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Nor-98 like atypical scrapie Wyoming but slaughtered in MI



R. Nonno1, E. Esposito1, G. Vaccari1, E. Bandino2, M. Conte1, B. Chiappini1,
S. Marcon1, M. Di Bari1, S.L.
Benestad3, U. Agrimi1
1 Istituto Superiore di Sanità, Department of Food Safety and Veterinary
Public Health, Rome, Italy ([email protected]); 2
Istituto Zooprofilattico della Sardegna, Sassari, Italy; 3 National
Veterinary Institute, Department of Pathology, Oslo, Norway

Molecular variants of PrPSc are being increasingly investigated in sheep
scrapie and are generally referred to as “atypical” scrapie, as opposed to “classical scrapie”. Among
the atypical group, Nor98 seems to be the best identified. We studied the molecular properties of
Italian and Norwegian Nor98 samples by WB analysis of brain homogenates, either untreated, digested with
different concentrations of proteinase K, or subjected to enzymatic deglycosylation. The identity of PrP
fragments was inferred by means of antibodies spanning the full PrP sequence.

We found that undigested brain homogenates contain a Nor98-specific PrP
fragment migrating at 11 kDa (PrP11), truncated at both the C-terminus and
the N-terminus, and not N-glycosylated. After mild PK digestion, Nor98 displayed full-length PrP
(FL-PrP) and N-glycosylated C-terminal fragments (CTF), along with increased levels of PrP11. Proteinase
K digestion curves (0,006-6,4 mg/ml) showed that FL-PrP and CTF are mainly digested above 0,01
mg/ml, while PrP11 is not entirely digested even at the highest concentrations, similarly to PrP27-30
associated with classical scrapie. Above 0,2 mg/ml PK, most Nor98 samples showed only PrP11
and a fragment of 17 kDa with the same properties of PrP11, that was tentatively identified as a dimer of
PrP11. Detergent solubility studies showed that PrP11 is insoluble in 2% sodium laurylsorcosine and is mainly
produced from detergentsoluble, full-length PrPSc.

Furthermore, among Italian scrapie isolates, we found that a sample with
molecular and pathological properties consistent with Nor98 showed plaque-like deposits of PrPSc in the
thalamus when the brain was analysed by PrPSc immunohistochemistry. Taken together, our results show
that the distinctive pathological feature of Nor98 is a PrP fragment spanning amino acids ~ 90-155. This fragment is
produced by successive N-terminal and C-terminal cleavages from a full-length and largely
detergent-soluble PrPSc, is produced in vivo and is extremely resistant to PK digestion. Intriguingly,
these conclusions suggest that some pathological features of Nor98 are reminiscent of
Gerstmann-Sträussler-Scheinker disease.


Summary of Selected Disease Events January—March 2007

Scrapie: Nor98-like—Wyoming On March 16, 2007, the USDA Animal and Plant
Health Inspection Service (APHIS) notified stakeholders that an aged female
sheep had tested positive for scrapie, consistent with the Nor98 type. The
ewe was slaughtered in Michigan, where it was tested as part of USDA’s
ongoing regulatory scrapie slaughter surveillance program. The ewe was
traced back to a flock in Wyoming. This is the first time this particular
type of scrapie has been found in the United States. The Nor98 type of
scrapie is uncommon even in Europe, with fewer than 300 similar cases
diagnosed since it was identified in 1998.

Published online before print October 20, 2005

Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0502296102
Medical Sciences

A newly identified type of scrapie agent can naturally infect sheep with
resistant PrP genotypes

( sheep prion | transgenic mice )

Annick Le Dur *, Vincent Béringue *, Olivier Andréoletti , Fabienne Reine *,
Thanh Lan Laï *, Thierry Baron , Bjørn Bratberg ¶, Jean-Luc Vilotte ||,
Pierre Sarradin **, Sylvie L. Benestad ¶, and Hubert Laude *
*Virologie Immunologie Moléculaires and ||Génétique Biochimique et
Cytogénétique, Institut National de la Recherche Agronomique, 78350
Jouy-en-Josas, France; Unité Mixte de Recherche, Institut National de la
Recherche Agronomique-Ecole Nationale Vétérinaire de Toulouse, Interactions
Hôte Agent Pathogène, 31066 Toulouse, France; Agence Française de Sécurité
Sanitaire des Aliments, Unité Agents Transmissibles Non Conventionnels,
69364 Lyon, France; **Pathologie Infectieuse et Immunologie, Institut
National de la Recherche Agronomique, 37380 Nouzilly, France; and
¶Department of Pathology, National Veterinary Institute, 0033 Oslo, Norway

Edited by Stanley B. Prusiner, University of California, San Francisco, CA,
and approved September 12, 2005 (received for review March 21, 2005)

Scrapie in small ruminants belongs to transmissible spongiform
encephalopathies (TSEs), or prion diseases, a family of fatal
neurodegenerative disorders that affect humans and animals and can transmit
within and between species by ingestion or inoculation. Conversion of the
host-encoded prion protein (PrP), normal cellular PrP (PrPc), into a
misfolded form, abnormal PrP (PrPSc), plays a key role in TSE transmission
and pathogenesis. The intensified surveillance of scrapie in the European
Union, together with the improvement of PrPSc detection techniques, has led
to the discovery of a growing number of so-called atypical scrapie cases.
These include clinical Nor98 cases first identified in Norwegian sheep on
the basis of unusual pathological and PrPSc molecular features and "cases"
that produced discordant responses in the rapid tests currently applied to
the large-scale random screening of slaughtered or fallen animals.
Worryingly, a substantial proportion of such cases involved sheep with PrP
genotypes known until now to confer natural resistance to conventional
scrapie. Here we report that both Nor98 and discordant cases, including
three sheep homozygous for the resistant PrPARR allele (A136R154R171),
efficiently transmitted the disease to transgenic mice expressing ovine PrP,
and that they shared unique biological and biochemical features upon
propagation in mice. These observations support the view that a truly
infectious TSE agent, unrecognized until recently, infects sheep and goat
flocks and may have important implications in terms of scrapie control and
public health.


Author contributions: H.L. designed research; A.L.D., V.B., O.A., F.R.,
T.L.L., J.-L.V., and H.L. performed research; T.B., B.B., P.S., and S.L.B.
contributed new reagents/analytic tools; V.B., O.A., and H.L. analyzed data;
and H.L. wrote the paper.

A.L.D. and V.B. contributed equally to this work.

To whom correspondence should be addressed.

Hubert Laude, E-mail: [email protected]

Colorado Surveillance Program for Chronic Wasting Disease
Transmission to Humans (TWO SUSPECT CASES, see GSS case)

SOMETHING ELSE TO PONDER HERE, lets go back and look and the other potential
cases of CWD transmission to humans and where it was explained away as
genetic too i.e. GSS ;

In 2001, the case of a 25-year-old man who reportedly died of a prion
disease after an illness lasting ?22 months was investigated (Table 2).
Although this man had hunted deer only rarely, his grandfather hunted deer
and elk throughout much of the 1980s and 1990s and regularly shared the
venison with the case-patient's family. The grandfather primarily hunted in
southeastern Wyoming, around the known CWD-endemic area. The case-patient's
illness began with a seizure and progressed to fatigue, poor concentration,
and depression. Memory loss, ataxia, speech abnormalities, combative
behavior, and recurrent seizures also developed. Histopathologic,
immunohistochemical, and Western blot testing of brain autopsy samples
confirmed a prion disease diagnosis. Analysis of the prion protein gene
indicated a P102L mutation coupled with valine at the polymorphic codon 129
in the mutant allele, confirming a diagnosis of
Gerstmann-Sträussler-Scheinker syndrome (GSS). This case-patient was
unusually young even for a person with a GSS P102L mutation. It remains
unknown whether the possible exposure of the case-patient to CWD-infected
venison potentially contributed to the early onset of his prion disease.

Table 2. Creutzfeldt-Jakob disease patients investigated for a possible
causal link of their illness with chronic wasting disease of deer and elk,
United Statesa


snip... full text ;

An evaluation of scrapie surveillance in the United States
From: Terry S. Singeltary Sr.
Date: Sun, 5 Aug 2007 13:05

Volume 43 Page 1687 - December 2003
Volume 43 Issue 12

Similar levels of infectivity in the blood of mice infected with
human-derived vCJD and GSS strains of transmissible spongiform

Larisa Cervenakova, Oksana Yakovleva, Carroll McKenzie, Svetlana Kolchinsky,
Lisa McShane, William N. Drohan, and Paul Brown
The possible transmission of variant CJD (vCJD) through blood transfusion or
use of plasma-derived products prompted this study comparing infectivity in
murine models of vCJD and Gerstmann-Sträussler-Scheinker (GSS) disease, a
non-vCJD form of transmissible spongiform encephalopathy (TSE).

RIII/Fa/Dk (RIII) or Swiss-Webster (Swiss) mice were inoculated
intracerebrally (IC) with mouse-adapted strains of vCJD or GSS (Fukuoka-1)
of similar infectivity. Groups of RIII mice were euthanized 17 weeks after
inoculation (during the incubation period), and another 23 weeks after
inoculation (when symptomatic). Blood was collected, separated into
components, and inoculated into groups of healthy mice; brains and spleens
from all mice were harvested and tested for the presence of PrPres by
Western blot using 6H4 MoAb.

Levels of 20-30 infectious doses per mL were present in buffy coat and
plasma during both the incubation and symptomatic stages of disease; PLT
pellet infectivity was lower (10 ID/mL) and RBCs were not infectious. The
disease was transmitted more efficiently by IV than IC inoculation of
plasma, but there was no difference observed with inoculation of buffy coat.
The incubation period was shorter after IC inoculation of GSS- than
vCJD-brain inocula. The amount of PrPres in spleens was similar for both TSE
agents, but was slightly lower in brains of vCJD than GSS mice.

Infectivity was detected in blood components of mice infected with a
human-derived strain of vCJD during both the preclinical and clinical phases
of disease in a similarly low range of concentrations as in mice infected
with a human-derived nonvariant strain (GSS, Fukuoka-1). Other measures of
virulence, including brain infectivity titers, incubation periods, and the
accumulation of PrPres in spleens and brains, were also comparable in both
experimental models.

It is clear that scrapie in sheep is important commercially and
for that reason alone effective measures to control it should be
devised as quickly as possible.

Recently the question has again been brought up as to whether
scrapie is transmissible to man. This has followed reports that the
disease has been transmitted to primates. One particularly lurid
speculation (Gajdusek 1977) conjectures that the agents of scrapie,
kuru, Creutzfeldt-Jakob disease and transmissible encephalopathy of
mink are varieties of a single "virus". The U.S. Department of
Agriculture concluded that it could "no longer justify or permit
scrapie-blood line and scrapie-exposed sheep and goats to be processed
for human or animal food at slaughter or rendering plants" (ARC 84/77)"
The problem is emphasized by the finding that some strains of scrapie
produce lesions identical to the once which characterize the human

Whether true or not. the hypothesis that these agents might be
transmissible to man raises two considerations. First, the safety
of laboratory personnel requires prompt attention. Second, action
such as the "scorched meat" policy of USDA makes the solution of the
scrapie problem urgent if the sheep industry is not to suffer



J Infect Dis 1980 Aug;142(2):205-8

Oral transmission of kuru, Creutzfeldt-Jakob disease, and scrapie to
nonhuman primates.

Gibbs CJ Jr, Amyx HL, Bacote A, Masters CL, Gajdusek DC.

Kuru and Creutzfeldt-Jakob disease of humans and scrapie disease of sheep
and goats were transmitted to squirrel monkeys (Saimiri sciureus) that were
exposed to the infectious agents only by their nonforced consumption of
known infectious tissues. The asymptomatic incubation period in the one
monkey exposed to the virus of kuru was 36 months; that in the two monkeys
exposed to the virus of Creutzfeldt-Jakob disease was 23 and 27 months,
respectively; and that in the two monkeys exposed to the virus of scrapie
was 25 and 32 months, respectively. Careful physical examination of the
buccal cavities of all of the monkeys failed to reveal signs or oral
lesions. One additional monkey similarly exposed to kuru has remained
asymptomatic during the 39 months that it has been under observation.


The successful transmission of kuru, Creutzfeldt-Jakob disease, and scrapie
by natural feeding to squirrel monkeys that we have reported provides further
grounds for concern that scrapie-infected meat may occasionally give rise in
humans to Creutzfeldt-Jakob disease. ...end
(from full text study pdf...TSS)

PMID: 6997404

see full text ;

Research Project: Transmission, Differentiation, and Pathobiology of
Transmissible Spongiform Encephalopathies
Location: Virus and Prion Diseases of Livestock

Title: Pathobiology and diagnosis of animal transmissible spongiform
encephalopathies: current knowledge, research gaps, and opportunities


Kehrli, Marcus
O`rourke, Katherine
Hamir, Amirali
Richt, Juergen
Nicholson, Eric
Silva, Christopher
Gay, Cyril

Submitted to: Government Publication/Report
Publication Type: Government Publication
Publication Acceptance Date: May 1, 2007
Publication Date: July 1, 2007

Citation: Kehrli, Jr., M.E., O'Rourke, K.I., Hamir, A.N., Richt, J.A.,
Nicholson, E.M., Silva, C.J., Edelman, D., Gay, C.G. 2007.

Pathobiology and diagnosis of animal transmissible spongiform
encephalopathies: current knowledge, research gaps, and opportunities
[government white paper]. Beltsville, MD: Interagency Working Group on Prion
Science, Subcommittee on Pathobiology and Diagnostics. USDA, Agriculture
Research Service. 33 p.

Technical Abstract:

Transmissible spongiform encephalopathies (TSEs) are fatal neurologic
diseases that can affect several animal species and human beings. There are
four animal TSE agents found in the United States: scrapie of sheep and
goats; chronic wasting disease (CWD) of deer, elk, and moose; transmissible
mink encephalopathy (TME) and bovine spongiform encephalopathy (BSE).
Although the animal TSEs do not cause major death losses among US livestock
populations, they are important because of international trade issues. The
experience of the United Kingdom and Europe in dealing with the vast
majority of the world's BSE cases, serves as a reminder of the need for
continuing vigilance in monitoring risks for public health and research to
answer remaining questions around the pathogenesis and transmission of these
diseases. There remain questions on 1) cross-species transmissibility of
TSEs in livestock and wildlife; 2) the pathobiology of TSEs in natural and
secondary hosts; pathogenesis and transmission of CWD; and 4) pathogenesis
and ante mortem detection of typical and atypical BSE. Our understanding of
the pathogenesis and transmission of these diseases continues to evolve as
ongoing, global TSE research efforts focus on defining tissue sites of
abnormal prion accumulation, routes of infection, methods of strain
differentiation, genetics of susceptibility and ante-mortem diagnostics. In
this paper, a Subcommittee on Pathobiology and Diagnostics of TSEs for an
Interagency Working Group on Prion Science summarizes the science of animal
TSEs in order to identify knowledge gaps for the purpose of prioritizing
animal prion research needs. Because of substantial losses involving
international trade and potential risk for interspecies transmission to
susceptible livestock and possibly humans, the presence of BSE, CWD, scrapie
and TME in the United States presents a liability to U.S. domestic and
alternative livestock industries. In addition, the proven risk of BSE to
agriculture and public health from subclinical or clinically sick animals
requires science-based surveillance for any silent, unrecognized epizootic
expansions of these diseases in populations of animals that could either
directly or indirectly affect food animals. CWD is an example of an
uncontrolled expanding epidemic that threatens not only cervids but possibly
other livestock. CWD also has elicited public health surveillance programs
to monitor for scientific evidence of a prion disease in humans that consume
venison. Therefore, some of the research needs are precautionary, but the
risks to animal and human health from being caught unaware are high. Efforts
are being made by both federal and state regulatory agencies to eradicate
scrapie and CWD, and to determine the prevalence of BSE. The effectiveness
of these programs will depend heavily on having accurate information about
the nature of these diseases, not only in the original hosts, but also in
other species that may be in contact with infected animals.

Summary of Selected Disease Events January—March 2007

Scrapie: Nor98-like—Wyoming On March 16, 2007, the USDA Animal and Plant
Health Inspection Service (APHIS) notified stakeholders that an aged female
sheep had tested positive for scrapie, consistent with the Nor98 type. The
ewe was slaughtered in Michigan, where it was tested as part of USDA’s
ongoing regulatory scrapie slaughter surveillance program. The ewe was
traced back to a flock in Wyoming. This is the first time this particular
type of scrapie has been found in the United States. The Nor98 type of
scrapie is uncommon even in Europe, with fewer than 300 similar cases
diagnosed since it was identified in 1998.

M. W. Miller
Colorado Division of Wildlife, Wildlife Research Center, Fort Collins,
Colorado, USA.
Chronic wasting disease (CWD) occurs naturally in North American deer
(Odocoileus spp.), wapiti,
and moose (collectively called “cervids”). CWD presently occurs in scattered
foci throughout North
America, both in the wild and in commercial facilities. CWD is contagious
among its natural hosts,
and epidemics can persist under both captive and free-ranging conditions,
resulting in remarkably
high infection rates. The precise mechanism of contagion remains unclear,
although accumulations of
disease-associated prion protein (PrPCWD) in lymphatic tissues associated
with the gastrointestinal
tract suggest shedding via feces and perhaps saliva. Analyses of epidemic
data suggest that indirect
(animal-environment-animal) transmission may be the dominant force in
epidemic dynamics, and the
CWD agent has been shown to persist in environments contaminated by excreta
or carcass remains
for years. Variation in cellular prion protein appears to influence CWD
pathogenesis, and may provide
a biological mechanism for emergence of variant strains within and among the
four naturally
susceptible species. The long-term implications of CWD for public,
livestock, and wildlife health
remain uncertain. Unfortunately, limitations of existing technology
available to combat prion diseases
make control of CWD ineffective or infeasible under most conditions.

K.M. Green*1, S.R. Browning*1,6, T.S. Seward2, M. Green4, E.A. Hoover5, G.C.
1Department of Microbiology, Immunology and Molecular Genetics, 2Sanders
Brown Center on
Aging,3Department of Neurology, 4UK Transgenic Facility University of
Kentucky, Lexington, Ky USA.
5Department of Microbiology, Immunology and Pathology, Colorado State
University, Fort Collins, Co, USA
6Present address: Department of Infectology, Scripps Research Institute,
Jupiter, Florida, USA
7To whom correspondence should be addressed:[email protected]
* These authors contributed equally
The threats to humans and livestock from interspecies prion transmission are
difficult to assess
because the factors controlling this process remain uncertain. To address
this we have used
transgenic mouse models to understand the roles played by PrP primary
structure, prion strains and
the species specificity of protein X in controlling interspecies prion
infection in the context of cervid
transmission barriers. Cervid prions are of particular concern because
chronic wasting disease
(CWD) of North American and South Korean cervids is the only recognized
prion disease of wild
animals and its increasing geographic range, contagious nature, and
environmental persistence have
raised concerns about prion dissemination and the potential for further
interspecies transmission. We
show that conformational compatibility of PrPSc in a prion strain and PrP
primary structure in a new
host is the most important determinant of interspecies prion transmission
barriers. Although prion
strains can acquire totally new host range properties following heterologous
conversion of PrPP
C in a
new host, the strain-related biochemical properties of PrPSc may remain
relatively stable. We also
show that the cervid PrP polymorphism at residue 132, which is equivalent to
the human PrP 129
polymorphism, is a crucial determinant of cervid prion transmission and has
a profound controlling
effect on PrPSc-related prion strain properties. Our transgenic approaches
modeling trans-species
prion susceptibility in cervids also speak to the possible origins of CWD
since cervid transgenic mice
are also vulnerable, to varying degrees, to sheep scrapie prions, the degree
of susceptibility being
strain related. One particularly well-characterized sheep scrapie isolate,
SSBP/1, caused disease as
efficiently as CWD prions from diseased deer or elk. Finally, while
transmissions in transgenic mice
based on the protein X model of prion propagation produced chimeric prions,
passage of which
resulted in novel cervid prions with an extended host range compared to
CWD-cervid prions, the
unexpected susceptibilities of such mice to CWD and mouse prions are
inconsistent with the
previously hypothesized role of protein X in prion propagation.

C. Johnson1, J. Johnson1, J.P. Vanderloo1, D. Keane2, P. Bochsler2, J.M.
Aiken1, D. McKenzie1
1Department of Animal Health and Biomedical Sciences and 2Wisconsin
Veterinary Diagnostic Laboratory,
University of Wisconsin, Madison, WI, USA [email protected]
The primary sequence of the prion protein affects susceptibility to
transmissible spongiform
encephalopathies (TSE; prion disease) in mice, sheep and humans. The Prnp
sequence of freeranging,
Wisconsin white-tailed deer was determined and the Prnp genotypes of
CWD-positive and -
negative deer compared. Six amino acid (AA) changes were identified; two of
which were located in
pseudogenes. Two alleles, a glutamine to lysine polymorphism at codon 226
and a single
octapeptide repeat insertion into the pseudogene, have not been previously
reported. The
predominant alleles, wild-type (glutamine at AA95, glycine at AA96 and
glutamine at AA226) and a
glycine to serine polymorphism at AA96 (G96S), comprise almost 98% of the
Prnp alleles in the
Wisconsin white-tailed deer population. Comparison of the allelic
frequencies in the CWD-positive
and -negative deer suggests that G96S and a glutamine to histidine
polymorphism at AA 95 (Q95H)
are linked to a reduced susceptibility to CWD. The G96S allele does not,
however, provide complete
resistance, as a CWD-positive G96S/G96S deer was identified. The G96S allele
is also linked to
slower progression of disease in CWD-positive deer based on the deposition
of PrPCWD in the obex
region of the medulla oblongata. To further determine the effect of
variations of the cervid Prnp alleles
on susceptibility, deer with known Prnp genotypes were orally dosed with CWD
inocula prepared from
wild-type/wild-type homozygous animals. The experimentally infected
wild-type/wild-type animals
have succumbed to disease, animals heterozygous for Prnp alleles have not.

R.C. Angers*1, S.R. Browning*1,6, T.S. Seward2, C.J. Sigurdson4, 7, M.W.
Miller5, E.A. Hoover4, G.C. Telling1,
2, 3, 8
1Department of Microbiology, Immunology and Molecular Genetics, University
of Kentucky, Lexington, KY 40536, 2Sanders
Brown Center on Aging, University of Kentucky, 3Department of Neurology,
University of Kentucky, 4Department of
Microbiology, Immunology and Pathology, Colorado State University, 5Colorado
Division of Wildlife, Wildlife Research Center,
Fort Collins, CO 80526; 6 Present address: Department of Infectology,
Scripps Research Institute, 5353 Parkside Drive, RF-2,
Jupiter, Florida, 33458; 7 Present address: Institute of Neuropathology,
University of Zurich, Schmelzbergstr 12, 8091 Zurich,
Switzerland; 8 To whom correspondence should be addressed: e-mail: [email protected]; * These authors contributed equally to
this work
The zoonotic potential of chronic wasting disease (CWD) has become a public
health concern since the
transmission of bovine spongiform encephalopathy (BSE) prions to humans
resulting in variant Creutzfeldt-
Jakob disease (vCJD). Studies in mice, sheep and humans indicated that PrPSc
could be detected in the skeletal
muscles. Since the most probable route of human exposure to CWD is through
consumption or handling of
meat from infected animals, it is important to assess whether skeletal
muscle from affected cervids harbors
prions. CWD-susceptible Tg(CerPrP) mice were intracranially inoculated with
brain and matched skeletal
muscle homogenates from moribund as well as non-infected control deer. Tg
mice inoculated with either brain
or muscle homogenates from CWD-infected deer developed clinical illness with
characteristic prion disease
symptoms and the brains of recipients accumulated cervid PrPSc. The mean
incubation times for animals
inoculated with brain material ranged between 231 and 283 days, whereas mice
receiving muscle tissue had
average incubation periods between 360 and 492 days. Tg mice inoculated with
material from CWD-negative
deer did not develop prion disease or accumulate PrPSc. Brain and muscle
samples used to inoculate Tg(CerPrP)
mice were analyzed for the presence of PrPSc. Brain samples producing the
shortest incubation times had levels
of PrPP
Sc detectable by Western blotting in 25 µg total protein, whereas PrPSc
P was detectable only after sodium
phosphotungstate (NaPTA) precipitation of 0.5 mg for isolates with the
longest incubation periods. No
protease-resistant material was detected in muscle when 50 mg total protein
was precipitated with NaPTA and
analyzed by Western blot. Although a possible role of prion strain
variability cannot currently be dismissed,
these results suggest variable prion titers in the CNS and skeletal muscle
from different CWD-infected deer in
the same phase of disease.