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1 UMR INRA ENVT 1225, Interactions Hôte Agent Pathogène, Ecole Nationale Vétérinaire de Toulouse, 23 Chemin des Capelles, 31076 Toulouse, France
2 INRA Domaine de Langlade, 31450 Pompertuzat, France
3 INRA Station d'Amélioration Génétique des Animaux, 31326 Castanet Tolosan, France
Correspondence
O. Andréoletti
o.andreoletti{at}envt.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The accumulation of an abnormal isoform (PrPSc) of a normal cellular protein (PrPC) in tissues from infected individuals correlates with the presence of infectivity and is currently considered to be the only reliable biochemical disease marker (McKinley et al., 1983; Race et al., 2001). According to the prion hypothesis, PrPSc is an infectious protein, thought to be the causative agent of TSEs (Prusiner, 1982).
Ovine susceptibility to TSE (classical natural scrapie and experimental BSE) is controlled mainly by polymorphisms of the PRP gene encoding the PrP protein. The major mutations associated with susceptibility or resistance are located at codons 136 (A or V), 154 (R or H) and 171 (R, Q or H) (Clouscard et al., 1995; Hunter et al., 1996). V136R154Q171/VRQ, ARQ/VRQ and ARQ/ARQ PRP genotype animals are the most susceptible to scrapie, whereas homozygous or heterozygous AHQ and heterozygous ARR animals show only marginal susceptibility to natural exposure. Similarly, under natural conditions, ARR/ARR sheep are considered to be highly resistant to classical scrapie (Hunter et al., 1996, 1997).
Placentae from TSE-infected ewes are known to carry infectivity and PrPSc (Race et al., 1998). Recently, it has been shown that PrPSc accumulation in placental structures seems to depend on the genotype of the fetus (Andréoletti et al., 2002b; Tuo et al., 2002; Alverson et al., 2006). Indeed, fetal placentae obtained by mating VRQ/VRQ scrapie-incubating ewes with susceptible-genotype rams accumulate PrPSc, whilst placentae of heterozygous ARR fetuses (VRQ/VRQ scrapie-incubating ewes mated with ARR/ARR rams) remain PrPSc negative. This is especially important with regard to the dissemination of scrapie in infected flocks.
In this study, our main objective was to explore the mechanisms underlying the genetic control of PrPSc placental accumulation at a cellular level during the gestation period.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Animals were cared for following EU recommendations for animal welfare and under the supervision of the local INRA ethics committee. The PRP gene polymorphism at codons 136 (A/V), 154 (R/H) and 171 (R/Q) were determined for each animal using a single-nucleotide polymorphism minor groove binding probe technique (Labogena) (van Poucke et al., 2005).
PrP-genotyped ewes (ARR/VRQ and VRQ/VRQ) were inseminated with either ARR/VRQ or VRQ/VRQ ram semen, according to their experimental groups. At no time did the rams have any contact with the ewes. As described previously, the presence of scrapie in these ewes was assessed by immunohistochemical (IHC) detection of PrPSc following palatine tonsil biopsy (Schreuder et al., 1996, 1998).
VRQ/VRQ ewes that had positive tonsil biopsies were allocated randomly into one of two experimental groups, A (n=12) and B (n=12). A third group, C (n=12) comprised ARR/VRQ dams with negative tonsil biopsies. Group A dams were inseminated with semen from a VRQ/VRQ ram to produce VRQ/VRQ fetuses. Group B ewes were inseminated with ARR/VRQ ram semen to obtain either VRQ/VRQ or ARR/VRQ fetuses. The probability of obtaining multiple fetal gestation of each genotype in individual ewes was increased because of the high fecundity rate in the Romanov breed (Freetly & Leymaster, 2004). Group C dams were inseminated with semen from the same VRQ/VRQ ram as that used in group A.
Three dams from each group were euthanized (exsanguination following 10 mg pentobarbital sodium salt kg–1 by intravenous injection) at 2, 3, 4 and 5 months of gestation (i.e. up to 140 days after artificial insemination; normal duration of gestation in Romanov sheep is 145–150 days). For each ewe, lymphoid tissues (mesenteric lymph node, tonsil, spleen, prescapular lymph node), brain (obex) and reproductive tract tissues (ovary, intercaruncular uterine wall at three different locations) were collected. For each fetus, three cotyledons, chorion, amniotic fluid and umbilical cord were sampled carefully. Additionally, a fetal ear sample was collected for DNA extraction and genotyping.
Each sample was divided into two equal parts. One part was fixed for 10 days in a 10 % formalin neutral buffered solution for immunochemical PrPSc detection using mouse monoclonal antibody 8G8 (IgG2a, raised against the human recombinant PrP protein and specifically recognizing the 95–108 aa sequence; kindly provided by J. Grassi, CEA Saclay, France) (Andréoletti et al., 2002b). The second part was frozen at –80 °C prior to PrPSc detection by ELISA (BSE detection test, Platelia BSE TM; Bio-Rad) (Andréoletti et al., 2002b, 2004).
Cotyledons from VRQ/VRQ fetuses obtained from PrPSc-negative ARR/VRQ ewes (as assessed by ELISA and IHC in all tissues investigated) were used for PrPC immunolabelling. Prior to this experiment, the absence of PrPSc in selected cotyledons was checked using both ELISA and Western blotting (not shown). PrPC labelling in situ was carried out with mouse monoclonal antibody BAR224 (IgG2a, raised against the ovine recombinant PrP protein; kindly provided by J. Grassi, CEA Saclay, France), which has a high affinity for sheep PrP (Féraudet et al., 2005), by incubation at a concentration of 4 µg ml–1 for 1 h at room temperature. Briefly, sections from paraffin-embedded cotyledons were treated for PrPSc detection (Andréoletti et al., 2002b) but treatment with formic acid was excluded. The specificity of PrPC labelling was assessed by controls in which the BAR224 antibody was replaced by isotype-matched mouse IgG2a.
Paraffin-embedded tissue blots were performed with sections from positive and control cotyledons (from ARR/VRQ PrPSc-negative ewes) as described previously (Schulz-Schaeffer et al., 2000; Andréoletti et al., 2004). These techniques enabled the distribution of PrPSc-positive structures to be visualized on tissue sections.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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In VRQ/VRQ ewes inseminated with VRQ/VRQ ram semen, PrPSc was detected in some fetal cotyledons as early as 2 months into gestation by ELISA, paraffin-embedded tissue blots and IHC (Figs 1a and 2a, d). From 3 months of gestation, all of the examined cotyledons were clearly positive and the level of PrPSc, as assessed by ELISA, increased exponentially (Fig. 1a).
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). Whilst all of the placentae of ARR/VRQ fetuses were negative, VRQ/VRQ fetal placentae were consistently positive at 3 months old. As expected, in some ewes (at 3, 4 and 5 months of gestation) both positive and negative placentae were detected, depending only on the fetal genotype (Fig. 1b).
The level of PrPSc accumulation in cotyledons from VRQ/VRQ fetuses in group A (Fig. 1a) and group B (Fig. 1b) at 3, 4 and 5 months was similar, indicating that the presence of an ARR/VRQ fetus in the same uterus did not interfere with PrPSc accumulation in VRQ/VRQ placentae.
PrPSc does not accumulate in placentae from VRQ/VRQ fetuses in ARR/VRQ scrapie-positive sheep
In the Langlade flock, the incidence of scrapie in ARR/VRQ sheep is extremely low (<5 %) (Elsen et al., 1999). Because of this low incidence, the ARR/VRQ ewes mated with VRQ/VRQ rams (group C) were initially included as controls in this study. However, at necropsy, three ARR/VRQ ewes were found to have accumulated low but consistent levels of abnormal PrPSc in various secondary lymphoid formations (ileal mesenteric lymph node, prescapular lymph node) and the enteric nervous system. PrPSc deposits were also detected in the obex of three animals, indicating that these ewes were in the later stages of scrapie incubation, but with an absence of clinical signs. Absence of PrPSc in ARR/VRQ scrapie-infected sheep tonsil has already been described on several occasions and explains the negative tonsil biopsies in these three animals (van Keulen et al., 1996a; Andréoletti et al., 2002a).
One of the ARR/VRQ scrapie-positive ewes was euthanized at 2 months of gestation and two at 4 months of gestation. From these last two ewes, eight VRQ/VRQ fetuses and one ARR/VRQ fetus were recovered (four fetuses from the first ewe and five from the second). Whilst group A and B ewes in late gestation (5 months) had associated high levels of PrPSc accumulation in the cotyledons of VRQ/VRQ fetuses (Fig. 1a, b), no PrPSc was observed in the placentae from the scrapie-incubating ARR/VRQ ewes, regardless of fetal genotype.
PrPSc accumulation in the placentome
In group A ewes, focal PrPSc deposits were observed in the cotyledons of two VRQ/VRQ fetuses at 2 months of gestation (Fig. 2a). By 3 months, the deposits were multifocal in the placentae from all fetuses. By 4 months, PrPSc deposits had extended and begun to coalesce (Fig. 2b). In most cases, about 50–60 % of the tissue surface was positive. By 5 months of gestation, about 90–100 % of the epithelial caruncular interface was positive (Fig. 2c).
In VRQ/VRQ fetuses from infected VRQ/VRQ ewes early in gestation, isolated multinucleated trophoblasts (maternal and trophoblastic hybrid cells) were PrPSc positive (Fig. 2d). This observation suggested strongly that PrPSc accumulation could originate in these cells. At 2 and 3 months of gestation, PrPSc began to accumulate in small uninucleated trophoblastic cell foci surrounding positive multinucleated trophoblasts (Fig. 2e).
In cotyledons collected from PrPSc-negative ARR/VRQ ewes (group C – the absence of PrPSc was assessed by ELISA and Western blotting from various tissues including cotyledon), and whatever the genotype of the lamb, a strong and specific PrPC labelling, using BAR224 monoclonal antibody, was observed only in multinucleated trophoblasts, suggesting overexpression of PrPC by this cellular subtype (Fig. 3a). No signal was observed in the controls where primary anti PrP antibody was replaced by an isotype-matched mouse Ig, thus confirming the specificity of the labelling (Fig. 3b).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Tuo et al., 2002). In the same uterus, two different conceptuses, exposed to a similar risk of contamination, accumulated levels of PrPSc depending solely on their PrP genotype. Similar findings were recently reported in four ARQ/ARQ ewes mated with heterozygous ARR rams (Alverson et al., 2006). Our study substantially consolidates this result, using a large number of animals and different experimental conditions. Interestingly, our data also indicated a total lack of interference in PrPSc accumulation dynamics of VRQ/VRQ fetal placentae linked to the presence of ARR heterozygous fetuses. These observations suggest that both conspectuses behave as independent organisms in term of scrapie contamination.
ARR animals are unable to accumulate PrPSc in the placenta
In scrapie-incubating heterozygous ARR ewes (at an advanced stage of the disease), PrPSc could not be detected in fetal placentae, even those with the VRQ/VRQ genotype. This observation has major implications for the genetic control of scrapie and the understanding of the epidemiology of scrapie infection. ARR/ARR genotype sheep are considered to be highly resistant to classical scrapie under natural conditions of exposure and are selected in order to eradicate scrapie from sheep flocks (Dawson et al., 2003).
Introducing the ARR allele into scrapie-infected flocks is designed to reduce scrapie genetic susceptibility by producing, in the short-term, heterozygous ARR animals and then predominantly ARR/ARR animals. These genetic programmes have reduced considerably the dissemination of the scrapie agent through the placenta of incubating ewes with highly susceptible genotypes. Our study extends and reinforces these results by indicating that heterozygous ARR ewes seem unable to disseminate the agent through their placentae and hence participate in lateral or environmental contamination at lambing. However, because of the number of scrapie-incubating ARR heterozygous ewes in our study and the type of TSE agent involved, further experiments are needed before drawing definitive conclusions from this observation.
Contamination of trophoblastic cells
Ovine placenta is of synepitheliochorial type and results from the imbrications of fetal chorionic villi (cotyledons) with maternal preformed endometrial crypts (caruncules). In the placentome, binucleated trophoblastic cells are known to fuse with the maternal caruncular epithelial cell membrane to form a feto-maternal hybrid trinucleate trophoblastic cell. The continuous binucleated trophoblastic cell migration and fusion into trinucleate trophoblastic cells produces hybrid feto-maternal syncytial plaques (Wooding, 1983, 1984).
In VRQ/VRQ dams carrying VRQ/VRQ fetuses, the syncytial trophoblast is the initial cellular subset accumulating PrPSc. Interestingly, using IHC, multinucleated trophoblast cells also appeared to overexpress PrPC when compared with trophoblastic uninucleated cells or uterine epithelial cells. However, rapidly after accumulation in syncytial plaques, PrPSc also accumulated in uninucleated trophoblast cells with a centrifugal pattern originating from multinucleated trophoblastic cells.
Trinucleate trophoblastic cells and syncytial plates are chimeric cells harbouring the PrP gene from both the fetus and the dam. In ARR/VRQ ewes carrying VRQ/VRQ fetuses, syncytial plates express both the ARR and the VRQ alleles, whilst uninucleated trophoblast cells express only the fetal VRQ allele. Although in VRQ/VRQ ewes bearing VRQ/VRQ fetuses, the uninucleated trophoblastic cells were strongly positive, the lack of PrPSc accumulation in uninucleated trophoblastic cells from ARR/VRQ scrapie-infected ewes cannot be explained by genetic factors.
Taken together, these data suggest that multinucleated trophoblasts are key elements for uninucleated trophoblastic cell contamination. Expression of the ARR allele in that particular cellular subset could avoid contamination of uninucleated trophoblastic cells expressing only the VRQ allele.
Route of placental contamination
The lack of PrPSc accumulation in the placentae of VRQ/VRQ fetuses carried by scrapie-contaminated ARR/VRQ dams could be linked to inefficient spread of prions to the cotyledons of ARR/VRQ ewes. In scrapie-incubating ewes, the agent might spread through the organism following different pathways, such as blood or blood-circulating cells (Houston et al., 2000). In ARR/VRQ scrapie-incubating animals, the limited involvement of secondary lymphoid tissues during disease pathogenesis (van Keulen et al., 1996b; Andréoletti et al., 2002a) could limit the circulation of the agent in blood from these tissues. In addition to this, the peripheral nervous system might also be an efficient dissemination pathway of the agent (Andréoletti et al., 2004; Thomzig et al., 2004). However, from the results of our experiment, it was not possible to draw conclusions on the role of these two possible dissemination routes in placental trophoblastic cell contamination. Specially designed studies will be needed to answer this question.
Conclusion
Our study confirms that the way scrapie spreads at lambing in an infected flock is very complex. Agent release into the environment is not a clear-cut process; however, it is certainly governed by interdependent factors such as (i) the presence and number of ewes bearing susceptible genotypes, (ii) their infectious status and (iii) the number and PrP genotype of fetuses from each ewe. The assimilation of these factors into a scrapie epidemiological model remains a challenge.
Our observations also provide new insights into in vivo genetic-linked cellular permissiveness to prion infection.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Andréoletti, O., Berthon, P., Levavasseur, E., Marc, D., Lantier, F., Monks, E., Elsen, J.-M. & Schelcher, F. (2002a). Phenotyping of protein-prion (PrPsc)-accumulating cells in lymphoid and neural tissues of naturally scrapie-affected sheep by double-labeling immunohistochemistry. J Histochem Cytochem 50, 1357–1370.
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Received 20 May 2006;
accepted 12 November 2006.
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