Journal of General Virology (2005), 86, 827–838
DOI 10.1099/vir.0.80299-0
Phenotype of disease-associated PrP
accumulation in the brain of bovine spongiform
encephalopathy experimentally infected sheep
Lorenzo González,1 Stuart Martin,1 Fiona E. Houston,2 Nora Hunter,3
Hugh W. Reid,4 Sue J. Bellworthy5 and Martin Jeffrey1
Correspondence
Lorenzo González
[email protected]
1,4
Veterinary Laboratories Agency (VLA-Lasswade)1 and Moredun Research Institute4,
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
5
VLA-Weybridge, Addlestone, Surrey KT15 3NB, UK
Received 21 May 2004
Accepted 17 November 2004
In view of the established link between bovine spongiform encephalopathy (BSE) and variant
Creutzfeldt–Jakob disease and of the susceptibility of sheep to experimental BSE, the detection
of potential cases of naturally occurring BSE in sheep has become of great importance. In this
study, the immunohistochemical (IHC) phenotype of disease-associated prion protein (PrPd)
accumulation has been determined in the brain of 64 sheep, of various breeds and PrP
genotypes, that had developed neurological disease after experimental BSE challenge with
different inocula by a range of routes. Sheep BSE was characterized by neuron-associated
intra- and extracellular PrPd aggregates and by conspicuous and consistent deposits in the
cytoplasm of microglia-like cells. The stellate PrPd type was also prominent in most brain areas
and marked linear deposits in the striatum and midbrain were distinctive. Sheep of the ARR/ARR
and ARQ/AHQ genotypes displayed lower levels of PrPd than other sheep, and intracerebral
BSE challenge resulted in higher levels of PrPd accumulating in the brain compared with other
routes. The PrP genotype and the route of challenge also appeared to affect the incubation period
of the disease, giving rise to complex combinations of magnitude of PrPd accumulation and
incubation period. Despite these differences, the phenotype of PrPd accumulation was found to
be very consistent across the different factors tested (notably after subpassage of BSE in sheep),
thus highlighting the importance of detailed IHC examination of the brain of clinically affected
sheep for the identification of potential naturally occurring ovine BSE.
INTRODUCTION
Sheep are the natural hosts of scrapie, the first transmissible
spongiform encephalopathy (TSE) to be described, and
are also susceptible to experimental infection by other TSE
agents, such as those causing transmissible mink encephalopathy (Hadlow et al., 1987) and bovine spongiform
encephalopathy (BSE) (Foster et al., 1993). The susceptibility of sheep to BSE, together with the widely accepted
theory of BSE being the origin of variant Creutzfeldt–Jakob
disease (vCJD; Collinge et al., 1996; Bruce et al., 1997; Hill
et al., 1997; Scott et al., 1999), have raised considerable
concern. In Great Britain in particular, the size of the BSE
epidemic and management practices suggest that sheep
could have been exposed to natural infection through
contaminated feedstuffs (Schreuder & Somerville, 2003).
Moreover, in experimental ovine BSE, disease-associated
prion protein (PrPd) and infectivity are widespread throughout tissues of the lymphoreticular system (LRS), the
0008-0299 Crown copyright
gastrointestinal tract (Foster et al., 2001; Jeffrey et al.,
2001b) and blood (Houston et al., 2000; Hunter et al., 2002).
In this respect, sheep BSE resembles sheep scrapie more
closely than cattle BSE and, thus, the possibility exists that
it could be transmitted between infected and uninfected
sheep and maintained even in the absence of the original
source of infection. Whether natural BSE in sheep is merely
a hypothetical possibility is open to debate (Schreuder &
Somerville, 2003) but, in any case, the search for methods
that may allow differentiation between scrapie and BSE in
sheep is a current priority.
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
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L. González and others
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 93–99 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 celltype-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
828
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
Animals and experimental procedures. IHC examination for
PrPd accumulation was performed in the brains of 64 sheep, all of
which showed neurological signs after experimental infection with
the BSE agent. These animals were gathered from several ongoing
and completed experiments and were grouped according to breed
and PrP genotype, source and type of inoculum and route of
challenge, as detailed in Table 1.
Sheep were of four different breeds [Cheviot (n=27), Poll-Dorset
(n=7), Suffolk (n=12) and Romney (n=18)] and five different
PrP genotypes [ARQ/ARQ (n=42), ARQ/AHQ (n=6), AHQ/AHQ
(n=3), VRQ/VRQ (n=5) and ARR/ARR (n=8)], which are indicated
as polymorphisms at codons 136, 154 and 171 for the two alleles (the
single-letter amino acid code is used). PrP genotyping was performed
by sequencing of PCR-amplified products (Baylis et al., 2000). The
animals had been exposed experimentally to BSE infection by one of
three routes [intracerebral (IC, n=40), intravenous (IV, n=11) or oral
(n=13)], using either blood (n=5) or brain homogenates (n=59)
from either of two host sources [cattle (n=40) or sheep (n=24)].
Details of experimental procedures have been given elsewhere [for IC
challenge by Foster et al. (1993), for oral challenge by Jeffrey et al.
(2001b) and for IV challenge by Hunter et al. (2002)] and were also
summarized by Martin et al. (2005). The protocol for inoculation of
sheep-passaged BSE inoculum was identical to that described for cattle
inoculum.
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 formal-
dehyde, 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 217–231
(R. Jackman, personal communication). Sections from positivecontrol 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
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Journal of General Virology 86
Phenotype of PrPd accumulation in sheep BSE
Table 1. Animals used for the study, incubation period of the disease and magnitude of total PrPd accumulation
For description of genotypes, see text.
Group
Breed
Genotype
Age*
Inoculum
Source
1
2
3
4
5
6
7
8
9
10
11
Cheviot
Poll-Dorset
Suffolk
Romney
Cheviot
Cheviot
Suffolk
Poll-Dorset
Cheviot
Romney
Poll-Dorset
Suffolk
Romney
Cheviot
Cheviot
ARQ/ARQ
ARQ/ARQ
ARQ/ARQ
ARQ/ARQ
VRQ/VRQ
ARR/ARR
ARR/ARR
ARR/ARR
AHQ/AHQ
ARQ/ARQ
ARQ/ARQ
ARQ/ARQ
ARQ/ARQ
ARQ/ARQ
ARQ/AHQ
736±41
768±50
1110
365
226±32
680±179
288±6
182
204±22
273±34
397±13
Cattle
Cattle
Cattle
Sheep
Cattle
Cattle
Cattle
Cattle
Cattle
Cattle
Sheep
Sheep
Sheep
Sheep
Cattle
TypeD
a
Brain
Braina
Braina
Brainb
Braina
Braina
Braina
Braina
Brainc
Braind
Braine
Braine
Braine
Bloodf
Braina
Number
Clinical
course*
Incubation
period*
PrPd score§
5
4
5
13||
5
5
2
1
3
4
2
3
1
5
6
9±2?5
2±0?4
19±16?4
14±2
22±7?8
68±17
609±16
499±2
550±13
586±32
1068±9
1333±86
17?6±0?6
15?6±1?2
15?2±1?1
14?0±0?5
16?7±0?7
6?1±0?6
11±4?7
15±5?1
20±5?9
626±21
923±75
736±20
7?9±0?9
10?3±1?4
12?3±1?1
14±5?1
56±11?4
566±16
715±18
9?8±0?7
5?8±0?8
Routed
IC
IC
IC
IC
IC
IC
IC
IC
OR
OR
OR
OR
OR
IV
IV
*Age at challenge, clinical course (from first signs to cull) and incubation period (from challenge to cull) in days (mean±SEM).
DThe different inocula are identified by different superscripts: a, c, d, brain pools of BSE-affected cattle titrated IC/IP in RIII mice (titres were
102?4, 103?5 and 102?2 LD50 g21, respectively); b, brain pool of Cheviot sheep (titre in RIII mice of 104?7 LD50 g21) that developed clinical TSE
after oral challenge with cattle BSE inoculum; e, brain pool from Romney sheep (titration pending) that developed clinical disease after being
dosed orally with inoculum d; f, non-titrated individual blood aliquots (either 400–450 ml whole blood or buffy coat extracted from 50 ml whole
blood) from sheep of group 7.
dIC, Intracerebral; IV, intravenous; OR, oral (5 g inoculum as a 10 % homogenate).
§Mean±SEM of individual sheep scores (see text).
||This group of sheep comprised five, five and three animals challenged with 1023, 1024 and 1025 dilutions of inoculum b, respectively.
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.
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RESULTS
Negative-control brain samples and normal serum-treated
tissue sections produced no immunoreactivity, whereas
positive-control sections gave specific immunolabelling of
comparable intensity in all IHC runs performed.
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
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L. González and others
Fig. 1. Scoring the magnitude of PrPd accumulation in the brain of sheep for construction of PrPd profiles. The example
shows different magnitudes of the stellate type of PrPd accumulating in the cerebral cortex of BSE-affected sheep. (a) Nil=0;
(b) mild or slight=1; (c) moderate=2; (d) prominent, severe or striking=3. All four images were obtained after IHC with
R145 antibody and haematoxylin counterstaining. Bars, 170 mm.
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.
Corpus striatum. The pattern of PrPd accumulation at
this site was characteristic (Fig. 2c, d). Intraneuronal
deposits were moderate in the caudate nucleus and more
prominent in the globus pallidus and putamen nuclei
and, in many instances, PrPd accumulated in neurons that
also showed perineuronal deposits. The main type of neuropil accumulation of PrPd was in the form of thick linear
threads, which often connected with the perineuronal
aggregates (Fig. 2e). Intramicroglial PrPd accumulation
was consistent and prominent, with affected cells appearing scattered in the neuropil and occasionally also within
intraneuronal vacuoles (Fig. 2f). Extracellular PrPd of the
stellate type was conspicuous (Fig. 2c, d), whilst the
presence of intra-astrocytic, perivacuolar and perivascular
PrPd was negligible. Because of the lack of the latter
two types, the white-matter bundles of the external and
internal capsules were characteristically devoid of PrPd
(Fig. 2d).
Fig. 2. Topographical description of the phenotype of PrPd accumulation in the brain of BSE-affected sheep. (a) Cerebral
cortex of an ARQ/ARQ Cheviot sheep challenged IC with cattle-brain BSE (group 1) showing stellate PrPd in grey matter (left
half) and perivacuolar PrPd in white matter at the junction with the grey matter. Bar, 170 mm. (b) Detail of perivascular
accumulation of PrPd, made up of coalescing perivacuolar aggregates in the white matter of the cerebral cortex of another
sheep of group 1. Bar, 85 mm. (c) Corpus striatum of a VRQ/VRQ Cheviot sheep challenged IC with cattle-brain BSE (group
5) showing conspicuous stellate and linear types of PrPd accumulation in the caudate nucleus. Bar, 420 mm. (d) In another
sheep of group 5, the same PrPd types as in (c) are prominent in the grey matter of the corpus striatum, but white-matter
bundles lack immunodeposits. Bar, 420 mm. (e) ARQ/ARQ Romney sheep challenged IC with sheep-brain BSE (group 4)
showing intraneuronal PrPd deposits coexisting with perineuronal and linear aggregates in a single neuron of the globus
pallidus. Bar, 28 mm. (f) Corpus striatum of another sheep of group 5; phagocytic cells appear to be removing PrPd from the
remains of the cytoplasm of a neuron (intramicroglial PrPd), whilst PrPd also accumulates around the perikaryon and neurites.
Bar, 28 mm. (g) Conspicuous intramicroglial PrPd and miniature plaque-like deposits in the neuropil (coalescing PrPd type) of
the ventrolateral thalamic nucleus; although the image corresponds to an ARQ/ARQ Suffolk sheep challenged IC with cattlebrain BSE (group 3), these plaque-like deposits were most frequent and prominent in ARR/ARR sheep of group 6. Bar,
28 mm. (h) Midbrain of an ARQ/ARQ Poll-Dorset sheep challenged IC with cattle-brain BSE (group 2) showing prominent
linear and particulate PrPd deposits in the substantia nigra and marked stellate PrPd accumulation in the tegmentum. Bar,
420 mm. (i) Cerebellum of a Cheviot sheep of group 1; prominent stellate PrPd accumulation in the molecular layer, mild
subpial aggregate and marked extracellular, mesh-like deposits in the Purkinje and molecular layers, resembling the profiles of
Bergmann glial cells. Bar, 85 mm. (j) Detail of intra-astrocytic (arrows) and intramicroglial (arrowheads) PrPd granules in the
cerebellar white matter of a Poll-Dorset sheep of group 2. Bar, 17 mm. All images were obtained after IHC with R145 antibody
and haematoxylin counterstaining.
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Phenotype of PrPd accumulation in sheep BSE
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L. González and others
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 whitematter 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
832
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 1023, 1024 and 1025 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. 3a) 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 1–3 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 PrPdaccumulation phenotype.
An unambiguous effect of the PrP genotype was found
when ARR/ARR sheep were compared with other groups,
but no differences were observed between ARQ/ARQ and
VRQ/VRQ sheep (Fig. 4a). Sheep bearing the AHQ allele
showed levels of PrPd similar to those in ARR homozygotes, but the influence of histidine at codon 154 was
problematic to evaluate, as other coincidental factors may
have influenced the PrPd phenotype of these nine sheep
(three were AHQ homozygotes, dosed orally, and six were
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Journal of General Virology 86
Phenotype of PrPd accumulation in sheep BSE
Fig. 4. Effect of PrP genotype on the PrPd profile. (a) Profiles
(mean±SEM) of sheep groups of different genotypes with most
other factors in common (all sheep inoculated IC with cattle
brain: groups 1–3 merged, 5 and 6) are similar, despite differences in magnitude. (b) Similarity of the profiles (mean±SEM)
between sheep groups of different genotypes is also remarkable when other concurrent factors intervene (all 64 sheep
examined: groups 1–4 merged, 5, 6, 7, 8–10 merged, and 11).
m, ARQ/ARQ; &, VRQ/VRQ; #, ARR/ARR; ,, AHQ/AHQ; n,
ARQ/AHQ. For description of PrPd types, refer to Fig. 3.
Fig. 3. Effect of breed of sheep and source of inoculum on
the PrPd profile. (a) Similarity of profiles (mean±SEM) between
sheep of different breeds with all other factors in common (all
ARQ-homozygous sheep inoculated IC with cattle brain, groups
1, 2 and 3). Breeds: m, Cheviot; &, Poll-Dorset; #, Suffolk.
(b) The PrPd profiles (mean±SEM) of sheep inoculated IC with
cattle (groups 1–3 merged)- and sheep (group 4)-brain homogenates are similar. (c) Similarity of profiles (mean±SEM)
between groups dosed orally with cattle (group 8)- and sheep
(group 9)-brain inocula. Observe that the relative magnitudes of
PrPd accumulation for cattle and sheep inocula are different
depending on the route of challenge. Host sources of inoculum:
m, cattle; &, sheep. PrPd types: ITNR, intraneuronal; ITAS,
intra-astrocytic; ITMG, intramicroglial; STEL, stellate; SBPL,
subpial; SBEP, subependymal; PRVS, perivascular; PVAC, perivacuolar; PRCO, particulate/coalescing; LINR, linear; PNER,
perineuronal; EPEN, ependymal.
ARQ/AHQ, infected IV). The eight ARR/ARR affected
sheep (group 6) and the six ARQ/AHQ sheep challenged
IV (group 11) showed little or no subpial, subependymal,
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perivascular, perineuronal or intra-astrocytic PrPd. Perivacuolar PrPd was virtually confined to the cerebellar white
matter and the stellate type was largely restricted to the
striatum and midbrain. Linear and particulate PrPd
accumulations in these 14 sheep were substantial only
in the hypothalamus and midbrain. They displayed little
intramicroglial PrPd anywhere in the brain, and intraneuronal PrPd was inconspicuous except for the deep cerebellar
nuclei and the DMNV. Despite these differences in total
magnitude (Table 1), the neuroanatomical distribution and
profile of PrPd in these animals were similar to those of
sheep of other PrP genotypes (Fig. 4b), although miniature
pseudoplaques in the neuropil, often arranged in rows, were
most conspicuous and widespread in ARR/ARR sheep.
The route of inoculation influenced the magnitude of total
PrPd accumulation and, to a lesser extent, the PrPd profile
(Fig. 5a, b). Sheep challenged by the IC route accumulated
almost 50 % more PrPd than those dosed orally and more
than twice than animals inoculated IV (Table 1). Within
the IV group, however, ARQ/ARQ animals receiving sheep
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833
L. González and others
Fig. 6. Magnitude of PrPd accumulation and incubation period
in groups of sheep of different PrP genotypes challenged by
different routes. The two factors, PrP genotype and route of
challenge, are associated with the two variables, but these are
unrelated to each other. m, ARQ/ARQ challenged IC; &, ARQ/
ARQ challenged orally; $, ARQ/ARQ challenged IV; n, VRQ/
VRQ challenged IC; %, AHQ/AHQ challenged orally; #, ARQ/
AHQ challenged IV; ,, ARR/ARR challenged IC. n, No. sheep
in each group. Results are expressed as means±SEM.
blood reached PrPd levels similar to those infected by the
oral route, whereas those infected with cattle brain (ARQ/
AHQ sheep) were in the ranges of the ARR/ARR sheep, as
already mentioned. The differences in magnitude of PrPd
deposition were not distributed evenly throughout the
brain. Sheep inoculated IC showed similar levels of PrPd to
those challenged by other routes in and caudal to the
midbrain, the differences being particularly noticeable in
the cerebral cortex (Fig. 5c). This was reflected in the PrPd
profile, so that orally and IV-infected sheep showed lower
levels of the PrPd types found commonly in the forebrain
(subpial, stellate, perivacuolar and perivascular) than did
IC-challenged sheep (Fig. 5a, b).
Effect of PrP genotype and route of infection
on incubation period and magnitude of total
PrPd accumulation
Fig. 5. Effect of route of challenge on the PrPd profile and
topographical distribution. (a) Profiles (mean±SEM) of sheep
groups infected IC (groups 1–3 merged), orally (group 8) and
IV (group 11, ARQ/AHQ genotype) with cattle-brain BSE are
similar, despite IC-challenged sheep showing higher overall
levels of PrPd accumulation. (b) The differences in magnitude
are less obvious between ARQ/ARQ sheep infected with
sheep-passaged BSE inoculum by the IC (group 4), oral
(group 9) and IV (group 10, infected with sheep blood) routes.
For description of PrPd types, refer to Fig. 3. (c) Whilst sheep
challenged orally and IV have a similar anatomical distribution
of PrPd accumulation, IC-inoculated sheep show notably higher
levels in the frontal and, to a lesser extent, middle, areas of
the brain (mean±SEM). Routes of infection: m, intracerebral;
&, oral; #, intravenous. Brain areas: CCTX, cerebral cortex;
STRT, striatum; THHT, thalamus/hypothalamus; MIDB, midbrain;
CRBL, cerebellum; OBEX, medulla oblongata.
834
Sheep of the ARQ/ARQ genotype and those carrying the
AHQ allele had significantly shorter incubation periods
than ARR/ARR and VRQ/VRQ allele-bearing animals
(Fig. 6). Amongst the ARQ/ARQ animals, those challenged
IC and IV had shorter incubation periods than sheep
dosed orally, among which one sheep showed a very protracted infection (1132 days). Conversely, AHQ homozygotes challenged orally had a shorter incubation period
than ARQ/AHQ sheep inoculated IV with cattle brain. The
incubation period, however, was not the factor determining
the magnitude of total PrPd accumulation in the brain, as
ARR sheep showed much lower levels of PrPd than did VRQ
animals. Also, AHQ sheep showed a lower magnitude of
PrPd accumulation than ARQ homozygotes, a difference
that was clear when groups challenged by the same route
were compared (Fig. 6).
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Journal of General Virology 86
Phenotype of PrPd accumulation in sheep BSE
Fig. 7. PrPd profiles of sheep BSE and four
scrapie sources obtained by IHC with
R486/R145 antibodies. There are similarities
between the profiles of BSE in two different
genotypes ($, VRQ/VRQ, n=5, group 5,
this study; #, ARQ/ARQ, n=14, groups 1,
2 and 3, this study) and differences
between these and two experimental scrapie
sources [%, SSBP/1 in VRQ/VRQ Cheviot
sheep, n=5, adapted from González et al.
(2002); m, CH1641 in ARQ/ARQ and
ARQ/AHQ Cheviot sheep challenged IC,
n=4; M. Jeffrey & L. González, unpublished
observations] and two natural scrapie
sources (X, in ARQ/ARQ Suffolk sheep,
n=5; &, in VRQ/VRQ Cheviot sheep,
n=5; L. Gonzalez, unpublished observations). For description of PrPd types, refer to
Fig. 3. VASC, Vascular plaques.
DISCUSSION
After passage in inbred mice, the ‘BSE signature’ can be
recognized by incubation period, vacuolar lesion profile
(Fraser et al., 1992; Bruce et al., 1994) and glycotyping
(Collinge et al., 1996; Hill et al., 1997). This signature is
the same regardless of the source of the original isolate, i.e.
cattle, sheep or other species, including humans (Bruce
et al., 1997; Hill et al., 1997). With this study, we show that,
by detailed IHC examination, a similar signature can be
identified in BSE-infected sheep irrespective of breed, PrP
genotype, route of inoculation or source, type and dose of
inoculum.
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 BSEinfected 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
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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).
Our results suggest that a systematic IHC assessment of
PrPd accumulation in the brain would be suitable for
identification of naturally occurring BSE in sheep. This is
supported by several findings: firstly, the consistency of the
profile across sheep breeds (see Fig. 3a) and, allowing for
magnitude differences, PrP genotypes (Fig. 4a, b); secondly,
the absence of effect of the host source of the inoculum on
the PrPd profile (Fig. 3b, c), which agrees with the stability
of cross-species-passaged BSE found in mice (Bruce et al.,
1994) and with the similarity of glycoprofiles and IHClabelling properties between cattle-derived and sheeppassaged ovine BSE (M. Stack and others, unpublished
observations); thirdly, the homogeneity of PrPd profiles of
individual sheep (data not shown) challenged with sheepderived BSE inoculum by either IC, oral or IV routes.
Overall, this phenotypic consistency of PrPd accumulation
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835
L. González and others
in the brain points towards a ‘BSE signature’ in sheep that
would persist after a potential recirculation of BSE in sheep
following the removal of the original source of infection.
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 BSEinfected 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.
836
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-tosheep 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.
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Journal of General Virology 86
Phenotype of PrPd accumulation in sheep BSE
ACKNOWLEDGEMENTS
González, L., Martin, S., Begara-McGorum, I., Hunter, N.,
Houston, F., Simmons, M. & Jeffrey, M. (2002). Effects of agent
This work was funded by the Department for Environment, Food and
Rural Affairs and by the Department of Health. We are grateful to
J. Foster (IAH-NPU, Edinburgh, UK) for provision of tissues from
some of the sheep in this study, to R. Jackman (VLA, Weybridge, UK)
for supply of R-486 and R-145 antibodies and to T. Martin (VLA,
Weybridge, UK) and W. Goldmann (IAH-NPU, Edinburgh, UK) for
PrP genotyping of sheep. Technical work by Lynn Fairlie and Stuart
Aspen (VLA, Lasswade, UK) is also acknowledged.
strain and host genotype on PrP accumulation in the brain of sheep
naturally and experimentally affected with scrapie. J Comp Pathol
126, 17–29.
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