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Knock-in Transgenic Mice
14
Running title: Unusual TSE properties in goat
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Rona Wilson1, Declan King1, Nora Hunter1, Wilfred Goldmann1 and Rona M Barron1*
Characterisation of an Unusual TSE in a Goat by Transmission in
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1Neurobiology
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Roslin, Midlothian, EH25 9RG, UK
Division, The Roslin Institute and R(D)SVS, University of Edinburgh,
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*Corresponding
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Neurobiology Division
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The Roslin Institute and R(D)SVS, University of Edinburgh
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Roslin
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Midlothian, EH25 9PS
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UK
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Tel: 0131 527 4200
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Fax: 0131 440 0434
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rona.barron@roslin.ed.ac.uk
Author
1
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Contents Category: TSE Agents
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Summary Word Count: 248
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Main Text Word Count: 5,068
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Transgenic Mice
Number of Tables and Figures: 7
Characterisation of an Unusual TSE in a Goat by Transmission in Knock-in
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Summary
41
Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disorder of
42
cattle, and its transmission to humans through contaminated food is thought to be the
43
cause of the variant form of Creutzfeldt-Jakob disease (vCJD). BSE is believed to have
44
spread from the recycling in cattle of ruminant tissue in meat and bone meal (MBM),
45
however during this time sheep and goats were also exposed to BSE-contaminated
46
MBM. Both sheep and goats are experimentally susceptible to BSE, and while there
47
have been no reported natural BSE cases in sheep, two goat BSE field cases have
48
been documented. While cases of BSE are rare in small ruminants, the existence of
49
scrapie in both sheep and goats is well established. In the UK, during 2006-2007, a
50
serious outbreak of clinical scrapie was detected in a large dairy goat herd.
51
Subsequently, 200 goats were selected for post-mortem examinations, one of which
52
showed biochemical and immunohistochemical features of the disease associated prion
53
protein (PrPTSE) which differed from all other infected goats. In the present study we
54
investigated this unusual case by performing bioassays into a panel of mouse lines.
55
Following characterisation, we found that strain properties such as the ability to transmit
2
56
to different mouse lines, lesion profile pattern, degree of PrP deposition in the brain and
57
biochemical features of this unusual goat case were neither consistent with goat BSE
58
nor with a goat scrapie herdmate control. However our results suggest this unusual
59
case has BSE-like properties and highlights the need for continued surveillance.
60
61
Introduction
62
The transmissible spongiform encephalopthies (TSEs) are a group of fatal infectious
63
neurodegenerative diseases that include scrapie in sheep and goats, bovine spongiform
64
encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in cervids, and
65
Creutzfeldt-Jakob disease (CJD) in humans. TSEs are characterised by the
66
accumulation in the brain of PrPTSE, which is a relatively protease resistant
67
conformational variant of the normal host encoded cellular prion protein (PrP C). Due to
68
the infectious nature of TSEs, these diseases can be transmitted via a number of
69
different routes and while TSEs tend to transmit more readily within species, they are
70
able to transmit between different species, although this is dependent on both the TSE
71
agent and the host.
72
73
The existence of naturally occurring classical scrapie in sheep and goats has been
74
known for more than 200 years and is not considered a public health risk according to
75
the “Opinion of the Scientific Panel on Biological Hazards on the request from the
76
European Commission on classification of atypical Transmissible Spongiform
77
Encephalopathy (TSE) cases in Small Ruminants” (Budka H, 2005). However, the
78
transmission of BSE to humans through contaminated food is thought to be the cause of
3
79
the variant form of Creutzfeldt-Jakob disease (vCJD) (Bruce et al., 1997; Hill et al.,
80
1997). This relationship reveals a potential risk of transmission of other ruminant TSEs
81
to humans. During the BSE epidemic both sheep and goats were exposed to BSE-
82
contaminated meat and bone meal (MBM) (Baylis et al., 2002). Studies have shown that
83
sheep and goats are experimentally susceptible to BSE (Baylis et al., 2002; Bellworthy
84
et al., 2008; Foster et al., 1993; Houston et al., 2003; Stack et al., 2009a). While no
85
natural cases of BSE have thus far been detected in sheep, two BSE cases in goats
86
have been confirmed, one in France (Eloit et al., 2005) and the other in the UK
87
(Spiropoulos et al., 2011). Furthermore, studies have shown the transmission of goat
88
and/or sheep BSE into transgenic mice expressing human PrP and have found
89
transmission of these isolates more efficient than cattle BSE (Padilla et al., 2011;
90
Plinston et al., 2011).
91
92
BSE was first reported in 1987 (Wells et al., 1987) and although it has been extensively
93
characterised, the true aetiology of infection remains unknown. Due to classical strain
94
typing in mice (incubation time, lesion profiles, patterns of PrP staining in the brain)
95
(Bruce, 1996; Bruce et al., 1997) and biochemical features of the proteinase K (PK)
96
resistant PrP in natural and experimental BSE (Collinge et al., 1996; Kuczius &
97
Groschup, 1999; Kuczius et al., 1998), TSE disease in cattle was originally believed to
98
be caused by a single prion strain, known as classical BSE (BSE-C). However, through
99
increased TSE surveillance, two atypical BSE agents have also recently (since 2004)
100
been reported (Biacabe et al., 2004; Casalone et al., 2004; Jacobs et al., 2007; Stack et
101
al., 2009b) and are identified as H-type BSE and bovine amyloidotic spongiform
102
encephalopathy (BASE, also named L-type BSE). These atypical bovine TSEs can be
4
103
distinguished by the electrophoretic positions of their PrPTSE isoforms (Biacabe et al.,
104
2004; Buschmann et al., 2006; Casalone et al., 2004; Jacobs et al., 2007) and different
105
glycoform pattern.
106
107
Due to concerns that BSE may also infect sheep and goats, active surveillance of small
108
ruminants has been considerably improved. Many countries now apply screening,
109
confirmatory and discriminatory tests based on the post mortem detection of PrPTSE in
110
the central nervous system. Following an outbreak of scrapie in a large goat herd in
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2006-2007, the whole herd was culled and 200 goats were examined for PrPTSE
112
(Gonzalez et al., 2009). One goat showed biochemical and immunohistochemical (IHC)
113
features of PrPTSE different from all other 71 infected goats in the herd. This particular
114
case was an eight-year old Anglo-Nubian goat, homozygous for the caprine wildtype
115
allele isoleucine at codon 142 (Gonzalez et al., 2009). Notably, the existence of atypical
116
scrapie was ruled out (Gonzalez et al., 2009). Initial analysis of this goat case identified
117
some similarities to BSE, such as a reduced P4 antibody signal following western
118
blotting and lack of 12B2 antibody staining of intracellular PrPTSE deposits, however
119
further analysis with additional antibodies also showed distinct differences with
120
experimental BSE in goats (Gonzalez et al., 2009). In the present study we aimed to
121
characterise this unusual goat TSE (ref V3698) by challenging knock-in (produced by
122
gene targeting) bovine (Bov6) or human PrP Tg (HuTg) mice, as well as 129/Ola wild-
123
type mice. One experimental goat BSE and a goat scrapie herdmate (goat scrapie HM)
124
were included as controls. Following neuropathological and biochemical analysis we
125
found that although V3698 presented some BSE-like properties, it differed from both
126
experimental goat BSE and goat scrapie HM.
5
127
128
Results
129
Transmissibility of V3698 differs from experimental goat BSE and goat scrapie
130
Using a panel of mouse lines (Bov6, HuTg and 129/Ola), animals were challenged
131
intracerebrally with isolate V3698, experimental goat BSE or goat herdmate V3981 with
132
confirmed classical scrapie (Gonzalez et al., 2009) (from here will be referred to as goat
133
scrapie HM). Following V3698 inoculations, a TSE was induced in 9/24 Bov6 and 1/22
134
129/Ola mice and was defined by the presence of PrP deposition (using
135
immunohistochemistry) in the brain and/or vacuolar pathology (Table 1). In contrast, the
136
majority of Bov6 mice (22/23) and almost 60% of 129/Ola mice (13/22) developed a
137
TSE following challenge with experimental goat BSE. Following goat scrapie HM
138
challenge, the majority of 129/Ola mice (20/24) were positive for TSE disease, however
139
interestingly 9/23 Bov6 mice were positive for TSE pathology (PrP deposition and/or
140
vacuolar pathology). While the majority of TSE positive mice challenged with either
141
experimental goat BSE or goat scrapie HM showed clinical signs of disease, this was
142
not the case for V3698 (Table 1). Furthermore, TSE neuropathology occurred around
143
150 days later in mice challenged with V3698, than in either Bov6 mice challenged with
144
experimental goat BSE or 129/Ola challenged with goat scrapie HM. Thus, our results
145
demonstrated that V3698 attack rates were not consistent with either experimental goat
146
BSE or goat scrapie HM. Full strain characteristics of V3698 are shown on
147
supplementary table 1.
148
149
Comparisons of Lesion Profiles from previous TSE agents transmissible to Bov6
150
mice
6
151
The lesion profiles, which define areas of vacuolation and their degree of severity in the
152
brain, were determined for Bov6 mice inoculated with V3698, experimental goat BSE
153
and goat scrapie HM (Fig. 1a). Although, the production of lesion profiles in pre-clinical
154
mice is not standard practice, all Bov6 mice challenged with V3698 or goat scrapie HM
155
which scored positively for vacuolation pathology were included to give an indication of
156
vacuolation profile in comparison to experimental goat BSE. Lesion profiles of V3698
157
and experimental goat BSE were clearly distinct, indicating different TSE isolates (Fig.
158
1a). A higher degree of vacuolar pathology was observed in several brain regions
159
(hippocampus, retrospinal cortex, cingulate and motor cortex and cerebellar white
160
matter) in Bov6 mice challenged with V3698. However, lesion profiles in Bov6 mice
161
challenged with V3698 or goat scrapie HM were strikingly similar. To further investigate
162
the vacuolar pattern of V3698, we also compared lesion profiles produced from previous
163
experiments in which cattle isolates of BASE, H-type BSE and BSE-C had been
164
transmitted to the Bov6 mice (Wilson et al., 2012a) (Fig. 1b). In these comparisons,
165
V3698 was found to display a similar pattern of vacuolar targeting to that observed in
166
Bov6 mice challenged with BASE, and was distinct from BSE-C and H-type BSE.
167
168
169
170
171
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Bov6 Mice Challenged with V3698 or goat scrapie show low levels of PrP
deposition
Pattern of PrP deposition
173
Widespread extensive PrP deposition, detected using immunohistochemistry, was
174
observed in Bov6 mice challenged with experimental goat BSE (Fig. 2a-b). In contrast,
175
Bov6 mice challenged with V3698 showed very little PrP deposition which was observed
176
in the thalamus (Fig. 2c-d), midbrain and medulla. While the majority of 129/Ola
7
177
challenged with goat scrapie HM showed heavy PrP deposition (Fig. 2e-f), only seven
178
Bov6 mice showed PrP deposition. However, similarly to V3698, we observed very little
179
PrP deposition in the Bov6 mice challenged with goat scrapie HM, which was also
180
confined to the thalamus (Fig. 2g-h), midbrain and medulla. Due to the low levels of PrP
181
deposition in Bov6 mice challenged with V3698 or goat scrapie HM, we could not
182
conclude similarities or differences to PrP distribution pattern compared to experimental
183
goat BSE or goat scrapie HM in 129/Ola mice. Using thioflavin-S fluorescence we were
184
unable to detect PrP amyloid plaques in Bov6 mice challenged with experimental goat
185
BSE, Bov6 mice challenged with V3698 and 129/Ola challenged with goat scrapie HM
186
(supplementary Fig. 1).
187
188
Glial Neuropathology
189
To control for age-related effects, gliosis was first assessed in brain tissue obtained
190
from a previous aging study of Bov6 mice (Wilson et al., 2012a). Mild gliosis was
191
present throughout the brains of aged Bov6 control mice (Fig. 3b-c). In contrast to
192
gliosis due to aging, an increase in the appearance of astrogliosis and microgliosis was
193
clearly evident throughout the brains of Bov6 mice inoculated with V3698, experimental
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goat BSE and goat scrapie HM and 129/Ola challenged with goat scrapie HM (all of
195
which showed signs of TSE pathology), and was most evident in the thalamus (Fig. 3e-f,
196
3h-i, 3k-l and 3n-o). While areas of PrP deposition in the thalamus clearly correlated
197
with gliosis in Bov6 mice challenged with experimental goat BSE and 129/Ola
198
challenged with goat scrapie HM (Fig. 3g, 3m), Bov6 mice challenged with V3698 or
199
goat scrapie HM showed gliosis despite the presence of very little visible PrP deposition
200
(Fig. 3d, 3j).
8
201
202
Molecular Profile of V3698 Differs from goat scrapie HM and experimental goat
203
BSE
204
Brains from Bov6 mice challenged with V3698, experimental goat BSE and goat scrapie
205
HM and also 129/Ola mice challenged with goat scrapie HM (2 or 3 brains per isolate)
206
were examined for the presence of PrPTSE by western blot (Fig. 4a-b). Brains from Bov6
207
mice challenged with BASE and BSE-C were also included as comparisons. All brains
208
selected
209
immunohistochemistry) in the brain, except for Bov6 mice challenged with V3698 and
210
goat scrapie HM which showed very little PrPTSE deposition. Due to the low levels of PrP
211
in these brains, precipitation of PrP using sodium phosphotungstic acid (NaPTA) was
212
performed to concentrate all samples. Following western blot analysis using anti-PrP Ab
213
6H4, distinct PrPTSE profiles were observed between Bov6 mice challenged with V3698
214
or experimental goat BSE and 129/Ola mice challenged with goat scrapie HM (Fig. 4a).
215
However, similar PrPTSE profiles were observed between Bov6 brains challenged with
216
V3698 or goat scrapie HM (Fig. 4c). The experimental goat BSE PrPTSE had a similar
217
glycopattern to BSE-C with the characteristic heavier diglycosylated band. Indeed,
218
previous studies have shown that glycoform pattern can be used to distinguish between
219
different TSE strains (Baron et al., 2007; Beringue et al., 2007; Capobianco et al., 2007;
220
Somerville, 1997; Somerville et al., 2005), however a number of phenotypic markers are
221
required to reliably discriminate between strains. Ratios of all three glycoforms (un-,
222
mono- and diglycosylated), calculated by a Kodak 440CF digital imager, were similar
223
between Bov6 mice brains challenged with BSE-C PrPTSE and experimental goat BSE
224
PrPTSE. These differed from V3698, which showed a PrP glycoform ratio similar to
for
analysis
showed
moderate
9
to
intense
PrPTSE
deposition
(by
225
BASE in Bov6 mice. All were distinct from 129/Ola mouse brains challenged with goat
226
scrapie HM (Fig. 5). Western blot analysis was also performed using anti-PrP Ab 12B2,
227
which can be used to discriminate between BSE and classical scrapie (Jacobs et al.,
228
2011; Jeffrey et al., 2006; Langeveld et al., 2006). A strong signal was detected only
229
from the 129/Ola mouse brains challenged with goat scrapie HM (Fig. 4b & d).
230
231
232
233
Transmission of Experimental Goat BSE into HuTg Mice
Previous studies have shown that experimental goat BSE and sheep BSE are
234
transmissible to Tg mice expressing codon 129 methionine human PrP (Padilla et al.,
235
2011; Plinston et al., 2011) and transmit to these mice more efficiently than cattle BSE
236
(Padilla et al., 2011). In the present study we also found transmission of experimental
237
goat
238
Neuropathological analysis of these mice showed PrP deposition was plaque-like and
239
restricted to the thalamus (Fig. 6a-b) and midbrain. Despite the presence of PrP
240
deposition, no HuTg mice were positive for vacuolation pathology, except for one which
241
had limited vacuolation in the cerebral and midbrain white matter. Due to the lack of
242
overexpression of PrP in these mice, they may more closely represent what happens in
243
nature and thus this finding is of particular concern. No transmission of either V3698 or
244
goat scrapie HM was detected in the HuTg mice, which further suggests that V3698 is
245
distinct from goat BSE (Table1).
246
247
248
249
BSE
into
HuTg
mice
(10/24
HuMM
mice
showed
PrP
deposition).
Discussion
In this study, we examined the transmission characteristics, neuropathology and
250
biochemical characteristics of an unusual TSE present in a goat, designated V3698.
251
Initial investigation of this case revealed unusual biochemical and immunohistochemical
10
252
features of PrPTSE compared to other scrapie infected goats in the herd (Gonzalez et al.,
253
2009), which presented both similarities and differences to both goat BSE and
254
previously studied isolates of goat scrapie. In order to help determine the nature of
255
V3698 and improve our understanding of the diversity of goat TSEs, we performed
256
transmission bioassays into a panel of mice. Along with V3698 we therefore also
257
inoculated an experimental goat BSE isolate and goat scrapie HM control into gene-
258
targeted bovine or human PrP Tg mice, as well as 129/Ola wildtype mice. As these
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transgenic mice are produced by gene replacement, they do not suffer from any
260
adverse phenotypes which are associated with overexpression or ectopic expression of
261
the transgene in standard transgenic lines, and express PrPC at the same level as wild
262
type mice (Manson et al., 1999; Moore et al., 1998). Furthermore, previous studies have
263
shown efficient transmission of TSE agents into gene targeted transgenic PrP mouse
264
lines, demonstrating that these mice do live long enough to show signs of infection,
265
supporting the use of targeted mouse models to analyse TSE disease transmission
266
(Bishop et al., 2010; Bishop et al., 2006; Plinston et al., 2011). While caprine transgenic
267
mouse lines may have been an alternative model system, as yet there are no available
268
knock-in caprine transgenic mice. Furthermore, in this study, the use of bovine and
269
human PrP knock-in transgenic lines allows direct comparison with archive data
270
produced following inoculation of these lines with a wide range of TSE agents, including
271
vCJD, BSE-C (Bishop et al., 2006), BASE and H-type BSE (Wilson et al., 2012a).
272
273
The data presented here showed that transmission, neuropathology and biochemical
274
properties of V3698 were not consistent with either experimental goat BSE or goat
275
scrapie HM. Lesion profiles between V3698 and experimental goat BSE were clearly
11
276
distinct, as were the amount of PrP deposition present in the brain, and ratios of PrPTSE
277
glycoforms. Furthermore, lack of binding to an anti-PrP antibody (12B2) suggested
278
V3698 was not a classical scrapie strain. This was consistent with the original study of
279
V3698 in which PrPTSE in brain tissue, but not retropharyngeal lymph node, was shown
280
to lack 12B2 binding (Gonzalez et al., 2009). Interestingly, although goat scrapie HM
281
inoculated 129/Ola mice produce a strong PrPTSE signal on immunoblot following
282
probing with 12B2, indicating classical scrapie, the PrPTSE in Bov6 mice inoculated with
283
goat scrapie HM was negative for 12B2 binding. We have previously observed a strong
284
12B2 signal on immunoblots from Bov6 mice challenged with H-type BSE (data not
285
shown), proving Bov6 PrPTSE can bind 12B2. These data are also consistent with
286
studies showing that while labelling for classical BSE or BASE with 12B2 is minimal or
287
absent, H-type BSE is strongly labelled with this antibody (Baron et al., 2011; Dudas et
288
al., 2010; Jacobs et al., 2011). Unlike goat scrapie HM or experimental goat BSE,
289
V3698 failed to transmit efficiently to 129/Ola mice. While evidence of scrapie
290
transmission to cattle has been described by others (Cutlip et al., 1994; Konold et al.,
291
2006), our previous studies have shown a lack of transmission of either classical or
292
atypical scrapie into Bov6 mice, (Plinston et al., 2011; Wilson et al., 2012b). Importantly,
293
all successful disease transmissions into Bov6 mice in our lab, as yet have been from
294
BSE-like strains (Bishop et al., 2006; Plinston et al., 2011; Wilson et al., 2012a). Hence,
295
the observed transmissibility of the goat scrapie HM to Bov6 mice and lack of 12B2
296
binding is perplexing. As we have not previously challenged our Bov6 mice with other
297
isolates of goat scrapie, we cannot rule out the possibility that scrapie in goats is
298
transmissible to this mouse line. However the results presented here may also suggest
299
that the same agent has been isolated from both goat field samples in the Bov6 mice
12
300
based on to the lack of 12B2 reactivity, similarities in both lesion and molecular profile,
301
the lack of PrP deposition, and the fact that both V3698 and the goat scrapie HM control
302
came from the same herd. Indeed, it is widely accepted that animals carrying TSE
303
infection in the field may be infected with more than one “strain”. Laboratory TSE strains
304
represent isolates which have been selected from field cases following serial
305
transmission in mice and probably represent selection from the field case of the isolate
306
that replicates most efficiently in that mouse genotype. The similarities of V3698 and the
307
goat scrapie HM inoculum in Bov6 mice may suggest that the same agent was isolated
308
from both goats. However the high transmission of goat scrapie HM, but not V3698, to
309
129/Ola mice indicates a difference either in the TSE agent, or the presence of more
310
than one TSE agent that may have been differentially selected in the different mouse
311
genotypes. Further transmission studies would need to be performed to determine
312
whether this was the case.
313
314
Interestingly, we found V3698 showed a number of similarities to earlier transmissions
315
investigating the susceptibility of Bov6 mice to BASE (Wilson et al., 2012a). As
316
previously discussed, V3698 failed to transmit efficiently into 129/Ola control mice,
317
which was similar to prior studies with BASE (Wilson et al., 2012a). In contrast, as was
318
the case for experimental goat BSE in these studies, others have shown that classical
319
cattle BSE transmits very efficiently to a variety of inbred mouse strains (Capobianco et
320
al., 2007; Wilson et al., 2012a). Despite signs of neuropathology, we did not observe
321
clinical signs in Bov6 mice challenged with V3698, which was also the case in Bov6
322
mice challenged with BASE (Wilson et al., 2012a). Indeed, several studies have shown
323
that cattle infected with atypical BSE were found to be healthy at slaughter (Biacabe et
13
324
al., 2004; Casalone et al., 2004). We also observed a similarity between the vacuolation
325
pattern of V3698 and BASE in the Bov6 mice. However, it is also important to highlight
326
that we observed properties of V3698 that were not consistent with BASE, such as the
327
mobility of the unglycosylated PrP fragment, lack of prominent, widespread PrP
328
deposition and amyloid plaques, which have been shown in previous studies in Bov6
329
mice (Wilson et al., 2012a). As BASE has not been previously transmitted into goats,
330
the strain characteristics of this TSE in goats is unknown. Furthermore, the existence of
331
BASE could be difficult to determine if mixed TSE infections were present in a single
332
host. Therefore, further studies would be necessary to identify whether V3698 was
333
BASE in a goat.
334
335
Although incubation periods or survival times are often used to distinguish between
336
TSEs, they may not be as reliable during first passage, especially when a TSE is only
337
diagnosed in a small number of animals due to low titre and/or limited transmission of
338
specific TSE strains in certain mouse lines. Therefore, while we still observed
339
transmission in 37% of Bov6 mice challenged with V3698, in this study we included
340
incubation and survival times as an observation but did not rely on them to discriminate
341
between TSE isolates.
342
343
As part of this study we also investigated the susceptibility of gene-targeted human PrP
344
Tg mice to V3698, experimental goat BSE and goat scrapie HM. Approximately 40% of
345
brains from HuMM Tg mice challenged with experimental goat BSE showed PrP
346
deposition. Our results support previous studies showing that goat and sheep BSE are
347
more efficient at transmitting to human Tg mice than cattle BSE (Padilla et al., 2011).
14
348
These findings are of concern with regards to the risk to human health and whereas in
349
cattle BSE infectivity is mainly confined in the central nervous system, in small
350
ruminants BSE has been detected in peripheral tissues and can be transmitted
351
horizontally (Bellworthy et al., 2005; Kao et al., 2002). Thus, the data presented here
352
highlight the requirement to prevent BSE from becoming established in small ruminants.
353
354
Overall, our results revealed that while showing some BSE-like properties on
355
transmission in a panel of transgenic mice, V3698 is neither goat BSE nor a classical
356
goat scrapie, and may rather be a BSE-like TSE or an unusual form of scrapie, however
357
further experiments are required to fully characterise this case. While atypical BSE has
358
been reported in cattle since 2004 (Biacabe et al., 2004; Casalone et al., 2004; Jacobs
359
et al., 2007; Stack et al., 2009b), no cases have been documented in small ruminants.
360
As yet, the origin of atypical BSE is unknown, however it has been suggested that it
361
may be a spontaneous TSE in cattle, thus it would be interesting to speculate whether
362
this was also possible in goats. We have also considered the possibility that V3698 is a
363
CH1641-like scrapie isolate. These isolates have been described in sheep and goats
364
both experimentally (Foster & Dickinson, 1988; Jeffrey et al., 2006) and naturally
365
(Bellworthy et al., 2005; Bencsik & Baron, 2011; Vulin et al., 2011), and show partial
366
similarities to experimental ovine BSE. Previous studies have used anti-PrP antibody
367
SAF84 to identify an additional lower molecular PrPTSE band which has been shown as
368
marker of CH1641 (Baron et al., 2008). Despite our western blot attempts using SAF84,
369
due to the extremely low levels of PrPTSE in the Bov6 mice challenged with V3698 we
370
were unable to fully investigate the V3698 PrPTSE glycoform. Furthermore, without
371
performing transmission studies into Bov6 mice, we cannot fully compare V3698 with
15
372
CH1641. As yet, the spectrum of TSE agents circulating in the goat population is
373
unknown. Other studies investigating a flock of scrapie affected goats in Greece have
374
shown the existence of a unique TSE (Bouzalas et al., 2011). These studies showed
375
that a number of clinically healthy goats were found to have a unique C- and N-
376
terminally truncated PrPTSE fragment, which was similar but not identical to atypical
377
scrapie. Similarly to our results which showed very little PrP deposition in Bov6 mice
378
challenged with V3698 these goats did not show any PrP deposits in the brain
379
(Bouzalas et al., 2011). Therefore, despite the relatively small UK goat population,
380
various TSE agents in goats may exist, however it is also possible that in certain
381
instances more than one agent may exist in an individual goat. As surveillance of small
382
ruminants continues, the ability to recognise natural goat TSE strains will be of particular
383
importance, not only to improve our knowledge on the variability of goat TSEs but to
384
assess any potential risk to human health.
385
386
Materials and Methods
387
Preparation of Inocula
388
Brain homogenate G8247 from an experimentally BSE-infected goat (2nd goat passage,
389
The Roslin Institute, unpublished), genotype GIRQP/GIRQS (Goldmann et al., 2011)
390
was used as a goat BSE control. The unusual goat TSE case V3698 and the goat
391
scrapie HM control (Ref V3981; a 9 year old Saanen goat) were both supplied by The
392
Animal Health Veterinary Laboratories Agency (AHVLA), Weybridge, UK. All brain
393
tissue supplied was from the thalamic region. All inocula were prepared from brain
394
tissue in sterile saline at a concentration of 10% (wt/vol).
16
395
396
Inoculation of Transgenic Mice
397
Gene-targeted Tg mice expressing bovine PrP (Bov6) or human PrP (HuMM, HuMV
398
and HuVV) have been described previously (Bishop et al., 2006). Wildtype 129/Ola
399
mice were used as controls (Bishop et al., 2006). Transgenic mice were grouped (24
400
mice each) prior to intracerebral inoculation (i.c.) and then injected with 0·02 mL of 10 -1
401
brain homogenate (experimental goat BSE, V3698 or goat scrapie HM) into the right
402
cerebral hemisphere under halothane anaesthesia.
403
maceration of the brain tissue in sterile saline to a dilution of 10 −1. From 100 days post
404
inoculation mice were scored each week for signs of disease. Mice were killed by
405
cervical dislocation at a pre-defined clinical endpoint, or due to welfare reasons
406
(Dickinson et al., 1968). Brains were recovered at post mortem. Half the brain was
407
snap-frozen in liquid nitrogen for biochemical analysis and the remaining half brain was
408
fixed for histological processing. All mouse experiments were reviewed and approved
409
by the Local Ethical Review Committee and performed under licence from the United
410
Kingdom Home Office in accordance with the United Kingdom Animals (Scientific
411
Procedures) Act 1986.
All inocula were prepared by
412
413
Vacuolation Scoring
414
Sections were cut (6µm) from each mouse brain and stained using hematoxylin and
415
eosin (H&E).
416
(medulla, cerebellum, superior colliculus, hypothalamus, thalamus, hippocampus,
417
septum, retrospinal cortex, cingulated and motor cortex) and three regions of white
418
matter (cerebellar white matter, midbrain white matter, and cerebral peduncle).
TSE-related vacuolation was assessed at nine grey-matter regions
17
419
Sections were scored on a scale of 0 (no vacuolation) to 5 (severe vacuolation) for the
420
presence and severity of vacuolation and mean vacuolation scores for each mouse
421
group in each experiment were calculated and plotted with standard errors of the mean
422
(SEM) against scoring areas to produce a lesion profile, as previously described (Bruce
423
et al., 1997; Fraser & Dickinson, 1967). While, the production of lesion profiles in pre-
424
clinical mice is not standard practice, all mice which scored positive for vacuolar
425
pathology were included whether or not clinical signs were present, due to the lack of
426
clinical signs observed in Bov6 mice challenged with V3698.
427
428
Immunohistochemical (IHC) analysis of PrP deposition and glial activation in the
429
brain
430
PrPTSE localisation in the brain was assessed using immunohistochemistry. Following
431
fixation in 10% formal saline, brains were treated for 1·5 h in 98% formic acid, dissected
432
to expose several brain regions, and embedded in paraffin. Sections (6µm) were then
433
autoclaved for 15 min at 121C and immersed in 95% formic acid for 10 minutes prior to
434
incubation with 0.44 g/ml anti-PrP monoclonal antibody (MAb) 6H4 (Prionics) at room
435
temperature overnight. Secondary anti-mouse biotinylated antibody (Jackson Immuno
436
Research Laboratories, UK) was added at 2.5 g ml-1 and incubated for 1 h at room
437
temperature. Immunolabelling was performed using the ABC Elite kit (Vector
438
Laboratories) and the signal was visualised by a reaction with hydrogen peroxidase-
439
activated diaminobenzidine (DAB). The presence of astrogliosis, a hallmark of prion
440
disease, was assessed by incubating brain sections (6µm) with 1.45 g ml-1 anti-glial
441
fibrillary acidic protein (GFAP; DAKO UK Ltd) antibody at room temperature for 1 hour.
18
442
To detect microglial activation, brain sections were pre-treated using hydrated
443
microwaving for 10 minutes prior to incubation with 0.05 g ml-1 anti-Iba1 antibody
444
(Wako Chemicals GmbH) at room temperature for 1 hour. For both GFAP and anti-Iba-
445
1 antibodies, 2.6 g ml-1 biotinylated secondary anti-rabbit antibody (Jackson Immno
446
Research Laboratories, UK) was added for 1 hour at room temperature.
447
astrocytes and microglia were visualized by a reaction with hydrogen peroxidise-
448
activated DAB.
Both
449
450
Detection of amyloid plaques by thioflavin fluorescence
451
Amyloid deposits in tissue sections were observed using Thioflavin-S. Briefly, following
452
haematoxylin staining, sections (6µm) were immersed in 1% Thioflavin-S (Sigma, UK)
453
solution as previously described (Piccardo et al., 1998). Sections were mounted and
454
viewed under a fluorescence microscope.
455
456
Identification of PrPTSE by immunoblotting
457
Frozen brain samples from Bov6 mice challenged with experimental goat BSE; V3698,
458
goat scrapie HM, cattle BASE, BSE-C, and 129/Ola challenged with goat scrapie HM,
459
were homogenised at 10-1 in an NP40 buffer (0·5% v/v NP40, 0·5% w/v sodium
460
deoxycholate, 0·9% w/v sodium chloride, 50mM Tris-HCl pH 7·5) and clarified at
461
11,000g for 15 minutes. Brain homogenate supernatant from the transgenic mice and
462
controls was incubated with 20 g proteinase K ml-1 for 1 hour at 37C. Due to the lack
463
of PrP deposition, brains from Bov6 mice challenged with V3698 or goat scrapie HM
464
were treated with phosphotungstic acid (NaPTA) to precipitate PrP and concentrate the
465
samples. Briefly, 100mg of brain tissue was incubated for 15 minutes at 37 0C in
19
466
phosphate buffered saline containing 1mM MgCl2. The sample were adjusted to 2%
467
(w/v) Sarcosyl and incubated for 5 minutes at 37 0C. The samples were then altered to a
468
final concentration of 20 g proteinase K ml-1 and incubated at 37 °C for 1 hour. PrPTSE
469
was precipitated with the addition of a final concentration of 0.3% NaPTA, incubated at
470
37°C for 30 minutes and centrifuged at 14,000 rpm for 20 minutes. The pellet was
471
dissolved in 15 μl phosphate-buffered saline, pH 7.4, containing 0.1% sarkosyl. All
472
products were denatured and separated on a 10-20% Novex Tris/Glycine gel
473
(Invitrogen, UK) before transfer to polyvinylidine difluoride (PVDF) membrane by
474
western blotting.
475
0.3mg and 50mg brain equivalent. PrP was identified with monoclonal antibody 6H4
476
(0.1 g ml-1) or 12B2 (0.2 g ml-1) and bands visualized using horseradish peroxidise
477
(HRP)-labelled
478
Laboratories, UK) and a chemiluminescence substrate (Roche). Images were captured
479
on radiographic film and with a Kodak 440CF digital imager.
The amount of brain tissue loaded onto the gels varied between
anti-mouse
secondary
antibody
(Jackson
Immuno
Research
480
481
PCR genotyping of mouse tail DNA
482
All mice were analysed by PCR post mortem to confirm PrP genotype. Mouse tail DNA
483
was extracted and genotyped as previously described (Bishop et al., 2006; Wemheuer
484
et al., 2011).
485
486
Acknowledgements
487
The authors would like to acknowledge Professor J. Manson for Bov6 and HuTg mouse
488
lines; S. Cumming, S. Carpenter, R. Greenan and K. Hogan for experimental setup,
20
489
care and scoring of the animals; A. Boyle, G. McGregor and D. Drummond for histology
490
processing and scoring; J. Foster and D. Parnham for the experimental goat challenge
491
to generate isolate G8247; J. Hope, L. González and S. Hawkins from the Animal
492
Health Veterinary Laboratories Agency (AHVLA) UK, for supply of V3698 and the goat
493
scrapie (V3981) and L. González for his comments on the paper. These studies were
494
partly funded by contract M03054 from the Food Standards Agency (FSA) UK and
495
contract SE1441 from the UK Department for Environment, Food and Rural Affairs
496
(Defra). The Roslin Institute receives Institute Strategic Grant (ISP) funding from the
497
Biotechnology and Biological Sciences Research Council (BBSRC), UK.
498
499
500
501
21
502
503
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673
25
674
Table 1. Bioassay results of primary passage of V3698, goat BSE and goat scrapie HM
675
into both bovine and human PrP Tg mice and 129/Ola mice.
TSE isolate
V3698
Experimental
Goat BSE
(G8247)
Goat Scrapie
HM
(V3981)
Mouse
Line
Survival*
Incubation†
Clinical
Signs
TSE
Pathology‡
Clin+/
TSE Path+§
Bov6
129/Ola
HuMM
HuMV
HuVV
Bov6
129/Ola
HuMM
HuMV
HuVV
Bov6
129/Ola
HuMM
HuMV
HuVV
598±20
447±14
379±18
625±12
646±12
439±6
-
572
453±15
385±18
443±6
-
1/24
3/22
1/23
8/24
2/23
18/23
12/22
3/24
6/23
5/23
0/23
18/24
1/22
3/24
1/24
9/24
1/22
0/23
0/24
0/23
22/23
13/22
10/24
0/23
0/23
9/23
20/24
0/22
0/24
0/24
0
1
18
12
0
18
-
676
677
* Measured as days ± SEM and calculated from mice showing vacuolar pathology and/or PrP deposition.
678
† Measured as days ± SEM and calculated from mice showing both clinical signs of disease and
679
neuropathology (vacuolar pathology and/or PrP deposition).
680
‡ Number of animals positive for PrP deposition and/or vacuolar pathology.
681
§ Number of animals with clinical signs and confirmed TSE pathology.
682
- Not applicable (No vacuolar pathology of PrP deposition observed).
683
684
685
686
687
688
689
690
26
691
Figure 1. Pattern of vacuolation observed in brains of Bov6 mice challenged with
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V3698, experimental goat BSE and goat scrapie HM (a). Pattern of vacuolation
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observed in brains of Bov6 mice challenged with V3698, BASE, H-type BSE and
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classical BSE (BSE-C) (b-d). Lesion profiles for BASE, H-type BSE and BSE-C (shown
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for comparison) originate from previously published work (Wilson et al, 2012a). A profile
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was produced from nine grey matter areas (1, medulla; 2, cerebellum; 3, superior
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colliculus; 4, hypothalamus; 5, thalamus; 6, hippocampus; 7-septum, 8-retrospinal
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cortex, 9-cingulate and motor cortex) and three white matter areas (1, cerebellar white
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matter; 2, midbrain white matter; 3, cerebral peduncle). Average scores were taken
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from a minimum of seven mice per group and plotted against brain area ± SEM.
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Figure 2. Comparative analysis of variation in intensity of PrPTSE in the thalamus region
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of brains from Bov6 mice challenged with experimental goat BSE, 456dpi (a, b), V3698,
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623dpi (c, d) or goat scrapie HM, 680dpi (g, h) and 129/Ola mice challenged with goat
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scrapie HM, 407dpi (e, f). Panel b, d and f are higher magnifications images of the
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boxed area in panel a, c and e respectively. Images obtained after staining with anti-PrP
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antibody 6H4 and counterstained with hematoxylin. Magnification is as shown.
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Figure 3. Comparative analysis of the thalamus of Bov6 mice challenged with V3698,
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experimental goat BSE and goat scrapie HM and 129/Ola mice challenged with goat
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scrapie HM. Micro- and astrogliosis is present in all mice, detected by anti-GFAP (e, h,
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k, n) and anti-Iba1 respectively (f, i, l, o). PrP deposition is visible by anti-6H4 antibody
27
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(g, m). Uninfected aged Bov6 mice showing mild gliosis (b, c) and no PrP deposition (a)
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were used as controls. Magnification 10x.
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Figure 4. (a) and (b); Comparative western blot analysis of the proteinase K-resistant
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fragment (PrPTSE) of the prion protein. Discrimination between BSE and scrapie is
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achieved using two monoclonal antibodies, 6H4 (a & c) and 12B2 (b & d). (a & b) lanes
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1-2, BSE-C in Bov6 mice; lanes 3-4, experimental goat BSE in Bov6 mice; lanes 5-6,
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V3698 in Bov6 mice; lanes 7-8, BASE in Bov6 mice, and lanes 9-10, goat scrapie HM in
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129/Ola mice. Each lane represents an individual mouse brain. Brain samples in lanes
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1-4 and 7-10 were loaded at 0.3 to 0.6 mg brain equivalent, and samples in lanes 5-6
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were loaded at 5 mg brain equivalent. (c & d) lanes 1-2, V3698 in Bov6 mice; lanes 3-4,
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goat scrapie HM in Bov6 mice (image cropped from a single blot to remove non-relevant
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samples). Samples loaded at 5mg brain equivalent. All samples PK-treated and NaPTA
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precipitated.
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Figure 5. PrP glycoform ratio of samples from Fig.4(a) quantified by Kodak Imager
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440CF and normalised as % total PrP. Samples 1-2, BSE-C; samples 3-4, experimental
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goat BSE; samples 5-6, V3698; samples 7-8, BASE, and samples 9-10, goat scrapie
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HM (129/Ola).
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28
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Figure 6. PrPTSE accumulation in the thalamus of a HuMM mouse (594dpi) challenged
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with experimental goat BSE. Panel (b) is a higher magnification image of the boxed area
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in panel (a). Images obtained after staining with anti-PrP antibody 6H4 and
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counterstained with hematoxylin. Magnification as shown.
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29