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SID 5
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Research Project Final Report
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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
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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)
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