Progress 02/06/02 to 01/31/07
Outputs
Progress Report Objectives (from AD-416) We will identify and develop novel methods for detection and destruction of infectious animal prions in meat animals, by-products, and processing facilities; in feed; and in the environment. We will also study molecular pathophysiology of prion replication to discover new means for detection and prevention of prion diseases. Approach (from AD-416) The threat of BSE continues to affect export economics for US meat. Meanwhile CWD, a related transmissible spongiform encephalopathy (TSE), is an emerging disease now spreading throughout North America. Thus US animal industry stakeholders have identified detection of the TSE infectious agent (PrPd or prions), in animals and animal products, as a priority biosecurity research issue essential for prevention of TSE diseases. We will develop methods for extraction, concentration, and amplification of PrPd in samples from feed, animals and animal products, and the environment. Sample processing approaches include solid and liquid phase partitioning, gravimetric analysis, electrophoresis, immunochemical methods, and mass spectroscopy. We will also explore in vitro replication of prions to identify surrogate analytes involved in TSE pathogenesis. New immunochemical methods and reagents will require development of novel PrP ablated mouse strains and cell lines to overcome limitations in existing monoclonal antibody technology. State-of-the-art assay platforms, e.g., time-resolved fluorescence, will be developed through collaborations. Decontamination of prions is an unusually challenging objective, against which we will employ agricultural waste products, such as fruit pomace containing non-toxic tannins and flavonoids; composting and bioremediation with special microorganisms; and chemical and enzymatic hydrolysis. Our work on molecular pathophysiology will utilize in vitro amplification of scrapie and CWD in tissue and defined cell culture systems from hamster and transgenic mouse models, to identify molecular partners involved in TSE pathogenesis. All work with infectious material will take place within our APHIS-approved BL2 biocontainment facilities labs at the Western Regional Research Center (WRRC), while mass spectrometry will be performed on non- infectious material under BL1 containment. Work on BSE will be performed outside WRRC, through collaborations.FY03 Program Increase $357,660. 1 SY's. FY06 Program Increase $630,000. Add 1 SY. Significant Activities that Support Special Target Populations This project has expired and is replaced with 5325-32000-007-00D. The milestones listed in this report were proposed in a Project Plan but not formally approved for this project. Accomplishments Chemical inactivation of prions in meat-and-bone meal (MBM). MBM is a nutritious beef by-product formerly used as a feed supplement, but recently prohibited from US cattle feed because of the difficulty of removing prion infectivity responsible for the BSE epidemic in Europe. We have shown, using a hamster model, that a simple chemical method to produce biodiesel directly from MBM also inactivates prions. At WRRC Foodborne Contaminants Research Unit scientists used a method learned from the ARS Scientistst ERRC to process MBM that was infected with hamster scrapie disease. The product of the reaction was tested in a sensitive animal model and found to be non-infective. This discovery should help increase the market value of MBM, a commodity whose value was adversely affected when BSE was discovered in the US. This accomplishment aligns with NP103 Action Plan Component 8: Countermeasures to Prevent and Control Transmissible Spongiform Encephalopathies. Identification of a novel prion conformer. Prion diseases such as BSE are caused by a poorly understood process of misfolding protein in the brain. Protein Misfolding Cyclic Amplification (PMCA) is a means of reproducing this protein misfolding in a test tube, instead of an animal. Scientist at WRRC, ALbany, CA in Foodborne Contaminants Research Unit used PMCA to characterize a novel conformation involved in the prion misfolding process. This observation is influencing the technology and understanding of in vitro prion conversion in other labs. This accomplishment aligns with NP103 Action Plan Component 8: Countermeasures to Prevent and Control Transmissible Spongiform Encephalopathies. Technology Transfer Number of New CRADAS and MTAS: 2 Number of Active CRADAS and MTAS: 2 Number of Invention Disclosures submitted: 3 Number of Patent Applications filed: 1 Number of Web Sites managed: 1 Number of Non-Peer Reviewed Presentations and Proceedings: 4 Number of Newspaper Articles,Presentations for NonScience Audiences: 2
Impacts
(N/A)
Publications
- Tian, J., Radma, M., Hnasko, R.M., Locker, J. 2006. Loss of Nkx2.8 deregulates progenitor cells in the large airways and leads to dysplasia. Cancer Research. 1:66(21):10399-407.
- Hnasko, R.M., Carter, J.M., Medina, F., Lisanti, M. 2006. Pv-1 labels trans-cellular openings in mouse endothelial cells and is negatively regulated by vegf. Cell Cycle. 5(17):2021-28.
- Hnasko, R.M., Philippe, F.G., Ben-Jonathan, N., Lisanti, M.P. 2006. Pv-1 is negatively regulated by vegf in the lung of cav-1, but not cav-2, null mice. Cell Cycle 5(17):2012-20.
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Progress 10/01/05 to 09/30/06
Outputs
Progress 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. 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., Alzheimers and Huntingtons 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.
Impacts
(N/A)
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.
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Progress 10/01/04 to 09/30/05
Outputs
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 in sheep, this TSE is 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 the health of humans and livestock is limited, losses and potential
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 falls under National Program 103 Animal Health. 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 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 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 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Validate GC/MS assay for cholesterol in various cattle feed formulations. Milestone Fully Met 2. Incorporate new prion MAb into Conformation-dependent Immunoassay. Milestone Fully Met 3. MS analysis of crosslinked, full-length scrapie PrP. Milestone Substantially Met 4. Develop cholesterol immunoassay. Milestone Not Met Redirection of Research focus due to change in priorities 5. Identify additional markers for meat and
bone meal Milestone Not Met Redirection of Research focus due to change in priorities 6. In vivo tests of polyketide anti-TSE compounds. Milestone Not Met Critical SY Vacancy 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? FY06 Establish rodent model for scrapie in mice or hamsters. Demonstrate MS-based quantitation of scrapie prions. Evaluate direct fluorescence polarization assay for prions. Establish in vitro conversion model for scrapie in hamster brain homogenate. Develop PrP null myeloma cell line. FY07 Establish transgenic mouse model for Chronic Wasting Disease. Evaluate in vitro conversion model for amplification of weak assay signals. Validate MS-based quantitation of scrapie prions. Establish standard immunoassay for prions. Receive Select Agent registration for BSE research. FY08 Establish rodent model for BSE in mice or hamsters.
Demonstrate MS-based quantitation of BSE prions. Begin study of commercial disinfectants for surface decontamination of prions. Evaluate recombinant or cell culture based prion assay. 4a What was the single most significant accomplishment this past year? Improved monoclonal antibody for prion detection. Existing methods are unable to detect transmissible spongiform encephalopathy (TSE) diseases in live animals. We used various animals and immunogens to develop new monoclonal antibodies, and filed a US Patent application for an antibody that significantly increases sensitivity of tests for the prions believed responsible for TSEs. The Patent application was filed by the University of California for the new antibody, which was discovered by an ARS scientist under a cooperative agreement with UC San Francisco (5325-32000-003-01S). Our new antibody reagent may be incorporated into the Conformation-Dependent Immunoassay produced by InPro Biotechnology. 4b List other significant
accomplishments, if any. Prions via mass spectroscopy. Existing tests are unable to detect TSE disease or disease agents at infectious levels. We developed a new method for detection of prions using nanospray liquid chromatography coupled to mass spectroscopy (LC-MS) and filed a US Provisional Patent application. This technique is exquisitely sensitive, more sensitive than any other method ever used for prions. In addition to offering a possibility for an antemortem BSE test, this technique may be generalized to detect other rare and difficult molecules. New mice strains for prion research. Common lab mice are ineffective for generation of monoclonal antibodies that recognize the misfolded proteins that characterize prion diseases. We finished development of two new strains of mice, BALB/c Jax and BALB/c Baily, both lacking the normal PrP protein. Because they do not normally have this protein, the animals generate a robust immune response against it upon immunization. The animals
were bred by an ARS scientist from animals developed under a cooperative agreement with UC San Francisco (5325-32000-003-01S). These mice, and a subsequent myeloma cell line product still under development, will be used for future generation of improved monoclonal antibodies for sensitive prion assays. 4d Progress report. This Project is scheduled to undergo OSQR in FY06. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. This project is three 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 three major products, of which two 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. 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., Alzheimers disease
and Type II diabetes. 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. They will be patented beginning in FY06, while we use them to develop a complementary PrP null myeloma cell line. They will generate powerful impact for scientists who perform prion research worldwide. 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 this year. 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). JM Carter, 2004, Prion/TSE Research in the Foodborne Contaminants Research Unit at WRRC, presented at Institute for Animal Health, Newbury, UK. J Requena, B Onisko, 2004, Probing PrPSc structure using chemical cross- linking, presented at University Santiago de Compostela, Spain. CG Gay, 2004, ARS battles mad cow, scrapie, and other TSEs, in Agricultural Research magazine, Vol 52 (12), p 2. K Kaplan, 2004, TSEs touch off ARS research, in Agricultural Research magazine, Vol 52 (12), p 4-9. C Silva, 2005, Scrapie: The very model of an infectious (protein) isoform, presented at the 96th
Annual Meeting and Expo of the American Oil Chemists' Society (AOCS) Feed Microscopy Division, Salt Lake City, UT. T Neeley, 2005, New cattle feed test streamlines process, published in The Record Stockman online newspaper, http://www.recordstockman.com/. JM Carter, 2005, Foodborne Contaminants Research Unit, report presented to BSE Industry Consultation Meeting, Washington, DC.
Impacts
(N/A)
Publications
- Carter, J.M., Onisko, B., Silva, C., Stanker, L. Requena, J.R., 2005. Prion research at the Western Regional Research Center [abstract]. International Symposium on the New Prion Biology: Basic Science, Diagnosis and Therapy, April 7-9, 2005, Venice, Italy. Poster P17.
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Progress 10/01/03 to 09/30/04
Outputs
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.
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