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From: TSS ()
Subject: Development of New Technologies for Detection and Decontamination of Prions in Meat Animals, Feed, and Environmental Samples
Date: January 3, 2007 at 11:23 am PST

Research Project: Development of New Technologies for Detection and Decontamination of Prions in Meat Animals, Feed, and Environmental Samples
Location: Foodborne Contaminants Research

2006 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? Why does it matter?
Prions are believed to be the 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 in sheep, this TSE is believed to be non-transmissible toward humans. Chronic Wasting Disease (CWD) 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 the health of humans and livestock is limited, economic losses due to embargoes are huge.

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 improve screening of food and feed to protect animal health. Thus this research is administered under National Program 103 Animal Health.

The primary focus of this project is detection of prions in animal tissue samples, feed additives, and environmental samples. Any new methods we develop must be highly sensitive and specific, with no 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 by year the currently approved milestones (indicators of research progress)
Year 1 (FY06) Demonstrate Protein Cyclic Misfolding Assay (PCMA) in mouse brain homogenate. Select neural cell line for TSE culture. Construct gene for tandem affinity purification of PrP protein. Validate new PrP null mouse system. Analysis of serum from infected animals using mass spectrometry without proteinase K. Identify hamster and mouse cell lines expressing PrP protein. Optimize PrP protein normal form expression. Manufacture PrP protein normal form on lab scale using cell culture. Validate hamster intracranial model for scrapie 263K and 3 additional TSE strains. Validate new hamster intraocular model for assay of clinical scrapie 263K. Perform alkaline methanolysis on brain homogenate and on Meat and Bone Meal after spiking with scrapie infected tissue. Screen results of scrapie alkaline methanolysis via immunoassay and Western blot. Initiate rodent assay of scrapie alkaline methanolysis. Screen 20 natural products for TSE disinfection via Western blot and cell culture. Screen 3 commercial decontamination methods for TSE disinfection via Western blot and cell culture. Start validation of commercial decontamination methods for scrapie strain 263K in hamster model.

Year 2 (FY07) Inhibition of PCMA in hamster brain homogenate. Transfection and TAP analysis of uninfected cells. Generate and characterize novel monoclonal antibody. MS detection of PrP protein diseased form in PCMA amplified sample. Inoculate cell cultures with brain homogenate from TSE infected animals. Characterize and optimize PrPd conversion in cell culture. Transfect cell cultures with genes for mouse PrP and FLAG-tagged PrP. Evaluate expression of transgenic PrP in cell culture using Western blot. Apply Hydrogel adjuvant to intraocular model. Finish rodent assay of scrapie alkaline methanolysis. Perform alkaline methanolysis on brain homogenate and on Meat and Bone Meal after spiking with BSE. Screen results of BSE alkaline methanolysis via immunoassay and Western blot. Screen 20 (more) natural products for TSE disinfection via Western blot and cell culture. Purify and characterize 3 lead decontamination compounds. Start validation of 3 lead compounds for scrapie strain 263K in hamster model.

Year 3 (FY08) Reconstitution of PCMA from pure PrP. Tandem affinity purification analysis of two TSE strains. Incorporate new monoclonal antibody into assays. Distinguish normal from diseased PrP in mouse brain homogenate using mass spectrometry without proteinase K. Transfect cell cultures with sheep and deer PrP genes. Inoculate animals with diseased form of FLAG-PrP protein. Track diseased form of FLAG-PrP protein in cell culture using immunocytochemistry. Validate intraocular model for preclinical 263K. Initiate rodent assay of BSE alkaline methanolysis. Validate 3 lead compounds for CWD in transgenic cervidized mouse model. Validate commercial decontamination method for CWD in transgenic cervidized mouse model.

Year 4 (FY09) Identify/select novel surrogate analytes via PCMA. Identify novel surrogate via mass spectrometry and begin validation. Validate new assays on infected hamster blood. Employ new proteinase K-free chemistry on infected hamster serum. Administer pharmacological reagents for modification of PrP processing in cell cultures. Validate targets identified in cell culture via PMCA and co-localization. Use immunohistochemistry to track PrP diseased form in intraocular model. Finish rodent assay of BSE alkaline methanolysis. Validate 3 lead compounds for BSE in transgenic bovinized mouse model. Validate commercial decontamination method for BSE in transgenic bovinized mouse model.

Year 5 (FY10) Validate novel analytes via immunochemistry and/or mass spectrometry assay development. Validate one novel surrogate via assay development for infected hamster brain homogenate. Validate new assays on BSE infected bovine blood. Validate mass spectrometry method on BSE infected bovine serum. Evaluate targets identified in cell culture as surrogate in vitro analytes. Evaluate cell culture pharmacological reagents as potential decontamination reagents. Technology transfer of novel cell culture technology for TSE detection. Evaluate natural products for disinfection of Meat and Bone Meal. Technology transfer of novel natural product derived decontamination methods for TSEs.


4a.List the single most significant research accomplishment during FY 2006.
Prions via mass spectroscopy: Existing tests are unable to detect TSE disease or disease agents at low levels of infectivity. We developed a new method for detection of prions using nanospray liquid chromatography coupled to mass spectroscopy (LC-MS-MS) and filed a US Patent application this year. This technique is exquisitely sensitive, more sensitive than any other method ever used for prions. In addition to offering a possibility for an antemortem blood test for BSE, this technique may be generalized to detect other rare and difficult molecules. This accomplishment aligns with NP103 Action Plan Component 8: Countermeasures to Prevent and Control Transmissible Spongiform Encephalopathies.


4b.List other significant research accomplishment(s), if any.
None.


4c.List significant activities that support special target populations.
None.


4d.Progress report.
The proposed Project Plan for this project (including the new milestones listed above) is currently under consideration by OSQR.


5.Describe the major accomplishments to date and their predicted or actual impact.
This project is four years old. Focused on detection and decontamination of prions, it recently evolved from a larger project involving detection of foodborne pathogens in general. We have four major products, of which three are currently undergoing technology transfer.

1. Our cholesterol assay is as effective as classical feed microscopy for detection of animal products in feed materials. Many beef and dairy producers feed their animals exclusively vegetable feeds, and they appreciate this tool. However, the beef and dairy industries also depend on the sale of meat and bone meal (MBM) for use as a feed supplement, e.g. in pet foods. So they are enthusiastic about a test for prohibited materials in feed (e.g., brain and spinal cord), or a test that could identify prion-contaminated feed. But they have not adopted our cholesterol-based test for animal material in general. Notwithstanding a change in economics of MBM, this product has no impact. This accomplishment aligns with NP103 Action Plan Component 8: Countermeasures to Prevent and Control Transmissible Spongiform Encephalopathies.

2. Our mass spectroscopy based prion detection method is the most sensitive method ever discovered for detection of prions. Ultimately it might be adopted by testing facilities for widespread use, once we validate it with biological specimens from live animals. This requires animal model testing – a relatively slow, expensive, and hazardous series of experiments we will undertake with collaborators in ARS. It is notable that the method may be generalized for use in detecting other diseases that involve protein misfolding, e.g., Alzheimer’s and Huntington’s diseases. This accomplishment aligns with NP103 Action Plan Component 8: Countermeasures to Prevent and Control Transmissible Spongiform Encephalopathies.

3. Our new PrP null mouse strains lack the PrP protein required for transmission and progression of prion disease, but possess a genetic background suitable for monoclonal antibody creation. We have filed a US Patent Application on them while we use them to develop a complementary PrP free cell line. Together these products will generate powerful impact for scientists who perform prion research worldwide. This accomplishment aligns with NP103 Action Plan Component 8: Countermeasures to Prevent and Control Transmissible Spongiform Encephalopathies.

4. We developed a new monoclonal antibody for PrP and filed a US Patent Application in FY05. The product is now being commercially developed and considered for licensing by co-inventors at the University of California. By including it in an already-commercialized assay for prions in animal tissues, the new antibody will provide increased sensitivity. Constraints for commercialization are based on market factors, especially limited growth in TSE testing, and cost-benefit of developmental costs versus incremental improvement in product competitive status. This accomplishment aligns with NP103 Action Plan Component 8: Countermeasures to Prevent and Control Transmissible Spongiform Encephalopathies.


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 developed a new monoclonal antibody and filed a US Patent Application in FY05. The product is now being commercially developed and considered for licensing by co-inventors. By including it in an already-commercialized assay for prions in animal tissues, the new antibody will provide increased sensitivity. Constraints for commercialization are based on market factors, especially growth of testing in the US, and cost-benefit of developmental costs versus improvement in product competitive status.


7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below).
None.


Review Publications
Zukas, A.A., Carter, J.M. 2006. Strain-specific prp-res in vitro amplification by the pmca technique. [Abstract]. Cambridge Healthtec Institute, Transmissible Spongiform Encephalopathies. Poster #12.

Zukas, A.A., Carter, J.M. 2006. Sonication induced intermediate in prion conversion. [Abstract]. ACS National Meeting. Poster BIOL55


Stanker, L.H., Serban, A.V., Safa, J., Prusiner, S.B. 2006. Isolation and characterization of new anti-prp monoclonal antibodies. [Abstract]. ACS National Meeting and Exposition. Platform Presentation AGFD 199.

Title: Isolation and Characterization of New Anti-Prp Monoclonal Antibodies


Authors


Stanker, Larry
Serban, Ana - UNIV CALIF SAN FRANCISCO
Safa, Jiri - UNIV CALIF SAN FRANCISCO
Prusiner, Stanley - UNIV CALIF SAN FRANCISCO


Submitted to: Meeting Abstract
Publication Type: Abstract
Publication Acceptance Date: May 31, 2006
Publication Date: June 21, 2006
Citation: Stanker, L.H., Serban, A.V., Safa, J., Prusiner, S.B. 2006. Isolation and characterization of new anti-prp monoclonal antibodies. [Abstract]. ACS National Meeting and Exposition. Platform Presentation AGFD 199.

Technical Abstract: The prion diseases (or transmissible spongiform encephalopathies) are fatal neurodegenerative illnesses caused by the accumulation of PrPSc, which is an alternatively folded isoform of the cellular prion protein (PrPC). These disorders are widespread and are found in humans (Creutzfeldt-Jakob disease), in sheep (scrapie), in elk and deer (chronic wasting disease), and in cattle (bovine spongiform encephalopathy) as well as in mink and cats. Detection of PrPSc commonly relies on immunochemical methods. In this study, we isolated antibodies that improved the performance of the conformation-dependent immunoassay (CDI) used to measure both the protease-resistant and -sensitive forms of PrPSc. Following immunization of Prnp-null mice, a multitiered screening strategy was developed and a panel of candidate antibodies identified. Monoclonal antibody binding to PrP from different species, to reduced versus non-reduced PrP, to synthetic peptides, and to denatured PrP suggest that antibodies with both continuous and discontinuous epitopes were isolated. At least one of the antibodies, F4-31, substantially improved performance of the CDI for detection of PrPSc in cattle.

http://www.ars.usda.gov/research/projects/projects.htm?ACCN_NO=405257&showpars=true&fy=2006

Research Project: Identification & Detection of Tse-Infected Tissues, Food Products, & Animals Using Fluorescent Spectroscopy

Location: Virus and Prion Diseases of Livestock

2006 Annual Report

4d.Progress report.
This report serves to document research under a reimbursable agreement between ARS and Iowa State University. Additional details of research can be found in the report for the parent project 3625-32000-073-00D, Transmission, Differentiation, and Pathobiology of Transmissible Spongiform Encephalopathies.

The objectives of this research are to: 1. Characterize the spectral properties of normal and scrapie-infected tissue from sheep. 2. Characterize the fluorescent spectral patterns of central nervous system (CNS) tissue. 3. Adapt prior carcass imaging technology to the detection of CNS tissue for use in a packing plant environment.

Our accomplishments to date include: 1. Conducted a correlative age and ophthalmic spectroscopy experiment in mice. 2. Secured additional supplies of healthy and scrapie-infected sheep tissue from collaborators in ARS and APHIS and sources outside USDA. 3. Obtained tissue from mice experimentally infected with scrapie and age-matched uninfected animals. 4. Confirmed scrapie-free status of sheep used as a source of uninfected control tissues. 5. Characterized the fluorescent spectral properties of scrapie infected tissue as compared to noninfected tissue. 6. Expanded a spectroscopy laboratory on the campus of Iowa State University, Department of Chemistry, which is capable of handling prion-infected tissue. 7. Maintained the necessary safety and importation permits for this laboratory and for shipment, chain of custody and receipt of experimental tissue. 8. Renewed an Institutional Animal Care and Use protocol and obtained approval from the NADC ACUC authorizing committee. 9. Trained collaborating university faculty and staff in appropriate measures for safe handling and disposal of prion-infected tissue. Relation to in-house CRIS objectives: Detection of prion infected animals through new technology will provide a means of diagnosis, a means to eradication and control for U.S. livestock producers.

http://www.ars.usda.gov/research/projects/projects.htm?ACCN_NO=408454&showpars=true&fy=2006

TSS





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