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Presence of Subclinical Infection in Gene-Targeted Human Prion
Protein Transgenic Mice Exposed to Atypical BSE
Running title: Subclinical Infection in HuTg Mice Exposed to BASE
Rona Wilson1, Karen Dobie1, Nora Hunter1, Cristina Casalone2, Thierry Baron3 and
Rona M Barron1*
1 Neurobiology Division, The Roslin Institute and R(D)SVS, University of Edinburgh, Roslin,
Midlothian, UK
2 Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Turin, Italy
3 Agence Nationale de Sécurité Sanitaire, Lyon, France
*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
Contents Category: TSE Agents
Word count summary: 220
Word count main text: 4806
Number of tables and figures: 6
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Presence of Subclinical Infection in Gene-Targeted Human Prion Protein
Transgenic Mice Exposed to Atypical BSE
Summary
The transmission of bovine spongiform encephalopathy (BSE) to humans, leading to variant
Creutzfeldt-Jakob disease (vCJD) has demonstrated that cattle transmissible spongiform
encephalopathies (TSEs) can pose a risk to human health. Until recently, TSE disease in
cattle was thought to be caused by a single agent strain, BSE, also known as classical BSE,
or BSE-C. However, due to the initiation of a large scale surveillance programme throughout
Europe, two atypical BSE strains, bovine amyloidotic spongiform encephalopathy (BASE,
also named BSE-L) and BSE-H have since been discovered. To model the risk to human
health, we previously inoculated these two forms of atypical BSE (BASE and BSE-H) into
gene-targeted transgenic (Tg) mice expressing the human prion protein (PrP) (HuTg) but
were unable to detect any signs of TSE pathology in these mice. However, despite the
absence of TSE pathology, upon subpassage of some BASE challenged HuTg mice, a TSE
was observed in recipient gene-targeted bovine PrP Tg (Bov6) mice, but not in HuTg mice.
Disease transmission from apparently healthy individuals indicates the presence of
subclinical BASE infection in mice expressing human PrP that cannot be identified by current
diagnostic methods. However, due to the lack of transmission to HuTg mice on subpassage,
the efficiency of mouse to mouse transmission of BASE appears to be low when mice
express human rather than bovine PrP.
Introduction
Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disorder of cattle,
and belongs to a group of diseases known as transmissible spongiform encephalopathies
(TSEs) or prion diseases. The main characteristic of BSE is the accumulation in the brain of
PrPTSE, which is a protease resistant conformational variant of the normal host encoded
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cellular prion protein (PrPC). BSE was first reported in the UK in 1987 (Bruce et al., 1997;
Wells et al., 1987) and its transmission to humans through the consumption of contaminated
food is thought to be the cause of the variant form of Creutzfeldt-Jakob disease (vCJD)
(Bruce et al., 1997; Hill et al., 1997). While a number of other animal TSEs exist, including
scrapie in sheep and goats and chronic wasting disease (CWD) in cervids, BSE is the only
TSE known to be naturally transmissible from animals to humans. Previously, TSE disease
in cattle was believed to be caused by a single prion strain, known as classical BSE (BSE-
C). However, due to the initiation of a large-scale surveillance programme throughout
Europe, two atypical BSE agents were reported (Biacabe et al., 2004; Casalone et al., 2004;
Jacobs et al., 2007; Stack et al., 2009), and identified as BSE-H and bovine amyloidotic
spongiform encephalopathy (BASE, also named BSE-L). BSE-H and BASE were originally
described in 2004 in France (Biacabe et al., 2004) and Italy (Casalone et al., 2004)
respectively, however these atypical BSE strains have since been identified in other
European countries (Jacobs et al., 2007), Japan (Hagiwara et al., 2007) and North America
(Dudas et al., 2010; Richt et al., 2007). Several studies have shown that following
transmission into transgenic mice that overexpress the bovine prion protein, these atypical
BSE agents show neuropathological and molecular phenotypes which are distinct from BSE-
C, indicating they are different BSE strains (Beringue et al., 2007; Béringue et al., 2006;
Buschmann et al., 2006; Capobianco et al., 2007; Okada et al., 2010). Indeed, BASE and
BSE-H can be distinguished by the electrophoretic migration of their protease-resistant
PrPTSE isoforms and their different patterns of glycosylation (Biacabe et al., 2004;
Buschmann et al., 2006; Casalone et al., 2004; Jacobs et al., 2007). Interestingly however,
studies have shown the conversion of both BASE and BSE-H to classical BSE when
passaged through wildtype mice (Baron et al., 2011; Capobianco et al., 2007).
Previously, we modelled the possible susceptibility of humans to BASE and BSE-H using
gene-targeted human PrP transgenic (HuTg) mice (Wilson et al., 2012b). In humans,
susceptibility to TSE infection is linked to a polymorphism in the human PrP gene at codon
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129 (Zeidler et al., 1997). The UK population is approximately 50% MV, 40% MM and 10%
VV, however all clinical cases of vCJD to date have occurred in codon 129 MM individuals.
Using gene targeting we have produced two unique lines of transgenic mice (HuTg) in which
the endogenous murine PrP gene has been replaced with the human PrP gene encoding
either methionine (HuMM) or valine (HuVV) at codon 129 (Bishop et al., 2006). By crossing
the homozygous 129MM and 129VV lines we can also produce a true 129MV heterozygote
(HuMV). Following inoculation of these atypical BSE agents in HuTg mice, we did not detect
any signs of TSE disease pathology (Wilson et al., 2012b), suggesting that the transmission
barrier was significant in the presence of human PrP, and that the risk of disease
transmission from atypical BSE was low. However we could not rule out the possibility that
disease transmission in the presence of human PrP may be inefficient, and that the HuTg
mice which survive to lifespan may be able to maintain low level agent replication in the
CNS. Indeed, primary transmission of prion diseases between different species is often
challenging, and although this subclinical infection does not result in TSE disease during the
lifespan of the animal, the replication of low levels of infectivity in an animal may pose a risk
of accidental transmission through routes such as surgery and blood transfusion. Therefore,
the presence of subclinical forms of TSE infection resulting from atypical BSE could present
a major public health risk, and warrants further investigation.
To address the potential for subclinical infection in animals expressing human PrP following
exposure to atypical BSE, we performed subpassage experiments from several HuTg brains
challenged with BASE or BSE-H into HuTg mice and Bov6 Tg mice (expressing bovine PrP).
In the present study we found evidence of subclinical infection in one HuMM and one HuVV
Tg mouse challenged with BASE. These findings suggest that that low level replication of
BASE can occur in hosts expressing human PrP, however the efficiency of any potential
mouse to mouse transmission in hosts expressing human PrP appears to be low.
Results
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Subpassage of brain tissue from BASE challenged HuPrP Tg mice into Bov6 Tg mice
Brain tissue harvested from previous transmission studies of BASE and BSE-H into HuTg
mice (Wilson et al., 2012b) were examined for evidence of TSE agent replication by
bioassay in HuTg or Bov6 mice. Several different tissues were analysed (see Table 1),
which included six BASE-challenged HuTg brains, three BSE-H-challenged HuTg brains and
two Bov6 brains challenged with either BASE or BSE-H (included as controls). All HuTg
tissues had previously been shown to lack any signs of TSE pathology by
immunohistochemistry and vacuolation profiling. Each brain homogenate was inoculated into
HuTg mice of the same genotype as the inoculum (or HuMM mice in the case of the bovine
control tissue) and Bov6 mice. Details of genotype, clinical status and age of the
subpassage tissues used are shown in Table 1. BASE-challenged HuMM Tg brain (C18985-
HuMM) transmitted a TSE to 4/10 Bov6 mice and was defined by the presence of either PrP
deposition (using immunohistochemistry) in the brain or vacuolar pathology (Table 2). No
clinical signs of TSE disease were detected in these mice. Unexpectedly, BASE-challenged
HuVV Tg brain (C19409-HuVV) also transmitted a TSE to 1/11 Bov6 mice (Table 2), and
again PrP deposition and vacuolar pathology were present without clinical signs of disease.
No other BASE- or BSE-H-challenged HuTg mouse brains transmitted disease in either
Bov6 or HuTg mice. As expected a TSE was identified, by the presence of PrPTSE deposition
and vacuolar pathology, in 10/11 and 11/11 of Bov6 Tg mice inoculated with BASE-
challenged Bov6 brain (C18275-Bov6) and BSE-H-challenged Bov6 brain (C19414-Bov6)
respectively (both controls).
The lesion profiles, which define areas of vacuolation and their degree of severity in the
brain, were determined for Bov6 mice inoculated with controls C18275-Bov6 and C19414-
Bov6 (Figure 1). Although the production of lesion profiles in pre-clinical mice is not
standard practice, all mice challenged with C18275-Bov6 and C19414-Bov6 which scored
positively for vacuolation pathology were included to give an indication of vacuolation profile.
Lesion profile patterns of primary and secondary passages of BASE or BSE-H in Bov6 mice
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were similar between the same BSE agent (Figure 1), although higher levels of vacuolation
were observed in areas 8 and 9 of the grey matter and in the while matter in BASE
subpassage mice. This difference may be due to these mice being non-clinical, and culled at
different stages of pre-clinical disease, or adaptation of the agent to the murine host on
subpassage. Due to low number of animals which scored positively for vacuolation
pathology and lack of statistical significance, lesion profiles from Bov6 mice challenged with
C18985-HuMM or C19409-HuVV were not included.
Neuropathology
PrP deposition in the thalamus, medulla (Figure 2a, 2b and Figure S1), midbrain and
caudate was detected using immunohistochemistry in Bov6 mice challenged with C18985-
HuMM. Similarly, one Bov6 mouse challenged with C19409-HuVV showed PrP deposition in
the same brain regions, however staining was not as heavy (Figure 2c, 2d). Control Bov6
mice challenged with C18275-Bov6 showed widespread heavy PrP deposition (Figure 2e, 2f
and Figure S1), unlike control Bov6 mice challenged with C19414-Bov6 which only showed
sparse PrP deposition (Figure 2g, 2h). Bov6 mice challenged with C18985-HuMM, C19409-
HuVV and C18275-Bov6 showed plaque-like PrP deposits, which were stained dark brown.
While Bov6 mice challenged with C19414-Bov6 also showed plaque-like PrP deposition,
these deposits were not as heavily stained. 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 these mice (Figure 3a 3b). In
contrast to gliosis due to aging, an obvious increase in the appearance of astrogliosis was
clearly evident throughout the brains of Bov6 mice inoculated with C18985-HuMM, C18275-
Bov6 and C19414-Bov6 (Figure 3d, 3g, 3j). While Bov6 mice challenged with C18275-Bov6
showed a marked increase in microgliosis (Figure 3h) as compared to the uninfected aged
Bov6 control, the increase in microgliosis in Bov6 challenged with C18985-HuMM was not as
pronounced (Figure 3e) and interestingly the microgliosis observed in Bov6 mice challenged
with C19414-Bov6 (Figure 3k) was not dissimilar to that seen in the uninfected aged Bov6
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control mice. Interestingly, the presence of PrP deposition correlated with astrogliosis and
microgliosis (Figure 3i) in Bov6 mice challenged with C18275-Bov6. However, despite
obvious astrogliosis, none or very little PrP deposition was observed in the hippocampus of
Bov6 mice challenged with C18985-HuMM (Figure 3f) or C19414-Bov6 (Figure 3l)
respectively.
Molecular Profile of PrPTSE in Bov6 mice inoculated with C18985, C18275 and C19414
Brains from Bov6 mice challenged with C18985-HuMM, C18275-Bov6 and C19414-Bov6 (2
brains per isolate) were examined for the presence of PrPTSE by western blot. Brains from
primary transmissions of Bov6 mice challenged with BASE, BSE-H or BSE-C were also
included for comparison. All brains selected for analysis were from mice which survived
challenge to ≥418 dpi and immunohistochemical analysis showed PrP deposition in all
selected animals. Following western blot analysis, proteinase-K resistant PrPTSE was
present in the brains of Bov6 mice challenged with C18275-Bov6, C19414-Bov6 (Fig 4a, b)
and C18985-HuMM (Fig 4b). Furthermore, these agents produced distinct PrPTSE profiles.
Similarly to primary transmissions of BASE into Bov6 mice, we found that the C18275-Bov6
PrPTSE unglycosylated isoform had a lower molecular weight than BSE-C and a distinct
PrPTSE glycoform pattern (Fig 4a). Likewise, we found that the C19414-Bov6 PrPTSE
unglycosylated isoform had a higher molecular weight than BSE-C or BASE and was similar
to the PrPTSE molecular profile from primary transmissions of BSE-H into Bov6 mice (Fig 4a).
These results are consistent with studies using transgenic overexpressing bovine PrP mice
(Beringue et al., 2007; Béringue et al., 2006; Buschmann et al., 2006; Capobianco et al.,
2007; Okada et al., 2010). Due to low levels of C18985-HuMM PrPTSE, it was difficult to fully
resolve the glycoform pattern, although we did observe a heavier diglycosylated band which
may be suggestive of a BSE-like PrPTSE profile (Fig 4b). However, further subpassage
experiments in Bov6 mice would be required to establish the presence of BSE-C or BASE.
Investigating presence of PrPTSE in the brains of BASE-challenged HuMM mice
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Previously we performed primary inoculations of BASE into HuMM mice, but did not detect
any signs of TSE neuropathology (Wilson et al., 2012b). Due to the observed transmission of
TSE from one of the HuMM mice selected for subpassage, and the possibility of subclinical
infection, brain tissue from 15 remaining HuMM mice that received the primary BASE
inoculum were assayed for accumulation of PrP using a rapid TSE diagnostic assay. The
IDEXX HerdChek* Bovine BSE Antigen Test Kit, which is an antigen capture enzyme
immunoassay (EIA), utilises a unique Seprion ligand capture technology (Microsens
Biotechnologies) to identify the presence of aggregated PrP in the brain, and has been used
successfully on HuTg tissue in previous experiments (Plinston et al., 2011). All assay
readouts for the 15 brain tissues examined were negative, indicating the lack of PrPTSE within
these brains.
Discussion
In this study, knock-in transgenic mice expressing human PrP were utilised to model the
potential risks posed to humans from exposure to atypical BSE agents. While the use of
transgenic mice does not accurately mimic infection of humans, they provide a model
system in which TSE infection in the presence of human PrP can be examined in
comparison with genetically identical control lines expressing either bovine PrP or wild-type
murine PrP. Data cannot be fully extrapolated to humans, but can provide some indication of
potential risk. Three gene targeted human PrP Tg (HuTg) mouse lines were utilised,
representing the genetic diversity in the human population due to the PrP codon 129-
methionine/valine polymorphism (HuMM, HuMV and HuVV). As these mice are produced by
gene replacement, they do not suffer from any adverse phenotypes observed in standard
transgenic lines and may more accurately model what happens in nature. Our previous
studies showed that following primary transmissions of BASE and BSE-H into HuTg mice,
we could not detect any pathological signs of TSE disease (PrP deposition and vacuolar
pathology) in any of the mice (Wilson et al., 2012b). In the current study we re-examined all
remaining tissues from the BASE inoculated HuMM Tg mice using a rapid TSE diagnostic
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assay (IDEXX) to investigate the presence of PrPTSE in the brains of HuTg mice challenged
with BASE. This assay was employed to ensure no PrP deposition was missed due to the
level of sectioning of the tissue block, and to assay for any forms of abnormal PrP which
may not have been identified by IHC. However, all IDEXX results on the 15 HuMM tissues
examined were negative. Although the identification of PrPTSE by IHC or IDEXX assay is an
indication of TSE disease, the only method by which TSE infectivity can be identified is
bioassay. To address the potential for subclinical infection and low level agent replication in
animals expressing human PrP, we performed subpassage experiments from HuTg brains
following primary challenge with BASE or BSE-H into HuTg mice and Bov6 Tg mice. Upon
subpassage we found evidence of subclinical infection in both a HuMM and a HuVV Tg
mouse challenged with BASE, demonstrated by the pathological signs of TSE disease (PrP
deposition and/or vacuolar pathology) observed in 5 Bov6 Tg mice (4 in MM challenge, 1 in
VV challenge). However no TSE disease transmission was observed in the HuTg mice
following subpassage.
The existence of subclinical TSE infection in humans and animals has been documented
previously (Hill & Collinge, 2003a, b; Race et al., 2001; Race et al., 2002). One such study
showed that the cross-species passage of hamster 263K scrapie into wild-type mice,
appeared not to transmit, with the mice appearing clinically normal with no PrPTSE detectable.
However upon subpassage of brain tissue from a PrPTSE negative, clinically normal mouse to
wild-type mice, PrPTSE was detectable in the brain tissue (Race et al., 2002). Indeed, while
less than 200 people have developed clinical vCJD, it is likely that millions were exposed by
consumption of BSE-contaminated beef and it is possible a number of these individuals may
act as asymptomatic carriers of TSE infectivity. In the present study we found evidence of
subclinical TSE infection in two HuTg mice challenged with BASE (C18985-HuMM, C19409-
HuVV; both negative for neuropathology and clinical signs of TSE disease). In these studies,
our experiments may suggest that during primary passage low level replication of BASE may
occur in the HuTg mice, however once within a susceptible host (Bov6) it is able to replicate
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efficiently. Indeed, the inefficient replication of the infectious TSE agent may explain why we
were unable to detect any signs of TSE pathology in our HuTg mice challenged with BASE.
Furthermore, our current experimental methods of detection may not be sensitive enough to
detect low levels of PrPTSE. However, it is also possible that PrPTSE is not the infectious agent
and that we are simply not searching for the correct markers for TSE infection in these
animals. Indeed, the dissociation between PrPTSE and TSE infectivity has been shown in
both natural and experimental cases (Andreoletti et al., 2011; Barron et al., 2007; Race et
al., 2002). Nevertheless, as it is known that PrP expression in the host is necessary for the
development of neurodegeneration, PrPTSE remains an important diagnostic marker of TSE
infection.
It is theoretically possible that residual inocula might be the cause of the TSE pathology
observed in our Bov6 mice challenged with C18985-HuMM or C19409-HuVV, however it
would seem unlikely in these studies. Indeed, if this were the case, the expectation would be
to see more cases of TSE pathology in all groups of inoculated mice. Furthermore, previous
studies have shown the rapid clearance of prions from the brain following intracerebral
inoculation (Safar et al., 2005a; Safar et al., 2005b), which would not agree with the retention
of significant levels of BASE 600 days post inoculation. In previous studies we observed that
following challenge of vCJD into Bov6 mice or HuTg mice, more clinical cases were
observed in Bov6 mice (personal communication, data unpublished). These findings would
support our data showing the lack of TSE pathology in the HuTg mice subpassaged with
BASE- or BSE-H-challenged HuTg mice.
Transmission of BASE to overexpressing human PrP Tg mice (homozygous for methionine
at codon 129) has been demonstrated following primary passage (Beringue et al., 2008;
Kong et al., 2008), however in the present study we were particularly interested in the
discovery of subclinical infection in a HuVV mouse challenged with BASE, which has not
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previously been documented. Indeed, very few studies have been published investigating
the transmission of TSEs into HuVV mice. Although all clinical cases of vCJD have been in
individuals who are methionine homozygous at codon 129, studies in HuTg mice have
shown that all three genotypes (MM, MV and VV) may be susceptible with differing
incubation times (Bishop et al., 2006). Indeed transmission experiments have shown that
only 1/16 HuVV challenged with vCJD displayed TSE pathology as compared to 11/17
HuMM Tg mice. However, although classical BSE and BASE are both cattle TSE strains, we
cannot assume that the susceptibility of different human PrP genotypes would be the same
between HuTg challenged with classical BSE or BASE. Indeed, other studies have
suggested that the phenotypic features and PrPTSE characteristics of BASE bear
resemblance to a subtype of sCJD (sCJDMV2) (Casalone et al., 2004). sCJDMV2 has been
found to affect individual who are methionine/valine heterozygous at codon 129 of the PrP
gene (Parchi et al., 1999), and this finding has raised the possibility that sCJDMV2 may
actually not be a truly sporadic disease but may be acquired from the consumption of BASE-
contaminated meat (Brown et al., 2006; Casalone et al., 2004). Although our previous
studies showed no TSE pathology on primary passage of BASE (Wilson et al., 2012b) or
classical cattle BSE (Bishop et al., 2006) to the HuTg mice, others have shown efficient
transmission of BASE in mice overexpressing 129-Met human PrP, with higher levels of
transmissibility than observed with classical cattle BSE. We have yet to perform subpassage
from HuTg mice that received BSE-C to determine whether low level agent replication also
occurs in these mice. However these data combined suggest that BASE may indeed
transmit more efficiently to HuTg mice than BSE-C (Beringue et al., 2008).
The potential existence of subclinical TSE infection in humans has several significant
implications for public health, especially regarding the possibility of iatrogenic transmission of
TSE disease from individuals who seem apparently healthy. Therefore, continued efforts
must be made to ensure public health. However, while our findings suggest that low levels
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replication of BASE can occur in hosts expressing human PrP, the efficiency of any potential
human to human transmission appears to be low.
Materials and Methods
Preparation of Inocula
Brain tissue from the frontal and parietal cortices of a 15 year old Piemontese cow (fallen
stock) infected with BASE was supplied by Istituto Zooprofilattico Sperimentale del
Piemonte, Liguria e Valle D'Aosta, Torino, Italy. The brainstem of a 15 year old BSE-H
infected Prim Holstein cow (identified from a rendering plant) was supplied by the French
TSE Reference Laboratory (Agence Nationale de Sécurité Sanitaire (Anses-Lyon), France).
Primary transmissions are described in Wilson et al., 2012a. Seven BASE challenged mouse
brains and four BSE-H-challenged mouse brains were selected for subpassage (Table 1). All
inocula were prepared from brain tissue in sterile saline at a concentration of 5% (wt/vol). In
order to prevent any possibility of cross-contamination of samples, all tissues were
homogenised in clean previously unused dounce glass homogenisers that were discarded
after single use. Samples were handled individually and the safety cabinet decontaminated
between each inoculum prep. Positive control bov6 tissues were prepared on a different day
from the HuMM and HuVV tissues (See Table 1). Full pathological characterisation of source
tissues (BASE, C.Casalone; BSE-H, T.Baron) was previously performed to confirm disease
status.
Subpassage 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). Tissues selected for
subpassage covered a range of genotypes, ages and clinical status and included 9 HuTg
mice inoculated with either BASE (6) or BSE-H (3) and 2 control Bov6 mice inoculated with
BASE or BSE-H (Table 1). Each of the 11 brains selected for subpassage was used to
prepare a 5% homogenate for use as inocula. Mice were injected by intracerebral inoculation
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(i.c.) into the right cerebral hemisphere under halothane anaesthesia. Each mouse received
0·02 mL of 5% brain homogenate and each homogenate was inoculated into a group of 12
HuTg mice of the same genotype as the inoculum, and 12 Bov6 mice as controls.
Homogenates from the 2 Bov6 control brains were each inoculated into groups of 12 HuMM
and 12 Bov6 mice. In order to prevent any possible cross-contamination, inoculations were
performed from one or two inocula only per day, with the safety cabinet cleaned and
decontaminated between each set of inoculations. Of note, the HuMM and HuVV samples in
Table 1 shown to transmit disease were inoculated on different days from the two Bov6
control tissues (C18275 and C19414; Table 1). From 100 days mice were scored each week
for signs of disease and were killed by cervical dislocation at a pre-defined clinical endpoint,
or due to welfare reasons (Dickinson et al., 1968). Due to the low number of culls for clinical
TSE disease, survival times only were calculated for mice showing both PrP deposition and
vacuolar pathology. Brains were recovered at post mortem and one half of 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). 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 means (SEM) against scoring areas to produce a lesion
profile, as previously described (Bruce et al., 1997; Fraser & Dickinson, 1967). While the
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production of lesion profiles in pre-clinical mice is not standard practice, all mice which
scored positively for vacuolation pathology were included whether or not clinical signs were
present, due to the lack of clinical signs observed in any of the mice.
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 peroxidise-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 pretreated using hydrated 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.
Identification of PrPTSE by immunoblotting
Frozen brain samples from Bov6 mice challenged with C18985-HuMM, C18275-Bov6 and
C19414-Bov6 (and also brains from primary inoculations of Bov6 mice challenged with
BASE, BSE-H and BSE-C) were homogenised at 10% 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
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clarified at 11,000g for 15 minutes. Brain homogenate supernatant from the transgenic mice
and controls was incubated with or without 20 g proteinase K ml-1 for 1 hour at 37C. The
products were denatured and separated on a 12% 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 0.6 and 3mg). PrP was
identified with monoclonal antibody 6H4 (0.1 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.
Immunoassay for detection of PrPTSE in the brain
The IDEXX HerdChek* Bovine Spongiform Encephalopathy (BSE) Antigen Test Kit is an
antigen capture enzyme immunoassay (EIA) for detection PrPTSE in post-mortem tissues.
Previously we performed primary inoculations of BASE into HuMM mice (Wilson et al.,
2012b). Brains derived from these mice were homogenised in sterile saline in a Rybolyser
(Hybaid, Middlesex, UK) to achieve a 30% homogenate. The protocol was performed
following manufacturer’s instructions.
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
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The authors would like to acknowledge J. Manson for the HuTg and Bov6 mice; S.
Cumming, S. Carpenter, R. Greenan and K. Hogan for experimental setup, care and scoring
of the animals; A. Boyle, S. Mack, D. Drummond and G. McGregor for histology processing
and scoring. These studies were funded by contract M03054 from the Food Standards
Agency (FSA) UK. The Roslin Institute receives Institute Strategic Programme Grant (ISP)
funding from the Biotechnology and Biological Sciences Research Council (BBSRC), UK.
Table 1. Tissues used for subpassage into HuTg and Bov6 Tg mice.
TSE Agent
Reference No.
Genotypeof mice
ClinicalScore* Pathology Survival
(days)1 BASE C17840 HuVV + - 395
2 BASE C18220 HuMM + - 539
3 BASE C18222 HuMM + - 527
4 BASE C18275 Bov6 + + 541
5 BASE C18985 HuMM - - 652
6 BASE C19170 HuMM - - 687
7 BASE C19409 HuVV - - 749
8 H-type C18252 HuMV + - 421
9 H-type C19414 Bov6 - + 623
10 H-type C19697 HuMM - - 708
11 H-type C19700 HuVV - - 708
*Animals in all primary passage experiments were scored blind. Animals with clinical TSE score but no confirmed TSE neuropathology were included to examine whether the phenotype was transmissible.
Reference numbers highlighted in bold represent the tissue from which disease transmission was observed (see Table 2)
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Table 2. Subpassage transmissions into HuTg and Bov6 Tg mice
InoculationSource
MouseLine
Survival* (Pathol
neg)
Survival†
(Pathol Pos)
ClinicalSigns
VacuolarPathology
PrPdeposition
C17840 Bov6HuVV
562±12464±33
--
0/120/12
0/120/12
0/120/12
C18220 Bov6HuMM
538±20542±14
--
0/120/12
0/120/12
0/120/12
C18222 Bov6HuMM
512±36472±42
--
0/110/12
0/110/12
0/110/12
C18275 Bov6HuMM
329554±21
588±8-
0/110/10
10/110/10
10/110/10
C18985 Bov6HuMM
526±35475±43
483±42-
0/100/10
3/100/10
4/100/10
C19170 Bov6HuMM
526±36498±31
--
0/110/12
0/110/12
0/110/12
C19409 Bov6HuVV
525±37507±28
589-
0/110/12
1/110/12
1/110/12
C18252 Bov6HuMV
502±27520±35
--
0/100/12
0/100/12
0/100/12
C19414 Bov6HuMM
-530±43
524±27-
0/110/12
10/110/12
11/110/12
C19697 Bov6HuMM
549±26451±40
--
0/110/12
0/110/12
0/110/12
C19700 Bov6HuVV
538±29501±25
--
1/110/12
0/110/12
0/110/12
* Measured as days ± SEM and calculated from mice with no signs of TSE neuropathology† Measured as days ± SEM and calculated from mice showing vacuolar pathology and/or PrP deposition.
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Figure 1. Pattern of vacuolation observed in brains of Bov6 mice derived from 1st and 2nd
passage of BASE or H-type BSE. 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 (11, cerebellar white matter; 12, midbrain white matter; 13, 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 PrPTSE deposition in the thalamus (a,c,e,g) and midbrain
(b,d,f,h) regions of brains from Bov6 mice subpassaged with C18985-HuMM (a,b), C19409-
HuVV (c,d), C18275-Bov6 (e,f) and C19414-Bov6 (g,h). Images obtained after staining with
anti-PrP antibody 6H4 and counterstained with hematoxylin. Magnification is as shown.
Figure 3. Comparative analysis of the hippocampus of Bov6 mice challenged with C18985-
HuMM, C18275-Bov6 and C19414-Bov6. Micro- and astrogliosis is present in all mice,
detected by anti-GFAP (d,g,i) and anti-Iba1 respectively (e,h,k). PrP deposition is visible by
anti-6H4 antibody (i,l). Uninfected aged Bov6 mice showing mild gliosis (a,b) and no PrP
deposition (c) were used as controls. Magnification 10x.
Figure 4. Comparative western blot analysis of the proteinase K-resistant fragment (PrPTSE)
of the prion protein in Bov6 mice challenged with C18275-Bov6 (lanes 3 and 4) and C19414-
Bov6 (lanes 7 and 8). Primary transmissions of BSE-C (lanes 1 and 2), BASE (lanes 5 and
6) and BSE-H (lanes 9 and 10) into Bov6 mice have also been included for comparison (a).
Western blot analysis of the proteinase K-resistant fragment (PrPTSE) of the prion protein in
Bov6 mice challenged with BSE-C (lane 1), C18275-Bov6 (lane 2), C18985-HuMM (lane 3)
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and C19414-Bov6 (lane 4) (b).All lanes show PK-treated brain homogenate. Anti-PrP mAb
6H4 was used to detect bands.
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