Institute for Animal Health, Neuropathogenesis Unit, Edinburgh EH9 3JF, UK1
Author for correspondence: James Foster. Fax +44 131 668 3872. e-mail Jim.Foster{at}BBSRC.AC.UK
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Abstract |
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Introduction |
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The presence of an abnormal form of the host Prion Protein (PrPSc) is regarded as a marker for infection of tissues in animals with TSEs. Immunodetection in tissue sections using anti-PrP antibodies has been used in rodents (Bruce et al., 1989 ; McBride & Beekes, 1999
) and ruminants (cattle Wells & Wilesmith, 1995
; sheep Miller et al., 1993
; Foster et al., 1996a
; van Keulen et al., 1996
, 1999
). Our previous studies of experimental BSE infection of sheep have shown that incubation period depends on PrP genotype, with ARQ/ARQ and AHQ/AHQ animals having the shortest incubation periods (Goldmann et al., 1994
; Foster et al., 2001
).
The objectives of the present pathogenesis study were to infect sheep with BSE by the oral route and then to establish which tissues showed signs of infection using immunocytochemical detection of PrPSc as a marker. To achieve this sheep were drawn from two studies. The first examined the possibility that BSE could be transmitted from an experimentally infected parturient ewe to her offspring, and the second was to trace the sequential development of BSE by infectivity bioassay and PrPSc diagnosis of various tissues. End-point cases were recorded in both experiments with some of these and some which so far have survived challenge, reported here. There were also a number of uninfected, control sheep. Previous transmission studies showed that brain and spleen of experimentally BSE-infected sheep were infected (Foster et al., 1996b ). This report gives details of animals at clinical phase, although our other studies in preclinical sheep also incorporate infectivity assays and analysis by Western blotting and will be reported when complete.
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Methods |
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BSE challenge and recording of clinical signs.
Experimental challenge was by the oral route into 13 sheep, which were part of two other experiments designed to examine the pathogenesis of experimental BSE in sheep (see Introduction). Seven sheep were between 7 and 10 months of age at challenge, two were 23 months, two were 46 months and two were 58 months of age. A dose of 50 ml of a 10% BSE cow brain homogenate (equivalent to 5 g of brain tissue) was administered into the buccal cavity of each sheep through a rigid plastic tube. A rigorous effort was made to ensure that every sheep received the same dose with no wastage. The source inoculum was tested by transmission to mice and produced characteristic BSE incubation periods (data not shown) and lesion profiles as seen with other BSE sources (Bruce et al., 1994 ). It was also titrated and found to contain 104 mouse i.c. ID50 units of infectivity per gram of tissue. Experimental animals were housed as a group in a dedicated facility, but segregated from other sheep. They were fed 1·5 kg of hay/straw and approximately 150 to 200 g of vegetable protein supplement daily. They had continual access to water and were monitored every day for signs of disease.
Approximately once weekly and during management procedures a more detailed inspection took place. Clinical signs were recorded as follows: being ataxic, especially if made to run around, pruritic, hyperasthaesic, fleece discoloration or feeding/drinking anomalies.
Animals were sacrificed on presentation of clinical signs with an overdose of barbiturate (according to Home Office regulations) and a range of tissues was harvested aseptically and frozen for transmission and Western blotting (to be reported elsewhere), as well as for fixation in formol saline for histology. It was not always possible to collect the same extensive list of tissues from every animal. Two of the BSE-challenged sheep were mated accidentally and produced foetal material. Tissues were also recovered from eight clinically healthy, unchallenged sheep, which provided suitable controls.
Immunohistology.
Tissues processed for immunocytochemistry were fixed in formol saline and pretreated in 98% formic acid for 2 h as tissue blocks. Tissue sections were subjected to hydrated autoclaving (120 °C for 15 min) followed by 20 min incubation at 37 °C in a 0·1% trypsin solution designed to enhance PrP recognition. Endogenous peroxidase was blocked by treating sections with 1% hydrogen peroxide in methanol. The primary antibody (BG4, supplied by Chris Birkett, Institute for Animal Health) was a monoclonal raised in mice against recombinant bovine PrP protein N terminus with amino acid sequence numbered 4757 and 8999, and was used at 1:100 dilution. The secondary antibody, biotinylated rabbit anti-mouse IgG, was then linked to streptavidinperoxidase and the chromagen was AEC (aminoethyl carbazole). Control slides included those treated with normal mouse serum instead of primary antibody and those from unchallenged, scrapie-free sheep. Sections of brainstem from natural scrapie cases were used for PrPSc positive immunostaining controls.
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Results and Discussion |
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Vacuolation was observed in the brains of all seven sheep. It was most prominent in the brainstem where the dorsal motor nucleus of the vagus nerve (DMNV) was clearly affected. In contrast vacuolation in the hypoglossal nucleus, which is adjacent to the DMNV, was absent or marginal. The reticular formation showed evidence of vacuolation as did the raphe and to a lesser extent the pontine nuclei. Vacuolation was also present in the mesencephalon, especially in and adjacent to the central grey matter, and the diencephalon. Here, the hypothalamus was sometimes affected while the thalamic nuclei had localized areas of low to moderate intensity vacuolation. In the basal ganglia there was a paucity of vacuolation and it could not be detected in either of the parietal or frontal cortices, nor in the hippocampus.
PrPSc immunostaining was identified in central and peripheral nervous tissue and in many lymphoid tissues throughout the animals (Table 2 gives all seven affected sheep). PrPSc deposition was widespread throughout the brainstem and in particular the DMNV (Fig. 1
), which immunostained in all seven sheep. The vagus is part of the parasympathetic, autonomic nervous system and has been implicated as a possible route of infection into the brain from the gut (Baldauf et al., 1997
). Mostly, PrPSc immunostaining took the form of an extracellular punctate deposition as well as perineuronal staining in which cell surface and projecting neurites were affected (Fig. 1
). There was no evidence of intracellular PrPSc deposits. Other areas showing prominent immunostaining in all affected sheep were the raphe, reticular formation, pontine nuclei, the granular layer of the cerebellum as localized deposits, mesencephalon, hypothalamus, medio-lateral thalamus and the basal ganglia. PrPSc immunostaining was also present as localized deposits in the inner layers of the parietal and frontal cortex.
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PrPSc immunohistology has shown that nearly all of the lymphoid tissue stained positively. For example, tonsil (Fig. 2) and retropharyngeal lymph node demonstrated strongly staining aggregates in germinal centres of lymphoid follicles in all sheep examined (seven and six respectively), probably as a result of an intimate association with follicular dendritic cells (Brown et al., 1999
). There did not appear to be any PrPSc immunostaining in the indigenous population of tingible body macrophages (TBM), which are a site for PrPSc accumulation in sheep scrapie and in mouse models (Jeffrey et al., 2000
). A possible explanation is that intracellular truncation of PrP within TBMs has removed the N-terminal epitopic sites recognized by the BG4 monoclonal antibody.
Other lymphoid tissue such as the ileo-caecal (four sheep) and mesenteric (seven sheep) lymph nodes associated with lymph drainage from the gut, also immunostained for PrPSc, although slightly less intensely, in all sheep examined. Nictitating membrane (Fig. 3) has been proposed as a possible site for diagnostic biopsy (ORourke et al., 2000
). However, it showed irregular PrPSc immunostaining, being observed in only two of the four sheep inspected. Mamillary and prefemoral lymph node showed even less intense PrPSc immunostaining, but appeared in all of the five sheep from which these tissues were recovered. In these peripheral lymphoid tissues it is not clear whether PrPSc arose through haematogenous spread of the infection (Houston et al., 2000
) or via innervative neuronal pathways. The bronchial-mediastinal lymph node also showed low levels of PrPSc immunostaining in five of the six sheep examined. Both this and prefemoral lymph node are known to harbour infectivity in natural scrapie cases from clinical and some preclinically affected sheep (Hadlow et al., 1982
). In spleen, PrPSc was observed as marginal immunostaining in four of the six sheep examined. The two remaining sheep showed nil staining, even although BSE infectivity has been detected in the spleens of two BSE-challenged, terminally affected sheep that were tested from another study (Foster et al., 1996a
) and from which PrPSc was also detected by Western blotting (not shown).
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Allied to the notion of absorption through the gut has been the identification of PrPSc immunostaining in nerve cells of the myenteric plexus in Meissners ganglia (Fig. 5 observed in five sheep). This phenomenon has already been observed in sheep with natural scrapie (van Keulen et al., 1999
) and in hamsters orally infected with scrapie (Beekes & McBride, 2000
). The vagus nerve, which is involved with visceral afferent and efferent nervous impulses of the parasympathetic nervous system, is a link between the gut and the brain and showed only mild PrPSc staining in three of the six sheep examined. The coeliac-mesenteric ganglion is part of the sympathetic nervous system, which innervates the viscera via the spinal cord. PrPSc immunostaining was present in both cases from which it was possible to recover this tissue (Fig. 6
). Certainly the possibility of infection being absorbed through the gut and becoming established in adjacent lymphoid and peripheral nervous tissue is supported by these observations of PrPSc immunostaining.
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The results of this study on PrPSc immunostaining of various tissues from BSE-challenged, terminally affected sheep have shown extensive staining in the brain, spinal cord and lymphoid tissues. Peripheral nervous tissues, such as the myenteric plexus and coeliac-mesenteric ganglion have also shown immunostaining, supporting the strong possibility that neuroinvasion could have been initiated via the alimentary tract. A recent study on the early pathogenesis of BSE in orally challenged sheep tracing PrPSc immunostaining substantiates this idea (Jeffrey et al., 2001 ).
At this stage it has not been possible to make any clear distinction between the extent and intensity of PrPSc immunostaining between cases of natural scrapie arising in the NPU flock, and these sheep orally challenged with BSE. So far, the number of natural scrapie cases with multiple tissue recovery has been limited, although preliminary evidence has tended to indicate slightly more intense staining of PrPSc in the brainstem of BSE-infected sheep compared to those with scrapie. For example in NPU natural scrapie cases, PrPSc is often observed as localized in the hypothalamic and/or habenula nuclei but with little immunostaining in either medial or lateral thalamic nuclei. An exception to this sometimes occurs with the appearance of densely staining perivascular deposits of PrPSc (Foster et al., 1996a ). With our BSE cases, however, PrPSc tends to be much more widespread with extracellular, punctate and perineuronal staining in the main thalamic nuclei, as well as the hypothalamus. Other transmission studies in sheep comparing BSE and a natural scrapie isolate have also indicated that BSE produces slightly stronger immunostaining in the brain of affected sheep (Foster et al., 2001
).
A further point of interest from these BSE oral challenge experiments is that there are sheep surviving beyond the longest incubation period recorded so far (Table 1). At the time of writing, three challenged sheep survive at 1631 days and three at 1333 days post-challenge. The longest reported BSE-induced experimental incubation period was in an AHQ/ARR sheep when disease developed 2353 days following intracerebral injection (Foster et al., 2001
). Although it is too early to conclude that these sheep will remain free of clinical disease these results indicate the possibility of sheep from known BSE-susceptible genotypes surviving a large dose of BSE infection.
The extent and intensity of PrPSc in lymphoid and nervous tissue from terminal sheep cases of experimental oral BSE infection are in direct contrast to clinical cases of BSE in cattle, which have minimal levels of PrPSc immunostaining in all but central nervous system tissue (Wells et al., 1995 ). There is also little or no peripheral infectivity in naturally occurring and experimentally induced BSE cattle cases (Fraser & Foster, 1994
; Wells et al., 1998
). This may be an indication that if BSE has penetrated the national sheep flock, methods for its detection through biopsy or bioassay of tissues from preclinical cases may prove to be more easily fulfilled in sheep than in cattle with BSE.
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Acknowledgments |
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References |
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Received 2 May 2001;
accepted 22 June 2001.