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From: TSS (
Subject: Neuronal accumulation of abnormal prion protein in sheep carrying a scrapie-resistant genotype (PrPARR/ARR) [FULL TEXT]
Date: August 13, 2004 at 7:19 am PST

-------- Original Message --------
Subject: Neuronal accumulation of abnormal prion protein in sheep carrying a scrapie-resistant genotype (PrPARR/ARR) [FULL TEXT]
Date: Fri, 13 Aug 2004 09:24:24 -0500
From: "Terry S. Singeltary Sr."
To: Bovine Spongiform Encephalopathy

Neuronal accumulation of abnormal prion protein in
sheep carrying a scrapie-resistant genotype

Anne Buschmann,1 Gesine Lu¨ hken,2 Julia Schultz,1 G. Erhardt2
and Martin H. Groschup1
Martin H. Groschup
1Federal Research Centre for Virus Diseases of Animals, Institute for
Novel and Emerging
Infectious Diseases, Boddenblick 5a, 17493 Greifswald  Insel Riems, Germany
2Department of Animal Breeding and Genetics, Justus-Liebig-University
Gießen, Ludwigstraße
21B, 35390 Gießen, Germany
Received 28 January 2004
Accepted 11 May 2004

The susceptibility of sheep to scrapie infection is influenced by prion
gene alleles, which are
modulated by polymorphic variations corresponding to amino acid
positions 136, 154 and 173
of the prion protein (PrP). As no unquestioned report of a diseased
sheep carrying homozygous
alleles encoding alanine, arginine and arginine (PrPARR) at these sites
has been published to date,
sheep of this genotype are believed to be scrapie resistant. After the
introduction of large-scale
rapid testing for scrapie, a number of so-called atypical scrapie
cases have been found in Germany
and elsewhere. Among those cases were two supposedly scrapie-resistant
sheep. Brain samples
from these animals tested positive for abnormal PrP (PrPSc) in one of
four rapid tests available.
Moreover, scrapie-associated fibril (SAF)-immunoblotting and
immunohistochemistry, which are
the generally accepted diagnostic techniques for scrapie, revealed
prominent PrPSc deposition
in the cerebellum. SAF immunoblotting also revealed PrPSc deposition in
the obex, frontal cortex
and brainstem. Transmission experiments to investigate the infectivity
of scrapie propagated
from these sheep have been initiated.


Scrapie in sheep is a transmissible spongiform encephalopathy
(TSE), a group of infectious conditions closely
associated with the deposition of abnormal prion protein
(PrPSc) in the central nervous system. Incubation times in
scrapie can be months or years before clinical symptoms
such as neurological dysfunction and chronic and fatal
wasting of the animal are observed. Clinical symptoms may
depend on the infectious scrapie strain involved and on the
breed and prion protein (PrP) genotype of the affected
sheep. To date, there is no live animal test that is applicable
during the scrapie incubation period to sheep of all PrP
genotypes. Moreover, the under-reporting of clinical cases is
thought to be high; therefore, the true prevalence of scrapie
can only be deduced vaguely from incidence rates found by
random testing of fallen stock and slaughtered animals.
The diagnostic methods currently applied to detect a TSE
infection (BSE rapid tests as well as confirmatory methods)
are based on the detection of PrPSc. In contrast to its cellular
counterpart (PrPC), PrPSc is partially proteinase K (PK)
resistant and forms scrapie-associated fibrils (SAFs) because
of its high hydrophobicity (Diringer et al., 1983; Hope et al.,
1986; Oesch et al., 1985; Lehmann & Harris, 1995). The four
commonly used rapid tests as well as the confirmatory
methods [SAF immunoblot and immunohistochemistry
(IHC)] that have been recommended by the Office International
des Epizooties (OIE) apply polyclonal or monoclonal
antibodies to detect PK-treated PrPSc accumulated in
the brains of TSE-affected animals.
In April 2002, an obligatory large-scale rapid-testing
programme on slaughtered and fallen sheep and goats was
implemented for monitoring purposes in the European
Union (EU) and has revealed considerable numbers of
scrapie cases in many member states. Because of the
theoretical risk of transmission of the BSE agent to sheep
and goats, the eradication of scrapie has become a high
priority in the EU. These two infections cannot be
distinguished by clinical symptoms or common diagnostic
methods alone. Unequivocal discrimination requires comparison
of the biochemical properties of the PrPSc or strain
typing by lesion-profile scoring, which is performed by
mouse bioassay in three conventional mouse lines (Bruce
et al., 1996; Fraser & Dickinson, 1968). It is generally
accepted that the susceptibility of sheep to scrapie is directly
linked to particular allelic polymorphisms of PrP. Sheep
carrying alleles encoding valine/arginine/glutamine
(PrPVRQ) or alanine/arginine/glutamine (PrPARQ) at
amino acid positions 136, 154 and 171 of PrP are highly
0007-9997 G 2004 SGM Printed in Great Britain 2727
Journal of General Virology (2004), 85, 27272733 DOI 10.1099/vir.0.79997-0
susceptible, whereas alleles encoding alanine/arginine/
arginine (PrPARR) seem to protect against this disease,
particularly when homozygous (Goldmann et al., 1990;
Hunter, 1996, 1997; Hunter et al., 1997). With one
questioned exception (Ikeda et al., 1995), scrapie has
never been diagnosed in a PrPARR homozygous sheep. In the
UK, France, The Netherlands and many other EU member
states, large genotyping and breeding programmes have
been started in order to increase the number of so-called
scrapie-resistant sheep (Arnold et al., 2002; Dawson et al.,
1998). In scrapie-affected sheep flocks, recent eradication
strategies rely on the removal of sheep that are considered
genetically susceptible and on the selective breeding of socalled
scrapie-resistant animals.


Rapid testing. Rapid tests for BSE with kits of the following
brands were done according to the manufacturers instructions:
Platelia (Bio-Rad); Enfer TSE kit version 2.0 (Enfer Scientific);
Prionics Check Western (Prionics); Prionics Check LIA (Prionics).
Confirmatory testing: SAF immunoblot. For preparation of
SAFs, a 10% (w/v) homogenate was prepared of 1 g brainstem
material from the obex region in 0?01 M sodium phosphate buffer,
pH 7?4, containing 10% (w/v) sarcosine, 0?5 mM PMSF and
0?5 mM N-ethylmaleimide. After a preliminary centrifugation for
30 min at 20 000 g to pellet residual detritus, the supernatant was
transferred into a new centrifuge tube and centrifuged for 135 min
at 220 000 g. Pellets were resuspended in 3 ml 0?015 M Tris/HCl
pH 7?4 and incubated for 15 min at 37 uC and twice the sample
volume of 15% potassium iodide/high-salt buffer containing 0?1 M
sodium thiosulphate pentahydrate, 0?3 M N-lauroylsarcosine and
0?01 M Tris/HCl was added. Samples were incubated at 37 uC for a
further 30 min. Samples were split into equal parts and 45 mg PK
was added to one of the aliquots and incubated for 60 min at 37 uC.
Afterwards, 4?5 ml 10% potassium iodide/high-salt buffer was
added to the digested and untreated aliquots. Finally, samples
were centrifuged through a 20% sucrose gradient for 60 min at
280 000 g. Pellets were resuspended in a sample buffer pH 6?8 containing
1% (w/v) SDS, 25 mM Tris/HCl pH 7?4, 0?5% mercaptoethanol
and 0?001% bromophenol blue, heat-denatured for 5 min
at 95 uC and loaded on SDS-polyacrylamide gels containing 13%
bisacrylamide. After electrophoresis, proteins were transferred to a
PVDF membrane in a semi-dry chamber. Membranes were blocked
in I-Block (Tropix) for 30 min and incubated with the PrP-specific
monoclonal detection antibody (mAb) L42 (specific for both PrPC
and PrPSc) (Harmeyer et al., 1998) for 90 min at room temperature.
Membranes were washed three times for 10 min with PBS containing
0?1% Tween 20 and then incubated with a secondary antibody
bound to alkaline phosphatase (goat anti-mouseAP; Dianova) for
60 min at room temperature. After washing, the chemiluminescence
substrate CDP-Star (Tropix) was applied and incubated on the
membrane for 5 min, before the light signals on the membrane were
detected directly in a camera.
PrPSc detection by IHC. Samples were processed as described previously
(Hardt et al., 2000). Briefly, 3 mm sections of the obex
region were fixed in 3?5% sodium-buffered formalin (SBF) for at
least 48 h. After a 60 min incubation in 98% formic acid, samples
were dehydrated automatically with pressure and vacuum at 35 uC
through a series of ethanol solutions and embedded in paraffin
blocks. Sections (3 mm) were then prepared and IHC staining with
the PrP-specific mAb L42, binding to an epitope at aa 145163 of
ovine PrP (Harmeyer et al., 1998), or SAF 70, binding to an epitope
at aa 142160 of hamster PrP (Demart et al., 1999), was done in an
automated stainer. This procedure included a pre-treatment for
15 min in 98% formic acid followed by a 5 min incubation in tap
water, 30 min in SBF and washing twice in PBS for 5 min before
placing the slides into a Ventana Discovery autostainer. The automated
staining protocol included a heat treatment at 95 uC for
12 min followed by a protease treatment for 12 min at 42 uC. After
blocking the slides in 30% goat serum, they were incubated with
primary antibody for 20 min at room temperature. This was followed
by a washing procedure, incubation for 2 min with biotinylated
Ig (Ventana), washing again and then incubation with
streptavidinhorseradish peroxidase for 8 min. Signals were visualized
with the diaminobenzidine detection system and hydrogen peroxide.
Finally, sections were counterstained with haematoxylin and
blueing reagent. All reagents used in this protocol were supplied by
Ventana. As controls, brain sections from the obex region and cerebellum
from a TSE-negative sheep and from a sheep with classical
scrapie were used.
Determination of PrP allele. PrP alleles of the diseased sheep
were determined by sequencing and by PCR-RFLP (Lu¨hken et al.,
2004). Briefly, genomic DNA was extracted from brain samples with
a commercial kit (QiaAmp DNA kit) followed by PCR amplification.
To generate templates for sequencing, primers 59-TGCCACTGCTATACAGTCATT-
39 (sense; nt 2217922199 of GenBank
sequence U67922) and 59-TGGTGGTGACTGTGTGTTGCT-39 (antisense;
nt 2284122861) for amplification of a 682 bp fragment and
primers 59-AACCAACATGAAGCATGTGGC-39 (sense; nt 22604
22624) and 59-AAGCAAGAAATGAGACACCACC-39 (antisense; nt
2312723148) for amplification of a 544 bp fragment were used in
reaction mixtures comprising, in a volume of 50 ml, approximately
200 ng genomic DNA, 20 pmol of each primer, 5 mM of each
dNTP, 2?0 mM (682 bp fragment) or 3?0 mM (544 bp fragment)
MgCl2 and 0?25 U Taq polymerase in onefold reaction buffer with
the following PCR conditions: denaturation at 94 uC for 1?5 min, 40
amplification cycles of denaturation at 94 uC for 15 s, annealing at
59 uC (682 bp fragment) or 60 uC (544 bp fragment) for 20 s and
extension at 72 uC for 45 s, followed by a final 5 min extension at
72 uC. The PCR fragments were used directly in sequencing reactions
or restriction enzyme digestions for determination of the DNA
codons at positions 136, 154 and 171 of the ovine PrP. For sequencing,
each of the four PCR primers was reused, which resulted in
sequences covering the complete coding region of exon 3 of ovine


In this paper we report the diagnosis of two scrapie cases in
sheep carrying the supposedly scrapie-resistant PrPARR/ARR
genotype. Both showed deposition of PrPSc in their brains.
In a more recent case, designated ARR II, a slaughtered 2-
year-old sheep that had not shown any clinical symptoms
was routinely tested in a regional state laboratory as part of
the German scrapie surveillance programme and was found
weakly reactive by the Bio-Rad Platelia test with absorbance
values of 0?205 (cut-off 0?234) and 0?342/0?392 (cut-off
0?241) in the obex. The sample was subsequently submitted
to the German National Reference Laboratory (NRL) for
confirmatory testing. So-called SAF immunoblotting (as
recommended by the OIE) (Anonymous, 2000) and the EU
Commission (Anonymous, 1994) was done. The obex
sample repeatedly gave a banding signal that resembled that
of scrapie PrPSc; however, at least five bands including the
different PrP glycosylation forms and additional PrP
2728 Journal of General Virology 85
A. Buschmann and others
fragments were visible (Fig. 1a). A band of approximately
12 kDa was also observed that has been described for
atypical scrapie cases in Norway (Fig. 1b) (Benestad et al.,
2003). It was also noted that the quantities of the three PrP
bands did not completely match those usually found in
typical scrapie cases with a dominant upper (diglycosylated)
band, a weaker middle (monoglycosylated) band and
only a faint lowest (unglycosylated) band. Instead, in the
case described here, the lowest band was more prominent
than the middle band, so that the signals of the
diglycosylated and the unglycosylated PrP bands were of
similar strengths. Moreover, the PrPSc depositions in this
atypical scrapie case were less PK-resistant than the PrPSc
found in classical cases (Fig. 2).
Neg. sheep Classical scrapie ARR II case
PK 0 0 5 10 0 20 100 500 0 10 50 250 2.5
Fig. 2. Comparison of PK sensitivity of PrPSc. To compare the resistance
of PrPSc derived from scrapie samples with
concordant results in all tests with that of samples with incongruent
results, SAF preparations were treated with PK
concentrations ranging from 10 to 500 mg ml1. PrPC from an uninfected
sheep control was completely digested by
10 mg ml1; PrPSc from a sample with concordant results (S3/02) still
gave a strong signal after incubation with 500 mg ml1
for 60 min at 37 6C; PrPSc from a sample with incongruent results
(S14/03) was almost completely digested after incubation
with 250 mg ml1. MAP L42 was used as the detection antibody.
Negative control (obex sheep)
Frontal cortex ARR II
Cerebellum ARR II
Scrapie (obex sheep)
ARR I atypical scrapie
ARR II atypical scrapie
Typical scrapie
PK _ + _ _ _ _ + + + +
(a) (b)
Fig. 1. Detection of PrPSc deposition in obex, frontal cortex and
cerebellum (a) of the second sheep of genotype PrPARR/ARR
(case ARR II) and the obex region of ARR cases I and II (b). Brain
samples (1 g) were processed according to the SAF
immunoblotting protocol with (+) or without () PK treatment, as
indicated. Samples were then separated by SDS-PAGE and
immunoblotted with mAb L42 as the detection antibody. Antibody binding
was visualized with goat anti-mouseAP and CDPStar
chemiluminescence substrate. Signals were visualized using the VersaDoc
Imaging system (Bio-Rad) with Quantity One
software. Positions of molecular mass markers are indicated (M).
Interestingly, the PrP banding pattern does not match that of
a typical case as at least five bands of PrP and its fragments,
including a 12 kDa band, are visible. This banding pattern is
reminiscent of the Nor98 scrapie cases. 2729

Scrapie in genetically resistant sheep

A sample from the obex region was also taken and subjected
to IHC staining with mAbs L42 and SAF 70 but no PrPspecific
signals were visible. Incidentally, several other brain
regions were available from this animal and were analysed by
rapid testing, SAF immunoblotting and IHC. For the obex
region, a different sample had to be taken for retesting with
the Bio-Rad Platelia test at the NRL, and gave an absorbance
value of 0?767 (Table 1). These studies revealed PrPSc
depositions, with the largest amounts in the cerebellum, and
smaller or borderline amounts in the frontal cortex,
diencephalon and obex regions (Table 1, Fig. 3). Most
strikingly, IHC with mAbs L42 and SAF 70 on the
cerebellum of this case revealed a very strong signal for
PrPSc deposition in the molecular cell layer, whereas no
staining was found in other regions of the central nervous
system (Fig. 3). This is in contrast to what has been
described for classical scrapie cases, where PrPSc deposition
is detectable mainly in association with cellular membranes
of neurons and astrocytes in the medulla oblongata and
pons but no staining is seen in the cerebellum (Van Keulen
et al., 1995), which is in accordance with what has recently
been described for atypical scrapie cases in Norway
designated Nor98 (Benestad et al., 2003). Surprisingly,
this case was not detected by the Prionics Check Western
test or the LIA rapid test, an observation that has also
been reported for a number of atypical scrapie cases
(Buschmann et al., 2004) recently found in Germany,
France, Norway (Benestad et al., 2003) and elsewhere. The
reason for this non-homogeneous detection has been
analysed for a number of German and French scrapie
cases (Buschmann et al., 2004) and it seems to be more
dependent on the sheep isolate than on the antibodies
applied for PrP detection. To get a second opinion on our
interpretation, a formalin-fixed obex sample and the SAF
immunoblotting and IHC testing results were submitted to
the Community Reference Laboratory (CRL) at the
Veterinary Laboratories Agencies, Weybridge, UK. The
CRL confirmed the absence of IHC staining of the obex, but
from the positive SAF immunoblotting and positive IHC
results in other brain areas concluded that this would be
sufficient to support the confirmation of this case as TSE
As the head of this animal was available for further sampling,
the lymphoid tissue adherent to the nictitating membrane as
well as the mandibular and retropharyngeal lymph nodes
were examined by IHC with mAbs L42 and SAF 70.
However, no PrP-specific staining was detectable in these
tissues (data not shown). This is in accordance with what has
been described previously for scrapie-affected sheep carrying
only one ARR allele (Van Keulen et al., 1996; Andre´oletti
et al., 2000).
Following this diagnosis, all animals in the flock from which
the affected sheep were derived were genotyped, and animals
carrying no PrPARR allele or the PrPVRQ allele in the
homozygous or the heterozygous form were culled. No
other scrapie case was found by rapid testing (Bio-Rad
Platelia) within this selected group. This may result at least
partly from the fact that no PrPSc was detectable in the
analysed lymphoid tissue of this animal, which reduces the
risk of horizontal scrapie transmission within a flock.
In the course of retrospective genotyping of all German
scrapie cases, DNA was prepared from brain tissue (three
independent samples) and from masseter muscle of the
scrapie-diagnosed animal and sequenced by RFLP analysis.
Surprisingly, all tests consistently gave the PrP genotype of
this animal to be PrPARR/ARR. Genotyping of this sheep was
repeated by another laboratory and the PrPARR/ARR allele
was again confirmed. Other than the amino acid substitution
at codon 171 (QQ/RR), no further nucleotide
variations were observed comparing the sequence obtained,
covering the complete coding region of exon 3 of PRNP,
with GenBank sequence U67922. This is the second time
that a PrPARR/ARR genotype has been found in a scrapie case
from Germany. A previous case, designated ARR I, which
was also discovered retrospectively by genotyping, occurred
in an 810-year-old female sheep that was sent for
rendering, and was tested using the Bio-Rad Platelia rapid
test as a routine sample. The only available brain-tissue
Table 1. Detection of PrPSc in different brain regions of the ARR II
case using rapid tests
and confirmatory methods at the German TSE reference laboratory
Note that a fresh obex sample was taken for the Bio-Rad test at the NRL
as no residual homogenate
was available from the initial testing at the regional laboratory.
Results are absorbances for the Bio-Rad
Platelia test (cut-off 0?294), PrPSc signal for the Prionics Check
Western and relative light units for the
Prionics Check LIA (cut-off 2791). Scores are: 2, no signal; +/2, very
weak signal; +, weak signal;
++, strong signal; +++, very strong signal.
Brain region Bio-Rad
Prionics Check
Check LIA
SAF immunoblot IHC
Obex 0?767 2 76 ++ 2
Cerebellum 1?424 2 74 +++ ++
Frontal cortex 1?206 2 91 ++ +/2
2730 Journal of General Virology 85
A. Buschmann and others
(e) (f) (g) (h)
(b) (c) (d)
(i) (j)
Fig. 3. IHC staining for PrPSc deposition in the obex (c), cerebellum
(eh), thalamus
(i) and frontal cortex (j) of case ARR II. Obex (a) and cerebellum (d)
sections from a
normal animal served as negative control, whereas an obex section of a
scrapie case (b) was used as positive control. mAb L42 was used
throughout the
experiment except for (f), which was stained with mAb SAF 70 in order to
the specificity of the staining. Note the heavy IHC staining, as
indicated by precipitation
of brown substrate (diaminobenzidine), indicating PrPSc deposition in the
molecular layer (eh) also extending into the granular layer (g, h) of
the cerebellum.
Bars, 50 mm. 2731
Scrapie in genetically resistant sheep
sample from this case was heavily autolysed and of a pastelike
consistency so that the obex region could scarcely be
determined. The Platelia readings on the assumed obex
sample were borderline reactive (absorbance in duplicate
readings 0?231/0?554) in the first set of experiments and low
reactive in the second set of experiments (absorbance in
duplicate readings 0?426/0?492). However, no positive
result was obtained when the Prionics Check Western or
LIA or the Enfer rapid test were used. Accumulation of PrPSc
was also found by SAF immunoblotting in two independent
experiments (Fig. 1b). Again, the banding patterns did not
match those of typical scrapie. IHC staining with mAbs L42
or SAF 70 on the assumed obex was negative, a result that
could also have been because of the heavy autolysis or a
sampling artefact. The suspicion of scrapie was eventually
confirmed on the basis of the Bio-Rad Platelia and the SAF
immunoblotting results. All sheep in the herd of origin were
culled irrespective of their genotype, but no other sheep was
found to be positive.
In this case, DNA purified from the reactive brain was
sequenced and PrPARR/ARR allele was determined. To
confirm this, another DNA preparation was genotyped,
again in the same laboratory and in parallel in an
independent laboratory. All investigations confirmed the
first genotyping results of ARR homozygosity. Again, no
nucleotide variations other than the amino acid substitution
at codon 171 (QQ/RR) could be observed by comparing the
sequence obtained with GenBank sequence U67922.
For both of the potential ARR scrapie cases, mouse bioassays
have now been initiated to investigate the presence of
inherent TSE infectivity. RIII, C57Bl, VM95 and ovine PrPC
(PrPARQ)-overexpressing transgenic mice have been inoculated
intracerebrally with cerebellar and/or obex homogenates
(10 %, w/v). Results from these studies are not
expected to be available before the middle of 2005.
It is well established that PrPARR allele homozygosity does
not confer absolute resistance to TSE experimental
challenge. PrPARR/ARR sheep experimentally exposed to
BSE by intracerebral inoculation develop clinical signs of a
TSE infection (Houston et al., 2003). PrPSc found in these
animals displayed the same glycoform pattern as found in
diseased sheep of the susceptible PrPARQ homozygous
genotype. Moreover, cell-free conversion of PrPARR using
typical scrapie as seed is possible, albeit at a much lower
efficiency (Bossers et al., 2000).
There has been no previous unquestioned report of a
natural scrapie case in a PrPARR/ARR sheep, although a
large number of diseased animals have been genotyped.
However, the large majority of scrapie cases have been
confirmed previously by IHC staining of PrPSc in the obex,
which is considered the most reliable diagnostic marker
of scrapie (Anonymous, 2004). Only in a few instances was
SAF immunoblotting carried out, because this requires a
larger quantity of sample and is more time-consuming.
After the introduction of the scrapie-monitoring programme
in the EU, in which obex samples are now being tested using
rapid tests, atypical scrapie cases are being detected more
frequently. It is intriguing to see that atypical scrapie cases
have been uncovered recently in which PrPSc deposition at
the level of the obex is faint or absent. As only the obex or
brainstem is sampled for this programme, reassessing these
cases by analysis of other brain areas is often impossible.
Therefore, atypical scrapie cases may have been previously
under-reported. However, it must be noted that the infectious
nature of this novel scrapie type still has to be con-
firmed by transmission experiments. A similar situation
may exist for yet unrecognized scrapie cases in PrPARR/ARR
animals, which may display a distinct PrPSc deposition
topology in the brain and a distinct biochemical glycotyping
pattern. This novel phenotype may originate from a
particular PrP genotype, from a peculiar scrapie strain or
from the combination of the two.
It is interesting to see that SAF immunoblotting of the obex
or cortex gave a positive result, like the Bio-Rad Platelia,
while IHC staining for PrPSc was negative. It could be argued
that IHC is much less sensitive at recognizing these
particular cases. Moreover, our own results indicate that
SAF preparation is in general more sensitive to dilution of
positive samples than IHC staining (A. Buschmann and
others, unpublished results). However, the heavy PrP
staining in the cerebellum proves that this method is in
principle equally suitable for detection of these cases.
Taken together, the findings reported here indicate that
sheep homozygous for the PrPARR allele may not be fully
resistant to natural scrapie infections and may exhibit
diagnostic features that fit the most recently discovered
atypical scrapie case definition. Atypical scrapie cases
occur predominantly in sheep carrying a scrapie-resistance
PrP allele in heterozygous or homozygous form. If
transmission studies indeed show that the PrPSc depositions
in these cases are infectious and that such infections are able
to spread from sheep under natural conditions, these
findings would question the large-scale sheep genotyping
and scrapie-resistant breeding programmes that have been
introduced in several EU member states over the last 5 years.


The authors wish to thank Dan Balkema and Gesine Kreplin for their
excellent technical support. Dr Marion Simmons at the Community
Reference Laboratory, Veterinary Laboratories Agency Weybridge, UK,
is acknowledged for her support in the diagnosis of these and other
atypical cases. We are indebted to Sylvie Benestad from the National
Veterinary Institute in Oslo, Norway, for supplying us with Nor98
material. This work was financially supported in parts by the German
Ministry for Consumer Protection, Nutrition and Agriculture, the
German Ministry for Education and Research and by funds from the
European Commission.
Andre´ oletti, O., Berthon, P., Marc, D., Sarradin, P., Grosclaude, J.,
Van Keulen, L., Schelcher, F., Elsen, J. M. & Lantier, F. (2000). Early
2732 Journal of General Virology 85
A. Buschmann and others
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