1 Veterinary Laboratories Agency (VLA-Lasswade), Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 0PZ, UK
2 Institute for Animal Health, Compton, Berkshire RG20 7NN, UK
3 Institute for Animal Health Neuropathogenesis Unit, Edinburgh EH9 3JF, UK
4 Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 0PZ, UK
5 VLA-Weybridge, Addlestone, Surrey KT15 3NB, UK
Correspondence
Lorenzo González
l.gonzalez{at}vla.defra.gsi.gov.uk
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The classical method of TSE strain typing involves transmission and serial passage of infectious material (isolate) in a panel of inbred mouse lines and its characterization by the incubation period of the disease in mice and the vacuolar lesion profile in brain (Fraser & Dickinson, 1973). This method has allowed discrimination between BSE and several sheep scrapie strains, leading to the conclusion that the BSE agent is a unique, single strain that is stable after passage in different species, including sheep (Bruce et al., 1994
, 1997
; Foster et al., 1996
).
Either after passage in mice or directly in original tissues, assessment of the biochemical profile of TSE isolates by immunoblotting (glycotyping) attempts the characterization of TSE strains by differences in the proportions of the three glycoforms of protease-resistant PrP (PrPres) and in the molecular mass (MM) of the aglycosyl moiety after protease digestion. Despite some variability in the results of the studies carried out so far (reviewed by Schreuder & Somerville, 2003), the general pattern is that the aglycosyl fraction runs more slowly (higher MM) in most scrapie sources tested than in experimental ovine BSE (lower MM). This probably reflects differences in the cleavage site within the N terminus of abnormal PrP, a notion that is confirmed by the absence or marked reduction of the bands when sheep BSE gels are incubated with mAb P4 (Stack et al., 2002
), which recognizes the 9399 PrP epitope (Thuring et al., 2004
). However, immunoblot patterns similar to those of sheep BSE have been described for CH1641, a classical experimental scrapie source (Hope et al., 1999
), and the biochemical properties of PrPres may depend not just on the TSE agent, but also on the tissue type and even area within the brain (Somerville, 1999
).
The concept of differential truncation of the N terminus of sheep BSE-derived PrP was first proposed by Jeffrey et al. (2001a), who used immunohistochemistry (IHC) with a panel of PrP antibodies to distinguish between experimental ovine BSE and several sheep scrapie sources. Further studies indicated that, unlike scrapie, the site of truncation within the flexible tail of ovine BSE PrPd was tissue- and even cell-type-dependent (Jeffrey et al., 2003
) and that differentiation between the two infections could be approached by examining neural and non-neural tissues, particularly those of the LRS. Whilst the initial studies were restricted to sheep of the ARQ/ARQ PrP genotype, recent extended examinations have pointed out that the pattern of immunolabelling is identical in experimental BSE of sheep of other genotypes (Martin et al., 2005
).
We have previously reported the possible usefulness of the PrPd profile for characterization of scrapie strains (González et al., 2002). Unlike the lesion profile, which addresses the magnitude of neuropil vacuolation in specific brain areas, the method is based on IHC recognition and scoring of different morphological and cell-associated types and patterns of PrPd accumulation in the brains of affected sheep. The PrPd profile appears to be mainly determined by the TSE agent or strain, with other factors, particularly the PrP genotype, producing only minor effects (González et al., 2003a
). It has been hypothesized that these distinct profiles can reflect differences in cellular tropism and in PrP processing by different TSE strains (González et al., 2003b
). In the present study, we have used a similar IHC-profiling method to characterize the phenotype of PrPd accumulation in the brains of sheep affected experimentally with BSE, and have assessed the effects of several factors. Our intention was to provide further tools for discriminating between scrapie and ovine BSE and to contribute to the understanding of the pathogenesis of sheep TSEs.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
All sheep were monitored closely and were killed humanely once clinical signs were considered to be highly suggestive of TSE (Table 1). The clinical period extended from 1 to 10 days in approximately 50 % of the sheep, from 11 to 30 days in another 25 % and from 1 to 5 months in the remaining sheep. Three of the eight ARR/ARR sheep succumbing to BSE IC challenge were those reported by Houston et al. (2003)
and the other five belonged to the same experimental series.
IHC examinations and PrPd profile.
Brains were fixed in formaldehyde, trimmed and embedded in paraffin wax according to standard procedures. A detailed account of the IHC protocol, including antigen retrieval and blocking steps, was given previously (González et al., 2002). Primary antibody R486 was used in 21 animals and PrP antibody R145 in the remaining 43; ten sheep were examined with both antibodies to ensure comparability of results. R486 and R145 are, respectively, a rabbit anti-PrP polyclonal antiserum and a rat mAb that recognize bovine PrP amino acid residues 217231 (R. Jackman, personal communication). Sections from positive-control tissue blocks were included in each IHC run to ensure consistency in the sensitivity of the method. Apart from internal negative controls of the IHC technique (substitution of primary antibody by normal rabbit serum or normal rat IgG), each run also included negative-control tissues from TSE-unexposed sheep.
Brains were examined at six different neuroanatomical sites: frontal cerebral cortex, corpus striatum, thalamus/hypothalamus, midbrain, cerebellum at the vermis and medulla oblongata at the obex. Most of the PrPd types and patterns considered at these sites corresponded to those already described in previous publications (González et al., 2002, 2003b
). Intracellular PrPd included intraneuronal and intraglial granular immunodeposits in the cell cytoplasm. Two types of intraglial PrPd were recognized: one as single or a few large granules in close proximity to microglia-like nuclei (hereafter referred to as intramicroglial) and the other as multiple, small granules scattered in the cytoplasm of astrocyte-resembling cells (hereafter referred to as intra-astrocytic). Extracellular accumulation of PrPd occurred in the grey-matter neuropil as linear, perineuronal and particulate/coalescing immunodeposits, and also in association with the astrocyte processes that form the glial limitans (subpial, subependymal and perivascular types) and with individual cells of uncertain glial origin. Of the latter, two types were identified: the stellate PrPd accumulations in the grey matter and the more ill-defined, mesh-like or perivacuolar PrPd agglomerations found in the white matter. The designations of the different PrPd types as intra- or extracellular are based on IHC and ultrastructural studies done in mice (Jeffrey et al., 1990
, 1994
) and sheep (M. Jeffrey and others, unpublished observations). Other PrPd types that were sought were those related to blood vessels (vascular plaques), ependymal and choroid plexus cells and oligodendrocytes.
Construction of the PrPd profiles has been described in detail previously (González et al., 2002). The magnitude of accumulation of the above PrPd types was scored from 0 to 3 (Fig. 1
) in the six neuroanatomical sites described, and mean values were obtained for each type. These values were added to provide the total PrPd score for each sheep, and their graphical representation constituted the individual PrPd profile. The profiles and total PrPd values for each of the different sheep groups were obtained as the respective means of the individual sheep that made up those groups.
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Topographical description of the PrPd profile
None of the animals investigated showed PrPd accumulation in the choroid plexus or in the form of vascular plaques. Detection of PrPd associated with oligodendrocytes was attempted in the corpus callosum and in the cerebellar white matter; although some immunolabelling was observed at these points, it was unclear whether it was associated with oligodendrocytes or with intermingled astrocytes. Therefore, no separate quantification of oligodendroglial PrPd was performed. Subependymal and ependymal PrPd deposits were generally mild and inconsistent and, except for a few IC-challenged sheep in which lateral ventricles were involved, they were restricted to the third ventricle.
Cerebral cortex.
The magnitude of PrPd accumulation in the cerebral cortex was in general lower than in other areas. The predominant types were the subpial and stellate in the grey matter and the perivacuolar in the white matter. The latter tended to appear at the margins of the gyral white-matter tips, subjacent to the grey-matter junction (Fig. 2a), and was often associated with perivascular PrPd accumulations that seemed to be made of coalescing perivacuolar deposits (Fig. 2b
). Intra-astrocytic, intraneuronal and intramicroglial PrPd accumulation in the grey matter was of low grade and inconsistent.
|
Thalamus and hypothalamus.
In most sheep, PrPd accumulation at this level was more prominent in the hypothalamus than in the thalamus. Granular PrPd aggregates in neuronal perikarya were consistently present and the most frequent and prominent PrPd type in the neuropil was the particulate/coalescing, followed by the linear. Occasionally, coalescing PrPd aggregates had a miniature plaque-like appearance (Fig. 2g), but were devoid of a homogeneous central core, and Congo red staining of semi-serial sections provided negative results. The predominant extracellular PrPd type relating to glial cells was again the stellate, whilst the magnitude of perivascular PrPd never reached high levels. Intra-astrocytic PrPd accumulation was generally low or negligible, but intramicroglial PrPd was very prominent and consistent (Fig. 2g
).
Midbrain.
The overall magnitude of PrPd accumulation was greatest at this site. The predominant intracellular types were the intraneuronal, particularly in the red nucleus, and the intramicroglial. The intra-astrocytic type, in contrast, was seldom found and only in low amounts. Marked and consistent PrPd deposits were found in the neuropil, particularly in the substantia nigra, where the linear and the particulate/coalescing types were characteristic (Fig. 2h). Stellate PrPd accumulation was also very prominent and frequent, whereas perivascular PrPd was seldom conspicuous.
Cerebellum.
This site also displayed significant amounts of PrPd. Almost all sheep showed very prominent intraneuronal PrPd in the deep cerebellar nuclei, but none did so in the Purkinje cells. Intramicroglial PrPd was conspicuous both in the white matter and in the granular and Purkinje cell layers, coinciding with extracellular PrPd collections. Perivacuolar PrPd accumulation in the white matter was frequently marked, but, unlike the cerebrum, these aggregates involved the core of white-matter bundles and were not associated with perivascular deposits, which were almost completely absent. Perineuronal PrPd around deep cerebellar nuclei was mild and inconsistent. Subpial PrPd was not as conspicuous as in the cerebrum, being generally negligible or low. Moderate or high stellate PrPd deposits were often found in the cortical molecular layer, but more marked and frequent were extracellular, coalescing collections of PrPd in the granular and Purkinje cell layers (Fig. 2i). Multigranular deposits of PrPd in the cytoplasm of astrocyte-like cells were evident in the white-matter tips of most sheep (Fig. 2j
).
Medulla oblongata (obex).
The three most prominent and consistent PrPd types in this area were the intraneuronal, the particulate/coalescing and the intramicroglial. Although there were some individual variations, all neuronal nuclei were affected to practically the same extent. Particulate PrPd aggegates were most evident in the dorsal motor nucleus of the vagus (DMNV) and in the spinal tract of the trigeminal nerve. Linear deposits of PrPd were only occasionally substantial, and perineuronal PrPd was confined to the ventral border of the DMNV. Perivascular and stellate accumulations of PrPd were inconsistent and sparse. Intramicroglial PrPd was very prominent throughout and intra-astrocytic deposits, without reaching the same levels as in the cerebellum, were often found in the spinocerebellar tracts.
Effect of different factors on the phenotype of PrPd accumulation
Three aspects or parameters were considered when comparing the phenotype of PrPd accumulation between sheep groups: the magnitude of total PrPd, its topographical distribution and the PrPd profile. All groups were similar in terms of topographical distribution and, particularly, relative proportions of the different PrPd types and patterns (PrPd profile); phenotypic differences mainly involved the magnitude of total PrPd accumulation.
The dose of inoculum did not affect any of these parameters. Within group 4 (Table 1), the incubation periods for the animals challenged with 103, 104 and 105 dilutions were 520±19·4, 554±35·0 and 756±48·0 days, respectively (two sheep out of five of the latter group were still alive at 1550 days post-infection). In spite of these differences, the PrPd phenotypes of the three subgroups were almost identical (results not shown). The absence of correlation between PrPd phenotype and incubation period extended to all sheep groups studied (see next section). The magnitude of total PrPd was also unrelated to the duration of the clinical disease, the incubation period or the age at challenge (Table 1
).
The breed of sheep did not affect either the magnitude of total PrPd accumulation (Table 1; compare groups 1, 2 and 3), its profile (Fig. 3
a) or its topographical distribution (data not shown). The host source of inoculum (cattle or sheep) did not influence the PrPd profile, but appeared to have a slight contradictory effect on the magnitude of total PrPd (Fig. 3b, c
). Thus, whilst sheep challenged IC with infected cattle brain accumulated slightly more PrPd than those of the same genotype infected with sheep BSE inoculum (Table 1
; compare groups 13 with group 4), the opposite was observed with animals challenged orally (groups 8 and 9). For these comparisons, sheep of different breeds were grouped, but, as described above and in comparisons made within group 9 (data not shown), the breed did not seem to have an effect on the PrPd-accumulation phenotype.
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PrPd phenotype of BSE in sheep
In spite of differences in overall magnitude and, to a lesser extent, topographical distribution of PrPd deposition in the brain, the PrPd profile was remarkably similar in all BSE-infected sheep examined. In our series, PrPd accumulated at the highest levels in the brainstem, thalamus/hypothalamus and cerebellum, and at the lowest levels in the cerebral cortex. After IC challenge, however, PrPd deposits in the cerebral cortex and particularly in the striatum were conspicuous (presumably reflecting its proximity to the injection point), whereas aggregates in the cerebellum were mild following IV infection (Fig. 5c). As a result, the magnitude of total PrPd deposition in the brain was highest in IC-challenged sheep and lowest in IV-inoculated animals. Another factor, the PrP genotype, also influenced the overall amount of PrPd in the brain, its effect being evident in ARR homozygotes, which showed low PrPd levels. Similarly, ARQ/AHQ sheep accumulated little PrPd, but, in this case, a combined effect of the route of challenge (IV) and the source and type of inoculum (cattle brain) could not be analysed separately.
The PrPd profile of BSE-affected sheep was characterized by conspicuous intraneuronal, intramicroglial and extracellular stellate and neuropil aggregates, relatively low or moderate astrocyte-associated PrPd, either intra- or extracellular, inconsistent ependymal PrPd deposits and absence of PrPd in choroid plexus cells or in the form of vascular plaques. Very characteristic was the appearance of PrPd deposition in the striatum, hypothalamus and substantia nigra. The PrPd profile of BSE-affected sheep was different from that seen previously in cases of natural scrapie in sheep of various breeds and genotypes and in experimental SSBP/1 infection (González et al., 2002, 2003b
; Fig. 7
). It was also very different from that generated by infection of sheep with the CH1641 scrapie strain (Fig. 7
; M. Jeffrey & L. González, unpublished observations), a finding of particular relevance in view of the reported biochemical similarities between both agents (Hope et al., 1999
; Stack et al., 2002
). Furthermore, the features of sheep BSE reported here are indistinguishable from those described following infection of Lacaune sheep with a French isolate of cattle BSE (Lezmi et al., 2004
), but very different from atypical scrapie cases, such as the recently reported Nor98 type (Benestad et al., 2003
; M. Jeffrey & L. González, unpublished observations).
|
PrPd phenotype of BSE in other species
The consistency of the BSE phenotype in sheep does not parallel the variation detected when different species are compared. Thus, the abundance of PrPd plaques in mice carrying the p7 Sinc allele (Fraser et al., 1992; Brown et al., 2003
), in experimentally infected macaque monkeys (Lasmézas et al., 1996
) and in vCJD patients (DeArmond & Ironside, 1999
) contrasts with the almost-complete absence of amyloid/PrP plaques in other species. In addition to sheep, as reported above, neither pigs (Ryder et al., 2000
), cats (Wyatt et al., 1991
) nor exotic ungulates (Jeffrey & Wells, 1988
) develop such plaques, and they are absent or very rare in BSE cases affecting British cattle (Wells & McGill, 1992
; Wells & Wilesmith, 1995
). Assessing similarities or differences for PrPd types other than plaques proves difficult, due to lack of detailed descriptions of the PrPd-accumulation patterns in other species. From the literature, however, accumulation of PrPd in cattle BSE targets grey matter rather than white matter and includes granular or particulate, linear and perineuronal types in the neuropil, as well as intraneuronal and stellate types (Wells & Wilesmith, 1995
). These descriptions are coincident with our observations on sheep BSE, as are the findings of intraneuronal, neuropil-associated and stellate PrPd accumulations in the brainstem and corpus striatum of experimentally infected pigs (Ryder et al., 2000
).
Influence of PrP genotype on the PrPd phenotype of BSE
The phenotype of PrPd accumulation in the brain of BSE-infected mice is influenced by PrP genotype, so that plaque formation is only observed in homo- or heterozygous Sinc p7, but not in Sinc s7s7, mice, which accumulate PrPd in a sparse, diffuse form (Fraser et al., 1992; Brown et al., 2003
). With the exception of the miniature pseudoplaques, which were found almost exclusively in ARR/ARR sheep, we have not observed similar qualitative effects of the PrP genotype in sheep, but rather one on the magnitude of PrPd accumulation. This effect was unambiguous in ARR/ARR sheep and probably also in those carrying the AHQ allele. In our study, the host PrP genotype also appeared to influence the incubation period, which was very lengthy for ARR and VRQ homozygotes and VRQ allele-bearing sheep (Table 1
; Fig. 6
). In contrast, ARQ homozygotes had much shorter incubation periods, although this was apparently modulated by the route of challenge. The two groups of sheep carrying the AHQ allele also had short incubation periods, but the route of infection appeared to have an opposite effect; this, however, could also reflect genotype differences (homo- and heterozygotes) or result from interaction between source and type of inoculum (cattle brain) and route of challenge.
Overall, four combinations of incubation period and magnitude of PrPd accumulation have been observed: (i) short incubation period and high PrPd levels in ARQ/ARQ sheep; (ii) short incubation period and low PrPd levels in AHQ sheep; (iii) long incubation period and high PrPd levels in VRQ sheep; and (iv) long incubation period and low PrPd levels in ARR homozygotes. This situation raises questions about the dynamics of accumulation of PrPd in the brain following BSE agent infection. In vitro studies have shown that PrP polymorphisms modulate the conversion of cellular PrP into its abnormal counterpart, which is more efficient for allotypes linked to highly susceptible genotypes and vice versa (Bossers et al., 1997, 2000
). These findings might explain our observations in ARQ/ARQ and ARR/ARR sheep, but not the inverse relationship between incubation period and PrPd levels found in VRQ and AHQ sheep. We hypothesize that conversion/accumulation of VRQ PrP is efficient, hence the high PrPd levels found in these sheep, but starts late after infection, hence the long incubation period, and that just the opposite situation (low efficiency but early start) could happen in AHQ sheep.
Another intriguing question, derived from the low PrPd levels found in ARR and AHQ sheep, regards the significance of PrPd to clinical disease, as it seems clear from the evidence shown that they are not proportionally related. This finding is not unique to sheep BSE and has also been described when comparing SSBP/1 with natural scrapie (González et al., 2002). We think that at least two explanations can be considered: firstly, that only some morphological types of PrPd give rise to neurological disease when accumulating in the brain, and, secondly, that PrPd of different polymorphisms has different damaging potential or toxicity for the brain. In the first case, intraneuronal PrPd and extracellular deposits in the neuropil would be the likely candidates, as these are the only types that reached moderate levels in ARR and AHQ sheep. In the second case, less ARR and AHQ PrPd of any cellular or morphological type would be needed to trigger the neurological manifestations than when accumulating PrPd is of the ARQ or VRQ polymorphisms. A third possibility would be that PrPd accumulation is either unrelated to or not the main event propitiating neurological deficit and disease.
Conclusion
Detailed assessment of the morphological features and neuroanatomical distribution of PrPd in the brain of sheep displaying TSE-like clinical signs is a useful means of approaching identification of BSE in sheep. The consistency of the IHC phenotype of PrPd accumulation after sheep-to-sheep passage and across a range of sheep breeds, routes of challenge and PrP genotypes shows the stability of the BSE agent, without having to resort to experimental bioassay methods. Whilst not a unique or definitive method, study of the PrPd phenotype, in conjunction with other IHC, biochemical and biological approaches, offers a realistic possibility for the confirmation of naturally occurring BSE in sheep.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Benestad, S. L., Sarradin, P., Thu, B., Schönheit, 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, 202208.[Medline]
Bossers, A., Belt, P. B. G. M., Raymond, G. J., Caughey, B., de Vries, R. & Smits, M. A. (1997). Scrapie susceptibility-linked polymorphisms modulate the in vitro conversion of sheep prion protein to protease-resistant forms. Proc Natl Acad Sci U S A 94, 49314936.
Bossers, A., de Vries, R. & Smits, M. A. (2000). Susceptibility of sheep for scrapie as assessed by in vitro conversion of nine naturally occurring variants of PrP. J Virol 74, 14071414.
Brown, D. A., Bruce, M. E. & Fraser, J. R. (2003). Comparison of the neuropathological characteristics of bovine spongiform encephalopathy (BSE) and variant CreutzfeldtJakob disease (vCJD) in mice. Neuropathol Appl Neurobiol 29, 262272.[CrossRef][Medline]
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. Philos Trans R Soc Lond B Biol Sci 343, 405411.[Medline]
Bruce, M. E., Will, R. G., Ironside, J. W. & 10 other authors (1997). Transmission to mice indicate that new variant CJD is caused by the BSE agent. Nature 389, 498501.[CrossRef][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. & Ironside, J. W. (1999). Neuropathology of prion diseases. In Prion Biology and Diseases, pp. 585652. Edited by S. B. Prusiner. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
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., Bruce, M., McConnell, I., Chree, A. & Fraser, H. (1996). Detection of BSE infectivity in brain and spleen of experimentally infected sheep. Vet Rec 138, 546548.[Medline]
Foster, J. D., Parnham, D. W., Hunter, N. & Bruce, M. (2001). Distribution of the prion protein in sheep terminally affected with BSE following experimental oral transmission. J Gen Virol 82, 23192326.
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]
Fraser, H., Bruce, M. E., Chree, A., McConnell, I. & Wells, G. A. H. (1992). Transmission of bovine spongiform encephalopathy and scrapie to mice. J Gen Virol 73, 18911897.[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]
González, L., Martin, S., Hunter, N., Houston, F., Simmons, M. M., Bellworthy, S. J., Ryder, S. J. & Jeffrey, M. (2003a). Distinct profiles of disease-specific PrP accumulation are present in the brains of sheep affected with BSE and different scrapie sources. In Recent Progress in Transmissible Spongiform Encephalopathies. Edited by J. R. Fraser. Neuropathol Appl Neurobiol 29, 207208 (available at http://www.blackwellpublishing.com/products/journals/suppmat/nan/nan477/NAN477sm.pdf).
González, L., Martin, S. & Jeffrey, M. (2003b). Distinct profiles of PrPd immunoreactivity in the brain of scrapie- and BSE-infected sheep: implications on differential cell targeting and PrP processing. J Gen Virol 84, 13391350.
Hadlow, W. J., Race, R. E. & Kennedy, R. C. (1987). Experimental infection of sheep and goats with transmissible mink encephalopathy virus. Can J Vet Res 51, 135144.[Medline]
Hill, A. F., Desbruslais, M., Joiner, S., Sidle, K. C. L., Gowland, I., Collinge, J., Doey, L. J. & Lantos, P. (1997). The same prion strain causes vCJD and BSE. Nature 389, 448450.[CrossRef][Medline]
Hope, J., Wood, S. C. E. R., Birkett, C. R., Chong, A., Bruce, M. E., Cairns, D., Goldmann, W., Hunter, N. & Bostock, C. J. (1999). Molecular analysis of ovine prion protein identifies similarities between BSE and an experimental isolate of natural scrapie, CH1641. J Gen Virol 80, 14.[Abstract]
Houston, F., Foster, J. D., Chong, A., Hunter, N. & Bostock, C. J. (2000). Transmission of BSE by blood transfusion in sheep. Lancet 356, 9991000.[CrossRef][Medline]
Houston, F., Goldmann, W., Chong, A., Jeffrey, M., González, L., Foster, J., Parnham, D. & Hunter, N. (2003). BSE in sheep bred for resistance to infection. Nature 423, 498.[CrossRef][Medline]
Hunter, N., Foster, J., Chong, A., McCutcheon, S., Parnham, D., Eaton, S., MacKenzie, C. & Houston, F. (2002). Transmission of prion diseases by blood transfusion. J Gen Virol 83, 28972905.
Jeffrey, M. & Wells, G. A. H. (1988). Spongiform encephalopathy in a nyala (Tragelaphus angasi). Vet Pathol 25, 398399.[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.[CrossRef][Medline]
Jeffrey, M., Martin, S., González, L., Ryder, S. J., Bellworthy, S. J. & Jackman, R. (2001a). Differential diagnosis of infections with the bovine spongiform encephalopathy (BSE) and scrapie agents in sheep. J Comp Pathol 125, 271284.[CrossRef][Medline]
Jeffrey, M., Ryder, S., Martin, S., Hawkins, S. A. C., Terry, L., Berthelin-Baker, C. & Bellworthy, S. J. (2001b). Oral inoculation of sheep with the agent of bovine spongiform encephalopathy (BSE). 1. Onset and distribution of disease-specific PrP accumulation in brain and viscera. J Comp Pathol 124, 280289.[CrossRef][Medline]
Jeffrey, M., Martin, S. & González, L. (2003). Cell-associated variants of disease-specific prion protein immunolabelling are found in different sources of sheep transmissible spongiform encephalopathy. J Gen Virol 84, 10331046.
Lasmézas, C. I., Deslys, J.-P., Demaimay, R., Adjou, K. T., Lamoury, F., Dormont, D., Robain, O., Ironside, J. & Hauw, J.-J. (1996). BSE transmission to macaques. Nature 381, 743744.[CrossRef][Medline]
Lezmi, S., Martin, S., Simon, S., Comoy, E., Bencsik, A., Deslys, J.-P., Grassi, J., Jeffrey, M. & Baron, T. (2004). Comparative molecular analysis of the abnormal prion protein in field scrapie cases and experimental bovine spongiform encephalopathy in sheep by use of Western blotting and immunohistochemical methods. J Virol 78, 36543662.
Martin, S., González, L., Chong, A., Houston, F. E., Hunter, N. & Jeffrey, M. (2005). Immunohistochemical characteristics of disease-associated PrP are not altered by host genotype or route of inoculation following infection of sheep with bovine spongiform encephalopathy. J Gen Virol 86, 839848.
Ryder, S. J., Hawkins, S. A. C., Dawson, M. & Wells, G. A. H. (2000). The neuropathology of experimental bovine spongiform encephalopathy in the pig. J Comp Pathol 122, 131143.[CrossRef][Medline]
Schreuder, B. E. C. & Somerville, R. A. (2003). Bovine spongiform encephalopathy in sheep? Rev Sci Tech 22, 103120.[Medline]
Scott, M. R., Will, R., Ironside, J., Nguyen, H.-O. B., Tremblay, P., DeArmond, S. J. & Prusiner, S. B. (1999). Compelling transgenetic evidence for transmission of bovine spongiform encephalopathy prions to humans. Proc Natl Acad Sci U S A 96, 1513715142.
Somerville, R. A. (1999). Host and transmissible spongiform encephalopathy agent strain control glycosylation of PrP. J Gen Virol 80, 18651872.[Abstract]
Stack, M., Chaplin, M. & Clark, J. (2002). Differentiation of prion protein glycoforms from naturally occurring sheep scrapie, sheep-passaged scrapie strains (CH1641 and SSBP1), bovine spongiform encephalopathy (BSE) cases and Romney and Cheviot breed sheep experimentally inoculated with BSE using two monoclonal antibodies. Acta Neuropathol (Berl) 104, 279286.[Medline]
Thuring, C. M. A., Erkens, J. H. F., Jacobs, J. G. & 8 other authors (2004). Discrimination between scrapie and bovine spongiform encephalopathy in sheep by molecular size, immunoreactivity, and glycoprofile of prion protein. J Clin Microbiol 42, 972980.
Wells, G. A. H. & McGill, I. S. (1992). Recently described scrapie-like encephalopathies of animals: case definitions. Res Vet Sci 53, 110.[Medline]
Wells, G. A. H. & Wilesmith, J. W. (1995). The neuropathology and epidemiology of bovine spongiform encephalopathy. Brain Pathol 5, 91103.[Medline]
Wyatt, J. M., Pearson, G. R., Smerdon, T. N., Gruffydd-Jones, T. J., Wells, G. A. H. & Wilesmith, J. W. (1991). Naturally occurring scrapie-like spongiform encephalopathy in five domestic cats. Vet Rec 129, 233236.[Medline]
Received 21 May 2004;
accepted 17 November 2004.