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From: TSS (
Date: August 6, 2004 at 8:11 am PST

-------- Original Message --------
Date: Thu, 5 Aug 2004 16:11:44 -0500
From: "Terry S. Singeltary Sr."
Reply-To: Bovine Spongiform Encephalopathy

Greetings Canadian Cooperative Wildlife Health Centre,

I find disturbing, the studies that have been omitted in this study below;


Prepared by:
Expert Scientific Panel on Chronic Wasting Disease
Dr. Trent Bollinger, CCWHC, SK, Canada
Dr. Peter Caley, CSIRO, Canberra, Australia
Dr. Evelyn Merrill, Univ. of Alberta, AB, Canada
Dr. François Messier, Chair, Univ. of Saskatchewan, SK, Canada
Dr. Michael W. Miller, Colorado Div. of Wildlife, CO, USA
Dr. Michael D. Samuel, Wisconsin Coop. Wildl. Res. Unit, WI, USA
Dr. Emmanuel Vanopdenbosch, Veterinary and Agrochemical Res. Centre, Belgium
Submitted to:
Canadian Cooperative Wildlife Health Centre
Western College of Veterinary Medicine
University of Saskatchewan
52 Campus Drive
Saskatoon, SK, S7N 5B4
July 2004
Final Report

Executive Summary
This document represents a summary of discussion, conclusions, and
recommendations of an
Expert Scientific Panel convened to: 1) provide a synopsis of chronic
wasting disease (CWD) in
free-living cervids in Canada, 2) evaluate the ecological and
socio-economic implications of
CWD in Canada, and 3) make recommendations on research and management
actions to
minimize and mitigate the effects of CWD in cervid species.
The emergence of chronic wasting disease, a transmissible spongiform
potentially affecting mule deer, white-tailed deer and elk, is arguably
the most important issue
in the management of free-living cervids in North America. The disease
has the potential to
reduce cervid populations in the long-term, and to create major
socio-economic impacts as
observed in other areas in North America.
CWD has been detected in western Canada only recently, first in 1996 in
farmed cervids and
subsequently in 2000 in free-living cervids in Saskatchewan.
Epidemiological investigations
and surveillance programs of farmed cervids identified 40 game farms in
Saskatchewan and 3
game farms in Alberta with the disease. CWD is thought to have been
introduced into farmed
cervids in Saskatchewan during the late 1980s by the importation of
CWD-infected elk from
South Dakota. Management programs to eradicate the disease in farmed
cervids appear to have
been successful and there are currently no known infected farms in
Canada. Environmental
contamination of some CWD-infected premises continues to pose a
potential threat to wildlife.
Of most significance, the presence of CWD in wild deer in some areas is
a potential source of
infection for farmed cervids and poses a continued threat to the
long-term economical viability
of cervid farming.
In Canada, CWD in free-living cervids appears restricted to three
relatively distinct geographic
foci in Saskatchewan, although surveillance efforts in many areas are
inadequate to detect the
disease at low prevalence. Hence, the disease may yet be detected in
other areas. Intense, riskbased
surveillance to determine the distribution of this disease should be a
high priority over the
next few years. Demonstration of a more widespread distribution of CWD
within Saskatchewan
or elsewhere in Canada would affect management response to this disease.
Results over the last two years in the Saskatchewan Landing area,
Saskatchewan, indicate CWD
is well established in the local mule deer population. In spite of
initial attempts to reduce deer
densities by increasing hunting harvest, deer densities in most areas of
western Canada are more
than sufficient to allow CWD to spread and increase in prevalence.
The range of species that may be infected with CWD is not known with
certainty. Information
from the USA would indicate all mule deer, white-tailed deer and elk are
susceptible to the
disease. Infection in moose has been recently confirmed experimentally,
but similar data for
caribou are not available. CWD does not appear to pose a risk to cattle
or bison. The risk to
humans appears to be extremely low. Nonetheless, the World Health
Organization and other
government health agencies recommend that any animals with a TSE disease
not be consumed
by humans.
The panel concludes that the emergence of CWD in free-living mule deer
and white-tailed deer
in Saskatchewan warrants an aggressive regional and national management
and research
response to prevent further spread of CWD and to control or eliminate
the disease in wild
cervids. The recent introduction of CWD in Canada, and its restricted
distribution, provides us
with a unique opportunity to manage CWD before it is too late.
Once established in a population of free-living cervids, control or
eradication of CWD is
extremely difficult. Preventing establishment of new foci of CWD should
be given the highest
priority, which entails preventing the movement of CWD-infected cervids
and infectious
material to new areas. To prevent natural spread from endemic areas, and
to reduce potential
environmental contamination with infectious prions, severe population
reductions of deer, to
levels of <1 animal/km2 of critical habitat, will likely be required for
at least a decade.
Complete removal of deer in local areas may eliminate focal
introductions of CWD. Deer
densities that can prevent spread of CWD, and sizes of buffer zones to
contain CWD, are largely
unknown at this time. Management programs will need to be developed
using a research
framework, and updated as we learn about this disease.
Canada is at a critical juncture in its response to CWD in free-living
cervids. The Panel
recognizes the success of the federal CWD program for game farms and
envisions a comparable
investment in the management of CWD in wildlife. Significant investment
in CWD
management and research by federal and provincial governments, within a
national framework,
is required and urgent in order to develop an effective response to this
emerging disease.


The prion strain thought to cause CWD has not been linked to cases of
human illness in either
Canada or the USA, and consuming venison from areas where CWD is present
does not appear
to increase the likelihood of people contracting sporadic
Creutzfeldt-Jakob disease (CJD; a
human prion disease). Moreover, experimental studies have demonstrated a
molecular barrier to conversion of normal human prion proteins in the
presence of CWD prion
proteins. Such a response is similar to the molecular barriers to human
prion protein
conversions by the prion strains that cause scrapie or bovine spongiform
encephalopathy (BSE).
Despite the reassuring nature of the findings in studies of human health
risks conducted to date,
there is public concern about the implications of human exposure to CWD
and other animal
prion diseases. This concern, based on experiences with massive exposure
of people to the BSE
prion in the UK and other European countries that apparently led to
about 150 cases of variant
CJD, will likely influence public attitudes toward CWD for the
foreseeable future. Regardless
of how unlikely human illness arising from CWD exposure may be, the
perception that CWD
could be a human pathogen will shape public attitudes toward hunting and
consuming deer and
elk in areas where CWD occurs. The panel recognizes and supports
international public health
officials recommendations against consuming any parts of animals known
to be infected with a
prion disease.


Current and evolving methods of testing
Diagnostic test procedures for detecting abnormal prion proteins in
sampled individuals are
constantly improving. Initially, diagnosis of CWD was based on observing
spongiform (i.e.
sponge-like) change in brain tissue with the light microscope.
However, these changes are
only observed in animals in later stages of the disease and therefore
this method does not detect
earlier preclinical cases. Immunohistochemical stains specific for
abnormal prion proteins
(PrPres) greatly improve the sensitivity and specificity of tests for
CWD and permit early
detections of CWD. Infection trials in mule deer and white-tailed deer
have shown that
abnormal prion proteins accumulate first in tonsil and retropharyngeal
lymph nodes, followed
by deposition in the dorsal motor nucleus of the vagal nerve in the obex
region of the brain. As
the disease progresses, abnormal prion proteins are found in other areas
of the brain stem as
well. A similar pattern of disease progression is observed in elk, but
whether this is consistent
among individual elk is still under study.
Sensitivity of the test procedure is dependant on which tissues are
tested; of the three tissues
most commonly sampled (retropharyngeal lymph node, tonsil, and medulla
oblongata at the
obex), retropharyngeal lymph nodes are the most sensitive (i.e.tests
performed on this tissue
detect earlier preclinical cases) and obex (i.e brainstem) is the least
sensitive. Surveillance
programs should clearly state the testing procedures and the criteria
used to classify an animal as
test negative. These criteria should be standardized and validated
amongst laboratories. In the
past, different criteria have been used to define an animal as test
negative. Hence, caution
should be used when interpreting historical surveillance results,
especially results from different
laboratories. Less sensitive tests reduce the probability of detecting
CWD, and consequently
negative results are less meaningful than they would be if more
sensitive tests were used.
The new rapid CWD tests detect abnormal prion proteins in unfixed
tissues by using Western
blot (WB) or enzyme-linked immunosorbent assay (ELISA) techniques. The
sensitivities of
these tests are similarly dependant on which tissues are analyzed.
Sensitivities and specificities
of these tests for a particular surveillance program should be
determined and clearly stated when
presenting results. The rapid tests have a high sensitivity but lower
specificity which leads to
false positives. Immunohistochemistry has high sensitivity and
specificity and is appropriate as
a secondary test in order to reject false positives identified via
initial screening. Appropriate
samples need to be collected to ensure that positives from rapid tests
can be confirmed with
immunohistochemistry. When CWD surveillance is based on testing only
lymph nodes, formalin fixed and frozen brain samples should still be
collected from each animal
in order to confirm infection in positive animals and allow for strain
typing of the abnormal
prion proteins. This information is needed to understand the
epidemiology of CWD in wild


It is imperative that a national plan is developed for monitoring,
managing and researching
CWD in wild cervids in Canada. The panel wishes to highlight the
following conclusions,
herein presented in point form for ease of understanding:
" The panel views the CWD issue to be of national importance.
" Unless some concerted and effective management action is undertaken in
the near future,
CWD will become widespread with the potential for major consequences to
game farming, and a variety of socio-economic interests in Canada.
" The panel recognizes the success of the federal CWD program for game
farms and
recommends a comparable investment in the management of CWD in wildlife.
" Notwithstanding the provincial jurisdiction over wildlife management,
the panel sees the
need for federal assistance in developing a national program to manage
CWD in
collaboration with provincial jurisdictions.
" Eradication is a desirable goal but extremely difficult to achieve in
wild populations
given current knowledge, technologies, and resources.
" Achieving a low or negligible level of prevalence of CWD is an
appropriate strategy to
reduce transmission rates, reduce the potential for spread, and to
minimize the amount of
transmissible prions in the environment.
" The panel recognizes the core elements for managing and preventing the
spread of CWD
to include:
o Implement comprehensive surveillance for CWD in wildlife and game farms.
o Prevent transmission of CWD between free-living cervids and animals in
o Avoid artificial animal concentrations (e.g., baiting and artificial
o Conduct scientific investigations that guide management of CWD
o Control populations of free-living cervids to achieve disease management
o Develop policies and regulations for animal translocations and other
activities to
prevent the spread of CWD.
o Conduct scientific investigations to understand the epidemiology of CWD in
wildlife populations.
" Recognizing the uncertainties associated with CWD, managing agencies
should adopt an
adaptive management approach to incorporate new information as it
becomes available.
The Panel feels that there is a sense of urgency in taking actions to
contain or eradicate CWD in
Canadian wild deer populations. The Panel members are unanimous in
supporting the following
recommendations; they are grouped in sections but presented in no
particular order of priority.
A: Management of game farms
1. Develop and implement policies to prevent transmission of CWD between
game farm
facilities and wildlife. Actions should include:
Do not permit new game farms in infected areas.
Use double fencing in infected areas.
Ensure previously infected farms are not accessible by wild cervids for a
minimum of 5 years.
Develop policies and regulations for animal translocations that may lead to
spread of CWD.
2. Maintain current surveillance and management programs for CWD in
farmed cervids.
3. Conduct additional retrospective epidemiological tracing of all farms
for more
comprehensive risk assessments in cooperation with US authorities.
4. Mandatory CWD testing of all cervid mortalities on game farms.
5. Mandatory participation in CFIA and provincial surveillance programs
for CWD.
6. Any transportation permit should be approved by both the import and
export authorities.
7. Share information on surveillance results and epidemiological
investigations among
agencies with jurisdictions over wildlife and game farm animals in a
timely fashion.
B: Management of free-living cervids
1. Develop and implement policies to minimize artificial aggregations of
free-living cervids
to reduce transmission of CWD. Actions should include:
Prevent access to hay stacks, salt blocks, and artificial water sources by
wildlife in high risk areas.
Ban baiting or artificial feeding for cervids in high risk areas.
2. Develop and evaluate management programs for reducing prevalence and
spread of CWD
in cervids by:
Eradicating sparks (i.e., new foci of infection) through local
and intensification of monitoring in surrounding areas.
Controlling CWD in infected areas through population reduction to a target
density of 1 cervid/km2 in critical habitat (i.e., winter range) with
reassessment based on surveillance results.
2. Monitoring and surveillance of CWD:
Develop and implement a risk-based surveillance program on a national
scale, e.g., SK and ON models.
Implement an aggressive surveillance program in the next 1-3 years to
document the distribution of CWD in free-ranging cervids in Canada.
To prevent the spread of CWD, collect sample sizes in areas adjacent to
infected areas that would allow the detection of prevalence at a level
of 0.5%
(5 infected individuals per 1000) with a 95% confidence level. The window
of sampling can be up to 3 years.
Adopt standardized diagnostic testing procedures at the national level.
C: Research needs
1. Evaluate the distribution of abnormal prion proteins (PrPres)
specific to CWD in different
body parts of infected animals, and its implication to infectivity
within a context of
2. Assess the potential for transmission of CWD within moose and caribou
3. Design an integrated research program to quantify the contribution of
various transmission
pathways within and among cervid species.
4. Develop spatially explicit models of CWD transmission and spread to
guide management
actions and monitoring in an adaptive management framework.
5. Collaborate in development and evaluation of diagnostic
epidemiological tools including
ante mortem tests, strain typing and environmental detection of prions.
D: Communications
1. Expand communication tools about the CWD issues and programs,
including regularly
maintained and linked websites, fact sheets about CWD distribution, and
media releases.
The targeted clientele should be broad based, including landowners,
scientists, hunters,
consumers, etc.

Greetings again Canadian Cooperative Wildlife Health Centre,

I am very concerned about some studies left out in this study below.
Please let me voice my concerns and submit my findings as follows;

> The range of species that may be infected with CWD is not known with
> certainty. Information
> from the USA would indicate all mule deer, white-tailed deer and elk
> are susceptible to the
> disease. Infection in moose has been recently confirmed
> experimentally, but similar data for
> caribou are not available. CWD does not appear to pose a risk to
> cattle or bison. The risk to
> humans appears to be extremely low. Nonetheless, the World Health
> Organization and other
> government health agencies recommend that any animals with a TSE
> disease not be consumed
> by humans.


-------- Original Message --------
Subject: Re: CWD TO CATTLE by inoculation (ok, is it three or four OR
Date: Mon, 23 Jun 2003 09:25:27 -0500
From: "Terry S. Singeltary Sr."
Reply-To: BSE-L


Greetings List Members,

i hear now that a 5th cow has gone done with CWD from the
studies of Amir Hamir et al. will Dr. Miller please
confirm or deny this please, and possibly explain why
this has not made the news, if in fact this is the case?

seems these cows infected with CWD/TSE did not display the
usual BSE symptoms. i wonder how many more are out there
in the field? course, we will never know unless someone
starts rapid TSE/BSE testing in sufficient numbers to find...

thank you,
kind regards,

Date: Sat, 23 Nov 2002 18:54:49 -0600
Reply-To: BSE
Sender: BSE
From: "Terry S. Singeltary Sr."
Subject: CWD TO CATTLE by inoculation (ok, is it three or four???)

1: J Vet Diagn Invest 2001 Jan;13(1):91-6

Preliminary findings on the experimental transmission of chronic wasting
disease agent of mule deer to cattle.

Hamir AN, Cutlip RC, Miller JM, Williams ES, Stack MJ, Miller MW,
O'Rourke KI, Chaplin MJ.

National Animal Disease Center, ARS, USDA, Ames, IA 50010, USA.

To determine the transmissibility of chronic wasting disease (CWD) to
cattle and to provide information about clinical course, lesions, and
suitability of currently used diagnostic procedures for detection of CWD
in cattle, 13 calves were inoculated intracerebrally with brain
suspension from mule deer naturally affected with CWD. Between 24 and 27
months postinoculation, 3 animals became recumbent and were euthanized.
Gross necropsies revealed emaciation in 2 animals and a large pulmonary
abscess in the third. Brains were examined for protease-resistant prion
protein (PrP(res)) by immunohistochemistry and Western blotting and for
scrapie-associated fibrils (SAFs) by negative-stain electron microscopy.
Microscopic lesions in the brain were subtle in 2 animals and absent in
the third case. However, all 3 animals were positive for PrP(res) by
immunohistochemistry and Western blot, and SAFs were detected in 2 of
the animals. An uninoculated control animal euthanized during the same
period did not have PrP(res) in its brain. These are preliminary
observations from a currently in-progress experiment. Three years after
the CWD challenge, the 10 remaining inoculated cattle are alive and
apparently healthy. These preliminary findings demonstrate that
diagnostic techniques currently used for bovine spongiform
encephalopathy (BSE) surveillance would also detect CWD in cattle should
it occur naturally.

Sat, Nov 23, 2002

Scientists unsure if CWD can jump species

By Jessica Bock Wausau Daily Herald


Janice Miller, a veterinarian in charge of the experiment, said she
believes previous research shows it is hard for the disease to be
transmitted naturally from whitetail deer to dairy cattle. "Our study
says nothing of how it could be transmitted in natural surroundings,"
she said.

Miller has been studying the transmission of CWD from mule deer to
cattle since 1997. Since then, chronic wasting disease was transmitted
to four out of 13 cattle injected with brain tissue from naturally
infected mule deer, she said.

In Wyoming, Williams has been studying cattle that were given a
concoction of diseased brain tissue orally, and five years into the
study the animals remain healthy, Miller said. No one knows if chronic
wasting disease could ever spread to another species through natural

"Our experience is that it's pretty hard to predict," Miller said.

greetings list,

> Since then, chronic wasting disease was

> transmitted to four out of 13 cattle

is this a typo by the media or has another cow gone down with CWD since
the preliminary findings were found?



-------- Original Message --------
Subject: Re: CWD TO CATTLE by inoculation (ok,is it three or four OR NOW
Date: Mon, 23 Jun 2003 12:36:59 -0500
From: "Janice M. Miller"
Reply-To: BSE-L


I am happy to provide an update on the experimental inoculation of
cattle and sheep with CWD. These are ongoing experiments and updates
are normally provided via presentations at meetings. Dr. Hamir has
prepared a poster of the following information that will be displayed at
4 upcoming meetings this summer and fall.

Experimental Transmission of Chronic Wasting Disease (CWD) to Cattle
and Sheep
Progress report - June 23, 2003

Experimental Transmission to Cattle

In 1997, 13 calves were inoculated intracerebrally with brain
suspension from mule deer naturally affected with CWD. During the first
3 years, 3 animals were euthanized 23, 24, and 28 months after
inoculation because of weight loss (2) or sudden death (1). Although
microscopic examination of the brains did not show classical lesions of
transmissible spongiform encephalopathy (TSE), a specific TSE marker
protein, PrPres, was detected by immunohistochemistry (IHC) and western
blot. Detailed information on these animals has been published
previously (A Hamir et al., J Vet Diagn Invest 13: 91-96, 2001).

During the 3rd, 4th and 6th years of observation, 7 additional animals
have been euthanized due to a variety of health concerns (primarily
chronic joint and foot problems). IHC and western blot results indicate
that 2 of these animals, necropsied 59 and 63 months after inoculation,
were positive for PrPres. One animal (# 1746) had not been eating well
for approximately 1 week prior to being found recumbent. At necropsy,
significant gross lesions consisted of an oblique fracture of L1
vertebral arch with extension into the body, and moderate multifocal
hemorrhagic ulceration in the abomasum. Microscopic examination of
brain revealed a few isolated neurons with single or multiple vacuoles,
but neither neuronal degeneration nor gliosis was observed. IHC
revealed the presence of PrPres in sections from several areas of the
brain. The other PrPres positive animal (#1742) was euthanized after
being found in lateral recumbency with a body temperature of 104.6 F.
It had not shown prior clinical signs except for some decreased appetite
for 2 days. Necropsy revealed only moderate hepatitis and a small renal
infarct due to intravascular thrombosis.

Summary of findings on all necropsied animals to date:

Ear tag Date of Survival Disease Clinical
Histo- IHC WB
no. necropsy period course signs
1745 8/18/99 23m 2m +
+/- + +
1768 9/22/99 24m 3m +
+/- + +
1744 1/29/00 28m 3d +/-
- + +
1749 5/20/01 44m NA -
- - -
1748 6/27/01 45m NA -
- - -
1743 8/21/02 59m NA -
- - -
1741 8/22/02 59m NA -
- - -
1746 8/27/02 59m 7d +/-
+/- + +
1765 11/27/02 62m 1d +/-
+/- - -
1742 12/28/02 63m 2d +/-
- + +
NT = not tested; IHC = immunohistochemistry for PrPres; SAF = scrapie
associated fibrils; NA = not applicable; WB = Western blot
(Prionics-Check); + = lesions or antigen present; - = lesions or
antigen absent; +/- = signs/lesions equivocal; i/c = intracerebral; m =
months; d = days.

After 5.75 years of observation we have 5 CWD transmissions to cattle
from a group of 13 inoculates. These animals, which were necropsied 23,
24, 28, 59, and 63 months after inoculation, did not show the clinical
signs or histopathologic lesions typical of a TSE, but PrPres was
detected in brain samples by both immunohistochemistry and western blot.
Five other animals necropsied during the 4th, 5th and 6th years of
observation have not shown evidence of PrPres and the remaining 3 cattle
are apparently healthy. Note that this study involved direct
intracerebral inoculation of cattle with the CWD agent, which is an
unnatural route of exposure. Likely, it would be more difficult to
infect cattle by the oral route. Cattle have been inoculated orally at
the University of Wyoming with the same inoculum used in this
experiment, and 5.75 years into the study the animals remain healthy
(personal communication, Dr. Beth Williams).

Experimental Transmission of CWD to sheep

Eight Suffolk sheep from the NADC scrapie-free flock were inoculated
intracerebrally with the CWD brain suspension used to inoculate cattle.
PRNP genotyping showed that 4 of the sheep were QQ at codon 171 and the
other four were QR. Two of the QQ sheep were euthanized during the 3rd
year of observation. At necropsy one of these animals had a urethral
obstruction and PrPres was not detected in brain or lymphoid tissues.
The other sheep, necropsied 35 months after inoculation, showed clinical
signs and histopathologic lesions that were indistinguishable from
scrapie. IHC tests showed typical PrPres accumulations in brain,
tonsil, and some lymph nodes. The 2 remaining QQ sheep and all 4 QR
sheep are apparently healthy 47 months after inoculation.

After 4 years of observation we have 1 transmission of CWD to a 171 QQ
sheep. This animal, which was necropsied 35 months after inoculation,
showed clinical signs and histopathologic lesions that were
indistinguishable from scrapie. Another QQ sheep that was necropsied
during the 3rd year showed no evidence of prion disease and all
remaining sheep (2 QQ and 4 QR) are apparently healthy.


> CANADA The prion strain thought to cause CWD has not been linked to
> cases of human illness in either Canada or the USA, and consuming
> venison from areas where CWD is present does not appear to increase
> the likelihood of people contracting sporadic Creutzfeldt-Jakob
> disease (CJD; a human prion disease). Moreover, experimental studies
> have demonstrated a substantial molecular barrier to conversion of
> normal human prion proteins in the presence of CWD prion proteins.
> Such a response is similar to the molecular barriers to human prion
> protein conversions by the prion strains that cause scrapie or bovine
> spongiform encephalopathy (BSE). Despite the reassuring nature of the
> findings in studies of human health risks conducted to date, there is
> public concern about the implications of human exposure to CWD and
> other animal prion diseases. This concern, based on experiences with
> massive exposure of people to the BSE prion in the UK and other
> European countries that apparently led to about 150 cases of variant
> CJD, will likely influence public attitudes toward CWD for the
> foreseeable future. Regardless of how unlikely human illness arising
> from CWD exposure may be, the perception that CWD could be a human
> pathogen will shape public attitudes toward hunting and consuming deer
> and elk in areas where CWD occurs. The panel recognizes and supports
> international public health officials recommendations against
> consuming any parts of animals known to be infected with a prion disease.


Chronic Wasting Disease and Potential Transmission to Humans

Ermias D. Belay,*Comments
Ryan A.
Maddox,* Elizabeth S. Williams, Michael W. Miller,! Pierluigi
Gambetti,§ and Lawrence B. Schonberger*
*Centers for Disease Control and Prevention, Atlanta, Georgia, USA;
University of Wyoming, Laramie, Wyoming, USA; !Colorado Division of
Wildlife, Fort Collins, Colorado, USA; and §Case Western Reserve
University, Cleveland, Ohio, USA

Suggested citation for this article: Belay ED, Maddox RA, Williams
ES, Miller MW, Gambetti P, Schonberger LB. Chronic wasting disease
and potential transmission to humans. Emerg Infect Dis [serial on
the Internet]. 2004 Jun [date cited]. Available from:


Chronic wasting disease (CWD) of deer and elk is endemic in a
tri-corner area of Colorado, Wyoming, and Nebraska, and new foci of
CWD have been detected in other parts of the United States. Although
detection in some areas may be related to increased surveillance,
introduction of CWD due to translocation or natural migration of
animals may account for some new foci of infection. Increasing
spread of CWD has raised concerns about the potential for increasing
human exposure to the CWD agent. The foodborne transmission of
bovine spongiform encephalopathy to humans indicates that the
species barrier may not completely protect humans from animal prion
diseases. Conversion of human prion protein by CWD-associated prions
has been demonstrated in an in vitro cell-free experiment, but
limited investigations have not identified strong evidence for CWD
transmission to humans. More epidemiologic and laboratory studies
are needed to monitor the possibility of such transmissions.



The lack of evidence of a link between CWD transmission and unusual
cases of CJD, despite several epidemiologic investigations, and the
absence of an increase in CJD incidence in Colorado and Wyoming suggest
that the risk, if any, of transmission of CWD to humans is low. Although
the in vitro studies indicating inefficient conversion of human prion
protein by CWD-associated prions raise the possibility of low-level
transmission of CWD to humans, no human cases of prion disease with
strong evidence of a link with CWD have been identified. However, the
transmission of BSE to humans and the resulting vCJD indicate that,
provided sufficient exposure, the species barrier may not completely
protect humans from animal prion diseases. Because CWD has occurred in a
limited geographic area for decades, an adequate number of people may
not have been exposed to the CWD agent to result in a clinically
recognizable human disease. The level and frequency of human exposure to
the CWD agent may increase with the spread of CWD in the United States.
Because the number of studies seeking evidence for CWD transmission to
humans is limited, more epidemiologic and laboratory studies should be
conducted to monitor the possibility of such transmissions. Studies
involving transgenic mice expressing human and cervid prion protein are
in progress to further assess the potential for the CWD agent to cause
human disease. Epidemiologic studies have also been initiated to
identify human cases of prion disease among persons with an increased
risk for exposure to potentially CWD-infected deer or elk meat (47
). If such cases
are identified, laboratory data showing similarities of the etiologic
agent to that of the CWD agent would strengthen the conclusion for a
causal link. Surveillance for human prion diseases, particularly in
areas where CWD has been detected, remains important to effectively
monitor the possible transmission of CWD to humans. Because of the long
incubation period associated with prion diseases, convincing negative
results from epidemiologic and experimental laboratory studies would
likely require years of follow-up. In the meantime, to minimize the risk
for exposure to the CWD agent, hunters should consult with their state
wildlife agencies to identify areas where CWD occurs and continue to
follow advice provided by public health and wildlife agencies. Hunters
should avoid eating meat from deer and elk that look sick or test
positive for CWD. They should wear gloves when field-dressing carcasses,
bone-out the meat from the animal, and minimize handling of brain and
spinal cord tissues. As a precaution, hunters should avoid eating deer
and elk tissues known to harbor the CWD agent (e.g., brain, spinal cord,
eyes, spleen, tonsils, lymph nodes) from areas where CWD has been

The EMBO Journal, Vol. 21, No. 23 pp. 6358-6366, 2002
© European Molecular Biology Organization

BSE prions propagate as either variant CJD-like or sporadic CJD-like
prion strains in transgenic mice expressing human prion protein

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 1
Corresponding author e-mail:

Received August 1, 2002; revised September 24, 2002; accepted October
17, 2002

Variant CreutzfeldtJakob 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 indistinguishable from that of
sporadic CJD with PrPSc type 2. These data suggest that more than one
BSE-derived 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.



Prion propagation involves recruitment and conversion of host PrPC into
PrPSc, and the degree of primary structural similarity between
inoculated PrPSc and host PrPC is thought to be a key component of
intermammalian transmission barriers (Prusiner et al., 1990 ). It is
clear, however, that prion strain type can also be crucial, as clearly
demonstrated by the very distinctive transmission properties of sporadic
CJD 129MM and vCJD 129MM prions (of identical PrP primary structure) in
either 129VV Tg152 (Hill et al., 1997 ; Collinge, 1999 ) or 129MM Tg35
mice. Prion strain type may also affect transmission barriers via an
effect on PrPSc tertiary structure and state of aggregation (Hill et
al., 1997 ; Collinge, 1999 ).

These 129MM Tg35 mice, in which human PrPSc types can be propagated,
have been used to study the BSE-to-human species barrier. The frequent
presence of sub-clinical prion disease in vCJD- and BSE-inoculated 129MM
Tg35 mice further argues for the need to reassess current definitions of
species or transmission barriers that limit prion transmission between
different hosts (Hill et al., 2000 ). Such barriers have hitherto been
quantitated on the basis of either comparative end-point titrations in
the two respective hosts, or by measuring the fall in incubation period
between primary and subsequent passage as the prion strain adapts to the
new host. Both methods rely on measurement of time to onset of a
clinical syndrome. Modelling the BSE-to-human barrier in 129MM Tg35 mice
would lead to the conclusion, on the basis of induced clinical disease,
that a substantial barrier existed. How ever, it is clear that human
PrPSc propagation can be efficiently induced by inoculation with BSE or
vCJD prions, suggesting a smaller barrier to infection (but not to
clinical disease) than hitherto thought (Collinge et al., 1995 ) in
humans of the PRNP 129MM genotype. Humans infected with BSE prions, but
who became asymptomatic carriers, may nevertheless pose a threat of
iatrogenic transmission via medical and surgical procedures.
Alternatively, it is possible that the lifespan of the laboratory mouse
is insufficient to allow expression of clinical disease in most
inoculated mice, whereas a higher proportion of infected humans might
survive the incubation period to develop clinical signs of disease.
Serial passage studies and titration of prions in these mice are in
progress to study this further.

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 florid PrP plaques, can be
recapitulated on BSE or vCJD transmission to these mice. However, the
most surprising aspect of the studies was the finding 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 sub-type of sporadic CJD. This finding 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
these 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 casecontrol studies with stratification 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 sub-types in both BSE-affected and unaffected countries
in the light of these findings. 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 passage.

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 identified 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 findings
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 progress.

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 indistinguishable from naturally occurring prion
strains. It remains of considerable concern whether BSE has transmitted
to, and is being maintained in, European sheep flocks. 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

THE new findings of BASE in cattle in Italy of Identification of a
second bovine amyloidotic spongiform encephalopathy: Molecular
similarities with sporadic Creutzfeldt-Jakob disease

Adaptation of the bovine spongiform encephalopathy agent to primates
and comparison with Creutzfeldt- Jakob disease: Implications for
human health

THE findings from Corinne Ida Lasmézas*, [dagger] , Jean-Guy Fournier*,
Virginie Nouvel*,

Hermann Boe*, Domíníque Marcé*, François Lamoury*, Nicolas Kopp [Dagger

] , Jean-Jacques Hauw§, James Ironside¶, Moira Bruce [||] , Dominique

Dormont*, and Jean-Philippe Deslys* et al, that 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;

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
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
CreutzfeldtJakob 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
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
involvement of the host genome in modulating prion strain selection and
in mice. It is possible that multiple disease phenotypes may also be
possible in
BSE prion infection in humans and other animals.



Several strains of sheep scrapie and human CJD have been
described, yet BSE in the UK is thought to be caused by a
single prion strain (Bruce et al., 1994, 1997; Collinge et al.,
Fig. 2. Bar graph showing relative proportions of di- (white), mono-
(black) and unglycosylated (grey) PrP following partial digestion
with proteinase K. Data are plotted as mean±SEM (n=5).
2474 Journal of General Virology 85
S. E. Lloyd and others
1996; Hill et al., 2003). However, recent data from French
and Italian cattle suggest that more than one strain of BSE
may exist (Biacabe et al., 2004; Casalone et al., 2004). Strain
typing may be carried out in many ways (Safar et al., 1998;
Peretz et al., 2002) but strains were originally defined and
classified by their characteristics on passage to particular
inbred lines of mice based on incubation time and patterns
of neuropathological targeting, and more recently have been
distinguished by PrPSc type on Western blots. A mouse
prion strain difference should only be assessed in the same
line of inbred mouse, as genetic background is known to
modulate all the defining features of a prion strain (Bruce,
1993; Moore et al., 1998). Based on these criteria, we have
isolated two distinct strains from cattle BSE, which we have
designated MRC1 and MRC2, respectively. MRC1 showed a
mono-glycosylated-dominant PrPSc type on Western blots.
It was derived from a primary BSE passage in SJL/OlaHsd
mice and had a relatively short incubation time and a
Fig. 3. Neuropathological analysis of brain from SJL mice inoculated
with BSE following two passages in C57BL/6 (a, c, e)
or SJL (b, d, f) mice. (a, b) Haematoxylin and eosin (H&E)-stained
sections of the hippocampus showing spongiform
neurodegeneration. Arrowheads in (a) indicate neuronal loss. (c, d) PrP
immunohistochemistry showing the distinct laminar
distribution pattern of abnormal PrP in the cortex of
C57BL/6RC57BL/6RSJL-passaged BSE (c), while the PrP
immunoreactivity in SJLRSJLRSJL-passaged BSE is uniformly distributed
(d). The cerebellum of the former group (e) mainly shows
plaques and very little diffuse staining, while the latter group (f)
predominantly shows diffuse staining and no plaques. Bars,
450 mm (ad), 110 mm (e, f). 2475
Two BSE-derived prion strains
generalized diffuse pattern of PrP immunostaining in the
brain. MRC2 shows a di-glycosylated-dominant PrPSc type
on Western blots, was derived from a primary BSE passage
in C57BL/6JOlaHsd mice and had a relatively long
incubation time and showed a distinctive pattern of PrP
immunoreactivity and neuronal loss.
SJL mice were able to support both the MRC1 and MRC2
strain patterns. However, it will be important to establish
whether these strains are stable on further passage in SJL and
other strains of mice. Both C57BL/6 and SJL mice share the
same PrP amino acid sequence and therefore the strain
selection must be a feature of other genetic loci. Within
the limits of resolution available from Western blotting,
the fragment sizes following proteinase K digestion were the
same for the PrPSc associated with both strains, raising the
possibility that the differences related to glycosylation and
not to gross conformational differences, at least as differentiated
by proteinase K digestion. Additional studies will
be required to characterize precisely the conformation and
physico-chemical properties of PrPSc associated with these
prion strains.
Although recent reports suggest that alternative strains of
BSE can be found in cattle, previous studies have suggested
that BSE is caused by a single strain of agent (Bruce et al.,
1994; Biacabe et al., 2004; Casalone et al., 2004). 301V
and 301C represent previously reported independent BSE
strains; however, these were propagated in different strains
of mice, which not only had very different genetic backgrounds
(VM/Dk and C57) but also had two amino acid
coding differences in PrP and therefore their strain characteristics
cannot be directly compared. It is possible that
MRC2 represents the same strain as 301C; however, formal
comparisons will be required to investigate this.
The strain characteristics associated with MRC1 resemble
those seen with passage of Chandler/RML scrapie in these
strains of mice with respect to incubation time (122±1 days),
histology and PrPSc type on Western blotting (unpublished
data). We are confident that our findings are not the result
of contamination with mouse scrapie as these transmissions
were performed in accordance with rigorous biosafety
protocols for preparation of inocula, inoculations and care
of mice. Disposable equipment was used for each inoculum
and each mouse was identified with a unique transponder.
In addition, the MRC1 strain was also seen on primary
passage to RIIIS mice and also on primary passage of vCJD
to SJL mice (Asante et al., 2002). vCJD is caused by a BSElike
prion strain (Hill et al., 1997; Bruce et al., 1997; Collinge
et al., 1996); therefore, it is not surprising that the same
phenomenon is seen with both vCJD and BSE prions
in the same strains of mice. Further transmission studies
are under way to characterize these vCJD-derived mouse
strains. A similar bifurcation of the BSE strain characteristics
was also observed with independent BSE inocula in
transgenic mice expressing human PrP with methionine
at codon 129, Tg(HuPrP129M+/+Prnp0/0)-35 (Asante
et al., 2002).
MRC2 and MRC1 were derived from different sources of
BSE prions, I038 and I783, respectively. I038 originated
from a pool of five infected cow brainstems. However, our
extensive transmission studies with these isolates excluded
the pooling of material from five cows as the source of
the strain variation that we observe in these transmissions.
I038 gave the classical BSE signature and the novel phenotype
was derived from I783, which originated from a single
cow brain. I783 has been used for transmissions in several
inbred lines of mice in our laboratory and consistently
produces the MRC2 strain (as determined by Western
blotting) in C57BL/6JOlaHsd, FVB/NHsd, NZW/OlaHsd,
SM/J and SWR/OlaHsd mice. However, it also consistently
produces the MRC1 strain (as determined by Western
blotting) in SJL/OlaHsd and RIIIS/J mice (Fig. 1b, lane 3
and Asante et al., 2002). DNA sequence analysis of the
bovine Prnp gene in I783 confirmed that the animal was
homozygous for PrP amino acids, thereby excluding PrP
heterozygosity as the source of the strain bifurcation. I038
and other independent BSE inocula have also been shown
to produce two strains in Tg(HuPrP129M+/+Prnp0/0)-35
transgenic mice, which are on a mixed genetic background.
We therefore believe that the prion strain selection or
Fig. 4. Schematic representation of spongiosis
(blue, a, b) and PrPSc deposition (red,
c, d) in the brain for SJL mice inoculated
with BSE following two passages in C57BL/
6 (a, c) or SJL (b, d) mice. For PrPSc
deposition, red represents areas of intense
staining and pink represents areas of lighter
2476 Journal of General Virology 85
S. E. Lloyd and others
mutation is a feature of the genetic background of the mice
and studies are under way to identify the genes involved.
Although recent data suggest that more than one strain of
BSE may exist in cattle (Biacabe et al., 2004; Casalone et al.,
2004), in this study, both I038 and I783 appeared to be the
same single strain. However, Western blotting was unable to
determine whether a mixed population of strains already
existed in either of these brains or whether the alternative
strains were generated de novo in the mice. The inbred lines
studied to date represent only a small proportion of the
allelic variation that exists in the mouse genome; therefore,
the study of other strains of mice, particularly those derived
from unrelated strains such as trapped wild mice, may reveal
additional BSE-derived strains.
The human population exposed to BSE has a much more
diverse genetic background than can be observed using
laboratory strains of inbred mice. Therefore, it is possible
that BSE infection may be revealed not only as the type 4
PrPSc and associated vCJD clinicopathological phenotype
seen to date (Collinge et al., 1996; Hill et al., 2003), but in
future, may also present with alternative PrPSc types, incubation
times and neuropathology that may not be distinguishable
from sporadic CJD (Hill et al., 1997; Asante et al.,
2002) or represent additional novel phenotypes (Collinge,
1999). BSE may also have infected sheep flocks in the UK
and, given the genetic diversity of sheep breeds, it is also
possible that in some breeds BSE propagates as a strain type
that is indistinguishable from natural sheep scrapie.

THE LANCET " Vol 363 " June 19, 2004 "

Identification of possible animal origins of
prion disease in human beings

The transmissible spongiform encephalopathies (TSE or
prion diseases) are fatal transmissible neurodegenerative
diseases of mammals that include sporadic and variant
Creutzfeldt-Jakob disease (sCJD and vCJD,
respectively) in human beings, bovine spongiform
encephalopathy (BSE or mad-cow disease) in cattle, and
scrapie in sheep. Definitive diagnosis of a TSE infection
largely depends on the detection of abnormal prion protein
(PrPSc), a conformational isoform of normal mammalian
prion protein (PrPC). PrPSc accumulates mainly in tissues of
the central nervous and lymphoreticular systems. Different
strains of TSE agent are defined by several properties of
brain-derived protease-digested PrPSceg, size, glycosylation,
and conformationand by in-vivo phenotypes such
as neuropathology, disease incubation times, and patterns
of PrPSc accumulation (figure).16 These criteria have been
used with epidemiological data to link vCJD to BSE, and to
identify a single strain of TSE agent as probably responsible
for both diseases.4,7,8

Cristina Casalone and colleagues report1 is the latest in a
flurry of studies2,3 that challenge the conclusion that there is
only one strain of BSE. These investigators analysed PrPSc
from the brains of cattle with BSE. An atypical form of the
disease was identified (bovine amyloidotic spongiform
encephalopathy or BASE) that had PrPSc molecules with
different biochemical properties and distribution patterns
within the brain compared with typical cases of BSE
(figure). A further comparison6 of BASE with sCJD in
people revealed that, at least in terms of PrPSc characteristics,
BASE was similar to a particular type of sCJD
sCJD(MV2) (figure). These researchers conclude not only
that BASE is a new strain of BSE, but also that characteristics
of human and animal PrPSc could help to identify
potential risk factors for disease in individuals with sCJD of
unknown origin.

For personal use. Only reproduce with permission from The Lancet
Publishing Group.

Determining whether or not some cases of sCJD are a
consequence of exposure to animal TSE diseases is very
important epidemiologically given concerns that animal
TSE diseases other than BSE might also cause disease in
human beings.9 As such, Casalone and colleagues study
brings to the forefront a long standing question within the
TSE community: can we trace the origin of some sCJD
cases to an animal source based solely on PrPSc characteristics?
In view of the strong link between vCJD and
BSE,4,7,8 the similarity of their PrPSc molecules,5 and the
assumption that PrPSc encodes strain phenotypes, the notion
seems plausible.

However, use of PrPSc characteristics as markers for
tracking transmission of TSE disease between species
depends on those characteristics remaining invariant for any
given TSE strain. There is no question that biologically
cloned or carefully characterised TSE strains maintain their
strain-specific phenotypes when passaged in defined mouse
strains.10 Unfortunately there is also no question that these
phenotypes can change in unpredictable ways once defined
TSE strains are put into different mouse strains or species.10
Such transmissions often yield new neuropathologies or
PrPSc that is different from the original PrPSc,11,12 or absent
altogether.13,14 Thus, once a TSE strain has crossed a
transmission barrier, nothing is certain. In view of this
uncertainty, assuming that cross-species similarities in PrPSc
characteristics are indicative of a common TSE strain is
rather like assuming that two identically wrapped boxes
with the same shape and size must have identical contents.

Far from being invariant, key characteristics of PrPSc,
such as size and glycosylation, can be easily altered by
changing the sequence of PrPC,11,12 its glycosylation state,15
or the infected cell type.15,16 Ironically, BSE is one of the best
examples of how PrPSc characteristics do not necessarily
predict in-vivo phenotypes once a species barrier has been
crossed. Although PrPSc molecules in all animal species
infected with BSE look similar,5 neuropathologically BSE in
cattle is different from vCJD in people4,8 or even BSE in
other mammalian species.8 Such results strongly suggest
that, independent of PrPSc, the infected host plays a large
part in determining the final TSE disease phenotype. Thus
careful transmission studies and meticulous neuropathological
analysis, with defined mouse strains, are
essential for identification of TSE strains.

The rarity of TSE diseases and their extremely long
incubation times make identification of possible exogenous
sources of infection for human sCJD
difficult.17 This situation will probably
remain unchanged until we have a better
understanding of how TSE strain phenotypes
are controlled. The data reported by
Casalone and colleagues1 and others2,3 reemphasise
the importance of BSE testing
to look not only for new strains that could
pose a danger to human beings but also to
understand the pathogenesis of BSE in
cattle. However, as Casalone and colleagues
rightfully state, caution is dictated
when only similarities between PrPSc
molecules are used to conclude that TSE
diseases in different species are related.

Atypical BSE BSE
BASE France Japan vCJD sCJD(MV2) sCJD(MM1)
Size (kDa) ~20 ~20 ~19 ~19 ~2021 ~21 ND
Yes Yes Yes ND No path No No Plaques
28 59 66 ~2 11, 15 8, 10, 15 ~5 Age (years)
Common Rare Rare ND ND ND Rare Peripheral tissue
Characteristics of different BSE and CJD isolates
Ü=approximate. Strains of TSE are defined in part by biochemical
properties of PrPSc
(glycosylation and size), pattern of PrPSc deposition in brain
(plaques), average age of disease
onset, and involvement of peripheral tissues, such as spleen and lymph
nodes. These
characteristics are shown for typical BSE and atypical BSE cases from
cattle in Italy (BASE),1
France,2 and Japan,3 and for three different human CJD strains, vCJD4,5
sCJD(MV2),6 and
sCJD(MM1).6 Glycosylation: Di=diglycosylated, Mono=monoglycosylated,
Height and intensity of bar (black=highest, light grey=lowest) indicate
relative abundance of
each band for given strain. Size: size of unglycosylated PrPSc in kDa.
ND=not done, No path=no
BSE-associated pathology in brain. Data are adapted and simplified from
references indicated.

We have no conflict of interest to declare.

*Suzette A Priola, Ina Vorberg
Laboratory of Persistent Viral Diseases, Rocky
Mountain Laboratories, National Institute of Allergy
and Infectious Diseases,
National Institutes of Health, Hamilton, MT 59840,
USA (SAP); and Institute of Virology, Technical
University of Munich, Munich, Germany (IV)

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second bovine amyloidotic spongiform encephalopathy:
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2 Biacabe A-G, Laplanche JL, Ryder S, Baron T. Distinct
molecular phenotypes in bovine prion diseases. EMBO Rep
2004; 5: 11014.
3 Yamakawa Y, Hagiwara K, Nohtomi K, et al. Atypical
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J Pathol 2004; 202: 000000
Published online in Wiley InterScience (
DOI: 10.1002/path.1580
Original Paper
Prevalence of lymphoreticular prion protein
accumulation in UK tissue samples
David A Hilton,1* Azra C Ghani,2 Lisa Conyers,1 Philip Edwards,1 Linda
McCardle,3 Diane Ritchie,3
Mark Penney,1 Doha Hegazy1 and James W Ironside3
1Department of Histopathology, Derriford Hospital, Plymouth, UK
2Department of Infectious Disease Epidemiology, Faculty of Medicine,
Imperial College, London, UK
3National CJD Surveillance Unit, University of Edinburgh, Edinburgh, UK
*Correspondence to:
Dr David A Hilton, Department
of Histopathology, Derriford
Hospital, Plymouth, PL6
8DH, UK.
Received: 19 February 2004
Revised: 15 March 2004
Accepted: 22 March 2004
This study aims to provide an estimate of the number of individuals in
the UK who may
be incubating variant Creutzfeldt-Jakob disease and at risk of causing
iatrogenic spread
of the disease. Lymphoreticular accumulation of prion protein is a
consistent feature of
variant Creutzfeldt-Jakob at autopsy and has also been demonstrated in
the pre-clinical
phase. Immunohistochemical accumulation of prion protein in the
lymphoreticular system
remains the only technique that has been shown to predict neurological
disease reliably
in animal prion disorders. In this study, immunohistochemistry was used
to demonstrate
the presence of prion protein, with monoclonal antibodies KG9 and 3F4,
in surgically
removed tonsillectomy and appendicectomy specimens. The samples were
collected from
histopathology departments across the UK and anonymised prior to
testing. Samples were
tested from 16 703 patients (14 964 appendectomies, 1739
tonsillectomies), approximately
60% of whom were from the age group 2029 years at operation.
Twenty-five per cent of the
samples were excluded from the final analyses because they contained
inadequate amounts
of lymphoid tissue. Three appendicectomy samples showed lymphoreticular
of prion protein, giving an estimated prevalence of 3/12 674 or 237 per
million (95%
CI 49692 per million).

The pattern of lymphoreticular accumulation in two of these
samples was dissimilar from that seen in known cases of variant
Creutzfeldt-Jakob disease.

Although it is uncertain whether immunohistochemical accumulation of
prion protein in
the lymphoreticular system is specific for variant Creutzfeldt-Jakob
disease, it has not been
described in any other disease, including other forms of human prion
disease or a range of
inflammatory and infective conditions. These findings reinforce the
importance of measures
taken by the UK Department of Health to reduce the risk of spread of
variant Creutzfeldt-
Jakob via blood products and surgical instruments, and of the urgency to
proceed with
large-scale screening of fresh tonsil specimens for the presence of
prion protein.
Copyright ? 2004 Pathological Society of Great Britain and Ireland.
Published by John
Wiley & Sons, Ltd.
Keywords: Creutzfeldt-Jakob disease (CJD); prion; screening;


Journal of Virological Methods 117 (2004) 2736

Atypical scrapie cases in Germany and France are identified
by discrepant reaction patterns in BSE rapid tests

A. Buschmanna, A.-G. Biacabe b, U. Ziegler a, A. Bencsik b, J.-Y. Madecb,
G. Erhardt c, G. Lühken c, T. Baron b, M.H. Groschup a,?
a Federal Research Centre for Virus Diseases of Animals, Institute for
Novel and Emerging Infectious Diseases,
Boddenblick 5a, 17493 Greifswald-Insel Riems, Germany
b AFSSA-Lyon Unité Virologie-ATNC, 31 Avenue Tony Garnier, 69364 Lyon
Cedex 07, France
c Department of Animal Breeding and Genetics, Justus-Liebig University
Giessen, Ludwigstr. 21B, 35390 Giessen, Germany
Received 27 August 2003; received in revised form 13 November 2003;
accepted 18 November 2003


The intensified surveillance of scrapie in small ruminants in the
European Union (EU) has resulted in a substantial increase of the number
of diagnosed cases. Four rapid tests which have passed the EU evaluation
for BSE testing of cattle are also recommended currently and used for
the testing of small ruminants by the EU authorities. These tests
include an indirect ELISA (cELISA), a colorimetric sandwich ELISA
(sELISA I), a chemiluminescent sandwich ELISA (sELISA II), and a Western
blot (WB). To this point, the majority of samples have been screened by
using either sELISA I (predominantly in Germany) or WB (predominantly in
France). In this study, it is shown that a number
of the German and French scrapie cases show inconsistent results using
rapid and confirmatory test methods. Forty-eight German sheep, 209
French sheep and 19 French goat transmissible spongiform encephalopathy
(TSE) cases were tested. All cases were recognised by the sELISA I and
either one of the confirmatory methods (scrapie-associated fibrils
(SAF)-immunoblot or immunohistochemistry). Surprisingly, three rapid
tests failed to detect a significant number of scrapie cases (29 in
France and 24 in Germany). The possible reasons for these inconsistent
reaction patterns of scrapie cases are discussed. Similar discrepancies
have not been observed during rapid testing of cattle for BSE, the
disease for which all diagnostic methods applied have been evaluated.
© 2003 Elsevier B.V. All rights reserved.
Keywords: Scrapie; Prion protein; Rapid test; Confirmatory method
1. Introduction


Richard T. Johnson
Pioneer neuroscientist says major improvements are needed in U.S. prion
research programs


Johnson stresses that it is especially important to do more research on
CWD, which is rapidly spreading among deer and elk populations.
Colleagues in Europe say, You are potentially sitting on a time bomb
[CWD] and doing nothing about it,  he says. He notes several reasons
why the disease is worrisome: It is more infectious in the field than
any of the other TSEs; deer can contract the disease merely from living
in a paddock where a CWD-infected deer lived previously. Also, CWD can
move across species barriers. When lab monkeys and mice are inoculated
with brain tissue from CWD-infected deer, they become infected and die,
Johnson says. Furthermore, hunters eat deer and elk, upscale restaurants
serve their meat, and cattle share fields with them, he says.

Researchers at the Centers for Disease Control & Prevention assume that
if CWD is transmissible to humans, it would cause vCJD. But Johnson
says, Actually, nobody has any idea what symptoms CWD might cause in
humans. It might be burning sensations in elderly ladies feet. The only
way we are going to find out is to increase autopsy rates in this
country. Autopsies, which are disappearing, should be done on all
people with chronic neurological diseases, he says.

It would be really useful to do strain typing on the prions from any new
cases of mad cow disease that show up in the U.S., Johnson says.
Scientists need to know whether the U.S. prion strain is the same
virulent strain that arose in England or if it is perhaps a milder form
that arose spontaneously in the U.S., he says. It could be a strain that
occurs intermittently and causes no disease in humans, he notes. That
would be reassuring and help to restore confidence in American beef.

Chemical & Engineering News
ISSN 0009-2347
Copyright © 2004

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 59840,
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 2ID-Lelystad,
Institute for Animal Science and Health, Lelystad, The Netherlands
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 PrP-sen
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 were
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 CWD
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 sheep
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 infectivity,
the susceptibility of humans may still be very low compared with
cattle, which would be consistent with the relatively inefficient
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 been
recommended for other animal TSEs.


Perceptions of Unconventional Slow Virus
disease of animals in the USA

Further CWD transmissions were carried out by Dick Marsh into
ferret, mink, and squirrel monkey. Transmission occured in all
of these species with the shortest incubation period in the


see full text;

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.


Since previous incidences of TME were associated with common or shared
feeding practices, we obtained a careful history of feed ingredients
used over the
past 12-18 months. The rancher was a "dead stock" feeder using mostly
(>95%) downer or dead dairy cattle and a few horses. Sheep had never
been fed.
Experimental Transmission. The clinical diagnosis of TME was confirmed
by histopaihologic examination and by experimental transmission to mink
after incubation periods of four months. To investigate the possible
involvement of cattle in this disease cycle, two six-week old castrated
Holstein bull
calves were inoculated intracerebrally with a brain suspension from
affected mink.
Each developed a fatal spongiform encephalopathy after incubation
periods of 18 and 19 months.

MAY 2002 431 NEWS

TSE threat to US increase

The US Department of Agriculture last month confirmed that two sheep
taken from a farm in Vermont were infected with a form of transmissible
spongiform encephalopathy (TSE). Further tests are being carried out to
determine whether the disease is bovine spongiform encephalopathy (BSE)
or scrapie. The sheep were imported from the Netherlands (Nature Med. 6,
1301; 2000). Analysis will take at least two years, and if the prion is
that which causes BSE, this would be the first case of disease in the US.

Aguzzi warns of CWD danger

The TSE family of diseases also includes chronic wasting disease (CWD)
in deer, a condition that has spread in the US in recent years (Nature
416, 569; 2002). Speaking at the Days of Molecular Medicine conference
in La Jolla in March, prion expert Adriano Aguzzi issued a strong
warning against underestimating this form of TSE.

"For more than a decade, the US has by-and-large considered mad cows
to be an exquisitely European problem. The perceived need to protect
US citizens from this alien threat has even prompted the deferral of
blood donors from Europe," he said. "Yet the threat-from-within
posed by CWD needs careful consideration, since the evidence that CWD
is less dangerous to humans than BSE is less-than-complete. Aguzzi
went on to point out that CWD is arguably the most mysterious of all
prion diseases.

"Its horizontal spread among the wild population is exceedingly
efficient, and appears to have reached a prevalence unprecedented even
by BSE in the UK at its peak. The pathogenesis of CWD, therefore,
deserves a vigorous research effort. Europeans also need to think
about this problem, and it would be timely and appropriate to increase
CWD surveillance in Europe too." Aguzzi has secured funding from the
National Institutes of Health to investigate CWD, and the effort will
be lead by Christina Sigurdson in his department at the University of

Simon Frantz, London

B) 2002 Nature Publishing Group NUMBER 5 R VOLUME 8

This quote from Dr. Gambetti is especially significant since he is the
rather cautious TSE researcher under contract with the Centers for Disease
Control to examine the brains of individuals who have died of CJD.


Pierluigi Gambetti, director of the National Prion Disease Pathology
Surveillance Center at Case Western Reserve University in Cleveland,
said all deer should be tested for chronic wasting disease before any
processing is done.

"There is no way around it," he said. "Nobody should touch that meat
unless it has been tested."

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

PMID: 6997404

RE-Monitoring the occurrence of emerging forms of Creutzfeldt-Jakob
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 reportable
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?

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

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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


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

Asante/Collinge et al, 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;

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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 SUBMISSION
TO DOCKET 2003N-0312]

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TSS 1/27/03 (0)

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Accepted - Volume 1

I am no Doctor, I have no PhDs and I am President/CEO of nothing...

Thank You,

I am sincerely,

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

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