| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of TSE Molecular Biology, Veterinary Laboratories Agency, New Haw, Addlestone, Surrey KT15 3NB, UK
2 Institute for Animal Health, Pirbright Laboratory, Woking, Surrey, UK
3 Veterinary Laboratories Agency Lasswade, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 0PZ, UK
Correspondence
J. Hope
j.hope{at}vla.defra.gsi.gov.uk
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
These authors contributed equally to this work. 
Supplementary material is available in JGV Online.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Wickner, 1994; Legname et al., 2004; Wickner et al., 2004). Frequently, the conversion of a normal cellular protein into a prion form involves aggregation, which changes its physico-chemical properties from those of a soluble protein into those of an amyloid, a sparingly-soluble, protease-resistant isoform rich in cross-
-pleated sheet structures (Dobson, 2005). Scrapie of sheep and goats is the most common prion disease of mammals and typically causes a progressive degeneration of the central nervous system (Fraser, 1976). The ovine cellular prion protein (oPrPC) is a glycosylphosphatidyl inositol-linked membrane glycoprotein with a molecular mass of 33–35 kDa and is normally widely expressed in the central nervous system, lymphoid and other tissues. During the development of scrapie, this protein accumulates in these tissues as a relatively proteinase K (PK)-resistant prion form (PrPSc) (Gilmour et al., 1986; van Keulen et al., 1995; Jeffrey et al., 1998; Hardt et al., 2000). Extraction of these tissues with mild non-denaturing detergents, limited proteolysis and differential ultracentrifugation allows the purification of fibril or rod-like aggregates of the PK-resistant, amyloid core structures of PrPSc, known as PrPres. PrPres has a molecular mass of 27–30 kDa and typically lacks the N-terminal 67–70 aa of oPrPC (Hope et al., 1986; Goldmann et al., 1990). Aggregates of PrP isoforms are common to all naturally occurring and most experimental prion diseases and, because they co-purify with high titres of infectivity, they are thought to represent at least one identity of an infectious agent formed from conformational isomers of PrP.
Conceptually, however, there may be other forms of prions in domestic animals (mammals) that do not have the amyloid properties of PrPres (or even its deleterious effects) and so may escape detection by screening methods (protease hydrolysis and analysis by Western blotting or ELISA) based on this single physical property of the abnormal protein. Even at the time of the first isolation of PrPres (Bolton et al., 1982; McKinley et al., 1983), doubts were raised over the quantitative correlation of infectivity and this PrP amyloid (Czub et al., 1986, 1988), and these reservations were reinforced by the finding that most abnormal PrP in infected brain was not PrP of 27–30 kDa but an isoform covalently identical to normal PrPC (Hope et al., 1986; Bolton et al., 1991). Subsequently, the introduction of analytical methods omitting PK has allowed quantification of this proteinase-sensitive component of PrPSc (Safar et al., 1998, 2005), and the use of several artificial transgenic models of transmissible, prion protein-related disorders has shown again the dissociation of infectivity titre and the classical, PK-resistant amyloid core of PrPres (Barron et al., 2003). Recently, the in vitro generation of PrPres failed to match the specific infectivity of an equivalent amount of naturally occurring PrPSc (Castilla et al., 2005). High-resolution amino acid sequencing of abnormal PrP isolated from human (Tagliavini et al., 1994) and mouse (Hope et al., 1988) brain has identified peptide fragments of PrPres diagnostic for cleavage within the amyloid core of PrPres, between residues 
140 and 170, and molecular analysis of scrapie strain Nor98 sheep brain has provided evidence recently for a similar co-existence of PrP core amyloid and less-stable PrPSc conformers in a naturally occurring sheep disease (Benestad et al., 2003; De Bosschere et al., 2004; Gavier-Widen et al., 2004; Onnasch et al., 2004).
This aspect of prion biology has taken on greater practical significance with the advent of rapid testing that relies on PrPres detection for transmissible spongiform encephalopathy (TSE) surveillance in sheep. Prior to 2005, these tests had only been evaluated extensively for their performance in detecting PrPres as a confirmatory marker for clinical disease in cattle and one in particular, the Bio-Rad Platelia test system, appeared to detect a PrP abnormality in apparently healthy sheep in the absence of the characteristic PK resistance of PrPSc. During 2002–2003, a programme of statutory EU surveillance for scrapie in sheep in Great Britain was conducted in the sheep population submitted for abattoir slaughter (Elliott et al., 2005) in which brain stems from cull sheep were screened for disease-associated PrPsc using the Bio-Rad Platelia test system (Moynagh & Schimmel, 1999; Grassi et al., 2001). From a total of 29 201 samples tested, 54 were reported to be positive in the screening assay. Of those available for testing, 24 were confirmed as scrapie by immunohistochemical (IHC) detection of disease-specific PrPsc deposition in fixed sections of the obex. A further 28 samples that were positive by screening, however, were negative by IHC examination at this limited anatomical site and thus could not be confirmed as scrapie cases. Here, we have described characterization of the prion protein associated with these cases.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
, 1988). Negative and positive control reference materials (CRMs) were prepared, respectively, from pooled homogenates of brains of Bio-Rad Platelia ELISA-negative sheep (CRM-A) and pooled homogenates of brains of PrP IHC-confirmed, Bio-Rad Platelia ELISA-positive sheep (CRM-B). An unclassified CRM (CRM-C) was prepared from four IHC-negative, Bio-Rad Platelia ELISA-positive sheep. The genotypes of the sheep used in CRM-A were not determined, but CRM-B contained a limited number of genotypes known to be susceptible to clinical scrapie: ARQ/AHQ, ARQ/ARQ, ARQ/VRQ and VRQ/VRQ. Of the four CRM-C Bio-Rad Platelia ELISA-positive brains, two were of the AHQ/VRQ genotype, one was ARR/VRQ and the PrP genotype of the fourth was unknown. These positive, negative and unclassified CRMs were included daily as controls in each ELISA and Western blot procedure.
Genotyping of PrP codons 136, 154 and 171.
PrP genotyping of the Bio-Rad Platelia ELISA-positive cases at the three codons associated with scrapie susceptibility and resistance (codons 136, 154 and 171) was undertaken as part of the British surveillance programme through partial sequencing of the PrP gene. DNA was extracted from 25 mg brain medulla tissue using the DNeasy 96 Tissue kit (Qiagen) and dissolved in a final volume of 150 µl. A hot-start PCR amplification was undertaken using 2·5 µl of this genomic DNA, 2x PCR master mix (Promega) and 30 pmol each of the primers 5'-ATGAGACACCACCACTACAGGGCT-3' and 5'-CATTTGATGCTGACACCCTCTTTA-3'. After 40 cycles, the PCR product of 900 bp was treated with shrimp alkaline phosphatase and exonuclease 1 according to the manufacturer's instructions (New England Biolabs). Cycle sequencing was undertaken with the reverse primer 5'-TCGCTCCATTATCTTGATGTCAGTTT-3' using the BigDye terminator kit following the manufacturer's instructions (PE Applied Biosystems). The cycle sequencing product was precipitated with ethanol and resuspended in 40 µl sample loading solution (PE Applied Biosystems) and 4 µl was loaded onto a 36 cm ABI Prism 377 DNA Sequencer gel. The full PrP ORF of sample DNAs from each case was independently sequenced and these genotypes were verified by a commercial contractor (Qiagen).
Bio-Rad Platelia ELISA.
The Bio-Rad Platelia Purification and Detection kits provided the reagents for the extraction and PK hydrolysis of PrPSc in our study and the subsequent detection of residual PrPres by a sandwich ELISA (Bio-Rad). The procedures set out in the manufacturer's kit insert were followed. Briefly, caudal medulla (0·35 g) was homogenized in a mild detergent buffer, proteins including PrPC were digested at 37 °C for 10 min using low concentrations of PK, and residual proteins including PrPres were precipitated by using alcohol. Following low-speed centrifugation, the sample pellet was denatured and solubilized, diluted and used for the assay using a microtitre plate-based colorimetric immunoassay with two monoclonal antibodies (mAbs) (one for capture and one for detection). This ELISA system, originally approved for the confirmation of disease in clinical cases of cattle bovine spongiform encephalopathy (BSE) (Grassi et al., 2001; Moynagh & Schimmel, 1999), has recently been evaluated for its ability to detect scrapie, Nor98 and experimental BSE in sheep (EFSA, 2005).
The detergents, mAbs, PK activity and buffers are subject to commercial confidentiality, but a key feature of this kit is the element of controlled PK digestion. Elimination of normal PrPC uses 4 µl of the kit PK reagent (ml homogenate)–1 and this level is designated 1x PK. The final concentration or protease activity of this reagent is undisclosed, but is probably at least 10 times less than concentrations of PK usually used to purify and characterize PrPres (Hope et al., 1986). The ELISA sample preparation was also performed without PK treatment (designated 0x PK) or using 20 µl of the kit reagent (ml homogenate)–1 (5x PK).
Western blotting.
Brain stems (caudal medulla) were extracted and the extract was treated with PK and alcohol precipitated as described above for the preparation of ELISA samples (see above). For Western blotting, the sample pellet was boiled in Laemmli SDS-PAGE sample buffer and loaded (0·05 g equivalents per track) onto a gel cassette (Bio-Rad). Electrophoresis was carried out at room temperature at 200 V and the dye front was allowed to migrate for no more than 50 min to prevent loss of polypeptides of <6 kDa running off the end of the gel. After electrophoresis, the gel proteins were blotted onto an activated PVDF membrane at 100 V for 1 h and, after rinsing and blocking, the membrane was incubated with either a mixture of mAbs SAF60 and BAR226 (specific for the core prion protein; referred to as the core blot) or the single mAb SAF34 (specific for the N-terminal octarepeat area; the N-blot) and PrP-related bands were visualized by using a horseradish peroxidase-coupled anti-mouse immunoglobulin secondary antibody conjugate (Bio-Rad) and the Amersham ECL developer kit. Each set of samples was flanked by MagicMark Western Protein Standards (eight proteins tagged with an IgG-binding sequence and covering the range 20–120 kDa; Invitrogen) to calibrate the blots for molecular mass.
mAb SAF60 binds to a linear peptide epitope sequence, YPNQVY, encoded by codons 160–165 of the oPrP gene, while BAR226 appears to be specific for an unidentified conformational epitope that is retained on Western blotting of the PK-resistant core of oPrPSc, encoded by codons 90–233 of the ovine PrP gene [J. Grassi, personnal communication, Commissariat a l'Energie Atomique (CEA) Pharmacology and Immunology Unit, CEA/Saclay, Gif sur Yvette, France]. A mixture of mAbs SAF60 and BAR226 was used in the core blot. The mAb SAF34 binds to the octapeptide repeat sequence of the ovine PrP gene, typically represented by the sequence PH/QGGG(G)WGQ which occurs five times in a tandem repeat encoded by codons 54–95 of the ovine PrP gene (Swiss-Prot accession number P23907
[GenBank]
; Goldmann et al., 1990). This single mAb was used in the N-blot.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Comparison of the ovine PrP genotypes for these confirmed and unclassified (atypical) Bio-Rad Platelia ELISA-positive cases indicated that the majority of the atypical cases occurred in genotypes with a reduced relative risk of developing clinical TSE (Fig. 1). For example, 8 % of atypical cases were found in ARR/ARR homozygous sheep in which natural cases of clinical TSE have never been reported, although they can develop TSE disease after intracerebral infection (Houston et al., 2003).
|
). These data are shown in Fig. 2, and presented with additional PrP genotype information in Supplementary Fig. S1(a–d) (available in JGV Online).
|
and Supplementary Fig. 2, available in JGV Online). The CRM-B and CRM-C standards gave values equivalent to the IHC-confirmed, Bio-Rad Platelia ELISA-positive survey samples and the IHC-unconfirmed, Bio-Rad Platelia ELISA-positive survey samples, respectively. All New Zealand-derived, scrapie-free samples and CRM-A samples (pooled negative-control material) were below the negative cut-off value of the kit when assayed at 1x and 5x PK levels.
Western immunoblotting of sheep brain CRMs
The immunoreactive PrP polypeptides in extracts of CRMs before and after low and high PK exposure are shown in Fig. 3. Normal PrPC, mostly present as the diglycosylated 33–35 kDa isoform, is abundant in brain and gave a strong signal in all three brain standards with both the N-blot and core blot mAbs. PrPC was destroyed even by limited (1x PK) proteolysis, as shown by the complete disappearance of bands from CRM-A (TSE-negative control) in both blots at high and low PK exposure levels (Fig. 3a and b; CRM-A).
|
). At the 1x PK level, the N-blot gave a good signal consistent with only partial cleavage of the N-terminal sequence at this low exposure, but, as expected from previous studies (Hope et al., 1986), the signal disappeared after 5x PK treatment (Fig. 3b).
CRM-C, the brain pool made from PrP IHC-negative, Bio-Rad Platelia ELISA-negative tissue, gave an almost identical PrP immunoreactive profile in both N-blot and core blot systems. At the 1x PK level, reduced but qualitatively similar banding to that seen without protease treatment was observed in the molecular mass range of PrPC using both N- and C-terminal PrP-specific mAbs. No 6–7 kDa band shift characteristic of the conversion of PrPSc to its N-terminally truncated amyloid core subunit, PrPres, was observed at either level of protease exposure. At the 5x PK exposure level, no banding was seen with either mAb system (Fig. 3a and b).
British survey samples
Representative core and N-blots of the British survey samples are shown in Fig. 4, and the patterns obtained with a wider range of genotypes are provided as Supplementary Fig. S2 (available in JGV Online). In general, the survey IHC-confirmed, Bio-Rad Platelia ELISA-positive samples gave patterns resembling CRM-B (Fig. 4a) and the survey IHC-unconfirmed, Bio-Rad Platelia ELISA-positive samples gave patterns resembling CRM-C (Fig. 4b). In some cases, intermediate banding patterns and the appearance of lower molecular mass PrP immunoreactive bands migrating between the lowest molecular mass standard (20 kDa) and the gel dye front were visualized in the core blots but not in the N-blot system (see Fig. 4c, asterisks, and samples 3 and 5 in Supplementary Fig. S2).
|
14 kDa (more precisely <20 kDa) band seemed related both to the degree of proteolysis, as judged by inter- and intra-analysis comparisons of the CRM standards, and to the nature of the sample (genotype, Bio-Rad Platelia response, IHC-confirmed or unconfirmed) (data not presented). This variability needs to be controlled further to allow higher-resolution molecular phenotyping of these classes of sheep PrP-related abnormalities but, notwithstanding this operational constraint, the two groups of IHC-confirmed and IHC-negative, Bio-Rad Platelia ELISA-positive cases could clearly be differentiated on the basis of the molecular phenotypes defined in Fig. 4(a and b). |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Hope et al., 1986, 1988). This oPrPSc had the same migration as PrPC on SDS-polyacrylamide gels, equivalent to a molecular mass of 33–35 kDa, but, unlike PrPC, oPrPSc aggregated into conformers that were relatively resistant to proteolytic hydrolysis. PK treatment of this abnormal prion protein in mild denaturing buffers (PrPSc : enzyme ratio of approx. 1 : 50) cleaved the N-terminal 60–70 aa from the more robust core structure to leave a large fragment of PrP, PrPres (codons 90–230, with a molecular mass of 27–30 kDa), under conditions where PrPC was completely hydrolysed. Consequently, this PrPres was seen even after stringent PK treatment (5x) using our core blot system, but was not seen using the N-blot molecular phenotyping system. No PrP signal was observed in either blotting system using mild (1x) or stringent (5x) PK digestion of PrPC standards (CRM-A; Fig. 3a and b). The characteristic molecular mass band shift of PrPSc to PrPres (7–8 kDa) was also seen in core blots but not in N-blots of this phenotype (CRM-B; Fig. 3a and b, and Fig. 4a). This is the molecular phenotype commonly associated with the case definition of scrapie in small ruminants (OIE, 2004) and these suspect cases were confirmed as TSE by PrP IHC detection at the level of the obex and by an independent Western blotting system (Prionics Check WB). The oPrP gene is highly polymorphic and the relative susceptibility or resistance of a sheep to clinical scrapie depends, amongst other factors, on its PrP genotype (Hunter, 1997; Goldmann et al., 2005). The most common dimorphisms are at codons 136, 154 and 171 and common alleles are denoted by the amino acids (in single-letter code) encoded by codons 136, 154 and 171. For instance, the ARR allele comprises A136, R154 and R171, and the ARR/ARR homozygous genotype is classed as the one most resistant to the development of scrapie (and experimental BSE). Conversely, the VRQ/VRQ genotype is recognized to be at high risk of developing natural scrapie. The PrP genotypes at codons 136, 154 and 171 in these confirmed cases broadly matched those previously described in association with clinical cases of scrapie with almost all carrying either a VRQ or an ARQ allele (National Scrapie Plan, http://www.defra.gov.uk/animalh/bse/othertses/scrapie/nsp/index.html).
By contrast, the remaining half of the suspect TSE cases were characterized by a form of the putative transmissible agent that appeared to have a less stable PrP core structure. Under stringent PK digestion conditions (5x), these samples were completely destroyed and were indistinguishable from normal, genotype-matched PrPC controls by ELISA (Fig. 2) or by either type of Western blot (Fig. 3 and Fig. 4b). Using mild (1x) PK conditions, these samples gave a diffuse banding pattern similar to undigested PrPC in both core and N-blot systems with no apparent band shift. These cases clearly represent a prion protein abnormality, as no similar signal was seen by ELISA or Western blotting of normal PrPC controls after mild PK treatment (n>100, Figs 2 and 3). There are some similarities between these cases and Nor98 and Nor98-like cases now described in Europe (Benestad et al., 2003; Buschmann et al., 2004; De Bosschere et al., 2004; Gavier-Widen et al., 2004; Madec et al., 2004; Orge et al., 2004; Onnasch et al., 2004; Moum et al., 2005), particularly their genotype distribution, which appears to favour AHQ and ARR (and AF141RQ; Moum et al., 2005) carriers (Fig. 1; Baylis & McIntyre, 2004). The L141F dimorphism typing of our samples is in progress and may provide further insight into these molecular phenotypes.
At the molecular level, these European cases are characterized by the accumulation in brain, particularly in the cerebrum and cerebellum, of a PrP immunoreactive peptide (or mixture of peptides) of molecular masses of 6·5–12 kDa in addition to higher molecular mass PrPres amyloid core markers for sheep scrapie. In the first case definition of Nor98, Benestadt and colleagues used highly stringent PK digestion conditions (100 µg PK ml–1 for 30 min at 37 °C) and the mAb P4 against a peptide fragment defined by codons 84–104 to define a molecular profile where there was mostly 12 kDa peptide and some 27–30/23–26 kDa banding, depending on the area of the brain sampled; other mAbs were used to map these fragments and it was reported that, as expected, the molecular mass of the lowest band detected varied with the mAb used, although the data were not shown (Benestad et al., 2003). Two Irish cases and a case from Belgium showed a range of immunoreactive PrPres (12–30 kDa) in cerebellum and/or cerebrum when compared with Norwegian Nor98 using the mAbs BAR226/SAF60 or 12F10/SAF60 (De Bosschere et al., 2004) or the Prionics Check WB (Onnasch et al., 2004), a system utilizing mAb 6H4, which is similar in specificity to SAF60. Seven Portuguese cases sampled from the brain stem and processed using a Bio-Rad kit (but otherwise undefined) also gave a profile of PrPres ranging from <20 to 30 kDa (Orge et al., 2004). German sheep TSE cases in ARR/ARR sheep had a more protease-sensitive PrPres than that of classical scrapie and a molecular mass range of <20–30 kDa was detected using the SAF tissue preparation procedure for Western blotting (Hope et al., 1986) and mAb L42 against codons 145–163 of oPrP (Harmeyer et al., 1998). A 6·5 kDa fragment observed in Swedish cases has been tentatively assigned to a 90–144 aa peptide (Ottinger et al., 2005) similar to that described in Gerstmann–Sträussler–Scheinker syndrome of humans (Tagliavini et al., 1994). Fig. 4(c) illustrates the same or a similar molecular phenotype with immunoreactive PrP bands at 27–30, 23–26, 18–21 and 10 kDa seen using the core blot, but, consistent with a 90–144 aa-containing peptide, not using the N-blot in a confirmed case in an ARR/VRQ sheep from the British survey. However, other cases from the British survey did not show a 6–12 kDa fragment, or in some cases any fragments, with either blotting system under the same conditions and so represent a wider range of sheep TSE phenotypes that requires further biological and biochemical definition to extend our understanding of mammalian prions.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Baylis, M. & McIntyre, K. M. (2004). Transmissible spongiform encephalopathies: scrapie control under new strain. Nature 432, 810–811.[CrossRef][Medline]
Benestad, S. L., Sarradin, P., Thu, B., Schonheit, J., Tranulis, M. A. & Bratberg, B. (2003). Cases of scrapie with unusual features in Norway and designation of a new type, Nor98. Vet Rec 153, 202–208.
Bolton, D. C., McKinley, M. P. & Prusiner, S. B. (1982). Identification of a protein that co-purifies with the scrapie prion. Science 218, 1309–1311.
Bolton, D. C., Rudelli, R. D., Currie, J. R. & Bendheim, P. E. (1991). Copurification of Sp33-37 and scrapie agent from hamster brain prior to detectable histopathology and clinical disease. J Gen Virol 72, 2905–2913.
Buschmann, A., Biacabe, A.-G., Ziegler, U., Bencsik, A., Madec, J.-Y., Erhardt, G., Lühken, G., Baron, T. & Groschup, M. H. (2004). Atypical scrapie cases in Germany and France are identified by discrepant reaction patterns in BSE rapid tests. J Virol Methods 117, 27–36.[CrossRef][Medline]
Castilla, J., Saá, P., Hetz, C. & Soto, C. (2005). In vitro generation of infectious scrapie prions. Cell 121, 195–206.[CrossRef][Medline]
Czub, M., Braig, H. R. & Diringer, H. (1986). Pathogenesis of scrapie: study of the temporal development of clinical symptoms, of infectivity titres and scrapie-associated fibrils in brains of hamsters infected intraperitoneally. J Gen Virol 67, 2005–2009.
Czub, M., Braig, H. R. & Diringer, H. (1988). Replication of the scrapie agent in hamsters infected intracerebrally confirms the pathogenesis of an amyloid-inducing virosis. J Gen Virol 69, 1753–1756.
De Bosschere, H., Roels, S., Benestad, S. L. & Vanopdenbosch, E. (2004). Scrapie case similar to Nor98 diagnosed in Belgium via active surveillance. Vet Rec 155, 707–708.
Defra Scrapie NSP (2005). http://www.defra.gov.uk/animalh/bse/othertses/scrapie/nsp.
Defra Scrapie Survey (2005). http://www.defra.gov.uk/animalh/bse/othertses/scrapie/scrapiesurvey.
Dobson, C. M. (2005). Structural biology: prying into prions. Nature 435, 747–749.[CrossRef][Medline]
EFSA (2005). Scientific Report of the European Food Safety Authority on the evaluation of rapid post mortem TSE tests intended for small ruminants. EFSA J 31, 1–17.
Elliott, H., Gubbins, S., Ryan, J., Ryder, S., Tongue, S., Watkins, G. & Wilesmith, J. W. (2005). Prevalence of scrapie in sheep in Great Britain estimated from abattoir surveys during 2002 and 2003. Vet Rec 157, 418–419.[Medline]
Fraser, H. (1976). The pathology of natural and experimental scrapie. In Slow Virus Diseases of Animals and Man, pp. 267–305. Edited by R. H. Kimberlin. Amsterdam: North-Holland Publishing Company.
Gavier-Widen, D., Noremark, M., Benestad, S., Simmons, M., Renstrom, L., Bratberg, B., Elvander, M. & Segerstad, C. H. (2004). Recognition of the Nor98 variant of scrapie in the Swedish sheep population. J Vet Diagn Invest 16, 562–567.
Gilmour, J. S., Bruce, M. E. & MacKellar, A. (1986). Cerebrovascular amyloidosis in scrapie-affected sheep. Neuropathol Appl Neurobiol 12, 173–183.[Medline]
Goldmann, W., Hunter, N., Foster, J. D., Salbaum, J. M., Beyreuther, K. & Hope, J. (1990). Two alleles of a neural protein gene linked to scrapie in sheep. Proc Natl Acad Sci U S A 87, 2476–2480.
Goldmann, W., Baylis, M., Chihota, C., Stevenson, E. & Hunter, N. (2005). Frequencies of PrP gene haplotypes in British sheep flocks and the implications for breeding programmes. J Appl Microbiol 98, 1294–1302.[CrossRef][Medline]
Grassi, J., Comoy, E., Simon, S. & 8 other authors (2001). Rapid test for the preclinical postmortem diagnosis of BSE in central nervous system tissue. Vet Rec 149, 577–582.
Hardt, M., Baron, T. & Groschup, M. H. (2000). A comparative study of immunohistochemical methods for detecting abnormal prion protein with monoclonal and polyclonal antibodies. J Comp Pathol 122, 43–53.[CrossRef][Medline]
Harmeyer, S., Pfaff, E. & Groschup, M. H. (1998). Synthetic peptide vaccines yield monoclonal antibodies to cellular and pathological prion proteins of ruminants. J Gen Virol 79, 937–945.[Abstract]
Hope, J., Morton, L. J. D., Farquhar, C. F., Multhaup, G., Beyreuther, K. & Kimberlin, R. H. (1986). The major protein of scrapie-associated fibrils (SAF) has the same size, charge distribution and N-terminal protein sequence as predicted for the normal brain protein (PrP). EMBO J 5, 2591–2597.[Medline]
Hope, J., Multhaup, G., Reekie, L. J. D., Kimberlin, R. H. & Beyreuther, K. (1988). Molecular pathology of scrapie-associated fibril protein (PrP) in mouse brain affected by the ME7 strain of scrapie. Eur J Biochem 172, 271–277.[Medline]
Houston, F., Goldmann, W., Chong, A., Jeffrey, M., González, L., Foster, J., Parnham, D. & Hunter, N. (2003). Prion diseases: BSE in sheep bred for resistance to infection. Nature 423, 498.[CrossRef][Medline]
Hunter, N. (1997). PrP genetics in sheep and the implications for scrapie and BSE. Trends Microbiol 5, 331–334.[CrossRef][Medline]
Jeffrey, M., Goodsir, C. M., Holliman, A., Higgins, R. J., Bruce, M. E., McBride, P. A. & Fraser, J. R. (1998). Determination of the frequency and distribution of vascular and parenchymal amyloid with polyclonal and N-terminal-specific PrP antibodies in scrapie-affected sheep and mice. Vet Rec 142, 534–537.
Legname, G., Baskakov, I. V., Nguyen, H.-O., Riesner, D., Cohen, F. E., DeArmond, S. J. & Prusiner, S. B. (2004). Synthetic mammalian prions. Science 305, 673–676.
Madec, J.-Y., Simon, S., Lezmi, S., Bencsik, A., Grassi, J. & Baron, T. (2004). Abnormal prion protein in genetically resistant sheep from a scrapie-infected flock. J Gen Virol 85, 3483–3486.
McKinley, M. P., Bolton, D. C. & Prusiner, S. B. (1983). A protease-resistant protein is a structural component of the scrapie prion. Cell 35, 57–62.[CrossRef][Medline]
Moum, T., Olsaker, I., Hopp, P., Moldal, T., Valheim, M., Moum, T. & Benestad, S. L. (2005). Polymorphisms at codons 141 and 154 in the ovine prion protein gene are associated with scrapie Nor98 cases. J Gen Virol 86, 231–235.
Moynagh, J. & Schimmel, H. (1999). Tests for BSE evaluated. Nature 400, 105.[Medline]
OIE Handbook (2004). Scrapie. In Manual of Standards for Diagnostic Tests and Vaccines. Publ. Office International des Epizooties, Paris, France.
Onnasch, H., Gunn, H. M., Bradshaw, B. J., Benestad, S. L. & Bassett, H. F. (2004). Two Irish cases of scrapie resembling Nor98. Vet Rec 155, 636–637.
Orge, L., Galo, A., Machado, C., Lima, C., Ochoa, C., Silva, J., Ramos, M. & Simas, J. P. (2004). Identification of putative atypical scrapie in sheep in Portugal. J Gen Virol 85, 3487–3491.
Ottinger, T., Simonsson, M., Klingeborn, M. & Linne, T. (2005). Western immunoblot mapping of a Swedish case of Nor 98 (abstract). In 2nd International Symposium on The New Prion Biology: basic science, diagnosis and therapy. Istituo Veneto di Science, Lettere di Arte, Venice.
Prusiner, S. B. (1982). Novel proteinaceous infectious particles cause scrapie. Science 216, 136–144.
Safar, J., Wille, H., Itrri, V., Groth, D., Serban, H., Torchia, M., Cohen, F. E. & Prusiner, S. B. (1998). Eight prion strains have PrPSc molecules with different conformations. Nat Med 4, 1157–1165.[CrossRef][Medline]
Safar, J. G., Geschwind, M. D., Deering, C. & 10 other authors (2005). Diagnosis of human prion disease. Proc Natl Acad Sci U S A 102, 3501–3506.
Tagliavini, F., Prelli, F., Porro, M. & 7 other authors (1994). Amyloid fibrils in Gerstmann–Sträussler–Scheinker disease (Indiana and Swedish kindreds) express only PrP peptides encoded by the mutant allele. Cell 79, 695–703.[CrossRef][Medline]
van Keulen, L. J. M., Schreuder, B. E. C., Meloen, R. H., Poelen-van den Berg, M., Mooij-Harkes, G., Vromans, M. E. W. & Langeveld, J. P. M. (1995). Immunohistochemical detection and localization of prion protein in brain tissue of sheep with natural scrapie. Vet Pathol 32, 299–308.[Abstract]
Wickner, R. B. (1994). [URE3] as an altered URE2 protein: evidence for a prion analog in Saccharomyces cerevisiae. Science 264, 566–569.
Wickner, R. B., Edskes, H. K., Roberts, B. T., Baxa, U., Pierce, M. M., Ross, E. D. & Brachmann, A. (2004). Prions: proteins as genes and infectious entities. Genes Dev 18, 470–485.
Received 9 March 2005;
accepted 14 October 2005.
This article has been cited by other articles:
|
|
 |
|
  J. Foster, L. Toovey, C. McKenzie, A. Chong, D. Parnham, D. Drummond, and N. Hunter Atypical scrapie in a sheep in a closed UK flock with endemic classical natural scrapie Vet Rec., May 31, 2008; 162(22): 723 - 724. [Full Text] [PDF]
|
|
|
|
|
|
M. P. Dagleish, S. M. Rodger, M. M. Simmons, J. Finlayson, D. Buxton, and F. Chianini Atypical scrapie in a sheep in Scotland Vet Rec., April 19, 2008; 162(16): 518 - 519. [Full Text] [PDF]
|
|
|
|
 |
|
 D. M. Green, V. J. del Rio Vilas, C. P. D. Birch, J. Johnson, I. Z. Kiss, N. D. McCarthy, and R. R. Kao Demographic risk factors for classical and atypical scrapie in Great Britain J. Gen. Virol., December 1, 2007; 88(12): 3486 - 3492. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. P. Owen, H. C. Rees, B. C. Maddison, L. A. Terry, L. Thorne, R. Jackman, G. C. Whitelam, and K. C. Gough Molecular Profiling of Ovine Prion Diseases by Using Thermolysin-Resistant PrPSc and Endogenous C2 PrP Fragments J. Virol., October 1, 2007; 81(19): 10532 - 10539. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Seuberlich, C. Botteron, S. L. Benestad, H. Brunisholz, R. Wyss, U. Kihm, H. Schwermer, M. Friess, A. Nicolier, D. Heim, et al. Atypical scrapie in a swiss goat and implications for transmissible spongiform encephalopathy surveillance J Vet Diagn Invest, January 1, 2007; 19(1): 2 - 8. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. C. Saunders, S. Cawthraw, S. J. Mountjoy, J. Hope, and O. Windl PrP genotypes of atypical scrapie cases in Great Britain. J. Gen. Virol., November 1, 2006; 87(Pt 11): 3141 - 3149. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
