Genetic resistance to Scrapie infection in experimentally challenged goats

JVI Accepts, published online ahead of print on 27 November 2013 J. Virol. doi:10.1128/JVI.02872-13 Copyright © 2013, American Society for Microbiolog...
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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.

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Genetic resistance to Scrapie infection in experimentally challenged goats

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Caroline Lacroux1†, Cécile Perrin-Chauvineau2†, Fabien Corbière1, Naima Aron1, Patricia Aguilar-Calvo3,

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Juan Maria Torres3, Pierrette Costes1, Isabelle Brémaud2, Séverine. Lugan1, François.Schelcher1, Francis

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Barillet3 and Olivier Andréoletti1 *

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Capelles, 31076 Toulouse Cedex, France

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2

AFSSA-Niort, Laboratoire d’études et de recherches caprines, BP 3081, 79012 Niort Cedex, France

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3

CISA- INIA, 28130 Valdeolmos, Madrid, Spain.

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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

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Running title: K222 goat carriers are resistant to Scrapie

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† both authors contributed equally to this work

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* Corresponding author: o. andreoletti

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[email protected]

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Abstract

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In goats, several field studies have identified coding mutations of the gene encoding for the Prion protein

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(I/M142, N/D146, S/D146, R/Q211 and Q/K222) that are associated with a lower risk of developing classical

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scrapie.

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However, the data related to the level of resistance to TSE of these different PRNP gene mutations are still

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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

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and/or heterozygous I/M142, R/H154, R/Q211 and Q/K222 goats with a goat natural scrapie isolate by either the

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oral or the intracerebral (IC) route. Our results indicate that the I/M142 PRNP polymorphism does not

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provide substantial resistance to scrapie infection following intracerebral or oral inoculation. They also

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demonstrate that the H154, Q211 and K222 PRNP allele carriers are all resistant to Scrapie infection following

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oral exposure. However in comparison to WT animals the H154 and the Q211 allele’s carriers displayed only a

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moderate increase in the incubation period following IC challenge. After IC challenge, heterozygous K222

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and a low proportion of the homozygous K222 goats also developed the disease but with incubation period

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that were 4-5 times longer than in WT animals.

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These results support the contention that K222 goat Prion protein variant provides a strong but not absolutely

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protective effect against classical scrapie.

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3 42

Introduction

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Transmissible spongiform encephalopathies (TSE), or prion diseases, are fatal neurodegenerative disorders

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occurring in small ruminants (scrapie), cattle (bovine spongiform encephalopathy - BSE), or humans

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(Creutzfeldt-Jakob disease - CJD). The key event in TSE is the conversion of a normal cellular protein

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(PrPC) into an abnormal isoform (PrPSc) which accumulates in tissues in infected individuals. According to

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the prion concept, abnormal PrP is the causative agent of TSEs (11).

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In sheep, the susceptibility to TSE is strongly modulated by polymorphisms of the prion protein gene

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(PRNP) and the nature of the prion disease agent (strain) (8). The A136R154R171 allele is associated with a

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highly protective effect against natural or experimental infection with classical scrapie and BSE agents,

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while the V136R154Q171 allele and the wild type A136R154Q171 allele are associated with susceptibility (16,

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26). However, in sheep the ARR allele does not provide any particular protection against atypical scrapie

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whereas the R/H154 or L/F141 amino acid substitutions are associated with an increased risk of occurrence of

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this TSE (9, 17, 28 , 29).

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At the European level, the selection of the ARR allele carriers was successfully applied for controlling and

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eradicating classical Scrapie in infected sheep flocks (30). At a population level, large scale selection

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programs were also implemented. They aimed to increase the frequency of the ARR allele in the general

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population making it less favourable to TSE agent circulation and spreading. This ‘Breeding for resistance

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policy’ in combination with the other eradication measures, resulted in a significant reduction of the

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classical Scrapie prevalence in populations where it was comprehensively applied (15, 18, 24).

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In goats, several field studies have identified coding mutations of the PRNP gene that are associated with a

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lower risk of developing classical scrapie; namely the I/M142, N/D146 and S146, R/Q211 and Q/K222 (1, 7, 22,

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31, 32, 2011 #11698, 35). However, the low frequency of these alleles in goat population limits the

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possibility of reaching an unequivocal conclusion about the resistance/susceptibility to infection associated

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with these different PRNP genotypes (1, 8, 35). In that context, experimental TSE inoculation in goats is a

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straightforward and robust approach to better assess the level of resistance associated to certain PRNP

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polymorphisms in this species (2, 34).

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In this study, we inoculated wild-type genotype goats and homozygous and/or heterozygous I/M142, R/H154,

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R/Q211 and Q/K222 goats either by the intracerebral or the oral route with a goat natural Scrapie isolate in

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order to characterize their relative resistance/susceptibility to infection.

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Methods

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Ethics Statement

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All animal experiments have been performed in compliance with institutional and French national

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guidelines, in accordance with the European Community Council Directive 86/609/EEC. The experimental

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protocol was approved by the INRA Toulouse/ENVT ethics committee.

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Scrapie inoculum

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The inoculum was derived from a single natural field Scrapie case (clinical) obtained in a 3.5 year old goat

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with the wild type PRNP genotype. This animal was necropsied under TSE sterile conditions and its central

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nervous system (brain and spinal cord) was used to prepare a 10% tissue homogenate in 5% glucose. The

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stock homogenate was aliquoted and stored at -80°C.

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Experimental animals’ production

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Goats kids intended to be used in the experiment were produced by direct mating of PRNP sequenced

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Alpine and Saanen female goats and bucks. Parents were selected from three herds that are managed by the

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French National Agronomic Institute (INRA). Selection was based on the PRNP polymorphism at codons

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142 (I/M), 154 (R/H), 211 (R/Q) and 222 (Q/K) which were identified by previous study to influence

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susceptibility to natural Scrapie (7, 14, 35). Animals were then naturally mated to produce the goats used for

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experimental inoculation. The Exon 3 of the PRNP gene of each goat kid was sequenced as previously

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described (6).

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Goat oral challenge experiments

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For the oral challenge experiment, gravid goats were relocated to ANSES Niort A2 facilities. Within 48

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hours after birth each goat kid received 1.5g brain equivalent material through natural suckling (1% diluted

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stock inoculum in glucose 5%). A second inoculation (same material and route) was performed at the age of

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30 days. Considering (i) the logistic constraints (housing of goats and goat kids) and (ii) the fact that the

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parent goats were only heterozygous for the alleles of interest, the oral inoculation of goat kids that would

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have been homozygous for the mutated PRNP alleles was not feasible in the framework of this experiment.

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Two separate oral inoculation experiments were performed. The first one aimed at establishing the PrPSc

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dissemination scheme and kinetics in animals with the wild type PRNP genotype. For that purpose, 3 or 4

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animals were culled 30, 90, 120, 360, 540 and 940 days post inoculation (dpi). A last group of animals

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(n=4) was kept until the occurrence of clinical signs.

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The second experiment aimed at establishing the relative susceptibility of goats harbouring various

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genotypes to scrapie following oral exposure. WT, heterozygous I/M142, R/Q211 and Q/K222 animals were

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orally challenged using the same isolate as the first experiment, and culled at 120, 360, 760, 1040 dpi. Five

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animals of each genotype were killed at each of the different time points.

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In addition a group of animals from each of these genotypes, and a group (n=6) of heterozygous R/H154

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animals that had also been challenged orally were kept alive for establishing the incubation period. Because

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of space constraint in the animal facilities, it was not possible to challenge a sufficient number of R/H154

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PRNP allele carriers to complete the time point experiment (see below).

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Goat intracerebral challenge

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After weaning, goat kids selected by genotype were transported to UMR INRA ENVT A2 animal facilities

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for intracerebral (IC) inoculation. When the animals were six months of age they were anesthetised

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(Ketamine/Valium) and 400µL of the stock inoculum was injected in temporal cortex.

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Clinical monitoring and sample collection

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Inoculated goats were clinically monitored on a daily basis. The animals that developed TSE were

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euthanased when exhibiting locomotor signs that impaired their feeding capacity. Animals that developed

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intercurrent health problems were treated by qualified veterinarians and euthanased if the condition was not

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curable.

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Dead animals were systematically necropsied and central nervous system (CNS), a variety of lymphoid

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(mesenteric lymph node, tonsil, Prescapular lymph node, Peyer’s patches) and non-lymphoid tissues were

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collected (Table 1). Half of the samples were formalin fixed while the other half was stored frozen (-20°C).

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PrPSc Immunohistochemestry (IHC) and PrPres ELISA detection

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PrPSc IHC detection was performed as described in Lacroux et al using 8G8 antibody raised against human

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recombinant PrP protein and specifically recognising the 95-108 amino acid sequence (SQWNKP) of the

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PrP protein (27).

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Western-blot (WB) of the abnormal PrP

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PK resistant abnormal PrP extraction (PrPres) and Western blot were performed as previously described (5),

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using a commercial extraction kit (Biorad, France). PrP immunodetection was performed using either Sha31

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monoclonal antibody (0,06 µg per mL, epitope: YEDRYYRE , amino acid 145-152) or 12B2 (4 µg/mL)

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(epitope WGQGG, amino acid sequences 93-97) (19).

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For glycoprofiling of the PrPres, signal volume and relative percentage associated with each band was

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established using Quantity One® software (Bio-Rad) following immunoblot. For each sample, three

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independent measures were realized on three different gels.

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Results

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Oral challenge in goats

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Our first oral challenge experiment in goats was designed to establish the scheme and kinetics of PrPSc

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dissemination in the tissues of Wild type (WT) PRNP genotype animals. For that purpose, goat kids

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obtained by natural mating of WT PRNP genotype goats and bucks were orally challenged within the first

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48 hours following birth. Groups of these animals were culled at different time point after inoculation

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(Table 1). PrPSc accumulation was first observed in the gut associated lymphoid tissue (Peyer’s patches) in

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animals more than 180 dpi. As already described in sheep, PrPSc progressively spread to all lymphoid organs

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before becoming detectable (between 180 and 360 dpi) in the enteric nervous system (ENS) and later

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(between 540 and 940 dpi) in the central nervous system (3).

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On the basis of these results, a second oral challenge experiment was designed. The goal of this experiment

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was to characterize the impact of the investigated polymorphisms on the susceptibility and the PrPSc

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dissemination in the tissues of orally exposed animals. Groups of wild type animals and heterozygous

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I/M142, R/Q211 and Q/K222 were produced by natural mating and orally challenged using the same procedure

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and Scrapie isolate than in the first experiment.

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In this second experiment, the PrPSc dissemination scheme observed in WT genotype animals was consistent

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with the results of the first experiment (Table 2). No PrPSc deposition was observed in the tissues collected

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from goats killed at 120dpi. At 360dpi, significant PrPSc deposition was observed in various lymphoid

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tissues (Peyer’s patches, mesentery lymph nodes and tonsil) of some of the challenged individuals. PrPSc

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deposition was observed in ENS, CNS, peripheral nervous tissues and skeletal muscles in four out of the

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five animals culled at 760 dpi.

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In the heterozygous I/M142 orally challenged animals a similar but slightly delayed PrPSc accumulation

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scheme was observed; PrPSc was first detected in the gut associated lymphoid tissue (Peyer’s patches) at

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360dpi but it was only detected at 1040 dpi in the CNS (Table 2).

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No PrPSc was observed in any of the tissues collected in the heterozygous R/Q211 and Q/K222 animals that

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had been orally challenged and killed at the different time points (5 animals per genotype per time point),

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and none of the animals bearing these genotype had developed a clinical TSE after more than 2500 dpi.

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In both oral challenge experiments a group of animals harbouring the different PRNP genotypes (I/M142,

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R/Q211 and Q/K222) was kept alive and clinically monitored for TSE development (table 3). Similarly, a

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group of heterozygous R/H154 animals (n=6) that had been orally challenged with the same inoculum was

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also monitored.

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In WT goats, incubation periods in the first (n=4) and the second experiment (n=5) were not different. All

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the challenged I/M142 goats (n=4) also developed a clinical TSE but with slightly longer incubation period

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(1490±126 dpi) than in WT animals (1141±93 dpi). In both genotypes, affected animals showed PrPSc

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deposition in CNS and lymphoid tissues (table 2).

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After more than 2500 days of incubation, none of the orally inoculated R/H154, R/Q211 and Q/K222

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heterozygous animals had developed a clinical TSE. Some of the heterozygous R/H154 (n=3) or Q/K222

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(n=2) goats and one homozygous K/K222 animal died from intercurrent disease (table 3). In these orally

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challenged animals none of the investigated tissues (lymphoid organs and CNS) displayed any detectable

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PrPSc deposition.

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Together, these findings support the contention that R/H154, R/Q211, and Q/K222 PrP mutated alleles have a

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strong protective effect against Scrapie infection following oral exposure.

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Intracerebral challenge in goats

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To further assess the resistance to Scrapie associated to I/M142, R/H154, R/Q211 and Q/K222 PRP alleles,

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groups of heterozygous and homozygous animals were intracerebrally inoculated using the same isolate as

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the one used for oral challenge (table 4). As expected, all the intracerebrally inoculated WT goats developed

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a clinical TSE. In those animals, PrPSc deposits were observed in both the central nervous system and in

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lymphoid tissues.

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In contrast to the oral challenge experiment, the heterozygous I/M142, R/H154, R/Q211, but also the Q/Q211

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homozygous animals developed a clinical TSE. Strikingly, the heterozygous R/Q211 individuals displayed a

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longer incubation period than the homozygous Q/Q211. PrPSc deposition was observed in the lymphoid

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tissues of the heterozygous I/M142 and homozygous Q/Q211 animals. No or limited PrPSc accumulation was

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observed in lymphoid tissues from R/H154 and R/Q211 animals. Using Sha31 antibody, the WB PrPres

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patterns observed in the brain of all the R/H154 scrapie affected individuals was identical and differed

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strikingly from those observed in individuals bearing other PRP genotypes (figure 1); the PrPres bands

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displayed an apparent lower molecular weight. Immunoblot probed with the 12B2 antibody indicated that in

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H/R154 goats PK digestion resulted in a N-terminal cleavage of PrPres (amino-acid sequences 93-97) that

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differed from the other genotypes groups.

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In the IC challenged heterozygous Q/K222 goats three animals died of intercurrent disease at 568, 898, 1062

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dpi. No PrPSc accumulation was observed in any of the investigated tissues from these goats. However, the

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two remaining animals developed a clinical TSE after 1980 and 2134 dpi respectively and in those two

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individuals PrPSc deposits were observed (IHC and WB) in the central nervous system but not in lymphoid

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organs.

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One out of the five IC challenged homozygous K222 animals developed a clinical TSE and was euthanased

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after 2101 dpi. Abnormal PrP deposition was detected (IHC and WB) in the central nervous system but not

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in the lymphoid tissues. The four remaining K/K222 animals are still apparently healthy at the time of writing

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(more than 2400 dpi).

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Using the Sha31 antibody, Q/K222 and K/K222 positive individuals displayed a similar PrPres glycoform ratio.

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The PrPres glycoprofyle of these individuals displayed a dominant mono-glycosylated bands which clearly

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differed from the patterns observed in the goats with other genotypes (Figure 2).

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More generally, the presence of apparently different PrPres WB signature in IC challenged goats that harbour

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different genotypes suggest that different TSE agent propagated in those animals. However it is our opinion

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that bioassays (which are currently ongoing) remain necessary before concluding on that point.

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Discussion

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Cases controls studies in classical scrapie affected herds (1, 7, 22, 31, 32, 35) and limited data from

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experimental challenge (intracerebral routes) (2, 36) supported the view that K222 PrP allele goats might be

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strongly resistant to classical scrapie infection. Rare cases of the disease (n=3) were reported in

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heterozygote K222 goats belonging to one single flock that displayed a high disease prevalence (27.4%), and

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no case was reported so far in homozygous K222 animals (7, 14).

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Our study indicated that a classical scrapie isolate failed to propagate in K222 heterozygous goats following

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oral challenge. However, it also demonstrated that the same classical scrapie isolate can propagate in

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heterozygous and in a proportion of homozygous K222 animals following IC challenge, but with incubation

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period that exceeded 4 to 5 times those observed in WT animals.

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These results in K222 goats are very evocative of those obtained in the A136R154R171 allele sheep carriers that

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were naturally or experimentally exposed to TSE agents. After oral experimental challenge in homozygous

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and heterozygous ARR sheep no or poorly efficient propagation of classical scrapie and BSE agents was

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observed (4, 20, 21). In heterozygous ARR sheep that were IC challenged with classical scrapie, the disease

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occurred but with significantly longer incubation periods than in homozygous ARQ (wild type PRNP

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genotype) sheep (21). A clinical TSE occurred in a proportion of homozygous ARR that were IC challenged

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with cattle BSE and occurrence of rare natural classical scrapie cases was reported in animals harboring this

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genotype (23, 25).

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All these results support the view that like the ARR allele in sheep the K222 allele is associated with a high

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but not absolute resistance to Scrapie.

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In the I/M142 allele carriers, the IC and oral challenge results indicate that this allele is not associated with a

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substantial resistance to the classical scrapie isolate we used. These observations are consistent with data

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collected in naturally infected goats herds (7, 14) and with the observations previously reported by Goldman

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et al. in goats challenged with cattle BSE, CH1641 and ME7 passaged in sheep (22).

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No transmission or PrPSc deposition could be observed in orally challenged H154, and Q211 heterozygous

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animals. However a 100% attack rate was observed in animals bearing those genotypes following IC

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challenge, albeit with longer incubation period than in WT animals.

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These results were similar to those observed in heterozygous K222 animals. However, unlike K222

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homozygous goats, the Q211 homozygous animals developed the disease with a 100% attack rate following

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IC challenge, with shorter incubation periods than in Q211 heterozygous animals. This indicates that the Q211

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allele cannot be considered to provide the same level of resistance against scrapie as the K222 allele.

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The lack of H154 homozygous goats in the intracerebral inoculation experiment clearly limits our capacity to

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draw final conclusions concerning the level of resistance/susceptibility to classical scrapie associated with

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this PRNP allele. Nevertheless, the risk of atypical scrapie occurrence has been shown to be significantly

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higher in both H154 allele carrier sheep and goat (same PrPC sequence in goats and sheep) than in WT

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animals (13, 28). This higher susceptibility to atypical scrapie represents a major argument against the

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selection of the H154 PRNP allele in goat population

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Beyond this, the main limitation of this experiment is the fact that only one classical scrapie goat isolate was

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used to test the relative susceptibility of the different genotypes. The diversity of TSE agents in small

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ruminants has been documented for several decades (3, 12, 33). In sheep, the susceptibility to TSE infection

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was shown to be influenced by both the nature of the TSE strain and the PRNP polymorphisms (10).

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Considering the time and the resources necessary to carry out bioassay in large animals, testing several

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classical scrapie agents in parallel in this model was simply not feasible. In that context, the inoculation of a

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variety of TSE agent in transgenic mice that express the WT and K222 PRNP goat alleles will play a pivotal

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role in confirming the apparent low susceptibility / high resistance associated with this last allele.

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Finally, it should be noticed that the experimental approach we used only allowed estimating the impact of

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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

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both Q211 and K222 allele).

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The development of PRNP genotype selection programs is now being considered by the EU authorities as a

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potential tool to control and eradicate scrapie in commercial goat populations. In sheep, the diffusion of the

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ARR allele in the general population and, in particular, its introduction in classical scrapie affected herds

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has proven its efficacy for the long term control of the disease (15, 18, 24). The data that we report concur

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with the view that the K222 allele in goats provides a similar level of resistance against scrapie infection as

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the ARR allele in sheep.

281 282

Acknowledgments

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The authors wish to thank Frédéric Bouvier and his team for producing goat with appropriate PRP

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genotypes (‘INRA domaine de la Sapinière’). This work was funded by GIS PRION grant 31B06134, by

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INRA grant AIP P00297, by the Poitou-Charentes region grants 04/RPC-A-103 and 05/RPC-A-13 and

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European Union (FOOD-CT-2006-36353 and 219235 FP7 ERA-NET EMIDA). The authors declare no

287

competing financial interests.

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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

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