1 Veterinary Laboratories Agency (VLA-Lasswade), Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 0PZ, UK
2 Institute for Animal Health Neuropathogenesis Unit, Edinburgh EH9 3JF, UK
3 Institute for Animal Health, Compton, Berkshire RG20 7NN, UK
Correspondence
Martin Jeffrey
m.jeffrey{at}vla.defra.gsi.gov.uk
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ABSTRACT |
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INTRODUCTION |
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Since the ban on the use of ruminant feed in the 1990s, the prevalence of BSE in cattle in the UK has declined steadily and it has recently been suggested that the incidence of vCJD cases may have peaked in 2000 (Andrews et al., 2003). However, sheep may also have been exposed to BSE-contaminated meat and bonemeal during the mid-1980s and mid-1990s (Butler, 1998
) and previous studies have shown that BSE may be transmitted readily to sheep by oral, intravenous and intracerebral routes (Foster et al., 1993
; Jeffrey et al., 2001c
; Houston et al., 2003
). The possibility that consumption of BSE-infected sheep meat may pose a risk to humans has prompted urgent research into this area. As sheep originally exposed to cattle BSE would not be in production at present and as the feeding of meat and bonemeal to ruminants is no longer legal, any ovine BSE infection could only be maintained by transmission between sheep. Whilst maternal and horizontal (except from contaminated feedstuffs) methods of transmission of cattle BSE do not occur or are epidemiologically insignificant (Wilesmith et al., 1997
), scrapie is maintained endemically in flocks through sheep-to-sheep contagion. However, as the distribution of PrPd in tissues of sheep infected orally with BSE (Jeffrey et al., 2001c
) resembles that of natural sheep scrapie more closely than that of cattle BSE (Van Keulen et al., 1996
, 2000
; Jeffrey et al., 2001b
; Heggebø et al., 2002
), the epidemiological features of BSE in cattle cannot be taken as an example for any potential naturally occurring sheep BSE.
Detectable infectivity in cattle with BSE is mainly confined to tissues that may be excised from the carcass, thus rendering the risk to man via the food chain low (Wells et al., 1998). Evidence obtained from mouse bioassay has shown that, in experimental cattle BSE, infectivity is present in the distal ileum between 6 and 18 months post-infection, in the central nervous system (CNS) and dorsal root ganglia at 32 months post-infection and in the peripheral nervous system between 32 and 40 months post-infection (Wells et al., 1998
), with the earliest onset of clinical signs taking place at 35 months post-infection. This pattern is in marked contrast to BSE infection of sheep. By using IHC detection of PrPd as a marker (Prusiner, 1999
), infectivity has been found in the intestinal Peyer's patches as early as 5 months post-infection (Jeffrey et al., 2001c
). Subsequently, spread to the enteric nervous system and spinal cord occurs after 10 months and widespread dissemination throughout the lymphoreticular and peripheral nervous systems by 21 months post-infection (Jeffrey et al., 2001c
). This widespread infectivity of tissues in experimentally BSE-infected sheep (and also in natural scrapie) would make successful excision of high-risk tissues from the carcass impractical.
In previous studies, we have shown that oral BSE in sheep of the ARQ/ARQ genotype can be distinguished from natural sheep scrapie by differences in the immunolabelling patterns of phagocytic cells and neurons with a panel of PrP antibodies (Jeffrey et al., 2001a, 2003
; González et al., 2003
). The intracellular aggregates of PrPd in tingible body macrophages (TBMs) and in glial cells are located in lysosomes (Jeffrey et al., 1994
, 2000
), where they are acted upon by endogenous proteases, so that the full-length protein will be truncated and partially digested. The site of truncation within the N terminus of PrP appears to be different for sheep scrapie and experimental sheep BSE, giving rise to distinct IHC patterns. These data have recently been supported by Western blot studies on brain (Stack et al., 2002
; Lezmi et al., 2004
; Thuring et al., 2004
), which suggest that, after digestion by exogenous enzymes, the BSE PrPres molecule is shorter than that obtained from all sheep scrapie sources so far tested. Preliminary studies of the brains of three BSE-affected ARR/ARR sheep also showed similar features (Houston et al., 2003
).
In order to determine whether the truncation patterns of PrPd and/or PrPres reported previously in ARQ/ARQ sheep challenged orally with BSE are influenced by route of infection or by the genotype of the host, we have examined six groups of sheep with different PrP genotypes that had been challenged orally, intracerebrally (IC) or intravenously (IV) with BSE agent. In the present study, we show that the route of infection and the host PrP genotype do not affect the immunolabelling and Western blotting properties of sheep BSE PrPd.
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METHODS |
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Immunohistochemistry.
After developing clinical signs of TSE, animals were euthanized and post-mortem examination was performed. Samples of medulla oblongata at the level of the obex, spleen, palatine tonsil and mesenteric lymph node (MsLN) were fixed in neutral 10 % phosphate-buffered formalin, trimmed, post-fixed and embedded according to standard procedures. Sections (5 µm thick) were cut on a microtome, mounted on treated glass slides (Superfrost Plus; Menzel-Glaser) and dried overnight at 37 °C.
IHC methods have been described previously (González et al., 2002, 2005
). Briefly, tissue sections were immersed in 98 % formic acid for 15 min, followed by 30 min autoclaving at 121 °C in 0·2 % citrate buffer, pH 6·4. Incubation with the primary antibody was performed overnight at 4 °C and the rest of the IHC protocol was performed by a commercial immunoperoxidase technique (VECTASTAIN Elite ABC; Vector Laboratories), tissue sections being finally counterstained with Mayer's haematoxylin. Serial sections of each tissue sample were immunolabelled with five different PrP antibodies, namely R145 (rat mAb at 1/4000 dilution), P4 (mouse mAb at 1/20 000 dilution), 521 and 505 (rabbit polyclonal antisera at 1/10 000 and 1/6000 dilutions, respectively) and BG4 (mouse mAb at 1/2000 dilution). The PrP epitope affinity of these antibodies has been referred to previously (Jeffrey et al., 2001a
, 2003
; González et al., 2003
). BG4 was raised against recombinant bovine PrP and mapped to a sequence between amino acids 45 and 57 by Pepscan analysis (Geysen et al., 1984
). The epitope recognized by the P4 antibody (amino acids 9399 of ovine PrP) has recently been determined precisely (Thuring et al., 2004
).
Control tissues used in each IHC run were as follows. For brain, positive controls included samples of obex from a clinically sick sheep previously challenged with ovine BSE and from a Suffolk sheep naturally affected with scrapie. For lymphoid tissues, positive controls consisted of samples of tonsil or MsLN from an ARQ/ARQ sheep challenged orally with BSE and from an ARQ/ARQ Suffolk sheep with natural scrapie. As negative controls, samples of obex and LRS tissues from a normal, unaffected ARQ/ARQ sheep were used.
Morphometric methods and statistical analysis.
Morphometric estimations of the amount of PrPd accumulation were performed for tonsil, MsLN and spleen on all sheep included in the study. To determine the distribution and intensity of PrPd accumulation in these tissues, two parameters were scored: the percentage of positive follicles and the magnitude and distribution of PrPd labelling within follicles. To avoid biases arising from variation in follicle size or in the site of PrPd accumulation, only follicles in which distinct light and dark zones could be seen were counted. The magnitude of labelling was scored as follows: 0, no labelling; 1, labelling confined to only one or two TBMs in the light zone; 2, TBM labelling throughout the light zone; 3, TBM labelling in both the light and dark zones; 4, TBM labelling in both the light and dark zones and weak labelling associated with follicular dendritic cells (FDCs) present in the light zone; 5, as for 4, but intense FDC-type labelling in the light zone. These labelling patterns are interpreted to indicate a progressive increase in the magnitude and/or duration of follicular infection. For each LRS tissue examined, a mean value was obtained from those follicles that actually showed PrPd immunolabelling.
Comparisons of morphometric data, both percentage of positive follicles and magnitude of immunolabelling, obtained from the different sheep groups examined were performed by Student's two-sample test using a commercially available statistical package (InStat; Graphpad Software).
Western blotting.
Western blotting was performed on samples of brain from all IC-challenged sheep in order to detect PrPres. PrPres was extracted by using modifications of the methods described by Hope et al. (1986, 1988)
as follows: 0·2 g brain tissue was homogenized in 2·0 ml 0·01 M sodium phosphate, pH 7·4, 10 % (w/v) N-lauryl sarcosinate containing 1 mM phenylmethylsulphonyl chloride and 1 mM N-ethyl maleimide. The suspension was centrifuged at 13 000 g for 20 min. After discarding the pellet, the supernatant was centrifuged at 215 000 g for 150 min at 10 °C. The supernatant was discarded and the pellet was resuspended in 0·6 ml deionized water and shaken at 37 °C for 30 min. The sample was then adjusted to a volume of 1·8 ml with deionized water and to an ionic concentration of 0·6 M potassium iodide, 6 mM sodium thiosulphate, 1 % (w/v) N-lauryl sarcosinate and 10 mM sodium phosphate, pH 8·5. The sample was then treated with 50 µg proteinase K (PK) ml1 and incubated with agitation at 37 °C for 60 min. The PK-treated fraction was sedimented through a 2 ml cushion of 20 % sucrose, 0·6 M potassium iodide, 6 mM sodium thiosulphate, 1 % (w/v) N-lauryl sarcosinate and 10 mM sodium phosphate, pH 8·5, by centrifugation at 285 000 g for 90 min at 10 °C. The pellets were dissolved in 23·4 M formic acid and dried under vacuum. Peptide-N-glycosidase F (PNGase)-treated PrPres was prepared by using the method described by Collinge et al. (1996)
.
Samples were run on 14 % Tris/glycine gels (Novex) and immunoblotted onto PVDF membranes. For detection of PrPres, two mAbs were used: 6H4 (Prionics, 2 mg ml1) diluted 1 : 5000 and P4 (R-Biopharm, 1 mg ml1) diluted 1 : 2500. Visualization of the blots was performed with a chemiluminescent substrate and Lumi-Film (both from Roche).
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RESULTS |
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Following IC infection, the proportion of LRS tissues accumulating PrPd varied according to PrP genotype, with ARQ/ARQ sheep showing the most widespread distribution of PrPd, ARR/ARR animals lacking detectable PrPd in LRS tissues and VRQ/VRQ sheep showing an intermediate situation (Table 1). Statistically significantly more positive follicles were found in ARQ/ARQ sheep than in VRQ/VRQ animals in tonsil, MsLN and spleen (Table 2
; Fig. 1
a). In sheep of these two genotypes, the percentage of PrPd-positive follicles was lower in the MsLN than in the tonsil (P<0·01), whereas the spleen did not show differences from either of the other two LRS tissues in this respect (Table 2
; Fig. 1a
).
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Following IV or oral inoculation, sheep of all three PrP genotypes examined showed a consistent involvement of the palatine tonsil but, in agreement with data from IC-challenged sheep, the MsLN and spleen were only affected consistently in ARQ/ARQ sheep (Table 1). Both groups of IV-challenged sheep had a greater intrafollicular intensity of immunolabelling in the spleen when compared with the other two LRS tissues examined and a greater percentage of follicles accumulating PrPd in spleen than in tonsil and/or MsLN (Table 2
). Conversely, orally challenged sheep had a lower percentage of follicles accumulating PrPd and a lower intrafollicular intensity of immunolabelling in the spleen when compared with the other two LRS tissues (Table 2
). Similarly, IV-challenged sheep had a greater percentage of follicles accumulating PrPd and intrafollicular intensity of immunolabelling in the spleen when compared with orally challenged sheep (Table 2
).
PrPd immunolabelling with different antibodies (peptide mapping) in LRS and CNS tissues
In previous studies, we have shown that, unlike scrapie, the intracellular truncation site of ovine BSE PrPd is influenced by the tissue and cell types in which it accumulates, giving rise to distinct patterns of intracellular immunolabelling when different PrP antibodies are employed. By using this IHC strategy, also called epitope mapping, the inferred shortest fragment of intracellular PrPd is detected in TBMs, followed by glial cells and neurons (Jeffrey et al., 2003). In contrast, in natural sheep scrapie, intracellular PrPd appears to be of the same length, regardless of the cell type in which it accumulates, and is always longer than any intracellular PrPd found in sheep BSE. In the present study, we aimed to determine whether the host PrP genotype or the route of infection could affect these IHC differential properties of ovine BSE and whether or not differentiation between ovine BSE and sheep scrapie would still be possible independently of such variables.
A summary of the intracellular labelling patterns for LRS tissues is shown in Table 3 and examples are shown in Fig. 2
. As described above (see Tables 1 and 2
), PrPd did not accumulate in the LRS tissues of all sheep studied, but when it did, intense, multifocal, granular accumulations could be identified throughout the light and dark zones of secondary follicles. This pattern of labelling is associated with intracellular accumulation of PrPd within TBMs. When tissues were immunolabelled with the C-terminal PrP antibody R145, the TBM-associated PrPd pattern could be seen in all positive follicles and LRS tissues. However, when serial sections of these tissues and secondary follicles were incubated with PrP N-terminal antibodies (BG4, P4, 521 and 505), there was little or no discernible labelling of TBMs. This antibody-dependent differential immunolabelling was not affected by the route of challenge or the host PrP genotype. In contrast to BSE-challenged sheep, LRS tissues from Suffolk scrapie-affected sheep showed labelling of TBMs with all the antibodies used except BG4 (Table 3
).
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In all BSE-affected sheep, intraneuronal accumulation of PrPd was not observed with the P4 or BG4 antibodies, but was found with all three other antibodies tested (Table 3; Fig. 3
). In contrast, Suffolk sheep with natural scrapie showed similar magnitudes of intraneuronal PrPd accumulation with antibodies R145, 505, 521 and P4. As described previously, the extreme N-terminal antibody BG4 did not label intracellular PrP accumulations in glia or in neurons.
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Western blotting profiles of PrPres accumulation in sheep challenged IC with BSE
The IHC epitope-mapping results described above suggest intracellular truncation of PrPd in phagocytic and non-phagocytic cells occurring at different sites of the N terminus of the abnormal protein when sheep BSE and natural scrapie are compared. Ultrastructural studies indicate that truncation takes place in lysosomes, where PrPd accumulates (Jeffrey et al., 1994, 2000
), and is therefore likely to result from enzymic activity. Western blotting and most other rapid testing methods for diagnosis of TSEs in ruminants use PK to reveal the presence of PrPres in tissue samples. Preliminary results from Western blotting analysis of experimental sheep BSE and natural scrapie indicate that the site of truncation by this exogenous enzyme differs between these two infections, allowing its differential diagnosis (Stack et al., 2002
). In the present study, we have addressed whether or not these differential immunochemical properties of sheep scrapie and ovine BSE PrPres depend on the host PrP genotype.
Samples of BSE-affected sheep brains analysed by Western blotting using the mAb 6H4 gave strong PrPres bands in all experimental BSE sheep (Fig. 4a). A range of intensities of labelling with 6H4 and some variation in the speed of migration of PrPres was observed, but these were probably related to protein-loading levels and not to differences in PrP genotype. The apparently lower molecular mass of the unglycosylated fragment of PrPres from sheep BSE samples compared with scrapie samples (Stack et al., 2002
) was observed consistently (Fig. 4a
) and could be demonstrated more clearly by removing the carbohydrate side chains following treatment of the samples with PNGase (Fig. 4b
). The main differential Western blot feature between sheep BSE and natural scrapie is the decreased signal found in blots labelled with mAb P4 (Stack et al., 2002
). Samples of BSE-affected sheep brains analysed by Western blotting using the mAb P4 showed no significant differences between sheep of different PrP genotypes, which could be distinguished readily from scrapie controls (Fig. 4c
), as they showed a much lower labelling intensity. As samples from each of the different sources, scrapie and BSE, were prepared and split into two identical preparations prior to loading onto gels and exposure times of the gels were the same, the differences in Western blotting profile were not attributed to differences in protein content.
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DISCUSSION |
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Genotype-associated variation in the distribution and magnitude (proportion of positive lymphoid follicles and intensity of immunolabelling) of PrPd accumulation in LRS tissues was observed in sheep infected with the BSE agent by the IC route. All of these parameters were greater in animals of the PrP ARQ/ARQ genotype than in VRQ/VRQ sheep. The most conspicuous genotype effect, however, was the absence of any LRS involvement in ARR/ARR sheep, which is in agreement with a previous report (Houston et al., 2003). Although peripheral replication is probably irrelevant to incubation period following IC challenge, there appears to be a genotype-related inverse relationship between LRS involvement and incubation period. Thus, an increased length of incubation period corresponds to a lower frequency and magnitude of PrPd accumulation in LRS tissues (Fig. 5
). The incubation period of sheep TSEs is often taken as a measure of susceptibility, and a greater susceptibility of ARQ/ARQ sheep to BSE infection has also been suggested in previous IC or oral experiments, although VRQ homozygotes were not represented in either of these studies (Foster et al., 1993
, 2001a
). All orally or IV-inoculated sheep homozygous for glutamine at codon 171 had levels of peripheral PrPd accumulation approximately similar to those of IC-challenged sheep of the ARQ/ARQ genotype. When the incubation periods and levels of peripheral tissue involvement are viewed together (Fig. 5
), this suggests that ARQ/ARQ, AHQ/AHQ and AHQ/ARQ sheep have similar susceptibilities to infection. Alternatively, the PrP of such sheep would have similar efficiencies of conversion from normal to abnormal forms and the accumulation of these would therefore cause disease in a similar time course. In contrast, sheep bearing the VRQ allele would be able to generate less abnormal PrP, as a result of a lower ability either to replicate infectivity or to convert PrPc to PrPd, than sheep of the ARQ/ARQ, AHQ/AHQ and AHQ/ARQ genotypes. Finally, ARR/ARR sheep would be the least able to replicate infectivity in LRS tissues or to support PrPc-to-PrPd conversion.
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In previous studies of sheep challenged orally with BSE, different intracellular antibody-labelling patterns of PrPd were found within brain when compared with LRS tissues (Jeffrey et al., 2003). As ultrastructural studies suggest that intracellular PrPd aggregates are found within lysosomes (Jeffrey et al., 1994
, 2000
), the differences in antibody-labelling patterns probably relate to variation in the truncation site of different cellular sources of PrPd. This variation might be related to cell-driven differences in PrPd conformation, to cell-specific variation in the enzymic content or to other biochemical conditions within lysosomes. These same patterns of between-cell variation of truncation were found in the present study, with the putative shortest fragment of intracellular BSE PrPd being found in TBMs and the longest in neurons, with glia occupying an intermediate position. As these patterns were common to all PrP genotypes, the present study shows that, in ovine BSE, PrPd processing and the resultant variable fragment sizes of PrPd in different cell types are not due to any conformational changes in PrP derived from amino acid substitutions at codons 136, 154 and 171.
In conclusion, this study shows that the antibody-labelling patterns of intracellular PrPd are the same in sheep of different genotypes infected with BSE by different routes and can be differentiated readily from those of all natural sheep scrapie sources so far examined. Wide-scale testing for sheep BSE based on proteinase-digested PrPres fragment sizes may therefore be feasible. In addition, these findings provide support for earlier studies and further suggest that sheep homozygous for alanine at codon 136 and glutamine codon 171 are more susceptible to BSE-agent infection than are other sheep genotypes.
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ACKNOWLEDGEMENTS |
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Received 15 June 2004;
accepted 17 November 2004.