Department of Morphology, Genetics and Aquatic Biology1 and Department of Biochemistry, Physiology and Nutrition2, Norwegian School of Veterinary Science, PO Box 8146 Dep., N-0033, Oslo, Norway
Department of Sheep and Goat Research, Norwegian School of Veterinary Science, Kyrkjevegen 332/334, 4300 Sandnes, Norway3
Federal Research Centre for Virus Diseases of Animals, Paul-Ehrlich-Str. 28, 72076 Tübingen, Germany4
Author for correspondence: Charles Press. Fax +47 22964764. e-mail Charles.Press{at}veths.no
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Abstract |
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Introduction |
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Another important experimental observation for the early pathogenesis of scrapie is the need for expression of the normal cellular prion protein, termed PrPC. Studies have shown that transgenic mice not expressing PrPC are resistant to infection by the scrapie agent (Büeler et al., 1993 ) and that PrPC-expressing tissue is required for transmission of infectivity from the periphery to the central nervous system (Blättler et al., 1997
). While biochemical and genetic investigations have mapped expression of PrPC at the tissue and organ level in sheep (Brown et al., 1990
; Horiuchi et al., 1995
; Goldmann et al., 1999
), few immunohistochemical studies of lymphoid tissues have addressed the distribution of PrPC. Most immunohistochemical protocols have been directed toward detecting the accumulation of PrPSc that occurs during disease (van Keulen et al., 1996
; Miller et al., 1993
). Indeed, the diagnostic usefulness of these procedures is based on the inability of conventional immunohistochemical protocols to detect normal cellular PrP.
Gut-associated lymphoid tissue refers to the solitary and aggregated lymphatic follicles, subepithelial lymphocytes, plasma cells and macrophages and intraepithelial lymphocytes that are distributed along the whole length of the alimentary tract (Landsverk et al., 1991 ; Landsverk & Press, 1998
). In sheep, during the first months of life, a major continuous aggregation of lymphatic follicles, termed the ileal Peyers patch, is present in the ileum and terminal jejunum but by 18 months of age this major lymphoid organ has undergone involution and is all but non-existent (Carlens, 1928
; Reynolds & Morris, 1983
). During its life, the ileal Peyers patch is responsible for generating the vast majority of B-lymphocytes in the circulation and peripheral lymphoid tissues (Reynolds & Morris, 1983
; Gerber et al., 1986
; Press et al., 1996
) and for diversification of the pre-immune antibody repertoire (Reynaud et al., 1991
; Lucier et al., 1998
). Accordingly, the lymphatic follicles of the ileal Peyers patch consist predominantly of B-lymphocytes supported by an extensive network of mesenchymal stromal cells including follicular dendritic cells and reticular cells along with a population of tingible body macrophages (Nicander et al., 1991
; Halleraker et al., 1990
; Press et al., 1992
). T-lymphocytes, macrophages and dendritic cells are a significant presence in the lymphoid tissues overlying and adjacent to the follicles, namely the dome and interfollicular areas (Halleraker et al., 1990
; Press et al., 1992
). The ileal Peyers patch also possesses another feature of possible relevance to the pathogenesis of scrapie, namely a follicle-associated epithelium that can take up a wide spectrum of macromolecules and particles (Landsverk, 1987
, 1988
). The uptake of pathological agents across this specialized epithelium has been implicated in a number of diseases including salmonellosis and paratuberculosis infection (Wolf & Bye, 1984
; Momotani et al., 1988
; Landsverk et al., 1990a
).
Infection with the scrapie agent is considered to occur in young sheep, less than 9 months of age (Hourrigan et al., 1979 ). The apparent susceptibility of lambs to infection is influenced by a number of factors including PrP genotype, strain of agent and environmental factors such as artificial or natural rearing (Hunter et al., 1996
; Elsen et al., 1999
). During this period of high susceptibility to infection, the ileal Peyers patch is the major gut-associated lymphoid tissue possessing an extensive bed of follicular dendritic cells and a specialized epithelium actively engaged in the uptake and transcytosis of macromolecules from the gut. However, studies of the distribution of PrPSc in the lymphoid tissues of sheep with scrapie have mostly focused on the spleen and lymph nodes or investigated mucosa-associated lymphoid tissues at readily accessible sites such as the tonsils (Schreuder et al., 1998
). The present study was undertaken to investigate the distribution of PrP in the ileal Peyers patch of sheep. The distribution of PrP in lambs that had been exposed to scrapie-infected material either naturally or experimentally was compared with the distribution of PrP in scrapie-free lambs.
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Methods |
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An experimental oral infection of 11 lambs of known PrP genotype (Table 3) was performed. Each lamb was given a single dose of 15 ml of a 30% (w/v) homogenate of brain tissue (5 g brain tissue) by stomach tube. The homogenate for each PrP genotype contained frozen sheep brain tissue (-20 °C; transverse section of cerebrum anterior to hippocampus) from clinical cases of scrapie with the appropriate PrP genotype. The frozen tissues were pooled and mixed with physiological saline in a stomacher. The brain tissue from clinical cases with the PrP genotypes VV136RR154QQ171 and AV136RR154QQ171 had shown extensive histopathological changes typical for scrapie. The brain tissue from the clinical case with the PrP genotype AA136RR154QQ171, which is a PrP genotype that is seldom associated with disease, had only minor histopathological changes, restricted to the cerebellum. In all clinical cases, the diagnosis of scrapie was confirmed using immunohistochemistry as described by van Keulen et al. (1995)
, with polyclonal antibodies kindly provided by L. J. M. van Keulen (DLO-Institute for Animal Science and Health, Lelystad, The Netherlands) and Western blotting (National Veterinary Institute, Oslo, Norway).
One week after oral exposure to the scrapie agent, four lambs aged 79 weeks were necropsied and tissue samples collected. Five lambs were necropsied and tissue samples collected 5 weeks after oral dosing at an age of 1113 weeks. Two lambs were necropsied 11 months after oral dosing. Tissue samples were collected from control lambs that were the same age and had the same PrP genotype as the lambs orally exposed to the scrapie agent. The control lambs received an oral dose of physiological saline on the same day and in the same manner as the lambs exposed to the scrapie agent and were necropsied on the same day after oral dosing, namely 1 week (four lambs), 5 weeks (five lambs) and 11 months (one lamb) (Table 3).
For the three groups of lambs, namely abattoir-collected control lambs, and naturally and experimentally exposed lambs, tissue samples were collected from the ileal Peyers patch near the insertion of the ileo-caecal fold. The tissue samples were frozen in 1,1,1-trifluoretane/pentafluoretane (R404A, Ausimont Via S. Pietro, Bollate, Italy), chilled in liquid nitrogen and stored at -70 °C. To protect the mucosa during freezing and sectioning, the tissues were placed with the mucosa down onto pieces of liver. Sections 8 µm in thickness were cut on a cryostat and stored at -70 °C.
PrP genotyping.
PrP genotyping was performed on blood or frozen tissues as described previously (Tranulis et al., 1999 ). Briefly, tissue samples were thawed and gently detached from the supporting liver tissues and thoroughly washed in lysis buffer (10 mM TrisHCl pH 8·0; 100 mM EDTA; 1%, w/v, SDS) by vortexing followed by centrifugation at 10000 g for 30 s and removal of the supernatant to allow addition of fresh lysis buffer. This procedure was repeated five times to ensure adequate removal of any contaminant DNA from the liver slices. PrP polymorphisms were detected by automated DNA sequencing, using dye-terminator cycle sequencing of a PCR-generated product covering codons 93 to 216 of the PrP open reading frame (GenBank accession no. M31313). Samples were analysed by capillary electrophoresis on an ABI Prism 310 Genetic Analyser (Perkin Elmer).
Immunhistochemistry.
An avidinbiotinperoxidase immunohistochemical method (Vectastain ABC Kit, Vector Laboratories) was used to detect PrP in frozen tissue sections. This protocol did not distinguish between PrPC and PrPSc. The protocol incorporated an avidinbiotin blocking step (Avidin/Biotin Blocking Kit, Vector Laboratories) and was combined with a tyramide signal amplification system (TSA-Indirect, NEN Life Science Products) to increase the sensitivity of the protocol over that of conventional immunohistochemical procedures.
The frozen sections were allowed to dry for at least 2 h and were fixed in 10% formol-calcium for 10 min. The sections were washed in Tris-buffered saline (TBS) pH 7·5 for 10 min and then incubated with a blocking reagent (TSA-Indirect) containing 17% avidin (Vector Laboratories) for 20 min to block endogenous biotin. The blocking reagent from the TSA kit was used as diluent in all further steps in the protocol. The blocking serum was tapped off the slides, and the primary antibody containing 17% biotin (Vector Laboratories) was applied directly to the sections. The mouse monoclonal anti-PrP antibodies used in the present study were P4 (Harmeyer et al., 1998 ), L42 (Hardt et al., 2000
) and 6H4 (Korth et al., 1997
), and all had an IgG1 isotype. P4 and 6H4 both recognize an epitope in the same domain of ruminant PrP while L42 recognizes a different epitope in the first
-helix of ruminant PrP. The sections were incubated overnight at 4 °C. After washing for 10 min in TBS, the sections were incubated with a biotinylated secondary antibody (Vectastain ABC Kit) for 30 min, washed and then incubated for 10 min with 1% H2O2 in methanol to inhibit endogenous peroxidase. Following washing, the sections were incubated for 30 min with peroxidase-conjugated biotinavidin complex (Vectastain ABC Kit). After washing, the sections were incubated with biotinyl tyramide (TSA-Indirect) for 5 min, washed and then incubated with streptavidinhorseradish peroxidase for 30 min. Peroxidase activity was detected using 3-amino-9-ethyl carbazole (Sigma) for 10 min. The reaction was stopped by washing the sections in distilled water. The sections were counterstained with haematoxylin, mounted in polyvinyl alcohol and cover-slipped before examination. As a control for the anti-PrP antibodies used, the primary antibody was replaced with the blocking reagent containing 17% biotin, or with an irrelevant antibody of the same isotype as the primary antibody. A mouse monoclonal IgG1 antibody against rainbow trout immunoglobulin (K. Falk, personal communication) was used. Furthermore, sections from a confirmed scrapie case in an adult sheep were included in each immunohistochemical run. As a control for the tissues, an IgG1 monoclonal antibody against a sheep T-lymphocyte marker (SBU-T1) (Beya et al., 1986
) was used.
For sections that were not subjected to tyramide signal amplification, the initial 30 min incubation with the peroxidase-conjugated biotinavidincomplex was followed by washing and incubation with the aminoethylcarbazole solution, as above.
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Results |
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Lambs naturally exposed to scrapie
As with the control scrapie-free lambs, in sections not subjected to tyramide signal amplification, staining for PrP was detected only in the peripheral nervous tissue of the enteric plexus. With tyramide signal amplification, a prominent feature in lambs from the naturally exposed group was staining in the dome and the neck region of follicles (Fig. 3A). Small focal areas of staining were also observed to be associated with the luminal border of some epithelial cells in the follicle-associated epithelium and in some regions of the adjacent absorptive epithelium. A granular punctate pattern of staining was evident in the domes as was strong staining in the cytoplasm of large mononuclear cells. Strong staining was also present in large, single mononuclear cells in the interfollicular areas (not shown). In some follicles, the granular pattern of staining extended into the follicle and staining was prominent throughout the light central zone of the follicle (Fig. 3B
). The dark peripheral zone of the follicle, close to the capsule, tended to show little or no staining. The level of staining varied between follicles and between individuals.
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Discussion |
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PrP was consistently detected within the enteric nervous system of the intestinal wall from the three groups of lambs examined in the present study. The enteric nervous system associated with the ileal Peyers patch includes a myenteric plexus between the muscle layers, a submucosal plexus between the lymphoid follicles and nerve fibres extending into interfollicular areas and domes of follicles and to the lamina propria (Krammer & Kühnel, 1993 ). PrPSc has been detected in the peripheral nervous system of scrapie-diseased sheep (Groschup et al., 1999
) and has recently been reported in the myenteric and submucosal plexus of sheep with natural scrapie (van Keulen et al., 1999
). The wide distribution of PrP described in the present study supports the suggestion that the autonomic nervous system of the gut wall may be involved in the early pathogenesis of scrapie. However, the cells and nerve fibres of the enteric nervous system were not the only sites of PrP in control scrapie-free lambs. Mononuclear cells, presumably macrophages within the lamina propria and dome and interfollicular areas, also showed staining. Interestingly, there was little staining for PrP in the follicles of the ileal Peyers patch. The detection of PrP in the follicle capsule and in cells associated with vascular structures in the light central zone suggests an association in scrapie-free lambs with certain subpopulations of reticular fibroblasts rather than follicular dendritic cells (Nicander et al., 1991
). A number of morphological and possibly functional differences have been described between follicular dendritic cell populations in the ileal Peyers patch and the populations in germinal centres of lymph nodes and spleen. Indeed, it is debatable whether the follicular dendritic cells of the ileal Peyers patch are true follicular dendritic cells (Nicander et al., 1991
).
An important observation in the present study was the prominence of staining in the dome and neck regions of follicles in lambs naturally exposed to the scrapie agent. The extent of the granular pattern of staining and the presence of staining in large mononuclear cells indicate that PrP was present in a diverse cell population, which could include nerves, smooth muscle cells, macrophages, dendritic cells and reticular fibroblasts. The presence of staining associated with the luminal border of cells in the follicle-associated epithelium suggests uptake of PrP from the gut lumen. Studies conducted predominantly in calves have shown that the follicle-associated epithelium of the ileal Peyers patch possesses a homogeneous population of specialized epithelial cells (Landsverk, 1981 ) that is capable of internalizing macromolecules and particles including pathological agents (Reynolds & Morris, 1983
; Landsverk, 1987
, 1988
; Landsverk et al., 1990b
). This specialized epithelium is also able to eliminate residual bodies by exocytosis, bringing transcytosed material into contact with the well-developed subepithelial rim of macrophages and other cell populations in the dome (Landsverk, 1981
). The ileal Peyers patch is a large organ in young lambs, extending for up to 2·5 m and estimated to contain over 100000 follicles (Reynolds & Morris, 1983
). In these young animals, the continuous Peyers patch in the region of the ileum accounts for about 90% of the aggregated follicles in the whole alimentary tract. Thus, the demonstration in the present study that the follicle-associated epithelium appears to be involved in the uptake of PrP from the gut lumen identifies a significant site of entry for the scrapie agent. The involution of the ileal Peyers patch at puberty (about 1218 months in sheep) (Reynolds & Morris, 1983
) and the accompanying drastic reduction in the number of follicles (and follicle-associated epithelia) may contribute to the reduced susceptibility to infection by the scrapie agent observed in older animals (Hourrigan et al., 1979
; Hunter et al., 1996
; Elsen et al., 1999
).
The extent of exposure of the naturally exposed group of lambs to the scrapie agent was unknown. The lambs had been in close contact with scrapie-infected sheep (Brotherson et al., 1968 ; Dickinson, 1974
) but whether the variation in staining observed between lambs was the result of differences in duration and levels of exposure or the ages of the lambs or their PrP genotype is unknown. The three confirmed cases of scrapie in the affected flock that were held in isolation with the lambs had PrP genotypes that were typical for Norwegian cases of scrapie, namely AV136RR154QQ171 and VV136RR154QQ171 (Tranulis et al., 1999
). However, none of the nine lambs from this flock carried either of these two PrP genotypes. Indeed, these lambs carried PrP genotypes not previously identified in scrapie cases in Norway (Tranulis et al., 1999
). While the resistant PrP genotype of these lambs probably reduces the likelihood of the development of clinical disease (Hunter et al., 1996
), the present results suggest that these animals have at least taken up the scrapie agent, as evidenced by changes in the distribution of PrP. Recent studies investigating the existence of carrier animals, that is, animals which have a latent infection and do not show signs of clinical scrapie during a natural lifespan but have the ability to pass on the infection to other sheep, have focused on sheep with resistant PrP genotypes (Clouscard et al., 1995
; Bosser et al., 1996
; ORourke et al., 1997
). The present study provides information on the accumulation of PrP in the extraneural tissues of naturally exposed sheep with QR171 and QQ171 genotypes (ORourke et al., 1997
). However, the presence of a carrier state of infection would need to be confirmed by bioassay.
To address under more controlled conditions the uptake of PrP in sheep with different combinations of PrP genotypes in donor and recipient, groups of lambs with PrP genotypes strongly associated with disease or seldom associated with disease were exposed experimentally to brain material from clinical scrapie-cases (Table 3). One and five weeks after a single oral administration of scrapie-infected brain material, staining for PrP was prominent in the dome and neck regions of follicles, albeit at lower levels than observed in some naturally exposed lambs. Nevertheless, this pattern of staining was not detected in the age- and PrP genotype-matched control lambs and supports the observations in the naturally exposed lambs. Even so, it should be noted that from the present study it is not known whether the prominent staining for PrP in the naturally and experimentally exposed lambs was associated with infectivity for the scrapie agent that would eventually lead to disease. Eleven months after oral exposure to brain material from clinical cases with homologous PrP genotypes strongly associated with disease, there was little or no staining in ileal Peyers patch follicles. Sigurdson et al. (1999)
used repeated oral exposure to a relatively large amount of inoculum over 5 days and were able to detect chronic wasting disease PrPres in the gut-associated lymphoid tissues of mule deer fawns 6 weeks after exposure. Whether the absence of follicular staining in ileal Peyers patch follicles 11 months after oral exposure to scrapie-infected brain material in the present study is the result of using a too small dose will need to be addressed in further experiments.
A feature that was more obvious in the experimentally exposed lambs than in the naturally exposed lambs was the variation in the staining for PrP between neighbouring follicles in the ileal Peyers patch. The reason for the variation in staining for PrP between follicles is not known, although these striking differences were not observed in the age- and PrP genotype-matched controls, suggesting an involvement of the scrapie agent. It is interesting to note that genetic analysis of immunoglobulin light-chain rearrangement in the ileal Peyers patch of sheep has shown that the follicles are colonized by only a limited number of B-lymphocyte clones having rearranged one light-chain allele (two to three per follicle) (Reynaud et al., 1991 ). Whether the immunoglobulin genotype of B-lymphocyte clones in Peyers patch follicles influences the distribution of PrP following exposure to the scrapie agent or whether other factors such as selective uptake (Landsverk, 1987
, 1988
; Wolf & Bye, 1984
), disturbances in PrP metabolism (Jeffrey et al., 1998
) or maturational status of mesenchymal stromal cells within the follicles (Nicander et al., 1991
) are more relevant will need to be addressed in future studies.
The present study did not distinguish between the normal cellular form of PrP (PrPC) and the abnormal isoform associated with disease (PrPSc). Both forms exist within an infected animal and both forms would have been transferred in the infected brain material used for the experimental exposures. Distinguishing between these two forms represents a challenge for immunohistochemical investigations of the early pathogenesis of scrapie and antibodies suitable for immunohistochemistry that recognize epitopes exclusively on the abnormal isoform are needed (Korth et al., 1997 ). Nevertheless, the present study has shown that enhancing the sensitivity of conventional immunohistochemical approaches may prove to be a useful tool in the investigation of the early pathogenesis of scrapie and argues for the wider application of these approaches to methods such as histoblotting and PET-blotting (Schulz-Schaeffer et al., 2000
). This study also shows that experimental infection of scrapie-free lambs of defined age and PrP genotype may yield further insights into the nature of the uptake and dissemination of PrP in scrapie-infected sheep.
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Acknowledgments |
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References |
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Received 21 February 2000;
accepted 16 May 2000.