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From: Terry S. Singeltary Sr. (216-119-143-74.ipset23.wt.net)
Subject: SCRAPIE 2004 ANNUAL REPORT ARS
Date: January 3, 2005 at 10:01 am PST

In Reply to: Research Project: Bse Pathogenesis Study Project Number: 3625-32000-073-06 posted by Terry S. Singeltary Sr. on January 1, 2005 at 2:38 pm:

-------- Original Message --------
Subject: SCRAPIE 2004 ANNUAL REPORT ARS
Date: Sun, 2 Jan 2005 11:28:18 -0600
From: "Terry S. Singeltary Sr."
Reply-To: Bovine Spongiform Encephalopathy
To: BSE-L@LISTSERV.KALIV.UNI-KARLSRUHE.DE


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

Research Project: Eradication of Ovine Scrapie Tse Through
Selective Genetics Using a Two-Flock Model


Location:

Animal
Diseases Research


2004 Annual Report

What were the most significant accomplishments this past year?
D. Susceptibility to scrapie is controlled by polymorphisms in the PRNP
gene, with susceptibility associated with an allele encoding valine (V)
at codon 136 (formerly strain A scrapie) or with homozygosity for the
allele encoding glutamine (Q) at codon 171 (formerly strain C scrapie).
Resistance is seen in adult animals subjected to natural or experimental
exposure. In addition, fetal placental tissue with the heterozygous
resistant genotype (QR) in bred susceptible ewes (QQ) does not contain
detectable PrP-Sc, the marker for disease. In collaboration with North
Dakota State University, we have characterized the epidemiology of a
major outbreak of scrapie in the valine-related scrapie strain, the only
such well-documented outbreak in the US. Transmission patterns suggest
efficient spread of the disease among adults. In addition, NDSU serves
as the site for a study investigating the placentas of susceptible (QQ)
fetuses in resistant (QR) ewes; the ewes themselves are progeny of
infected QQ ewes. The experiment will test the hypothesis that the ewes
are free from disease, rather than silent carriers. The QR progeny of
infected dams are removed to the QR breeding study at Hettinger, ND.
These ewes will be bred to susceptible (QQ) rams, producing both
resistant (QR) and susceptible (QQ) progeny. All placentas from these
ewes will be examined for PrP-Sc and all QQ progeny will be held in long
term quarantine and monitored for scrapie. This report serves to
document research conducted under a specific cooperative agreement
between ARS and North Dakota State University. Additional details of
research can be found in the report for the parent project
5348-32000-019-00D Animal Health.


Project Team
O'Rourke, Katherine

Dale Redmer - Professor 509-335-6020

Project Annual Reports
FY 2004

Related National Programs


Rhttp://www.ars.usda.gov/research/projects/projects.htm?ACCN_NO=407203&fy=2004

Research Project: Scrapie
Control Through Prp Genetics: Production Traits and Economic
Implications


Location:

Animal
Diseases Research


2004 Annual Report

What were the most significant accomplishments this past year?
D. Breeding sheep for a resistant diploid genotype encoding arginine at
position 171 in the deduced PrP amino acid sequence is the most
effective way to prevent scrapie. We have shown that scrapie resistance
did not influence lamb production (birth weight and weaning weight) in
many purebred sheep of many breeds of commercial importance. This report
serves to document research conducted under a specific cooperative
agreement between ARS and University of Wyoming. Additional details of
research can be found in the report for the parent project
5348-32000-019-00D Animal Health.

http://www.ars.usda.gov/research/projects/projects.htm?ACCN_NO=405525&fy=2004

Research Project: Studies
to Further Understanding of the Biological Strain Typing of Tse Isolates


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 Veterinary
Laboratories Agency, New Haw, Addlestone, Surrey, 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. The work is a 2-year project
that will focus on the evaluation of variables involved in the use of
traditional mouse strain typing systems for characterization of
transmissible spongiform encephalopathy (TSE) isolates. This research is
directly related to a major objective of the parent project, which is to
apply the mouse strain typing system to TSEs found in the United States
(scrapie in sheep, transmissible mink encephalopathy and chronic wasting
disease in deer and elk). One objective is to determine how the amount
of prion protein (PrP) that is inoculated affects transmission success
(attack rate) and incubation period. The cooperators have identified
groups of scrapie inocula, consisting of scrapie-affected sheep brain
homogenates which encompass a range of transmissibility properties in
inoculated mice, to be used in the experimental work. The cooperators
are working on the development and interpretation of ELISA data to
determine the total PrP in the brain homogenates used as inocula. Work
has been completed on control brain tissues to determine efficacy of the
Proteinase K digestion. A finding was that diluted brain inocula have to
be centrifuged prior to use in the standard assay kit. Results have
shown good agreement with published data, but some adjustment is
required for the diluted inocula. Collaborators are awaiting arrival of
new kits to finalize development and perform analyses of the test
inocula. A second line of investigation focuses on the evaluation of
immunohistochemical PrP localization as an additional parameter for
differentiation of TSE strains. Cooperators have optimized the protocol
for PrP IHC staining, using rabbit polyclonal antibody Rb486. They have
determined that improved immunostaining can be achieved for Rb486 using
a modified epitope demasking protocol. This has been only superficially
assessed on BSE inoculated mice and requires assessment on scrapie
inoculated mice before it can be used routinely. In addition, the
cooperators have supplied negative control slides to the ADODR to check
efficiency of transport and for preliminary assessment of staining
techniques.

http://www.ars.usda.gov/research/projects/projects.htm?ACCN_NO=407025&showpars=true&fy=2004

Research Project: Studies
to Further Understanding of the Biological Strain Typing of Tse Isolates


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 Veterinary
Laboratories Agency, New Haw, Addlestone, Surrey, 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. The work is a 2-year project
that will focus on the evaluation of variables involved in the use of
traditional mouse strain typing systems for characterization of
transmissible spongiform encephalopathy (TSE) isolates. This research is
directly related to a major objective of the parent project, which is to
apply the mouse strain typing system to TSEs found in the United States
(scrapie in sheep, transmissible mink encephalopathy and chronic wasting
disease in deer and elk). One objective is to determine how the amount
of prion protein (PrP) that is inoculated affects transmission success
(attack rate) and incubation period. The cooperators have identified
groups of scrapie inocula, consisting of scrapie-affected sheep brain
homogenates which encompass a range of transmissibility properties in
inoculated mice, to be used in the experimental work. The cooperators
are working on the development and interpretation of ELISA data to
determine the total PrP in the brain homogenates used as inocula. Work
has been completed on control brain tissues to determine efficacy of the
Proteinase K digestion. A finding was that diluted brain inocula have to
be centrifuged prior to use in the standard assay kit. Results have
shown good agreement with published data, but some adjustment is
required for the diluted inocula. Collaborators are awaiting arrival of
new kits to finalize development and perform analyses of the test
inocula. A second line of investigation focuses on the evaluation of
immunohistochemical PrP localization as an additional parameter for
differentiation of TSE strains. Cooperators have optimized the protocol
for PrP IHC staining, using rabbit polyclonal antibody Rb486. They have
determined that improved immunostaining can be achieved for Rb486 using
a modified epitope demasking protocol. This has been only superficially
assessed on BSE inoculated mice and requires assessment on scrapie
inoculated mice before it can be used routinely. In addition, the
cooperators have supplied negative control slides to the ADODR to check
efficiency of transport and for preliminary assessment of staining
techniques.

http://www.ars.usda.gov/research/projects/projects.htm?ACCN_NO=407025&fy=2004


Research Project:
Development of New Technologies for Detection of Prions in Animal Feed
and Environmental Samples


Location:


Foodborne Contaminants 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?
Prions are believed to be the infectious agents responsible for
Transmissible Spongiform Encephalopathy diseases (TSEs), including
Bovine Spongiform Encephalopathy (BSE, aka mad cow disease). TSEs are
generally transmitted orally, through ingestion of infected/contaminated
feed. Upon eating BSE-infected beef, humans may contract a TSE called
variant Creutzfeldt-Jakob Disease (vCJD).

TSEs remain rare in humans and cattle, although most countries are
endemic for scrapie, a sheep TSE believed to be non-transmissible toward
humans. Chronic Wasting Disease is a more recently recognized TSE, which
is now spreading among North American deer and elk. All TSEs are 100%
fatal, with no effective treatment known. Although the threat to lives
of humans or even livestock is limited, losses and potential economic
losses due to embargoes are substantial, as evidenced by Japans recent
trade policy toward American beef.

Detection of prions in food, feed, animal tissues, and the environment
is currently very difficult. The best tests available are not able to
detect low levels of infection. Improved sensitivity will increase
confidence in screening of food and feed. Thus this research falls under
National Program 108 Food Safety.

The primary focus of this CRIS is investigation and development of novel
technologies for detection, quantitation, and strain characterization of
TSE infectious agents in environmental samples as well as animal feed
and feed additives. The new detection methods must be highly sensitive
and specific, with minimal levels of false negative or false positive
results. In addition, they must be rapid, cost effective, and easily
interpreted. Specific goals include.
1)significant improvements in sensitivity for prion assays, including
methods for strain-specific detection. In addition to immunochemistry
and traditional analytical chemistry, hybrid methods combining the best
features of both approaches are being investigated. Additional goals
include.
2)effective sampling methods for capturing, concentrating, and detecting
prions from samples of air, water, soil, feed, and feed additives; and.
3)inactivation/destruction of prions for decontamination. Useful methods
must be appropriate for sampling and decontamination of food processing
surfaces and animal holding pens. Thus they must be: free from chemical
and physical extremes; safe, with no generally toxic residue; and
relatively inexpensive, for use on large areas.

2. List the milestones (indicators of progress) from your Project Plan.
Year 1 (FY03) Develop GC/MS assay for cholesterol Immunize mice with
Campylobacter and Mycobacteria strains Generate cholesterol haptens and
immunize mice Immunize mice with PrP Establish Actinomycetes cultures

Year 2 (FY04) MS analysis of crosslinked, proteolyzed scrapie PrP
protein Purify polyketides from Actinomycetes cultures Culture analysis
of polyketides for TSE neutralization Develop improved monoclonal
antibodies (MAb) versus PrP New ELISA for Campylobacter strain analysis
New ELISA for Mycobacteria strain analysis Year 3 (FY05) Validate GC/MS
assay for cholesterol in various cattle feed formulations Develop
cholesterol immunoassay Identify additional markers for meat and bone
meal (MBM) Incorporate new prion MAb into Conformation-dependent
Immunoassay MS analysis of crosslinked, full-length scrapie PrP In vivo
tests of polyketide anti-TSE compounds Validation of new Campylobacter
and Mycobacteria strain-specific ELISAs

This Project is scheduled to undergo OSQR during FY05.

3. Milestones:
The following four milestones for FY04 were fully met: MS analysis of
crosslinked, proteolyzed scrapie PrP protein Develop improved monoclonal
antibodies (MAb) versus PrP New ELISA for Campylobacter strain analysis
New ELISA for Mycobacteria strain analysis

The following two milestones for FY04 were not met, due to unmet
staffing needs (two vacant SYs). They may be incorporated in the new
Project Plan, to be submitted in FY05. Purify polyketides from
Actinomycetes cultures Culture analysis of polyketides for TSE
neutralization

The following seven milestones were scheduled for FY05, -06, and -07:

Validate GC/MS assay for cholesterol in various cattle feed
formulations. This objective will be completed and published in FY05.

Develop cholesterol immunoassay. This objective will be studied
intensively during FY05. If our MAb are not specific for cholesterol
(i.e., if they cross-react with similar phytosterols), this objective
will not be continued.

Identify additional markers for meat and bone meal (MBM). Many
scientists have published in this area since the Project was originally
proposed. But we will identify novel markers in FY05, and develop
specific immunoassays for them in FY06.

Incorporate new prion MAb into Conformation-dependent Immunoassay. This
objective is on schedule for achievement in FY05. Also in FY05, we will
develop a new strain of mouse to use for further attempts at even better
MAb in FY06.

MS analysis of crosslinked, proteolyzed scrapie PrP. In addition to the
originally-proposed crosslinking method, we will explore a novel
approach for identification and detection of specific protein fragments
via MS.

In vivo tests of polyketide anti-TSE compounds. This objective will be
replaced by a more general bioremediation approach to decontamination in
our next Project proposal.

Validation of new Campylobacter and Mycobacteria strain-specific ELISAs.
Completed ahead of schedule, in FY04.

4. What were the most significant accomplishments this past year?
Prions are believed to be the infectious agents responsible for
Transmissible Spongiform Encephalopathy diseases (TSEs), including
Bovine Spongiform Encephalopathy (BSE, aka mad cow disease) and the
detection of prions in food, feed, animal tissues, and the environment
is currently very difficult. FCR scientists in WRRC developed an assay
for detection of cholesterol in animal feed, as a marker for animal
products. An assay which is based on GC-MS analysis of food/feed
extracts can detect meat and bone meal contamination of feed at levels
of 0.1 - 5%. The assay is being converted into a rapid, field portable
immunoassay that can be applied to feed or meal samples and will help
USDA regulators in monitoring the food/feed ban policy to prevent TSEs.
B. Other significant accomplishment(s), if any.

One reason that existing immunoassays for TSEs are not sensitive is that
they are based on antibodies with relatively weak binding. FCR
scientists and collaborators at the University of California San
Francisco have generated many new antibodies that bind PrP prion
protein, including some with significantly higher affinity than
currently available commercial products. These new antibodies were
developed by immunization of PrP ablated mice and a modified cell fusion
protocol. They have broad specificity and improved sensitivity in
immunoassay.

C. Significant activities that support special target populations.

Since the discovery of BSE in the US, Unit members have frequently
participated in national level discussions of strategies and tactics for
addressing this crisis.

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).

This project is currently under re-organization at the Unit level.
During FY05 we will develop separate Project Plans for CRIS
5325-32000-003-00D and 5325-42000-027-00D, and submit them both for
evaluation and review under OSQR.

5. Describe the major accomplishments over the life of the project,
including their predicted or actual impact.
(Since this is a new Project, see #4, above.)

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 GC-MS method for detecting prions and prion strains has been
approved for filing as an application for US Patent. Once issued, this
technology can be licensed, making the method readily available to
producers and regulators. This technique is many times more sensitive
than existing immunoassays. Although current instrumentation for the
method is neither robust nor affordable, rapid advances in MS technology
are already proceeding apace.

7. List your most important publications in the popular press and
presentations to organizations and articles written about your work.
Silva C, Stanker LH 2003 Detection of meat and bone meal using
cholesterol as a general marker, Assoc. Am. Feed Control Officials
Annual Meeting.

Onisko B 2004 Prion quantitation and strain-typing, ARS BSE Tactical
Planning Meeting.

Onisko B 2004 Prion quantitation and strain-typing, ARS BSE Strategic
Planning Meeting.

Silva C 2004 Inactivation of prions, ARS BSE Tactical Planning Meeting.

Scientific Publications:

Brandon DL, Bates AH, and Friedman M 2004 Immunoassays for Bowman-Birk
and Kunitz soybean trypsin inhibitors in infant formula, J. Food Sci.,
Vol 69, pp 45-49. Onisko B 2003 Analysis of peptides and proteins by
mass spectrometry, Invited lecture.

Onisko BC 2003 The application of mass spectrometry to analysis of
peptides, American Soc. Mass Spectrometry.

Onisko BC 2003 Analysis of recombinant hamster prion protein by mass
spectrometry, American Soc. Mass Spectrometry.

Stanker LH, Ravva SV 2003 Survival of E. coli O157:H7 in aerated dairy
manure lagoons, In: Proceedings of the 31st U.S. and Japan Natural
Resources (UJNR) Protein Resources Panel Meeting.

Weeks BL, Camarero J, Noy A, Miller AE, Stanker LH, DeYoreo JJ 2003 A
microcantilever-based pathogen detector, Symposium Proceedings of
Nanotechnology Conference.

Anis N, Gotthardt J, Thomas M, VonBredow J, Khall M, Stanker LH, Menking
D, Valdes J, Park J 2004 Solid-phase immunosensor for quantitative rapid
detection of antibiotic residues in raw unprocessed milk, FDA Science:
The Critical Path from Concept to Consumer (best poster award).

Brandon DL, Mandrell RE, Kerr PG, Traynor IM, Elliott CT 2004 Detection
of E. coli 0157 by surface plasmon resonance, Association of Analytical
Chemists International 118th annual meeting.

Silva C 2004 Prions the twisted tale of infectious protein isoforms,
invited lecture to the California Section of the American Chemical Society.

Stanker L, Carter JM 2004 Techniques and challenges in the diagnosis of
prion diseases, EU-US Task Force on Biotechnology Research.

http://www.ars.usda.gov/research/projects/projects.htm?ACCN_NO=405257&fy=2004

Research Project:
Development of New Technologies for Rapid Identification of
Pathogen/pathogen Products


Location:


Foodborne Contaminants 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?
Many important diseases of livestock and humans are transmitted orally,
through consumption of contaminated feed or food. This project is
concerned with foodborne diseases caused by bacteria and prions. Each
year in the US, food-borne microbial diseases cause 76 million illnesses
and 5,000 deaths, costing $7 billion in lost productivity. These numbers
are steadily increasing.

While culture methods exist for identification of foodborne pathogens,
we are developing sensitive and specific tests that provide rapid strain
typing, to help in monitoring the food supply and in tracking outbreaks.
Our immunoassay capabilities can be focused on new and emerging
pathogens to provide timely new detection methods.

Transmissible Spongiform Encephalopathies (TSEs), including bovine
spongiform encephalopathy (BSE, aka "mad cow disease"), are a class of
diseases that are generally transmitted orally, by ingestion of
infected/contaminated feed. The US has avoided epidemic BSE by careful
control of feed and food. There are two components to food/feed control.
Feed must not be contaminated with animal parts, and Specified Risk
Materials (SRM), such as brain and spinal cord tissues, where TSE
contamination is especially concentrated, must be kept out of food.
Maintaining a BSE-free food supply is thus mostly a matter of enforcing
the ban on bovine products in feed and SRM in food. This CRIS involves
identification and detection of surrogate markers for bovine products
and SRM.

This research falls under National Program 108, Food Safety.
Specifically this research addresses the following goals in the National
Program Action Plan: 1.1.1 Sampling, isolation, identification, and
quantification of pathogens in animal fluids and tissues, manure; and
the environment, including feed, water, and wild animals. 1.2.1 Ecology
and assessment of risk factors of pathogens in food producing animals,
including those carrying antibiotic resistance, outside of the host
animal. 2.1.1 Pathogen detection methodologies that have regulatory,
industry and research use. 2.2.1 Elucidate the ecology of pathogens on
animal products, seafood, fruit, and vegetables, and within the
processing environment. 2.6.1 Quantitative measurement of pathogens at
all critical points during food processing.

2. List the milestones (indicators of progress) from your Project Plan.
(The Project Plan developed for this CRIS is shared with CRIS
5325-32000-003-00D "Development of new technologies for detection of
prions in animal feed and environmental samples.")

Year 1 (FY03) Develop GC/MS assay for cholesterol Obtain Campylobacter
and Mycobacteria strains and immunize mice Generate cholesterol haptens
and immunize mice Immunize mice with PrP Establish Actinomycetes cultures

Year 2 (FY04) MS analysis of crosslinked, proteolyzed normal PrPc
protein Purify polyketides from Actinomycetes cultures Culture analysis
of polyketides for TSE neutralization Develop improved monoclonal
antibodies (MAb) versus PrP New ELISA for Campylobacter strain analysis
New ELISA for Mycobacteria strain analysis Year 3 (FY05) Validate GC/MS
assay for cholesterol in various cattle feed formulations Develop
cholesterol immunoassay Identify additional markers for meat and bone
meal (MBM) Incorporate new prion MAb into Conformation-dependent
Immunoassay MS analysis of crosslinked, proteolyzed scrapie PrP In vivo
tests of polyketide anti-TSE compounds Validation of new Campylobacter
and Mycobacteria strain-specific ELISAs

This Project is scheduled to undergo OSQR during FY05.

3. Milestones:
The following four milestones for FY04 were fully met: MS analysis of
crosslinked, proteolyzed normal PrPc protein Develop improved monoclonal
antibodies (MAb) versus PrP New ELISA for Campylobacter strain analysis
New ELISA for Mycobacteria strain analysis

The following two milestones for FY04 were not met, due to unmet
staffing needs. They may be rolled into the new Project Plan, to be
prepared in FY05. Purify polyketides from Actinomycetes cultures Culture
analysis of polyketides for TSE neutralization

B. The following seven milestones are planned for FY05, -06, and -07:

Validate GC/MS assay for cholesterol in various cattle feed
formulations. This objective is ahead of schedule. It will be completed
and published in FY05.

Develop cholesterol immunoassay. Although technical problems may prevent
success of the chosen approach, this objective will be studied
intensively during FY05. If our MAb are not specific for cholesterol
(i.e., if they cross-react with similar phytosterols), this objective
will not be continued.

Identify additional markers for meat and bone meal (MBM). Many
scientists have published in this area since the Project was originally
proposed. But we will identify novel markers in FY05, and develop
specific immunoassays for them in FY06.

Incorporate new prion MAb into Conformation-dependent Immunoassay. This
objective is on schedule for achievement in FY05. Also in FY05, we will
develop a new strain of mouse to use for further attempts at even better
MAb in FY06.

MS analysis of crosslinked, proteolyzed scrapie PrP. In addition to the
originally-proposed crosslinking method, we will explore a novel
approach for identification and detection of specific protein fragments
via MS.

In vivo tests of polyketide anti-TSE compounds. This objective will be
replaced by a more general bioremediation approach to decontamination in
our next Project proposal.

Validation of new Campylobacter and Mycobacteria strain-specific ELISAs.
Completed ahead of schedule, in FY04.

4. What were the most significant accomplishments this past year?
Because sensitive detection of prions is difficult, and the USDA has
developed a food/feed ban policy to prevent TSEs, we developed an assay
for detection of cholesterol in animal feed, as a marker for animal
products. FCR scientists in WRRC laboratories developed an assay based
on GC-MS analysis of food/feed extracts. The method can be used to
detect meat and bone meal contamination of feed at levels of 0.1 - 5%.
The assay is being converted into a rapid, field portable immunoassay
that can be applied to feed or meal samples.

B. Other significant accomplishment(s), if any.

One reason that existing immunoassays for TSEs are not sensitive is that
they are based on antibodies with relatively weak binding. FCR
scientists and collaborators at the University of California San
Francisco have generated many new antibodies that bind PrP prion
protein, including some with significantly higher affinity than
currently available commercial products. These new antibodies were
developed by immunization of PrP ablated mice and a modified cell fusion
protocol. They have broad specificity and improved sensitivity in
immunoassay.

C. Significant activities that support special target populations.

Since the discovery of BSE in the US, Unit members have frequently
participated in national level discussions of strategies and tactics for
addressing this crisis.

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).

This project is currently under re-organization at the Unit level.
During FY05 we will develop separate Project Plans for CRIS
5325-32000-003-00D and 5325-42000-027-00D, and submit them both for
evaluation and review under OSQR.

5. Describe the major accomplishments over the life of the project,
including their predicted or actual impact.
Upon a request from the Food Safety Inspection Service (FSIS) of the
USDA, we developed an assay for residues of Ceftiofur, a broad spectrum
cephalosporin antibiotic used in beef production. The immunoassay we
created is faster and cheaper than previous bioassay methods and is now
routinely employed by FSIS for regulation

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?
We have developed an assay for residues of Ceftiofur, a regulated
antibiotic found in beef. Our immunoassay is faster and cheaper than
previous methods. In FY04 technology transfer to FSIS was formally
completed, along with reagents and training/consultation.

Immunoassay technology for Bowman-Birk Inhibitor, a naturally-occurring
anti-nutritional component of soybeans, was originally developed under a
separate project. Transferred to this Project, the technology has now
been patented and licensed to an immunodiagnostics manufacturer, Agdia,
Inc. (Elkhart, IN).

Our MS method for detecting prions and prion strains has been approved
for filing as an application for US Patent. Once issued, this technology
can be licensed, making the method available to producers and
regulators. This technique is many times more sensitive than existing
immunoassays. Although current instrumentation for the method is neither
robust nor affordable, rapid advances in MS technology are already
proceeding apace.

7. List your most important publications in the popular press and
presentations to organizations and articles written about your work.
Silva C, Stanker LH (2003) "Detection of meat and bone meal using
cholesterol as a general marker", Assoc. Am. Feed Control Officials
Annual Meeting.

Onisko B (2004) "Prion quantitation and strain-typing", ARS BSE Tactical
Planning Meeting.

Onisko B (2004) "Prion quantitation and strain-typing", ARS BSE
Strategic Planning Meeting.

Silva C (2004) "Inactivation of prions", ARS BSE Tactical Planning Meeting.

Scientific Publications:

Brandon DL, Bates AH, and Friedman M (2004) "Immunoassays for
Bowman-Birk and Kunitz soybean trypsin inhibitors in infant formula", J.
Food Sci., Vol 69, pp 45-49.

Abstracts:

Onisko B (2003) "Analysis of peptides and proteins by mass
spectrometry", Invited lecture.

Onisko BC (2003) "The application of mass spectrometry to analysis of
peptides", American Soc. Mass Spectrometry.

Onisko BC (2003) "Analysis of recombinant hamster prion protein by mass
spectrometry", American Soc. Mass Spectrometry.

Stanker LH, Ravva SV (2003) "Survival of E. coli O157:H7 in aerated
dairy manure lagoons", In: Proceedings of the 31st U.S. and Japan
Natural Resources (UJNR) Protein Resources Panel Meeting.

Weeks BL, Camarero J, Noy A, Miller AE, Stanker LH, DeYoreo JJ (2003) "A
microcantilever-based pathogen detector", Symposium Proceedings of
Nanotechnology Conference.

Anis N, Gotthardt J, Thomas M, VonBredow J, Khall M, Stanker LH, Menking
D, Valdes J, Park J (2004) "Solid-phase immunosensor for quantitative
rapid detection of antibiotic residues in raw unprocessed milk", FDA
Science: The Critical Path from Concept to Consumer (best poster award).

Brandon DL, Mandrell RE, Kerr PG, Traynor IM, Elliott CT (2004)
"Detection of E. coli 0157 by surface plasmon resonance", Association of
Analytical Chemists International 118th annual meeting.

Silva C (2004) "Prions, the twisted tale of infectious protein
isoforms", invited lecture to the California Section of the American
Chemical Society.

Stanker L, Carter JM (2004) "Techniques and challenges in the diagnosis
of prion diseases", EU-US Task Force on Biotechnology Research.

http://www.ars.usda.gov/research/projects/projects.htm?ACCN_NO=403289&fy=2004

esearch >
Title: Experimental Transmission of Sheep Scrapie by Intracerebral and
Oral Routes to Genetically Susceptible Suffolk Sheep in the United
States Authors
item Hamir, Amirali

item Kunkle, Robert - bob

item Richt, Juergen

item Miller, Janice - ARS RETIRED
item Cutlip, Randall - ARS RETIRED
item Jenny, Allen - USDA-VS-APHIS-NVSL

http://www.ars.usda.gov/research/publications/publications.htm?seq_no_115=161349

Submitted to: Journal Of Veterinary Diagnostic Investigation
Publication Acceptance Date: June 1, 2004
Publication Date: N/A
Interpretive Summary: Scrapie is a naturally occurring fatal disease of
sheep and goats. It affects nervous system of the animal. Susceptibility
to the disease is dependent upon genetic makeup of the host and
infectious agent. This study documents findings in Suffolk sheep
affected with experimental disease. Four-month-old lambs were utilized
for the study. They were administered (5 in the brain and 19 orally)
scrapie-infected sheep brains. All animals that were administered the
infected material directly into the brain revealed signs of scrapie and
were euthanized between 13'24 months after administration. In sheep
given the material orally, signs of scrapie were seen between 27 and 43
months in 5 of 9 animals. Three of the 4 clinically normal sheep were
found to be positive for scrapie by laboratory tests at 15, 20, and 49
months after administration of infected material. There is lack of
information on experimental transmission of US scrapie agent in
genetically diverse flocks of sheep. This study attempts to partially
fill this void by documenting findings of this disease in Suffolks.
Since similar investigations are also needed for other breeds of sheep
in this country, the present study will serve as a foundation to compare
results of other future studies.

Technical Abstract: Scrapie is a naturally occurring fatal
neurodegenerative disease of sheep and goats. Susceptibility to the
disease is partly dependent upon genetic makeup of the host. This study
documents clinicopathological findings and distribution in tissues of
abnormal prion proteins (PrPres) by immunohistochemical (IHC) and
Western blot (WB) techniques, in tissues of genetically susceptible
sheep inoculated with US sheep scrapie agent. Four-month-old Suffolk
lambs (QQ or HQ at codon 171) were utilized for the study. They were
inoculated (5 intracerebrally and 19 orally) with an inoculum (No. 13-7)
consisting of a pool of scrapie-infected sheep brains. Intracerebrally
inoculated animals were euthanized when advanced clinical signs of
scrapie were observed. Orally inoculated animals were euthanized at
pre-determined time-points (4, 9, 12, 15 and 21 months post inoculation,
PI) and thereafter when the animals had terminal signs of disease. A
detailed postmortem examination was conducted on each carcass and
tissues were examined for microscopic lesions and for the presence of
PrPres by IHC and WB techniques. All intracerebrally inoculated animals
exhibited clinical signs of scrapie and were euthanized between 13 ' 24
months PI. Spongiform lesions in the brains and PrPres deposits in
central nervous system (CNS) and lymphoid tissues were seen in these
sheep. In orally inoculated sheep, clinical signs of scrapie were seen
between 27 and 43 months PI in 5/9 animals. The earliest detectable
PrPres was observed in brainstem and lymphoid tissues of a clinically
normal sheep at 15 months PI. Three of the 4 clinically normal sheep
were found to be positive at 15, 20, and 49 months PI by either
histopathology or the PrPres tests. This study was done to partially
fill a void in information on experimental studies of US scrapie
transmission (by intracerebral and oral routes) in genetically
susceptible sheep.


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