J.; Landon,M.; Mayer R,J. (1994) - Expression of polyubiquitin and heat-shock protein-70 genes increase in the later stages of disease progression in scrapie- infected mouse brain - Journal of Neurochemistry, 62: 1870-1877
Manson,J.C.; Clarke,A.R.; Hooper,M,L.; Aitchison,L.; McConnell,I.; Hope,J. (1994) - 129/Ola mice carrying a null mutation in PrP that abolishes mRNA production are developmentally normal - Molecular Neurobiology, 8: 121-127
Manson,J.; Clarke,A.; McBride,P.; McConnell,I.; Bard,J.; Hope,J. (1994) - Transgenic mice deficient in PrP gene expression allow analysis of scrapie disease mechanisms - Abstracts, 9th International Workshop on Molecular Genetics of the Mouse, G10
Manson,J.; Clarke,A.; McBride,P.; McConnell,I.; Bard,J.; Hope,J. (1994) - The use of PrP null mice to study PrP normal function and mechanisms of the transmissible spongiform encephalopathies - Abstracts, Neurodegenerative Disorders: Common Molecular Mechanisms, Ocho Rios
Scott,J.R. (1994) - Unsuspected early neuronal loss in scrapie-infected mice revealed by morphometric analysis - Annals of the New York Academy of Sciences, 724: 338-343
Scott,J.R.; Jeffrey,M.; Halliday,W.G. (1994) - Unsuspected early neuronal loss in scrapie- infected mice revealed by morphometric analysis - Annals of the New York Academy of Sciences, 724: 338-343
Taylor,D.M.; McConnell,I.; Brown,D.A.; Brown,K.L. (1994) - New inactivation data for the agents of bovine spongiform encephalopathy. (BSE) and scrapie - Abstracts, Neuropathology and Applied Neurobiology, 10
Williams,A.E.; Lawson,L.J.; Perry,V.H.; Fraser,H. (1994) - Characterization of the microglial response in murine scrapie - Neuropathology and Applied Neurobiology, 20: 47-55
Williams,A.E.; van Dam,A.M.; Berkenbosch,F.; Eikelenboom,P.; Fraser,H. (1994) - Cytokines, prostaglandins and lipocortin 1 are present in the brains of scrapie-infected mice - Neurobiology of Aging, 15: 89
Williams,A.E.; van Dam,A.M.; Berkenbosch,F.; Eikelenboom,P.; Fraser,H. (1994) - Cytokines, prostaglandins and lipocortin-1 are present in the brains of scrapie-infected mice - Neurobiology of Aging, 15(Supplement 1): 366
Williams,A.E.; van Dam,A.M.; Berkenbosch,F.; Eikelenboom,P.; Fraser,H. (1994) - Cytokines, prostaglandins and lipocortin-1 are present in the brains of scrapie-infected mice - Brain Research Association Abstracts, 11: 8.2
Williams,A.E.; van Dam,A.M.; Berkenbosch,F.; Eikelenboom,P.; Fraser,H. (1994) - Cytokines, prostaglandins and lipocortin-1 are present in the brains of scrapie-infected mice - Abstracts, 1st European Meeting on Glial Cell Function in Health and
Williams,A.; van Dam,A.M.; Berkenbosch,F.; Fraser,H. (1994) - Cytokines, prostaglandins and lipocortin-1 are present in the brains of scrapie-infected mice - Journal of Neuroimmunology, 54: P02.17
Williams,A.E.; van Dam,A.M.; Man-A-Hing,W.K.H.; Berkenbosch,F.; Eikelenboom,P.; Fraser,H. (1994) - Cytokines, prostaglandins and lipocortin-1 are present in the brains of scrapie-infected mice - Brain Research, 654: 200-206

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