Institute for Animal Health, Compton, Newbury, Berkshire RG20 7NN, UK1
BBSRC/MRC Neuropathogenesis Unit, Ogston Building, West Mains Road, Edinburgh EH9 3JF, UK2
VLA Lasswade, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 0PZ, UK3
Author for correspondence: Fiona Houston. Fax +44 1635 577263. e-mail fiona.houston{at}bbsrc.ac.uk
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The sheep were Cheviots imported from NZ by the Department for Environment, Food and Rural Affairs (DEFRA; formerly the Ministry of Agriculture, Fisheries and Food), or were the offspring of a scrapie-free breeding flock (the DEFRA/SF flock) established from the imported animals. They were held in strict isolation before being transported to IAH, Compton. Six groups of five DEFRA/SF animals 611 months of age and with PrP genotypes VRQ/VRQ, VRQ/ARQ, VRQ/ARR, ARQ/ARQ, ARQ/ARR and ARR/ARR, were inoculated subcutaneously with SSBP/1 (2 ml 10% brain homogenate). This dose is known to induce scrapie in 100% of inoculated UK Cheviots carrying the VRQ allele with incubation periods that are not related to age (J. D. Foster & W. Goldmann, unpublished). In a related experiment Poll Dorset sheep (VRQ/ARQ and VRQ/ARR) from a flock (PD1) imported from NZ and maintained free of scrapie (Hunter & Cairns, 1998 ) were also inoculated subcutaneously with SSBP/1.
All animals were housed under strict isolation and fed on a diet of dried lucerne pellets and hay, with free access to a mineral supplement. Care was taken to source the lucerne and hay from farms/land that had been free of livestock for at least 20 years and to ensure that the diet did not contain any animal protein.
When clinical signs consistent with scrapie were clearly present, the animals were humanely destroyed according to Home Office regulations. The brains were removed at necropsy and divided longitudinally. One portion was fixed in neutral buffered formalin (NBF) and processed for histopathological examination according to standard protocols. Samples of lymphoid tissues (spleen, tonsil, prescapular lymph node, mesenteric lymph node, Peyers patch) were also taken and fixed in NBF. Immunohistochemical staining with the avidinbiotin complex system was carried out on paraffin-embedded sections using a method involving hydrated autoclaving, based on the technique described for detection of PrPSc by Haritani et al. (1994) . Immunostaining for PrPSc in the brain was carried out using a polyclonal antibody raised in rabbits to amino acids 217231 of the bovine PrP sequence (R482) and also with R521 (van Keulen et al., 1995
), which provide good staining of sheep scrapie-affected brains from a range of breeds and genotypes. Immunostaining for PrPSc in the lymphoid tissues was carried out using the mouse monoclonal antibody FH11 (Foster et al., 1996
).
All DEFRA/SF Cheviot sheep in the PrP genotype groups VRQ/VRQ, VRQ/ARQ and VRQ/ARR developed clinical signs suggestive of scrapie at various intervals following inoculation with SSBP/1 while ARQ/ARQ, ARQ/ARR and ARR/ARR Cheviots remain alive and healthy at the time of writing (1108, 1110 and 729 days post-inoculation, respectively). It is intended that animals in this experiment are allowed to survive for lifespan because although expected to be resistant to SSBP/1 challenge, this needs to be confirmed. In the scrapie-affected VRQ sheep, the first sign to be observed was pruritus, manifesting as rubbing of the hindquarters and flanks (often accompanied by nibbling movements and licking the lips) and/or nibbling and biting of the lower limbs. All the animals also showed ataxia and incoordination, initially affecting the hind limbs, but progressing to involve the forelimbs in some cases. This appeared to be associated with proprioceptive deficits, exhibited by wide base stance, stumbling, swaying, greater abduction/adduction of the hind limbs and a tendency to fall when turning sharply. There were no marked abnormalities of mentation or behaviour, e.g. increased nervousness, aggression or hyperaesthesia, although some animals appeared mildly depressed. These clinical signs are consistent with those previously reported in sheep infected with SSBP/1, where pruritus, incoordination of gait and weight loss were the predominant clinical signs (Wilson et al., 1950 ; Dickinson et al., 1968
).
The diagnosis of scrapie was confirmed by the demonstration of mild, though characteristic, lesions on histological examination of the brain and by immunostaining for PrPSc. The patterns of vacuolation and PrPSc accumulation were similar in all three genotype groups. Sparse or mild neuropil vacuolation was present in the grey matter of a restricted range of neuroanatomical nuclei; the olives, ventral thalamic nucleus, the dorso-medial thalamic nucleus, caudate nucleus and accumbens. This limited range of vacuolation is similar to that previously found with this experimental scrapie strain in UK Cheviot and Swaledale sheep (Foster et al., 1996 ).
Immunostaining indicated widespread disease-specific PrPSc accumulation in brain. In addition to the vacuolated sites mentioned above, PrPSc accumulations were also found in dorsal brainstem nuclei (including the dorsal motor nucleus of the vagal nerve), cerebellar, midbrain and cerebrocortical sites, which did not show vacuolation. Several patterns of disease-specific PrPSc accumulation were found including intraneuronal PrPSc accumulation, stellate accumulations (thought to be associated with astrocytes) and diffuse granular patterns of immunolabelling. These patterns of PrPSc accumulation in brain are also typical of natural sheep scrapie (van Keulen et al., 1995 ).
Immunostaining was also performed on sections of prescapular lymph node (PSLN), mesenteric lymph node (MLN), tonsil, spleen, jejunal and ileal Peyers patch (JPP and IPP respectively) from each animal. In general, the pattern of PrPSc deposition was similar to that seen in natural scrapie, i.e. diffuse reticular staining within follicular germinal centres characteristic of accumulation on follicular dendritic cells (FDCs). There were clear differences between the genotypes in the distribution and relative levels of PrPSc accumulation (see Table 1). In VRQ/VRQ sheep, PrPSc was consistently detected in all the lymphoid tissues of every animal examined, and the percentage of positive follicles and intensity of staining were higher than in the other genotypes. In VRQ/ARQ sheep, PrPSc was most consistently detected in the PSLN (5/5 animals), tonsil (5/5 animals) and spleen (4/5 animals). The distribution of PrPSc was much more restricted, and the staining much weaker, in VRQ/ARR sheep than in the other genotypes, although 4/5 animals showed weak staining in the tonsil and 3/5 were positive in the PSLN. In general, these observations are similar to natural scrapie cases. However, the intensity of the staining was weaker, and the distribution of PrPSc in VRQ/ARQ sheep was more restricted, than would be expected in naturally infected animals. In natural cases, PrPSc is usually widely distributed in lymphoid tissues of both VRQ/VRQ and VRQ/ARQ animals by the time clinical signs develop. Interestingly, in the rare natural cases of scrapie in VRQ/ARR sheep, PrPSc is not found in lymphoid tissues (van Keulen et al., 1996
; Andréoletti et al., 2000
; M. Jeffrey, unpublished), but all of the SSBP/1-infected sheep showed positive staining, albeit weak, in at least one of the tissues examined. A similar study of lymphoid tissues from SSBP/1-inoculated Cheviots from the IAH Neuropathogenesis Unit (NPU) flock is still in progress, and a full comparison of the pathogenesis of SSBP/1 scrapie in sheep inoculated by several different routes of injection will be presented in a separate publication.
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Received 2 October 2001;
accepted 7 January 2002.