Veterinary Laboratories Agency (VLA-Lasswade), Pentlands Science Park, Bush Loan, Midlothian EH26 0PZ, UK
Correspondence
Lorenzo González
l.gonzalez{at}vla.defra.gsi.gov.uk
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ABSTRACT |
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INTRODUCTION |
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The PrPres fragments generated during TSE infections have physical and chemical properties, such as electrophoretic mobility, glycoform profile and degree of proteinase resistance, which may vary between different TSE agents (Bessen & Marsh, 1994; Collinge et al., 1996
; Somerville et al., 1997
; Kuczius et al., 1998
). The biochemical characterization of PrPres can therefore aid in the discrimination between TSE isolates, although it has been reported that some biochemical properties of PrPres are most likely to result from the interaction between the agent and the host (Somerville, 1999
). A further antibody-based test, the conformation-dependent immunoassay technique, may be able to discriminate between different TSE agents by detecting specific hidden epitopes within PrPres, which are only revealed after denaturation (Safar et al., 1998
).
Transmission and serial passage of scrapie sources in inbred mice have been employed to identify different murine scrapie strains, which may be distinguished by their incubation periods and their patterns of vacuolation in brain (the lesion profile; Fraser & Dickinson, 1973). Although murine scrapie strains have been well characterized, the degree to which strain diversity might exist in sheep has not yet been determined. Experimental transmission of two highly passaged inocula derived from different sheep scrapie sources, SSBP/1 and CH1641, target most efficiently sheep of different PrP genotypes (Hunter, 1998
), and this contrasting behaviour has been considered indicative of the potential existence of different scrapie strains in the field. However, the patterns of vacuolation that have been described in natural sheep scrapie cases are highly variable (Wood et al., 1997
), and factors other than the PrP gene appear to have an effect on the vacuolar lesion profile in sheep (Begara-McGorum et al., 2002
). On present evidence therefore, it is unlikely that vacuolar lesion profiling per se can allow distinction between natural sheep scrapie sources.
In contrast to other methods for the detection of abnormal PrP, immunohistochemical (IHC) demonstration of PrPd in tissue sections allows a detailed assessment of its cellular localization and morphological characteristics of accumulation. Brains of sheep with scrapie show marked variation in the morphology of PrPd deposits (Miller et al., 1993; van Keulen et al., 1995
; Foster et al., 1996
; Hardt et al., 2000
; Ryder et al., 2001
). We have recently indicated that these different PrPd types and patterns can be used to obtain PrPd profiles, which appear to be mainly influenced by the source of scrapie agent and very little, if at all, by the host breed and PrP genotype (González et al., 2002
). Based on these histological and other ultrastructural observations (Jeffrey et al., 1990
, 1994
), the different PrPd patterns are thought to represent infection and subsequent intracytoplasmic accumulation and/or release into the adjacent extracellular space of PrPd by different cell types. Apart from providing a means of characterizing sheep TSE sources, the diversity of PrPd profiles could also be indicative of variation in cell tropism by different agents.
Differences in neuronal tropism have been indicated in human and murine TSEs and can account for the observed differences in histopathological phenotypes. As an example, while parvalbumin-containing gamma-aminobutyric acid interneurons are preferentially lost in CreutzfeldtJakob disease (Guentchev et al., 1997), glutamatergic neurons appear to be affected in murine scrapie after infection with the Rocky Mountain Laboratory strain (Díez et al., 2001
). Also, specific patterns of PrPd accumulation characterize infection of mice of the same PrP genotype with different TSE sources (Bruce, 1993
; Bruce et al., 1991
) and such differential targeting occurs following cross-species transmission with particular TSE agents (Bruce et al., 1994
; Telling et al., 1996
).
The differential diagnosis of ovine infections with a limited number of scrapie sources and the BSE agent can be achieved by IHC examination of brain and lymphoreticular tissues using a range of antibodies directed at different peptide sequences of PrP (Jeffrey et al., 2001). The differences observed in the immunoreactivity of intramacrophage and intraglial PrPd, presumably phagocytosed from the extracellular space, were attributed to intracellular truncation of the protein occurring at different amino acid residues of the PrPd molecule in the two infections. It was further suggested that these differences in PrPd processing were probably due to differences in conformation of the PrPd produced during sheep scrapie and ovine BSE.
In the present study we examined possible effects of the use of different antibodies on the PrPd profile. In so doing we have attempted further validation of the PrPd profiling as a system to characterize sheep TSE sources. In addition, the results are suggestive of differential TSE agent-specific cellular targeting and PrPd processing, these possibly being pathogenetic mechanisms contributing to the variation in pathological phenotype of different sheep TSE infections.
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METHODS |
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(i) Naturally infected ARQ/ARQ Suffolk sheep from a single flock in Scotland that had been kept as a closed flock for many years; the five animals examined developed clinical disease within a 5 week period.
(ii) Naturally infected VRQ/VRQ Welsh Mountain sheep from a single commercial flock in Wales; the five animals examined developed clinical disease over a 20 month period.
(iii) VRQ/VRQ Cheviot sheep experimentally infected with the SSBP/1 inoculum by the subcutaneous route, as described elsewhere (Goldmann et al., 1994).
(iv) Three Poll-Dorset and two Romney sheep, all of the ARQ/ARQ genotype and experimentally infected with BSE agent by the intracerebral route, as previously reported (Foster et al., 1993).
The genotypes given indicate the polymorphisms of the PrP protein at codons 136, 154 and 171 in turn for each allele, in which amino acids are indicated by the appropriate single letter code. PrP genotyping was performed by sequencing with an ABI Prism 377 DNA sequencer according to the manufacturer's instructions (PE Applied Biosystems). The diagnosis of TSE in these sheep was based on clinical signs of neurological disease and histopathological confirmation of typical vacuolar pathology and PrPd accumulation in the brain. Negative control sheep, either uninfected or of resistant ARR/ARR PrP genotype, were included in the study.
Immunohistochemistry.
The brains of the sheep were fixed, trimmed and embedded following standard procedures, and subjected to IHC labelling for PrPd according to previously described protocols (González et al., 2002). Serial sections of each brain tissue sample were immunolabelled with four PrP antibodies, namely P4 (mouse monoclonal raised against the 89104 amino acid sequence of ovine PrP; Hardt et al., 2000
), 521.7 and 505.2 (rabbit polyclonals raised against the 94105 and 100111 amino acid sequence of ovine PrP, respectively; van Keulen et al., 1996
) and R486 (rabbit polyclonal raised against the 221234 amino acid sequence of bovine PrP; R. Jackman, unpublished).
Immunohistochemical examinations for a total of 11 different morphological types of PrPd accumulation were done at five different neuroanatomical sites, i.e. frontal cerebral cortex and corpus striatum (telencephalon), thalamus/hypothalamus (diencephalon), midbrain (mesencephalon) and medulla oblongata at the obex (myelencephalon). Those types were grouped in six PrPd patterns that included two intracellular (intraneuronal and intraglial) and four extracellular [glia-associated (stellate, perivascular and subpial PrPd types), neuropil (coarse particulate, coalescing, linear and perineuronal PrPd types), vascular and ependymal] accumulations of PrPd. The designations as intra- or extracellular and the grouping of some types into particular patterns are based on previous immunohistochemical and ultrastructural studies (Jeffrey et al., 1990, 1994
). The PrPd profile of each animal represented the relative magnitudes of the six PrPd patterns. A detailed description of these types and patterns, with the exception of the ependymal PrPd, and of the scoring system to obtain the PrPd profile has been given previously (González et al., 2002
). The ependymal type refers to PrPd deposits on the apical border of the ependymal cells, and is different from and does not always coexist with the subependymal type previously reported (González et al., 2002
), which was not considered in this study. In order to ascertain eventual differences of intraneuronal PrPd accumulation between specific neurone nuclei, this type was assessed in 15 neuroanatomical sites. These were: in the myelencephalon, the lateral cuneate, dorsal motor of the vagus, olivary, hypoglossal and midline raphe nuclei; in the mesencephalon, the red, dorsolateral geniculate, oculomotor, superior colliculus and tectum mesencephali nuclei; in the diencephalon, the ventrolateral thalamic nuclei; in the telencephalon, the caudate, putamen and globus pallidus nuclei, and the cerebral cortex neurones.
Statistical analysis.
The scores obtained for the different PrPd patterns, i.e. the PrPd profiles, were compared between sheep groups and between antibodies by means of unpaired t-tests, using a statistics computer package (InStat; GraphPad Software). Parametric t-tests were used when the groups under comparison presented similar standard deviations (Bartlett test) and the data had Gaussian distributions (Kolmogorov and Smirnov test); otherwise, a non-parametric t-test (MannWhitney) was used.
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RESULTS |
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SSBP/1 scrapie was characterized by high levels of intracellular PrPd, both intraneuronal and intraglial, significantly lower levels of extracellular glia-associated, neuropil and ependymal PrPd patterns and absence of vascular plaques. By comparison with the other groups, it showed a significantly lower neuropil accumulation of PrPd.
Suffolk sheep scrapie resulted in the accumulation of high amounts of glia-associated PrPd, significantly lower but still moderate levels of intracellular and neuropil PrPd patterns, low ependymal PrPd deposits and very rare vascular plaques (a single plaque detected in the thalamus of a single sheep). By comparison with the other groups, it showed a significantly higher glia-associated accumulation of PrPd.
Sheep BSE produced moderate to high levels of intracellular, glia-associated and neuropil PrPd patterns, significantly lower ependymal PrPd and no vascular plaques. It showed a significantly lower glia-associated accumulation of PrPd than Suffolk sheep, but higher than Welsh Mountain and SSBP/1-affected sheep.
Intraneuronal PrPd accumulation
As expected from the antibody-dependent variation observed in the magnitude of intraneuronal PrPd accumulation (Table 1), the neuroanatomical distribution of this PrPd type also showed substantial differences depending on the antibody used (results not shown). In order to test any eventual differences in neurone nuclei tropism between the four TSE sources studied, a single intraneuronal PrPd profile was obtained for each of the sheep groups, by selecting the scores provided by the most efficient antibody in each case. These neuroanatomical profiles of intraneuronal PrPd accumulation were compiled from 15 sites and are shown in Fig. 6
, in which the scores for each neurone nucleus are expressed as percentages of the total intraneuronal PrPd score. In all the four sheep groups, PrPd deposits were more consistent and conspicuous in the neurones of the myelencephalon, diencephalon and mesencephalon than in those of the telencephalon. Some relative differences were found in specific neurone nuclei, the most evident being in the olivary nuclei, in which Welsh Mountain sheep showed lower levels of intraneuronal PrPd than the other groups, particularly the Suffolk sheep, and in the dorsal motor nucleus of the vagus, where the opposite situation was observed.
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DISCUSSION |
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PrPd profile and cell tropism
To evaluate differences in tropism or cell targeting by different TSE agents it is assumed that PrPd accumulation in and/or extracellular release by a particular cell type reflects infection and therefore susceptibility of that cell type. Due to differences of reactivity of different PrP antibodies, the assessment of tropism can most accurately be performed when comparing the PrPd profiles obtained with the most efficient antibody for each TSE source and cell-associated PrPd pattern (optimal PrPd profiles; see Fig. 5). Examples of differential cell tropism can be qualitative, as with infection of endothelial cells (revealed as vascular PrPd plaques) by the scrapie agent affecting Welsh Mountain sheep, which is negligible in infection by any of the other three sources examined. However, differences are more often quantitative, the best example being the high affinity for glial cells, particularly astrocytes, shown by the scrapie agent infecting the Suffolk sheep group (highest glia-associated PrPd pattern score). This scrapie agent would show higher tropism for astrocytes than the other three TSE sources examined and also higher affinity for this cell type than for neurones, ependymal or endothelial cells. In contrast, the scrapie agent affecting Welsh Mountain sheep would show a similar tropism for all cell types, while the BSE agent would show high tropism for neurones and glial cells but very low or none for ependymal and endothelial cells, respectively. These results, therefore, show differential targeting of CNS cells of different lineage by different TSE sources, in contrast with previous reports, in which differences in pathological phenotypes of PrPd (or PrPsc) accumulation were attributed almost exclusively to differences in neuronal targeting (DeArmond et al., 1997
, 1999
). However, these latter reports suggested that selective neuronal targeting would be a function of brain region and in that respect are in agreement with our own results. While we did not find differences in the targeting of specific neuroanatomical nuclei between TSE sources, regional tropic effects were evident when the myelencephalon, mesencephalon and diencephalon were compared with the telencephalon, irrespective of the TSE source (see Fig. 6
).
PrPd profile and PrP processing
Not all the variation in the PrPd profile can be readily attributed to a greater or lesser tropism of different TSE agents for particular cell types. The significantly lowest neuropil PrPd pattern score shown by SSBP/1 could initially be interpreted as indicative of a low tropism of this scrapie source for neurones, but this is difficult to reconcile with the high levels of intraneuronal PrPd seen in SSBP/1 infection. Our interpretation is rather that SSBP/1 is highly neurone-tropic, but that PrP processing during this infection is different from that occurring during infections by the other three TSE sources studied. In SSBP/1 infection, most of the PrPd produced would remain intraneuronal, with very little release to the neuropil (statistical differences shown in Table 1 and in Fig. 5
), whereas in the other three infections there would be a balance between intraneuronal and extracellular neuropil PrPd (lack of statistical differences shown in Table 1
and in Fig. 5
).
Variability in PrP processing by neurones would seem to be the result not only of infection by different TSE sources, as discussed above, but could also be cell-dependent. Thus, because of the high levels of neuropil PrPd in the dorsal motor nucleus of the vagus of Suffolk sheep (results not shown), the low levels of intraneuronal PrPd at this site (see Fig. 6) are probably reflecting a quick and efficient release (or a low re-internalization) of PrPd by infected neurones, rather than a low tropism of the corresponding scrapie agent for this neurone nucleus. A similar situation would take place in the olivary nuclei during scrapie infection of Welsh Mountain sheep, while the opposite (high intraneuronal and low neuropil PrPd accumulation) would occur in the olivary nuclei of Suffolk sheep and in the dorsal motor nucleus of the vagus of Welsh Mountain sheep.
Cell tropism PrP processing and PrPd conformation
The precise mechanisms that account for the differences in PrP processing cannot be determined from the present data. However, our results suggest that they might be at least partially due to differences in conformation of the PrPd that is produced after infection by different TSE agents. Differences in PrPres glycosylation patterns have been described in infections by different TSE agents (Kascsak et al., 1985; Somerville et al., 1997
) and the use of glycoform analysis for TSE agent typing has been advocated (Collinge et al., 1996
; Hill et al., 1998
). In our study, the variation in PrPd conformation is revealed by the different affinity of the protein to the four antibodies used. Thus, while scrapie infection of Welsh Mountain sheep would lead to the production of a PrPd conformer that reacts similarly with all the antibodies used, the PrPd produced during SSBP/1 infection would be of a different conformation, showing consistently low affinity for the 505.2 antibody and high for the P4 antibody. The differences in PrPd conformation between the four TSE sources studied are particularly evident with intracellular accumulations, both intraneuronal and intraglial. Intraneuronal PrPd produced during SSBP/1 infection showed a higher affinity for P4 antibody than the proteins produced as a result of the other three infections, and a lower affinity for 505.2 antibody when compared with Suffolk scrapie and sheep BSE. Also, BSE infection would lead to the production and intraneuronal accumulation of a PrPd that reacts differently with P4 and R486 antibody than the intraneuronal protein produced during scrapie infection of Suffolk sheep. PrPd accumulating in the cytoplasm of glial cells during SSBP/1 infection showed much higher affinity for P4 and 521.7 antibodies than intraglial PrPd in the other three infections. Moreover, intraglial PrPd in BSE-infected sheep was almost undetectable with 521.7 antibody, while it was readily detected with this antibody in infections with the other three sources.
We have evidence from separate studies to suggest that intraglial and intraneuronal PrPd represent truncated, intralysosomal forms of the prion protein (Jeffrey et al., 2001, 2003
). Truncation at different amino acid sequences of the N terminus could explain the almost complete lack of detectable intraglial PrPd with P4 in Suffolk scrapie and BSE-affected sheep, and also with 521.7 in the latter group. It would also account for the absence of intraneuronal PrPd with P4 in the same BSE-affected sheep. Differences in intracellular truncation could be explained not only by differences in conformation of the PrPd produced by, but also by differences in enzymatic ability of different cells during infections by different TSE sources. However, the detection of higher immunoreactivity for intracellular PrPd with some of the antibodies to the N terminus than with R486, as is the case for intraneuronal PrPd in Suffolk scrapie or for both intraneuronal and intraglial in SSBP/1 infection, is difficult to explain as a result of truncation. We consider that these findings are more likely to be due to differences in PrPd conformation and not in enzymatic capability.
Variation in PrPd conformation appears to depend not only on the infecting agent, but also on the infected cell type. This could explain why glia-associated PrPd showed a consistently higher immunoreactivity with R486 than with any of the other three antibodies, regardless of the infecting agent. Another example would be the differences in intracellular PrPd of BSE-infected sheep, in which intraglial PrPd lacks both the 521.7 and P4 recognition epitope, while intraneuronal PrPd lacks only the P4 recognition site. A dual effect on PrPd conformation by the TSE agent and the cell type producing and/or releasing PrPd as a result of infection would be in agreement with previous studies on PrP glycosylation (Somerville, 1999). These pointed out differences in both PrPc and PrPsc glycoforms obtained from different brain sites, and others have made similar observations when comparing different tissues, such as brain and spleen (Rubenstein et al., 1991
) or brain and tonsil (Hill et al., 1999
). We think that these differences are best explained as being cell-dependent and that differences in PrPd glycosylation are probably associated with differences in protein conformation, as suggested in the present report.
Conclusion
In conclusion, the results of this study indicate that different TSE sources show differences in their tropism for different cell lineages in the brain of affected sheep. It also appears that infection of sheep with different TSE agents results in differences in processing of the abnormal PrP, including greater or lesser release to the extracellular space and different intracytoplasmic truncation of the prion protein. We suggest that variation in PrPd processing results from differences in conformation of the abnormal protein produced during infection by different TSE agents, although different conformers of abnormal PrP may exist for a single TSE agent depending on the cell type in which infection takes place.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bessen, R. A. & Marsh, R. F. (1994). Distinct PrP properties suggest the molecular basis of strain variation in transmissible mink encephalopathy. J Virol 68, 78597868.[Abstract]
Bruce, M. E. (1993). Scrapie strain variation and mutation. Br Med Bull 49, 822838.[Abstract]
Bruce, M. E., McConnell, I., Fraser, H. & Dickinson, A. G. (1991). The disease characteristics of different strains of scrapie in Sinc congenic mouse lines: implications for the nature of the agent and host control of pathogenesis. J Gen Virol 72, 595603.[Abstract]
Bruce, M. E., 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. Philos Trans R Soc Lond Ser B Biol Sci 343, 405411.[Medline]
Collinge, J., Sidle, K. C. L., Meads, J., Ironside, J. & Hill, A. F. (1996). Molecular analysis of prion strain variation and the aetiology of "new variant" CJD. Nature 383, 685690.[CrossRef][Medline]
DeArmond, S. J., Sánchez, H., Yehiely, F. & 12 other authors (1997). Selective neuronal targeting in prion disease. Neuron 19, 13371348.[Medline]
DeArmond, S. J., Qiu, Y., Sánchez, H., Spilman, P. R., Ninchak-Casey, A., Alonso, D. & Daggett, V. (1999). PrPc glycoform heterogeneity as a function of brain region: implications for selective targeting of neurons by prion strains. J Neuropathol Exp Neurol 58, 10001009.[Medline]
Díez, M., DeArmond, S. J., Groth, D., Prusiner, S. B. & Hökfelt, T. (2001). Decreased MK-801 binding in discrete hippocampal regions of prion-infected mice. Neurobiol Dis 8, 692699.[CrossRef][Medline]
Foster, J. D., Hope, J. & Fraser, H. (1993). Transmission of bovine spongiform encephalopathy to sheep and goats. Vet Rec 133, 339341.[Medline]
Foster, J. D., Wilson, M. & Hunter, N. (1996). Immunolocalisation of the prion protein (PrP) in the brains of sheep with scrapie. Vet Rec 139, 512515.[Medline]
Fraser, H. & Dickinson, A. G. (1973). Scrapie in mice: agent strain differences in the distribution and intensity of grey matter vacuolation. J Comp Pathol 83, 2940.[Medline]
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. J Gen Virol 75, 989995.[Abstract]
González, L., Martin, S., Begara-McGorum, I., Hunter, N., Houston, F., Simmons, M. & Jeffrey, M. (2002). Effects of agent strain and host genotype on PrP accumulation in the brain of sheep naturally and experimentally affected with scrapie. J Comp Pathol 126, 1729.[CrossRef][Medline]
Guentchev, M., Hainfellner, J. A., Trabattoni, G. R. & Budka, H. (1997). Distribution of parvalbumin-immunoreactive neurons in brain correlates with hippocampal and temporal cortical pathology in CreutzfeldtJakob disease. J Neuropathol Exp Neurol 56, 11191124.[Medline]
Hardt, M., Baron, T. & Groschup, M. H. (2000). A comparative study of immuno-histochemical methods for detecting abnormal prion protein with monoclonal and polyclonal antibodies. J Comp Pathol 122, 4353.[CrossRef][Medline]
Hill, A. F., Sidle, K. C., Joiner, S., Keyes, P., Martin, T. C., Dawson, M. & Collinge, J. (1998). Molecular screening of sheep for bovine spongiform encephalopathy. Neurosci Lett 255, 159162.[CrossRef][Medline]
Hill, A. F., Butterworth, R. J., Joiner, S. & 12 other authors (1999). Investigation of variant CreutzfeldtJakob disease and other human prion diseases with tonsil biopsy samples. Lancet 353, 183189.[CrossRef][Medline]
Hunter, N. (1998). Scrapie. Mol Biotechnol 9, 225234.[Medline]
Jeffrey, M., Wells, G. A. H. & Bridges, A. W. (1990). An immunohistochemical study of the topography and cellular localization of three neural proteins in the sheep nervous system. J Comp Pathol 103, 2335.[Medline]
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. Neurosci Lett 174, 3942.[Medline]
Jeffrey, M., Martin, S., González, L., Ryder, S. J., Bellworthy, S. J. & Jackman, R. (2001). Differential diagnosis of infections with the bovine spongiform encephalopathy (BSE) and scrapie agents in sheep. J Comp Pathol 125, 271284.[CrossRef][Medline]
Jeffrey, M., Martin, S. & L. González, L. (2003). Cell-associated variants of disease-specific protein immunolabelling are found in different sources of sheep transmissible spongiform encephalopathy. J Gen Virol 84, 10331046.
Kascsak, R. J., Rubenstein, R., Merz, P. A., Carp, R. I., Wisinewski, H. M. & Diringer, H. (1985). Biochemical differences among scrapie-associated fibrils support the biological diversity of scrapie agents. J Gen Virol 66, 17151722.[Abstract]
Kuczius, T., Haist, I. & Groschup, M. H. (1998). Molecular analysis of bovine spongiform encephalopathy and scrapie strain variation. J Infect Dis 178, 639699.
Miller, J. M., Jenny, A. L., Taylor, W. D., Marsh, R. F., Rubenstein, R. & Race, R. E. (1993). Immunohistochemical detection of prion protein in sheep with scrapie. J Vet Diagn Invest 5, 309316.[Medline]
Pan, K., Baldwin, M., Nguyen, J. & 8 other authors (1993). Conversion of -helices into
-sheets features in the formation of the scrapie prion proteins. Proc Natl Acad Sci U S A 90, 1096210966.[Abstract]
Rubenstein, R., Merz, P. A., Kascsak, R. J., Scalici, C. L., Papini, M. C., Carp, R. I. & Kimberlin, R. H. (1991). Scrapie-infected spleens: analysis of infectivity, scrapie-associated fibrils, and protease-resistant proteins. J Infect Dis 164, 2935.[Medline]
Ryder, S. J., Spencer, Y. I., Bellerby, P. J. & March, S. A. (2001). Immunohistochemical detection of PrP in the medulla oblongata of sheep: the spectrum of staining in normal and scrapie-affected sheep. Vet Rec 148, 713.[Medline]
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, 11571165.[CrossRef][Medline]
Somerville, R. A. (1999). Host and transmissible spongiform encephalopathy agent strain control glycosylation of PrP. J Gen Virol 80, 18651872.[Abstract]
Somerville, R. A., Chong, A., Mulqueen, O. U., Birkett, C. R., Wood, S. C. E. R. & Hope, J. (1997). Biochemical typing of scrapie strains. Nature 386, 564.[Medline]
Telling, G. C., Parchi, P., DeArmond, S. J. & 7 other authors (1996). Evidence for the conformation of the pathological isoform of the prion protein enciphering and propagating prion diversity. Science 274, 20792082.
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, 299308.[Abstract]
van Keulen, L. J. M., Schreuder, B. E. C., Meloen, R. H., Mooij-Harkes, G., Vromans, M. E. W. & Langeveld, J. P. M. (1996). Immunohistochemical detection of prion protein in lymphoid tissues of sheep with natural scrapie. J Clin Microbiol 34, 12281231.[Abstract]
Wood, J. L. N., McGill, I. S., Done, S. H. & Bradley, R. (1997). Neuropathology of scrapie: a study of the distribution of brain lesions in 222 cases of natural scrapie in sheep, 19821991. Vet Rec 140, 167174.[Medline]
Received 28 August 2002;
accepted 13 January 2003.