UMR 959 INRA-ENVT, Physiopathologie Infectieuse et Parasitaire des Ruminants, Ecole Nationale Vétérinaire, 23 Chemin des Capelles, 31076 Toulouse Cedex 3, France1
INRA, Laboratoire de Pathologie Infectieuse et Immunologie, Nouzilly, France2
INRA, Domaine de Langlade, Pompertuzat, France3
NVI, Department of Pathology, Oslo, Norway4
INRA, Station dAmélioration Génétique des Animaux, Auzeville, France5
Author for correspondence: Olivier Andréoletti. Fax +33 5 61 19 38 34. e-mail o.andreoletti{at}envt.fr
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Abstract |
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Introduction |
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Ovine susceptibility to TSE is controlled mainly by polymorphism at 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
). Animals with PrP genotypes V136R154Q171/VRQ, ARQ/VRQ and ARQ/ARQ are the most susceptible to scrapie, whereas homozygous or heterozygous AHQ and heterozygous ARR animals show only marginal susceptibility. ARR/ARR sheep are considered to be fully clinically resistant (Hunter et al., 1996
, 1997
).
Under natural conditions, the transmission of the TSE agent between individuals is still poorly understood. A case control study in BSE-affected cattle has suggested that maternal transmission may occur at low levels. The existence of post-natal transmission was not evaluated (Wilesmith et al., 1994 , 1997
) but no infectivity was detected in the placentas of clinically BSE-affected cows (Wrathall, 1997
). Concerning human CJD (sporadic and variant), transmission from mother to child has never been observed until now (Berrebi et al., 1997
), despite the detection of placenta infectivity in one case (Tamai et al., 1992
).
Scrapie in small ruminants is certainly the best-documented model for TSE transmission. Numerous epidemiological data support the possibility of maternal transmission. Offspring from scrapie-affected ewes show a higher risk of developing scrapie than offspring from asymptomatic ewes (reviewed by Detwiler, 1992 ; Hoinville, 1996
). Unfortunately, most of the original data were collected from dams and sires with a possibly misclassified infectious status and unknown PrP genotype (Dickinson et al., 1965
). However, recent and reliable observations argue strongly for maternal transmission, as lambs born from scrapie-incubating ewes present a higher risk of developing scrapie (Hoinville, 1996
; Elsen et al., 1999
).
The hypothesis of scrapie transmission via germinal cells has been assessed in experimental infection models using the technique of embryo transfer. Results obtained from scrapie in sheep (Foster et al., 1992 , 1996
; Foote et al., 1993
) or BSE in goats (Foster et al., 1999
) did not lead to any striking evidence on this possible pathway of transmission of the TSE agent in small ruminants.
The presence of the scrapie agent in placentas from clinically scrapie-affected goats (Pattison et al., 1972 , 1974
) and ewes (Onodera et al., 1993
) was demonstrated by mouse bioassays. PrPSc accumulation was detected in foetal chorion from pregnant scrapie-incubating ewes. However, the placenta from a scrapie-incubating ewe can be found to be PrPSc-positive in its first gestation and negative in the following one (Race et al., 1998
; Tuo et al., 2001
). Until now, no clear explanation for this phenomenon has been proposed and the transmission pathway of scrapie from dams to offspring remains uncertain as there is no direct evidence for scrapie infectivity in lambs either in utero or post-partum.
Using sheep naturally exposed to scrapie, we identified the PrPSc-accumulating cell subsets in the placenta and demonstrated the role of the foetal PrP genotype in the control of PrPSc accumulation in this organ. We also investigated the presence of the abnormal PrP isoform in susceptible VRQ/VRQ lambs born from either scrapie-incubating dams accumulating PrPSc in their placentas or scrapie-negative dams with PrPSc-negative placentas. Our results suggest that the most likely pathway of scrapie transmission from ewes to lambs is an early lateral post-natal event rather than in utero transmission.
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Methods |
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The PrP gene polymorphism at codons 136 (A/V), 154 (R/H) and 171 (R/Q) was determined for each animal used in this study (Labogena) by the RFLPPCR method (Clouscard et al., 1995 ).
Ewes with the chosen PrP genotypes (ARR/ARR, ARR/VRQ, ARQ/VRQ and VRQ/VRQ) were inseminated with either ARR/ARR or VRQ/VRQ ram semen, according to the experimental group. No natural mating or contact occurred with the rams.
As described previously, the presence of the scrapie agent in all ewes was investigated by immunohistochemical (IHC) detection of PrPSc in palatine tonsil biopsies (Dickinson et al., 1965 ; Schreuder et al., 1996
, 1998
; Roels et al., 1999
). Finally, 80 lambing ewes were included in this study and were sampled twice, 1 month prior to artificial insemination (AI) and 1 month after lambing. Each animal showed an identical PrPSc accumulation result in the two consecutive tonsil biopsies. No clinical signs of scrapie were observed during the experimental period in any of the animals. At lambing, all ARQ/VRQ and VRQ/VRQ ewes were between 15 and 20 months old (first lambing), while ARR/VRQ and ARR/ARR ewes were between 18 and 48 months old (first to fourth lambing). In this flock, VRQ/VRQ and ARQ/VRQ scrapie-affected sheep usually expressed clinical signs around 24 and 32 months, respectively (unpublished data).
Experimental group and tissue collection.
The influence of foetal PrP genotype on PrPSc accumulation in the placenta was investigated by producing groups of ARQ/VRQ, VRQ/VRQ, ARR/VRQ and ARR/ARR lambs. Dams (n=36) were assessed for PrPSc deposition in their peripheral lymphoid tissues (tonsil biopsy) and rams with the appropriate PrP genotype were used for AI purposes (Table 1). At lambing, at least two cotyledons and intercotyledonary foetal chorion for each placenta were sampled and fixed in a 10% neutral-buffered formalin solution for 10 days. Thereafter, paraffin-embedded samples were processed for IHC. Similar samples from the same location were frozen at -80 °C before PrPSc detection by ELISA.
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In order to investigate the influence of placental PrPSc accumulation on the timing of lamb infection, VRQ/VRQ lambs were produced from either ARR/VRQ ewes (PrPSc-negative tonsils, group 1, n=13 ewes) or VRQ/VRQ ewes (PrPSc-positive tonsils, group 2, n=8 ewes). A third group, used as a control, comprised ARR/VRQ lambs born from VRQ/VRQ ewes (PrPSc-positive tonsils, n=8) that had been inseminated with ARR/ARR ram semen.
Three lambs from each group were euthanized (exsanguination following 10 mg/kg pentobarbital sodium salt intravenous injection) at birth (before any suckling) and at 10, 21, 64, 104 and 144 days of age. For each lamb, 23 different tissues were sampled, including the umbilical system and the digestive tract (Table 2). IHC analysis for PrPSc detection was performed on all tissues.
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PrPSc single-labelling protocol.
Single PrPSc immunolabelling was carried out using mouse monoclonal antibody (mAb) 8G8 (IgG2a, raised against the human recombinant PrP protein and specifically recognizing the 95108 aa sequence, kindly provided by J. Grassi, CEA Saclay, France), used at a 1:2000 dilution of the ascites fluid (Krasemann et al., 1996 ).
Non-specific binding sites were blocked by incubating sections with 20% normal goat serum in 0·1 M TBS (pH 7·6) for 20 min. Primary antibody was then applied for 60 min at room temperature. A 30 min incubation with a biotinylated secondary goat antibody (1:100 dilution) specific for mouse immunoglobulins was performed before applying a streptavidinalkaline phosphatase complex (1:100 dilution) for 30 min. Immunostaining was achieved using BCIP/NBT (Dako).
PrPSc double-labelling protocol.
PrPSc-positive cell phenotyping was achieved using anti-CD and anti-cytokeratin antibodies. Antibodies were mAbs specific for: human CD68 [clone Ki-M6 (monocytes/macrophages), used at a dilution of 1:150; Serotec]; human CD20-like [clone BLA36 (B lymphocytes), used at a dilution of 1:50; Novocastra] or a rabbit anti-human CD3 antiserum [A0452 (T lymphocytes), used at a dilution of 1:100; Dako]. An anti-cytokeratin rabbit serum [A0575 (large spectrum cytokeratin), used at a dilution of 1:1600; Dako] was used for uninucleated trophoblastic epithelial cell characterization (Lee et al., 1990 ).
In the PrPSc double-labelling protocol, anti-PrP antibodies were either mAb 8G8 or a rabbit polyclonal antibody (pAb), R521-7 (van Keulen et al., 1995 ), diluted 1:1000 (raised against ovine PrP sequence peptide 94105 and kindly provided by L. van Keulen, CIDC, Lelystad, The Netherlands). A two-step method based on the simultaneous application of two primary antibodies followed by a simultaneous incubation with both secondary antibodies was applied as described previously (Andréoletti et al., 2000
). Revelation was performed sequentially using first the alkaline phosphatase substrate BCIP/NBT followed by the peroxidase chromogen using the AEC+ red end product (Dako).
Controls.
In order to characterize non-specific immunolabelling, each IHC run included negative serum controls in which the primary antibody was either omitted or replaced by normal rabbit or mouse serum. In addition, mouse mAbs were replaced by isotype-matched mAbs irrelevant to the tissue under investigation. Reproducibility of immunostaining was assessed using serial tissue sections from the same sample included in two successive IHC runs. For double-labelling, cross-reactivity controls were performed for each couple of primary antibodies and each sample in order to verify the absence of interspecies reactivity of secondary antibodies toward primary antibodies. The absence of a possible affinity between the two secondary antibodies was also checked.
PrPSc detection by ELISA.
A commercially available BSE detection test (Platelia BSE; Bio-Rad) was used according to the manufacturers recommendations. This test is used currently for the post-mortem diagnosis of BSE in bovine brain tissue but is also suitable for the diagnosis of scrapie in sheep: mAbs used in this immunometric assay also recognize the ovine PrP protein. The procedure established for bovine brain tissue was applied directly to sheep tissue. Briefly, 350±40 mg tissue was subjected first to three cycles of homogenization of 45 s each (maximum speed in an Ribolyser; Coger) in 1·5 ml of 5% glucose solution. Of the homogenate, 500 µl was filtered through a 25 gauge needle and incubated for 10 min at 37 °C with 500 µl of buffer A containing proteinase K. PrPSc was recovered as a pellet after the addition of 500 µl of buffer B and centrifugation for 5 min at 20000 g at room temperature. Supernatants were discarded and the pellets were dried. Finally, the pellet was dissolved in buffer C1 (5 min at 100 °C) and diluted 1:6 in R6 reagent. A synthetic PrP peptide specifically recognized by the two mAbs included with the immunometric assay was used as an internal standard to compare interassay results and to quantify ovine PrPSc in samples.
A total of 18 placentas was assessed for PrPSc by this method. Eight were collected from ARQ/VRQ ewes (PrPSc-positive tonsils) inseminated with VRQ/VRQ ram semen. Three others were sampled from ARQ/VRQ ewes (PrPSc-positive tonsils) inseminated with semen from an ARR/ARR ram and the last seven were from ARR/ARR ewes (PrPSc-negative tonsils) inseminated with semen from ARR/ARR rams. Moreover, tissues from five clinically scrapie-affected ARQ/VRQ ewes (obex, spleen, mesenteric lymph node, ileal Peyers patch and tonsil) were tested as positive controls and results were compared to accumulation levels of PrPSc in the placenta.
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Results |
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The absence of any detectable PrPSc accumulation in the tonsils of dams (n=22/36, Table 1) was associated with PrPSc-negative results in placental structures and, thus, irrespective of the PrP genotype of the foetus (n=38) or dam. PrPSc accumulation in the placenta was only identified in ewes with PrPSc-positive tonsils (n=14/36, Table 1
) and carrying a foetus with a susceptible PrP genotype. PrPSc deposition was detected by IHC in the placentas of all genetically susceptible offspring (n=13) from these ewes (n=9). Scrapie-incubating susceptible ewes (n=5) inseminated with scrapie-resistant ram semen resulted in heterozygous ARR foetuses (n=8) and no PrPSc accumulation was observed in the corresponding placenta (Table 1
).
PrPSc-accumulating cell subsets in the placenta
In the placenta, using both IHC and ELISA techniques, PrPSc accumulation was found to be restricted to cotyledonary tissues. In the intercotyledonary foetal chorion or the maternal uterine wall, no PrPSc was detected. PrPSc deposition in placentomes was multifocal (Fig. 1A) and localized within epithelial uni- (Fig. 1B
) and multinucleated cells (Fig. 1D
). Deposits were granular, intracytoplasmic and often massive. These epithelial cells were identified as trophoblasts using PrPSc/cytokeratin double-labelling (Fig. 1C
). No apparent cellular modification was associated with PrPSc accumulation in trophoblastic cells.
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Levels of PrPSc within cotyledons
According to ELISA data obtained from eight PrPSc-positive placentas of ARQ/VRQ ewes and different organs from five ARQ/VRQ ewes at the clinical stage of scrapie (Fig. 2), the relative concentration of PrPSc from 47 mg fresh cotyledon was found to be equivalent to 1 mg obex. Similarly, 8 and 4 mg positive cotyledon appeared, respectively, to be equivalent to 1 mg tonsil and 1 mg spleen from clinically scrapie-affected sheep.
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Scrapie infection of lambs in relation to PrPSc accumulation in the placenta
VRQ/VRQ lambs could be divided into two groups depending on the genotype of the ewe. Group 1 lambs were born from ARR/VRQ ewes (PrPSc-negative tonsil biopsies) inseminated with a VRQ/VRQ ram, whereas those of group 2 were born from VRQ/VRQ ewes (PrPSc-positive tonsil biopsies) inseminated with a VRQ/VRQ ram. No PrPSc accumulation was detected in tissues from offspring at 8 days before lambing, 0 days or 10 days post-partum, whatever their group. At 21 days of age, PrPSc was detected in ileal Peyers patches from one of three lambs in both groups, suggesting a primary site of entry through ileal gut-associated lymphoid tissues (GALT).
At 64 days of age, all animals in each group were PrPSc-positive for at least one tissue sample. PrPSc-positive tissues were more numerous in animals from group 2 than from group 1 but no major discrepancy was observed in the tissue distribution of the abnormal protein between these groups. From 104 days of age, PrPSc deposits extended progressively to all GALT formations and then to other secondary lymphoid organs, such as hepatic, prescapular, mediastinal lymph nodes and spleen, with no observable differences between the two groups (Table 2). Over the same period, there was no PrPSc accumulation detected in any of the tissues sampled from ARR/VRQ control lambs (group 3).
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Discussion |
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The apparent absence of PrPSc accumulation in placentas from scrapie-incubating ewes carrying ARR lambs, as assessed by IHC and ELISA, is consistent with and extends the similar observations reported recently from Suffolk sheep in the USA (Tuo et al., 2002 ) and is of primary importance for the control of scrapie. The introduction of the ARR PrP allele in infected flocks should not only reduce the incidence of the disease by making the animals less genetically susceptible (Dawson et al., 1998
) but should also prevent the environmental spread of the scrapie agent by the placental route from infected ewes. Moreover, a PrP genotype genetic control from the conceptus on placental PrPSc accumulation is a coherent explanation for previous observations (Race et al., 1998
) from scrapie-incubating ewes, in which the placentas were found to be either PrPSc-positive or -negative according to the lambing year. However, these results need confirmation on a larger scale before any final conclusion can be drawn. Furthermore, in rodent models, some authors have reported infectivity in spite of any detectable PrPSc accumulation (Somerville & Dunn, 1996
; Lasmezas et al., 1997
; Manousis et al., 2000
; Race et al., 2001
).
Ovine placenta is of the synepitheliochorial type (Fig. 3). It results from the imbrication of foetal chorionic villi (cotyledons) with maternal preformed endometrial crypts (caruncles). Cotyledons and caruncles combine to form placentomes, while in the intercotyledonary space, the chorion remains smooth, its superficial trophoblastic layer being immediately adjacent to the uterine epithelium. Sheep chorion is also characterized by the presence of binucleated trophoblastic cells (BNCs), which constitute 1520% of the trophoblastic epithelial cells (Wooding, 1983
, 1984
). At the level of the placentome, BNCs have been shown to fuse with the maternal caruncular epithelial cell membrane to form a foetomaternal hybrid trinucleate cell (TNC). The continuous migration of BNCs and fusion into TNCs produces hybrid foetomaternal syncytial plaques of limited extent, which completely replace the uterine epithelium in the placentomes throughout the gestation period. The trophoblast-derived nuclei, whether in uninucleated cells, BNCs, TNCs or syncytial plaques, are genotypically identical (Wooding et al., 1997
). In our study, PrPSc-accumulating epithelial cells were only placentomal multi- and uninucleated trophoblasts. The influence of the foetal PrP genotype on PrPSc accumulation in the placenta is consistent with the PrP genotype of the foetal trophoblastic cells.
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In PrPSc-accumulating placentas, CD68/PrPSc-positive cells were identified in caruncular endometrial chorion. Some of these CD68-positive cells were in very close contact with positive trophoblastic cells. Previous observations in lymphoid organs from scrapie-incubating sheep showed some CD68-positive cells accumulating PrPSc and circulating via the lymphoid system (Andréoletti et al., 2000 ). Studies in mice have pointed to a role for dendritic (CD68-positive) cells in the spread of the scrapie agent also through the lymphoid system (Beringue et al., 2000
; Huang et al., 2002
). However, the lack of PrPSc-positive cells within the placental chorion from scrapie-incubating ewes with PrPSc-negative placentas, and the absence of CD68/PrPSc-positive cells in uterine chorion intercotyledonary spaces from ewes with PrPSc-positive placentas, does not sustain the hypothesis of trophoblastic cell contamination by CD68-positive cells. BNCs produce and deliver proteins through granule secretions (Wooding, 1992
; Wooding et al., 1993
), which can be phagocyted by maternal uterine mucosal chorion CD68-positive cells, making PrPSc accumulation within this cell subset possible. In this case, the contamination pathway of trophoblastic cells, via peripheral nerve fibres or circulating cells, remains to be determined.
Endometrial epithelial and trophoblastic cells share the same environment in the uterus of a pregnant ewe. However, one set of cells (endometrial epithelium) remains PrPSc-negative, while the other (trophoblast) accumulates large amounts of PrPSc. This question of cellular permissiveness to PrPSc accumulation and the comparison of trophoblastic cells with other epithelial cells (permissive/non-permissive) will be interesting to investigate.
In late gestation, no apparent PrPSc was found within VRQ/VRQ foetuses, while PrPSc deposition was observed within the placentas of their VRQ/VRQ dams. Further experiments (bioassays) are required to assess definitively the absence of scrapie infectivity in the foetus. This result suggests the absence of in utero transmission via foetal blood circulation or amniotic liquid, which are the two most common routes of foetus infection. Placental connections in sheep ensure total isolation of the foetus from the chorionic structure (trophoblast) and is a possible explanation for non-contamination of lambs in utero (Foote et al., 1993 ; Tuo et al., 2001
). However, no definitive statement can be made for transplacental PrPSc dissemination in case of placentitis or accidental loss of the continuity of the foetomaternal barrier.
Placentation in cattle and humans differs from sheep. In the bovine species, there is no fusion of embryonic trophoblastic cells with maternal uterine cells. The absence of contact between trophoblastic cells and maternal chorion (Guillomot, 1995 ; Wooding et al., 1997
), combined to the apparent absence of the prion agent in peripheral tissues of BSE-affected cattle, could explain the absence of PrPSc accumulation in the placentas in this species. In humans, the placenta is characterized by a total fusion between the embryonic and maternal structures (haemochorial) (Enders & Blankenship, 1999
). Such a structure would be more likely to (i) allow contamination of the embryonic placenta and (ii) facilitate possible embryonic contamination in utero. Infectivity studies on a placenta from a pregnant CJD patient has been reported once (Tamai et al., 1992
). However, no effective transmission of the disease has been noticed yet in children (who are now 29, 17, 14 and 10 years of age) born from CJD-affected mothers (Berrebi et al., 1997
), and previous studies in an experimental primate model argued against a vertical transmission in case of haemochorial placentation (Amyx et al., 1981
).
No difference was observed in the spatial PrPSc dissemination pattern in genetically susceptible VRQ/VRQ lambs, born from ewes with (group 2) or without (group 1) PrPSc deposition in their placentas and tonsils. Ages at first detection of PrPSc in ileal Peyers patches did not differ between the two groups of lambs, suggesting that these lambs could have been infected after birth by contact with infectious material in the surroundings rather than by in utero contamination. However, TSE infections have been reported to occur in the absence of detectable PrPSc (Somerville & Dunn, 1996 ; Lasmezas et al., 1997
; Manousis et al., 2000
). According to this hypothesis, our data do not allow the dismissal of the theoretical possibility that lambs of group 1 could have been infected by their dams. Nevertheless, until now, no infectivity from such PrPSc-negative ARR/VRQ ewes was detected by mouse bioassay (unpublished data).
Previous epidemiological studies involving separation of lambs from their dams at different ages after lambing demonstrated clearly the influence of dam-to-offspring contact length on lamb scrapie contamination efficiency (Hourrigan et al., 1979 ; Hoinville, 1996
; Elsen et al., 1999
).
Our data highlight that lambing scrapie-infected ewes mated with genetically susceptible rams are a source of early and highly efficient lateral contamination, even for susceptible lambs born from scrapie-free ewes. The use of genetically resistant (ARR/ARR) rams in infected flocks will not only improve the ovine genetic resistance to scrapie on a long-term basis but also directly and dramatically reduce the factors of exposure.
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Acknowledgments |
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Received 30 April 2002;
accepted 21 June 2002.