JVI Accepts, published online ahead of print on 27 November 2013 J. Virol. doi:10.1128/JVI.02872-13 Copyright © 2013, American Society for Microbiology. All Rights Reserved.
1 1
Genetic resistance to Scrapie infection in experimentally challenged goats
2
Caroline Lacroux1†, Cécile Perrin-Chauvineau2†, Fabien Corbière1, Naima Aron1, Patricia Aguilar-Calvo3,
3
Juan Maria Torres3, Pierrette Costes1, Isabelle Brémaud2, Séverine. Lugan1, François.Schelcher1, Francis
4
Barillet3 and Olivier Andréoletti1 *
5 6
1
7
Capelles, 31076 Toulouse Cedex, France
8
2
AFSSA-Niort, Laboratoire d’études et de recherches caprines, BP 3081, 79012 Niort Cedex, France
9
3
CISA- INIA, 28130 Valdeolmos, Madrid, Spain.
10
4
INRA, UR 631, Station d’amélioration génétique des animaux, BP 52627, 31326 Castanet-Tolosan Cedex, France
INRA, UMR 1225, Interactions Hôtes Agents Pathogènes, Ecole Nationale Vétérinaire de Toulouse, 23 chemin des
11 12 13 14 15 16 17
Running title: K222 goat carriers are resistant to Scrapie
18
† both authors contributed equally to this work
19
* Corresponding author: o. andreoletti
20
[email protected]
2 21
Abstract
22 23
In goats, several field studies have identified coding mutations of the gene encoding for the Prion protein
24
(I/M142, N/D146, S/D146, R/Q211 and Q/K222) that are associated with a lower risk of developing classical
25
scrapie.
26
However, the data related to the level of resistance to TSE of these different PRNP gene mutations are still
27
considered insufficient for developing large scale genetic selection against Scrapie in this species.
28 29
In this study, we inoculated wild type (WT) PRNP genotype (I142R154R211Q222) goats and homozygous
30
and/or heterozygous I/M142, R/H154, R/Q211 and Q/K222 goats with a goat natural scrapie isolate by either the
31
oral or the intracerebral (IC) route. Our results indicate that the I/M142 PRNP polymorphism does not
32
provide substantial resistance to scrapie infection following intracerebral or oral inoculation. They also
33
demonstrate that the H154, Q211 and K222 PRNP allele carriers are all resistant to Scrapie infection following
34
oral exposure. However in comparison to WT animals the H154 and the Q211 allele’s carriers displayed only a
35
moderate increase in the incubation period following IC challenge. After IC challenge, heterozygous K222
36
and a low proportion of the homozygous K222 goats also developed the disease but with incubation period
37
that were 4-5 times longer than in WT animals.
38 39
These results support the contention that K222 goat Prion protein variant provides a strong but not absolutely
40
protective effect against classical scrapie.
41
3 42
Introduction
43
Transmissible spongiform encephalopathies (TSE), or prion diseases, are fatal neurodegenerative disorders
44
occurring in small ruminants (scrapie), cattle (bovine spongiform encephalopathy - BSE), or humans
45
(Creutzfeldt-Jakob disease - CJD). The key event in TSE is the conversion of a normal cellular protein
46
(PrPC) into an abnormal isoform (PrPSc) which accumulates in tissues in infected individuals. According to
47
the prion concept, abnormal PrP is the causative agent of TSEs (11).
48 49
In sheep, the susceptibility to TSE is strongly modulated by polymorphisms of the prion protein gene
50
(PRNP) and the nature of the prion disease agent (strain) (8). The A136R154R171 allele is associated with a
51
highly protective effect against natural or experimental infection with classical scrapie and BSE agents,
52
while the V136R154Q171 allele and the wild type A136R154Q171 allele are associated with susceptibility (16,
53
26). However, in sheep the ARR allele does not provide any particular protection against atypical scrapie
54
whereas the R/H154 or L/F141 amino acid substitutions are associated with an increased risk of occurrence of
55
this TSE (9, 17, 28 , 29).
56 57
At the European level, the selection of the ARR allele carriers was successfully applied for controlling and
58
eradicating classical Scrapie in infected sheep flocks (30). At a population level, large scale selection
59
programs were also implemented. They aimed to increase the frequency of the ARR allele in the general
60
population making it less favourable to TSE agent circulation and spreading. This ‘Breeding for resistance
61
policy’ in combination with the other eradication measures, resulted in a significant reduction of the
62
classical Scrapie prevalence in populations where it was comprehensively applied (15, 18, 24).
63 64
In goats, several field studies have identified coding mutations of the PRNP gene that are associated with a
65
lower risk of developing classical scrapie; namely the I/M142, N/D146 and S146, R/Q211 and Q/K222 (1, 7, 22,
66
31, 32, 2011 #11698, 35). However, the low frequency of these alleles in goat population limits the
4 67
possibility of reaching an unequivocal conclusion about the resistance/susceptibility to infection associated
68
with these different PRNP genotypes (1, 8, 35). In that context, experimental TSE inoculation in goats is a
69
straightforward and robust approach to better assess the level of resistance associated to certain PRNP
70
polymorphisms in this species (2, 34).
71
In this study, we inoculated wild-type genotype goats and homozygous and/or heterozygous I/M142, R/H154,
72
R/Q211 and Q/K222 goats either by the intracerebral or the oral route with a goat natural Scrapie isolate in
73
order to characterize their relative resistance/susceptibility to infection.
74 75
5 76
Methods
77
Ethics Statement
78
All animal experiments have been performed in compliance with institutional and French national
79
guidelines, in accordance with the European Community Council Directive 86/609/EEC. The experimental
80
protocol was approved by the INRA Toulouse/ENVT ethics committee.
81 82
Scrapie inoculum
83
The inoculum was derived from a single natural field Scrapie case (clinical) obtained in a 3.5 year old goat
84
with the wild type PRNP genotype. This animal was necropsied under TSE sterile conditions and its central
85
nervous system (brain and spinal cord) was used to prepare a 10% tissue homogenate in 5% glucose. The
86
stock homogenate was aliquoted and stored at -80°C.
87 88
Experimental animals’ production
89
Goats kids intended to be used in the experiment were produced by direct mating of PRNP sequenced
90
Alpine and Saanen female goats and bucks. Parents were selected from three herds that are managed by the
91
French National Agronomic Institute (INRA). Selection was based on the PRNP polymorphism at codons
92
142 (I/M), 154 (R/H), 211 (R/Q) and 222 (Q/K) which were identified by previous study to influence
93
susceptibility to natural Scrapie (7, 14, 35). Animals were then naturally mated to produce the goats used for
94
experimental inoculation. The Exon 3 of the PRNP gene of each goat kid was sequenced as previously
95
described (6).
96 97
Goat oral challenge experiments
98
For the oral challenge experiment, gravid goats were relocated to ANSES Niort A2 facilities. Within 48
99
hours after birth each goat kid received 1.5g brain equivalent material through natural suckling (1% diluted
100
stock inoculum in glucose 5%). A second inoculation (same material and route) was performed at the age of
6 101
30 days. Considering (i) the logistic constraints (housing of goats and goat kids) and (ii) the fact that the
102
parent goats were only heterozygous for the alleles of interest, the oral inoculation of goat kids that would
103
have been homozygous for the mutated PRNP alleles was not feasible in the framework of this experiment.
104
Two separate oral inoculation experiments were performed. The first one aimed at establishing the PrPSc
105
dissemination scheme and kinetics in animals with the wild type PRNP genotype. For that purpose, 3 or 4
106
animals were culled 30, 90, 120, 360, 540 and 940 days post inoculation (dpi). A last group of animals
107
(n=4) was kept until the occurrence of clinical signs.
108
The second experiment aimed at establishing the relative susceptibility of goats harbouring various
109
genotypes to scrapie following oral exposure. WT, heterozygous I/M142, R/Q211 and Q/K222 animals were
110
orally challenged using the same isolate as the first experiment, and culled at 120, 360, 760, 1040 dpi. Five
111
animals of each genotype were killed at each of the different time points.
112
In addition a group of animals from each of these genotypes, and a group (n=6) of heterozygous R/H154
113
animals that had also been challenged orally were kept alive for establishing the incubation period. Because
114
of space constraint in the animal facilities, it was not possible to challenge a sufficient number of R/H154
115
PRNP allele carriers to complete the time point experiment (see below).
116 117
Goat intracerebral challenge
118
After weaning, goat kids selected by genotype were transported to UMR INRA ENVT A2 animal facilities
119
for intracerebral (IC) inoculation. When the animals were six months of age they were anesthetised
120
(Ketamine/Valium) and 400µL of the stock inoculum was injected in temporal cortex.
121 122
Clinical monitoring and sample collection
123
Inoculated goats were clinically monitored on a daily basis. The animals that developed TSE were
124
euthanased when exhibiting locomotor signs that impaired their feeding capacity. Animals that developed
7 125
intercurrent health problems were treated by qualified veterinarians and euthanased if the condition was not
126
curable.
127
Dead animals were systematically necropsied and central nervous system (CNS), a variety of lymphoid
128
(mesenteric lymph node, tonsil, Prescapular lymph node, Peyer’s patches) and non-lymphoid tissues were
129
collected (Table 1). Half of the samples were formalin fixed while the other half was stored frozen (-20°C).
130 131
PrPSc Immunohistochemestry (IHC) and PrPres ELISA detection
132
PrPSc IHC detection was performed as described in Lacroux et al using 8G8 antibody raised against human
133
recombinant PrP protein and specifically recognising the 95-108 amino acid sequence (SQWNKP) of the
134
PrP protein (27).
135 136
Western-blot (WB) of the abnormal PrP
137
PK resistant abnormal PrP extraction (PrPres) and Western blot were performed as previously described (5),
138
using a commercial extraction kit (Biorad, France). PrP immunodetection was performed using either Sha31
139
monoclonal antibody (0,06 µg per mL, epitope: YEDRYYRE , amino acid 145-152) or 12B2 (4 µg/mL)
140
(epitope WGQGG, amino acid sequences 93-97) (19).
141
For glycoprofiling of the PrPres, signal volume and relative percentage associated with each band was
142
established using Quantity One® software (Bio-Rad) following immunoblot. For each sample, three
143
independent measures were realized on three different gels.
144
8 145
Results
146 147
Oral challenge in goats
148
Our first oral challenge experiment in goats was designed to establish the scheme and kinetics of PrPSc
149
dissemination in the tissues of Wild type (WT) PRNP genotype animals. For that purpose, goat kids
150
obtained by natural mating of WT PRNP genotype goats and bucks were orally challenged within the first
151
48 hours following birth. Groups of these animals were culled at different time point after inoculation
152
(Table 1). PrPSc accumulation was first observed in the gut associated lymphoid tissue (Peyer’s patches) in
153
animals more than 180 dpi. As already described in sheep, PrPSc progressively spread to all lymphoid organs
154
before becoming detectable (between 180 and 360 dpi) in the enteric nervous system (ENS) and later
155
(between 540 and 940 dpi) in the central nervous system (3).
156
On the basis of these results, a second oral challenge experiment was designed. The goal of this experiment
157
was to characterize the impact of the investigated polymorphisms on the susceptibility and the PrPSc
158
dissemination in the tissues of orally exposed animals. Groups of wild type animals and heterozygous
159
I/M142, R/Q211 and Q/K222 were produced by natural mating and orally challenged using the same procedure
160
and Scrapie isolate than in the first experiment.
161
In this second experiment, the PrPSc dissemination scheme observed in WT genotype animals was consistent
162
with the results of the first experiment (Table 2). No PrPSc deposition was observed in the tissues collected
163
from goats killed at 120dpi. At 360dpi, significant PrPSc deposition was observed in various lymphoid
164
tissues (Peyer’s patches, mesentery lymph nodes and tonsil) of some of the challenged individuals. PrPSc
165
deposition was observed in ENS, CNS, peripheral nervous tissues and skeletal muscles in four out of the
166
five animals culled at 760 dpi.
167
In the heterozygous I/M142 orally challenged animals a similar but slightly delayed PrPSc accumulation
168
scheme was observed; PrPSc was first detected in the gut associated lymphoid tissue (Peyer’s patches) at
169
360dpi but it was only detected at 1040 dpi in the CNS (Table 2).
9 170
No PrPSc was observed in any of the tissues collected in the heterozygous R/Q211 and Q/K222 animals that
171
had been orally challenged and killed at the different time points (5 animals per genotype per time point),
172
and none of the animals bearing these genotype had developed a clinical TSE after more than 2500 dpi.
173
In both oral challenge experiments a group of animals harbouring the different PRNP genotypes (I/M142,
174
R/Q211 and Q/K222) was kept alive and clinically monitored for TSE development (table 3). Similarly, a
175
group of heterozygous R/H154 animals (n=6) that had been orally challenged with the same inoculum was
176
also monitored.
177
In WT goats, incubation periods in the first (n=4) and the second experiment (n=5) were not different. All
178
the challenged I/M142 goats (n=4) also developed a clinical TSE but with slightly longer incubation period
179
(1490±126 dpi) than in WT animals (1141±93 dpi). In both genotypes, affected animals showed PrPSc
180
deposition in CNS and lymphoid tissues (table 2).
181
After more than 2500 days of incubation, none of the orally inoculated R/H154, R/Q211 and Q/K222
182
heterozygous animals had developed a clinical TSE. Some of the heterozygous R/H154 (n=3) or Q/K222
183
(n=2) goats and one homozygous K/K222 animal died from intercurrent disease (table 3). In these orally
184
challenged animals none of the investigated tissues (lymphoid organs and CNS) displayed any detectable
185
PrPSc deposition.
186
Together, these findings support the contention that R/H154, R/Q211, and Q/K222 PrP mutated alleles have a
187
strong protective effect against Scrapie infection following oral exposure.
188 189
Intracerebral challenge in goats
190 191
To further assess the resistance to Scrapie associated to I/M142, R/H154, R/Q211 and Q/K222 PRP alleles,
192
groups of heterozygous and homozygous animals were intracerebrally inoculated using the same isolate as
193
the one used for oral challenge (table 4). As expected, all the intracerebrally inoculated WT goats developed
10 194
a clinical TSE. In those animals, PrPSc deposits were observed in both the central nervous system and in
195
lymphoid tissues.
196
In contrast to the oral challenge experiment, the heterozygous I/M142, R/H154, R/Q211, but also the Q/Q211
197
homozygous animals developed a clinical TSE. Strikingly, the heterozygous R/Q211 individuals displayed a
198
longer incubation period than the homozygous Q/Q211. PrPSc deposition was observed in the lymphoid
199
tissues of the heterozygous I/M142 and homozygous Q/Q211 animals. No or limited PrPSc accumulation was
200
observed in lymphoid tissues from R/H154 and R/Q211 animals. Using Sha31 antibody, the WB PrPres
201
patterns observed in the brain of all the R/H154 scrapie affected individuals was identical and differed
202
strikingly from those observed in individuals bearing other PRP genotypes (figure 1); the PrPres bands
203
displayed an apparent lower molecular weight. Immunoblot probed with the 12B2 antibody indicated that in
204
H/R154 goats PK digestion resulted in a N-terminal cleavage of PrPres (amino-acid sequences 93-97) that
205
differed from the other genotypes groups.
206
In the IC challenged heterozygous Q/K222 goats three animals died of intercurrent disease at 568, 898, 1062
207
dpi. No PrPSc accumulation was observed in any of the investigated tissues from these goats. However, the
208
two remaining animals developed a clinical TSE after 1980 and 2134 dpi respectively and in those two
209
individuals PrPSc deposits were observed (IHC and WB) in the central nervous system but not in lymphoid
210
organs.
211
One out of the five IC challenged homozygous K222 animals developed a clinical TSE and was euthanased
212
after 2101 dpi. Abnormal PrP deposition was detected (IHC and WB) in the central nervous system but not
213
in the lymphoid tissues. The four remaining K/K222 animals are still apparently healthy at the time of writing
214
(more than 2400 dpi).
215
Using the Sha31 antibody, Q/K222 and K/K222 positive individuals displayed a similar PrPres glycoform ratio.
216
The PrPres glycoprofyle of these individuals displayed a dominant mono-glycosylated bands which clearly
217
differed from the patterns observed in the goats with other genotypes (Figure 2).
11 218
More generally, the presence of apparently different PrPres WB signature in IC challenged goats that harbour
219
different genotypes suggest that different TSE agent propagated in those animals. However it is our opinion
220
that bioassays (which are currently ongoing) remain necessary before concluding on that point.
221 222
12 223
Discussion
224 225
Cases controls studies in classical scrapie affected herds (1, 7, 22, 31, 32, 35) and limited data from
226
experimental challenge (intracerebral routes) (2, 36) supported the view that K222 PrP allele goats might be
227
strongly resistant to classical scrapie infection. Rare cases of the disease (n=3) were reported in
228
heterozygote K222 goats belonging to one single flock that displayed a high disease prevalence (27.4%), and
229
no case was reported so far in homozygous K222 animals (7, 14).
230
Our study indicated that a classical scrapie isolate failed to propagate in K222 heterozygous goats following
231
oral challenge. However, it also demonstrated that the same classical scrapie isolate can propagate in
232
heterozygous and in a proportion of homozygous K222 animals following IC challenge, but with incubation
233
period that exceeded 4 to 5 times those observed in WT animals.
234
These results in K222 goats are very evocative of those obtained in the A136R154R171 allele sheep carriers that
235
were naturally or experimentally exposed to TSE agents. After oral experimental challenge in homozygous
236
and heterozygous ARR sheep no or poorly efficient propagation of classical scrapie and BSE agents was
237
observed (4, 20, 21). In heterozygous ARR sheep that were IC challenged with classical scrapie, the disease
238
occurred but with significantly longer incubation periods than in homozygous ARQ (wild type PRNP
239
genotype) sheep (21). A clinical TSE occurred in a proportion of homozygous ARR that were IC challenged
240
with cattle BSE and occurrence of rare natural classical scrapie cases was reported in animals harboring this
241
genotype (23, 25).
242
All these results support the view that like the ARR allele in sheep the K222 allele is associated with a high
243
but not absolute resistance to Scrapie.
244 245
In the I/M142 allele carriers, the IC and oral challenge results indicate that this allele is not associated with a
246
substantial resistance to the classical scrapie isolate we used. These observations are consistent with data
13 247
collected in naturally infected goats herds (7, 14) and with the observations previously reported by Goldman
248
et al. in goats challenged with cattle BSE, CH1641 and ME7 passaged in sheep (22).
249
No transmission or PrPSc deposition could be observed in orally challenged H154, and Q211 heterozygous
250
animals. However a 100% attack rate was observed in animals bearing those genotypes following IC
251
challenge, albeit with longer incubation period than in WT animals.
252
These results were similar to those observed in heterozygous K222 animals. However, unlike K222
253
homozygous goats, the Q211 homozygous animals developed the disease with a 100% attack rate following
254
IC challenge, with shorter incubation periods than in Q211 heterozygous animals. This indicates that the Q211
255
allele cannot be considered to provide the same level of resistance against scrapie as the K222 allele.
256
The lack of H154 homozygous goats in the intracerebral inoculation experiment clearly limits our capacity to
257
draw final conclusions concerning the level of resistance/susceptibility to classical scrapie associated with
258
this PRNP allele. Nevertheless, the risk of atypical scrapie occurrence has been shown to be significantly
259
higher in both H154 allele carrier sheep and goat (same PrPC sequence in goats and sheep) than in WT
260
animals (13, 28). This higher susceptibility to atypical scrapie represents a major argument against the
261
selection of the H154 PRNP allele in goat population
262
Beyond this, the main limitation of this experiment is the fact that only one classical scrapie goat isolate was
263
used to test the relative susceptibility of the different genotypes. The diversity of TSE agents in small
264
ruminants has been documented for several decades (3, 12, 33). In sheep, the susceptibility to TSE infection
265
was shown to be influenced by both the nature of the TSE strain and the PRNP polymorphisms (10).
266
Considering the time and the resources necessary to carry out bioassay in large animals, testing several
267
classical scrapie agents in parallel in this model was simply not feasible. In that context, the inoculation of a
268
variety of TSE agent in transgenic mice that express the WT and K222 PRNP goat alleles will play a pivotal
269
role in confirming the apparent low susceptibility / high resistance associated with this last allele.
270
Finally, it should be noticed that the experimental approach we used only allowed estimating the impact of
271
individual PRNP polymorphisms on the susceptibility to the disease. For obvious material reasons, it was
14 272
not possible to investigate the effect of PRNP haplotypes combinations (like individuals that would bear
273
both Q211 and K222 allele).
274 275
The development of PRNP genotype selection programs is now being considered by the EU authorities as a
276
potential tool to control and eradicate scrapie in commercial goat populations. In sheep, the diffusion of the
277
ARR allele in the general population and, in particular, its introduction in classical scrapie affected herds
278
has proven its efficacy for the long term control of the disease (15, 18, 24). The data that we report concur
279
with the view that the K222 allele in goats provides a similar level of resistance against scrapie infection as
280
the ARR allele in sheep.
281 282
Acknowledgments
283
The authors wish to thank Frédéric Bouvier and his team for producing goat with appropriate PRP
284
genotypes (‘INRA domaine de la Sapinière’). This work was funded by GIS PRION grant 31B06134, by
285
INRA grant AIP P00297, by the Poitou-Charentes region grants 04/RPC-A-103 and 05/RPC-A-13 and
286
European Union (FOOD-CT-2006-36353 and 219235 FP7 ERA-NET EMIDA). The authors declare no
287
competing financial interests.
288
15 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336
References 1.
2.
3.
4.
5.
6.
7.
8.
9. 10. 11. 12.
13.
14.
15.
Acutis, P. L., A. Bossers, J. Priem, M. V. Riina, S. Peletto, M. Mazza, C. Casalone, G. Forloni, G. Ru, and M. Caramelli. 2006. Identification of prion protein gene polymorphisms in goats from Italian scrapie outbreaks. J Gen Virol 87:1029-1033. Acutis, P. L., F. Martucci, A. D'Angelo, S. Peletto, S. Colussi, C. Maurella, C. Porcario, B. Iulini, M. Mazza, L. Dell'atti, F. Zuccon, C. Corona, N. Martinelli, C. Casalone, M. Caramelli, and G. Lombardi. 2012. Resistance to classical scrapie in experimentally challenged goats carrying mutation K222 of the prion protein gene. Vet Res 43:8. Andreoletti, O., P. Berthon, D. Marc, P. Sarradin, J. Grosclaude, L. van Keulen, F. Schelcher, J. M. Elsen, and F. Lantier. 2000. Early accumulation of PrP(Sc) in gut-associated lymphoid and nervous tissues of susceptible sheep from a Romanov flock with natural scrapie. J Gen Virol 81 Pt 12:3115-3126. Andreoletti, O., N. Morel, C. Lacroux, V. Rouillon, C. Barc, G. Tabouret, P. Sarradin, P. Berthon, P. Bernardet, J. Mathey, S. Lugan, P. Costes, F. Corbiere, J. C. Espinosa, J. M. Torres, J. Grassi, F. Schelcher, and F. Lantier. 2006. Bovine spongiform encephalopathy agent in spleen from an ARR/ARR orally exposed sheep. J Gen Virol 87:1043-1046. Andreoletti, O., L. Orge, S. L. Benestad, V. Beringue, C. Litaise, S. Simon, A. Le Dur, H. Laude, H. Simmons, S. Lugan, F. Corbiere, P. Costes, N. Morel, F. Schelcher, and C. Lacroux. 2011. Atypical/Nor98 scrapie infectivity in sheep peripheral tissues. PLoS Pathog 7:e1001285. Arsac, J. N., O. Andreoletti, J. M. Bilheude, C. Lacroux, S. L. Benestad, and T. Baron. 2007. Similar biochemical signatures and prion protein genotypes in atypical scrapie and Nor98 cases, France and Norway. Emerg Infect Dis 13:58-65. Barillet, F., D. Mariat, Y. Amigues, R. Faugeras, H. Caillat, K. Moazami-Goudarzi, R. Rupp, J. M. Babilliot, C. Lacroux, S. Lugan, F. Schelcher, C. Chartier, F. Corbiere, O. Andreoletti, and C. Perrin-Chauvineau. 2009. Identification of seven haplotypes of the caprine PrP gene at codons 127, 142, 154, 211, 222 and 240 in French Alpine and Saanen breeds and their association with classical scrapie. J Gen Virol 90:769-776. Baylis, M., C. Chihota, E. Stevenson, W. Goldmann, A. Smith, K. Sivam, S. Tongue, and M. B. Gravenor. 2004. Risk of scrapie in British sheep of different prion protein genotype. J Gen Virol 85:2735-2740. Benestad, S. L., J. N. Arsac, W. Goldmann, and M. Noremark. 2008. Atypical/Nor98 scrapie: properties of the agent, genetics, and epidemiology. Vet Res 39:19. Beringue, V., J. L. Vilotte, and H. Laude. 2008. Prion agent diversity and species barrier. Vet Res 39:47. Bolton, D. C., M. P. McKinley, and S. B. Prusiner. 1982. Identification of a protein that purifies with the scrapie prion. Science 218:1309-1311. Bruce, M. E., A. Boyle, S. Cousens, I. McConnell, J. Foster, W. Goldmann, and H. Fraser. 2002. Strain characterization of natural sheep scrapie and comparison with BSE. J Gen Virol 83:695-704. Colussi, S., G. Vaccari, C. Maurella, C. Bona, R. Lorenzetti, P. Troiano, F. Casalinuovo, A. Di Sarno, M. G. Maniaci, F. Zuccon, R. Nonno, C. Casalone, M. Mazza, G. Ru, M. Caramelli, U. Agrimi, and P. L. Acutis. 2008. Histidine at codon 154 of the prion protein gene is a risk factor for Nor98 scrapie in goats. J Gen Virol 89:3173-3176. Corbiere, F., C. Perrin-Chauvineau, C. Lacroux, P. Costes, M. Thomas, I. Bremaud, S. Martin, S. Lugan, C. Chartier, F. Schelcher, F. Barillet, and O. Andreoletti. 2013. PrPassociated resistance to scrapie in five highly infected goat herds. J Gen Virol 94:241-245. Dawson, M., R. C. Moore, and S. C. Bishop. 2008. Progress and limits of PrP gene selection policy. Vet Res 39:25.
16 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384
16.
17.
18.
19.
20.
21.
22.
23.
24.
25. 26. 27.
28.
29.
30.
31.
Elsen, J. M., Y. Amigues, F. Schelcher, V. Ducrocq, O. Andreoletti, F. Eychenne, J. V. Khang, J. P. Poivey, F. Lantier, and J. L. Laplanche. 1999. Genetic susceptibility and transmission factors in scrapie: detailed analysis of an epidemic in a closed flock of Romanov. Arch Virol 144:431-445. Fediaevsky, A., C. Maurella, M. Noremark, F. Ingravalle, S. Thorgeirsdottir, L. Orge, R. Poizat, M. Hautaniemi, B. Liam, D. Calavas, G. Ru, and P. Hopp. 2010. The prevalence of atypical scrapie in sheep from positive flocks is not higher than in the general sheep population in 11 European countries. BMC Vet Res 6:9. Fediaevsky, A., S. C. Tongue, M. Noremark, D. Calavas, G. Ru, and P. Hopp. 2008. A descriptive study of the prevalence of atypical and classical scrapie in sheep in 20 European countries. BMC Vet Res 4:19. Feraudet, C., N. Morel, S. Simon, H. Volland, Y. Frobert, C. Creminon, D. Vilette, S. Lehmann, and J. Grassi. 2005. Screening of 145 anti-PrP monoclonal antibodies for their capacity to inhibit PrPSc replication in infected cells. J Biol Chem 280:11247-11258. Foster, J. D., D. Parnham, A. Chong, W. Goldmann, and N. Hunter. 2001. Clinical signs, histopathology and genetics of experimental transmission of BSE and natural scrapie to sheep and goats. Vet Rec 148:165-171. Goldmann, W., N. Hunter, G. Smith, J. Foster, and J. Hope. 1994. PrP genotype and agent effects in scrapie: change in allelic interaction with different isolates of agent in sheep, a natural host of scrapie. J Gen Virol 75 ( Pt 5):989-995. Goldmann, W., T. Martin, J. Foster, S. Hughes, G. Smith, K. Hughes, M. Dawson, and N. Hunter. 1996. Novel polymorphisms in the caprine PrP gene: a codon 142 mutation associated with scrapie incubation period. J Gen Virol 77 ( Pt 11):2885-2891. Groschup, M. H., C. Lacroux, A. Buschmann, G. Luhken, J. Mathey, M. Eiden, S. Lugan, C. Hoffmann, J. C. Espinosa, T. Baron, J. M. Torres, G. Erhardt, and O. Andreoletti. 2007. Classic scrapie in sheep with the ARR/ARR prion genotype in Germany and France. Emerg Infect Dis 13:1201-1207. Hagenaars, T. J., M. B. Melchior, A. Bossers, A. Davidse, B. Engel, and F. G. van Zijderveld. 2010. Scrapie prevalence in sheep of susceptible genotype is declining in a population subject to breeding for resistance. BMC Vet Res 6:25. Houston, F., W. Goldmann, A. Chong, M. Jeffrey, L. Gonzalez, J. Foster, D. Parnham, and N. Hunter. 2003. Prion diseases: BSE in sheep bred for resistance to infection. Nature 423:498. Hunter, N., L. Moore, B. D. Hosie, W. S. Dingwall, and A. Greig. 1997. Association between natural scrapie and PrP genotype in a flock of Suffolk sheep in Scotland. Vet Rec 140:59-63. Lacroux, C., F. Corbiere, G. Tabouret, S. Lugan, P. Costes, J. Mathey, J. M. Delmas, J. L. Weisbecker, G. Foucras, H. Cassard, J. M. Elsen, F. Schelcher, and O. Andreoletti. 2007. Dynamics and genetics of PrPSc placental accumulation in sheep. J Gen Virol 88:1056-1061. Moreno, C. R., K. Moazami-Goudarzi, P. Laurent, G. Cazeau, O. Andreoletti, S. Chadi, J. M. Elsen, and D. Calavas. 2007. Which PrP haplotypes in a French sheep population are the most susceptible to atypical scrapie? Arch Virol 152:1229-1232. Moum, T., I. Olsaker, P. Hopp, T. Moldal, M. Valheim, T. Moum, and S. L. Benestad. 2005. Polymorphisms at codons 141 and 154 in the ovine prion protein gene are associated with scrapie Nor98 cases. J Gen Virol 86:231-235. Nodelijk, G., H. J. van Roermund, L. J. van Keulen, B. Engel, P. Vellema, and T. J. Hagenaars. 2011. Breeding with resistant rams leads to rapid control of classical scrapie in affected sheep flocks. Vet Res 42:5. Papasavva-Stylianou, P., M. Kleanthous, P. Toumazos, P. Mavrikiou, and P. Loucaides. 2007. Novel polymorphisms at codons 146 and 151 in the prion protein gene of Cyprus goats, and their association with natural scrapie. Vet J 173:459-462.
17 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402
32.
33. 34.
35.
36.
Papasavva-Stylianou, P., O. Windl, G. Saunders, P. Mavrikiou, P. Toumazos, and C. Kakoyiannis. 2011. PrP gene polymorphisms in Cyprus goats and their association with resistance or susceptibility to natural scrapie. Vet J 187:245-250. Pattison, I. H., and G. C. Millson. 1962. Distribution of the scrapie agent in the tissues of experimentally inoculated goats. J Comp Pathol 72:233-244. Tabouret, G., C. Lacroux, S. Lugan, F. Corbiere, J. L. Weisbecker, P. Costes, F. Schelcher, and O. Andreoletti. 2011. Relevancy of oral experimental challenge with classical scrapie in sheep. J Gen Virol. Vaccari, G., M. A. Di Bari, L. Morelli, R. Nonno, B. Chiappini, G. Antonucci, S. Marcon, E. Esposito, P. Fazzi, N. Palazzini, P. Troiano, A. Petrella, G. Di Guardo, and U. Agrimi. 2006. Identification of an allelic variant of the goat PrP gene associated with resistance to scrapie. J Gen Virol 87:1395-1402. White, S. N., J. O. Reynolds, D. F. Waldron, D. A. Schneider, and K. I. O'Rourke. 2012. Extended scrapie incubation time in goats singly heterozygous for PRNP S146 or K222. Gene 501:49-51.
18 403 404
Figures:
405
Figure 1: PrPres Western Blot pattern in the brain of goat intracerebrally challenged with a classical
406
scrapie isolate
407
10% tissue homogenates were prepared using brain from goats that had been intracerebrally challenged with
408
a classical scrapie goat isolate and developed the disease (see table 3). Abnormal PK resistant PrP (PrPres)
409
was detected following western blot using Sha31 antibody (epitope YEDRYYRE) and 12B2 (epitope
410
WGQGG). On each gel a classical scrapie sheep isolate (WB control) and the original isolate (orig. isolate)
411
were used as a control.
412 413
19 414
Figure 2: PrPres glycoprofyle in the brain of goat intracerebrally challenged with a classical scrapie
415
isolate
416
10% tissue homogenates were prepared using posterior brain stem from goats that had been intracerebrally
417
challenged with a classical scrapie goat isolate and developed the disease (see table 3). Abnormal PK
418
resistant PrP (PrPres) was detected following western blot using Sha31 antibody (epitope YEDRYYRE).
419
Signal volume and relative percentage associated with mono-glycosylated, bi-glycosylated and un-
420
glycosylated band were established using Quantity One® software (Bio-Rad).
421
*: original isolate,
422 423 424 425
: H/R154
: wild type genotype,
: I/M142,
: Q/K222,
: K/K222,
: R/Q211,
: Q/Q211,
20 26
Sc
Table 1: PrP detection in tissues of wild type (WT) PRNP genotype goat orally challenged with scrapie and sequentially killed
Organ/Age
27 28 29
30 dpi
90 dpi
Obex
0/3
Spinal cord cervical
ND
ND
Spinal cord thoracic
ND
ND
Spinal cord lumbar
ND
Tonsil
0/3
-
0/4
180 dpi -
ND -
0/4
0/3
360 dpi -
940 dpi
-
0/4
-
4/4
ND
0/3
-
0/4
-
4/4
+/++
ND
0/3
-
0/4
-
4/4
+/+++
0/3
-
0/4
-
4/4
+/+++
+/+++
4/4
+++
ND -
540 dpi
0/3
0/3
-
1/3 (a)
Parotid LN
0/3
-
0/4
-
0/3
-
1/3 (a)
Retropharyngeal LN
0/3
-
0/4
-
0/3
-
2/3 (a-b)
++
2/4 (a-b)
+/+++
+
3/4 (a-c)
++/+++
4/4
+++
+/++
3/4 (a -c)
++/+++
4/4
++++
Spleen
0/3
-
0/4
-
0/3
-
0/3
-
Duodenal PP
0/3
-
0/4
-
0/3
-
3/3
+++
1/4 (a) 4/4
+
4/4
+/+++
+++
4/4
+++
Jejunum PP
0/3
-
0/4
-
2/3 (a-b)
+
3/3
+++
4/4
+++
4/4
+++
Ileum PP
0/3
-
0/4
-
2/3 (a-b)
+/++
3/3
+++
4/4
+++
4/4
+++
Caecum PP
0/3
-
0/4
-
0/3
-
1/3 (a)
++
4/4
++/+++
4/4
++/+++
Jejunal MLN
0/3
-
0/4
-
2/3 (a-b)
+
3/3
+/+++
4/4
+++
4/4
+++
+++
Ileal MLN
0/3
-
0/4
-
1/3 (a)
+
3/3
4/4
++++
4/4
+++
Médiastinal LN
0/3
-
0/4
-
0/3
-
1/3 (a)
+
2/4 (a-b)
+/+++
4/4
+++
Prescapular LN
0/3
-
0/4
-
0/3
-
1/3 (a)
+
0/4
-
4/4
+/+++
Retro hepatic LN
0/3
-
0/4
-
0/3
-
1/3 (a)
++
4/4
++/+++
4/4
++/+++
Duodenum (ENS)
0/3
-
0/4
-
0/3
-
0/3
-
4/4
+/++
4/4
+/++
Jejunum (ENS)
0/3
-
0/4
-
0/3
-
1/3 (a)
+
4/4
+/++
4/4
+/++
Ileum (ENS)
0/3
-
0/4
-
0/3
-
1/3 (a)
+
4/4
+/++
4/4
+/++
Caecum (ENS)
0/3
-
0/4
-
0/3
-
0/3
-
3/4 (a-c)
+/++
4/4
+/++
Colon (ENS)
0/3
-
0/4
-
0/3
-
0/3
-
3/4 (a-c)
+/++
4/4
+/++
Sciatic nerve
0/3
-
0/4
-
0/3
-
0/3
-
0/4
-
4/4
+
Brachial nerve
0/3
-
0/3
-
0/3
-
0/3
-
0/4
-
4/4
+
External ocular muscle
0/3
-
0/3
-
0/3
-
0/3
-
0/4
-
4/4
+
Goats with a positive reaction in the examined tissues are indicated as the number of positive individuals out of the number of goats examined at each time point. At each time point the positive goats are identified (in brackets), arbitrarily by the letter a, b, c or d. For spinal cord C3-C4 (cervical) , Th7-8 (Thoracic) and L3-4
30 31 32
21 (Lumbar) segments were analysed. PP: Peyer’s Patches. ENS: enteric nervous system. PrPSc-labelling intensity are indicated as negative (-), minimal to slight (+), moderate (++) or strong (+++) as previously described (3).
22 33
Sc
Table 2: PrP detection in tissues of wild type (WT) and I/M142 PRNP genotype goats orally challenged with scrapie and sequentially killed
120 dpi
Organ/Age WT
34
Obex
0/5
Spinal cord cervical
ND
Spinal cord thoracic
ND
360 dpi I/M142
-
0/5
WT -
760 dpi I/M142
WT
1040 dpi I/M142
WT
I/M142
0/5
-
0/5
-
4/5 (a-d)
+/+++
0/5
-
5/5
ND
0/5
-
0/5
-
1/5 (a)
+
0/5
-
4/5 (a-d)
+/+++
0/5
-
ND
0/5
-
0/5
-
1/5 (a)
++
0/5
-
5/5
++/+++
0/5
-
-
5/5
++/+++
0/5
-
Spinal cord lumbar
ND
0/5
-
0/5
-
1/5 (a)
+/++
0/5
Tonsil
0/5
-
ND 0/5
-
1/5 (a)
+
0/5
-
4/5 (a-d)
+/+++
5/5
Parotid LN
0/5
-
0/5
-
0/5
-
0/5
-
4/5 (a-d)
+/++
3/5 (a-c)
Retropharyngeal LN
0/5
-
0/5
-
0/5
-
0/5
-
4/5 (a-d)
+/+++
5/5
Spleen
0/5
-
0/5
-
0/5
-
0/5
-
4/5 (a-d)
+/++
2/5 (a, b)
Duodenal PP
0/5
-
0/5
-
1/5 (a)
Jejunum PP
0/5
-
0/5
-
0/5
Ileum PP
0/5
-
0/5
-
2/5 (a-b)
Caecum PP
0/5
-
0/5
-
0/5
-
Jejunal MLN
0/5
-
0/5
-
1/5 (a)
+++
2/5 (a,b)
+/++
5/5
3/5 (a-c)
+/+++
+/+++
5/5
3/5 (a-c)
+/+++
+/+++
5/5
3/5 (a-c)
+/+++
+
5/5
+/+++
2/5 (a, b)
+
++
0/5
-
5/5
+++
5/5
++/+++
5/5
++/+++
4/5 (a-d)
++/+++
-
0/5
-
5/5
+++
5/5
++/+++
5/5
+/+++
4/5 (a-d)
++/+++
1/5 (a)
+
5/5
+++
5/5
++/+++
5/5
+++
4/5 (a-d)
++/+++
0/5
-
5/5
++/+++
4/5 (a-d)
+/+++
5/5
++/+++
4/5 (a-d)
+/+++
+
1/5 (a)
+
5/5
++
5/5
++
5/5
+++
4/5 (a-d)
+/+++ +++
++
Ileal MLN
0/5
-
0/5
-
2/5 (a-b
++
0/5
-
5/5
+++
5/5
++
5/5
+++
4/5 (a-d)
Médiastinal LN
0/5
-
0/5
-
0/5
-
0/5
-
4/5 (a-d)
+/++
4/5 (a-d)
+
5/5
++/+++
2/5 (a, b)
++/+++
Prescapular LN
0/5
-
0/5
-
0/5
-
0/5
-
4/5 (a-d)
+/++
0/5
-
5/5
++/+++
2/5 (a, b)
+/++
Retro hepatic LN
0/5
-
0/5
-
0/5
-
0/5
-
5/5
++/+++
3/5(a-c)
+/++
5/5
++/+++
4/5 (a-d)
++/+++
Duodenum (ENS)
0/5
-
0/5
-
0/5
-
0/5
-
5/5
+/++
4/5 (a-d)
+/++
5/5
+/++
4/5 (a-d)
+/++
Jejunum (ENS)
0/5
-
0/5
-
0/5
-
0/5
-
5/5
+/++
5/5
+/++
5/5
+/++
4/5 (a-d)
+/++
Ileum (ENS)
0/5
-
0/5
-
0/5
-
0/5
-
5/5
+/++
5/5
+/++
5/5
+/++
4/5 (a-d)
+/++
Caecum (ENS)
0/5
-
0/5
-
0/5
-
0/5
-
5/5
+/++
5/5
+/++
5/5
+/++
4/5 (a-d)
+/++
Colon (ENS)
0/5
-
0/5
-
0/5
-
0/5
-
4/5(a-d)
+/++
4/5 (a-d)
+/++
5/5
+/++
4/5 (a-d)
Sciatic nerve
0/5
-
0/5
-
0/5
-
0/5
-
1/5 (a)
+
0/5
-
5/5
+
0/5
+/++ -
Brachial nerve
0/5
-
0/5
-
0/5
-
0/5
-
1/5 (a)
+
0/5
-
5/5
+
0/5
-
External ocular muscle
0/5
-
0/5
-
0/5
-
0/5
-
1/5 (a)
+
0/5
-
5/5
+
0/5
-
35 36 37 38 39
23 Goats with a positive reaction in the examined tissues are indicated as the number of positive individuals out of the number of goats examined at each time point. At each time point the positive goats are identified (in brackets), arbitrarily by the letter a, b, c or d. For spinal cord C3-C4 (cervical) , Th7-8 (Thoracic) and L3-4 (Lumbar) segments were analysed. PP: Peyer’s Patches. ENS: enteric nervous system. PrPSc-labelling intensity are indicated as negative (-), minimal to slight (+), moderate (++) or strong (+++) as previously described (3).
24 40 41 42
Table 3: Scrapie incubation periods and PrPSc deposition in the central nervous system and the lymphoid tissues in goats inoculated by the oral route according to their genotypes at codons 142, 154, 211, 222 and 240 of the PRNP gene. Genotype
43 44 45 46
Intercurrent disease death
Scrapie affected animals
Scrapie incubation period in dpi (mean ±SD)
Number of goats
IRRQS/IRRQS (wild type genotypê)
9/9
1141±93
M142RQP240/IRRQS
4/4
1490±126
Time to death (dpi)
PrPSc accumulation Central Nervous system
Lymphoid tissues*
-
9/9
9/9
-
4/4
4/4
IH154RQS/IRRQS
0/6
-
3/6
966, 1002, 1853
0/3
0/3
IRQ211QS/IRRQS
0/5
-
2/5
1234, 1678
0/2
0/2
IRRK222S/IRRQS
0/5
-
1/5
1815
0/1
0/1
*: tonsil, prescapular lymph node, ileal/jejunal Peyer’s patches and mesenteric lymph node >2500 days post inoculation
25 47 48 49 50
Table 4: Scrapie incubation periods and PrPSc deposition in central nervous system and lymphoid tissues in intracerebrally inoculated goats according to their genotypes at codon 142, 154, 211, 222 and 240 of the PRNP gene.
Genotype
TSE clinically affected animals
Scrapie incubation period in dpi (mean±SD)
I142R154R211Q222/IRRQ (wild type genotypê)
5/5
486±21
Intercurrent disease death Number of goats
Time to death in dpi
PrPSc accumulation Central Nervous system
Lymphoid tissues*
-
5/5
5/5
M142RQ/IRRQ
5/5
788±99
-
5/5
5/5
IH154RQ/IRRQ
5/5
624±148
-
5/5
0/5
IRQ211Q/IRRQ
5/5
1291±325
-
5/5
5/5
IRQ211Q/ IRRQ211Q
10/10
770±139
-
10/10
1/10
IRRK222/IRRQ
2/5
1900, 2174
3/5
2/5†
0/5
IRRK222/ IRRK222
1/5
2101
-
1/1
0/1
568, 898, 1062
51 52 53 54 55 56 57
Groups of five goats were intracerebrally challenged in temporal cortex with the same classical scrapie isolate used for oral challenge. Animals that are still alive are more than 2400 days post inoculation at the time of writing. *: tonsil, prescapular lymph node, ileal/jejunal Peyer’s patches and mesenteric lymph node † the two PrPSc positive animals were clinically affected