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General enquiries on this form should be made to: Defra, Science Directorate, Management Support and Finance Team, Telephone No. 020 7238 1612 E-mail: [email protected] SID 5 Research Project Final Report Note In line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects. 1. Defra Project code 2. Project title This form is in Word format and the boxes may be expanded or reduced, as appropriate. 3. ACCESS TO INFORMATION The information collected on this form will be stored electronically and may be sent to any part of Defra, or to individual researchers or organisations outside Defra for the purposes of reviewing the project. Defra may also disclose the information to any outside organisation acting as an agent authorised by Defra to process final research reports on its behalf. Defra intends to publish this form on its website, unless there are strong reasons not to, which fully comply with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000. Defra may be required to release information, including personal data and commercial information, on request under the Environmental Information Regulations or the Freedom of Information Act 2000. However, Defra will not permit any unwarranted breach of confidentiality or act in contravention of its obligations under the Data Protection Act 1998. Defra or its appointed agents may use the name, address or other details on your form to contact you in connection with occasional customer research aimed at improving the processes through which Defra works with its contractors. SID 5 (Rev. 3/06) Project identification SE1952 Studies of the immune system during infection of sheep with scrapie and BSE agent Contractor organisation(s) Moredun Research Institute Pentlands Science Park Bush Loan Penicuik, Midlothian EH26 0PZ 54. Total Defra project costs (agreed fixed price) 5. Project: Page 1 of 8 £ 816,669.00 start date ................ 01 September 2001 end date ................. 31 August 2006 6. It is Defra’s intention to publish this form. Please confirm your agreement to do so. ................................................................................... YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow. Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer. In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000. (b) If you have answered NO, please explain why the Final report should not be released into public domain Executive Summary 7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work. It is usually stated that there is no immunological response to scrapie infection. However, early studies showed alterations in globulin levels during scrapie infection and more recently it has been demonstrated that scrapie infection replicates in the lymphoreticular system (LRS) and that there are structural alterations to secondary follicles of lymph nodes and spleen. We wished to determine the role of the lymphoreticular system and the immunological alterations in scrapie infected sheep and whether these are influenced by the sheep gentoype as well as the dynamics of infection in lymph and blood and the nature of the cells responsible for the dissemination of infection throughout the LRS. Information obtained from this project could be important in any control strategy that might potentially utilise a vaccine based approach and provide new insights in the development of techniques for the early diagnosis of sheep scrapie. These studies will also foster our understanding of the pathogenesis of scrapie infection and can allow future studies to correlate the presence of prion protein (PrP) in blood and tissues with infectivity. In the first study we investigated the relationship between the immune response to scrapie infection and genetic susceptibility to the disease in sheep, immune cell subsets and prion protein (PrP) expression in susceptible and resistant Suffolk sheep in the pre-clinical phase of infection. At six months of age 12 ARQ/ARQ (susceptible) and 9 ARR/ARR (resistant) scrapie-free Suffolk lambs were subcutaneously challenged with scrapie inoculum. Prefemoral lymphadenectomies were carried out at 14 and 180 days post inoculation (d.p.i.) and serial bleeds were collected at monthly intervals up to one year p.i. An indirect double labelling procedure was carried out on peripheral blood mononuclear cells (PBMCs) and lymph node cell preparations and analysed using flow cytometry. Prior to scrapie challenge significantly more PrP+ cells were detected in PBMCs from the susceptible sheep. Furthermore, following challenge significantly more CD8+ and γδ+ T cells were detected in the PBMCs of the resistant sheep. At both 14 and 180 d.p.i, CD21+ cell expression was significantly higher in the lymph node preparations of the susceptible sheep however; in contrast more CD4+ cells were detected in the lymph nodes of the resistant sheep at both time points. It is concluded that significant differences in immune cells subsets and PrP expression do occur between ARQ/ARQ and ARR/ARR Suffolk sheep in the pre-clinical phase of infection. We also developed a method to simultaneously detect PrP, CD marker and major histocompatibility class II antigens on ovine mononuclear cells. The main aim of this second study was to describe the early changes in PrP distribution and mononuclear cell subsets in lymph nodes from sheep experimentally SID 5 (Rev. 3/06) Page 2 of 8 challenged with scrapie. Five scrapie-free sheep were subcutaneously inoculated with scrapie brain homogenate in the draining areas of the left submandibular, prescapular, prefemoral and popliteal lymph nodes at different time points. The contralateral draining area received normal brain inoculum. Triple staining on all lymph nodes was performed prior to flow cytometry analysis. No major variation in percentages of PrP, cell subset or major histocompatibility class II were found between scrapie brain challenged lymph nodes and the contralateral normal brain control. Considerable individual variation was observed between contralateral lymph nodes of different animals and different time point lymph nodes of the same animal. However, the question which remains to be addressed is: why are there differences in the immune cell subsets of infected animals given that PrPsc is not recognised as a foreign antigen and a normal immune response fails to be initiated by the host following scrapie infection. Further work is required to define the very early immune responses immediately after scrapie infection. In addition, research in immune cells from different sheep breeds, using a larger number of animals, alternative routes of inoculation and different infected tissue inocula is required to provide a greater understanding into the nature of immune system involvement in scrapie. Project Report to Defra 8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer). Scientific objectives set out in the contract: To determine for BSE agent infection and for natural sheep scrapie which cells, if any, of efferent lymph (and blood) contain or transport infectivity and at which stages of disease they can be recognised. To determine for BSE agent infection and for natural sheep scrapie the onset and distribution of system LRS infection following s.c inoculation and whether neuroinvasion can be prevented by regional lymphectomy following local s.c. infection. To determine how scrapie infection affects germinal centre formation and maturation To determine how scrapie infection affects lymphocyte populations of lymph nodes. To compare whether different strain of scrapie agent demonstrate different immunopathological phenotypes. To determine how sheep infected with scrapie respond to conventional antigenic challenge SID 5 (Rev. 3/06) Page 3 of 8 Objective set out in the contract have been met: During the course of the project it became apparent that it would not be possible to generate significant data comparing the immune response to different strain of scrapie and BSE. The objectives were therefore modified by agreement to focus on the comparison of the response of genetically resistant and susceptible sheep to a standard peripheral challenge. Materials and Methods, results, including statistical analysis Experimental design Twelve ARQ/ARQ (susceptible) and 9 ARR/ARR (resistant) female or castrated male Suffolk lambs were obtained from the DEFRA scrapie-free flock. At 6 months of age each lamb received 1 ml of 10 % clarified Suffolk scrapie brain pool homogenate (see below) injected subcutaneously in the drainage area of the right prefemoral lymph node. The lambs were divided into two groups determined by the time of prefemoral lymphadenectomy, with six susceptible and four resistant lambs in group 1 and six susceptible and five resistant lambs in group 2. The ipsi- and contralateral prefemoral lymph nodes were surgically removed from group 1 animals at 14 days post inoculation (d.p.i.) and from group 2 animals at 180 d.p.i.. Serial bleeds were carried out at monthly intervals up to one year post inoculation for use in flow cytometry. All animal procedures complied with the Animals (Scientific Procedures) Act 1986 and were approved by the ethics committee at Moredun Research Institute. Inocula preparation A pool of brains from clinical cases of scrapie, from a naturally infected Suffolk flock, confirmed positive by immunohistochemistry, was homogenised in four times their volume of 0.32M sucrose solution to produce a 20 % homogenate. The pooled homogenate was then centrifuged at 850g for 20 minutes. The clarified homogenate was further diluted by the addition of an equal volume of 0.32M sucrose to give a 10 % solution. Sample Collection Fresh whole blood was obtained by jugular venipucture and collected in evacuated tubes containing lithium heparin. The ipsi- and contra lateral prefemoral lymph nodes were removed and a representative sample of each node collected into Hanks’ balanced salt solution (HBSS) with 5 % heat-inactivated foetal calf serum (FCS) and penicillin/streptomycin (100 units ml-1 and 100 μg ml-1 respectively). Flow Cytometry The samples of lymph node were finely minced in HBSS solution supplemented as above with 5 % per cent FCS containing penicillin/streptomycin (100 units ml-1 and 100 μg ml-1 respectively) disaggregated with a stomacher (Seward stomacher 80 biomaster, UK) for 120 seconds at high power before being filtered through lens tissue paper (Whatman 100 x 150 mm). Red cells from the lymph node cell preparations and heparinised blood were lysed by mixing with an equal volume of sterilised Tris ammonium chloride (0.144 M NH4Cl, 0.017 M Tris, pH 7.65) solution at 37 °C. Cells were then counted using a cell counter (Beckman Coulter) and their concentration adjusted to 1 x107 cells ml-1. Cell labelling An indirect double labelling procedure was carried out on the blood and lymph node samples as previously described. Using the monoclonal antibodies listed in table 1 cells were first labelled with a combination of cluster domain (CD) specific antibody and either an antibody raised against the N terminal residues 23-30 of PrP or ovine MHC Class II. The CD markers were detected with an anti-mouse IgG1 antibody conjugated with alexa fluor 488 (Cambridge biosciences, Cambridge), and the FH11 antibody to PrP and the sw73.2 antibody to the MHC class II antibody were detected with an anti-mouse IgG2b antibody conjugated with r-phycoerythrin and an anti-rat IgG conjugated with r-phycoerythrin respectively (Caltag MedSystems Ltd, Towcester, UK). All incubation steps were carried out at 4 °C for 30 minutes and cells were washed (to remove excess unbound antibody) between incubations with PBS containing 5 % foetal calf serum and 0.02 % sodium azide. Cells were fixed in 1 % paraformaldehyde in PBS and stored in the dark at 4 °C until analysed. Table 1 Mab Isotype Target Antigen Target cell/Protein Reference FH11 IgG2b PrP N-terminal residues 23-90 Prion protein Foster et al, 1996 17D IgG1 Ovine CD4 T helper cells Mackay et al, 1988 86D IgG1 Ovine γδ TCR γδ T Cells Mackay et al, 1989 CC21 IgG1 Bovine CD21 B Cells/ CR2 Jan Naessens et al, 1997 ILA-51 IgG1 Ovine CD8 Cytotoxic T cells Jan Naessens et al, 1997 VPM65 IgG1 Ovine CD14 Macrophages, monocytes Gupta et al, 1996 Sw73.2 IgG2b Ovine MHC Class II, β Chain B cells, activated T cells, Hopkins et al, 1986 macrophages, monocytes, dendritic cells SID 5 (Rev. 3/06) Page 4 of 8 Cell Acquisition and Analysis Data was acquired on a FACSCalibur flow cytometer and analysed using CellQuest software (both Becton Dickinson, Oxford, UK). The live gates were set on a linear forward/side scatter axis which allowed elimination of debris and dead cells from the analysis and gating of the PBMCs. At least 10,000 gated cells were acquired for each sample. The fluorescence of the selected lymphocyte population was analysed on a logarithmic scale with results expressed as the percentage of the gated double labelled cells. Follow-up of Infection Tonsil biopsy samples (Jeffrey et al., 2001) were collected at 1 year intervals after infection until sheep were confirmed as PrPsc positive by immunohistochemistry (IHC). Statistical methods For the lymph node data, a mixed-effect model was used with genotype, node of challenge and time of lymphadenectomy included as fixed effects and sheep as a random effect. For the cells obtained from blood, a repeated-measures model was fitted for each cell marker with genotype, bleed and time of lymphadenectomy fitted as fixed effects. Time of lymphadenectomy appeared to have no effect on the responses in the blood and was subsequently removed from this model. To allow for the lack of independence between repeated measurements on the same animal an autoregressive type 1 correlation structure was also included in the model. Only bleeds from the first nine months post inoculation were included in the analysis because of the sparsity of data in the later stages of the study. Results were expressed as estimated mean percentages with approximate 95 % confidence intervals determined, in each case, as the mean plus or minus the standard error multiplied by the 97.5 % point of the associated t-distribution. A false discovery-control method, was used to aid the interpretation of the numerous individual tests that were carried out on both sets of data. All statistical analyses were undertaken using Genstat (version 8 for windows, VSN International, Herts, UK) apart from the false discovery rate procedure which were implemented within Excel. Results One year post-infection (p.i.) 9 out of 12 ARQ/ARQ sheep were PrPsc positive by tonsil biopsy and by two years p.i. one further ARQ/ARQ sheep was confirmed positive. The two ARQ/ARQ animals with negative tonsil biopsy results were clinically normal when culled at 18 month p.i.. All samples from ARR/ARR sheep were negative for PrPsc by IHC in tonsil biopsy at 12 and 24 months p.i.. The results shown below only describe the statistically significant findings however every cell subset dually labelled with either PrP or MHC II was analysed. Phenotyping of Prefemoral lymph node cells at 14 and 180 days post inoculation PrP surface labelling was detected on each cell phenotype investigated (CD4+, CD8+, γδ T cell, CD21+ and CD14+) within the prefemoral lymph node population. In the analyses, for each cell phenotype, only those effects that had an associated P-value of less than 0.02 were judged to be of interest, and these are summarised in table 2. Table 2 Lymph Node Challenged Unchallenged Marker 14 dpi 180 dpi 14 dpi 180 dpi Average SE ARQ 48.8 50.5 48.7 56.9 2.61 gxtxc =0.006 ARR 51.6 55.6 59.4 54.9 ARQ 9.1 6.9 9.3 8.5 2.30 g <0.001 ARR 12.5 19.1 17.4 20.4 ARQ 8.6 5.3 8.8 6.7 1.28 g =0.012 ARR 6.4 2.6 8.6 1.5 ARQ 4.5 3.0 5.1 4.6 ARR 5.0 3.4 5.5 3.4 ARQ 28.1 28.2 24.3 17.4 ARR 21.1 12.6 15.1 12.6 ARQ 25.2 25.4 22.7 15.8 ARR 20.5 12.3 14.9 12.5 Genotype CD4 CD4/MHC II CD8/MHC II CD8/Prion CD21 CD21/MHC II SID 5 (Rev. 3/06) Page 5 of 8 P-values* t <0.001 1.15 c =0.019 2.95 g <0.001 c=0.002 2.70 g <0.001 c =0.009 This cut-off point for the P-value was based on a false discovery rate (FDR) of 20 %. The FDR was set at a relatively high level so that potentially interesting results were not excluded. However, it does mean that some of the apparent effects may have arisen by chance. CD21+ cells The mean CD21+ percentages from the susceptible sheep ARQ/ARQ were significantly higher than those from the resistant animals in the ipsi- and contralateral lymph nodes at 14 and 180 d.p.i. (mean difference of about 9 %, P<0.001). The challenged node was found to have significantly more CD21+ cells (P=0.002) than the unchallenged node. A similar pattern of results was observed for the CD21+/MHC class II+ cell percentages. CD4+ cells A significant correlation between PrP genotype, days post infection and lymph node (challenged or not) was detected in the CD4+ cells (P=0.006) from the resected lymph nodes. More CD4+ cells were detected in the resistant sheep than in susceptible sheep at both 14 and 180 days post infection. The greatest difference in the CD4+ cells was detected at 14 d.p.i. with 10 % more CD4+ cells detected in the resistant sheep (estimated mean of 59 %) when comparing the resistant and susceptible animals in unchallenged lymph node cell preparations. At 180 d.p.i. the number of CD4+ cells was found to be higher in the challenged node of the resistant sheep although in the unchallenged node similar numbers of CD4+ cells were detected in both genotypes. CD8+/PrP cells The mean percentage of cells dually labelled with CD8 and PrP was found to be significantly higher in the unchallenged than in the ipsi-lateral lymph node (P=0.019), at 14 and 180 d.p.i. in both the susceptible and resistant sheep. Peripheral Blood Phenotyping PrP was detected on every cell type tested (CD4+, CD8+, γδ T cell, CD21+ & CD14+) in the mononuclear fraction of the blood. The pattern of responses over time for PrP+ cells, single or double labelled with any of the immune cell subset markers, appeared to be influenced by time post inoculation and PrP genotype. According to the FDR analysis the observed differences in the pattern of responses between the two genotypes were likely to be genuine differences even though the results for different markers were not all independent of each other; the raw P-values for the genotype. PrP+ cells Prior to scrapie challenge, the number of PrP+ cells was significantly higher in the susceptible (estimated mean of 60 %) compared to the resistant sheep (mean of approximately 40 %, P<0.001). Similar differences were also detected in CD4+, CD8+ and CD14+ cells (P<0.001) dually labelled with PrP. After challenge the numbers of PrP+ labelled peripheral blood mononuclear cells (PBMCs) was approximately 40 % irrespective of the sheep PrP genotype for the nine month period p.i.. Gamma delta TCR+/PrP+ cells The number of gamma delta (γδ) cells labelled with PrP (Fig. 2b) was very similar for both the susceptible and resistant sheep (mean of approximately 7 %) prior to inoculation. After inoculation the number of γδ+/PrP+ cells decreased significantly to an estimated mean of 2 % in the susceptible sheep whereas they remained constant in the resistant animals up to 2 months p.i.. After 2 months p.i. the γδ+/PrP+ cell numbers in the resistant sheep declined steadily until 5 months p.i. where the estimated mean number of γδ+/PrP+ cells for both genotypes was approximately 2 %. From 5 to 9 months p.i. the mean number of γδ+/PrP+ cells remained fairly constant in the susceptible and resistant sheep. CD8+/PrP+ cells A significant interaction (P<0.001) between PrP genotype and time was detected in the CD8+/PrP+ labelled PBMC’s (Fig. 2c). Before inoculation the mean number of CD8+/PrP+ cells was significantly higher in the susceptible sheep with a mean of 11 % compared to a mean of 3 % in the resistant sheep. After challenge with the scrapie infected inoculum the numbers of CD8+/PrP+ labelled cells detected decreased in the susceptible animals to a mean of 7 %. Conversely, the mean percentage of CD8+/PrP+ cells detected in the blood of the resistant sheep increased significantly until 2 months p.i. when the highest number of cells were detected (mean of 14 %). After 3 months p.i. both the susceptible and resistant cell numbers decreased and a relatively constant percentage of cells were detected in both genotypes groups from 5 month p.i. until 9 month p.i.. Discussion This study has shown differences in mononuclear cell subsets both in lymph node cell preparations and in blood between Suffolk sheep of susceptible and resistant PrP genotypes following subcutaneous challenge with scrapie inocula. A higher percentage of PrP labelled cells was found in the blood from sheep of susceptible PrP genotype sheep at 6 months of age prior to inoculation with scrapie. The CD21+ population was higher in the resected prefemoral lymph nodes of susceptible animals at 14 and 180 d.p.i.. Interestingly, at 14 and 180 d.p.i. the CD4+ population was greater in lymph nodes from the resistant animals and the CD8+/PrP+ population was higher in the challenged lymph nodes irrespective of the sheep’s PrP genotype. In addition, the proportion of CD8+ and γδ T cells expressing PrP in blood from resistant sheep was significantly higher than in the susceptible animals in the months immediately following infection. Before inoculation there were significantly more PrP labelled cells in sheep of a susceptible genotype. These data support the findings of previous studies from another group who suggested that susceptible animals SID 5 (Rev. 3/06) Page 6 of 8 may have higher numbers of cells expressing PrP and therefore a greater likelihood for these cells to convert to the disease-associated PrP form, resulting in successful infection. CD21+ B cells have been highlighted as being important in pre-clinical disease in deer calves orally infected with Chronic Wasting Disease as well as in mice peripherally inoculated with scrapie. In our study the CD21+ population, which is considered to represent the B cell population, increased in genetically susceptible sheep following inoculation with scrapie brain homogenate. This increase was detected in MHC Class II labelled blood and lymph node cells which are considered to be activated. However, no genotypic differences were found on PrP expressing B cells following scrapie infection which is contrary to the findings of other authors. As suggested previously, PrP antibodies targeting discrete regions in the PrP sequence may give rise to different levels of detection. Other authors used a mAb to PrP which targets an epitope located approximately 40 amino acids downstream from the epitope that the FH11 mAb recognises. Another potentially important difference is in the breed of sheep used, Cheviot sheep were used by the other authors whilst Suffolk sheep were used in the study we describe, and at codon 136, the former encode the amino acid valine whilst the latter encode alanine. However, the difference between results of these two studies could also be due to the stage of infection at which the animals were investigated, with our study assessing pre-clinical findings as opposed to differences at the onset of clinical symptoms. Studies in mice and humans have shown that TSE infection results in an infiltration of CD8 and CD4 T cells into the brain and, in mice, where these phenotypes are missing the incubation period is significantly increased. In our study the number of CD4+, CD8+ and gamma delta T cells in blood, and to some extent in the lymph node cell preparations, varied markedly between the susceptible and resistant sheep genotypes. At 14 d.p.i. the percentage of the CD4+ cells in the lymph node cell preparations was higher in the group of resistant sheep and this finding was even more pronounced at 180 d.p.i.. CD4+ cells, which are considered to represent T helper/regulatory cells, are thought to be directly involved in B cell activation in secondary lymphoid tissues. These cells, as well as macrophages and dendritic cells, are able to present antigen and release defined cytokines. In our study, B cells were more abundant in the challenged lymph nodes of susceptible sheep, although a lower number of T helper cells were detected. The B cells in the challenged nodes may have been preferentially activated by migratory macrophages and dendritic cells, which are known to transport and present PrPsc, instead of T helper cells. CD8+ T cells, otherwise defined as cytotoxic, are important in the successful eradication of most intracellular pathogens and after the resolution of an infection their numbers rapidly decrease. In the current study an appreciable increase of CD8+ T cells expressing PrP was only observed in the blood shortly after inoculation and only in resistant sheep. It is tempting to suggest that the cytotoxic T cells in these animals were recognising cells expressing the abnormal form of PrP and therefore inducing apoptosis in these cells. The increase in the percentage of CD8+/PrP+ cells may be directly related to the high proportion of γδ T cells detected as some cells can co-express CD8 and γδTCR epitopes on their cell surface. Gamma delta T cells are one of the first cells to be detected at a site of inflammation and it has been suggested that they may play a major role in cell mediated immunity. In our study, a higher percentage of γδ T cells, which also expressed PrP, were present in the circulation of the resistant sheep following inoculation. However, γδ T cells were detected at low levels in the lymph node cell preparations both in resistant and susceptible animals. This is probably due to the physiological tendency to localise in blood instead of lymph nodes. It is therefore intriguing to question whether sheep of a resistant genotype are able to more effectively remove the scrapie agent after inoculation. This study has detected significant changes in CD21+, CD4+, CD8+ and γδ T cells in the pre-clinical phase of infection following administration of scrapie at a peripheral site and has also shown variation in these parameters in sheep of different PrP genotypes. Our results, which support previous findings, conflict with the traditional dogma that an immune response is not generated during scrapie infection and furthermore they suggest that the type of immune response differs depending upon the PrP genetics of the animal. However, the question which remains to be addressed is: why are there differences in the immune cell subsets of infected animals given that PrPsc is not recognised as a foreign antigen and a normal immune response fails to be initiated by the host following scrapie infection. Further work is required to define the very early immune responses immediately after scrapie infection. In addition, research in immune cells from different sheep breeds, using a larger number of animals, alternative routes of inoculation and different infected tissue inocula is required to provide a greater understanding into the nature of immune system involvement in scrapie. References to published material 9. This section should be used to record links (hypertext links where possible) or references to other published material generated by, or relating to this project. SID 5 (Rev. 3/06) Page 7 of 8 1. Three-colour flow cytometric detection of PrP in ovine leukocytes. Mara S. Rocchi, Mary-Jo Anderson, Samantha L. Eaton, Scott Hamilton, Jeanie Finlayson, Philip Steele, Robin Barclay and Francesca Chianini Submitted to Vet Immunology Immunopathology 2. Immunological differences between susceptible and resistant sheep during the pre-clinical phase of scrapie infection S.L.Eaton , M.Rocchi , L.González, S.Hamilton, J.Finlayson, J.Sales, M.Jeffrey, P.J.Steele, M.P.Dagleish, S.M.Rodger, H.W.Reid & F.Chianini Submitted to General Journal of Virology 3. PrP detection and immunophenotyping of lymph nodes, lymph and blood cells from sheep experimentally challenged with scrapie. Eaton S.L., Chianini F., Gonzalez L., Rocchi M., Jeffrey M. & Reid H.W. Prion 2004, First international conference of the European Network of excellence Neuroprion 24-28 May 2004, Paris, France. 4. Immune system involvement in sheep experimentally challenged with scrapie. F Chianini, S.L. Eaton, L. Gonzalez, M Rocchi, M Jeffrey, S Hamilton, J Finlayson & H.W. Reid. Joint Funders’ Workshop 1 st-3rd September 2004. University of York, York, UK. 5. Experimental studies of the early events in ovine peripheral lymph nodes after scrapie challenge. Anderson MJ., Rocchi M., Eaton SL., Gonzalez L., Jeffrey M., Hamilton S., Reid HW & F. Chianini. British Society for Immunology Annual Congress, Harrogate. Immunology 113 Suppl 1 p136 British Society for Immunology annual meeting. 7-10 Dec 2004 , Harrogate, UK 6. Immunological differences between susceptible and resistant sheep during the pre-clinical phase of scrapie infection. F.Chianini, S.L.Eaton , M.Rocchi , L.González , S.Hamilton, J.Finlayson, J.Sales, M.Jeffrey, P.J.Steele, M.P.Dagleish, S.M.Rodger, H.W.Reid. Joint Funders’ Workshop 2006. Warwick, UK 7. PrP distribution in blood and lymph nodes from experimentally infected ARQ/ARQ Suffolk lambs. Chianini F., Eaton SL., Rocchi M., Gonzalez L., Finlayson J., Macaldowie C., Dagleish M.P., Reid H.W., Jeffrey M.. Proc 21st Annual Meeting of the European Society of Veterinary Pathology – 10-13 Sept. 2003 Dublin, Ireland 8. S. Sisó, M. Jeffrey, H. Baird, S. Martin, S. Bellworthy, F. Chianini, L. González (2006). The patterns of accumulation of PrP during preclinical sheep scrapie and BSE suggest different pathways of neuroinvasion. European Society of Veterinary Pathologist 24th Annual Meeting. 31st August-2nd September. Edinburgh, UK 9. S. Sisó, M. Jeffrey, H. Baird, S. Martin, S. Bellworthy, F. Chianini, L. González. The topographical distribution of prion protein in the brains of sheep challenges the current hypothesis of neuroinvasion. Neuroprion Conference, 4-6th October 2006, Torino, Italy SID 5 (Rev. 3/06) Page 8 of 8