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From: Terry S. Singeltary Sr. (216-119-143-74.ipset23.wt.net)
Subject: CWD 2004 Annual Report
Date: January 3, 2005 at 10:03 am PST

In Reply to: SCRAPIE 2004 ANNUAL REPORT ARS posted by Terry S. Singeltary Sr. on January 3, 2005 at 10:01 am:

-------- Original Message --------
Subject: CWD 2004 Annual Report
Date: Sun, 2 Jan 2005 11:43:59 -0600
From: "Terry S. Singeltary Sr."
Reply-To: Bovine Spongiform Encephalopathy
To: BSE-L@LISTSERV.KALIV.UNI-KARLSRUHE.DE


##################### Bovine Spongiform Encephalopathy #####################

Research Project: Prp-Cwd Profiles in White Tailed Deer and Elk
with Naturally Occuring Chronic Wasting Disease


Location:

Animal
Diseases Research


2004 Annual Report

What were the most significant accomplishments this past year?
D. What were the most significant accomplishments this past year? This
report serves to document research conducted under a specific
cooperative agreement between ARS and Colorado State University. Chronic
wasting disease has traditionally been diagnosed by histological
examination of the brain, although in TSE-infected sheep and mule deer,
the brain accumulates the marker prion protein months to years after the
lymph nodes. In collaboration with Colorado State University (Spraker),
the Canadian Food Inspection Agency, and the Nebraska Division of Game
and Parks, we established that the tonsils and retropharyngeal lymph
nodes of white tailed deer are a reliable early indicator of infection.
However, we demonstrated that a relatively high percentage of elk with
positive tests on brain tissue have negative tests of the
retropharyngeal lymph node. Therefore, accurate diagnosis of elk
requires examination of both the brain and the lymph node. This report
serves to document research conducted under a specific cooperative
agreement between ARS and Colorado State University. Additional details
of research can be found in the report for the parent project
5348-32000-021-00D Animal Health.

http://www.ars.usda.gov/research/projects/projects.htm?ACCN_NO=405528&fy=2004

Research Project: The Role of Environmental Contamination in
Transmission of Chronic Wasting Disease


Location:

Animal
Diseases Research


2004 Annual Report

What were the most significant accomplishments this past year?
D. This report serves to document research conducted under a specific
cooperative agreement between ARS and Edward's Ranch. Transmission
routes of CWD are not known. We cannot rule out the possibility of
transmission through contact with contaminated feed or watering areas
(premises contamination). A long-term specific cooperative agreement
(Edward's) has been initiated in a fenced facility in Nebraska to
address this possibility. All animals (white tailed deer, mule deer, and
elk) were removed from the facility, which consists of several
separately fenced pastures. CWD was diagnosed in all 3 species;
approximately 50% of the deer in one of the fenced areas were positive
for CWD. This area has been securely double fenced and has been
repopulated with a sentinel species (Elk) to test the hypothesis that
under the environmental conditions of Edward's Ranch, CWD is
transmissible from the environment. Elk harvested to date have tested
negative for CWD. Additional details of research can be found in the
report for the parent project 5438-32000-021-00D Animal Health.

http://www.ars.usda.gov/research/projects/projects.htm?ACCN_NO=405855&fy=2004

Research Project: A Transgenic Mouse Model for Chronic Wasting Disease


Location:

Animal
Diseases Research


2004 Annual Report

What were the most significant accomplishments this past year?
D. This report serves to document research conducted under a specific
cooperative agreement between ARS and the University of Washington. Our
goal is the development of transgenic CWD mice for testing the
relationship between 132L in elk and resistance to CWD infection. We've
cloned a full copy of the Elk prion gene from an Elk with CWD into
pBeloBACC11 and have DNA insert constructs. The constructs are being
amplified and readied for embryo injection. Secondly, as part of the
agreement we are testing the hypothesis that Elk CWD is transmissible to
mink. This experiment was initiated one year ago and we are observing
for clinical signs. This experiment serves to determine the
transmissibility between these two natural occurring TSEs. Robert
Harrington, DVM was funded by NIH (salary and limited supplies, KO8 with
Don Knowles serving as his sponsor) to carry out this work. Additional
details of research can be found in the report for the parent project
5348-32000-021-00D Animal Health.

http://www.ars.usda.gov/research/projects/projects.htm?ACCN_NO=405724&fy=2004

Research Project: Transmissibility of Chronic Wasting Disease to Fallow
Deer and to Elk with Different Prion Protein Genotypes


Location:


Virus
and Prion Diseases of Livestock


2004 Annual Report

What were the most significant accomplishments this past year?
D. Progress Report. This report serves to document research conducted
under a reimbursable agreement between ARS and the USDA-Animal and Plant
Health Inspection Service, Veterinary Services. Additional details of
research can be found in the report for the parent project
3625-32000-066-00D, Bovine Spongiform Encephalopathy and Other
Transmissible Spongiform Encephalopathies. This is a 5-year study with 2
objectives. The first is to determine if fallow deer are susceptible to
chronic wasting disease (CWD). As an initial experiment, 13 fallow deer
were inoculated intracerebrally with CWD agent from either elk or
white-tailed deer and the animals are currently under observation. Thus
far, no fallow deer have shown symptoms or lesions of CWD. The second
objective of this project is to evaluate whether differences in a
specific DNA sequence of the prion protein (PrP) gene will affect
susceptibility to development of CWD. A group of 8 elk with different
PrP amino acid genotypes at codon 132 (MM, LM, or LL) were inoculated
orally and are currently under study. Thus far both MM elk developed
CWD, both LM elk are showing clinical signs of wasting (consistent with
CWD), and all 4 LL elk are clinically normal at this time. This work is
part of a larger effort within the parent project that is devoted to
studies of the pathobiology of transmissible spongiform encephalopathy
(TSE) agents in both natural and secondary host species.

http://www.ars.usda.gov/research/projects/projects.htm?ACCN_NO=405933&fy=2004

Research Project: Characterization of Strains of Chronic Wasting Disease
in North American Cervids


Location:


Virus
and Prion Diseases of Livestock


2004 Annual Report

What were the most significant accomplishments this past year?
D. Progress Report. This report serves to document research conducted
under a specific cooperative agreement between ARS and the Institutes
for Animal Health (IAH), Edinburgh, UK. Additional details of research
can be found in the report for the parent project 3625-32000-066-00D,
Bovine Spongiform Encephalopathy and Other Transmissible Spongiform
Encephalopathies. Three brain samples of chronic wasting disease (CWD)
have been received at the IAH's Neuropathogenesis Unit: two from mule
deer (ID nos 12776 and 12783) and one from a North American Elk (ID no.
98RA265). Initial bacteriological screening indicated that two of these
(12776 and 98RA265) were suitable for injection into mice. The third (ID
no 12783) was bacterially contaminated to a degree that precluded
injection into mice. The two suitable samples were injected into panels
of inbred mouse strains (RIII, C57BL, VM and C57BLxVM) in September 2003
(98RA265) and November 2003 (12776). The mice are being maintained and
clinically monitored and remain healthy at the time of writing. Four
other samples from mule deer, white tailed deer and elk have been
collected at the ARS's National Animal Disease Center for inclusion in
the study. Shipment to the IAH's Neuropathogenesis Unit is awaiting the
issue of an importation permit.

http://www.ars.usda.gov/research/projects/projects.htm?ACCN_NO=405904&fy=2004

Title: Experimental Cross-Species Transmission of Chronic Wasting
Disease (Cwd) at the National Animal Disease Center (Nadc), Ames, Iowa:
An Update Author
item Hamir, Amirali

http://www.ars.usda.gov/research/publications/publications.htm?seq_no_115=167763

Research Project: Bse and Other Tses Ruminant Herbivors: Diagnosis,
Transmission and Genetics


Location:

Animal
Diseases Research


2004 Annual Report

1. What major problem or issue is being resolved and how are you
resolving it (summarize project aims and objectives)? How serious is the
problem? What does it matter?
Bovine spongiform encephalopathy (BSE) is a fatal disease of cattle,
first reported in the United Kingdom and now found in many European
countries and Japan, with isolated cases in Canada and the United
States. The disorder is related to consumption of feed contaminated with
the carcasses of BSE-infected cattle. Bans on recycling ruminant
material in ruminant feed are effective in reducing the number of
affected animals. However, identification of even a single case results
in economically devastating restriction in international trade, limits
the availability of some byproducts derived through rendering carcasses,
and increases the need for costly surveillance. Chronic wasting disease
(CWD) is another member of the TSE family of disorders, with a more
limited host range and no evidence to date of a zoonotic potential. CWD
is reported in free ranging and farm raised deer and elk in the U.S. and
Canada. CWD is a relatively uncharacterized TSE with novel patterns of
transmission and agent distribution, as well as providing the
considerable challenge of a controlling a persistent disease in free
ranging wildlife.

The objectives of the project are to develop inexpensive, high capacity
live animal and postmortem diagnostic tests, prion detection methods for
monitoring environmental contamination, and pathogenesis/transmission
experiments that provide direction to intervention techniques and
reagents. Control of all domestic prion diseases is important in
reducing trade barriers for U.S. sheep and germplasm and for assuring
the present and future global markets for cattle.

The goals of the project are (i) to develop diagnostic strategies that
contribute to disease control in captive and free ranging animals, (ii)
to identify genetic factors involved in resistance to disease and
reduction of transmission from infected animals, and (iii) to determine
routes of transmission and disease persistence in the population and the
environment.

These goals will be met through the following specific objectives: (1)
development of higher throughput, very inexpensive PrPd detection
methods, to be used diagnostically for postmortem and live animal
testing and in transmission and environmental contamination studies (2)
an efficient biological assay model using a panel of species-specific
transgenic mice, in which the murine gene is replaced by the appropriate
cervid PrP gene for genetic resistance and transmission studies; and (3)
characterization of the cervid PrP genes for identification of alleles
associated with genetic resistance

Our research concerning the transmissible encephalopathies addresses the
elements of our National Program in Animal Production, Product Value and
Safety. 103 Animal Health 100%, Pathogen Detection and Diagnostics,
Genetic Resistance to Disease, Epidemiology of Disease, and Strategies
to Control Infectious and Non-infectious diseases.. It also relates to
National Program 108, Food composition and Intake Food Safety.

The TSEs present a serious problem to US agriculture. Identification of
a single imported cow with BSE in December 2003 resulted in devastating
direct and indirect losses to the cattle, rendering, and pharmaceutical
industries with long term changes in handling of meat and byproducts
implemented by USDA, the Food and Drug Administration, and the Food
Safety Inspection Service. Although CWD affects a far smaller segment of
the agricultural community, the losses of domestic and foreign markets,
restrictions on trade and movement of live animals, and the substantial
costs associated with surveillance of hunter-killed animals represent
major economic losses for the cervid farming and hunting industries.
Agriculture and public health will benefit from reduced disease
prevalence, cost-effective diagnostic and surveillance methods,
prevention measures, and appropriate handling of facilities to reduce
the possibility of re-infection.

2. List the milestones (indicators of progress) from your Project Plan.
Year 1 (FY2003)

Develop monoclonal antibody and target binding molecules for PrP-d
detection.

Transgenic mouse (elk gene) development: construct preparation and
embryonic cell development.

Define the PRNP alleles in white tailed deer.

Identify a potentially contaminated cervid holding area, provide
appropriate biosecurity to insure that no wild ruminant animals enter or
leave the facility from the surrounding area, and restock with
susceptible species

Year 2 (FY2004)

Develop suitable assays for PrP-d detection.

Transgenic mouse (elk gene) development: characterization of homozygous
founder stock.

Describe the organization of the mule deer PRNP gene.

Prepare construct for white tailed deer transgenic mouse.

Monitor livestock in potentially contaminated facility.

Initiate oral exposure of mink to the CWD agent from elk.

Year 3 (FY2005)

Develop applications for PrP-d detection assays in animal tissues and
environmental samples.

Transgenic mouse development (elk gene): assay selected mouse lines.

Prepare construct for mule deer transgenic mouse.

Continue to monitor animals in potentially contaminated facility.

3. Milestones:
A. List the milestones that were scheduled to be addressed in FY 2004.
Which were fully or substantially met?

The milestones below were scheduled for FY2004 and have been partially met.

Develop suitable assays for PrP-d detection. This work is in progress.
We have developed assays using immunohistochemistry and enzyme-linked
immunosorbent assay. These techniques have not been shown to be
sensitive or specific enough to detect a marker in the blood of
naturally infected sheep. Additional work with more sensitive techniques
including mass spectroscopy and infrared spectroscopy will be performed
in year 2.

Transgenic mouse (elk gene) development: characterization of homozygous
founder stock. Prepare construct for white tailed deer transgenic mouse

Transgenic mouse development is progressing but is still in stage 1
(development of defined constructs and embryic stem cells.

The following milestones were described for FY 2004 and have been fully met.

Describe the organization of the mule deer PRNP gene.

Monitor livestock in potentially contaminated facility.

Initiate oral exposure of mink to the CWD agent from elk.

B. List the milestones that you expect to address over the next three
years. What do you expect to accomplish year by year?

Development and application of assays for diagnostic testing (FY2005)
and field testing of candidate assays (FY2006 and FY2007).

Development of assays for environmental contamination (FY2005 and 2006)
with remedial measures evaluated (FY2007).

Transgenic mouse development (elk and gene) with founder stock developed
in FY2005 and bioassays performed in FY2006.

Elk in potentially contaminated facility monitored in every year of the
project.

The potential for transmission of BSE through the placenta of
experimentally infected sheep will be evaluated (FY2005).

4. What were the most significant accomplishments this past year?
A. Genetics can be an important component of TSE control programs if
some polymorphisms in the prion gene are associated with reduced disease
incidence. In conjunction with Colorado State University, private
landowners (the Richard Edwards family), the Canadian Food Inspection
Agency, and the Nebraska Department of Game and Parks, the Animal
Disease Research Unit identified susceptible prion genotypes in elk,
white tailed deer and mule deer, characterized the first reported prion
pseudogene in placental mammals, and developed protocols for
differentiating between the functional gene and the pseudogene. We
demonstrated that all reported genotypes of mule deer are susceptible,
identified the single genotype in white tailed deer associated with a
reduced susceptibility to disease and demonstrated that elk of the most
resistant genotype can be affected by disease under some farm
conditions. This accomplishment supports the state and federal
regulatory agencies' control programs, and allows research laboratories
to correctly genotype cervid livestock.

B. Diagnostic testing is performed on a limited number of tissues and
correct identification of the most appropriate target tissue for each
species is critical for accurate surveillance. In collaboration with
Colorado State University, the Canadian Food Inspection Agency, and the
Nebraska Division of Game and Parks, the Animal Disease Research Unit
described the distribution of PrP-Sc in large populations of captive elk
and free ranging white tailed deer. This study demonstrated that the
most accurate testing of elk requires examination of the both the brain
and the retropharyngeal lymph node but testing of white tailed deer can
be accomplished by examination of the retropharyngeal lymph node alone.
This work provides the scientific basis for tissue selection for
diagnostic testing of elk and white tailed deer, complementing the
previous study on mule deer testing.

2) Although the transmission routes of CWD are unknown, placentas shed
by infected deer or elk are potential sources of environmental
contamination. Testing of reproductive tissues for assessing prion
contamination is performed in collaboration with Colorado State
University and the University of Idaho. These studies have shown no
evidence that CWD is transmitted by contact with the placenta of
infected females, as is seen in sheep scrapie.

3) Large and small carnivores feed on dead deer and elk in the CWD
endemic area. If these animals are susceptible to a TSE following oral
exposure to the agent in the tissues of dead deer or elk, they represent
another reservoir of disease. Under a cooperative agreement with the
University of Washington, mink were exposed to tissues from CWD-positive
elk. The incubation period is expected to exceed 18 months and results
are expected in FY2005.

C. Significant activities that support special target populations.

None

D. Progress Report opportunity to submit additional programmatic
information to your Area Office and NPS (optional for all in-house (¿D¿)
projects and the projects listed in Appendix A; mandatory for all other
subordinate projects).

None

5. Describe the major accomplishments over the life of the project,
including their predicted or actual impact.
Diagnostic test development: Postmortem diagnostic test methodology was
developed and transferred to the regulatory agencies for use in the U.S.
The test was modified for use in live deer, although the test requires
general anesthesia and re-trapping of deer found to be positive.
Monoclonal antibodies useful in assays on routinely formalin fixed
tissue from infected sheep, deer, elk, cattle, humans, mink, domestic
cats and a wide variety of captive wildlife potentially exposed to prion
diseases were developed. The technology was transferred to USDA, Animal
Plant Health Inspection Service, which contracts with a network of 20
veterinary diagnostic laboratories to provide surveillance for CWD in
the US. Gold standard testing of cattle for BSE is conducted only at the
National Veterinary Services Laboratory in Ames, IA. The technology and
antibodies are also used by the Canadian Food Inspection Agency in
Nepean, Ontario, and the Foreign Animal Disease Diagnostic Laboratory in
Winnipeg.

CWD genetics: We provided the first report of a processed prion
pseudogene in livestock, allowing research laboratories and wildlife
managers to correctly identify the prion functional gene in wildlife.
Using the appropriate testing technology, we demonstrated that none of
the reported genotypes of cervids are associated with disease resistance
and genetic measures cannot be a component of the federal CWD control
program. However, one genotype in elk is associated with very low
disease prevalence and it is possible that selection for this genotype
in captive elk with very minor potential exposure to CWD would reduce
the disease incidence.

6. What science and/or technologies have been transferred and to whom?
When is the science and/or technology likely to become available to the
end-user (industry, farmer, other scientists)? What are the constraints,
if known, to the adoption and durability of the technology products?
The antiprion monoclonal antibodies have been licensed to private
companies for development of immunohistochemistry and enzyme linked
immunosorbent assays. An IHC test for BSE using one of the monoclonal
antibodies is licensed and marketed internationally. In the US, TSE
testing is conducted by the federal government or by federally approved
laboratories. All the IHC testing performed in the US for livestock TSEs
is performed with the technology and antibody reagents developed by this
project.

7. List your most important publications in the popular press and
presentations to organizations and articles written about your work.
1. BSE diagnostic testing in the US. Invited presentation to the 12th
Annual Food Safety Farm to Table Conference, May 2004, Moscow, ID

2. BSE Testing Technology. Invited presentation to the Beef Industry
Food Safety Council Industry Summit on Bovine Spongiform Encephalopathy,
Fort Worth, TX, April 26, 2004

3. Is BSE in sheep at threat to the US sheep industry? Invited
presentation to the Sheep and Goat Health Committee Seminar, National
Institute for Animal Agriculture, April 4, 2004, Salt Lake City, UT

4. Prion susceptibility genetics in three cervid species, invited
presentation to the Annual meeting of the American College of Veterinary
Pathologists, Banff, Canada, November 15, 2003

Review Publications
O'Rourke, K.I., Spraker, T.R., Hamburg, L.K., Besser, T.E., Brayton,
K.A., Knowles, D.P. 2004. Polymorphisms in the prion precursor
functional gene but not the pseudogene are associated with susceptibilty
to chronic wasting disease in white-tailed deer. Journal of General
Virology. 85:1339-1346.


Brayton, K.A., O'Rourke, K.I., Lyda, A.K., Miller, M.W., Knowles, D.P. A
processed pseudogene contributes to apparent mule deer prion gene
heterogeneity. Gene. 2003. v. 326. p. 167-173.

Hamir, A.N., Miller, J.M., O'Rourke, K.I., Bartz, J.C., Stack, M.J.,
Chaplin, M.J. 2004. Transmission of transmissible mink encephalopathy
(TME) to raccoons (Procyon lotor) by intracerebral inoculation. Journal
of Veterinary Diagnostic Investigation. 16(1):57-63.

http://www.ars.usda.gov/research/projects/projects.htm?ACCN_NO=404978&showpars=true&fy=2004

Title: Experimental Transmission of Chronic Wasting Disease Agent to
Cattle by Intracerebral Route: Final Outcome of the Study Authors
item Hamir, Amirali

item Kunkle, Robert - bob

item Richt, Juergen

item Cutlip, Randall - ARS RETIRED
item Miller, Janice - ARS RETIRED
item O'Rourke, Katherine

item Williams, Elizabeth - UNIVERSITY OF WYOMING
item Miller, M - COLORADO DIV WILDLIFE
item Stack, Mick - VLA-WEYBRIDGE, UK
item Chaplin, Melanie - VLA-WEYBRIDGE, UK


http://www.ars.usda.gov/research/publications/publications.htm?SEQ_NO_115=167759


TSS
Submitted to: Meeting Abstract
Publication Acceptance Date: September 29, 2004
Publication Date: N/A
Publisher's URL: http://www.ustse2004.com/
Technical Abstract: Thirteen calves were inoculated intracerebrally with
brain suspension from mule deer naturally affected with CWD. Three other
calves were kept as uninoculated controls. The experiment was terminated
at 6 years post inoculation (PI). During that time, prion protein
(PrPres) was found in the central nervous system (CNS) of 5 cattle. None
of these animals had shown any specific clinical signs that were common
to all. Microscopic lesions suggestive of spongiform encephalopathy in
the brains of these PrPres positive animals were subtle in the first 3
and absent in the latter 2 cases. However, all 5 animals were positive
for PrPres by both immunohistochemistry and Western blot. The 3
uninoculated control cattle and 8 other inoculated animals euthanized
during this time did not have PrPres in their CNS. Degenerative changes
indicative of neuroaxonal dystrophy (NAD) were seen in dorsal medulla
oblongata and appeared to be related to advancing age in both inoculated
and control cattle. Analysis of the gene encoding bovine PRNP revealed
similar findings, i.e., homozygosity for alleles encoding 6 octapeptide
repeats, serine (S) at codon 46 and S at codon 146 in all samples.
Findings of this study show that although PrPres amplification occurred
following direct inoculation into the brain, none of the affected
animals had classical histopathological lesions of spongiform
encephalopathy. Furthermore, only 38% of the inoculated cattle
demonstrated amplification of PrPres. Although intracerebral inoculation
is an unnatural route of exposure, and is the most severe challenge
possible, this experiment shows that CWD transmission in cattle can have
long incubation periods (up to 5 years). This finding suggests that oral
inoculation of cattle with CWD may not result in amplification of PrPres
within CNS tissues during the normal lifespan of cattle. It is possible
that a second bovine passage of material (brain infected with CWDmd)
from this study may result in a larger proportion of affected cattle
with a shortened incubation time, and may produce different clinical and
pathological findings. Such a study is now in progress. Also,
experimental inoculations of cattle with CWD isolates from white-tailed
deer and elk are needed to compare clinicopathological findings with the
present study and these studies will be initiated in the near future.


Submitted to: Meeting Abstract
Publication Acceptance Date: September 29, 2004
Publication Date: N/A
Technical Abstract: Experimental cross-species transmission of
transmissible spongiform encephalopathies (TSEs) provides valuable
information for identification of potential host ranges, and generates
much needed prion-infected tissues for research. At NADC, studies
utilizing CWD agent(s) were initiated in 1997. However, since these
studies involve long incubation periods under BL-2 conditions, to date
only one study has been completed. Initially our studies were restricted
to farm livestock (cattle and sheep). However, as a result of increased
demand from our stakeholders, we now also conduct research on wildlife
(herbivores and carnivores). Following are some of the significant
findings of past and on-going experiments at NADC: Completed study:
CATTLE: Intracerebral inoculation of CWD-mule-deer resulted in
amplification of PrPres in a small number of inoculated cattle (5 of 13;
38%). However, none of the animals with PrPres had classic
histopathologic lesions of spongiform encephalopathy. Studies utilizing
CWD-elk and CWD-white-tailed-deer in cattle are in progress. Preliminary
findings of ongoing CWD experiments in other species indicate that: 1.
SHEEP: CWD-mule-deer can be transmitted intracerebrally to sheep (1 of
8; 5 yrs PI). 2. WHITE-TAILED DEER: CWD-elk, CWD-white-tailed-deer, and
CWD-mule-deer are pathogenic for white-tailed deer (60%; 2 yrs PI). 3.
FALLOW DEER: Compared with other cervids studies, appear to be resistant
to intracerebral inoculation of CWD-elk and CWD-white-tailed-deer (0%; 2
yrs PI). White-tailed deer with CWD-mule-deer are pending. 4. RACCOONS:
TME and scrapie can be transmitted within 6 months and 2 years,
respectively, whereas CWD cannot. (Therefore, may be possible to
differentiate these 3 TSE agents in raccoons). Study utilizing BSE in
raccoons is pending.


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