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Characterisation of an Unusual TSE in a Goat by Transmission in
Knock-in Transgenic Mice
Running title: Unusual TSE properties in goat
Rona Wilson1, Declan King1, Nora Hunter1, Wilfred Goldmann1 and Rona M Barron1*
1Neurobiology Division, The Roslin Institute and R(D)SVS, University of Edinburgh,
Roslin, Midlothian, EH25 9RG, UK
*Corresponding Author
Neurobiology Division
The Roslin Institute and R(D)SVS, University of Edinburgh
Roslin
Midlothian, EH25 9PS
UK
Tel: 0131 527 4200
Fax: 0131 440 0434
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Contents Category: TSE Agents
Summary Word Count: 248
Main Text Word Count: 5,068
Number of Tables and Figures: 7
Characterisation of an Unusual TSE in a Goat by Transmission in Knock-in
Transgenic Mice
Summary
Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disorder of
cattle, and its transmission to humans through contaminated food is thought to be the
cause of the variant form of Creutzfeldt-Jakob disease (vCJD). BSE is believed to have
spread from the recycling in cattle of ruminant tissue in meat and bone meal (MBM),
however during this time sheep and goats were also exposed to BSE-contaminated
MBM. Both sheep and goats are experimentally susceptible to BSE, and while there
have been no reported natural BSE cases in sheep, two goat BSE field cases have
been documented. While cases of BSE are rare in small ruminants, the existence of
scrapie in both sheep and goats is well established. In the UK, during 2006-2007, a
serious outbreak of clinical scrapie was detected in a large dairy goat herd.
Subsequently, 200 goats were selected for post-mortem examinations, one of which
showed biochemical and immunohistochemical features of the disease associated prion
protein (PrPTSE) which differed from all other infected goats. In the present study we
investigated this unusual case by performing bioassays into a panel of mouse lines.
Following characterisation, we found that strain properties such as the ability to transmit
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to different mouse lines, lesion profile pattern, degree of PrP deposition in the brain and
biochemical features of this unusual goat case were neither consistent with goat BSE
nor with a goat scrapie herdmate control. However our results suggest this unusual
case has BSE-like properties and highlights the need for continued surveillance.
Introduction
The transmissible spongiform encephalopthies (TSEs) are a group of fatal infectious
neurodegenerative diseases that include scrapie in sheep and goats, bovine spongiform
encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in cervids, and
Creutzfeldt-Jakob disease (CJD) in humans. TSEs are characterised by the
accumulation in the brain of PrPTSE, which is a relatively protease resistant
conformational variant of the normal host encoded cellular prion protein (PrPC). Due to
the infectious nature of TSEs, these diseases can be transmitted via a number of
different routes and while TSEs tend to transmit more readily within species, they are
able to transmit between different species, although this is dependent on both the TSE
agent and the host.
The existence of naturally occurring classical scrapie in sheep and goats has been
known for more than 200 years and is not considered a public health risk according to
the “Opinion of the Scientific Panel on Biological Hazards on the request from the
European Commission on classification of atypical Transmissible Spongiform
Encephalopathy (TSE) cases in Small Ruminants” (Budka H, 2005). However, the
transmission of BSE to humans through contaminated food is thought to be the cause of
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the variant form of Creutzfeldt-Jakob disease (vCJD) (Bruce et al., 1997; Hill et al.,
1997). This relationship reveals a potential risk of transmission of other ruminant TSEs
to humans. During the BSE epidemic both sheep and goats were exposed to BSE-
contaminated meat and bone meal (MBM) (Baylis et al., 2002). Studies have shown that
sheep and goats are experimentally susceptible to BSE (Baylis et al., 2002; Bellworthy
et al., 2008; Foster et al., 1993; Houston et al., 2003; Stack et al., 2009a). While no
natural cases of BSE have thus far been detected in sheep, two BSE cases in goats
have been confirmed, one in France (Eloit et al., 2005) and the other in the UK
(Spiropoulos et al., 2011). Furthermore, studies have shown the transmission of goat
and/or sheep BSE into transgenic mice expressing human PrP and have found
transmission of these isolates more efficient than cattle BSE (Padilla et al., 2011;
Plinston et al., 2011).
BSE was first reported in 1987 (Wells et al., 1987) and although it has been extensively
characterised, the true aetiology of infection remains unknown. Due to classical strain
typing in mice (incubation time, lesion profiles, patterns of PrP staining in the brain)
(Bruce, 1996; Bruce et al., 1997) and biochemical features of the proteinase K (PK)
resistant PrP in natural and experimental BSE (Collinge et al., 1996; Kuczius &
Groschup, 1999; Kuczius et al., 1998), TSE disease in cattle was originally believed to
be caused by a single prion strain, known as classical BSE (BSE-C). However, through
increased TSE surveillance, two atypical BSE agents have also recently (since 2004)
been reported (Biacabe et al., 2004; Casalone et al., 2004; Jacobs et al., 2007; Stack et
al., 2009b) and are identified as H-type BSE and bovine amyloidotic spongiform
encephalopathy (BASE, also named L-type BSE). These atypical bovine TSEs can be
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distinguished by the electrophoretic positions of their PrPTSE isoforms (Biacabe et al.,
2004; Buschmann et al., 2006; Casalone et al., 2004; Jacobs et al., 2007) and different
glycoform pattern.
Due to concerns that BSE may also infect sheep and goats, active surveillance of small
ruminants has been considerably improved. Many countries now apply screening,
confirmatory and discriminatory tests based on the post mortem detection of PrPTSE in
the central nervous system. Following an outbreak of scrapie in a large goat herd in
2006-2007, the whole herd was culled and 200 goats were examined for PrPTSE
(Gonzalez et al., 2009). One goat showed biochemical and immunohistochemical (IHC)
features of PrPTSE different from all other 71 infected goats in the herd. This particular
case was an eight-year old Anglo-Nubian goat, homozygous for the caprine wildtype
allele isoleucine at codon 142 (Gonzalez et al., 2009). Notably, the existence of atypical
scrapie was ruled out (Gonzalez et al., 2009). Initial analysis of this goat case identified
some similarities to BSE, such as a reduced P4 antibody signal following western
blotting and lack of 12B2 antibody staining of intracellular PrPTSE deposits, however
further analysis with additional antibodies also showed distinct differences with
experimental BSE in goats (Gonzalez et al., 2009). In the present study we aimed to
characterise this unusual goat TSE (ref V3698) by challenging knock-in (produced by
gene targeting) bovine (Bov6) or human PrP Tg (HuTg) mice, as well as 129/Ola wild-
type mice. One experimental goat BSE and a goat scrapie herdmate (goat scrapie HM)
were included as controls. Following neuropathological and biochemical analysis we
found that although V3698 presented some BSE-like properties, it differed from both
experimental goat BSE and goat scrapie HM.
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Results
Transmissibility of V3698 differs from experimental goat BSE and goat scrapie
Using a panel of mouse lines (Bov6, HuTg and 129/Ola), animals were challenged
intracerebrally with isolate V3698, experimental goat BSE or goat herdmate V3981 with
confirmed classical scrapie (Gonzalez et al., 2009) (from here will be referred to as goat
scrapie HM). Following V3698 inoculations, a TSE was induced in 9/24 Bov6 and 1/22
129/Ola mice and was defined by the presence of PrP deposition (using
immunohistochemistry) in the brain and/or vacuolar pathology (Table 1). In contrast, the
majority of Bov6 mice (22/23) and almost 60% of 129/Ola mice (13/22) developed a
TSE following challenge with experimental goat BSE. Following goat scrapie HM
challenge, the majority of 129/Ola mice (20/24) were positive for TSE disease, however
interestingly 9/23 Bov6 mice were positive for TSE pathology (PrP deposition and/or
vacuolar pathology). While the majority of TSE positive mice challenged with either
experimental goat BSE or goat scrapie HM showed clinical signs of disease, this was
not the case for V3698 (Table 1). Furthermore, TSE neuropathology occurred around
150 days later in mice challenged with V3698, than in either Bov6 mice challenged with
experimental goat BSE or 129/Ola challenged with goat scrapie HM. Thus, our results
demonstrated that V3698 attack rates were not consistent with either experimental goat
BSE or goat scrapie HM. Full strain characteristics of V3698 are shown on
supplementary table 1.
Comparisons of Lesion Profiles from previous TSE agents transmissible to Bov6
mice
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The lesion profiles, which define areas of vacuolation and their degree of severity in the
brain, were determined for Bov6 mice inoculated with V3698, experimental goat BSE
and goat scrapie HM (Fig. 1a). Although, the production of lesion profiles in pre-clinical
mice is not standard practice, all Bov6 mice challenged with V3698 or goat scrapie HM
which scored positively for vacuolation pathology were included to give an indication of
vacuolation profile in comparison to experimental goat BSE. Lesion profiles of V3698
and experimental goat BSE were clearly distinct, indicating different TSE isolates (Fig.
1a). A higher degree of vacuolar pathology was observed in several brain regions
(hippocampus, retrospinal cortex, cingulate and motor cortex and cerebellar white
matter) in Bov6 mice challenged with V3698. However, lesion profiles in Bov6 mice
challenged with V3698 or goat scrapie HM were strikingly similar. To further investigate
the vacuolar pattern of V3698, we also compared lesion profiles produced from previous
experiments in which cattle isolates of BASE, H-type BSE and BSE-C had been
transmitted to the Bov6 mice (Wilson et al., 2012a) (Fig. 1b). In these comparisons,
V3698 was found to display a similar pattern of vacuolar targeting to that observed in
Bov6 mice challenged with BASE, and was distinct from BSE-C and H-type BSE.
Bov6 Mice Challenged with V3698 or goat scrapie show low levels of PrP
deposition
Pattern of PrP deposition
Widespread extensive PrP deposition, detected using immunohistochemistry, was
observed in Bov6 mice challenged with experimental goat BSE (Fig. 2a-b). In contrast,
Bov6 mice challenged with V3698 showed very little PrP deposition which was
observed in the thalamus (Fig. 2c-d), midbrain and medulla. While the majority of
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129/Ola challenged with goat scrapie HM showed heavy PrP deposition (Fig. 2e-f), only
seven Bov6 mice showed PrP deposition. However, similarly to V3698, we observed
very little PrP deposition in the Bov6 mice challenged with goat scrapie HM, which was
also confined to the thalamus (Fig. 2g-h), midbrain and medulla. Due to the low levels of
PrP deposition in Bov6 mice challenged with V3698 or goat scrapie HM, we could not
conclude similarities or differences to PrP distribution pattern compared to experimental
goat BSE or goat scrapie HM in 129/Ola mice. Using thioflavin-S fluorescence we were
unable to detect PrP amyloid plaques in Bov6 mice challenged with experimental goat
BSE, Bov6 mice challenged with V3698 and 129/Ola challenged with goat scrapie HM
(supplementary Fig. 1).
Glial Neuropathology
To control for age-related effects, gliosis was first assessed in brain tissue obtained
from a previous aging study of Bov6 mice (Wilson et al., 2012a). Mild gliosis was
present throughout the brains of aged Bov6 control mice (Fig. 3b-c). In contrast to
gliosis due to aging, an increase in the appearance of astrogliosis and microgliosis was
clearly evident throughout the brains of Bov6 mice inoculated with V3698, experimental
goat BSE and goat scrapie HM and 129/Ola challenged with goat scrapie HM (all of
which showed signs of TSE pathology), and was most evident in the thalamus (Fig. 3e-
f, 3h-i, 3k-l and 3n-o). While areas of PrP deposition in the thalamus clearly correlated
with gliosis in Bov6 mice challenged with experimental goat BSE and 129/Ola
challenged with goat scrapie HM (Fig. 3g, 3m), Bov6 mice challenged with V3698 or
goat scrapie HM showed gliosis despite the presence of very little visible PrP deposition
(Fig. 3d, 3j).
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Molecular Profile of V3698 Differs from goat scrapie HM and experimental goat
BSE
Brains from Bov6 mice challenged with V3698, experimental goat BSE and goat scrapie
HM and also 129/Ola mice challenged with goat scrapie HM (2 or 3 brains per isolate)
were examined for the presence of PrPTSE by western blot (Fig. 4a-b). Brains from Bov6
mice challenged with BASE and BSE-C were also included as comparisons. All brains
selected for analysis showed moderate to intense PrPTSE deposition (by
immunohistochemistry) in the brain, except for Bov6 mice challenged with V3698 and
goat scrapie HM which showed very little PrPTSE deposition. Due to the low levels of PrP
in these brains, precipitation of PrP using sodium phosphotungstic acid (NaPTA) was
performed to concentrate all samples. Following western blot analysis using anti-PrP Ab
6H4, distinct PrPTSE profiles were observed between Bov6 mice challenged with V3698
or experimental goat BSE and 129/Ola mice challenged with goat scrapie HM (Fig. 4a).
However, similar PrPTSE profiles were observed between Bov6 brains challenged with
V3698 or goat scrapie HM (Fig. 4c). The experimental goat BSE PrPTSE had a similar
glycopattern to BSE-C with the characteristic heavier diglycosylated band. Indeed,
previous studies have shown that glycoform pattern can be used to distinguish between
different TSE strains (Baron et al., 2007; Beringue et al., 2007; Capobianco et al., 2007;
Somerville, 1997; Somerville et al., 2005), however a number of phenotypic markers are
required to reliably discriminate between strains. Ratios of all three glycoforms (un-,
mono- and diglycosylated), calculated by a Kodak 440CF digital imager, were similar
between Bov6 mice brains challenged with BSE-C PrPTSE and experimental goat BSE
PrPTSE. These differed from V3698, which showed a PrP glycoform ratio similar to BASE
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in Bov6 mice. All were distinct from 129/Ola mouse brains challenged with goat scrapie
HM (Fig. 5). Western blot analysis was also performed using anti-PrP Ab 12B2, which
can be used to discriminate between BSE and classical scrapie (Jacobs et al., 2011;
Jeffrey et al., 2006; Langeveld et al., 2006). A strong signal was detected only from the
129/Ola mouse brains challenged with goat scrapie HM (Fig. 4b & d).
Transmission of Experimental Goat BSE into HuTg Mice
Previous studies have shown that experimental goat BSE and sheep BSE are
transmissible to Tg mice expressing codon 129 methionine human PrP (Padilla et al.,
2011; Plinston et al., 2011) and transmit to these mice more efficiently than cattle BSE
(Padilla et al., 2011). In the present study we also found transmission of experimental
goat BSE into HuTg mice (10/24 HuMM mice showed PrP deposition).
Neuropathological analysis of these mice showed PrP deposition was plaque-like and
restricted to the thalamus (Fig. 6a-b) and midbrain. Despite the presence of PrP
deposition, no HuTg mice were positive for vacuolation pathology, except for one which
had limited vacuolation in the cerebral and midbrain white matter. Due to the lack of
overexpression of PrP in these mice, they may more closely represent what happens in
nature and thus this finding is of particular concern. No transmission of either V3698 or
goat scrapie HM was detected in the HuTg mice, which further suggests that V3698 is
distinct from goat BSE (Table1).
Discussion
In this study, we examined the transmission characteristics, neuropathology and
biochemical characteristics of an unusual TSE present in a goat, designated V3698.
Initial investigation of this case revealed unusual biochemical and immunohistochemical
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features of PrPTSE compared to other scrapie infected goats in the herd (Gonzalez et al.,
2009), which presented both similarities and differences to both goat BSE and
previously studied isolates of goat scrapie. In order to help determine the nature of
V3698 and improve our understanding of the diversity of goat TSEs, we performed
transmission bioassays into a panel of mice. Along with V3698 we therefore also
inoculated an experimental goat BSE isolate and goat scrapie HM control into gene-
targeted bovine or human PrP Tg mice, as well as 129/Ola wildtype mice. As these
transgenic mice are produced by gene replacement, they do not suffer from any
adverse phenotypes which are associated with overexpression or ectopic expression of
the transgene in standard transgenic lines, and express PrPC at the same level as wild
type mice (Manson et al., 1999; Moore et al., 1998). Furthermore, previous studies have
shown efficient transmission of TSE agents into gene targeted transgenic PrP mouse
lines, demonstrating that these mice do live long enough to show signs of infection,
supporting the use of targeted mouse models to analyse TSE disease transmission
(Bishop et al., 2010; Bishop et al., 2006; Plinston et al., 2011). While caprine transgenic
mouse lines may have been an alternative model system, as yet there are no available
knock-in caprine transgenic mice. Furthermore, in this study, the use of bovine and
human PrP knock-in transgenic lines allows direct comparison with archive data
produced following inoculation of these lines with a wide range of TSE agents, including
vCJD, BSE-C (Bishop et al., 2006), BASE and H-type BSE (Wilson et al., 2012a).
The data presented here showed that transmission, neuropathology and biochemical
properties of V3698 were not consistent with either experimental goat BSE or goat
scrapie HM. Lesion profiles between V3698 and experimental goat BSE were clearly
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distinct, as were the amount of PrP deposition present in the brain, and ratios of PrPTSE
glycoforms. Furthermore, lack of binding to an anti-PrP antibody (12B2) suggested
V3698 was not a classical scrapie strain. This was consistent with the original study of
V3698 in which PrPTSE in brain tissue, but not retropharyngeal lymph node, was shown
to lack 12B2 binding (Gonzalez et al., 2009). Interestingly, although goat scrapie HM
inoculated 129/Ola mice produce a strong PrPTSE signal on immunoblot following
probing with 12B2, indicating classical scrapie, the PrPTSE in Bov6 mice inoculated with
goat scrapie HM was negative for 12B2 binding. We have previously observed a strong
12B2 signal on immunoblots from Bov6 mice challenged with H-type BSE (data not
shown), proving Bov6 PrPTSE can bind 12B2. These data are also consistent with
studies showing that while labelling for classical BSE or BASE with 12B2 is minimal or
absent, H-type BSE is strongly labelled with this antibody (Baron et al., 2011; Dudas et
al., 2010; Jacobs et al., 2011). Unlike goat scrapie HM or experimental goat BSE,
V3698 failed to transmit efficiently to 129/Ola mice. While evidence of scrapie
transmission to cattle has been described by others (Cutlip et al., 1994; Konold et al.,
2006), our previous studies have shown a lack of transmission of either classical or
atypical scrapie into Bov6 mice, (Plinston et al., 2011; Wilson et al., 2012b). Importantly,
all successful disease transmissions into Bov6 mice in our lab, as yet have been from
BSE-like strains (Bishop et al., 2006; Plinston et al., 2011; Wilson et al., 2012a). Hence,
the observed transmissibility of the goat scrapie HM to Bov6 mice and lack of 12B2
binding is perplexing. As we have not previously challenged our Bov6 mice with other
isolates of goat scrapie, we cannot rule out the possibility that scrapie in goats is
transmissible to this mouse line. However the results presented here may also suggest
that the same agent has been isolated from both goat field samples in the Bov6 mice
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based on to the lack of 12B2 reactivity, similarities in both lesion and molecular profile,
the lack of PrP deposition, and the fact that both V3698 and the goat scrapie HM control
came from the same herd. Indeed, it is widely accepted that animals carrying TSE
infection in the field may be infected with more than one “strain”. Laboratory TSE strains
represent isolates which have been selected from field cases following serial
transmission in mice and probably represent selection from the field case of the isolate
that replicates most efficiently in that mouse genotype. The similarities of V3698 and the
goat scrapie HM inoculum in Bov6 mice may suggest that the same agent was isolated
from both goats. However the high transmission of goat scrapie HM, but not V3698, to
129/Ola mice indicates a difference either in the TSE agent, or the presence of more
than one TSE agent that may have been differentially selected in the different mouse
genotypes. Further transmission studies would need to be performed to determine
whether this was the case.
Interestingly, we found V3698 showed a number of similarities to earlier transmissions
investigating the susceptibility of Bov6 mice to BASE (Wilson et al., 2012a). As
previously discussed, V3698 failed to transmit efficiently into 129/Ola control mice,
which was similar to prior studies with BASE (Wilson et al., 2012a). In contrast, as was
the case for experimental goat BSE in these studies, others have shown that classical
cattle BSE transmits very efficiently to a variety of inbred mouse strains (Capobianco et
al., 2007; Wilson et al., 2012a). Despite signs of neuropathology, we did not observe
clinical signs in Bov6 mice challenged with V3698, which was also the case in Bov6
mice challenged with BASE (Wilson et al., 2012a). Indeed, several studies have shown
that cattle infected with atypical BSE were found to be healthy at slaughter (Biacabe et
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al., 2004; Casalone et al., 2004). We also observed a similarity between the vacuolation
pattern of V3698 and BASE in the Bov6 mice. However, it is also important to highlight
that we observed properties of V3698 that were not consistent with BASE, such as the
mobility of the unglycosylated PrP fragment, lack of prominent, widespread PrP
deposition and amyloid plaques, which have been shown in previous studies in Bov6
mice (Wilson et al., 2012a). As BASE has not been previously transmitted into goats,
the strain characteristics of this TSE in goats is unknown. Furthermore, the existence of
BASE could be difficult to determine if mixed TSE infections were present in a single
host. Therefore, further studies would be necessary to identify whether V3698 was
BASE in a goat.
Although incubation periods or survival times are often used to distinguish between
TSEs, they may not be as reliable during first passage, especially when a TSE is only
diagnosed in a small number of animals due to low titre and/or limited transmission of
specific TSE strains in certain mouse lines. Therefore, while we still observed
transmission in 37% of Bov6 mice challenged with V3698, in this study we included
incubation and survival times as an observation but did not rely on them to discriminate
between TSE isolates.
As part of this study we also investigated the susceptibility of gene-targeted human PrP
Tg mice to V3698, experimental goat BSE and goat scrapie HM. Approximately 40% of
brains from HuMM Tg mice challenged with experimental goat BSE showed PrP
deposition. Our results support previous studies showing that goat and sheep BSE are
more efficient at transmitting to human Tg mice than cattle BSE (Padilla et al., 2011).
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These findings are of concern with regards to the risk to human health and whereas in
cattle BSE infectivity is mainly confined in the central nervous system, in small
ruminants BSE has been detected in peripheral tissues and can be transmitted
horizontally (Bellworthy et al., 2005; Kao et al., 2002). Thus, the data presented here
highlight the requirement to prevent BSE from becoming established in small ruminants.
Overall, our results revealed that while showing some BSE-like properties on
transmission in a panel of transgenic mice, V3698 is neither goat BSE nor a classical
goat scrapie, and may rather be a BSE-like TSE or an unusual form of scrapie, however
further experiments are required to fully characterise this case. While atypical BSE has
been reported in cattle since 2004 (Biacabe et al., 2004; Casalone et al., 2004; Jacobs
et al., 2007; Stack et al., 2009b), no cases have been documented in small ruminants.
As yet, the origin of atypical BSE is unknown, however it has been suggested that it
may be a spontaneous TSE in cattle, thus it would be interesting to speculate whether
this was also possible in goats. We have also considered the possibility that V3698 is a
CH1641-like scrapie isolate. These isolates have been described in sheep and goats
both experimentally (Foster & Dickinson, 1988; Jeffrey et al., 2006) and naturally
(Bellworthy et al., 2005; Bencsik & Baron, 2011; Vulin et al., 2011), and show partial
similarities to experimental ovine BSE. Previous studies have used anti-PrP antibody
SAF84 to identify an additional lower molecular PrPTSE band which has been shown as
marker of CH1641 (Baron et al., 2008). Despite our western blot attempts using SAF84,
due to the extremely low levels of PrPTSE in the Bov6 mice challenged with V3698 we
were unable to fully investigate the V3698 PrPTSE glycoform. Furthermore, without
performing transmission studies into Bov6 mice, we cannot fully compare V3698 with
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CH1641. As yet, the spectrum of TSE agents circulating in the goat population is
unknown. Other studies investigating a flock of scrapie affected goats in Greece have
shown the existence of a unique TSE (Bouzalas et al., 2011). These studies showed
that a number of clinically healthy goats were found to have a unique C- and N-
terminally truncated PrPTSE fragment, which was similar but not identical to atypical
scrapie. Similarly to our results which showed very little PrP deposition in Bov6 mice
challenged with V3698 these goats did not show any PrP deposits in the brain
(Bouzalas et al., 2011). Therefore, despite the relatively small UK goat population,
various TSE agents in goats may exist, however it is also possible that in certain
instances more than one agent may exist in an individual goat. As surveillance of small
ruminants continues, the ability to recognise natural goat TSE strains will be of
particular importance, not only to improve our knowledge on the variability of goat TSEs
but to assess any potential risk to human health.
Materials and Methods
Preparation of Inocula
Brain homogenate G8247 from an experimentally BSE-infected goat (2nd goat passage,
The Roslin Institute, unpublished), genotype GIRQP/GIRQS (Goldmann et al., 2011)
was used as a goat BSE control. The unusual goat TSE case V3698 and the goat
scrapie HM control (Ref V3981; a 9 year old Saanen goat) were both supplied by The
Animal Health Veterinary Laboratories Agency (AHVLA), Weybridge, UK. All brain
tissue supplied was from the thalamic region. All inocula were prepared from brain
tissue in sterile saline at a concentration of 10% (wt/vol).
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Inoculation of Transgenic Mice
Gene-targeted Tg mice expressing bovine PrP (Bov6) or human PrP (HuMM, HuMV
and HuVV) have been described previously (Bishop et al., 2006). Wildtype 129/Ola
mice were used as controls (Bishop et al., 2006). Transgenic mice were grouped (24
mice each) prior to intracerebral inoculation (i.c.) and then injected with 0·02 mL of 10 -1
brain homogenate (experimental goat BSE, V3698 or goat scrapie HM) into the right
cerebral hemisphere under halothane anaesthesia. All inocula were prepared by
maceration of the brain tissue in sterile saline to a dilution of 10−1. From 100 days post
inoculation mice were scored each week for signs of disease. Mice were killed by
cervical dislocation at a pre-defined clinical endpoint, or due to welfare reasons
(Dickinson et al., 1968). Brains were recovered at post mortem. Half the brain was
snap-frozen in liquid nitrogen for biochemical analysis and the remaining half brain was
fixed for histological processing. All mouse experiments were reviewed and approved
by the Local Ethical Review Committee and performed under licence from the United
Kingdom Home Office in accordance with the United Kingdom Animals (Scientific
Procedures) Act 1986.
Vacuolation Scoring
Sections were cut (6µm) from each mouse brain and stained using hematoxylin and
eosin (H&E). TSE-related vacuolation was assessed at nine grey-matter regions
(medulla, cerebellum, superior colliculus, hypothalamus, thalamus, hippocampus,
septum, retrospinal cortex, cingulated and motor cortex) and three regions of white
matter (cerebellar white matter, midbrain white matter, and cerebral peduncle).
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Sections were scored on a scale of 0 (no vacuolation) to 5 (severe vacuolation) for the
presence and severity of vacuolation and mean vacuolation scores for each mouse
group in each experiment were calculated and plotted with standard errors of the mean
(SEM) against scoring areas to produce a lesion profile, as previously described (Bruce
et al., 1997; Fraser & Dickinson, 1967). While, the production of lesion profiles in pre-
clinical mice is not standard practice, all mice which scored positive for vacuolar
pathology were included whether or not clinical signs were present, due to the lack of
clinical signs observed in Bov6 mice challenged with V3698.
Immunohistochemical (IHC) analysis of PrP deposition and glial activation in the
brain
PrPTSE localisation in the brain was assessed using immunohistochemistry. Following
fixation in 10% formal saline, brains were treated for 1·5 h in 98% formic acid, dissected
to expose several brain regions, and embedded in paraffin. Sections (6µm) were then
autoclaved for 15 min at 121C and immersed in 95% formic acid for 10 minutes prior to
incubation with 0.44 g/ml anti-PrP monoclonal antibody (MAb) 6H4 (Prionics) at room
temperature overnight. Secondary anti-mouse biotinylated antibody (Jackson Immuno
Research Laboratories, UK) was added at 2.5 g ml-1 and incubated for 1 h at room
temperature. Immunolabelling was performed using the ABC Elite kit (Vector
Laboratories) and the signal was visualised by a reaction with hydrogen peroxidase-
activated diaminobenzidine (DAB). The presence of astrogliosis, a hallmark of prion
disease, was assessed by incubating brain sections (6µm) with 1.45 g ml-1 anti-glial
fibrillary acidic protein (GFAP; DAKO UK Ltd) antibody at room temperature for 1 hour.
To detect microglial activation, brain sections were pre-treated using hydrated
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microwaving for 10 minutes prior to incubation with 0.05 g ml-1 anti-Iba1 antibody
(Wako Chemicals GmbH) at room temperature for 1 hour. For both GFAP and anti-Iba-
1 antibodies, 2.6 g ml-1 biotinylated secondary anti-rabbit antibody (Jackson Immno
Research Laboratories, UK) was added for 1 hour at room temperature. Both
astrocytes and microglia were visualized by a reaction with hydrogen peroxidise-
activated DAB.
Detection of amyloid plaques by thioflavin fluorescence
Amyloid deposits in tissue sections were observed using Thioflavin-S. Briefly, following
haematoxylin staining, sections (6µm) were immersed in 1% Thioflavin-S (Sigma, UK)
solution as previously described (Piccardo et al., 1998). Sections were mounted and
viewed under a fluorescence microscope.
Identification of PrPTSE by immunoblotting
Frozen brain samples from Bov6 mice challenged with experimental goat BSE; V3698,
goat scrapie HM, cattle BASE, BSE-C, and 129/Ola challenged with goat scrapie HM,
were homogenised at 10-1 in an NP40 buffer (0·5% v/v NP40, 0·5% w/v sodium
deoxycholate, 0·9% w/v sodium chloride, 50mM Tris-HCl pH 7·5) and clarified at
11,000g for 15 minutes. Brain homogenate supernatant from the transgenic mice and
controls was incubated with 20 g proteinase K ml-1 for 1 hour at 37C. Due to the lack
of PrP deposition, brains from Bov6 mice challenged with V3698 or goat scrapie HM
were treated with phosphotungstic acid (NaPTA) to precipitate PrP and concentrate the
samples. Briefly, 100mg of brain tissue was incubated for 15 minutes at 370C in
phosphate buffered saline containing 1mM MgCl2. The sample were adjusted to 2%
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(w/v) Sarcosyl and incubated for 5 minutes at 370C. The samples were then altered to a
final concentration of 20 g proteinase K ml-1 and incubated at 37 °C for 1 hour. PrPTSE
was precipitated with the addition of a final concentration of 0.3% NaPTA, incubated at
37°C for 30 minutes and centrifuged at 14,000 rpm for 20 minutes. The pellet was
dissolved in 15 μl phosphate-buffered saline, pH 7.4, containing 0.1% sarkosyl. All
products were denatured and separated on a 10-20% Novex Tris/Glycine gel
(Invitrogen, UK) before transfer to polyvinylidine difluoride (PVDF) membrane by
western blotting. The amount of brain tissue loaded onto the gels varied between
0.3mg and 50mg brain equivalent. PrP was identified with monoclonal antibody 6H4
(0.1 g ml-1) or 12B2 (0.2 g ml-1) and bands visualized using horseradish peroxidise
(HRP)-labelled anti-mouse secondary antibody (Jackson Immuno Research
Laboratories, UK) and a chemiluminescence substrate (Roche). Images were captured
on radiographic film and with a Kodak 440CF digital imager.
PCR genotyping of mouse tail DNA
All mice were analysed by PCR post mortem to confirm PrP genotype. Mouse tail DNA
was extracted and genotyped as previously described (Bishop et al., 2006; Wemheuer
et al., 2011).
Acknowledgements
The authors would like to acknowledge Professor J. Manson for Bov6 and HuTg mouse
lines; S. Cumming, S. Carpenter, R. Greenan and K. Hogan for experimental setup,
care and scoring of the animals; A. Boyle, G. McGregor and D. Drummond for histology
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processing and scoring; J. Foster and D. Parnham for the experimental goat challenge
to generate isolate G8247; J. Hope, L. González and S. Hawkins from the Animal
Health Veterinary Laboratories Agency (AHVLA) UK, for supply of V3698 and the goat
scrapie (V3981) and L. González for his comments on the paper. These studies were
partly funded by contract M03054 from the Food Standards Agency (FSA) UK and
contract SE1441 from the UK Department for Environment, Food and Rural Affairs
(Defra). The Roslin Institute receives Institute Strategic Grant (ISP) funding from the
Biotechnology and Biological Sciences Research Council (BBSRC), UK.
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Table 1. Bioassay results of primary passage of V3698, goat BSE and goat scrapie HM
into both bovine and human PrP Tg mice and 129/Ola mice.
TSE isolate Mouse Line Survival* Incubation†
Clinical Signs
TSE Pathology‡
Clin+/TSE Path+§
V3698
Bov6129/OlaHuMMHuMVHuVV
598±20----
-572
---
1/243/221/238/242/23
9/241/220/230/240/23
01---
ExperimentalGoat BSE(G8247)
Bov6129/OlaHuMMHuMVHuVV
447±14379±18625±12
--
453±15385±18
---
18/2312/223/246/235/23
22/2313/2210/240/230/23
18120--
Goat Scrapie HM
(V3981)
Bov6129/OlaHuMMHuMVHuVV
646±12439±6
---
-443±6
---
0/2318/241/223/241/24
9/2320/240/220/240/24
-18---
* Measured as days ± SEM and calculated from mice showing vacuolar pathology and/or PrP deposition.
† Measured as days ± SEM and calculated from mice showing both clinical signs of disease and
neuropathology (vacuolar pathology and/or PrP deposition).
‡ Number of animals positive for PrP deposition and/or vacuolar pathology.
§ Number of animals with clinical signs and confirmed TSE pathology.
- Not applicable (No vacuolar pathology of PrP deposition observed).
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Figure 1. Pattern of vacuolation observed in brains of Bov6 mice challenged with
V3698, experimental goat BSE and goat scrapie HM (a). Pattern of vacuolation
observed in brains of Bov6 mice challenged with V3698, BASE, H-type BSE and
classical BSE (BSE-C) (b-d). Lesion profiles for BASE, H-type BSE and BSE-C (shown
for comparison) originate from previously published work (Wilson et al, 2012a). A profile
was produced from nine grey matter areas (1, medulla; 2, cerebellum; 3, superior
colliculus; 4, hypothalamus; 5, thalamus; 6, hippocampus; 7-septum, 8-retrospinal
cortex, 9-cingulate and motor cortex) and three white matter areas (1, cerebellar white
matter; 2, midbrain white matter; 3, cerebral peduncle). Average scores were taken
from a minimum of seven mice per group and plotted against brain area ± SEM.
Figure 2. Comparative analysis of variation in intensity of PrPTSE in the thalamus region
of brains from Bov6 mice challenged with experimental goat BSE, 456dpi (a, b), V3698,
623dpi (c, d) or goat scrapie HM, 680dpi (g, h) and 129/Ola mice challenged with goat
scrapie HM, 407dpi (e, f). Panel b, d and f are higher magnifications images of the
boxed area in panel a, c and e respectively. Images obtained after staining with anti-PrP
antibody 6H4 and counterstained with hematoxylin. Magnification is as shown.
Figure 3. Comparative analysis of the thalamus of Bov6 mice challenged with V3698,
experimental goat BSE and goat scrapie HM and 129/Ola mice challenged with goat
scrapie HM. Micro- and astrogliosis is present in all mice, detected by anti-GFAP (e, h,
k, n) and anti-Iba1 respectively (f, i, l, o). PrP deposition is visible by anti-6H4 antibody
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(g, m). Uninfected aged Bov6 mice showing mild gliosis (b, c) and no PrP deposition (a)
were used as controls. Magnification 10x.
Figure 4. (a) and (b); Comparative western blot analysis of the proteinase K-resistant
fragment (PrPTSE) of the prion protein. Discrimination between BSE and scrapie is
achieved using two monoclonal antibodies, 6H4 (a & c) and 12B2 (b & d). (a & b) lanes
1-2, BSE-C in Bov6 mice; lanes 3-4, experimental goat BSE in Bov6 mice; lanes 5-6,
V3698 in Bov6 mice; lanes 7-8, BASE in Bov6 mice, and lanes 9-10, goat scrapie HM in
129/Ola mice. Each lane represents an individual mouse brain. Brain samples in lanes
1-4 and 7-10 were loaded at 0.3 to 0.6 mg brain equivalent, and samples in lanes 5-6
were loaded at 5 mg brain equivalent. (c & d) lanes 1-2, V3698 in Bov6 mice; lanes 3-4,
goat scrapie HM in Bov6 mice (image cropped from a single blot to remove non-relevant
samples). Samples loaded at 5mg brain equivalent. All samples PK-treated and NaPTA
precipitated.
Figure 5. PrP glycoform ratio of samples from Fig.4(a) quantified by Kodak Imager
440CF and normalised as % total PrP. Samples 1-2, BSE-C; samples 3-4, experimental
goat BSE; samples 5-6, V3698; samples 7-8, BASE, and samples 9-10, goat scrapie
HM (129/Ola).
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Figure 6. PrPTSE accumulation in the thalamus of a HuMM mouse (594dpi) challenged
with experimental goat BSE. Panel (b) is a higher magnification image of the boxed
area in panel (a). Images obtained after staining with anti-PrP antibody 6H4 and
counterstained with hematoxylin. Magnification as shown.
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