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Detection of PrPSc in Lymphoid Tissues of Lambs Experimentally Exposed to the Scrapie Agent
J. Comp. Path. 2003, Vol. 128, 172±181 doi:10.1016/S0021-9975(02)00140-8
Detection of PrPSc in Lymphoid Tissues of Lambs Experimentally Exposed to the Scrapie Agent R. Heggebù, C. McL. Press, G. Gunnes, M. J. Ulvundy, M. A. Tranulis and T. Landsverk Department of Morphology, Genetics and Aquatic Biology and Department of Biochemistry, Physiology and Nutrition, Norwegian School of Veterinary Science, P.O. Box 8146 Dep. N-0033, Oslo and yDepartment of Sheep and Goat Research, Norwegian School of Veterinary Science, Kyrkjevegen 332/334, N-4325 Sandnes, Norway Summary Histoblotting and immunohistochemistry were used to detect disease-associated prion protein (PrPSc) in lymphoid tissues of lambs of known PrP genotype infected with the scrapie agent by stomach tube at the age of 2 months. The ileal and jejunal Peyer's patches and retropharyngeal and distal jejunal lymph nodes were studied 1 week, 5 weeks, 5 months and 11 months after inoculation. Other lymphoid tissues examined included superficial cervical lymph node, tonsil and spleen. PrPSc was not detected in any tissue of any lamb at 1 week post-inoculation. At 5 weeks, PrPSc was detected in tissues of lambs of susceptible PrP genotypes (AV136QQ171 and VV136QQ171), but not lambs of other PrP genotypes (AA136QQ171, AA136QR171 and AV136QR171). PrPSc was present in the germinal centres of tonsils, distal jejunal and retropharyngeal lymph nodes, and spleen. In the nodules of ileal and jejunal Peyer's patches, only occasional solitary cells showed the presence of PrPSc. At 5 months post-inoculation, increased accumulations of PrPSc were detected in ileal and jejunal Peyer's patches, as well as in the retropharyngeal and distal jejunal lymph nodes of a single lamb inoculated with the agent from a sheep of the same susceptible PrP genotype. Eleven months after exposure to the scrapie agent, PrPSc was detected in all lymphoid tissues examined from sheep of susceptible PrP genotypes. These studies show that PrPSc was detectable in lymphoid tissues 5 weeks after exposure to the scrapie agent by stomach tube in lambs as young as 3 months of age and indicate that the PrP genotype is a significant factor for the rapid uptake and spread of the agent through lymphoid tissues.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction Scrapie is a transmissible neurodegenerative disorder that causes a fatal spongiform encephalopathy in sheep and goats. As with other prion diseases, the infectious agent responsible for the disease is considered to be the post-translationally modified form of a host-encoded membrane glycoprotein PrPC, termed PrPSc (Prusiner et al., 1982). The conformational changes in the disease-associated form PrPSc confer a relative resistance to proteinase digestion, which is widely accepted as a basis for distinguishing between PrPC and PrPSc. Various techniques are used to detect Correspondence to: Dr C. McL. Press. 0021±9975/03/$ ± see front matter
PrPSc, including the Western blot and histoblot techniques and immunohistochemistry. For studies on pathogenesis, the histoblot technique and immunohistochemistry have the advantage that they retain anatomical resolution and are therefore capable of revealing the distribution of PrPSc within tissues. While it is possible to treat conventional immunohistochemical sections with proteinase K to detect PrPSc, few research groups perform this treatment as it has a destructive effect on tissue sections on glass slides. Furthermore, fixation of tissues and pre-treatment of the tissue sections are critical for assessment of the degree of immunohistochemical reactivity (Haritani et al., 1994; van Keulen et al., 1996). The histoblot technique (Taraboulos et al., 1992) has the advantage # 2003 Elsevier Science Ltd. All rights reserved.
Detection of PrPSc in Lambs with Scrapie
for prion research that it combines sensitive protein detection after treatment with proteinase K and guanidine thiocyanate (Gnd-SCN) with some degree of preservation of tissue anatomy. A deeper understanding of the morphological distribution of the PrP forms detected by these different methods might provide insight into the early pathogenesis of scrapie. A recent investigation in Suffolk sheep revealed a wide distribution of PrPSc in peripheral lymphoid tissues of scrapie-affected animals (Heggebù et al., 2002). However, studies of the early pathogenesis of scrapie in rodents (Maignien et al., 1999; Beekes and McBride, 2000) and sheep (Andreoletti et al., 2000) emphasized the importance of the oral route of infection and the early accumulation of PrPSc in gutassociated lymphoid tissues (GALT). To investigate the uptake and dissemination of the scrapie agent from the gut of sheep, Heggebù et al. (2000) gave lambs of known PrP genotype, by stomach tube, a dose of scrapie-infected brain material from sheep with a homologous or a heterologous PrP genotype. Subsequently, a sensitive immunohistochemical technique was used to investigate the anatomical distribution of prion protein (PrP) in the ileal Peyer's patch of these lambs. The technique did not distinguish between PrPC and PrPSc, but relied on superior anatomical resolution to identify changes in the distribution of PrP. The study revealed a distribution of PrP in lambs 1 and 5 weeks after dosing similar to that in the ileal Peyer's patch of naturally exposed lambs. These findings were used to argue that the ileal Peyer's patch in young sheep is an important site for uptake of the scrapie agent. However, information on the presence of PrPSc in the ileal Peyer's patch and in a wider range of lymphoid tissues from these experimentally infected lambs is relevant to the understanding of the early pathogenesis of scrapie and the distribution of PrPSc in the preclinical phase of disease. In the present study, lambs of defined PrP genotype were exposed to the scrapie agent and examined 1 week to 11 months later by histoblotting and immunohistochemical techniques to detect PrPSc in the lymphoid tissues. Materials and Methods Sheep and Tissue Collection The 35 lambs used (18 experimental and 17 uninfected controls), aged 2 months, were of the Norwegian White breed (Rygja) and their PrP genotypes (Table 1) were designated according to the major disease-linked polymorphisms at codons 136 (alanine/valine), 154 (arginine/histidine) and 171
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(glutamine/arginine) (Tranulis et al., 1999). The PrP genotypes of confirmed natural cases of scrapie that served as donors in the experimental infection model are also listed in Table 1. Note that codon 154 is not mentioned as the PrP genotype of all lambs and donors was the same at this codon, namely RR154. Experimental infection by stomach tube of these 18 lambs of designated PrP genotype has already been described in detail by Heggebù et al. (2000). Briefly, a single dose of confirmed scrapie brain homogenate (15 ml, containing 5 g of brain tissue) was given to each lamb through a stomach tube passed down the oesophagus. At various times after exposure, namely 1 week (four lambs), 5 weeks (10 lambs), 5 months (two lambs) and 11 months (two lambs), the animals were anaesthetized, killed by exsanguination and subjected to necropsy. Age- and PrP genotype-matched control lambs, kept in separate isolated boxes, were given saline by stomach tube. In all infected and control lambs, tissue samples were collected from the ileal Peyer's patch (IPP), jejunal Peyer's patch ( JPP), distal jejunal lymph node (DJLN), retropharyngeal lymph node (RPLN), superficial cervical lymph node (SCLN), tonsil and spleen. Spleen and IPP samples from clinically affected field cases of scrapie were used as positive controls. Samples for histoblotting were frozen in 1,1,1-trifluorethane/pentafluorethane (R404A; Ausimont, Bollate, Italy), chilled in liquid nitrogen and stored at 70 C until use. Samples for immunohistochemistry were placed in formalin fixative for at least 24 h before paraffin wax-embedding.
Histoblotting Frozen sections (9 mm) were mounted on nitrocellulose membranes of 045 mm pore size (Sigma-Aldrich Chemie, Steinheim, Germany) and handled as described by Taraboulos et al. (1992), with minor modifications (Heggebù et al., 2002). In brief, the tissue sections were subjected to proteolysis by proteinase K (Serva Electrophoresis GmbH, Heidelberg, Germany) 400 mg/ml, at 55 C for 4 h. After rinsing in Trisbuffered saline (TBS) pH 75, sections were incubated for 20 min with 3 mM phenyl-sulphonylfluoride, followed by denaturation for 10 min in 3 M guanidine isothiocyanate (Sigma-Aldrich Chemie). The membrane was blocked in 5% non-fat milk for 1 h and then incubated overnight at 4 C with monoclonal anti-PrP antibody L42 (Hardt et al., 2000) diluted 1 in 200 in TBS, followed by rinsing in TBS. The membranes were next incubated with a secondary anti-mouse antibody (Vectastain1 ABC Kit; Vector Laboratories, Burlingame, CA, USA) for 30 min, followed by treatment for 30 min with streptavidin alkaline
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Table 1 Distribution of PrPSc as shown by immunohistochemistry (IHC) and histoblotting (H) in lymphoid tissues of 18 PrP-genotyped lambs inoculated with brain tissue from natural cases of scrapie by stomach tube, and 17 uninfected control lambs (age- and genotype-matched) Time after inoculation
Lamb no.
Recipient genotype
Donor genotype
Presence of PrPSc as shown by the stated methods in IPP IHC
1 week
5 weeks
5 months
11 months
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
VV136QQ 171 VV136QQ 171 AV136QQ 171 AV136QQ 171 AV136QR171 AV136QR171 AA136QR171 AA136QR171 VV136QQ 171 VV136QQ 171 VV136QQ 171 VV136QQ 171 VV136QQ 171 VV136QQ 171 AV136QQ 171 AV136QQ 171 AV136QR171 AV136QR171 AV136QQ 171 AV136QQ 171 AV136QR171 AV136QR171 AA136QQ 171 AA136QQ 171 AA136QQ 171 AA136QQ 171 AA136QR171 AA136QR171 AV136QQ 171 AV136QQ 171 AV136QR171 AV136QR171 AV136QQ 171 AV136QQ 171 AV136QQ 171
VV136QQ 171 AV136QQ 171 VV136QQ 171
JPP H
nd nd nd nd
IHC
spleen H
nd nd nd nd
IHC
tonsil H
nd nd nd nd
IHC
H
IHC
nd nd nd nd
nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd
nd nd nd nd
VV136QQ 171 VV136QQ 171
VV136QQ 171
VV136QQ 171 AV136QQ 171 AV136QQ 171 VV136QQ 171 VV136QQ 171 AA136QQ 171 VV136QQ 171 VV136QQ 171 AV136QQ 171
nl
VV136QQ 171 AV136QQ 171 AV136QQ 171
DJLN
SCLN H
IHC
RPLN H
nd nd nd nd
IHC
H
nd nd nd nd
nd
IPP, ileal Peyer's patch; JPP, jejunal Peyer's patch; DJLN, distal jejunal lymph node; SCLN, superficial cervical lymph node; RPLN, retropharyngeal lymph node. nd, Not done; nl, no lymphoid nodules.
phosphatase conjugate (Amersham Pharmacia Biotech, Buckinghamshire, England). The reaction product was developed with BCIP/NBT Pre-mixed Solution (Zymed Laboratories, San Francisco, CA, USA) for 10 min. Images of the histoblots were made with a Leica DC100 digital-camera mounted on a Leica MZ 125 stereomicroscope. Immunohistochemistry Avidin±biotin complex immunohistochemical labelling for disease-associated accumulations of PrP was
carried out by means of a modification of the methods described by Haritani et al. (1994). Formalin-fixed tissue was subjected to formic acid pre-treatment 1 h before embedding in paraffin wax. In the following procedure, tissue was subjected to hydrated autoclaving at 121 C for 30 min in citrate-buffer (001 M citric acid, pH 60). Immunohistochemical detection of PrP was performed overnight with the L42 antibody diluted 1 in 150 in TNB blocking buffer (01 M Tris-HCl, pH 75, 015 M NaCl, blocking reagent 05%; supplied in the TSATM-indirect kit of NEN Life Science Products,
Detection of PrPSc in Lambs with Scrapie
Boston, MA, USA) (Hardt et al., 2000). The control sections included in each run were incubated with either L42 or an irrelevant antibody of the same isotype as L42 (IgG1). The control tissues were from the IPP of a sheep with verified scrapie (positive control) and IPP tissue from one of the control animals from the inoculation model (negative control). After washing with TBS, all sections were incubated with a biotinylated secondary anti-mouse antibody (Amersham Biosciences, NJ, USA), followed by peroxidase-conjugated avidin±biotin complex (Dako, Glostrup, Denmark), each for 30 min. From each tissue sample, two sections were examined. One of the sections was subjected to tyramide signal amplification (TSA) (Heggebù et al., 2000) while the other section was kept in TBS. All sections were then developed with a single diamino-benzidine solution (ImmunoPure1 Metal enhanced DAB substrate; Pierce, Rockford, IL, USA). The sections were counterstained with haematoxylin and mounted in polyvinyl alcohol under coverslips before examination. Results Control Lambs Histoblot and immunohistochemical examination of the 17 control lambs, matched for age and PrP genotype, did not show PrPSc in any of the tissues examined from the alimentary canal (IPP, JPP and tonsil) or associated lymph nodes (DJLN and RPLN), or from non-gut-associated lymphoid tissues (SCLN and spleen). In some control animals, weak immunohistochemical labelling of the myenteric plexus was occasionally present in tissues from the alimentary canal following TSA-enhancement, but labelling was not present in parallel sections treated by the conventional immunohistochemical procedure for demonstrating PrPSc. Scrapie-inoculated Lambs PrPSc was not detected in any of the animals examined one week after exposure to scrapie-infected material (Table 1). Five weeks after exposure, PrPSc was detected in three of the five lambs with PrP genotypes most often associated with scrapie in Norway (AV136QQ 171 and VV136QQ 171) (Tranulis et al., 1999). Of the lambs with more susceptible PrP genotypes that did not show the presence of PrPSc, one (VV136QQ 171) had received donor brain material with a homologous PrP genotype, and the other (AV136QQ 171) had received donor material with a heterologous PrP genotype (VV136QQ 171). PrPSc was not found at this timepoint
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in tissues from lambs with other PrP genotypes (AA136QQ 171, AA136QR171and AV136QR171). Of the 10 infected lambs examined for PrPSc 5 weeks after inoculation, only three and two gave positive results with histoblotting and immunohistochemistry, respectively (Table 1). With the histoblot method, PrPSc was observed in the RPLN of lambs 9 and 11 (Fig. 1A), in the DJLN of lambs 9 and 15 (Fig. 1B) and in the spleen of lamb 9. The pattern of proteinaseresistant PrP labelling in the tissues from these lambs was consistent with the localization of PrPSc in the nodules of lymphoid tissues. Immunohistochemical examination revealed PrPSc in the RPLN of lamb 11 but in none of the other histoblot-positive tissues. However, unlike histoblotting, it revealed PrPSc in the IPP of lambs 9 and 11, and in the JPP of lamb 11; in addition the tonsils of lambs 9 and 11, which were not examined with histoblots, gave a positive result. Immunohistochemical examination of the tonsils showed a granular accumulation of PrPSc in cells of the nodules (Fig. 1C), while a more reticular pattern was observed in nodules in the cortex of the RPLN (Fig. 1D). The immunolabelling was prominent both with and without TSA-enhancement. In the IPP and JPP, PrPSc was detected in the myenteric plexus and in solitary cells in several nodules (Fig. 1E,F ). This immunolabelling was evident with or without TSAenhancement, but the labelling of cells in the nodules was more distinct with enhancement. Five months after inoculation, lamb 29, with homology between its PrP genotype (AV136QQ 171) and that of the donor brain material, showed PrPSc in a limited range of tissues. In this animal histoblotting showed a limited but clear granular labelling in lymphoid nodules in the cortex of the DJLN and RPLN; it also showed PrPSc in lymphoid nodules in the JPP (Fig. 2A) and IPP (Fig. 2B), and in locations that probably corresponded to myenteric plexuses of the IPP. The second inoculated lamb (No. 31; Table 1), whose PrP genotype (AV136QR171) was different from that of the donor brain material (VV136QQ 171), showed no PrPSc in any tissues examined by either of the two methods. Immunohistochemical examination of lamb 29 did not demonstrate PrPSc in lymph nodes or Peyer's patch nodules but showed some PrPSc accumulation in the myenteric plexuses of IPP and JPP; the labelling was visible both with or without TSA-enhancement but was weak without. Eleven months after inoculation, histoblot examination of lambs 33 and 35, both of which had the PrP genotype AV136QQ 171 and homology with the PrP genotype of the donor, showed a strong presence of PrPSc in all six tissues. There were intense, granular accumulations of PrPSc in the lymphoid nodules of both the IPP and JPP (Fig. 3A), and PrPSc was also
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Fig. 1A±F. Histoblot and immunohistochemical analysis of PrPSc accumulation in lymphoid tissues from lambs with PrP genotype VV136QQ171 5 weeks after inoculation with the scrapie agent by stomach tube. (A) Retropharyngeal lymph node. Numerous greyish accumulations of PrPSc (arrows) are present in an oblique section through the lymph node cortex. Capsule indicated by arrowhead. Histoblot. Bar, 1 mm. (B) Distal jejunal lymph node. Accumulations of PrPSc (arrows) are present in nodules in the lymph node cortex. Capsule indicated by arrowhead. Histoblot. Bar, 03 mm. (C) Palatine tonsil. Granular labelling (brown) for PrPSc is present in cells of the lymphoid nodules (n). Epithelium indicated by arrowhead. Immunohistochemistry. Bar, 200 mm. (D) Retropharyngeal lymph node. A reticular pattern of labelling (brown) for PrPSc is present in the centre of a nodule (n). Immunohistochemistry. Bar, 50 mm. (E) Ileal Peyer's patch. Only a few cells (arrows) show labelling (brown) for PrPSc in a lymphoid nodule. Capsule indicated by arrowhead. Immunohistochemistry. Bar, 50 mm. (F) Ileal Peyer's patch. Labelling (brown; arrow) for PrPSc is present in cells of a ganglion in the myenteric plexus lying between the muscle layers (m) of the gut wall. Immunohistochemistry. Bar, 50 mm.
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Fig. 2A,B. Histoblot analysis of PrPSc accumulation in lymphoid tissues from a lamb with PrP genotype AV136QQ171 5 months after inoculation with the scrapie agent by stomach tube. (A) Jejunal Peyer's patch. Granular accumulations of PrPSc in a lymphoid nodule (n). Bar, 01 mm. (B) Ileal Peyer's patch. Granular accumulations of PrPSc are present in two lymphoid nodules (arrows). Bar, 015 mm.
Fig. 3A±D. Histoblot analysis of PrPSc accumulation in lymphoid tissues from a lamb with PrP genotype AV136QQ171 11 months after inoculation with the scrapie agent by stomach tube. (A) Jejunal Peyer's patch. Lymphoid nodules (arrows) contain dense accumulations of PrPSc. Bar, 04 mm. (B) Retropharyngeal lymph node. Many lymphoid nodules in the lymph node cortex show the strong presence of PrPSc (arrows). Capsule indicated by arrowhead. Bar, 065 mm. (C) Spleen. Accumulations of PrPSc are present in lymphoid nodules (long arrows) and in the marginal zone (short arrows). Arrowhead indicates area shown in Fig. 3D. Bar, 065 mm. (D) Spleen. Higher magnification of the marked area in Fig. 3C, showing accumulation of PrPSc in lymphoid nodules (long arrow) and in the marginal zone (short arrow). Bar, 018 mm.
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Fig. 4A±C. Immunohistochemical analysis of PrPSc accumulation in lymphoid tissues from a lamb with PrP genotype AV136QQ171 11 months after inoculation with the scrapie agent by stomach tube. These tissues are from the same lamb as that illustrated in Fig. 3. (A) Spleen. Strong labelling (brown) for PrPSc is present in a lymphoid nodule (n) while little or no immunolabelling is evident in the marginal zone (m). Bar, 50 mm. (B) Jejunal Peyer's patch. There is strong labelling (brown) for PrPSc in lymphoid nodules (n). Some immunolabelling for PrPSc (arrows) is present in the dome (d) and interfollicular areas (i). Bar, 200 mm. (C) Retropharyngeal lymph node. Numerous lymphoid nodules (n) contain PrPSc. Labelling (brown) for PrPSc is present both in granular accumulations (arrows) in cells distributed throughout the nodule and in a reticular pattern (arrowheads) at the nodule centre. Bar, 200 mm.
present in the myenteric plexuses. The three lymph nodes (RPLN, DJLN and SCLN) all showed intense, granular labelling in nodules of the cortex (Fig. 3B). The spleen clearly showed PrPSc in nodules and dense accumulations were also present in locations consistent with the marginal zone (Fig. 3C, D). On immunohistochemical examination, all but one (SCLN) of the examined tissues revealed PrPSc (Table 1). Immunolabelling gave a less prominent and less abundant demonstration of PrPSc than that given by histoblotting, but it nonetheless showed characteristic granular accumulations of PrPSc in the nodules (Fig. 4A±C). In the IPP and JPP, PrPSc was present in myenteric plexuses. In the spleen, the accumulations of PrPSc were generally less intense than in the lymph nodes,
and were restricted to the nodules (Fig. 4A), the marginal zone lacking the clear presence of PrPSc seen with the histoblot method. Discussion In this study, the distribution of early accumulations of disease-associated PrPSc in the lymphoid tissues was examined in lambs dosed by stomach tube with scrapie-infected material. PrPSc was detected 5 weeks after inoculation in lymph nodes associated with the alimentary canal, spleen, and tonsil. Only limited PrPSc was demonstrated in the organized lymphoid tissue of the gut wall (the Peyer's patches). In previous studies, the earliest accumulation of pathological PrP,
Detection of PrPSc in Lambs with Scrapie
detected in GALT (including Peyer's patches and mesenteric lymph nodes draining the gut), occurred at 8 weeks of age in sheep (Andreoletti et al., 2000) and 10 weeks after infection in rodents (Beekes and McBride, 2000). The efficiency of detection of PrPSc differed between histoblotting and immunohistochemistry. The PrPSc detected, albeit in small amounts, in Peyer's patches 5 weeks after inoculation was most likely attributable to the preservation of tissue anatomy associated with the immunohistochemical technique. The limited ability of immunohistochemistry, however, to detect the significant levels of PrPSc revealed by histoblotting in the nodules of Peyer's patches, lymph nodes and spleen 5 weeks and 5 months after inoculation is more difficult to explain. The immunoreactivity of PrP is altered after exposure to protein-denaturing agents such as formic acid, proteinase K, various guanidine components and sodium dodecyl sulphate (Haritani et al., 1994). The difference between the detection of proteinase K-resistant PrP and PrP resistant to hydrated autoclaving and formic acid may be the result of (1) differences in sensitivity of the methods, or (2) inadequacies in tissue sampling procedures needed to identify a limited distribution of initial sites of uptake and accumulation within tissues. Alternatively, the existence of different forms of PrP in tissues has been proposed (Bruce and Fraser, 1991; Bruce et al., 1991; Safar et al., 1998) and the present results suggest that PrP variations at different stages in the pathogenesis of scrapie should be considered. The use of both histoblotting and immunohistochemistry enabled PrPSc to be detected in all tissues examined 5 weeks after inoculation, except for the superficial cervical lymph nodes. The initial sites of detection of PrPSc were the germinal centres of the lymph nodes associated with the alimentary canal, tonsil and spleen. However, the initial detection of PrPSc in these sites after inoculation by stomach tube raises issues in relation to the dissemination of the scrapie agent within the host. Firstly, given the method of inoculation, the presence of PrPSc in retropharyngeal lymph node and tonsil would appear to have required transport of the agent via the blood. Haematogenous dissemination of infectivity to lymphoreticular sites is an accepted feature of the early pathogenesis of experimental scrapie in rodents infected extra-cranially (Kimberlin and Walker, 1988; Scott, 1993). However, if PrPSc in lymphoid tissue associated with the oral cavity was the result of haematogenous dissemination, why was it not found in the superficial cervical lymph node until 11 months after inoculation? It is possible that regurgitation, which is partly established in lambs of the age of those used in the study, resulted in the exposure of the oral
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cavity to the inoculum. It is also possible that remnants of the inoculum present in the stomach tube at the time of withdrawal were deposited in the mouth. Several routes of transmission of transmissible spongiform encephalopathies (TSEs) to the central nervous system (CNS) have been described (Brown, 2001), including transmission from dental and buccal tissues in hamsters (Ingrosso et al., 1999). Further issues raised by the present study are the presence of PrPSc in germinal centres of lymph nodes and tonsil 5 weeks after inoculation, and the restricted amounts in the lymphoid nodules of Peyer's patches. It has been speculated that the follicle-associated epithelium of the Peyer's patches represents the main route of entry for the scrapie agent from the gut (Bruce et al., 2000; Heggebù et al., 2000). However, the present results suggest that while transport of the agent to the nodules of the Peyer's patches occurs, transport of the agent away from the gut via lymph and blood plays an important role. Alternatively, but not inconsistent with this interpretation, processes responsible for the trapping of the scrapie agent and transport into germinal centres in lymph nodes and spleen may be more effective than similar processes in the Peyer's patches. The size of the infecting dose and the duration of exposure to the scrapie agent may also influence this process. In some sheep infected experimentally with the agent of bovine spongiform encephalopathy (BSE), neuroinvasion occurred in the absence of detectable PrP accumulations ( Jeffrey et al., 2001b). Nevertheless, once established in the Peyer's patch nodules, the Peyer's patches represent an important site of PrPSc accumulation, which has been associated with neuroinvasion (van Keulen et al., 1999). Oral inoculation studies of TSEs, including BSE, in sheep have been performed in recent years (O'Rourke et al., 1997; Foster et al., 2001; Jeffrey et al., 2001b). Comparisons between them are made difficult by differences in method of infection and age at inoculation. Pathogenesis studies of scrapie in sheep have mostly been performed on animals from natural scrapie-affected flocks (van Keulen et al., 1999, 2000; Andreoletti et al., 2000; Jeffrey et al., 2001a). Although few young lambs have been examined, results indicate that uptake and spread of PrPSc from intestines and GALT occur at an age of 2±5 months (Andreoletti et al., 2000). Jeffrey et al. (2001a) detected PrPSc accumulations in tonsillar biopsy specimens at an age of 8 months, and PrPSc was detected in secondary lymphoid organs and myenteric plexuses at about 9 months of age (Andreoletti et al., 2000; van Keulen et al., 2000). At the time of initial detection of PrPSc, the sheep in the present study were 3 months of age. The presence of PrPSc in the tonsils in two of five lambs inoculated with agent from sheep of homologous
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susceptible PrP genotype at this early timepoint suggests that tonsillar biopsy would have a role in the diagnosis of preclinical cases of scrapie. Furthermore, the detection of PrPSc in RPLNs at this age indicates that, in abattoir surveillance programmes, this easily accessible lymph node should be considered for sampling. In a previous investigation of the ileal Peyer's patch from the present groups of experimental lambs, Heggebù et al. (2000) used a sensitive immunohistochemical technique that did not distinguish between PrPC and PrPSc. The altered distribution of PrP detected within the compartments of the ileal Peyer's patch at 1 and 5 weeks post-inoculation suggested changes associated with the uptake of the scrapie agent. In the present study, PrPSc was not detected in lymphoid nodules of the IPP at 1 week postinoculation and only solitary cells showed labelling for PrPSc at 5 weeks. The strong presence of PrPSc in Peyer's patch follicles in the present study 11 months after inoculation contrasts with the weak immunohistochemical presence of PrP reported by Heggebù et al. (2000). Interestingly, a recent study of the in-vivo conversion of PrPC to pathogenic isoforms (Yokoyama et al., 2001) revealed that in the CNS sites in mice, PrPC decreased as PrPSc was deposited. Taken together, these observations suggest that the sensitive immunohistochemical approach adopted by Heggebù et al. (2000) mainly detected changes in the presence of PrPC. In addition to the timing and location of PrPSc deposition, the present study examined the relationship between PrP genotype of the recipient lamb and the donor inoculum. PrPSc was detected only in the tissues of lambs of the most susceptible PrP genotypes, namely VV136QQ 171 and AV136QQ 171 (Tranulis et al., 1999), and only when there was homology between the PrP genotype of the donor inoculum and the recipient. However, such homology did not guarantee the presence of PrPSc at 5 weeks postinoculation, (e.g. lambs 13 and 23; Table 1). Prusiner et al. (1990) showed that matching of donor and recipient PrP genotypes resulted in a short incubation period. The present study draws attention to the early distribution of PrPSc in lymphoid tissues, showing that as soon as 5 weeks after exposure lambs may have PrPSc deposits in lymph nodes, tonsil and spleen. Moreover, the method of examination, time after infection, tissue distribution and PrP genotype may all influence the detection of PrPSc in sheep. Acknowledgments The authors are grateful to Eli Bruntland of the Department of Sheep and Goat Research, for
performing the PrP genotyping, and to Birgit Rùe of the Department of Morphology, Genetics and Aquatic Biology for performing the immunohistochemistry. This study was supported by grants 116073/122 (R.H.), 119688/110 (M.J.U) and 118091/122 (G.G. and C.McL.P.) from the Norwegian Research Council. References Andreoletti, O., Berthon, P., Marc, D., Sarradin, P., Grosclaude, J., van Keulen, L., Schelcher, F., Elsen, J. M. and Lantier, F. (2000). Early accumulation of PrP(Sc) in gut-associated lymphoid and nervous tissues of susceptible sheep from a Romanov flock with natural scrapie. Journal of General Virology, 81, 3115±3126. Beekes, M. and McBride, P. A. (2000). Early accumulation of pathological PrP in the enteric nervous system and gut-associated lymphoid tissue of hamsters orally infected with scrapie. Neuroscience Letters, 278, 181±184. Brown, P. (2001). The pathogenesis of transmissible spongiform encephalopathy: routes to the brain and the erection of therapeutic barricades. Cellular and Molecular Life Sciences, 58, 259±265. Bruce, M. E., Brown, K. L., Mabbott, N. A., Farquhar, C. F. and Jeffrey, M. (2000). Follicular dendritic cells in TSE pathogenesis. Immunology Today, 21, 442±446. Bruce, M. E. and Fraser, H. (1991). Scrapie strain variation and its implications. Current Topics in Microbiology and Immunology, 172, 125±138. Bruce, M. E., McConnell, I., Fraser, H. and Dickinson, A. G. (1991). The disease characteristics of different strains of scrapie in Sinc congenic mouse lines: implications for the nature of the agent and host control of pathogenesis. Journal of General Virology, 72, 595±603. Foster, J. D., Parnham, D., Chong, A., Goldmann, W. and Hunter, N. (2001). Clinical signs, histopathology and genetics of experimental transmission of BSE and natural scrapie to sheep and goats. Veterinary Record, 148, 165±171. Hardt, M., Baron, T. and Groschup, M. H. (2000). A comparative study of immunohistochemical methods for detecting abnormal prion protein with monoclonal and polyclonal antibodies. Journal of Comparative Pathology, 122, 43±53. Haritani, M., Spencer, Y. I. and Wells, G. A. (1994). Hydrated autoclave pre-treatment enhancement of prion protein immunoreactivity in formalin-fixed bovine spongiform encephalopathy-affected brain. Acta Neuropathologica, 87, 86±90. Heggebù, R., Press, C. McL., Gunnes, G., GonzaÂlez, L. and Jeffrey, M. (2002). Distribution and accumulation of PrP in gut-associated and peripheral lymphoid tissue of scrapie-affected Suffolk sheep. Journal of General Virology, 83, 479±489. Heggebù, R., Press, C. McL., Gunnes, G., Lie, K. I., Tranulis, M. A., Ulvund, M., Groschup, M. H. and Landsverk, T. (2000). Distribution of prion protein in the ileal Peyer's patch of scrapie-free lambs and lambs naturally and experimentally exposed to the scrapie agent. Journal of General Virology, 81, 2327±2337.
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Ingrosso, L., Pisani, F. and Pocchiari, M. (1999). Transmission of the 263K scrapie strain by the dental route. Journal of General Virology, 80, 3043±3047. Jeffrey, M., Martin, S., Thomson, J. R., Dingwall, W. S., Begara-McGorum, I. and GonzaÂlez, L. (2001a). Onset and distribution of tissue PrP accumulation in scrapieaffected Suffolk sheep as demonstrated by sequential necropsies and tonsillar biopsies. Journal of Comparative Pathology, 125, 48±57. Jeffrey, M., Ryder, S., Martin, S., Hawkins, S. A., Terry, L., Berthelin-Baker, C. and Bellworthy, S. J. (2001b). Oral inoculation of sheep with the agent of bovine spongiform encephalopathy (BSE). 1. Onset and distribution of disease-specific PrP accumulation in brain and viscera. Journal of Comparative Pathology, 124, 280±289. Kimberlin, R. H. and Walker, C. A. (1988). Pathogenesis of experimental scrapie. Ciba Foundation Symposium, 135, 37±62. Maignien, T., Lasmezas, C. I., Beringue, V., Dormont, D. and Deslys, J. P. (1999). Pathogenesis of the oral route of infection of mice with scrapie and bovine spongiform encephalopathy agents. Journal of General Virology, 80, 3035±3042. O'Rourke, K. I., Holyoak, G. R., Clark, W. W., Mickelson, J. R., Wang, S., Melco, R. P., Besser, T. E. and Foote, W. C. (1997). PrP genotypes and experimental scrapie in orally inoculated Suffolk sheep in the United States. Journal of General Virology, 78, 975±978. Prusiner, S. B., Bolton, D. C., Groth, D. F., Bowman, K. A., Cochran, S. P. and McKinley, M. P. (1982). Further purification and characterization of scrapie prions. Biochemistry, 21, 6942±6950. Prusiner, S. B., Scott, M., Foster, D., Pan, K. M., Groth, D., Mirenda, C., Torchia, M., Yang, S. L., Serban, D., Carlson, G. A., Hoppe, P.C., Westaway, D. and DeArmond, S. J. (1990). Transgenetic studies implicate interactions between homologous PrP isoforms in scrapie prion replication. Cell, 63, 673±686.
Safar, J., Wille, H., Itri, V., Groth, D., Serban, H., Torchia, M., Cohen, F. E. and Prusiner, S. B. (1998). Eight prion strains have PrP(Sc) molecules with different conformations. Nature Medicine, 4, 1157±1165. Scott, J. R. (1993). Scrapie pathogenesis. British Medical Bulletin, 49, 778±791. Taraboulos, A., Jendroska, K., Serban, D., Yang, S. L., DeArmond, S. J. and Prusiner, S. B. (1992). Regional mapping of prion proteins in brain. Proceedings of the National Academy of Sciences of the USA, 89, 7620±7624. Tranulis, M. A., Osland, A., Bratberg, B. and Ulvund, M. J. (1999). Prion protein gene polymorphisms in sheep with natural scrapie and healthy controls in Norway. Journal of General Virology, 80, 1073±1077. van Keulen, L. J. M., Schreuder, B. E. C., Meloen, R. H., Mooij-Harkes, G., Vromans, M. E. W. and Langeveld, J. P. M. (1996). Immunohistochemical detection of prion protein in lymphoid tissues of sheep with natural scrapie. Journal of Clinical Microbiology, 34, 1228±1231. van Keulen, L. J. M., Schreuder, B. E., Vromans, M. E., Langeveld, J. P. and Smits, M. A. (1999). Scrapieassociated prion protein in the gastrointestinal tract of sheep with natural scrapie. Journal of Comparative Pathology, 121, 55±63. van Keulen, L. J. M., Schreuder, B. E. C., Vromans, M. E. W., Langeveld, J. P. M. and Smits, M. A. (2000). Pathogenesis of natural scrapie in sheep. In: Prion Diseases Diagnosis and Pathogenesis, M. H. Groschup and H. A. Kretzschmar, Eds, Springer-Verlag, Vienna, pp. 57±71. Yokoyama, T., Kimura, K. M., Ushiki, Y., Yamada, S., Morooka, A., Nakashiba, T., Sassa, T. and Itohara, S. (2001). In vivo conversion of cellular prion protein to pathogenic isoforms, as monitored by conformationspecific antibodies. Journal of Biological Chemistry, 276, 11265±11271.
Received, May 20th, 2002 Accepted, October 26th, 2002
Detection of PrPSc in Lymphoid Tissues of Lambs Experimentally Exposed to the Scrapie Agent R. Heggebù, C. McL. Press, G. Gunnes, M. J. Ulvundy, M. A. Tranulis and T. Landsverk Department of Morphology, Genetics and Aquatic Biology and Department of Biochemistry, Physiology and Nutrition, Norwegian School of Veterinary Science, P.O. Box 8146 Dep. N-0033, Oslo and yDepartment of Sheep and Goat Research, Norwegian School of Veterinary Science, Kyrkjevegen 332/334, N-4325 Sandnes, Norway Summary Histoblotting and immunohistochemistry were used to detect disease-associated prion protein (PrPSc) in lymphoid tissues of lambs of known PrP genotype infected with the scrapie agent by stomach tube at the age of 2 months. The ileal and jejunal Peyer's patches and retropharyngeal and distal jejunal lymph nodes were studied 1 week, 5 weeks, 5 months and 11 months after inoculation. Other lymphoid tissues examined included superficial cervical lymph node, tonsil and spleen. PrPSc was not detected in any tissue of any lamb at 1 week post-inoculation. At 5 weeks, PrPSc was detected in tissues of lambs of susceptible PrP genotypes (AV136QQ171 and VV136QQ171), but not lambs of other PrP genotypes (AA136QQ171, AA136QR171 and AV136QR171). PrPSc was present in the germinal centres of tonsils, distal jejunal and retropharyngeal lymph nodes, and spleen. In the nodules of ileal and jejunal Peyer's patches, only occasional solitary cells showed the presence of PrPSc. At 5 months post-inoculation, increased accumulations of PrPSc were detected in ileal and jejunal Peyer's patches, as well as in the retropharyngeal and distal jejunal lymph nodes of a single lamb inoculated with the agent from a sheep of the same susceptible PrP genotype. Eleven months after exposure to the scrapie agent, PrPSc was detected in all lymphoid tissues examined from sheep of susceptible PrP genotypes. These studies show that PrPSc was detectable in lymphoid tissues 5 weeks after exposure to the scrapie agent by stomach tube in lambs as young as 3 months of age and indicate that the PrP genotype is a significant factor for the rapid uptake and spread of the agent through lymphoid tissues.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction Scrapie is a transmissible neurodegenerative disorder that causes a fatal spongiform encephalopathy in sheep and goats. As with other prion diseases, the infectious agent responsible for the disease is considered to be the post-translationally modified form of a host-encoded membrane glycoprotein PrPC, termed PrPSc (Prusiner et al., 1982). The conformational changes in the disease-associated form PrPSc confer a relative resistance to proteinase digestion, which is widely accepted as a basis for distinguishing between PrPC and PrPSc. Various techniques are used to detect Correspondence to: Dr C. McL. Press. 0021±9975/03/$ ± see front matter
PrPSc, including the Western blot and histoblot techniques and immunohistochemistry. For studies on pathogenesis, the histoblot technique and immunohistochemistry have the advantage that they retain anatomical resolution and are therefore capable of revealing the distribution of PrPSc within tissues. While it is possible to treat conventional immunohistochemical sections with proteinase K to detect PrPSc, few research groups perform this treatment as it has a destructive effect on tissue sections on glass slides. Furthermore, fixation of tissues and pre-treatment of the tissue sections are critical for assessment of the degree of immunohistochemical reactivity (Haritani et al., 1994; van Keulen et al., 1996). The histoblot technique (Taraboulos et al., 1992) has the advantage # 2003 Elsevier Science Ltd. All rights reserved.
Detection of PrPSc in Lambs with Scrapie
for prion research that it combines sensitive protein detection after treatment with proteinase K and guanidine thiocyanate (Gnd-SCN) with some degree of preservation of tissue anatomy. A deeper understanding of the morphological distribution of the PrP forms detected by these different methods might provide insight into the early pathogenesis of scrapie. A recent investigation in Suffolk sheep revealed a wide distribution of PrPSc in peripheral lymphoid tissues of scrapie-affected animals (Heggebù et al., 2002). However, studies of the early pathogenesis of scrapie in rodents (Maignien et al., 1999; Beekes and McBride, 2000) and sheep (Andreoletti et al., 2000) emphasized the importance of the oral route of infection and the early accumulation of PrPSc in gutassociated lymphoid tissues (GALT). To investigate the uptake and dissemination of the scrapie agent from the gut of sheep, Heggebù et al. (2000) gave lambs of known PrP genotype, by stomach tube, a dose of scrapie-infected brain material from sheep with a homologous or a heterologous PrP genotype. Subsequently, a sensitive immunohistochemical technique was used to investigate the anatomical distribution of prion protein (PrP) in the ileal Peyer's patch of these lambs. The technique did not distinguish between PrPC and PrPSc, but relied on superior anatomical resolution to identify changes in the distribution of PrP. The study revealed a distribution of PrP in lambs 1 and 5 weeks after dosing similar to that in the ileal Peyer's patch of naturally exposed lambs. These findings were used to argue that the ileal Peyer's patch in young sheep is an important site for uptake of the scrapie agent. However, information on the presence of PrPSc in the ileal Peyer's patch and in a wider range of lymphoid tissues from these experimentally infected lambs is relevant to the understanding of the early pathogenesis of scrapie and the distribution of PrPSc in the preclinical phase of disease. In the present study, lambs of defined PrP genotype were exposed to the scrapie agent and examined 1 week to 11 months later by histoblotting and immunohistochemical techniques to detect PrPSc in the lymphoid tissues. Materials and Methods Sheep and Tissue Collection The 35 lambs used (18 experimental and 17 uninfected controls), aged 2 months, were of the Norwegian White breed (Rygja) and their PrP genotypes (Table 1) were designated according to the major disease-linked polymorphisms at codons 136 (alanine/valine), 154 (arginine/histidine) and 171
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(glutamine/arginine) (Tranulis et al., 1999). The PrP genotypes of confirmed natural cases of scrapie that served as donors in the experimental infection model are also listed in Table 1. Note that codon 154 is not mentioned as the PrP genotype of all lambs and donors was the same at this codon, namely RR154. Experimental infection by stomach tube of these 18 lambs of designated PrP genotype has already been described in detail by Heggebù et al. (2000). Briefly, a single dose of confirmed scrapie brain homogenate (15 ml, containing 5 g of brain tissue) was given to each lamb through a stomach tube passed down the oesophagus. At various times after exposure, namely 1 week (four lambs), 5 weeks (10 lambs), 5 months (two lambs) and 11 months (two lambs), the animals were anaesthetized, killed by exsanguination and subjected to necropsy. Age- and PrP genotype-matched control lambs, kept in separate isolated boxes, were given saline by stomach tube. In all infected and control lambs, tissue samples were collected from the ileal Peyer's patch (IPP), jejunal Peyer's patch ( JPP), distal jejunal lymph node (DJLN), retropharyngeal lymph node (RPLN), superficial cervical lymph node (SCLN), tonsil and spleen. Spleen and IPP samples from clinically affected field cases of scrapie were used as positive controls. Samples for histoblotting were frozen in 1,1,1-trifluorethane/pentafluorethane (R404A; Ausimont, Bollate, Italy), chilled in liquid nitrogen and stored at 70 C until use. Samples for immunohistochemistry were placed in formalin fixative for at least 24 h before paraffin wax-embedding.
Histoblotting Frozen sections (9 mm) were mounted on nitrocellulose membranes of 045 mm pore size (Sigma-Aldrich Chemie, Steinheim, Germany) and handled as described by Taraboulos et al. (1992), with minor modifications (Heggebù et al., 2002). In brief, the tissue sections were subjected to proteolysis by proteinase K (Serva Electrophoresis GmbH, Heidelberg, Germany) 400 mg/ml, at 55 C for 4 h. After rinsing in Trisbuffered saline (TBS) pH 75, sections were incubated for 20 min with 3 mM phenyl-sulphonylfluoride, followed by denaturation for 10 min in 3 M guanidine isothiocyanate (Sigma-Aldrich Chemie). The membrane was blocked in 5% non-fat milk for 1 h and then incubated overnight at 4 C with monoclonal anti-PrP antibody L42 (Hardt et al., 2000) diluted 1 in 200 in TBS, followed by rinsing in TBS. The membranes were next incubated with a secondary anti-mouse antibody (Vectastain1 ABC Kit; Vector Laboratories, Burlingame, CA, USA) for 30 min, followed by treatment for 30 min with streptavidin alkaline
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Table 1 Distribution of PrPSc as shown by immunohistochemistry (IHC) and histoblotting (H) in lymphoid tissues of 18 PrP-genotyped lambs inoculated with brain tissue from natural cases of scrapie by stomach tube, and 17 uninfected control lambs (age- and genotype-matched) Time after inoculation
Lamb no.
Recipient genotype
Donor genotype
Presence of PrPSc as shown by the stated methods in IPP IHC
1 week
5 weeks
5 months
11 months
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
VV136QQ 171 VV136QQ 171 AV136QQ 171 AV136QQ 171 AV136QR171 AV136QR171 AA136QR171 AA136QR171 VV136QQ 171 VV136QQ 171 VV136QQ 171 VV136QQ 171 VV136QQ 171 VV136QQ 171 AV136QQ 171 AV136QQ 171 AV136QR171 AV136QR171 AV136QQ 171 AV136QQ 171 AV136QR171 AV136QR171 AA136QQ 171 AA136QQ 171 AA136QQ 171 AA136QQ 171 AA136QR171 AA136QR171 AV136QQ 171 AV136QQ 171 AV136QR171 AV136QR171 AV136QQ 171 AV136QQ 171 AV136QQ 171
VV136QQ 171 AV136QQ 171 VV136QQ 171
JPP H
nd nd nd nd
IHC
spleen H
nd nd nd nd
IHC
tonsil H
nd nd nd nd
IHC
H
IHC
nd nd nd nd
nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd
nd nd nd nd
VV136QQ 171 VV136QQ 171
VV136QQ 171
VV136QQ 171 AV136QQ 171 AV136QQ 171 VV136QQ 171 VV136QQ 171 AA136QQ 171 VV136QQ 171 VV136QQ 171 AV136QQ 171
nl
VV136QQ 171 AV136QQ 171 AV136QQ 171
DJLN
SCLN H
IHC
RPLN H
nd nd nd nd
IHC
H
nd nd nd nd
nd
IPP, ileal Peyer's patch; JPP, jejunal Peyer's patch; DJLN, distal jejunal lymph node; SCLN, superficial cervical lymph node; RPLN, retropharyngeal lymph node. nd, Not done; nl, no lymphoid nodules.
phosphatase conjugate (Amersham Pharmacia Biotech, Buckinghamshire, England). The reaction product was developed with BCIP/NBT Pre-mixed Solution (Zymed Laboratories, San Francisco, CA, USA) for 10 min. Images of the histoblots were made with a Leica DC100 digital-camera mounted on a Leica MZ 125 stereomicroscope. Immunohistochemistry Avidin±biotin complex immunohistochemical labelling for disease-associated accumulations of PrP was
carried out by means of a modification of the methods described by Haritani et al. (1994). Formalin-fixed tissue was subjected to formic acid pre-treatment 1 h before embedding in paraffin wax. In the following procedure, tissue was subjected to hydrated autoclaving at 121 C for 30 min in citrate-buffer (001 M citric acid, pH 60). Immunohistochemical detection of PrP was performed overnight with the L42 antibody diluted 1 in 150 in TNB blocking buffer (01 M Tris-HCl, pH 75, 015 M NaCl, blocking reagent 05%; supplied in the TSATM-indirect kit of NEN Life Science Products,
Detection of PrPSc in Lambs with Scrapie
Boston, MA, USA) (Hardt et al., 2000). The control sections included in each run were incubated with either L42 or an irrelevant antibody of the same isotype as L42 (IgG1). The control tissues were from the IPP of a sheep with verified scrapie (positive control) and IPP tissue from one of the control animals from the inoculation model (negative control). After washing with TBS, all sections were incubated with a biotinylated secondary anti-mouse antibody (Amersham Biosciences, NJ, USA), followed by peroxidase-conjugated avidin±biotin complex (Dako, Glostrup, Denmark), each for 30 min. From each tissue sample, two sections were examined. One of the sections was subjected to tyramide signal amplification (TSA) (Heggebù et al., 2000) while the other section was kept in TBS. All sections were then developed with a single diamino-benzidine solution (ImmunoPure1 Metal enhanced DAB substrate; Pierce, Rockford, IL, USA). The sections were counterstained with haematoxylin and mounted in polyvinyl alcohol under coverslips before examination. Results Control Lambs Histoblot and immunohistochemical examination of the 17 control lambs, matched for age and PrP genotype, did not show PrPSc in any of the tissues examined from the alimentary canal (IPP, JPP and tonsil) or associated lymph nodes (DJLN and RPLN), or from non-gut-associated lymphoid tissues (SCLN and spleen). In some control animals, weak immunohistochemical labelling of the myenteric plexus was occasionally present in tissues from the alimentary canal following TSA-enhancement, but labelling was not present in parallel sections treated by the conventional immunohistochemical procedure for demonstrating PrPSc. Scrapie-inoculated Lambs PrPSc was not detected in any of the animals examined one week after exposure to scrapie-infected material (Table 1). Five weeks after exposure, PrPSc was detected in three of the five lambs with PrP genotypes most often associated with scrapie in Norway (AV136QQ 171 and VV136QQ 171) (Tranulis et al., 1999). Of the lambs with more susceptible PrP genotypes that did not show the presence of PrPSc, one (VV136QQ 171) had received donor brain material with a homologous PrP genotype, and the other (AV136QQ 171) had received donor material with a heterologous PrP genotype (VV136QQ 171). PrPSc was not found at this timepoint
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in tissues from lambs with other PrP genotypes (AA136QQ 171, AA136QR171and AV136QR171). Of the 10 infected lambs examined for PrPSc 5 weeks after inoculation, only three and two gave positive results with histoblotting and immunohistochemistry, respectively (Table 1). With the histoblot method, PrPSc was observed in the RPLN of lambs 9 and 11 (Fig. 1A), in the DJLN of lambs 9 and 15 (Fig. 1B) and in the spleen of lamb 9. The pattern of proteinaseresistant PrP labelling in the tissues from these lambs was consistent with the localization of PrPSc in the nodules of lymphoid tissues. Immunohistochemical examination revealed PrPSc in the RPLN of lamb 11 but in none of the other histoblot-positive tissues. However, unlike histoblotting, it revealed PrPSc in the IPP of lambs 9 and 11, and in the JPP of lamb 11; in addition the tonsils of lambs 9 and 11, which were not examined with histoblots, gave a positive result. Immunohistochemical examination of the tonsils showed a granular accumulation of PrPSc in cells of the nodules (Fig. 1C), while a more reticular pattern was observed in nodules in the cortex of the RPLN (Fig. 1D). The immunolabelling was prominent both with and without TSA-enhancement. In the IPP and JPP, PrPSc was detected in the myenteric plexus and in solitary cells in several nodules (Fig. 1E,F ). This immunolabelling was evident with or without TSAenhancement, but the labelling of cells in the nodules was more distinct with enhancement. Five months after inoculation, lamb 29, with homology between its PrP genotype (AV136QQ 171) and that of the donor brain material, showed PrPSc in a limited range of tissues. In this animal histoblotting showed a limited but clear granular labelling in lymphoid nodules in the cortex of the DJLN and RPLN; it also showed PrPSc in lymphoid nodules in the JPP (Fig. 2A) and IPP (Fig. 2B), and in locations that probably corresponded to myenteric plexuses of the IPP. The second inoculated lamb (No. 31; Table 1), whose PrP genotype (AV136QR171) was different from that of the donor brain material (VV136QQ 171), showed no PrPSc in any tissues examined by either of the two methods. Immunohistochemical examination of lamb 29 did not demonstrate PrPSc in lymph nodes or Peyer's patch nodules but showed some PrPSc accumulation in the myenteric plexuses of IPP and JPP; the labelling was visible both with or without TSA-enhancement but was weak without. Eleven months after inoculation, histoblot examination of lambs 33 and 35, both of which had the PrP genotype AV136QQ 171 and homology with the PrP genotype of the donor, showed a strong presence of PrPSc in all six tissues. There were intense, granular accumulations of PrPSc in the lymphoid nodules of both the IPP and JPP (Fig. 3A), and PrPSc was also
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Fig. 1A±F. Histoblot and immunohistochemical analysis of PrPSc accumulation in lymphoid tissues from lambs with PrP genotype VV136QQ171 5 weeks after inoculation with the scrapie agent by stomach tube. (A) Retropharyngeal lymph node. Numerous greyish accumulations of PrPSc (arrows) are present in an oblique section through the lymph node cortex. Capsule indicated by arrowhead. Histoblot. Bar, 1 mm. (B) Distal jejunal lymph node. Accumulations of PrPSc (arrows) are present in nodules in the lymph node cortex. Capsule indicated by arrowhead. Histoblot. Bar, 03 mm. (C) Palatine tonsil. Granular labelling (brown) for PrPSc is present in cells of the lymphoid nodules (n). Epithelium indicated by arrowhead. Immunohistochemistry. Bar, 200 mm. (D) Retropharyngeal lymph node. A reticular pattern of labelling (brown) for PrPSc is present in the centre of a nodule (n). Immunohistochemistry. Bar, 50 mm. (E) Ileal Peyer's patch. Only a few cells (arrows) show labelling (brown) for PrPSc in a lymphoid nodule. Capsule indicated by arrowhead. Immunohistochemistry. Bar, 50 mm. (F) Ileal Peyer's patch. Labelling (brown; arrow) for PrPSc is present in cells of a ganglion in the myenteric plexus lying between the muscle layers (m) of the gut wall. Immunohistochemistry. Bar, 50 mm.
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Fig. 2A,B. Histoblot analysis of PrPSc accumulation in lymphoid tissues from a lamb with PrP genotype AV136QQ171 5 months after inoculation with the scrapie agent by stomach tube. (A) Jejunal Peyer's patch. Granular accumulations of PrPSc in a lymphoid nodule (n). Bar, 01 mm. (B) Ileal Peyer's patch. Granular accumulations of PrPSc are present in two lymphoid nodules (arrows). Bar, 015 mm.
Fig. 3A±D. Histoblot analysis of PrPSc accumulation in lymphoid tissues from a lamb with PrP genotype AV136QQ171 11 months after inoculation with the scrapie agent by stomach tube. (A) Jejunal Peyer's patch. Lymphoid nodules (arrows) contain dense accumulations of PrPSc. Bar, 04 mm. (B) Retropharyngeal lymph node. Many lymphoid nodules in the lymph node cortex show the strong presence of PrPSc (arrows). Capsule indicated by arrowhead. Bar, 065 mm. (C) Spleen. Accumulations of PrPSc are present in lymphoid nodules (long arrows) and in the marginal zone (short arrows). Arrowhead indicates area shown in Fig. 3D. Bar, 065 mm. (D) Spleen. Higher magnification of the marked area in Fig. 3C, showing accumulation of PrPSc in lymphoid nodules (long arrow) and in the marginal zone (short arrow). Bar, 018 mm.
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Fig. 4A±C. Immunohistochemical analysis of PrPSc accumulation in lymphoid tissues from a lamb with PrP genotype AV136QQ171 11 months after inoculation with the scrapie agent by stomach tube. These tissues are from the same lamb as that illustrated in Fig. 3. (A) Spleen. Strong labelling (brown) for PrPSc is present in a lymphoid nodule (n) while little or no immunolabelling is evident in the marginal zone (m). Bar, 50 mm. (B) Jejunal Peyer's patch. There is strong labelling (brown) for PrPSc in lymphoid nodules (n). Some immunolabelling for PrPSc (arrows) is present in the dome (d) and interfollicular areas (i). Bar, 200 mm. (C) Retropharyngeal lymph node. Numerous lymphoid nodules (n) contain PrPSc. Labelling (brown) for PrPSc is present both in granular accumulations (arrows) in cells distributed throughout the nodule and in a reticular pattern (arrowheads) at the nodule centre. Bar, 200 mm.
present in the myenteric plexuses. The three lymph nodes (RPLN, DJLN and SCLN) all showed intense, granular labelling in nodules of the cortex (Fig. 3B). The spleen clearly showed PrPSc in nodules and dense accumulations were also present in locations consistent with the marginal zone (Fig. 3C, D). On immunohistochemical examination, all but one (SCLN) of the examined tissues revealed PrPSc (Table 1). Immunolabelling gave a less prominent and less abundant demonstration of PrPSc than that given by histoblotting, but it nonetheless showed characteristic granular accumulations of PrPSc in the nodules (Fig. 4A±C). In the IPP and JPP, PrPSc was present in myenteric plexuses. In the spleen, the accumulations of PrPSc were generally less intense than in the lymph nodes,
and were restricted to the nodules (Fig. 4A), the marginal zone lacking the clear presence of PrPSc seen with the histoblot method. Discussion In this study, the distribution of early accumulations of disease-associated PrPSc in the lymphoid tissues was examined in lambs dosed by stomach tube with scrapie-infected material. PrPSc was detected 5 weeks after inoculation in lymph nodes associated with the alimentary canal, spleen, and tonsil. Only limited PrPSc was demonstrated in the organized lymphoid tissue of the gut wall (the Peyer's patches). In previous studies, the earliest accumulation of pathological PrP,
Detection of PrPSc in Lambs with Scrapie
detected in GALT (including Peyer's patches and mesenteric lymph nodes draining the gut), occurred at 8 weeks of age in sheep (Andreoletti et al., 2000) and 10 weeks after infection in rodents (Beekes and McBride, 2000). The efficiency of detection of PrPSc differed between histoblotting and immunohistochemistry. The PrPSc detected, albeit in small amounts, in Peyer's patches 5 weeks after inoculation was most likely attributable to the preservation of tissue anatomy associated with the immunohistochemical technique. The limited ability of immunohistochemistry, however, to detect the significant levels of PrPSc revealed by histoblotting in the nodules of Peyer's patches, lymph nodes and spleen 5 weeks and 5 months after inoculation is more difficult to explain. The immunoreactivity of PrP is altered after exposure to protein-denaturing agents such as formic acid, proteinase K, various guanidine components and sodium dodecyl sulphate (Haritani et al., 1994). The difference between the detection of proteinase K-resistant PrP and PrP resistant to hydrated autoclaving and formic acid may be the result of (1) differences in sensitivity of the methods, or (2) inadequacies in tissue sampling procedures needed to identify a limited distribution of initial sites of uptake and accumulation within tissues. Alternatively, the existence of different forms of PrP in tissues has been proposed (Bruce and Fraser, 1991; Bruce et al., 1991; Safar et al., 1998) and the present results suggest that PrP variations at different stages in the pathogenesis of scrapie should be considered. The use of both histoblotting and immunohistochemistry enabled PrPSc to be detected in all tissues examined 5 weeks after inoculation, except for the superficial cervical lymph nodes. The initial sites of detection of PrPSc were the germinal centres of the lymph nodes associated with the alimentary canal, tonsil and spleen. However, the initial detection of PrPSc in these sites after inoculation by stomach tube raises issues in relation to the dissemination of the scrapie agent within the host. Firstly, given the method of inoculation, the presence of PrPSc in retropharyngeal lymph node and tonsil would appear to have required transport of the agent via the blood. Haematogenous dissemination of infectivity to lymphoreticular sites is an accepted feature of the early pathogenesis of experimental scrapie in rodents infected extra-cranially (Kimberlin and Walker, 1988; Scott, 1993). However, if PrPSc in lymphoid tissue associated with the oral cavity was the result of haematogenous dissemination, why was it not found in the superficial cervical lymph node until 11 months after inoculation? It is possible that regurgitation, which is partly established in lambs of the age of those used in the study, resulted in the exposure of the oral
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cavity to the inoculum. It is also possible that remnants of the inoculum present in the stomach tube at the time of withdrawal were deposited in the mouth. Several routes of transmission of transmissible spongiform encephalopathies (TSEs) to the central nervous system (CNS) have been described (Brown, 2001), including transmission from dental and buccal tissues in hamsters (Ingrosso et al., 1999). Further issues raised by the present study are the presence of PrPSc in germinal centres of lymph nodes and tonsil 5 weeks after inoculation, and the restricted amounts in the lymphoid nodules of Peyer's patches. It has been speculated that the follicle-associated epithelium of the Peyer's patches represents the main route of entry for the scrapie agent from the gut (Bruce et al., 2000; Heggebù et al., 2000). However, the present results suggest that while transport of the agent to the nodules of the Peyer's patches occurs, transport of the agent away from the gut via lymph and blood plays an important role. Alternatively, but not inconsistent with this interpretation, processes responsible for the trapping of the scrapie agent and transport into germinal centres in lymph nodes and spleen may be more effective than similar processes in the Peyer's patches. The size of the infecting dose and the duration of exposure to the scrapie agent may also influence this process. In some sheep infected experimentally with the agent of bovine spongiform encephalopathy (BSE), neuroinvasion occurred in the absence of detectable PrP accumulations ( Jeffrey et al., 2001b). Nevertheless, once established in the Peyer's patch nodules, the Peyer's patches represent an important site of PrPSc accumulation, which has been associated with neuroinvasion (van Keulen et al., 1999). Oral inoculation studies of TSEs, including BSE, in sheep have been performed in recent years (O'Rourke et al., 1997; Foster et al., 2001; Jeffrey et al., 2001b). Comparisons between them are made difficult by differences in method of infection and age at inoculation. Pathogenesis studies of scrapie in sheep have mostly been performed on animals from natural scrapie-affected flocks (van Keulen et al., 1999, 2000; Andreoletti et al., 2000; Jeffrey et al., 2001a). Although few young lambs have been examined, results indicate that uptake and spread of PrPSc from intestines and GALT occur at an age of 2±5 months (Andreoletti et al., 2000). Jeffrey et al. (2001a) detected PrPSc accumulations in tonsillar biopsy specimens at an age of 8 months, and PrPSc was detected in secondary lymphoid organs and myenteric plexuses at about 9 months of age (Andreoletti et al., 2000; van Keulen et al., 2000). At the time of initial detection of PrPSc, the sheep in the present study were 3 months of age. The presence of PrPSc in the tonsils in two of five lambs inoculated with agent from sheep of homologous
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susceptible PrP genotype at this early timepoint suggests that tonsillar biopsy would have a role in the diagnosis of preclinical cases of scrapie. Furthermore, the detection of PrPSc in RPLNs at this age indicates that, in abattoir surveillance programmes, this easily accessible lymph node should be considered for sampling. In a previous investigation of the ileal Peyer's patch from the present groups of experimental lambs, Heggebù et al. (2000) used a sensitive immunohistochemical technique that did not distinguish between PrPC and PrPSc. The altered distribution of PrP detected within the compartments of the ileal Peyer's patch at 1 and 5 weeks post-inoculation suggested changes associated with the uptake of the scrapie agent. In the present study, PrPSc was not detected in lymphoid nodules of the IPP at 1 week postinoculation and only solitary cells showed labelling for PrPSc at 5 weeks. The strong presence of PrPSc in Peyer's patch follicles in the present study 11 months after inoculation contrasts with the weak immunohistochemical presence of PrP reported by Heggebù et al. (2000). Interestingly, a recent study of the in-vivo conversion of PrPC to pathogenic isoforms (Yokoyama et al., 2001) revealed that in the CNS sites in mice, PrPC decreased as PrPSc was deposited. Taken together, these observations suggest that the sensitive immunohistochemical approach adopted by Heggebù et al. (2000) mainly detected changes in the presence of PrPC. In addition to the timing and location of PrPSc deposition, the present study examined the relationship between PrP genotype of the recipient lamb and the donor inoculum. PrPSc was detected only in the tissues of lambs of the most susceptible PrP genotypes, namely VV136QQ 171 and AV136QQ 171 (Tranulis et al., 1999), and only when there was homology between the PrP genotype of the donor inoculum and the recipient. However, such homology did not guarantee the presence of PrPSc at 5 weeks postinoculation, (e.g. lambs 13 and 23; Table 1). Prusiner et al. (1990) showed that matching of donor and recipient PrP genotypes resulted in a short incubation period. The present study draws attention to the early distribution of PrPSc in lymphoid tissues, showing that as soon as 5 weeks after exposure lambs may have PrPSc deposits in lymph nodes, tonsil and spleen. Moreover, the method of examination, time after infection, tissue distribution and PrP genotype may all influence the detection of PrPSc in sheep. Acknowledgments The authors are grateful to Eli Bruntland of the Department of Sheep and Goat Research, for
performing the PrP genotyping, and to Birgit Rùe of the Department of Morphology, Genetics and Aquatic Biology for performing the immunohistochemistry. This study was supported by grants 116073/122 (R.H.), 119688/110 (M.J.U) and 118091/122 (G.G. and C.McL.P.) from the Norwegian Research Council. References Andreoletti, O., Berthon, P., Marc, D., Sarradin, P., Grosclaude, J., van Keulen, L., Schelcher, F., Elsen, J. M. and Lantier, F. (2000). Early accumulation of PrP(Sc) in gut-associated lymphoid and nervous tissues of susceptible sheep from a Romanov flock with natural scrapie. Journal of General Virology, 81, 3115±3126. Beekes, M. and McBride, P. A. (2000). Early accumulation of pathological PrP in the enteric nervous system and gut-associated lymphoid tissue of hamsters orally infected with scrapie. Neuroscience Letters, 278, 181±184. Brown, P. (2001). The pathogenesis of transmissible spongiform encephalopathy: routes to the brain and the erection of therapeutic barricades. Cellular and Molecular Life Sciences, 58, 259±265. Bruce, M. E., Brown, K. L., Mabbott, N. A., Farquhar, C. F. and Jeffrey, M. (2000). Follicular dendritic cells in TSE pathogenesis. Immunology Today, 21, 442±446. Bruce, M. E. and Fraser, H. (1991). Scrapie strain variation and its implications. Current Topics in Microbiology and Immunology, 172, 125±138. Bruce, M. E., McConnell, I., Fraser, H. and Dickinson, A. G. (1991). The disease characteristics of different strains of scrapie in Sinc congenic mouse lines: implications for the nature of the agent and host control of pathogenesis. Journal of General Virology, 72, 595±603. Foster, J. D., Parnham, D., Chong, A., Goldmann, W. and Hunter, N. (2001). Clinical signs, histopathology and genetics of experimental transmission of BSE and natural scrapie to sheep and goats. Veterinary Record, 148, 165±171. Hardt, M., Baron, T. and Groschup, M. H. (2000). A comparative study of immunohistochemical methods for detecting abnormal prion protein with monoclonal and polyclonal antibodies. Journal of Comparative Pathology, 122, 43±53. Haritani, M., Spencer, Y. I. and Wells, G. A. (1994). Hydrated autoclave pre-treatment enhancement of prion protein immunoreactivity in formalin-fixed bovine spongiform encephalopathy-affected brain. Acta Neuropathologica, 87, 86±90. Heggebù, R., Press, C. McL., Gunnes, G., GonzaÂlez, L. and Jeffrey, M. (2002). Distribution and accumulation of PrP in gut-associated and peripheral lymphoid tissue of scrapie-affected Suffolk sheep. Journal of General Virology, 83, 479±489. Heggebù, R., Press, C. McL., Gunnes, G., Lie, K. I., Tranulis, M. A., Ulvund, M., Groschup, M. H. and Landsverk, T. (2000). Distribution of prion protein in the ileal Peyer's patch of scrapie-free lambs and lambs naturally and experimentally exposed to the scrapie agent. Journal of General Virology, 81, 2327±2337.
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Received, May 20th, 2002 Accepted, October 26th, 2002