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SID 5Research Project Final Report
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Project identification
1.Defra Project code
SE0248
2.Project title
A Retrospective Search for Atypical Scrapie in Sheep
and Goats Submitted to VLA Between 1980 and 1989
3.Contractororganisation(s)
Veterinary Laboratories Agency
Woodham Lane
New Haw
Addlestone
Surrey
KT15 3NB
UK
54.Total Defra project costs
£98 776
(agreed fixed price)
5.Project:start date
01 December 2006
end date
01 July 2008
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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.
Atypical scrapie is a relatively recent discovery and it is
unknown whether it is a new phenomenon or whether it has existed
undetected in the national flock for some considerable time. The
majority of cases in the UK have so far occurred in older sheep of
PrP genotypes that are associated with relative resistance to
classical scrapie (Konold et al., 2007; Everest et al., 2006;
Konold et al., 2006; Saunders et al., 2006; Simmons et al.,
unpublished observations). It must also be considered that if
atypical scrapie has existed for some time, then it has not been
detected. Before 1998, the routine statutory diagnosis of TSE in
sheep relied on the presence of TSE vacuolation in the brain. This
method would not have been effective for the detection of atypical
scrapie. Immunohistochemistry (IHC) and Western blot are commonly
used to confirm atypical scrapie. The IHC pattern of atypical
scrapie is very different from that in classical scrapie using the
same antibody. For example, in the medulla at the level of the
obex, PrPd deposits are restricted to the spinal tract nucleus of
the trigeminal nerve in most cases of atypical scrapie. However,
using the same antibody, classical scrapie PrPd deposits are
located throughout the obex, with minimal cases showing deposition
in the dorsal vagal nucleus only. Although the trigeminal nucleus
is also affected in classical scrapie, it is never the first area
to become so. In addition, the morphology of the PrP deposits in
classical scrapie and atypical scrapie are very different. Atypical
scrapie also gives a different Western blot profile when compared
to classical scrapie, (Everest et al., 2006; EFSA, 2005). It is
thus possible that historic cases of atypical scrapie were
misdiagnosed, due to a lack of suitable diagnostic techniques and a
lack of awareness of this form of the disease.
The VLA holds a large stock of formalin-fixed, paraffin-embedded
tissues from a number of species, collected over a large number of
years, including material from sheep and goats. The first phase of
project SE0248 centralised and catalogued this material. The
earliest known case of atypical scrapie in the UK, found
retrospectively, was in a sheep presented for confirmation of
clinical scrapie in 1989 (Bruce et al, 2007). It was therefore
appropriate to test retrospectively the samples available from
animals submitted prior to 1990 for atypical scrapie to determine
whether or not it had existed before then. Over 1300 animals
submitted between 1980 and 1989 have been further investigated
using IHC. These were a mixture of scrapie negative animals and
those of unknown status, due to most of the original paperwork
being discarded in accordance with agency records management
procedures. We sought here to assess if any of these animals
(diagnosed by histopathological changes alone) were early cases of
atypical scrapie. There was at least one piece of brain material
available from each animal in the form of a formalin-fixed,
paraffin-embedded tissue block. There were multiple pieces of brain
tissue available for some cases, but no parity in terms of
available material for the entire group. Medulla at the level of
the obex and/or cerebellum were the most highly desired tissues,
with availability of both representing the ideal. If obex and/or
cerebellum were not available, then thalamus or basal ganglia were
the next tissues of choice. If none of the above were available,
then mesencephalon was selected. Each case was assessed by IHC
using antibody 2G11, which is particularly effective at detection
of atypical scrapie (Moore et al., 2008).
The vast majority of cases tested, 1155, were TSE negative. 57
were unsuitable for diagnosis. This was for a number of reasons,
including no representation of the target areas. 173 were IHC
positive with 2G11. Each of these were tested with R145 (the UK
statutory TSE diagnostic IHC antibody) and P4 in order to reveal
more information about the TSE strain involved. All were identified
as being scrapie-like. One case was identified as atypical scrapie
positive. Further immunohistochemical examinations were performed
on additional brain areas (basal ganglia, midbrain, cerebrum and
spinal cord) using 2G11, R145 and P4. All observations were
consistent with a diagnosis of atypical scrapie. A protocol for
obtaining nucleic acid from formalin-fixed, paraffin-embedded
tissues was developed and material from the atypical scrapie case
sequenced in order to obtain the PrP genotype which was AHQ/AHQ.
This animal was a scrapie suspect from 1987, but diagnosis was not
confirmed at the time by available techniques. Further information,
such as flock of origin and scrapie status of that flock were also
investigated.
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).
The first part of this report repeats exactly what has already
been submitted to Defra when the first phase of SE0248 was
completed. The second part details the immunohistochemical and PrP
genotype analyses.
The primary aim of this project was to audit VLA stocks of ovine
and caprine neural tissue held in tissue archives with a view to
enabling a debate about their suitability for a retrospective
investigation into whether or not atypical scrapie is a recent
phenomenon or has existed undetected in the national flock for
considerable time. Such a debate is essential before a decision can
be taken about whether or not such a study should be carried
out.
Briefly, the scientific objectives were:
1. Collect all VLA stocks of ovine and caprine neural tissues at
VLA Thirsk, unless already located at VLA Weybridge.
2. Catalogue material and assess for suitability for future
study.
3. Centralise all suitable material at VLA Weybridge.
Results from Audit at VLA Weybridge
1094 cases in total were assessed at VLA Weybridge. 588 of these
were older cases dating from 1980-1994. Of these 95 were scrapie
negative, with all the others being confirmed as scrapie positive
by assessment of vacuolar pathology. All 95 of these scrapie
negatives would be suitable for further assessment. There was no
indication that there was any frozen material for any of these
cases and no information on PrP genotype as they predate the
routine use of genotyping within statutory programmes. A selection
of the scrapie positives from this subset would serve as useful
positive controls to examine the potential effect of environmental
factors and long-term storage on PrP IHC. These data are summarised
in Table 1.
Table 1. Number of animals with formalin-fixed, paraffin wax
embedded tissue blocks available for further study, originating
from sheep and goats submitted between 1980 and 1994 and archived
at VLA Weybridge.
Year of Submission
Total Number of Animals
Number of Animals Not Diagnosed with Scrapie
1980
56
2
1981
5
0
1982
29
0
1983
27
1
1984
59
1
1985
44
0
1986
61
7
1987
57
8
1988
71
19
1989
97
33
1990
46
10
1991
29
13
1992
5
0
1993
1
0
1994
1
1
TOTAL
588
95
The majority of non-scrapie diagnoses for animals in the group
were listeriosis or cerebrocortical necrosis. Some animals were
reported as having no significant lesions and a very small number
of animals showed vacuolation in the CNS but no diagnosis was
reached.
The other 506 cases assessed at VLA Weybridge resulted from
searching the VLA TSE Database using the
following criteria:
· Overall status scrapie negative and all PrP genotypes EXCEPT
VRQ – either as a homozygote or a heterozygote.
· Date of submission was 1996-2004 (atypical scrapie was first
detected in UK in July 2004 and all cases subsequent to this date
will have been diagnosed using current methodology and therefore
would have been correctly diagnosed).
· All possible VLA projects were included in the search, so as
well as scrapie suspects, there are among this subset culls from
the VLA Ripley flock (SE0230) animals killed in order to provide
reference material for the TSE Archive. One case of atypical
scrapie has already been identified in this flock in an elderly
cull animal.
All 506 animals identified in this group have formalin-fixed,
paraffin wax embedded tissues suitable for further study. However,
equivalent material is not available for each animal. 461 cases
from this group had material frozen at the time of post-mortem.
Much of the material was taken for the purpose of supplying the TSE
Archive. This may be available to any future study but any work
would require Independent Archive Advisory Group (IAAG) approval
prior to release. The majority of cases had brainstem or caudal
medulla taken for Western blotting. Some of these cases will have
residual homogenate stored which, if present in sufficient
quantity, may be centrifuged to pellet abnormal PrP and then
analysed using the Bio-Rad Western blot system. Other work
requiring frozen material will depend on release of material from
the TSE archive and will be subject to IAAG approval. Frozen
material is not available for the remaining 45 cases. The number of
animals per year for this subset is shown in Table 2. The number of
cases for each PrP genotype in the 1996-2004 subset is given in
Table 3.
Table 2. Number of scrapie negative animals with formalin-fixed,
paraffin wax embedded tissue blocks available for further study,
originating from sheep and goats submitted between 1996 and 2004
and archived at VLA Weybridge.
Year of Submission
Number of Cases
1996
2
1997
101
1998
8
1999
43
2000
54
2001
52
2002
89
2003
64
2004
93
TOTAL
506
Table 3. Distribution of PrP genotypes in scrapie negative
animals with formalin-fixed, paraffin wax embedded tissue blocks
available for further study, originating from sheep and goats and
submitted between 1996 and 2004 and archived at VLA Weybridge.
PrP Genotype
Number of Cases
ARR/ARR
115
ARR/AHQ
42
ARR/ARH
11
ARR/ARQ
146
AHQ/AHQ
7
AHQ/ARH
2
AHQ/ARQ
48
ARH/ARH
4
ARH/ARQ
9
ARQ/ARQ
122
TOTAL
506
A small number of goats were identified in the audit at VLA
Weybridge. There were 32 goats identified in all. Only one of these
was from the 1996-2004 group, with the remaining 31 coming from the
1980-1994 group.
Results from Audit at VLA Thirsk
Wax blocks of formalin-fixed, paraffin-embedded tissues archived
at VLA Luddington, VLA Starcross, VLA Aberystwyth and VLA
Carmarthen were sent to VLA Thirsk where they were assessed for
suitability. In total, wax blocks from 3808 animals were deemed
suitable for further study. There is no frozen material available
for any of these animals and PrP genotype is not known. There is
little historical documentation available for the majority of these
animals – information on TSE status, age at death etc are not
always available. These data are summarised in Table 4.
Table 4. Number of animals with formalin-fixed, paraffin wax
embedded tissue blocks available for further study, originating
from sheep and goats submitted between 1980 and 2004, now archived
at VLA Thirsk.
Year of Submission
Number of Cases Suitable
1980
0
1981
1
1982
19
1983
91
1984
155
1985
161
1986
183
1987
230
1988
230
1989
229
1990
217
1991
332
1992
294
1993
248
1994
187
1995
291
1996
232
1997
200
1998
206
1999
141
2000
98
2001
23
2002
23
2003
6
2004
11
TOTAL
3808
Composite Results
In total, CNS material from a total of 4902 animals has been
assessed for suitability for a future retrospective
immunohistochemical study into atypical scrapie. The composite data
are shown in Table 5.
Table 5. Total number of animals with suitable material
available grouped by year of submission.
Year of Submission
Total Number of Suitable Animals
1980
56
1981
6
1982
48
1983
118
1984
214
1985
205
1986
244
1987
287
1988
301
1989
326
1990
263
1991
361
1992
299
1993
249
1994
188
1995
291
1996
234
1997
301
1998
214
1999
184
2000
152
2001
75
2002
112
2003
70
2004
104
TOTAL
4902
Of these 4902 animals, PrP genotype data is available for
506.
Discussion
We have determined that there is material from 4902 animals, of
which 150 were goats and the remainder sheep, from 1980-2004 that
are suitable for a future retrospective search for atypical
scrapie. Information on the majority of these animals is scanty and
varied. However, all have at least one piece of brain material in
the form of a formalin-fixed, paraffin embedded wax tissue block.
There are multiple pieces of material available for some animals,
but there is no parity in terms or available material for the
entire group. 506 animals in this group are from 1996-2004 and
there is more detailed information for this sub-group available.
This information includes PrP genotype and availability of frozen
material.
Recommendations for further examination in view of sample
availability
Most of the material from the animals identified in this audit
is likely to be scrapie negative. Alternative diagnoses include
listeriosis and cerebrocortical necrosis – material from animals
with such diagnoses is unlikely to have been examined for scrapie,
but that does not mean that they will in fact be scrapie negative.
It is possible that some of these animals with alternative
diagnoses may also have been scrapie positive. IHC using current
methodology would allow the identification of any such animals and
enable a more thorough and qualitative examination to proceed.
We recommend that material from each of the animals identified
here is immunolabelled using current methodology. The VLA’s routine
diagnostic antibody for atypical scrapie is R145. However, antibody
2G11 has been shown to be marginally more sensitive for atypical
scrapie and this would be the antibody of choice. A very small
number of cases of atypical scrapie show minimal immunolabelling in
the obex with 2G11 while immunolabelling in the cerebellum is
widespread and intense. Thus, if at all possible both of these
areas would be examined. Any cases that give a positive result
would be investigated further immunohistochemically using a panel
of antibodies to various epitopes on the PrP molecule (Gonzalez et
al., 2003).
It is likely that a great majority of these cases will in fact
be classical scrapie negative. However, it is this population that
are likely to be most interesting. Sheep confirmed as scrapie
negative prior to 2004 would have been confirmed using methodology
that we know was not fully optimised to detect atypical scrapie.
Any positive results will also yield information on whether or not
atypical scrapie has existed in the national flock undetected for
some time or if it is a new phenomenon. Frozen material, if
available, could be used to perform Western blot using the Bio-Rad
method. Material located in the TSE Archive may also be available
by its’ use would require prior approval from IAAG. Any positive
cases could also be used for strain typing in a mouse bioassay. We
know it is possible to extract material from a formalin-fixed,
paraffin-embedded wax tissue block and use it to inoculate and
transmit TSE to mice. The resulting strain profile would yield
information on how long a particular TSE strain has been present in
the UK.
The following details the second part of the project.
The earliest case of atypical scrapie in the UK, found
retrospectively, was in 1989 (Bruce et al, 2007). The primary aim
of the second part of this project was therefore to assess VLA
stocks of ovine and caprine neural tissue submitted prior to 1989
for atypical scrapie by immunohistochemistry (IHC). Briefly, the
scientific objectives were:
1.Centralise all suitable VLA stocks of ovine and caprine neural
tissues at VLA Weybridge
2.Perform and interpret IHC.
3.Develop PCR technique to enable PrP genotyping of nucleic acid
recovered from formalin-fixed, paraffin-embedded tissues.
4.Apply PrP genotyping technique to any cases of atypical
scrapie identified.
Immunohistochemistry Results
1385 of cases in total were assessed by IHC, using the VLA
routine diagnostic PrP IHC protocol. Details of this protocol can
be found at
http://www.defra.gov.uk/vla/science/sci_tse_rl_diagnosis.htm. This
is in excess of the 1370 expected, since several historical uses of
the word “case” in fact meant “case study” and material from
several animals formed a case. The actual number of cases assessed
would have been greater, but some wax blocks that were originally
deemed suitable were not suitable upon closer examination. A
diagnosis of classical scrapie required PrP deposition in the
tissue section in the correct neuroanatomical sites, for example
the dorsal motor nucleus of the vagus. A diagnosis of atypical
scrapie required PrP deposition in the trigeminal nerve of the
spinal tract in the medulla obex. Other indications of atypical
scrapie were observed as detailed in EFSA (2005) and from our
knowledge of atypical scrapie pathology (Moore et al., 2008).
Table 6 shows that 1155 cases were PrP IHC negative and 173 were
PrP IHC positive by 2G11. All of these positives were re-tested
using antibodies R145 and P4 in order to reveal more information
about the TSE strain involved (Gonzalez et al., 2003). All positive
cases, apart from one, were shown to be classical scrapie positive,
as shown by PrP deposition in one or more of the target sites
according to EFSA (2005). One case (07/42203) proved to be atypical
scrapie positive. This case showed extremely limited PrP deposition
in the trigeminal nucleus of the obex. A small area of the
cerebellum contained PrP deposits consistent with atypical scrapie.
See Figure 1. The PrP IHC on this case was repeated in order to
confirm the original observation using 2G11, but also R145 and P4.
2G11 and R145 gave similar patterns of immunolabelling in obex and
cerebellum. The P4 immunolabelling was impossible to interpret due
to high background staining. This is a common artefact associated
with P4. The dark background staining would mask any of the fine
atypical scrapie-associated PrP and made interpretation impossible.
All further material from this case was submitted for PrP IHC using
antibodies 2G11 and R145. Cerebrum and two pieces of spinal cord
were part of this case that had been archived at VLA Thirsk. There
was only a small piece of cerebrum available for analysis and this
made interpretation difficult as no neuroanatomical sites were
discernable. Observations of 2G11 and R145 immunolabelling revealed
some vacuolation and some fine particulate PrP immunolabelling in
cerebrum of the sort associated with atypical scrapie in the grey
matter. Both thoracic and cervical spinal cord showed very fine
particulate immunolabelling of the sort associated with atypical
scrapie, particularly in the substantia gelatinosa and scattered
throughout the ventral horn. Some caution may have to be urged in
interpretation of the fine immunolabelling in the substantia
gelatinosa, as this is an area that is commonly affected with
non-disease specific immunolabelling in PrP IHC, even in negative
cases. The P4 immunolabelling in both the cerebrum and the spinal
cord was impossible to interpret for the reasons outlined above. At
the time of original examination, the case was referred to the
Central Veterinary Laboratory (CVL) at Weybridge (see Discussion
for original Pathology observations). Material that was referred
included spinal cord (2 blocks), obex, cerebellum, rostral medulla,
midbrain (2 blocks) and cerebrum (3 blocks). All were
immunolabelled with 2G11, R145 and P4. 2G11 and R145 gave very
similar results. In the spinal cord (Figure 1F), some very faint
PrP deposits were observed. Widespread PrP deposits in both the
granular and molecular layers of the cerebellum (Figure 1D) were
observed. Very faint immunolabelling in the trigeminal nucleus of
the obex (Figure 1G and 1H) was observed. Interestingly, although
deposits in cerebellum and obex were weak, the immunolabelling in
these blocks processed and wax-embedded at CVL were stronger than
observed in blocks processed at VLA Thirsk. This may of course be
an artefact and it is not usually appropriate to compare intensity
of immunolabelling between IHC runs, but it is an interesting
observation. The rostral medulla (Figure 1E) showed some very
intensely stained spheroids in the white matter. These are commonly
observed in contemporary atypical scrapie and while they do not
seem to be associated with any neuroanatomical structure (other
than that they are in white matter only), their precise nature
remains unclear. Fine PrP immunolabelling was also observed in the
trigeminal nucleus at the level of the rostral medulla (Figure 1I
and 1J). The midbrain showed fine particulate immunolabelling in
the area of the substantia nigra (Figure 1C). This area is commonly
affected in contemporary atypical scrapie. The three blocks of
cerebrum contained various neuroanatomical nuclei (Figure 1A and
1B). The thalamus, hippocampus, parietal cortex, basal ganglia,
accumbens and septal nuclei all contained fine particulate
immunolabelling and the cortical layers of the cortex showed the
banding pattern associated with atypical scrapie (Moore et al.,
2008). None of the P4 immunolabelling was interpretable, due to the
reasons highlighted above. The high background was useful in
identifying certain neuroanatomical locations, but it masked any
PrP specific immunolabelling. Contemporary atypical scrapie from an
AHQ/AHQ homozygote is shown in Figure 1K (trigeminal nerve of the
spinal cord) and Figure 1L (cerebellum) for comparison. Examples of
PrP immunolabelling in classical scrapie are given in Figure 2. The
types, intensity and distribution of immunolabelling in classical
scrapie are quite different to those observed in atypical scrapie.
The dorsal vagal nucleus at the level of the obex (Figure 2A), the
trigeminal nerve of the spinal tract (Figure 2B) and two examples
of immunolabelling in the cerebellum (Figure 2C and 2D) in
classical scrapie are shown.
The atypical scrapie case identified 07/42203, originated from
1987 and was submitted as a scrapie suspect. An H&E section of
the obex was examined, but scrapie was not confirmed. Atypical
scrapie had not been described at this time, so any vacuoloar
pathology would not have been consistent with the definition of
scrapie applicable at the time. Only more modern immunochemical
tests are able to confirm atypical scrapie – none of which were
available in 1987.
Table 6. Number of animals with formalin-fixed, paraffin wax
embedded tissue blocks tested by immunohistochemistry originating
from sheep and goats submitted between 1980 and 1989
Year of Submission
Total Number of Scrapie Negative Animals
Total Number of Scrapie Positive Animals
Total Number of Cases Unsuitable for IHC Diagnosis
Total Number of Cases Assessed
1980
3
14
0
17
1981
1
0
0
1
1982
10
5
2
17
1983
69
10
3
82
1984
139
19
4
162
1985
132
15
2
149
1986
145
24
12
181
1987
197
34
17
248
1988
223
23
4
250
1989
236
29
13
278
TOTAL
1155
173
57
1385
Figure 1. PrP deposition in 07/42203, atypical scrapie case from
1987. Basal ganglia (A, magnification ×4), banding in cerebral
cortical layers (B, magnification ×4), substantia nigra in midbrain
(C, magnification ×10), cerebellum (D, magnification ×4), white
matter granules in rostral medulla (E, magnification ×40), spinal
cord (F, magnification ×20), trigeminal nerve of spinal tract in
obex (G, magnification ×10 and H, magnification ×20), trigeminal
nerve in spinal tract in rostral medulla (I, magnification ×10 and
J, magnification ×20). Contemporary atypical scrapie from an
AHQ.AHQ homozygote shown for comparison; trigeminal nerve in spinal
tract (K, magnification ×20) and cerebellum (L, magnification
×4).
Figure 2. PrP deposition in classical scrapie. Dorsal vagal
nucleus of obex (A, magnification (40), trigeminal nerve of spinal
tract (B, magnification (10), cerebellum (C and D magnification
(4)
Development of PrP Genotyping of Material from Formalin-Fixed,
Paraffin-Embedded Material
To allow PrP genotyping of sheep and goat material submitted to
VLA between 1980 and 1990, techniques were developed and optimised
for PrP genotyping from formalin-fixed paraffin embedded (FFPE)
tissue. The development of the techniques utilised material from
cases not required or not suitable for the IHC component of this
study. The brain areas evaluated were obex and cerebellum.
Many studies have shown that highly fragmented DNA is obtained
from FFPE tissue due to the fixation and embedding processes.
Fixation is the most critical factor since during formalin
fixation, protein-protein and protein-DNA cross-links occur. The
formaldehyde within the tissue gradually changes to formic acid,
hydrolysing the DNA and damaging DNA integrity. Storage
characteristics of the FFPE tissue such as duration, temperature
and humidity also affect DNA integrity (Gillio-Tos et al.,
2007).
Evaluation of DNA extraction methods
In this study, four different DNA extraction methods were
evaluated; two using tissue sections and two using de-waxed blocks
as starting material. Material was fixed and processed at VLA
Weybridge and ranged from 1980 to 1989. The tissue references used
were 80/0503, 81/0068, 81/0072, 82/03073, 82/2982, 83/4333,
84/00273, 84/4956, 85/4417, 85/03488, 86/3436, 86/03719, 87/04638,
88/02853 and 89/06960. The two digit prefix refers to the year of
submission with the rest of the number being the unique identifier
for that year. The DNA integrity (agarose gel electrophoresis),
yield (spectroscopy), quality (spectroscopy) and ability to amplify
the extracted nucleic acids using the polymerase chain reaction
(PCR) were all assessed. Where possible, obex and cerebellum were
both examined.
Tissue sections
DNA extraction was performed from tissue in FFPE sections (3x
20(m thick sections) using a high throughput commercial DNA
extraction kit (TrimGen Corporation, WaxFree( DNA kit-standard
protocol). The manufacturer’s instructions were followed and the
tissue was allowed to disrupt overnight (with occasional
vortexing). This ensured complete disruption of the tissue,
potentially recovering a higher DNA yield. The DNA profile was
analysed by electrophoresis using a 1% agarose gel (Figure 3).
A boiling/freezing technique was also used to remove the
paraffin from FFPE sections (3x 20(m thick sections) prior to DNA
extraction using proteinase K digestion. This method has previously
been successfully applied to PrP genotyping fixed tissue (Kunkle et
al., 2006). The DNA profile was analysed using a 1% agarose gel
(Figure 4).
Figure 3. 1% agarose electrophoresis of DNA extracted from FFPE
tissue sections using TrimGen Corporation, WaxFree( DNA
kit-standard protocol prior to clean up. (Sample number suffix
01=obex, 02=cerebellum).
Figure 4. 1% agarose electrophoresis of DNA extracted from FFPE
tissue sections using a boiling/freezing technique (Kunkle et al.,
2006) before and after clean-up using Qiagen, QiaAMP DNA micro kit-
Protocol for genomic DNA clean up. (Sample number suffix, 01=obex,
02=cerebellum).
For both extraction methods from tissue sections, the
supernatant containing the DNA also contained proteins as shown by
a reduced absorbance ratio (A260/280). DNA from the extracts was
therefore purified to remove the residual tissue proteins and also
to potentially restore the suitability of the DNA for PCR (Qiagen,
QiaAMP DNA micro kit- Protocol for genomic DNA clean up). This led
to an increase in A260/280 (Appendix-Table 10) in line with DNA
extracts free from protein (A260/280 1.8 – 2.0). In general, the
cerebellum yielded more DNA than obex; this reflected the
concentrations determined by absorbance ratios after the DNA had
been cleaned-up. The mass of the DNA was less than 1000 base pairs
(bp) and more concentrated below 100 bp.
De-waxed blocks
DNA from FFPE tissue blocks was also evaluated after the removal
of paraffin using the organic solvent xylene. The FFPE block was
incubated at 60oC to remove as much adherent wax as possible. The
sample was placed in a sterile glass container with x50 volumes of
xylene. After 3hrs incubation fresh xylene was added and the tissue
left overnight at room temperature. The xylene was replaced with
100% ethanol and changed three times at 30 minute intervals. This
was repeated with 90% ethanol, 70% ethanol and finally, sterile
saline. The block was stored in sterile saline at 4oC until DNA
extraction.
DNA was extracted from the tissue, after this de-waxing and
re-hydration process, using two DNA extraction methods, TrimGen
Corporation, WaxFree( DNA kit-short protocol and Qiagen, DNeasy
blood and tissue kit. The manufacturer’s protocols were followed,
again with overnight incubations (and occasional vortexing) to
allow maximum tissue disruption. The Qiagen method yielded DNA free
from contaminating proteins, however, the DNA extracted using
TrimGen Corporation, WaxFree( DNA kit-short protocol was cleaned up
as before. The concentrations and purity of DNA extracted using
these methods are also shown in Appendix- Table 10. The DNA profile
was analysed using a 1% agarose gel (Figure 5).
Figure 5. 1% agarose electrophoresis of DNA extracted from FFPE
de-waxed blocks using A) Qiagen, DNeasy blood and tissue kit and B)
TrimGen Corporation, WaxFree( DNA kit-short protocol, prior to
clean up. (01=obex, 02=cerebellum).
10,000bp
3000bp
1000bp
10,000bp
3000bp
1000bp
1Kbp ladder80/00555
-
01
81/00072
-
01
82/03073
-
01
83/05030
-
01
84/00273
-
02
85/03488
-
01
86/03719
-
01
87/04638
-
01
88/02853
-
01
Ext negative
10,000bp
3000bp
1000bp
10,000bp
3000bp
1000bp
10,000bp
3000bp
1000bp
10,000bp
3000bp
1000bp
1Kbp ladder80/00555
-
01
81/00072
-
01
82/03073
-
01
83/05030
-
01
84/00273
-
02
85/03488
-
01
86/03719
-
01
87/04638
-
01
88/02853
-
01
Ext negative
The cerebellum, generally, yielded a higher concentration of DNA
using all the extraction methods. However, the Qiagen extraction
method showed a slightly higher molecular mass profile when
compared to the other methods, despite the potentially harsher
deparaffinisation technique using xylene compared to
boiling/freezing or the TrimGen Corporation, WaxFree( DNA
kit-standard protocol. The mass of the DNA extracted was less than
3000 bp and more concentrated below 1000 bp. The DNA profile on a
1% agarose gel showed fragmentation most probably due to the
formalin fixation and embedding processes.
In conclusion, all 4 DNA extraction methods yielded highly
fragmented DNA, on average from 100 to 1000 bps; three of the four
methods required an additional DNA clean-up step to remove
impurities such as proteins that could interfere with down-stream
PCR amplification. DNA extracted from FFPE tissue sections using a
boiling/freezing technique (Kunkle et al., 2006) was not taken any
further. There was no advantage to the quality or quantity of the
DNA extracted using this technique, which was a more time consuming
process and not easily adapted for high throughput processing.
Partial PrP genotyping
High quality genomic DNA is hard to obtain from FFPE tissues.
DNA extracted from FFPE tissue can be suitable for PCR, as long as
the short DNA targets can be utilised. Generation of a 301bp PRNP
amplicon covering bases 312-612 of the ovine PRNP open reading
frame (ORF) equivalent to codons 104-204 will allow partial
sequencing of the PRNP ORF (Appendix–Figure 9). This covers the
most polymorphic region of the PRNP ORF and allows detection of
amino acid variants associated with atypical scrapie
susceptibility, namely codons 136, 141, 154 and 171 (Benestad et
al., 2003; Moum et al., 2005).
2(l and 10(l DNA extracts from three extraction methods were
added to a 25(l PCR reaction mix containing 1(M forward (G40) and
reverse (G41) primers (Appendix-Table 11), 0.2mM dNTP mix, 0.03U/(l
AmpliTaq gold DNA polymerase (Applied Biosystems) and 1x PCR buffer
I with 1.5mM MgCl2 (Applied Biosystems). The thermal cycling
conditions were 95oC for 10 minutes, 40 cycles of 95oC for 30
seconds, 63.6oC for 30 seconds and 72oC for 1 minute, followed by
72oC for 10 minutes. A second round PCR, using 2(l PCR product as
template, was performed at a reduced primer concentration of
0.5(M.
From the first round PCR, 2(l of DNA template was more
successful in producing a PCR product than 10(l, with DNA extracted
from the de-waxed blocks showing a higher PCR success that the
method using the FFPE tissue sections. After the second round PCR,
again extracts from de-waxed blocks were more successfully
amplified (Table 7). The failure of PCR using 10(l of template at
the first stage may be attributed to too high a DNA concentration
inhibiting the reaction.
In conclusion, greater success from first round PCR
amplification was obtained when the initial template volume was 2(l
in a 25(l reaction volume. However PCR product from 2(l and 10(l
initial starting template was suitable for amplification in the
second round PCR. The success or failure of amplifying the 301bp
PCR product during first round or second round PCR could not be
correlated directly with initial DNA concentration or purity
(Appendix-Table 12). The greater success of PCR amplification at
this stage was determined by de-paraffinisation of the sample using
xylene.
Table 7. Summary of partial PRNP gene amplification of DNA
extracted from FFPE sections using TrimGen Corporation, WaxFree(
DNA kit-standard protocol and of DNA extracted from de-waxed tissue
blocks using Qiagen, DNeasy blood and tissue kit and TrimGen
Corporation, WaxFree( DNA kit-short protocol.
DNA extraction method
% PCR Positive
First round PCR
Second round PCR using
2µl template
10µl template
2µl PCR amplicon
10µl PCR amplicon
TrimGen Corporation, WaxFree( DNA kit-standard protocol
(n=10)
10
0
30
40
Qiagen, DNeasy blood and tissue kit (n=10)
40
0
60
50
TrimGen Corporation, WaxFree( DNA kit-short protocol (n=10)
40
0
80
80
A selection of the 301bp PCR products, generated by
amplification of DNA from FFPE tissue, was purified using Qiagen,
QIAquick PCR purification kit and sequenced bi-directionally. DNA
sequence data was analysed using GAP4 software (Staden package,
version 1.7.0). Polymorphisms were identified by comparison to a
wild-type PRNP reference sequence from an ARQ/ARQ sheep, GenBank
accession number AY0350267. The resulting PrP genotypes are shown
in the Appendix-Table 13.
All three DNA extraction methods were suitable for determining
partial PrP genotype from FFPE tissue. The results for sample
84/4956 were inconclusive due to the genotype from the obex being
different to that from the cerebellum. This may have been due to
allelic dropout, the failure to detect one of the two alleles
within a sample or failure to amplify an allele during PCR, a
common problem associated with the amplification of low levels of
highly degraded DNA.
Also for this sample, the sequence generated from DNA extracted
from the obex showed an additional polymorphism at nucleotide 373
bp G>A. This is not a known ovine PRNP gene polymorphism and as
it was only seen in one of the sequences generated from this sample
is most probably an artefact of the fixation and processing
process. The effects of formalin fixation and processing to wax on
DNA are uncertain. Base damage may impair PCR by halting polymerase
elongation but another possible outcome is error prone translation
synthesis across sites of damage, producing in vitro, artificial
mutations with an incorrect base inserted opposite a damaged base
(Quach et al., 2004). Formalin damaged or cross-linked cytosine
nucleotides are not correctly recognised by the Taq DNA polymerase
and instead of the correct guanosine base being incorporated an
adenosine is added (the so-called A rule). An artificial C>T or
G>A mutation could therefore be created (Williams et al., 1999).
These artificial mutations are a known result of formalin fixation,
Williams et al., (1999) reported that of the 28 artificial
mutations detected in archival formalin fixed specimens, 27 of the
28 mutations were C>T or G>A. Sample 84/4956 showed the
lowest DNA concentration (26.06ng/(l) and Williams et al., (1999)
have shown that the chance of non-reproducible sequence alterations
detected in formalin-fixed tissues was greater when there was less
template. The genotype of all other samples was consistent, even
when the template from different first round PCRs was used.
This evaluation process highlighted the fact that artificial
mutations and allelic drop out were a probable occurrence in the
study of archived fixed tissue and the possibility of one or both
of these events taking place should be taken into account before a
final genotype assignment is made.
Evaluation of PrP genotype in retrospective atypical scrapie
cases
From the IHC component of this study, one sample, 07/42203, was
identified as an atypical scrapie case. Originally, tissue was
available from this case that had been formalin-fixed and processed
to wax at VLA Thirsk - later in this study tissue was located that
had been fixed and processed at VLA Weybridge. Differences could
exist between the two procedures, not least the use of a different
type of paraffin wax that could impact upon the quality of DNA
obtained and the final PCR product. The atypical scrapie IHC
profile of this case was stronger in the tissues fixed and
processed at VLA Weybridge than those fixed and processed at VLA
Thirsk.
DNA was extracted from 4 different brain areas (obex, cerebrum,
cerebellum and spinal cord) of case 07/42203 formalin-fixed and
processed at VLA Thirsk. DNA was also extracted from the midbrain
and cerebrum of case 07/42203 that was formalin fixed and processed
at VLA Weybridge and a control sample of known genotype, 08/0055
(obex). All samples were extracted using the Qiagen, DNeasy blood
and tissue kit following de-waxing and re-hydration as described
above.
The DNA profile is shown in Figure 6. DNA from case 07/42203 has
undergone severe fragmentation compared to case 08/0055. The
majority of the DNA from the atypical case fixed and processed at
VLA Thirsk has a molecular mass of less than 1000 bp with the
majority of the fragments being less than 500 bp in size. The
positive control sample (08/0055), in contrast, has DNA of a high
molecular mass from 3000 to 10,000 bp and above. When DNA from the
other brain areas from 07/42203 were observed, a profile similar to
that of the cerebellum was seen (Figure 7). The DNA profile of
extracts from tissues fixed at Weybidge shows DNA ranging from
approximately 500 to 10,000 bps (Figure 8), more in line with that
seen in the evaluation study.
Figure 6. Agarose (1%) electrophoresis of DNA extracted from A)
Atypical scrapie case 07/42203 cerebellum, fixed and processed at
VLA Thirsk, in triplicate and B) Control sample 08/0055 obex, fixed
and processed at VLA Weybridge, in triplicate.
10,000bp
3000bp
1000bp
1500bp
1000bp
500bp
100bp A B ext neg1Kbp
ladder ladder
10,000bp
3000bp
1000bp
1500bp
1000bp
500bp
100bp A B ext neg1Kbp
ladder ladder
Figure 7. Agarose (1%) electrophoresis of DNA extracted from the
cerebellum, spinal cord, cerebrum and obex of atypical scrapie
sample 07/42203, fixed and processed at VLA Thirsk.
10,000bp
3000bp
1000bp
500bp
ext. negative
Obex
Cerebrum
Spinal Cord
Cerebellum
100bp ladder
1Kbp ladder
10,000bp
3000bp
1000bp
500bp
ext. negative
Obex
Cerebrum
Spinal Cord
Cerebellum
100bp ladder
1Kbp ladder
The DNA concentrations (ng/(l) and absorbance (A260/280) ratios
of DNA extracted from the atypical case and the control case are
shown in Table 8. For the atypical scrapie case, 07/42203, the area
that yielded most DNA was the midbrain and the area that yielded
the least was the spinal cord. Both of the Weybridge fixed and
processed tissues gave the highest concentration of genomic DNA.
Genomic DNA from the obex of control sample 08/0055 had the highest
concentration of all DNA extracts.
Figure 8. Agarose (1%) electrophoresis of DNA extracted from the
midbrain and cerebrum of atypical scrapie sample 07/42203, fixed
and processed at VLA Weybridge.
10,000bp
3000bp
1000bp
500bp
Midbrain
Cerebrum
ext. negative
1Kbp ladder
10,000bp
3000bp
1000bp
500bp
Midbrain
Cerebrum
ext. negative
1Kbp ladder
Midbrain
Cerebrum
ext. negative
1Kbp ladder
Table 8. Concentrations (ng/µl) and absorbance (A260/280) ratios
of genomic DNA extracted from samples 07/42203 and 08/0055. (Tissue
code-01=obex, 02=cerebellum, 03=cerebrum. 04= spinal cord,
05=midbrain).a Fixed and processed at VLA Thirsk. b Fixed and
processed at VLA Weybridge.
Sample-tissue code
Genomic DNA concentration (ng/(l).
Absorbance (A260/280).
07/42203-02 a
100.09
2.00
07/42203-02 a
70.28
1.98
07/42203-02 a
74.49
1.96
07/42203-02 a
49.68
2.11
07/42203-02 a
76.51
1.99
07/42203-02 a
81.94
1.64
07/42203-01 a
36.09
1.82
07/42203-03 a
48.73
1.94
07/42203-04 a
17.07
1.77
07/42203-05 b
290.82
1.96
07/42203-03 b
214.6
1.94
08/0055-01 b
463.45
1.97
08/0055-01 b
357.86
1.97
08/0055-01 b
297.76
1.96
2(l and 10(l DNA extracts were used in the G40/G41 PCR as
described above. The results are shown in Table 9. As originally
only the samples fixed and processed at VLA Thirsk were available,
alternate sequencing strategies were employed in an attempt to
improve results, involving amplification of shorter sequences
(171F/R= 90 bp, 154F/R= 93 bp, 136F/R= 124 bp, 136F/154R= 216bp and
136F/171R=259 bp amplicons). The primer sequences are shown in
Appendix-Table 11. 2(l DNA extracts were added to a 25(l PCR
reaction mix containing 0.3(M forward and reverse primers, 0.2mM
dNTP mix, 0.03U/(l AmpliTaq gold DNA polymerase (Applied
Biosystems) and 1x PCR buffer I with 1.5mM MgCl2 (Applied
Biosystems). The thermal cycling conditions were 95oC for 10
minutes, 40 cycles of 95oC for 30 seconds, 60oC for 30 seconds and
72oC for 1 minute and followed by 72oC for 10 minutes. A second
round PCR with 2(l PCR product was also performed under the same
conditions.
The success of the PCR using DNA from the samples fixed and
processed at VLA Thirsk depended on the size of the amplicon. The
shorter PCRs had 100% amplification success in first round PCRs
(90bp, 93bp and 124bp), however as the amplicon size increased
(216bp and 259bp) the PCR success decreased (Table 9). The PCR
products greater than 100bp were sequenced bi-directionally using
the appropriate PCR primers (Table 9). Full analysis of the
sequencing data is shown in Appendix-Table 14.
The sequence data for the sample of known genotype, 08/0055 was
a conclusive homozygous ARR. This matches the result originally
obtained from blood containing ethylenediaminetetraacetic acid
(EDTA) as anti-coagulant.
In spite of 23 good quality sequences being generated for case
07/42203 using samples fixed and processed at VLA Thirsk, variable
results in terms of sequence data were obtained. In the DNA
extracted from tissue fixed and processed at VLA Thirsk, ten
different non-specific artificial mutations were detected, with up
to four being detected in a single sequencing reaction
(Appendix-Table 14). Electropherograms showing two of the
non-specific artificial mutations observed are shown in
Appendix-Figure 10. All ten of these artificial mutations were
reproducible only when the same 1st round PCR product was
re-amplified in the 2nd round, and were not reproducible when a
different 1st round PCR was generated from the same DNA extract.
This confirms that the mutations are artificial and mutated
templates are selected and amplified as an early event in the 1st
round PCR. Such a high rate of artificial mutations in these
samples suggests that the DNA has been very severely damaged by
fixation and processing.
Excluding the artificial mutations, three PrP alleles were
detected in the samples fixed and processed at VLA Thirsk, namely
AF141RQ, ARQ and AHQ. The ARQ allele was generated from one DNA
extract and from three 1st round PCR products, no artificial
mutations were associated with the ARQ allele, the AF141RQ allele
was generated from one DNA extract and one 1st round PCR product
and the AHQ allele was generated from three DNA extracts and three
1st round PCR products. As the C>T mutation at base 421 that
results in the leucine to phenylalanine codon change at position
141 was only detected as a result of a single 1st round PCR
product, it is possible that this mutation is an artificial
mutation. Also on occasion possible bovine PrP mutations
(highlighted in grey, Appendix-Table 14) were detected and so this
data may have been generated from contamination at the time of
sample preparation or a spurious amplification due to a large
number of PCR cycles. This was seen despite employing a number of
control measures, at every stage of the process to eradicate and
monitor for potential false results. Due to the variability
observed, the sequencing data generated from the samples fixed and
processed at VLA Thirsk from 07/42203 were considered
unreliable.
Using the DNA extracted from the two tissues of sample 07/42203
fixed and processed at VLA Weybridge, twelve readable DNA sequences
were obtained, five of which were of good quality throughout. Two
non-specific artificial mutations were detected in two of the
twelve sequences generated, suggesting that these tissues were not
as severely damaged by the fixation and processing processes as
those fixed and processed at VLA Thirsk. In all, twelve sequences
(eight from the midbrain sample and four from the cerebrum sample)
but only one PrP allele, namely AHQ, was detected, the mutation
G>A at base 461 specific for the codon 154 R>H was clear in
all twelve sequences. Based on results from the two tissues fixed
and processed at VLA Weybridge, the PrP genotype of sample 07/42203
is AHQ/AHQ.
Conclusion
The DNA of the atypical scrapie case 07/42203 extracted from
samples fixed and processed at VLA Thirsk was severely fragmented
and of a low concentration. The high numbers of artificial
mutations generated in the sequence data obtained from these
tissues suggests that the fixation or subsequent storage of this
sample was less than ideal for preservation of DNA and subsequent
analysis. The PrP genotype generated from the tissues fixed and
processed at VLA Thirsk samples were deemed unreliable. However,
the DNA from the same case fixed and processed at VLA Weybridge
showed less fragmentation, gave a greater yield of DNA and the
conclusive sequence data generated showed fewer formalin mediated
sequence artefacts, allowing a more confident PrP genotype result
to be obtained.
The DNA extraction and sequencing techniques optimised in this
study were successfully applied to FFPE samples and using two VLA
Weybridge-fixed and processed tissue samples identified the
genotype of the retrospective atypical case 07/42203 as AHQ/AHQ.
This is one of the PrP genotypes commonly associated with atypical
scrapie.
Table 9. Partial PRNP amplification and PrP genotyping of DNA
extracted from 07/42203 and 08/0055. (Tissue code-01=obex,
02=cerebellum, 03=cerebrum 04=midbrain. * DNA diluted 1:10 prior to
PCR and genotype obtained from first round 2(l PCR. AXX= 154 codon
and 171 codon not covered by amplification, AHX= 171 codon not
covered by amplification). a Sample fixed and processed at VLA
Thirsk. b Sample fixed and processed at VLA Weybridge. ND not
determined.
PCR
Genotype
Primers
G40/G41
G40/G41
136F/R
154F/R
171F/R
136F/154/R
136F/171
G40/G41
136F/R
136F/154/R
136F/171
Amplicon (bp)
301
301
124
93
90
216
259
301
124
216
259
Sample-code
Conc (ng/µl).
First round*
First round / second round
First round 2µl
First round 2µl
First round 2µl
First round 2µl/ second round
First round 2µl/ second round
Allele 1/ Allele 2
Allele 1/ Allele 2
Allele 1/ Allele 2
Allele 1/ Allele 2
07/42203-02 a
100.09
-
+
+
+
+
-
+
ARQ/ARQ
AXX/AXX
ND
ARQ/ARQ
07/42203-02 a
70. 28
-
+
+
+
+
+
+
AFRQ/AFRQ
AXX/AXX
AHX/AHX
fail
07/42203-02 a
74.49
-
+
ND
ND
ND
ND
ND
AHQ/AHQ
ND
ND
ND
07/42203-02 a
49.68
-
-
ND
ND
ND
ND
ND
ND
ND
ND
ND
07/42203-02 a
76.51
-
-
ND
ND
ND
ND
ND
ND
ND
ND
ND
07/42203-02a
81.94
-
-
+
+
+
-
+
ND
AXX/AXX
ND
AHQ/AHQ
07/42203-01 a
36.09
-
-
+
+
+
-
-
ND
ND
ND
ND
07/42203-03 a
48.73
-
-
+
+
+
-
-
ND
AXX/AXX
ND
ND
07/42203-04 b
290.82
+
+
ND
ND
ND
ND
ND
AHQ/AHQ
ND
ND
ND
07/42203-03 b
214.6
+
+
ND
ND
ND
ND
ND
AHQ/AHQ
ND
ND
ND
08/0055-01 b
357.86
+
+
+
+
+
+
+
ARR/ARR
AXX/AXX
fail
ARR/ARR
Discussion
We have assessed 1385 sheep and goats which were submitted to
VLA between 1980 and 1989 by IHC for the presence of atypical
scrapie. Some of these animals were originally submitted as scrapie
suspects, while others were submitted for confirmation of other
diseases such as listeriosis. Those originally assessed for scrapie
would have been done so by assessment of histopathological
appearance in a haematoxylin & eosin (H&E) tissue section
as modern diagnostic IHC techniques were not available at the time.
1155 animals were scrapie negative, 57 were unsuitable for
diagnosis, due for example, to an absence of the target areas and
173 were scrapie positive. Of those positives, a single animal was
shown to be atypical scrapie positive. This animal was a scrapie
suspect sheep from 1987 that was submitted to VLA Thirsk. No
diagnosis was reached by the pathologist who interpreted the
original H&E tissue section. This is not unexpected, since the
vacuolar pathology of atypical scrapie is very different to
classical scrapie, and any pathology observed would not have fitted
with the description of scrapie as it was known in 1987. The IHC we
have performed in this project shows quite weak immunolabelling,
even for a case of atypical scrapie. This may be due to either this
being a genuine weak case, or maybe due to possible detrimental
effects of long term storage in wax. Although older tissues that
have been embedded in wax for some time can be revisited and PrP
IHC successfully performed, a slide generated at the time of
embedding is required for comparison. Obviously, this is not
available. It is therefore impossible to rule out that this case
may have been affected by long term storage in wax. We have
observed that some PrP epitopes are degraded, either partially or
fully, during both during long term fixation or long term storage
in paraffin wax (unpublished observations). We have also observed
that the nature of the IHC product in cases of atypical scrapie can
sometimes deteriorate in the slide, even when it has been stored
optimally. There are obviously many unanswered questions relating
to the nature of PrP involved in atypical scrapie.
Some, but not all, of the original paperwork for this case of
atypical scrapie was located at VLA Thirsk and were reviewed. A ewe
of unknown age was originally submitted as showing “bizarre
behavioural changes” with a suspected diagnosis of scrapie or
listeriosis. The pathologist at VLA Thirsk who first examined the
brain noted:
“severe spongiform changes in some folia of cerebellum but
medulla largely unaffected, mainly in the molecular layer. Cerebrum
diffusely affected with spongiform encephalopathy – not scrapie.
Spinal cord – more spongy appearance than usual to white matter?”
The pathologist concluded that the animal had a spongiform
encephalopathy, but referred the case to the Consultancy Pathology
Unit at the Central Veterinary Laboratory (now the Department of
Pathology, VLA Weybridge). The second pathology opinion stated the
following.
Microscopic observations
Cerebrum x 3
Occasional mild lymphocytic meningeal infiltrates. Occasional
focal microglial nodules. Fine vacuolation of the neuropil in grey
matter and marked hypertrophy and hyperplasia of astrocytes.
Midbrain x 2
Occasional mild lymphoblastic perivascular cuffs
Medulla x 2
No visible lesions
Cerebellum
Patchy vacuolar changes in the neuropil at the molecular
later
Spinal Cord x 2
No visible lesions
Diagnosis
1. Diffuse neuropil vacuolation, grey matter, cerebrum
2. Astrogliosis, diffuse grey matter, cerebrum
3. Multifocal, milk? Non-suppurative meningoencephalitis
Remarks
The changes in the cerebrum are indicative of widespread
cytotoxic oedema of astrocytes. The cause is not apparent.
The mild non-suppurative meningitis is probably incidental.
There were no changes in the brain stem or spinal cord and
therefore no indication of scrapie
Most of the paperwork at VLA Thirsk has long been discarded in
accordance with organisational records management procedures. There
is also no indication of breed. However, all other ovine
submissions from this flock were Swaledale. Classical scrapie was
diagnosed at this farm in 1984, 1991, 1992 and 1995. There have
been no further ovine submissions to VLA of any sort since
1998.
These observations fit in with what we now understand about
atypical scrapie (Moore et al., 2008). To further characterise this
early case of atypical scrapie, it may be of interest to use mouse
bioassay, or even attempt transmission to the natural host. This
would not only yield further information on this case, but also
inform on whether or not this early case of atypical scrapie is
similar to strains in existence today or whether it was something
that has not been observed more recently, in terms of strain
involved.
Despite initial success in method development using material
fixed and processed at VLA Weybridge, initial attempts to obtain a
PrP genotype from 07/42203, the atypical scrapie case identified in
this study, were not successful. After a search at VLA Thirsk, it
was identified that additional material from this cases was sent to
VLA Weybridge for second opinion in 1987. This material was fixed
at VLA Thirsk, but processed to paraffin wax at VLA Weybridge. It
is impossible to state with any certainty, but it is likely that
there were differences in the processing procedures used, including
the possible use of different types of wax for embedding. Nucleic
acid extracted from material that had been processed at Weybridge
was suitable for obtaining a PrP genotype. The genotype obtained
was AHQ/AHQ. The process used also correctly identified the PrP
genotype of the positive control.
The other cases identified in this project as scrapie positive
by IHC were all re-immunolabelled with further antibodies in order
to reveal more information about the TSE strain involved. Rat
monoclonal anti-PrP R145 and mouse monoclonal anti-PrP P4 were
selected. R145 is the UK’s antibody used in confirmation of TSE’s
by IHC. Comparisons of serial tissue sections immunolabelled with
R145 or P4 were made according to Gonzalez et al (2003). All of the
cases examined were indistinguishable from classical scrapie. The
PrP genotype of these cases was not determined, since the original
project proposed only to obtain the genotype of any atypical
scrapie positives. If it is of interest to Defra, the methodology
exists to obtain the PrP genotypes of all these animals, but
funding would have to be made available.
This study supports the recently published observation that
atypical scrapie has been present in the UK sheep population for at
least two decades (Bruce et al., 2007). However, due to only a
single case being identified, it is not possible to extrapolate the
data to make any sort of estimate of prevalence. The
immunopathology in the single atypical scrapie positive case was
consistent with our observations in contemporary atypical scrapie
(Moore et al., 2008). Bioassay would provide conclusive evidence
about whether the biological properties of this case are the same
as contemporary atypical scrapie.
Appendix 1
Table 10. Concentrations (ng/µl) and absorbance (A260/280)
ratios of DNA extracted from FFPE method evaluation tissues (Tissue
code-01=obex, 02=cerebellum).
Extraction method – sample type
Sample-tissue code.
Prior to clean-up
After clean up
Concentration (ng/(l).
Absorbance
(A260/280).
Concentration (ng/l).
Absorbance
(A260/280).
TrimGen Corporation, WaxFree( DNA kit-standard protocol – tissue
sections
83/4333-01
422.32
0.95
34.81
1.77
83/4333-02
1123.65
1.13
140.08
1.72
84/4956-01
247.05
0.96
26.06
1.71
84/4956-02
1638.14
1.28
234.17
1.86
85/4417-01
1930.33
1.25
262.62
1.86
85/4417-02
398.36
0.99
34.32
1.71
86/3436-01
1409.67
1.26
322.11
1.84
86/3436-02
288.66
0.95
32.07
1.85
82/2982-01
228.49
0.92
23.76
1.8
82/2982-02
1298.09
1.22
260.64
1.77
Extraction negative
37.34
0.64
2.98
2.4
Boiling/freezing technique (Kunkle et al., 2006) – tissue
sections
80/0503-01
192.83
1.11
51.93
1.86
80/0503-02
832.84
1.21
324.15
1.88
81/0068-01
206.22
1.20
53.77
1.76
81/0068-02
619.99
1.21
212.59
1.86
Extraction negative
1.59
1.45
Qiagen, DNeasy blood and tissue kit - de-waxed tissue blocks
80/0555-01
56.1
1.79
81/0072-01
97.82
1.77
82/3073-01
61.86
1.66
83/5030-01
31.96
1.79
84/0273-02
239.01
1.85
85/3488-01
105.77
1.81
86/3719-01
73.51
1.82
87/4638-01
92.62
1.68
88/2853-01
70.44
1.82
89/6960-01
37.54
1.91
Extraction negative
3.88
1.23
TrimGen Corporation, WaxFree( DNA kit-short protocol - de-waxed
tissue blocks
80/0555-01
529.45
0.97
49.75
1.73
81/0072-01
760.48
0.95
100.48
1.8
82/3073-01
538.17
1
91.58
1.71
83/5030-01
360.1
0.93
38.44
1.8
84/0273-02
1047.96
1.19
423.47
1.75
85/3488-01
850.01
1.01
107.05
1.81
86/3719-01
495.81
0.99
89.82
1.78
87/4638-01
635.96
0.89
65.39
1.7
88/2853-01
660.57
0.9
81.38
1.8
89/6960-01
906.27
0.95
52.32
1.78
Extraction negative
32.78
0.69
-0.41
1.4
Figure 9. Ovine PRNP open reading frame- amplification strategy
using G40 (forward primer) and G41 (reverse primer).
Table 11. Primer sequences used for partial PrP genotyping.
Primer
Sequence 5’-3’
G40F
gcccagtaagccaaaaacca
G41R
agtttcggtgaagttctcccc
136F
GCCAAAAACCAACATGAAGCA
136R
TCCTCATAGTCATTGCCAAAATGTA
154F
CCTCTTATACATTTTGGCAATGACTATG
154R
ATCCACTGGTCTGTAGTACACTTGGTT
171F
GAAAACATGTACCGTTACCC
171R
TGTTGACACAGTCATGCAC
Table 12. Partial PRNP gene amplification of DNA extracted from
FFPE tissue. (Tissue code-01=obex, 02=cerebellum).
Extraction method – sample type
Sample-tissue code.
DNA template concentration (ng/(l).
First round
PCR (template variable volume)
Second round
PCR (template 2(l 1st round PCR product)
2(l DNA
10(l DNA
2(l PCR amplicon
10(l PCR amplicon
TrimGen Corporation, WaxFree( DNA kit-standard protocol – tissue
sections
83/4333-01
34.81
-
-
-
-
83/4333-02
140.08
-
-
-
-
84/4956-01
26.06
+
-
+
+
84/4956-02
234.17
-
-
-
+
85/4417-01
262.62
-
-
+
+
85/4417-02
34.32
-
-
-
-
86/3436-01
322.11
-
-
+
+
86/3436-02
32.07
-
-
-
-
82/2982-01
23.76
-
-
-
-
82/2982-02
260.64
-
-
-
-
Extraction negative
2.98
-
-
-
-
Qiagen, DNeasy blood and tissue kit - de-waxed tissue blocks
80/0555-01
56.1
-
-
-
-
81/0072-01
97.82
+
-
+
+
82/3073-01
61.86
-
-
-
-
83/5030-01
31.96
+
-
+
+
84/0273-02
239.01
+
-
+
+
85/3488-01
105.77
-
-
+
-
86/3719-01
73.51
-
-
+
+
87/4638-01
92.62
-
-
-
-
88/2853-01
70.44
+
-
+
+
89/6960-01
37.54
-
-
-
-
Extraction negative
3.88
-
-
-
-
TrimGen Corporation, WaxFree( DNA kit-short protocol - de-waxed
tissue blocks
80/0555-01
49.75
-
-
+
+
81/0072-01
100.48
+
-
+
+
82/3073-01
91.58
-
-
+
-
83/5030-01
38.44
+
-
+
+
84/0273-02
423.47
-
-
-
+
85/3488-01
107.05
-
-
+
+
86/3719-01
89.82
-
-
+
+
87/4638-01
65.39
-
-
-
-
88/2853-01
81.38
+
-
+
+
89/6960-01
52.32
+
-
+
+
Extraction negative
-0.41
-
-
-
-
Table 13. Partial PrP genotyping of DNA extracted from FFPE
sections using a) TrimGen Corporation, WaxFree( DNA kit-standard
protocol and of DNA extracted from de-waxed tissue blocks using b)
Qiagen, DNeasy blood and tissue kit and c) TrimGen Corporation,
WaxFree( DNA kit-short protocol. Red indicates nucleotide base
changes most probably caused by the fixation processes. (Tissue
code-01=obex, 02=cerebellum).
Amino acid variant
125 V>I
136 A>V
141 L>F
154 R>H
171 Q>R
Nucleotide variant
PrP genotype
Sample-code
Extraction method
First round PCR (template volume)
Second round PCR
2 and 10 refers to DNA in 1st round
373 bp
G>A/R
407 bp
C>T/Y
421 bp
C>T/Y
461 bp
A>G/R
512 bp
A>G/R
Allele 1
Allele 2
83/5030-01
c
2
N/A
G
Y
C
G
A
ARQ
VRQ
83/5030-01
c
2
2
G
Y
C
G
A
ARQ
VRQ
83/5030-01
c
10
10
G
Y
C
G
A
ARQ
VRQ
84/0273-02
b
2
N/A
G
T
C
G
A
VRQ
VRQ
84/0273-02
b
2
2
G
T
C
G
A
VRQ
VRQ
84/0273-02
b
2
10
G
T
C
G
A
VRQ
VRQ
84/0273-02
c
10
10
G
T
C
G
A
VRQ
VRQ
84/4956-01
a
2
N/A
A
T
C
G
A
VRQ
VRQ
84/4956-01
a
2
2
A
T
C
G
A
VRQ
VRQ
84/4956-02
a
10
10
G
C
C
G
A
ARQ
ARQ
85/3488-01
c
2
2
G
C
C
G
A
ARQ
ARQ
85/3488-01
c
2
2
G
C
C
G
A
ARQ
ARQ
85/4417-01
a
2
2
G
T
C
G
A
VRQ
VRQ
86/3436-01
a
2
2
G
C
C
G
A
ARQ
ARQ
86/3719-01
b
2
2
G
T
C
G
A
VRQ
VRQ
86/3719-01
b
10
10
G
T
C
G
A
VRQ
VRQ
Table 14. Mutation report and genotype from partial PRNP
sequencing of DNA extracted from 15/S506/7/87 and PG0055/08. Tissue
code-01=obex, 02=cerebellum, 03=cerebrum. 04= midbrain. Red
indicates nucleotide base changes most probably caused by the
fixation processes. a Formalin-fixed and processed at VLA Thirsk, b
formalin-fixed and processed at VLA Weybridge. AXX= 154 and 171
codons not covered by amplification, AHX=171 codon not covered by
amplification
Amino acid variant
118 A>T
124 V>M
132 M>I
133 silent L
141 L>F
149 silent E
150 D>N
151 G>A
154 R>H
164 V>M
167 silent R
171 Q>R
196 silent T
Nucleotide variant
PrP genotype
Sample code
Conc (ng/µl)
Primer
1st round format
2nd round format
352 bp G>A/R
370 bp G>A/R
396 bp G>A/R
399 bp G>A/R
421 bp C>T/Y
447 bp G>A/R
448 bp G>A/R
452 bp G>A/R
461 bp G>A/R
490 bp G>A/R
501 bp A>G/R
512 bp A>G/R
588 bp C>T/Y
Allele 1
Allele 2
07/42203-02 a
100.09
G41
Neat DNA 10(l
1(M
ARQ
ARQ
07/42203-02 a
100.09
G40
Neat DNA 10(l
1(M
ARQ
ARQ
07/42203-02 a
100.09
G41
Neat DNA 10(l
0.25(M
ARQ
ARQ
07/42203-02 a
100.09
G40
Neat DNA 10(l
0.25(M
ARQ
ARQ
07/42203-02 a
100.09
171R
2(l DNA 0.3(M primer
2(l PCR 0.3(M primers
ARQ
ARQ
07/42203-02 a
70.28
136F
2(l DNA 0.3(M primer
2(l PCR 0.3(M primers
A
A
No coverage
AHX
AHX
07/42203-02 a
70.28
136F
2(l DNA 0.3(M primer
2(l PCR 0.3(M primers
G
07/42203-02 a
70.28
171R
2(l DNA 0.3(M primer
2(l PCR 0.3(M primers
G
07/42203-02 a
70.28
G41
Neat DNA 10(l
1(M
A
T
AFRQ
AFRQ
07/42203-02 a
70.28
G40
Neat DNA 10(l
1(M
A
T
T
AFRQ
AFRQ
07/42203-02 a
70.28
G41
Neat DNA 10(l
0.5(M
A
T
AFRQ
AFRQ
07/42203-02 a
70.28
G40
Neat DNA 10(l
0.5(M
A
T
T
AFRQ
AFRQ
07/42203-02 a
70.28
G41
Neat DNA 10(l
0.25 (M
A
T
AFRQ
AFRQ
07/42203-02 a
70.28
G40
Neat DNA 10 (l
0.25(M
A
T
T
AFRQ
AFRQ
07/42203-02 a
74.49
G41
1:10 DNA 10(l
1(M
A
A
AHQ
AHQ
07/42203-02 a
74.49
G40
1:10 DNA 10(l
1(M
A
A
AHQ
AHQ
07/42203-02 a
74.49
G40
1:100 DNA 10(l
1(M
ARQ
ARQ
07/42203-02 a
74.49
G41
1:100 DNA 10(l
1(M
ARQ
ARQ
07/42203-02 a
74.49
G40
1:10 DNA 10(l
0.5(M
A
A
AHQ
AHQ
07/42203-02 a
74.49
G41
1:10 DNA 10(l
0.25(M
A
A
AHQ
AHQ
07/42203-02 a
74.49
G40
1:10 DNA 10(l
0.25(M
A
A
AHQ
AHQ
07/42203-02 a
81.94
136F
2(l DNA 0.3(M primer
2(l PCR 0.3(M primers
No coverage
A
A
A
A
AHQ
AHQ
07/42203-02 a
81.94
171R
2(l DNA 0.3(M primer
2(l PCR 0.3(M primers
A
A
A
A
A
A
AHQ
AHQ
07/42203-03 a
48.73
136F
2(l DNA 0.3(M primer
No coverage
AXX
AXX
07/42203-03 a
48.73
136R
2(l DNA 0.3(M primer
AXX
AXX
07/42203-04 b
290.82
G41
1:10 DNA 2(l
A
AHQ
AHQ
07/42203-03 b
214.6
G40
1:10 DNA 2(l
A
AHQ
AHQ
07/42203-04 b
290.82
G40
Neat DNA 2(l
0.5(M
A
AHQ
AHQ
07/42203-04 b
290.82
G41
Neat DNA 2(l
0.5(M
A
AHQ
AHQ
07/42203-03 b
214.6
G40
Neat DNA 2(l
0.5(M
A
AHQ
AHQ
08/0055-01 b
357.86
136F
2(l DNA 0.3(M primer
No coverage
AXX
AXX
08/0055-01 b
357.86
136R
2(l DNA 0.3(M primer
AXX
AXX
08/0055-01 b
357.86
G41
1:10 DNA 2(l
G
ARR
ARR
08/0055-01 b
357.86
G40
1:10 DNA 2(l
G
ARR
ARR
08/0055-01 b
357.86
136F
2(l DNA 0.3(M primer
G
ARR
ARR
08/0055-01 b
357.86
171R
2(l DNA 0.3(M primer
G
ARR
ARR
Figure 10. A) Bi-directionally sequencing of DNA amplfied from
samples from 07/42203 fixed and processed at VLA Thirsk. The
sequence data was analysed using GAP4 software (Staden package,
version 1.7.0). B) Chromatogram sequence files showing non-specific
artificial mutations at 396 bp and 399 bp, i) Wild-type base (G) at
positions 396 bp and 399 bp, ii) non-specific artificial mutations
at 396 bp (G>A), iii) non-specific artificial mutations at 399
bp (G>A). Polymorphisms were identified by comparison to a
wild-type PRNP gene reference sequence from an ARQ/ARQ sheep,
GenBank accession number AY0350267.
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.
Everest, S. J., Thorne, L., Barnicle, D. A., Edwards, J. C.,
Elliot, H., Jackman, R. and Hope, J. 2006. Atypical prion protein
in sheep brain collected during the British scrapie-surveillance
programme. J Gen Virol. 87. 471-7
Gonzalez, L., Martin, S. and Jeffrey, M. 2003. Distinct profiles
of PrP(d) immunoreactivity in the brain of scrapie and BSE infected
sheep: implications for differential cell targeting and PrP
processing. J Gen Virol. 84. 1339-50
Konold, T., Davis, A., Bone, G. E., Simmons, M.M., Kahn, J.,
Blake-Dyke, M.C., Bracegirdle, J. and Shimwell, C.J. 2006. Atypical
scrapie cases in the UK. Vet Rec. 158. 280
Konold, T., Davis, A., Bone, G., Bracegirdle, J., Everitt, S.,
Chaplin, M., Saunders, G. C., Cawthraw, S. and Simmons, M.M. 2007.
Clinical findings in two cases of atypical scrapie in sheep: a case
report. BMC Vet Res. 3. 2
Moore, S.J., Simmons, M., Chaplin, M. and Spiropoulos, J. 2008.
Neuroanatomical distribution of abnormal prion protein in naturally
occurring atypical scrapie cases in Great Britain. Acta
Neuropathol. 116. 547-59.
Saunders, G. C., Cawthraw, S., Mountjoy, S. J., Hope, J. and
Windl, O. 2006. PrP genotypes of atypical scrapie cases in Great
Britain. J Gen Virol. 87. 3141-9
Opinion of the Scientific Panel on Biological Hazards on the
request from the European Commission on classification of atypical
Transmissible Spongiform Encephalopathy (TSE) cases in Small
Ruminants. 2005. The EFSA Journal. 276. 1-30
�
A
B
C
D
E
F
G
H
I
J
K
L
A
B
C
D
10,000 bp
3000 bp
1000 bp
83/4333-01
83/4333-02
84/4956-01
84/4956-02
85/4417-01
1 Kbp ladder
85/4417-02
86/3436-01
86/3436-02
82/2982-01
82/2982-02
Ext negative
1 Kbp ladder
10,000 bp
3000 bp
1000 bp
Before After
80/0503-01
80/0503-02
81/0068-01
81/0068-02
80/0503-01
80/0503-02
81/0068-01
81/0068-02
1 Kbp ladder
TAG stop
G40
G41
312bp612bp
136 154 171aa
137 141aa
ATG start
TAG stop
G40
G41
312bp612bp
136 154 171aa
137 141aa
ATG start
A
B
i
ii
iii
396
399
396
399
396
399
SID 5 (Rev. 3/06)Page 1 of 31
