Follow Ups | Post Followup | Back to Discussion Board | VegSource
See spam or
inappropriate posts?
Please let us know.

From: TSS ()
Subject: Re: Public consultation SCENIHR Opinion on the Safety of Human-derived Products with regard to Variant Creutzfeldt- Jakob Disease
Date: December 22, 2005 at 1:52 pm PST

In Reply to: Public consultation SCENIHR Opinion on the Safety of Human-derived Products with regard to Variant Creutzfeldt- Jakob Disease posted by TSS on December 22, 2005 at 1:17 pm:


Coexistence of multiple PrPSc types in individuals with

Creutzfeldt-Jakob disease

Magdalini Polymenidou, Katharina Stoeck, Markus
Glatzel, Martin Vey, Anne Bellon, and Adriano Aguzzi


Background The molecular typing of sporadic
Creutzfeldt-Jakob disease (CJD) is based on the size
and glycoform

ratio of protease-resistant prion protein (PrPSc), and
on PRNP haplotype. On digestion with proteinase K, type
1 and

type 2 PrPSc display unglycosylated core fragments of
21 kDa and 19 kDa, resulting from cleavage around amino

acids 82 and 97, respectively.

Methods We generated anti-PrP monoclonal antibodies to
epitopes immediately preceding the differential proteinase

K cleavage sites. These antibodies, which were
designated POM2 and POM12, recognise type 1, but not
type 2, PrPSc.

Findings We studied 114 brain samples from 70 patients
with sporadic CJD and three patients with variant CJD.

Every patient classified as CJD type 2, and all variant
CJD patients, showed POM2/POM12 reactivity in the

cerebellum and other PrPSc-rich brain areas, with a
typical PrPSc type 1 migration pattern.

Interpretation The regular coexistence of multiple
PrPSc types in patients with CJD casts doubts on the
validity of

electrophoretic PrPSc mobilities as surrogates for
prion strains, and questions the rational basis of
current CJD



The above results set the existing CJD classifications

into debate and introduce interesting questions about

human CJD types. For example, do human prion types

exist in a dynamic equilibrium in the brains of affected

individuals? Do they coexist in most or even all CJD

cases? Is the biochemically identified PrPSc type simply

the dominant type, and not the only PrPSc species?

Published online October 31, 2005

Neuropathology and Applied Neurobiology



, 565-579 doi: 10.1111/j.1365-2990.2005.00697.x

© 2005 Blackwell Publishing Ltd


Blackwell Science, LtdOxford, UKNANNeuropathology and
Applied Neurobiology0305-1846Blackwell Publishing Ltd, 2005


Review article

Phenotypic variability in human prion diseases

J. W. Ironside, D. L. Ritchie and M. W. Head

National Creutzfeldt-Jakob Disease Surveillance Unit,
Division of Pathology, University of Edinburgh,
Edinburgh, UK

J. W. Ironside, D. L. Ritchie and M. W. Head (2005)

Neuropathology and Applied Neurobiology



Phenotypic variability in human prion diseases

Human prion diseases are rare neurodegenerative disorders

that can occur as sporadic, familial or acquired disorders.

Within each of these categories there is a wide range

of phenotypic variation that is not encountered in other

neurodegenerative disorders. The identification of the

prion protein and its key role in the pathogenesis of this

diverse group of diseases has allowed a fuller

of factors that influence disease phenotype. In particular,

the naturally occurring polymorphism at codon 129

in the prion protein gene has a major influence on the

phenotype in sporadic, familial and acquired prion

diseases, although the underlying mechanisms remain

unclear. Recent technical advances have improved our

ability to study the isoforms of the abnormal prion protein

in the brain and in other tissues. This has lead to the

of molecular strain typing, in which different isoforms

of the prion protein are proposed to correspond to

individual strains of the transmissible agent, each with

specific biological properties. In sporadic

disease there are at least six major combinations of codon

129 genotype and prion protein isotype, which appear to

relate to distinctive clinical subgroups of this disease.

However, these relationships are proving to be more complex

than first considered, particularly in cases with more

than a single prion protein isotype in the brain. Further

work is required to clarify these relationships and to

explain the mechanism of neuropathological targeting of

specific brain regions, which accounts for the diversity of

clinical features within human prion diseases.

© 2005 Blackwell Publishing Ltd, Neuropathology and
Applied Neurobiology, 31, 565-579

BSE prions propagate as either variant CJD-like or

sporadic CJD-like prion strains in transgenic mice

expressing human prion protein

The EMBO Journal Vol. 21 No. 23 pp. 6358±6366, 2002

Emmanuel A.Asante, Jacqueline M.Linehan,

Melanie Desbruslais, Susan Joiner,

Ian Gowland, Andrew L.Wood, Julie Welch,

Andrew F.Hill, Sarah E.Lloyd,

Jonathan D.F.Wadsworth and

John Collinge1

MRC Prion Unit and Department of Neurodegenerative Disease,

Institute of Neurology, University College, Queen Square,

London WC1N 3BG, UK

1Corresponding author


Variant Creutzfeldt±Jakob disease (vCJD) has been

recognized to date only in individuals homozygous for

methionine at PRNP codon 129. Here we show that

transgenic mice expressing human PrP methionine

129, inoculated with either bovine spongiform

encephalopathy (BSE) or variant CJD prions, may

develop the neuropathological and molecular phenotype

of vCJD, consistent with these diseases being

caused by the same prion strain. Surprisingly, however,

BSE transmission to these transgenic mice, in

addition to producing a vCJD-like phenotype, can also

result in a distinct molecular phenotype that is

from that of sporadic CJD with PrPSc

type 2. These data suggest that more than one BSEderived

prion strain might infect humans; it is therefore

possible that some patients with a phenotype consistent

with sporadic CJD may have a disease arising

from BSE exposure.


These studies further strengthen the evidence that vCJD

is caused by a BSE-like prion strain. Also, remarkably, the

key neuropathological hallmark of vCJD, the presence of

abundant ¯orid PrP plaques, can be recapitulated on BSE

or vCJD transmission to these mice. However, the most

surprising aspect of the studies was the ®nding that an

alternate pattern of disease can be induced in 129MM

Tg35 mice from primary transmission of BSE, with a

molecular phenotype indistinguishable from that of a

of sporadic CJD. This ®nding has important potential

implications as it raises the possibility that some humans

infected with BSE prions may develop a clinical disease

indistinguishable from classical CJD associated with type 2

PrPSc. This is, in our experience, the commonest molecular

sub-type of sporadic CJD. In this regard, it is of interest

that the reported incidence of sporadic CJD has risen
in the

UK since the 1970s (Cousens et al., 1997). This has been

attributed to improved case ascertainment, particularly as

much of the rise is reported from elderly patients and

similar rises in incidence were noted in other European

countries without reported BSE (Will et al., 1998).

However, it is now clear that BSE is present in many

European countries, albeit at a much lower incidence than

was seen in the UK. While improved ascertainment is

likely to be a major factor in this rise, that some of

additional cases may be related to BSE exposure cannot be

ruled out. It is of interest in this regard that a 2-fold

increase in the reported incidence of sporadic CJD in 2001

has recently been reported for Switzerland, a country that

had the highest incidence of cattle BSE in continental

Europe between 1990 and 2002 (Glatzel et al., 2002). No

epidemiological case±control studies with strati®cation of

CJD cases by molecular sub-type have yet been reported.

It will be important to review the incidence of sporadic

CJD associated with PrPSc type 2 and other molecular

in both BSE-affected and unaffected countries in the

light of these ®ndings. If human BSE prion infection can

result in propagation of type 2 PrPSc, it would be expected

that such cases would be indistinguishable on clinical,

pathological and molecular criteria from classical CJD. It

may also be expected that such prions would behave

biologically like those isolated from humans with sporadic

CJD with type 2 PrPSc. The transmission properties of

prions associated with type 2 PrPSc from BSE-inoculated

129MM Tg35 mice are being investigated by serial


We consider these data inconsistent with contamination

of some of the 129MM Tg35 mice with sporadic CJD

prions. These transmission studies were performed according

to rigorous biosafety protocols for preparation of

inocula and both the inoculation and care of mice, which

are all uniquely identi®ed by sub-cutaneous transponders.

However, crucially, the same BSE inocula have been used

on 129VV Tg152 and 129MM Tg45 mice, which are

highly sensitive to sporadic CJD but in which such

transmissions producing type 2 PrPSc were not observed.

Furthermore, in an independent experiment, separate

inbred lines of wild-type mice, which are highly resistant

to sporadic CJD prions, also propagated two distinctive

PrPSc types on challenge with either BSE or vCJD. No

evidence of spontaneous prion disease or PrPSc has been

seen in groups of uninoculated or mock-inoculated aged

129MM Tg35 mice.

While distinctive prion isolates have been derived from

BSE passage in mice previously (designated 301C and

301V), these, in contrast to the data presented here, are

propagated in mice expressing different prion proteins

(Bruce et al., 1994). It is unclear whether our ®ndings

indicate the existence of more than one prion strain in

individual cattle with BSE, with selection and preferential

replication of distinct strains by different hosts, or that

`mutation' of a unitary BSE strain occurs in some types of

host. Western blot analysis of single BSE isolates has not

shown evidence of the presence of a proportion of

monoglycosylated dominant PrPSc type in addition to the

diglycosylated dominant pattern (data not shown).

Extensive strain typing of large numbers of individual

BSE-infected cattle either by biological or molecular

methods has not been reported.

Presumably, the different genetic background of the

different inbred mouse lines is crucial in determining

which prion strain propagates on BSE inoculation. The

transgenic mice described here have a mixed genetic

background with contributions from FVB/N, C57BL/6 and

129Sv inbred lines; each mouse will therefore have a

different genetic background. This may explain the

differing response of individual 129MM Tg35 mice, and

the difference between 129MM Tg35 and 129MM Tg45

mice, which are, like all transgenic lines, populations

derived from single founders. Indeed, the consistent

distinctive strain propagation in FVB and C57BL/6 versus

SJL and RIIIS lines may allow mapping of genes relevant

to strain selection and propagation, and these studies
are in


That different prion strains can be consistently isolated

in different inbred mouse lines challenged with BSE

prions argues that other species exposed to BSE may

develop prion diseases that are not recognizable as being

caused by the BSE strain by either biological or molecular

strain typing methods. As with 129MM Tg35 mice, the

prions replicating in such transmissions may be

from naturally occurring prion strains. It

remains of considerable concern whether BSE has transmitted

to, and is being maintained in, European sheep

¯ocks. Given the diversity of sheep breeds affected by

scrapie, it has to be considered that some sheep might have

become infected with BSE, but propagated a distinctive

strain type indistinguishable from those of natural sheep

scrapie. ...

The EMBO Journal Vol. 21 No. 23 pp. 6358±6366, 2002

6358 ãEuropean Molecular Biology Organization

Personal Communication

-------- Original Message --------

Subject: re-BSE prions propagate as

either variant CJD-like or sporadic CJD Date: Thu, 28
Nov 2002 10:23:43

-0000 From: "Asante, Emmanuel A" To:

Dear Terry,

I have been asked by Professor Collinge to respond to
your request. I am

a Senior Scientist in the MRC Prion Unit and the lead
author on the

paper. I have attached a pdf copy of the paper for your
attention. Thank

you for your interest in the paper.

In respect of your first question, the simple answer
is, yes. As you

will find in the paper, we have managed to associate
the alternate

phenotype to type 2 PrPSc, the commonest sporadic CJD.

It is too early to be able to claim any further
sub-classification in

respect of Heidenhain variant CJD or Vicky Rimmer's
version. It will

take further studies, which are on-going, to establish
if there are

sub-types to our initial finding which we are now
reporting. The main

point of the paper is that, as well as leading to the
expected new

variant CJD phenotype, BSE transmission to the
129-methionine genotype

can lead to an alternate phenotype which is
indistinguishable from type

2 PrPSc.

I hope reading the paper will enlighten you more on the
subject. If I

can be of any further assistance please to not hesitate
to ask. Best wishes.

Emmanuel Asante

<> ____________________________________

Dr. Emmanuel A Asante MRC Prion Unit & Neurogenetics
Dept. Imperial

College School of Medicine (St. Mary's) Norfolk Place,

Tel: +44 (0)20 7594 3794 Fax: +44 (0)20 7706 3272 email: (until 9/12/02)

New e-mail: (active from now)


Human Prion Protein with

Valine 129 Prevents Expression

of Variant CJD Phenotype

Jonathan D. F. Wadsworth, Emmanuel A. Asante,

Melanie Desbruslais, Jacqueline M. Linehan, Susan Joiner,

Ian Gowland, Julie Welch, Lisa Stone, Sarah E. Lloyd,

Andrew F. Hill,* Sebastian Brandner, John Collinge.

Variant Creutzfeldt-Jakob disease (vCJD) is a unique
and highly distinctive

clinicopathological and molecular phenotype of human
prion disease

associated with infection with bovine spongiform
encephalopathy (BSE)-like

prions. Here, we found that generation of this
phenotype in transgenic mice

required expression of human prion protein (PrP) with
methionine 129.

Expression of human PrP with valine 129 resulted in a
distinct phenotype and,

remarkably, persistence of a barrier to transmission of
BSE-derived prions on

subpassage. Polymorphic residue 129 of human PrP
dictated propagation of

distinct prion strains after BSE prion infection. Thus,
primary and secondary

human infection with BSE-derived prions may result in
sporadic CJD-like or

novel phenotypes in addition to vCJD, depending on the
genotype of the prion

source and the recipient.



Characterization of two distinct prion strains

derived from bovine spongiform encephalopathy

transmissions to inbred mice

Sarah E. Lloyd, Jacqueline M. Linehan, Melanie Desbruslais,

Susan Joiner, Jennifer Buckell, Sebastian Brandner,

Jonathan D. F. Wadsworth and John Collinge


John Collinge

MRC Prion Unit and Department of Neurodegenerative
Disease, Institute of Neurology,

University College, London WC1N 3BG, UK

Received 9 December 2003

Accepted 27 April 2004

Distinct prion strains can be distinguished by
differences in incubation period, neuropathology

and biochemical properties of disease-associated prion
protein (PrPSc) in inoculated mice.

Reliable comparisons of mouse prion strain properties
can only be achieved after passage in

genetically identical mice, as host prion protein
sequence and genetic background are known

to modulate prion disease phenotypes. While multiple
prion strains have been identified in

sheep scrapie and Creutzfeldt-Jakob disease, bovine
spongiform encephalopathy (BSE) is

thought to be caused by a single prion strain. Primary
passage of BSE prions to different lines

of inbred mice resulted in the propagation of two
distinct PrPSc types, suggesting that two

prion strains may have been isolated. To investigate
this further, these isolates were

subpassaged in a single line of inbred mice (SJL) and
it was confirmed that two distinct prion

strains had been identified. MRC1 was characterized by
a short incubation time (110±3 days),

a mono-glycosylated-dominant PrPSc type and a
generalized diffuse pattern of PrP-immunoreactive

deposits, while MRC2 displayed a much longer incubation
time (155±1 days),

a di-glycosylated-dominant PrPSc type and a distinct
pattern of PrP-immunoreactive deposits

and neuronal loss. These data indicate a crucial
involvement of the host genome in modulating

prion strain selection and propagation in mice. It is
possible that multiple disease phenotypes

may also be possible in BSE prion infection in humans
and other animals.


Journal of General Virology (2004), 85, 2471-2478 DOI

Medical Sciences
Identification of a second bovine amyloidotic
spongiform encephalopathy: Molecular similarities with
sporadic Creutzfeldt-Jakob disease

Cristina Casalone *, Gianluigi Zanusso , Pierluigi
Acutis *, Sergio Ferrari , Lorenzo Capucci , Fabrizio
Tagliavini ¶, Salvatore Monaco ||, and Maria Caramelli *

*Centro di Referenza Nazionale per le Encefalopatie
Animali, Istituto Zooprofilattico Sperimentale del
Piemonte, Liguria e Valle d'Aosta, Via Bologna, 148,
10195 Turin, Italy; Department of Neurological and
Visual Science, Section of Clinical Neurology,
Policlinico G.B. Rossi, Piazzale L.A. Scuro, 10, 37134
Verona, Italy; Istituto Zooprofilattico Sperimentale
della Lombardia ed Emilia Romagna, Via Bianchi, 9,
25124 Brescia, Italy; and ¶Istituto Nazionale
Neurologico "Carlo Besta," Via Celoria 11, 20133 Milan,

Edited by Stanley B. Prusiner, University of
California, San Francisco, CA, and approved December
23, 2003 (received for review September 9, 2003)

Transmissible spongiform encephalopathies (TSEs), or
prion diseases, are mammalian neurodegenerative
disorders characterized by a posttranslational
conversion and brain accumulation of an insoluble,
protease-resistant isoform (PrPSc) of the host-encoded
cellular prion protein (PrPC). Human and animal TSE
agents exist as different phenotypes that can be
biochemically differentiated on the basis of the
molecular mass of the protease-resistant PrPSc
fragments and the degree of glycosylation.
Epidemiological, molecular, and transmission studies
strongly suggest that the single strain of agent
responsible for bovine spongiform encephalopathy (BSE)
has infected humans, causing variant Creutzfeldt-Jakob
disease. The unprecedented biological properties of the
BSE agent, which circumvents the so-called "species
barrier" between cattle and humans and adapts to
different mammalian species, has raised considerable
concern for human health. To date, it is unknown
whether more than one strain might be responsible for
cattle TSE or whether the BSE agent undergoes
phenotypic variation after natural transmission. Here
we provide evidence of a second cattle TSE. The
disorder was pathologically characterized by the
presence of PrP-immunopositive amyloid plaques, as
opposed to the lack of amyloid deposition in typical
BSE cases, and by a different pattern of regional
distribution and topology of brain PrPSc accumulation.
In addition, Western blot analysis showed a PrPSc type
with predominance of the low molecular mass glycoform
and a protease-resistant fragment of lower molecular
mass than BSE-PrPSc. Strikingly, the molecular
signature of this previously undescribed bovine PrPSc
was similar to that encountered in a distinct subtype
of sporadic Creutzfeldt-Jakob disease.


C.C. and G.Z. contributed equally to this work.

||To whom correspondence should be addressed.

E-mail: .


Phenotypic Similarities Between BASE and sCJD. The

of CJD brains was initially demonstrated in primates
(27), and

classification of atypical cases as CJD was based on
this property

(28). To date, no systematic studies of strain typing
in sCJD have

been provided, and classification of different subtypes
is based

on clinical, neuropathological, and molecular features
(the polymorphic

PRNP codon 129 and the PrPSc glycotype) (8, 9, 15, 19).

The importance of molecular PrPSc characterization in

the identity of TSE strains is underscored by several

showing that the stability of given disease-specific
PrPSc types is

maintained upon experimental propagation of sCJD, familial

CJD, and vCJD isolates in transgenic PrP-humanized mice (8,

29). Similarly, biochemical properties of BSE- and

PrPSc molecules remain stable after passage to mice

expressing bovine PrP (30). Recently, however, it has been

reported that PrP-humanized mice inoculated with BSE

may also propagate a distinctive PrPSc type, with a

dominant'' pattern and electrophoretic mobility of the

unglycosylated fragment slower than that of vCJD and
BSE (31).

Strikingly, this PrPSc type shares its molecular
properties with the

a PrPSc molecule found in classical sCJD. This
observation is at

variance with the PrPSc type found in MV2 sCJD cases and in

cattle BASE, showing a monoglycosylated-dominant
pattern but

faster electrophoretic mobility of the
protease-resistant fragment

as compared with BSE. In addition to molecular properties

of PrPSc, BASE and MV2 sCJD share a distinctive pattern of

intracerebral PrP deposition, which occurs as
plaque-like and

amyloid-kuru plaques. Differences were, however,
observed in

the regional distribution of PrPSc. While inMV2 sCJD
cases the

largest amounts of PrPSc were detected in the cerebellum,

brainstem, and striatum, in cattle BASE these areas
were less

involved and the highest levels of PrPSc were recovered
from the

thalamus and olfactory regions.

In conclusion, decoding the biochemical PrPSc signature of

individual human and animal TSE strains may allow the

of potential risk factors for human disorders with

unknown etiology, such as sCJD. However, although BASE and

sCJD share several characteristics, caution is dictated
in assessing

a link between conditions affecting two different mammalian

species, based on convergent biochemical properties of

PrPSc types. Strains of TSE agents may be better

characterized upon passage to transgenic mice. In the

until this is accomplished, our present findings
suggest a strict

epidemiological surveillance of cattle TSE and sCJD
based on

molecular criteria.

Published online before print March 20, 2001,

Adaptation of the bovine spongiform encephalopathy
agent to primates and comparison with Creutzfeldt-
Jakob disease: Implications for human health
Corinne Ida Lasmézas*,, Jean-Guy Fournier*, Virginie
Nouvel*, Hermann Boe*, Domíníque Marcé*, François
Lamoury*, Nicolas Kopp, Jean-Jacques Hauw§, James
Ironside¶, Moira Bruce, Dominique Dormont*, and
Jean-Philippe Deslys*

* Commissariat à l'Energie Atomique, Service de
Neurovirologie, Direction des Sciences du
Vivant/Département de Recherche Medicale, Centre de
Recherches du Service de Santé des Armées 60-68, Avenue
du Général Leclerc, BP 6, 92 265 Fontenay-aux-Roses
Cedex, France; Hôpital Neurologique Pierre Wertheimer,
59, Boulevard Pinel, 69003 Lyon, France; § Laboratoire
de Neuropathologie, Hôpital de la Salpêtrière, 83,
Boulevard de l'Hôpital, 75013 Paris, France; ¶
Creutzfeldt-Jakob Disease Surveillance Unit, Western
General Hospital, Crewe Road, Edinburgh EH4 2XU, United
Kingdom; and Institute for Animal Health,
Neuropathogenesis Unit, West Mains Road, Edinburgh EH9
3JF, United Kingdom

Edited by D. Carleton Gajdusek, Centre National de la
Recherche Scientifique, Gif-sur-Yvette, France, and
approved December 7, 2000 (received for review October
16, 2000)


There is substantial scientific evidence to support the
notion that bovine spongiform encephalopathy (BSE) has
contaminated human beings, causing variant
Creutzfeldt-Jakob disease (vCJD). This disease has
raised concerns about the possibility of an iatrogenic
secondary transmission to humans, because the
biological properties of the primate-adapted BSE agent
are unknown. We show that (i) BSE can be transmitted
from primate to primate by intravenous route in 25
months, and (ii) an iatrogenic transmission of vCJD to
humans could be readily recognized pathologically,
whether it occurs by the central or peripheral route.
Strain typing in mice demonstrates that the BSE agent
adapts to macaques in the same way as it does to humans
and confirms that the BSE agent is responsible for vCJD
not only in the United Kingdom but also in France. The
agent responsible for French iatrogenic growth
hormone-linked CJD taken as a control is very different
from vCJD but is similar to that found in one case of
sporadic CJD and one sheep scrapie isolate. These data
will be key in identifying the origin of human cases of
prion disease, including accidental vCJD transmission,
and could provide bases for vCJD risk assessment.


Characterization of the CJD and Scrapie Strains.
Controls were set up by transmitting one French and one
U.S. scrapie isolate from ruminants as well as French
sCJD and iCJD cases from humans. None of these revealed
a lesion profile or transmission characteristics
similar or close to those of BSE or vCJD, respectively,
thus extending to the present French scrapie isolate
the previous observation that the BSE agent was
different from all known natural scrapie strains (4, 24).

The lesion profiles of sCJD and iCJD differed only
slightly in severity of the lesions, but not in shape
of the profile, revealing the identity of the causative
agents. One of us reported the absence of similarity
between sCJD (six cases) and U.K. scrapie (eight cases)
in transmission characteristics in mice (4). Herein, we
made the striking observation that the French natural
scrapie strain (but not the U.S. scrapie strain) has
the same lesion profile and transmission times in
C57BL/6 mice as do the two human TSE strains studied.
This strain "affiliation" was confirmed biochemically.
There is no epidemiological evidence for a link between
sheep scrapie and the occurrence of CJD in humans (25).
However, such a link, if it is not a general rule,
would be extremely difficult to establish because of
the very low incidence of CJD as well as the existence
of different isolates in humans and multiple strains in
scrapie. Moreover, scrapie is transmissible to nonhuman
primates (26). Thus, there is still a possibility that
in some instances TSE strains infecting humans do share
a common origin with scrapie, as pointed out by our


1: 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.

PMID: 6997404


Interspecies Transmission of Chronic Wasting Disease
Prions to

Squirrel Monkeys (Saimiri sciureus)

Richard F. Marsh,1? Anthony E. Kincaid,2 Richard A.
Bessen,3 and Jason C. Bartz4*

Department of Animal Health and Biomedical Sciences,
University of Wisconsin, Madison 537061; Department of

Physical Therapy2 and Department of Medical
Microbiology and Immunology,4 Creighton University, Omaha,

Nebraska 68178; and Department of Veterinary Molecular
Biology, Montana

State University, Bozeman, Montana 597183

Received 3 May 2005/Accepted 10 August 2005

Chronic wasting disease (CWD) is an emerging prion
disease of deer and elk. The risk of CWD transmission

to humans following exposure to CWD-infected tissues is
unknown. To assess the susceptibility of nonhuman

primates to CWD, two squirrel monkeys were inoculated
with brain tissue from a CWD-infected mule deer. The

CWD-inoculated squirrel monkeys developed a progressive
neurodegenerative disease and were euthanized at

31 and 34 months postinfection. Brain tissue from the
CWD-infected squirrel monkeys contained the abnormal

isoform of the prion protein, PrP-res, and displayed
spongiform degeneration. This is the first reported

transmission of CWD to primates.


JOURNAL OF VIROLOGY, Nov. 2005, p. 13794-13796 Vol.
79, No. 21


Copyright © 2005, American Society for Microbiology.
All Rights Reserved.


The EMBO Journal, Vol. 19, No. 17 pp. 4425-4430, 2000
© European Molecular Biology Organization

Evidence of a molecular barrier limiting
susceptibility of humans, cattle and sheep to
chronic wasting disease

G.J. Raymond1, A. Bossers2, L.D. Raymond1, K.I. O?Rourke3,
L.E. McHolland4, P.K. Bryant III4, M.W. Miller5, E.S.
Williams6, M.
and B. Caughey1,7

1NIAID/NIH Rocky Mountain Laboratories, Hamilton, MT
3USDA/ARS/ADRU, Pullman, WA 99164-7030, 4USDA/ARS/ABADRL,
Laramie, WY 82071, 5Colorado Division of Wildlife,
Wildlife Research
Center, Fort Collins, CO 80526-2097, 6Department of
Veterinary Sciences,
University of Wyoming, Laramie, WY 82070, USA and
Institute for Animal Science and Health, Lelystad, The
7Corresponding author e-mail: Received
June 7, 2000;
revised July 3, 2000; accepted July 5, 2000.


Chronic wasting disease (CWD) is a transmissible
spongiform encephalopathy (TSE) of deer and elk,
and little is known about its transmissibility to other
species. An important factor controlling
interspecies TSE susceptibility is prion protein (PrP)
homology between the source and recipient
species/genotypes. Furthermore, the efficiency with which
the protease-resistant PrP (PrP-res) of one
species induces the in vitro conversion of the normal PrP
(PrP-sen) of another species to the
protease-resistant state correlates with the cross-species
transmissibility of TSE agents. Here we
show that the CWD-associated PrP-res (PrPCWD) of cervids
readily induces the conversion of recombinant cervid
molecules to the protease-resistant state in accordance
with the known transmissibility of CWD between cervids.
In contrast,
PrPCWD-induced conversions of human and bovine PrP-sen
much less efficient, and conversion of ovine PrP-sen was
intermediate. These results demonstrate a barrier at the
molecular level that should limit the susceptibility of
these non-cervid
species to CWD.


Clearly, it is premature to draw firm conclusions about
passing naturally into humans, cattle and sheep, but
the present
results suggest that CWD transmissions to humans would
be as
limited by PrP incompatibility as transmissions of BSE
or sheep
scrapie to humans. Although there is no evidence that
scrapie has affected humans, it is likely that BSE has
caused variant
CJD in 74 people (definite and probable variant CJD
cases to
date according to the UK CJD Surveillance Unit). Given the
presumably large number of people exposed to BSE
the susceptibility of humans may still be very low
compared with
cattle, which would be consistent with the relatively
conversion of human PrP-sen by PrPBSE. Nonetheless, since
humans have apparently been infected by BSE, it would
seem prudent
to take reasonable measures to limit exposure of humans
(as well as sheep and cattle) to CWD infectivity as has
recommended for other animal TSEs.


Neurology 1999;52:1757
© 1999 American Academy of Neurology


Expedited Publication

A subtype of sporadic prion disease mimicking fatal
familial insomnia
P. Parchi, MD, S. Capellari, MD, S. Chin, MD, PhD, H.
B. Schwarz, MD, N. P. Schecter, MD, J. D. Butts, MD, P.
Hudkins, MD, D. K. Burns MD, J. M. Powers, MD and P.
Gambetti, MD

Volume 43 Issue 12 Page 1687 - December 2003

Similar levels of infectivity in the blood of mice
infected with
human-derived vCJD and GSS strains of transmissible
Larisa Cervenakova, Oksana Yakovleva, Carroll McKenzie,
Kolchinsky, Lisa McShane, William N. Drohan, and Paul Brown


The possible transmission of variant CJD (vCJD) through
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
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.






Summary of SEAC's discussion on the second presumed
case of blood

transfusion-associated infection with vCJD

Transmission of Creutzfeldt-Jakob Disease from Blood
and Urine Into Mice

The Lancet, November 9, 1985

Sir,--Professor Manuelidis and his colleagues (Oct 19,
p896) report
transmission to animals of Creutzfeldt-Jakob disease
(CJD) from the
buffy coat from two patients. We also transmitted the
disease from
whole blood samples of a patient (and of mice) infected
with CJD.1
Brain, Cornea, and urine from this patient were also
infectious, and
the clinicopathological findings2 are summarised as

A 70-year-old man was noted to have a slowing of speech
and writing
and some disorientation, all of which progressed
rapidly. Decorticate
rigidity, forced grasping, positive snout reflex, and
appeared within 2 months. Electroencephalogram revealed
periodic synchronous discharge, and he died of
pneumonia and upper
gastrointestinal haemorrhage, about 3 months after
onset of the
symptoms. The Brain weighed 1290g and showed severe
changes diagnostic of CJD, including spongiform change,
loss of
nerve cells, and diffuse proliferation of astrocytes.
There were no
inflammatory cells, microglia, neurofibrillary tangles,
amyloid plaques, although virus-like particles were
detected by
electron microscopy.

Results of innoculation in Mice

Inocula NO* Incubation period (days)+
Brain 7/10 (4) 789 (+ or - 112)
Cornea 1/6 (0) 1037
Blood 2/13 (0) 1080 (+ or - 69)
Urine 5/10 (1) 880 (+ or - 55)
CSF 0/10

* Number of mice with CJD change/number examined
Number with amyloid plaques shown in parentheses.

+ means + or - SD

Samples were taken aseptically at necropsy. 10% crude
of brain and cornea in saline, whole blood (after
crushing a clot),
and untreated CSF and urine were innoculated
intracerebrally into
CF1 strain mice (20 ul per animal). Some mice showed
bradykinesia, rigidity of the body and tail, and
sometimes tremor
after long incubation periods. Tissues obtained after
the animal
died (or was killed) were studied histologically
(table). Animals
infected by various inocula showed common pathological
consisting of severe spongiform changes, glial
proliferation, and
a moderate loss of nerve cells. A few mice inoculated
with brain
tissue or urine had the same amyloid plaques found in
patients and
animals with CJD.3

In our long-term experiments, inoculating materials
taken from
twenty patients with CJD or
disease (GSS) into rodents, positive results were
obtained in
seventeen cases, including this patient. Brain tissue
the disease most frequently within the shortes
incubation period,
except for one case where the lymph node was the most
Transmission through the cornea has been noted in man4
and in
guineapigs.5 Whole blood samples taken from three
patients were
inoculated and a positive transmission occured only in
the case
recorded here. Mouse-to-mouse transmission through blood
inoculation was successful after a mean incubation
period of 365
days.1 Transmission through urine was positive in this
only, and negative in one other patient and in many
infected animals.
Transmission through the CSF from eight patients was
negative, yet
transmission via the CSF of infected rats was positive.1

As viraemia has been proved in guineapigs,6 mice,1,7
and lately
in patients with CJD, blood for transfusion or blood
products for
medical use must be tested for unconventional
pathogens. For this
purpose, we inoculated blood products inot rodents.8
pathogen was not found in the products examined.
However, this
approach takes too long to be of practical value. More
methods must be developed to detect pathogens and to
them from blood. One proposal9 is to apply membrane
filtration to
the pruification protocol of human growth hormone
suspected of
being contaminated with CJD. Similar methods are needed
for blood

Department of Neuropathology,
Neurological Institute,
Faculty of Medicine,
Kyushu University,
Fukuoka812, Japan


1. Tateishi J, Sato Y, Kaga M. Doi H, Ohta M.
Experimental transmission
of human subacute spongiform encephalopathy to small
rodents 1: Clinical and histological observations.
Acta Neuropathol (Berl) 1980; 51: 127.

2. Shibayama Y, Sakaguchi Y, Nakata K, et al,
disease with demonstration of virus-like particles.
Acta pathol Jpn 1982;32: 695.

3. Tateishi J, Nagara H, Hikita K, Sato Y. Amyloid
plaques in the
brains of mice with Creutzfeldt-Jakob disease.
Ann Neurol 1984; 15: 278.

4. Duffy P, Wolf J, Colings G, DeVoe AG, Streeten B,
Cowen D.
Possible person-to-person transmission of
Creutzfeldt-Jakob disease.
N Engl J Med 1974; 290: 692.

5. Manuelidis EE, Angelo JN, Gorgacz EJ, Kim JH,
Manuelidis L.
Experimental Creutzfeldt-Jakob disease transmitted via
the eye
with infected cornea. N Engl J Med 1977; 296: 1334.

6. Manuelidis EE, Gorgacz EJ, Manuelidis L. Viremia in
Creutzfeldt-Jakob disease. Science 1978: 200: 1069.

7. Kuroda Y, Gibbs CJ Jr, Amyx HL, Gajdusek DC.
Creutzfeldt-Jakob disease in mice. Persistent viremiam and
preferential replication of virus in low-density
Infect Immun 1983; 41: 154.

8. Tateishi J, Tsuji S. Unconventional pathogens
causing spongiform
encephalopathis absent in blood products. J Med Virol
1985; 15: 11.

9. Tateishi J, Kitamoto T, Hiratani H.
Creutzfeldt-Jakob disease
pathogen in growth hormone preparations is eliminatable.
Lancet (in press).


Volume 3, Number 8 01 August 2003


Tracking spongiform encephalopathies in North America

Xavier Bosch

My name is Terry S Singeltary Sr, and I live in
Bacliff, Texas. I lost

my mom to hvCJD (Heidenhain variant CJD) and have been
searching for

answers ever since. What I have found is that we have
not been told the

truth. CWD in deer and elk is a small portion of a much
bigger problem.

49-year-old Singeltary is one of a number of people who
have remained

largely unsatisfied after being told that a close
relative died from a

rapidly progressive dementia compatible with spontaneous

Creutzfeldt-Jakob disease (CJD). So he decided to
gather hundreds of

documents on transmissible spongiform encephalopathies
(TSE) and

realised that if Britons could get variant CJD from
bovine spongiform

encephalopathy (BSE), Americans might get a similar
disorder from

chronic wasting disease (CWD) the relative of mad cow
disease seen among

deer and elk in the USA. Although his feverish search
did not lead him

to the smoking gun linking CWD to a similar disease in
North American

people, it did uncover a largely disappointing situation.

Singeltary was greatly demoralised at the few attempts
to monitor the

occurrence of CJD and CWD in the USA. Only a few
states have made CJD

reportable. Human and animal TSEs should be reportable
nationwide and

internationally, he complained in a letter to the
Journal of the

American Medical Association (JAMA 2003; 285: 733). I
hope that the CDC

does not continue to expect us to still believe that
the 85% plus of all

CJD cases which are sporadic are all spontaneous,
without route or source.

Until recently, CWD was thought to be confined to the
wild in a small

region in Colorado. But since early 2002, it has been
reported in other

areas, including Wisconsin, South Dakota, and the
Canadian province of

Saskatchewan. Indeed, the occurrence of CWD in states
that were not

endemic previously increased concern about a widespread
outbreak and

possible transmission to people and cattle.

To date, experimental studies have proven that the CWD
agent can be

transmitted to cattle by intracerebral inoculation and
that it can cross

the mucous membranes of the digestive tract to initiate
infection in

lymphoid tissue before invasion of the central nervous
system. Yet the

plausibility of CWD spreading to people has remained

Part of the problem seems to stem from the US
surveillance system. CJD

is only reported in those areas known to be endemic
foci of CWD.

Moreover, US authorities have been criticised for not
having performed

enough prionic tests in farm deer and elk.

Although in November last year the US Food and Drug

issued a directive to state public-health and
agriculture officials

prohibiting material from CWD-positive animals from
being used as an

ingredient in feed for any animal species,
epidemiological control and

research in the USA has been quite different from the
situation in the

UK and Europe regarding BSE.

Getting data on TSEs in the USA from the government is
like pulling

teeth, Singeltary argues. You get it when they want
you to have it,

and only what they want you to have.

Norman Foster, director of the Cognitive Disorders
Clinic at the

University of Michigan (Ann Arbor, MI, USA), says
that current

surveillance of prion disease in people in the USA is
inadequate to

detect whether CWD is occurring in human beings;
adding that, the

cases that we know about are reassuring, because they
do not suggest the

appearance of a new variant of CJD in the USA or
atypical features in

patients that might be exposed to CWD. However, until
we establish a

system that identifies and analyses a high proportion
of suspected prion

disease cases we will not know for sure. The USA should
develop a

system modelled on that established in the UK, he
points out.

Ali Samii, a neurologist at Seattle VA Medical Center
who recently

reported the cases of three hunters two of whom were
friends who died

from pathologically confirmed CJD, says that at present
there are

insufficient data to claim transmission of CWD into
humans; adding that

[only] by asking [the questions of venison consumption
and deer/elk

hunting] in every case can we collect suspect cases and
look into the

plausibility of transmission further. Samii argues that
by making both

doctors and hunters more aware of the possibility of
prions spreading

through eating venison, doctors treating hunters with
dementia can

consider a possible prion disease, and doctors treating
CJD patients

will know to ask whether they ate venison.

CDC spokesman Ermias Belay says that the CDC will not
be investigating

the [Samii] cases because there is no evidence that the
men ate

CWD-infected meat. He notes that although the
likelihood of CWD

jumping the species barrier to infect humans cannot be
ruled out 100%

and that [we] cannot be 100% sure that CWD does not
exist in humans&

the data seeking evidence of CWD transmission to humans
have been very


>>>he complained in a letter to the Journal of the
American Medical

Association (JAMA 2003; 285: 733). I hope that the CDC
does not

continue to expect us to still believe that the 85%
plus of all CJD

cases which are sporadic are all spontaneous, without
route or source.<<<

actually, that quote was from a more recent article in
the Journal of

Neurology (see below), not the JAMA article...

Re: RE-Monitoring the occurrence of emerging forms of

disease in the United States

Email Terry S. Singeltary:

I lost my mother to hvCJD (Heidenhain Variant CJD). I
would like to

comment on the CDC's attempts to monitor the occurrence
of emerging

forms of CJD. Asante, Collinge et al [1] have reported
that BSE

transmission to the 129-methionine genotype can lead to
an alternate

phenotype that is indistinguishable from type 2 PrPSc,
the commonest

sporadic CJD. However, CJD and all human TSEs are not

nationally. CJD and all human TSEs must be made
reportable in every

state and internationally. I hope that the CDC does not
continue to

expect us to still believe that the 85%+ of all CJD
cases which are

sporadic are all spontaneous, without route/source. We
have many TSEs in

the USA in both animal and man. CWD in deer/elk is
spreading rapidly and

CWD does transmit to mink, ferret, cattle, and squirrel
monkey by

intracerebral inoculation. With the known incubation
periods in other

TSEs, oral transmission studies of CWD may take much
longer. Every

victim/family of CJD/TSEs should be asked about route
and source of this

agent. To prolong this will only spread the agent and
needlessly expose

others. In light of the findings of Asante and Collinge
et al, there

should be drastic measures to safeguard the medical and
surgical arena

from sporadic CJDs and all human TSEs. I only ponder
how many sporadic

CJDs in the USA are type 2 PrPSc?

Vol. 285 No. 6, February 14, 2001

Diagnosis and Reporting of Creutzfeldt-Jakob Disease

Since this article does not have an abstract, we have
provided the first 150 words of the full text and any
section headings.

To the Editor: In their Research Letter, Dr Gibbons and
colleagues1 reported that the annual US death rate due
to Creutzfeldt-Jakob disease (CJD) has been stable
since 1985. These estimates, however, are based only on
reported cases, and do not include misdiagnosed or
preclinical cases. It seems to me that misdiagnosis
alone would drastically change these figures. An
unknown number of persons with a diagnosis of Alzheimer
disease in fact may have CJD, although only a small
number of these patients receive the postmortem
examination necessary to make this diagnosis.
Furthermore, only a few states have made CJD
reportable. Human and animal transmissible spongiform
encephalopathies should be reportable nationwide and

Terry S. Singeltary, Sr
Bacliff, Tex

1. Gibbons RV, Holman RC, Belay ED, Schonberger LB.
Creutzfeldt-Jakob disease in the United States:
1979-1998. JAMA. 2000;284:2322-2323. FULL TEXT

To the Editor: At the time of my mother's death,
various diagnoses were advanced such as "rapid
progressive Alzheimer disease," psychosis, and
dementia. Had I not persisted and personally sought and
arranged . . . [Full Text of this Article]

Full Text
Diagnosis and Reporting of Creutzfeldt-Jakob Disease
Singeltary, Sr et al.
JAMA.2001; 285: 733-734.


Animal Prion Diseases Relevant to Humans (unknown types?)
Thu Oct 27, 2005 12:05

About Human Prion Diseases /
Animal Prion Diseases Relevant to Humans

Bovine Spongiform Encephalopathy (BSE) is a prion
disease of cattle. Since 1986, when BSE was recognized,
over 180,000 cattle in the UK have developed the
disease, and approximately one to three million are
likely to have been infected with the BSE agent, most
of which were slaughtered for human consumption before
developing signs of the disease. The origin of the
first case of BSE is unknown, but the epidemic was
caused by the recycling of processed waste parts of
cattle, some of which were infected with the BSE agent
and given to other cattle in feed. Control measures
have resulted in the consistent decline of the epidemic
in the UK since 1992. Infected cattle and feed exported
from the UK have resulted in smaller epidemics in other
European countries, where control measures were applied

Compelling evidence indicates that BSE can be
transmitted to humans through the consumption of prion
contaminated meat. BSE-infected individuals eventually
develop vCJD with an incubation time believed to be on
average 10 years. As of November 2004, three cases of
BSE have been reported in North America. One had been
imported to Canada from the UK, one was grown in
Canada, and one discovered in the USA but of Canadian
origin. There has been only one case of vCJD reported
in the USA, but the patient most likely acquired the
disease in the United Kingdom. If current control
measures intended to protect public and animal health
are well enforced, the cattle epidemic should be
largely under control and any remaining risk to humans
through beef consumption should be very small. (For
more details see Smith et al. British Medical Bulletin,
66: 185. 2003.)

Chronic Wasting Disease (CWD) is a prion disease of elk
and deer, both free range and in captivity. CWD is
endemic in areas of Colorado, Wyoming, and Nebraska,
but new foci of this disease have been detected in
Nebraska, South Dakota, New Mexico, Wisconsin,
Mississippi Kansas, Oklahoma, Minnesota, Montana, and
Canada. Since there are an estimated 22 million elk and
deer in the USA and a large number of hunters who
consume elk and deer meat, there is the possibility
that CWD can be transmitted from elk and deer to
humans. As of November 2004, the NPDPSC has examined 26
hunters with a suspected prion disease. However, all of
them appeared to have either typical sporadic or
familial forms of the disease. The NPDPSC coordinates
with the Centers for Disease Control and state health
departments to monitor cases from CWD-endemic areas.
Furthermore, it is doing experimental research on CWD
transmissibility using animal models. (For details see
Sigurdson et al. British Medical Bulletin. 66: 199.
2003 and Belay et al. Emerging Infectious Diseases.
10(6): 977. 2004.)

1997 TO 2004. SPORADIC CJD CASES TRIPLED, and that is
with a human TSE surveillance system that is terrible
flawed. in 1997 cases of the _reported_ cases of cjd
were at 54, to 163 _reported_ cases in 2004. see stats

p.s. please note the 47 PENDING CASES to Sept. 2005

p.s. please note the 2005 Prion D. total 120(8)
8=includes 51 type pending, 1 TYPE UNKNOWN ???

p.s. please note sporadic CJD 2002(1) 1=3 TYPE UNKNOWN???

p.s. please note 2004 prion disease (6) 6=7 TYPE


AS implied in the Inset 25 we must not _ASSUME_ that
transmission of BSE to other species will invariably
present pathology typical of a scrapie-like disease.



Infected and Source Flocks

As of August 31, 2005, there were 115 scrapie infected
and source flocks (figure 3). There were 3 new infected
and source flocks reported in August (Figure 4) with a
total of 148 flocks reported for FY 2005 (Figure 5).
The total infected and source flocks that have been
released in FY 2005 are 102 (Figure 6), with 5 flocks
released in August. The ratio of infected and source
flocks released to newly infected and source flocks for
FY 2005 = 0.69 :
1. In addition, as of August 31, 2005, 574 scrapie
cases have been confirmed and reported by the National
Veterinary Services Laboratories (NVSL), of which 122
were RSSS cases (Figure 7). This includes 55 newly
confirmed cases in August 2005 (Figure 8). Fifteen
cases of scrapie in goats have been reported since 1990
(Figure 9). The last goat case was reported in May 2005.


full text ;

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:





[Docket No. 03-025IFA] FSIS Prohibition of the Use of
Specified Risk Materials for Human Food and Requirement
for the Disposition of Non-Ambulatory Disabled Cattle

Terry S. Singeltary

Page 1 of 17

From: Terry S. Singeltary Sr. []

Sent: Thursday, September 08, 2005 6:17 PM


Subject: [Docket No. 03-025IFA] FSIS Prohibition of the
Use of Specified Risk Materials for Human Food and

for the Disposition of Non-Ambulatory Disabled Cattle

Greetings FSIS,

I would kindly like to submit the following to [Docket
No. 03-025IFA] FSIS Prohibition of the Use of Specified
Risk Materials for Human Food and

Requirements for the Disposition of Non-Ambulatory
Disabled Cattle

THE BSE/TSE SUB CLINICAL Non-Ambulatory Disabled Cattle

Broken bones and such may be the first signs of a sub
clinical BSE/TSE Non-Ambulatory Disabled Cattle ;



Issued: Monday, 28 August 2000



Terry S. Singeltary Sr.

P.O. Box 42

Bacliff, Texas USA 77518


Importation of Whole Cuts of Boneless Beef from Japan
[Docket No. 05-004-1] RIN 0579-AB93 TSS SUBMISSION

----- Original Message -----
From: Terry S. Singeltary Sr.
To: ;
Sent: Thursday, August 25, 2005 8:16 AM
Subject: Importation of Whole Cuts of Boneless Beef
from Japan [Docket No. 05-004-1] RIN 0579-AB93 TSS

Greetings Dr. Colgrove and Miss Johnson,

Thank you for taking this submission via email. i have
had trouble submitting via the comment page due to the
length of my submission. I was not sure that my file
attachment that i submitted via the ;


I submitted yesterday, just did not know if the file
reached anyone. so to make sure, I am sending to you to
submit for me.

many thanks,


From: TSS ()
Subject: Importation of Whole Cuts of Boneless Beef
from Japan [Docket No. 05-004-1] RIN 0579-AB93 TSS
Date: August 24, 2005 at 2:47 pm PST

August 24, 2005

Importation of Whole Cuts of Boneless Beef from Japan
[Docket No. 05-004-1] RIN 0579-AB93 TSS SUBMISSION

Greetings APHIS ET AL,

My name is Terry S. Singeltary Sr.

I would kindly like to comment on [Docket No. 05-004-1]
RIN 0579-AB93 ;

Exportation and importation of animals and animal
Whole cuts of boneless beef from-
48494-48500 [05-16422]

[Federal Register: August 18, 2005 (Volume 70, Number 159)]
[Proposed Rules]
[Page 48494-48500]
From the Federal Register Online via GPO Access


Greetings again APHIS ET AL,

THIS is not correct. IN fact, there are several factors
i would like to kindly address.

Muscle tissue has recently been detected with PrPSc
in the peripheral nerves (sciatic nerve, tibial nerve,
vagus nerve) of the 11th BSE
cow in Japan (Yoshifumi Iwamaru et al). also recently,
Aguzzi et al Letter to the Editor
Vet Pathol 42:107-108 (2005), Prusiner et al CDI test
is another example of detection
of the TSE agent in muscle in sCJD, Herbert Budka et al
CJD and inclusion body myositis:
Abundant Disease-Associated Prion Protein in Muscle,
and older studies from Watson
Meldrum et al Scrapie agent in muscle - Pattison I A
(1990), references as follow ;

PrPSc distribution of a natural case of bovine
spongiform encephalopathy

Yoshifumi Iwamaru, Yuka Okubo, Tamako Ikeda, Hiroko
Hayashi, Mori-
kazu Imamura, Takashi Yokoyama and Morikazu Shinagawa

Priori Disease Research Center, National Institute of
Animal Health, 3-1-5
Kannondai, Tsukuba 305-0856 Japan


Bovine spongiform encephalopathy (BSE) is a disease of
cattle that causes
progressive neurodegeneration of the central nervous
system. Infectivity
of BSE agent is accompanied with an abnormal isoform of
prion protein

The specified risk materials (SRM) are tissues
potentially carrying BSE
infectivity. The following tissues are designated as
SRM in Japan: the
skull including the brain and eyes but excluding the
glossa and the masse-
ter muscle, the vertebral column excluding the
vertebrae of the tail, spinal
cord, distal illeum. For a risk management step, the
use of SRM in both
animal feed or human food has been prohibited. However,
PrPSc distribution remains obscure in BSE cattle and it
has caused con-
troversies about definitions of SRM. Therefore we have
examined PrPSc
distribution in a BSE cattle by Western blotting to
reassess definitions of

The 11th BSE case in Japan was detected in fallen stock
The carcass was stocked in the refrigerator. For the
detection of PrPSc,
200 mg of tissue samples were homogenized. Following
treatment, samples were digested with proteinase K.
After digestion,
PrPSc was precipitated by sodium phosphotungstate
(PTA). The pellets
were subjected to Western blotting using the standard
Anti-prion protein monoclonal antibody (mAb) T2
conjugated horseradish
peroxidase was used for the detection of PrPSc.

PrPSc was detected in brain, spinal cord, dorsal root
ganglia, trigeminal
ganglia, sublingual ganglion, retina. In addition,
PrPSc was also detected
in the peripheral nerves (sciatic nerve, tibial nerve,
vagus nerve).

Our results suggest that the currently accepted
definitions of SRM in
BSE cattle may need to be reexamined. ...


T. Kitamoto (Ed.)
Food and Drug Safety

ALSO from the International Symposium of Prion Diseases
held in Sendai, October 31, to
November 2, 2004;

Bovine spongiform encephalopathy (BSE) in Japan


"Furthermore, current studies into transmission of
cases of BSE that are
atypical or that develop in young cattle are expected
to amplify the BSE

NO. Date conf. Farm Birth place and Date Age at diagnosis

8. 2003.10.6. Fukushima Tochigi 2001.10.13. 23

9. 2003.11.4. Hiroshima Hyogo 2002.1.13. 21

Test results

# 8b, 9c cows Elisa Positive, WB Positive, IHC
negative, histopathology

b = atypical BSE case

c = case of BSE in a young animal

b,c, No PrPSc on IHC, and no spongiform change on histology

International Symposium of Prion Diseases held in
Sendai, October 31, to
November 2, 2004.

The hardback book title is 'PRIONS' Food and Drug Safety
T. Kitamoto (Ed.)

Tetsuyuki Kitamoto
Professor and Chairman
Department of Prion Research
Tohoku University School of Medicine
2-1 SeiryoAoba-ku, Sendai 980-8575, JAPAN
TEL +81-22-717-8147 FAX +81-22-717-8148
Symposium Secretariat
Kyomi Sasaki
TEL +81-22-717-8233 FAX +81-22-717-7656



Vet Pathol 42:107-108 (2005)

Letters to the Editor


Absence of evidence is not always evidence of absence.

In the article ''Failure to detect prion protein
(PrPres) by

immunohistochemistry in striated muscle tissues of animals

experimentally inoculated with agents of transmissible

encephalopathy,'' recently published in Veterinary

Pathology (41:78-81, 2004), PrPres was not detected in

muscle of experimentally infected elk, cattle, sheep, and

raccoons by immunohistochemistry (IHC). Negative IHC,

however, does not exclude the presence of PrPSc. For

PrPres was detected in skeletal muscle in 8 of 32

humans with the prion disease, sporadic Creutzfeldt-Jakob

disease (CJD), using sodium phosphotungstic acid (NaPTA)

precipitation and western blot.1 The NaPTA precipitation,

described by Wadsworth et al.,3 concentrates the abnormal

isoform of the prion, PrPres, from a large tissue

volume before western blotting. This technique has

the sensitivity of the western blot up to three orders

of magnitude and could be included in assays to detect

PrPres. Extremely conspicuous deposits of PrPres in muscle

were detected by IHC in a recent case report of an

with inclusion body myositis and CJD.2 Here, PrPres was

detected in the muscle by immunoblotting, IHC, and paraf-

fin-embedded tissue blot. We would therefore caution that,

in addition to IHC, highly sensitive biochemical assays and

bioassays of muscle are needed to assess the presence or

absence of prions from muscle in experimental and natural

TSE cases.

Christina Sigurdson, Markus Glatzel, and Adriano Aguzzi

Institute of Neuropathology

University Hospital of Zurich

Zurich, Switzerland


1 Glatzel M, Abela E, et al: Extraneural pathologic prion

protein in sporadic Creutzfeldt-Jakob disease. N Engl J

Med 349(19):1812-1820, 2003

2 Kovacs GG, Lindeck-Pozza E, et al: Creutzfeldt-Jakob

disease and inclusion body myositis: abundant

prion protein in muscle. Ann Neurol 55(1):

121-125, 2004

3 Wadsworth JDF, Joiner S, et al: Tissue distribution
of protease

resistant prion protein in variant CJD using a highly

sensitive immuno-blotting assay. Lancet 358:171-180,



Corinna Kaarlela, News Director
Source: Jennifer O'Brien
14 February 2005

Diagnosis of prions in patients should utilize novel
strategy, team says

A technique for detecting prions in tissue, developed
in recent years by
UCSF scientists, is significantly more sensitive than
the diagnostic
procedures currently used to detect the lethal
particles in samples of
brain tissue from patients, according to a study
performed by a UCSF team.

The finding indicates that the diagnostic technique,
known as the
conformation-dependent immunoassay (CDI), should be
established as the
standard approach for brain biopsies of patients
suspected of having the
disease, they say. The team is exploring whether the
CDI might be
adapted to detect prions in blood and muscle.

The finding suggests that reliance on the current
methods for detecting
prions in human brain tissue -- microscopic examination
of tissue for
the telltale vacuoles that form in brain cells and
(IHC), which involves detecting prions in brain
sections using prion
protein-specific antibodies -- may have led to an under
diagnosis of the
disease in patients in recent years, they say. (A
definitive diagnosis
of the disease in humans is made only on autopsy, when a
neuropathologist can analyze multiple brain regions for
vacuoles and
evidence of prions by IHC, and it is estimated that
only 50 percent of
human cases are autopsied, in part because many
pathologists do not want
to risk infection during the autopsy.)

In the study, the team compared the ability of the CDI
and the two
traditional diagnostic techniques to detect prions in
various brain
samples from 28 patients diagnosed on autopsy as having
one of several
human forms of the disease -- sporadic, familial or
Creutzfeldt-Jakob disease (CJD). While the CDI detected
the biochemical
signal for prions in 100 percent of the samples
studied, the traditional
tests failed to detect the prion in a high proportion
of cases. For
example, in an experiment that focused on 18 brain
regions from eight
patients with sporadic CJD, the CDI detected prions in
100 percent of
the samples, while IHC detected them in 22 percent and
routine tissue
examination in 17 percent.

"In about 80 percent of the different brain regions
examined, prions
were not consistently detected by either IHC or routine
histology that
measure vacuolation. In contrast, the CDI was always
positive in all
regions of the brain," says the lead author of the
study, Jiri Safar,
MD, associate adjunct professor of neurology and a
member of the UCSF
Institute for Neurodegenerative Diseases, which is
directed by senior
author Stanley B. Prusiner, MD, UCSF professor of
neurology and

"These findings indicate that histology and
immunohistochemistry should
no longer be used to rule out prion disease in
single-site biopsy
samples," says Safar. "The superior performance of the
CDI in diagnosing
prion disease suggests that the CDI be used in future
evaluations of prion disease, particularly for
single-site brain
biopsies during life"

"If the traditional techniques are used at autopsy,
they must be applied
to many cortical and subcortical samples," says
co-author Stephen J.
DeArmond, MD, PhD, UCSF professor of neuropathology.

Moreover, while the study examined the efficacy of the
CDI in comparison
to the two techniques routinely used by
neuropathologists to detect
prions in human brain tissue, previous studies at UCSF
indicate that the
CDI is also significantly more sensitive than Western
blot analysis, the
technology used with IHC to detect prions in brain
tissue from cattle
suspected of having bovine spongiform encephalopathy
(BSE). That IHC and
Western blot analysis are relatively insensitive
methods, the
researchers say, supports their ongoing assertion that
the CDI should
also be used to evaluate the brain tissue of cattle.

"The studies reported here are likely to change
profoundly the approach
to the diagnosis of prion disease in both humans and
livestock," says Safar.

More broadly, the scientists say, the high sensitivity
of the CDI
suggests that CDI-like tests could also prove useful
for diagnosing
other neurodegenerative diseases, such as Alzheimer's
Parkinsons's disease and fronto-temporal dementias, all
of which, like
prion diseases, involve various forms of protein
misprocessing. These
diseases currently are diagnosed by neuropathological
analysis and

"Whether immunohistochemistry underestimates the
incidence of one or
more of these common neurodegenerative diseases is
unknown, but the CDI
could shed light on these diseases," says co-author
Bruce Miller, MD,
UCSF A.W. and Mary Margaret Clausen Distinguished
Professor of Neurology
and director of the UCSF Memory and Aging Center.

The finding will be printed on-line and in print on
March 1, 2005 in
Proceedings of the National Academy of Sciences.

The study brings into high relief the different
detection strategies of
immunohistochemistry and the CDI, both of which involve
revealing the
presence of prions, known as PrPsc, by applying
antibodies to brain tissue.

Standard immunohistochemistry, developed in the
DeArmond lab 20 years
ago, involves using an enzyme known as a protease, or a
combination of
harsh acid and high temperature treatment, to destroy
normal prion
protein (PrPC), which is ubiquitous in brain tissue.
Once this occurs,
scientists apply fluorescently lit antibodies that
react with residues
of the relatively resistant abnormal prion protein
(PrPSc), thereby
highlighting it.

The limitation of this technique is that scientists
have since learned
that there is a large part of the abnormal prion
protein that is
protease sensitive, and that portion escapes detection
by the standard
technique. Thus, this traditional method underestimates
the level of
PrPSc in tissue.

The CDI addresses this limitation by revealing the
region of PrPSc that
is exposed in the normal PrPC but is buried in
infectious PrPSc, using
high affinity, newly generated antibodies that identify
PrPSc through
the distinct shape of the molecule, independent of
treatments. This makes it possible to detect
potentially large
concentrations of protease sensitive PrPSc molecules.

Detractors would say that it is not necessary to detect
the minute level
of infectious agent that the CDI is capable of
revealing, as it would be
unlikely to be lethal, says Safar. But Prusiner and his
maintain that any risk is too great when it comes to
having prions in
the food supply. In addition, because even low levels
of prions are
extremely resistant to inactivation, they may
contaminate the
environment for many years.

Prusiner won the 1997 Nobel Prize in Physiology or
Medicine for
discovering that a class of neurodegenerative diseases
known as
spongiform encephalopathies was caused by prions. Prion
diseases develop
in humans, cattle, sheep, deer, elk and mink.

The CDI was developed by members of the Prusiner lab.
methodology has been licensed to InPro Biotechnology, Inc.

Prusiner, Safar, DeArmond and other members of the
Institute for
Neurodegenerative Diseases are scientific advisors to,
or own stock in,

Other co-authors of the study were Michael D.
Geschwind, Camille
Deering, Svetlana Didorenko, Mamta Sattavat, Henry
Sanchesz, Ana Serban,
Kurt Giles, of UCSF, and Martin Vey, of Behring,
Marburg, Germany, and
Henry Baron, of Behring, Paris.

The study was funded by the National Institutes of
Health, the John
Douglas French Foundation for Alzheimer's research, the
Foundation, the State of California, Alzheimer's
Disease Research Center
of California and the RR00079 General Clinical Research

The UCSF Institute for Neurodegenerative Diseases:


Explanation as to why the CDI is more sensitive than
Western blot
analysis: Studies at UCSF during development of the CDI
showed that CDI
could detect prions in brain homogenates at levels that
fail to produce
disease in animals (bioassay for prions). Therefore,
the CDI is more
sensitive than the bioassay method, which was
considered to be the most
sensitive technique for detecting prions. In contrast,
Western blot
analysis for prions is significantly less sensitive
than the bioassay
and is, therefore, significantly less sensitive than
the CDI. Currently,
the USDA uses a combination of Western blot analysis of
homogenates and immunohistochemistry of the medulla to
test cattle
suspected of having bovine spongiform encephalopathy
("mad cow
disease"). The relative insensitivity of IHC and
Western blot analysis,
says DeArmond, supports the UCSF scientists' ongoing
assertion that the
CDI should also be used to evaluate the brain tissue of

DeArmond cites additional evidence about Western blot
analysis from a
World Health Organization (WHO) study group, which
compared the CDI
method with Western blots for detection of prions in
sporadic and
variant CJD brains. Based on the smallest amount of
prions that could
detected by the two techniques, they found that the CDI
was from 1000-
to 100,000-fold more sensitive than Western blot
analysis performed in
six different research laboratories (Minor et al.
Standards for the
assay of Creutzfeldt-Jakob disease specimens. J. Gen.
Virol. 85:
1777-1784, 2004).

Explanation as to why IHC for prions is less sensitive
than the CDI: IHC
is routinely performed on formalin-fixed,
paraffin-embedded samples of
brain. Formalin fixation markedly decreases the ability
of antibodies to
bind to proteins in general, which greatly weakens the
IHC signal for
prions (PrPSc). In contrast, homogenates for the CDI
are not treated
with reagents that decrease prion antigenicity.
Moreover, to concentrate
the PrPSc for measurement by the CDI, the homogenates
are exposed to
phosphotungstic acid, which selectively precipitates both
protease-sensitive and protease-resistant PrPSc that
comprise prions,
but not the normal prion protein conformer found in
uninfected animals,
PrPC. This step results in a higher concentration of
PrPSc for detection
by the CDI. Because the PrPSc was not exposed to
proteases, the CDI
measures all forms of abnormally folded PrPSc molecules.
Protease-sensitive PrPSc can account for 50 percent of
the total PrPSc.
For Western analysis, homogenates of brain are treated
with protease to
eliminate PrPC; however, this step also eliminates
PrPSc leaving only protease-resistant PrPSc for Western
blot detection
and decreasing the PrPSc signal at least in half.


PNAS | March 1, 2005 | vol. 102 | no. 9 | 3501-3506


Diagnosis of human prion disease

Jiri G. Safar *, , Michael D. Geschwind , , Camille Deering
*, Svetlana Didorenko *, Mamta Sattavat ¶, Henry Sanchez ¶,
Ana Serban * , Martin Vey ||, Henry Baron **, Kurt Giles *,
, Bruce L. Miller , , Stephen J. DeArmond * , ¶ and Stanley
B. Prusiner *, , ,

*Institute for Neurodegenerative Diseases, Memory and Aging
Center, and Departments of Neurology, ¶Pathology, and
Biochemistry and Biophysics, University of California, San
Francisco, CA 94143; ||ZLB Behring, 35041 Marburg, Germany;
and **ZLB Behring, 75601 Paris, France

Contributed by Stanley B. Prusiner, December 22, 2004


With the discovery of the prion protein (PrP),
immunodiagnostic procedures were applied to diagnose
Creutzfeldt-Jakob disease (CJD). Before development of the
conformation-dependent immunoassay (CDI), all immunoassays
for the disease-causing PrP isoform (PrPSc) used limited
proteolysis to digest the precursor cellular PrP (PrPC).
Because the CDI is the only immunoassay that measures both
the protease-resistant and protease-sensitive forms of
PrPSc, we used the CDI to diagnose human prion disease. The
CDI gave a positive signal for PrPSc in all 10-24 brain
regions (100%) examined from 28 CJD patients. A subset
of 18
brain regions from 8 patients with sporadic CJD (sCJD) was
examined by histology, immunohistochemistry (IHC), and the
CDI. Three of the 18 regions (17%) were consistently
positive by histology and 4 of 18 (22%) by IHC for the 8
sCJD patients. In contrast, the CDI was positive in all 18
regions (100%) for all 8 sCJD patients. In both gray and
white matter, 90% of the total PrPSc was protease-sensitive
and, thus, would have been degraded by procedures using
proteases to eliminate PrPC. Our findings argue that
the CDI
should be used to establish or rule out the diagnosis of
prion disease when a small number of samples is
available as
is the case with brain biopsy. Moreover, IHC should not be
used as the standard against which all other
immunodiagnostic techniques are compared because an
immunoassay, such as the CDI, is substantially more



The clinical diagnosis of human prion disease is often
difficult until the patient shows profound signs of
neurologic dysfunction. It is widely accepted that the
clinical diagnosis must be provisional until a tissue
diagnosis either confirms or rules out the clinical
assessment. Before the availability of Abs to PrP, a tissue
diagnosis was generally made by histologic evaluation of
neuropil vacuolation. IHC with
anti-glial-fibrillary-acidic-protein Abs in combination
H&E staining preceded the use of anti-PrP Ab staining.

Recently, the role of IHC in the diagnosis of scrapie
in the
brains of eight clinically affected goats inoculated with
the SSBP1 prion isolate has been challenged (14). Thalamic
samples taken from seven of eight goats with scrapie were
positive for PrPSc by Western blotting but negative by IHC.
The eighth goat was negative by Western blotting and IHC.
Consistent with these findings in goats are the data
reported here, in which IHC of formalin-fixed,
paraffin-embedded human brain samples was substantially
sensitive than the CDI.

The CDI was developed to quantify PrPSc in tissue samples
from mammals producing prions. Concerned that limited PK
digestion was hydrolyzing some or even most of the
PrPSc, we
developed a CDI that does not require PK digestion. The CDI
revealed that as much as 90% of PrPSc is sPrPSc; thus, it
was being destroyed during limited proteolytic digestion
used to hydrolyze PrPC. sPrPSc comprises 80% of PrPSc
in the
frontal lobe and in the white matter (Fig. 4).

The CDI detected HuPrPSc with a sensitivity comparable to
the bioassay for prion infectivity in Tg(MHu2M) mice (Fig.
1). The high sensitivity achieved by the CDI is due to
several factors (8, 10, 11, 15). First, both sPrPSc and
rPrPSc conformers are specifically precipitated by PTA
(Table 5) (8, 9). PTA has also been used to increase the
sensitivity of Western blots enabling the detection of
rPrPSc in human muscle and other peripheral tissues (16,
17). Second, a sandwich protocol was used with the
high-affinity MAR1 mAb (11) to capture HuPrPSc and
Eu-labeled 3F4 mAb to detect HuPrPSc (12). Third, the CDI
detects PrPSc by Ab-binding to native and denatured
forms of
the protein and, therefore, does not depend on proteolytic
degradation of PrPC. We chose not to perform Western blots
on most of the samples used in this study because such
immunoblots require denaturation of the sample, which
eliminates measurement of the native signal
corresponding to
PrPC (Table 5). Moreover, a comparison between the CDI and
Western blotting on brain samples from sCJD and variant CJD
patients showed that the CDI was 50- to 100-fold more
sensitive (15). Additionally, Western blots combined with
densitometry are linear over a 10- to 100-fold range of
concentrations, whereas the CDI is linear over a >104-fold
range. The CDI has been automated, which not only improves
accuracy and reproducibility (10) but also allows numerous
samples to be analyzed, as reported here. Western blots are
difficult to automate and are labor intensive.

Our studies show that only the CDI detected PrPSc in all
regions examined in 24 sCJD and 3 fCJD(E200K) brains (Figs.
2 and 6). Comparative analyses demonstrated that the
CDI was
vastly superior to histology and IHC. When 18 regions of 8
sCJD and 2 fCJD(E200K) brains were compared, we discovered
that histology and IHC were unreliable diagnostic tools
except for samples from a few brain regions. In contrast,
the CDI was a superb diagnostic procedure because it
detected PrPSc in all 18 regions in 8 of 8 sCJD and 2 of 2
fCJD(E200K) cases (Tables 1 and 2).

Histologic changes in prion disease have been shown to
follow the accumulation of prions as measured by
bioassay of
infectivity and by PrPSc accumulation (18-22). Because low
levels of PrPSc are not associated with neuropathologic
changes, some discrepancy between vacuolation and PrPSc was
expected. In contrast to histology, IHC measures PrP
immunostaining after autoclaving tissue sections exposed to
formic acid. Because IHC measures PrP, we expected the
sensitivity of this procedure might be similar to the CDI,
but that proved not to be the case. Whether exposure of
formic acid-treated tissue sections to elevated temperature
destroys not only PrPC but also sPrPSc and only denatures
rPrPSc remains to be determined. Such a scenario could
account for the lower sensitivity of IHC compared with CDI
or bioassay (Tables 1 and 2).

Studies of the white matter in CJD brains were particularly
informative with respect to the sensitivity of the CDI,
where PrPSc levels were low but readily detectable, 10- to
100-fold above the threshold value (Fig. 4). Because animal
studies have shown that PrPSc and infectivity are
transported anterogradely from one brain region to another
along neuroanatomical pathways (23-25), we expected to find
PrPSc in white matter as demonstrated by the CDI but not
IHC. Axonal transport of PrPSc is also suggested by
diffusion-weighted MRI scans of CJD cases, which show
high-intensity signals in analogous neocortical regions of
the right and left cerebral hemispheres (26). This symmetry
of neuroradiological abnormalities is consistent with
of PrPSc to the contralateral cortex by means of callosal
commissural pathways.

Most immunoassays that detect HuPrPSc do so only after
subjecting the sample to limited proteolysis to form PrP
27-30, followed by denaturation. Because the CDI measures
the immunoreactivity before and after denaturation to an
epitope that is exposed in native PrPC but buried in PrPSc,
limited proteolysis to eliminate PrPC is unnecessary.
based on limited proteolysis underestimate the level of
PrPSc because they digest sPrPSc, which represents
80-90% of
PrPSc in CJD and scrapie brains (Fig. 4 and Table 5).

Gerstmann-Sträussler-Scheinker, an inherited human prion
disease, is caused by the P102L mutation in the PRNP gene.
In mice expressing the Gerstmann-Sträussler-Scheinker
PrP transgene, the CDI detected high levels of
as well as low levels of rPrPSc(P101L) long before
neurodegeneration and clinical symptoms occurred (9).
sPrPSc(P101L) as well as low concentrations of
previously escaped detection (27). Whether a similar
situation applies in other genetic forms of prion disease,
sCJD, or variant CJD remains to be determined. Because most
of the PrPSc in the brains of sCJD patients is
protease-sensitive (Fig. 4), it is likely that the lower
sensitivity of IHC is due to its inability to detect
Presently, we have no information about the kinetics of
either sPrPSc or rPrPSc accumulation in human brain.
information on the kinetics of PrPSc accumulation in
livestock comes from studies of cattle, sheep, and goats
inoculated orally, but most of the bioassays were performed
in non-Tg mice (28-30) in which prion titers were
underestimated by as much as a factor of 104 (10).

The studies reported here are likely to change profoundly
the approach to the diagnosis of prion disease in both
humans and livestock (31-33). The superior performance of
the CDI in diagnosing prion disease compared to routine
neuropathologic examination and IHC demands that the CDI be
used in future diagnostic evaluations of prion disease.
Prion disease can no longer be ruled out by routine
histology or IHC. Moreover, the use of IHC to confirm cases
of bovine spongiform encephalopathy after detection of
bovine PrPSc by the CDI (10) seems an untenable approach in
the future. Clearly, the CDI for HuPrPSc is as sensitive or
more sensitive than bioassays in Tg(MHu2M) mice (Fig. 1).

Our results suggest that using the CDI to test large
of samples for human prions might alter the epidemiology of
prion diseases. At present, there is limited data on the
frequency of subclinical variant CJD infections in the U.K.
population (34). Because appendixes and tonsils were
evaluated only by IHC, many cases might have escaped
detection (Tables 1 and 2). Equally important may be
the use
of CDI-like tests to diagnose other neurodegenerative
disorders, such as Alzheimer's disease, Parkinson's
and the frontotemporal dementias. Whether IHC
the incidence of one or more of these common degenerative
diseases is unknown. Moreover, CDI-like tests may help
determine the frequency with which these disorders and the
prion diseases occurs concomitantly in a single patient



Volume 349:1812-1820 November 6, 2003 Number 19

Extraneural Pathologic Prion Protein in Sporadic
Creutzfeldt-Jakob Disease

Background In patients with sporadic Creutzfeldt-Jakob
disease, pathologic disease-associated prion protein
(PrPSc) has been identified only in the central nervous
system and olfactory-nerve tissue. Understanding the
distribution of PrPSc in Creutzfeldt-Jakob disease is
important for classification and diagnosis and perhaps
even for prevention.

Methods We used a highly sensitive method of detection
- involving the concentration of PrPSc by differential
precipitation with sodium phosphotungstic acid, which
increased the sensitivity of Western blot analysis by
up to three orders of magnitude - to search for PrPSc
in extraneural organs of 36 patients with sporadic
Creutzfeldt-Jakob disease who died between 1996 and 2002.

Results PrPSc was present in the brain tissue of all
patients. In addition, we found PrPSc in 10 of 28
spleen specimens and in 8 of 32 skeletal-muscle
samples. Three patients had PrPSc in both spleen and
muscle specimens. Patients with extraneural PrPSc had a
significantly longer duration of disease and were more
likely to have uncommon molecular variants of sporadic
Creutzfeldt-Jakob disease than were patients without
extraneural PrPSc.

Conclusions Using sensitive techniques, we identified
extraneural deposition of PrPSc in spleen and muscle
samples from approximately one third of patients who
died with sporadic Creutzfeldt-Jakob disease.
Extraneural PrPSc appears to correlate with a long
duration of disease.

Source Information

From the Institute of Neuropathology and National
Reference Center for Prion Diseases, University
Hospital of Zurich, Zurich, Switzerland.

Dr. Glatzel and Mr. Abela contributed equally to the

Address reprint requests to Dr. Aguzzi at the Institute
of Neuropathology, University Hospital of Zurich,
Schmelzbergstr. 12, CH-8091 Zurich, Switzerland, or at .

Creutzfeldt-Jakob disease and inclusion body myositis:
Abundant disease-associated prion protein in muscle

Gabor G. Kovacs, MD PhD 1 2, Elisabeth Lindeck-Pozza,
MD 1, Leila Chimelli, MD, PhD 3, Abelardo Q. C. Araújo,
MD, PhD 4, Alberto A. Gabbai, MD, PhD 5, Thomas
Ströbel, PhD 1, Markus Glatzel, MD 6, Adriano Aguzzi,
MD, PhD 6, Herbert Budka, MD 1 *
1Institute of Neurology, University of Vienna, and
Austrian Reference Centre for Human Prion Diseases,
Vienna, Austria
2National Institute of Psychiatry and Neurology,
Budapest, Hungary
3Department of Pathology, School of Medicine, Federal
University of Rio de Janeiro
4Department of Neurology, School of Medicine, Federal
University of Rio de Janeiro
5Department of Neurology, School of Medicine, Federal
University of Sao Paulo, Brazil
6Institute of Neuropathology, University Hospital of
Zürich, Zürich, Switzerland
email: Herbert Budka ( )

*Correspondence to Herbert Budka, Institute of
Neurology, AKH 4J, Wühringer Gürtel 18-20, POB 48,
A-1097 Vienna, Austria

Funded by:
European Union (EU) Project; Grant Number: TSELAB
EU Concerted Action PRIONET; Grant Number:


Pathologicalprion protein (PrPSc) is the hallmark of
prion diseases affecting primarily the central nervous
system. Using immunohistochemistry, paraffin-embedded
tissue blot, and Western blot, we demonstrated abundant
PrPSc in the muscle of a patient with sporadic
Creutzfeldt-Jakob disease and inclusion body myositis.
Extraneural PrPC-PrPSc conversion in Creutzfeldt-Jakob
disease appears to become prominent when PrPC is
abundantly available as substrate, as in inclusion body
myositis muscle.


Received: 16 June 2003; Revised: 11 September 2003;
Accepted: 11 September 2003
Digital Object Identifier (DOI)

10.1002/ana.10813 About DOI

snip... full text;




Docket No, 04-047-l Regulatory Identification No. (RIN)
091O-AF46 NEW BSE SAFEGUARDS (comment submission)


Docket No. 2003N-0312 Animal Feed Safety System [TSS

Docket Management Docket: 02N-0273 - Substances
Prohibited From Use in

Animal Food or Feed; Animal Proteins Prohibited in
Ruminant Feed

Comment Number: EC -10

Accepted - Volume 2



File Format: PDF/Adobe Acrobat -

Page 1. J Freas, William From: Sent: To: Subject: Terry
S. Singeltary

Sr. [] Monday, January 08,200l 3:03 PM
freas ...

Asante/Collinge et al, that BSE transmission to the

genotype can lead to an alternate phenotype that is

from type 2 PrPSc, the commonest _sporadic_ CJD;

Docket Management Docket: 96N-0417 - Current Good
Manufacturing Practice
in Manufacturing, Packing, or Holding Dietary Ingredients a
Comment Number: EC -2
Accepted - Volume 7

[PDF] Appendices to PL107-9 Inter-agency Working Group
Final Report 1-1
File Format: PDF/Adobe Acrobat - View as HTML
Agent, Weapons of Mass Destruction Operations Unit
Federal Bureau of
those who provided comments in response to Docket No. ...
Meager 8/18/01 Terry S. Singeltary Sr ...

Docket No. 2003N-0312 Animal Feed Safety System [TSS
TO DOCKET 2003N-0312]

# Docket No: 02-088-1 RE-Agricultural Bioterrorism
Protection Act of
TSS 1/27/03 (0)

Docket Management

Docket: 02N-0276 - Bioterrorism Preparedness;
Registration of Food Facilities, Section 305
Comment Number: EC-254 [TSS SUBMISSION]

Dockets Entered On October 2, 2003 Table of Contents,
Docket #,
Title, 1978N-0301,

OTC External Analgesic Drug Products, ... EMC 7, Terry
S. Singeltary Sr.
Vol #: 1, ...

Daily Dockets Entered on 02/05/03

DOCKETS ENTERED on 2/5/03. ... EMC 4 Terry S.
Singeltary Sr. Vol#: 2.
.. Vol#: 1.

03N-0009 Federal Preemption of State & Local Medical
Device Requireme. ...

Docket Management

Docket: 02N-0370 - Neurological Devices; Classification
of Human Dura Mater

Comment Number: EC -1

Accepted - Volume 1

Daily Dockets - 04/10/03

.. 00D-1662 Use of Xenotransplantation Products in Humans.
EMC 98 Terry S. Singeltary Sr. Vol#: 3. 01F ...

- 05-20-2003
- Cached

Guidance for Industry: Use of Material From Deer and
Elk In Animal Feed

Terry S. Singeltary Sr.
Vol #:

Guidance for Industry: Use of Material From Deer and
Elk In Animal Feed

Terry S. Singeltary Sr.
Vol #:

Guidance for Industry: Use of Material From Deer and
Elk In Animal Feed

Terry S. Singeltary Sr.
Vol #:

01N-0423 Substances Prohibited from use in animal
food/Feed Ruminant

APE 5 National Renderers Association, Inc. Vol#: 2

APE 6 Animal Protein Producers Industry Vol#: 2

APE 7 Darling International Inc. Vol#: 2

EMC 1 Terry S. Singeltary Sr. Vol#: 3

ALL animals for human/animal consumption must be tested
for TSE.

ALL human TSEs must be made reportable Nationally and
Internationally, OF ALL AGES...TSS

Terry S. Singeltary Sr.
P.O. Box 42
Bacliff, Texas USA 77518

Follow Ups:

Post a Followup

E-mail: (optional)


Optional Link URL:
Link Title:
Optional Image URL: