Progress 04/07/08 to 02/28/13
Outputs
Progress Report Objectives (from AD-416): We will develop highly sensitive diagnostic tests to detect transmissible spongiform encephalopathy (TSE) in livestock and wildlife animal species prior to the onset of clinical disease. We will also develop tests to confirm the presence or absence of TSE disease agents in ingredients of animal origin and decontaminated environments. Approach (from AD-416): The threat of BSE continues to affect export economics for US meat. Meanwhile scrapie continues to influence sheep profits and herd biosecurity, and CWD is spreading throughout North America. Thus U.S. animal industry stakeholders have identified detection of the TSE infectious agent (prions) as a priority biosecurity research issue essential for prevention of TSE diseases. We will build on our previous successes using mass spectrometry (MS) for high-sensitivity and specificity in detection of PrPsc as a marker for TSE infectivity in blood using a hamster scrapie model. We will also develop a novel PrP-null mouse strain and related myeloma cell culture system for production of monoclonal antibodies (MAb), which may be specific for PrPsc. We will then choose MS or MAb and validate our novel diagnostic for preclinical diagnosis of scrapie in sheep blood. Whereas MS and MAb methods rely on dissolved samples, contamination of agricultural products and environmental surfaces is associated with solid samples. So we will produce a cell culture based assay for TSE infectivity that is surface-adsorbed. After using the relatively convenient hamster model for early development, we will validate this technology for detection of scrapie in sheep brain on meat-and-bone meal and stainless steel. 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. Replacing 5325-32000-007-00D (3/19/2008). This is the final report for this project which terminated in April of 2013 and was replaced with 5325-32000-009-00D, "Innumodiagnostics to Detect Prions and Other Important Animal Pathogens". Substantial results were realized over the project period. New analytical techniques and materials were developed for detection of infectious prions with improved results compared to conventional methods. A prion gene knockout mouse was developed and immunized with purified infectious prions resulting in the generation of sensitive anti-prion monoclonal antibodies. The impact of this research was the production of distinct anti-prion monoclonal antibodies that bind novel prion epitopes for use in the development of commercial immunoassays. The binding properties of these monoclonal antibodies were extensively characterized and their utility in a variety of immunoassay formats were demonstrated. A patent has been filed for this technology and three of these antibodies have been transferred to academic and government partners including the National Veterinary Services Laboratory (NVSL). License negotiations are currently ongoing with a commercial partner. The slow accumulation of prions and limited concentration in target tissues confounds detection. Sample preparative methods were defined that significantly enrich prions in tissue extract using isolation with lipid raft. The resulting increase in prion sample concentration results in improved immunoassay detection. The impact of this research is a method to detect prions from infected asymptomatic animal tissue. A patent has been filed and papers published on this methodology. Enzyme-linked immunosorbent assays (ELSIA) are frequently used for the detection of prions from target tissues. A chaotropic agent was effectively used in a modified immunohistochemical assay and an ELISA format to increase the sensitivity of prion detection. This technical modification resulted in a in a seminal paper with a broad impact on antigen detection by immunoassay. Infectious prion strains reflect alternate abnormal tertiary protein structure that stably propagate and produce distinct disease pathology. Analytical mass spectrometry has been used to elucidate structural differences in prion protein strains and quantitate the concentration of prion in a given sample. The impact of this research has been to further the understanding of prion strain differences and identify potential anti-prion binding epitopes for selective modification. This knowledge can be used to build immunoassays capable of discriminating prion strains. The overall impact of these accomplishments is improved prion immunoassay technologies for use in ongoing prion research and surveillance efforts with commercial application. Accomplishments 01 Improved anti-prion monoclonal antibodies for prion detection. Using purified prions from infected animals, ARS scientist in Albany, California, have generated anti-prion DRM monoclonal antibodies from immunized prion knockout mice. This approach effectively overcame the poor immune response to prion immunization in normal mice and our research efforts defined an improved method to yield a purified infectious prion immunogen used to generate high-titer antibody response in a new mouse model. A novel differential screening methods based on preferential binding to infectious prion antigen was used in the identification and selection of hybridoma cell lines producing anti- prion monoclonal antibodies. The properties of these antibodies have been extensively characterized, their binding epitopes defined and their utility in a number of immunoassay formats validated. These antibodies have been transferred to both academic and national laboratories for use in prion immunoassays. A patent has been filed (US Patent # 13/157,216) and a manuscript has been published describing these anti-prion monoclonal antibodies. Negotiations with a major international biotechnology company are in progress to issue a license for commercialization of this technology. 02 Novel peptide sequences for specific binding to infectious prions. Using a proteomic peptide array consisting of peptides corresponding to functional domains of endogenous proteins involved in protein-protein interaction ARS scientists in Albany, California, have identified specific amino acid sequences that bind only the infectious form of prion proteins. Using these peptides as part of a modified immunoassay for the selective capture of infectious prion protein from animal tissues, ARS scientists have demonstrated the ability to discriminate prion infected from uninfected samples using conventional anti-prion antibodies for detection. This approach allows for the direct capture and detection of infectious prions without the interference of the normal endogenous prion protein that confounds traditional assay techniques. The identification of several prion specific binding peptides represents important components of new peptide-based detection assays and important targets to facilitate research toward understanding the mechanism of prion conformational conversion. A patent has been issued (US Patent #12/571,275; Aug 2013) for this technology. 03 Novel methods to isolate prions in non-diagnostic tissues to improve detection of scrapie and chronic wasting disease (CWD). ARS scientists in Albany, California, along with an international consortium of scientists have developed an efficient means of isolating and detecting prions in non-diagnostic portions of brain tissue from sheep naturally infected with scrapie and elk infected with CWD. Conventional methods rely on accumulation of infectious prions in a brainstem tissue called the obex for diagnosis of disease. Other areas of the brain tend to have less prion and consequently have been excluded from sampling and analysis. The small size and lipid rich aspect of the obex has limited the application of prion detection assays. ARS scientists have devised new methods to enhance prion detection from brain regions that have been previously ignored for use in confirmatory diagnostic testing. These results have been published in the scientific literature and provide alternate techniques to augment current prion testing protocols.
Impacts
(N/A)
Publications
- Stanker, L.H., Scotcher, M.C., Lin, A.V., Mcgarvey, J.A., Prusiner, S., Hnasko, R.M. 2012. Novel epitopes identified by Anti-PrP monoclonal antibodies produced following immunization of Prnp0/0 Balb/cJ mice with purified scrapie prions. Hybridoma. 31(5):312-324. doi:10.1089/hyb.2012. 0022.
- Vasquez-Fernandez, E., Alonso, J., Pastrana, M.A., Ramos, A., Stitz, L., Vidal, E., Dynin, I.A., Petsch, B., Silva, C.J., Requena, J.R. 2012. Structural organization of mammalian prions as probed by limited proteolysis. PLoS One. 7(11):e50111. doi:10.1371/journal.pone.0050111.
- Silva, C.J., Dynin, I.A., Erickson-Beltran, M.L., Requena, J.R., Balachandran, A., Onisko, B.C., Hui, C., Carter, J.M. 2013. Using mass spectrometry to detect prions and oxidized prions in scrapie-infected sheep and CWD-infected elk. Biochemistry. 52:2139-2147. doi:10.1021/ bi3016795.
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): We will develop highly sensitive diagnostic tests to detect transmissible spongiform encephalopathy (TSE) in livestock and wildlife animal species prior to the onset of clinical disease. We will also develop tests to confirm the presence or absence of TSE disease agents in ingredients of animal origin and decontaminated environments. Approach (from AD-416): The threat of BSE continues to affect export economics for US meat. Meanwhile scrapie continues to influence sheep profits and herd biosecurity, and CWD is spreading throughout North America. Thus U.S. animal industry stakeholders have identified detection of the TSE infectious agent (prions) as a priority biosecurity research issue essential for prevention of TSE diseases. We will build on our previous successes using mass spectrometry (MS) for high-sensitivity and specificity in detection of PrPsc as a marker for TSE infectivity in blood using a hamster scrapie model. We will also develop a novel PrP-null mouse strain and related myeloma cell culture system for production of monoclonal antibodies (MAb), which may be specific for PrPsc. We will then choose MS or MAb and validate our novel diagnostic for preclinical diagnosis of scrapie in sheep blood. Whereas MS and MAb methods rely on dissolved samples, contamination of agricultural products and environmental surfaces is associated with solid samples. So we will produce a cell culture based assay for TSE infectivity that is surface-adsorbed. After using the relatively convenient hamster model for early development, we will validate this technology for detection of scrapie in sheep brain on meat-and-bone meal and stainless steel. 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. Transmissible spongiform encephalopathies (TSEs) are caused by mis-folded prion proteins that infect animals and contaminate their byproducts. TSEs represent a threat to agricultural livestock populations, global economic trade and human health. Diagnosis of TSE diseases as part of USDA animal surveillance efforts are confounded by the slow accumulation of the prion protein in the brain, late onset of clinical disease symptoms and multiple prion protein conformations. ARS scientist in Albany, California along with multi-institutional academic partners have generated novel anti-prion monoclonal antibodies and developed sensitive prion immunoassays that exploit the biochemical enrichment of prion protein with lipids and amplification of prions by cells in culture. To distinguish the multiple strains of mis-folded prions responsible for a range of TSE diseases ARS scientists in Albany, California along with their European colleagues have developed sample preparation methods that use chemical modification to uniquely tag prion strains. These chemical tags have been used to distinguish prion strain type by immunoassay and mass spectrometry. We have patented, published and transferred this technology to our USDA-APHIS partners and have established relationships with industrial partners for potential commercialization of these prion immunoassays. Accomplishments 01 New anti-prion monoclonal antibodies for diagnosis of Transmissible Spongiform Encephalopathy (TSEs). Detection of prion protein is central to the diagnosis of TSE diseases. Researchers at the Foodborne Contaminants Research Units in Albany, California, along with academic partners at the University of California San Francisco have generated an characterized three new anti-prion monoclonal antibodies (mAb) using pri knock-out mice and purified infectious prions. They have demonstrated th specificity and binding sensitivity of these mAb to prion proteins from multiple species, defined their bovine prion binding epitopes, and shown their utility for prion detection using multiple prion immunoassay platforms. These antibodies have been patented and should be of interest to food safety industry. 02 Amplification and detection of transmissible prion proteins by In-Cell Western (ICW). Researchers at the ARS in Albany, California, have developed a cell culture method that uses a prion susceptible cell line for the amplification and detection of infectious prion protein from biological and environmental samples. This method combines high-throughp cell culture-based bioassay amplification of prions from biological or environmental samples with near-infrared immunofluorescent detection and quantitation of accumulated prions. The development of this sensitive ne prion ICW provides a method to evaluate the occurrence of low-level prio contamination by providing a high-throughput quantitative assay with the added capability of low-level prion amplification from samples. A patent application has been filed for this immunoassay. 03 Distinguishing among strains of prion disease by identifying changes in protein shape. Changes in the shape of the prion protein are responsibl for the many strains of prion disease that can infect a single species, such as the three strains of �mad cow� disease. The nature of these shap differences is poorly understood and this lack of understanding makes it difficult to identify the different prion strains. ARS scientists in Albany, California, have developed new approaches to study these shape changes by reaction with synthetic chemical molecules. Animals infected with different prion strains were distinguished by their characteristic differences in chemical modification, as determined by changes in antibo binding. Such information can be used to develop antibody-based detectio methods and thereby facilitate the detection of different prion strains and the control of prion diseases. 04 Determination of the shape of high and low-density prion aggregates. Changes in the shape of the prion protein are responsible for prion disease such as �mad cow� and scrapie. These changes result in the formation two kinds of aggregates, the high-density aggregate and the lo density aggregate. ARS scientists in Albany, California, in collaboratio with their European colleagues, used a battery of tests to analyze the shape of the high-density and low-density aggregates. They were able to show that both forms cause disease and share a common shape, which means that a single antibody will be able to recognize both the high and low- density aggregates. This information can be used to develop antibody-bas detection methods and thereby facilitate the detection and control of prion diseases.
Impacts
(N/A)
Publications
- Sajnani, G., Silva, C.J., Ramos, A., Pastrana, M.A., Onisko, B.C., Erickson-Beltran, M.L., Antaki, E.M., Sigurdson, C.J., Carter, J.M., Requena, J.R. 2012. PK-sensitive PrPSc is infectious and shares basic structural features with PK-resistant PrPSc. PLoS Pathogens. 8(3):e1002547. doi:10.1371/journal.ppat.1002547.
- Silva, C.J. 2012. Using small molecule reagents to selectively modify epitopes based on their conformation. Prion. 6:(2)165-175.
- Ching, K.H., Lin, A.V., Mcgarvey, J.A., Stanker, L.H., Hnasko, R.M. 2012. Rapid and selective detection of botulinum neurotoxin serotypes-A and �B with a single immunochromatographic test strip. Journal of Immunological Methods. 380:23-29.
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) We will develop highly sensitive diagnostic tests to detect transmissible spongiform encephalopathy (TSE) in livestock and wildlife animal species prior to the onset of clinical disease. We will also develop tests to confirm the presence or absence of TSE disease agents in ingredients of animal origin and decontaminated environments. Approach (from AD-416) The threat of BSE continues to affect export economics for US meat. Meanwhile scrapie continues to influence sheep profits and herd biosecurity, and CWD is spreading throughout North America. Thus U.S. animal industry stakeholders have identified detection of the TSE infectious agent (prions) as a priority biosecurity research issue essential for prevention of TSE diseases. We will build on our previous successes using mass spectrometry (MS) for high-sensitivity and specificity in detection of PrPsc as a marker for TSE infectivity in blood using a hamster scrapie model. We will also develop a novel PrP-null mouse strain and related myeloma cell culture system for production of monoclonal antibodies (MAb), which may be specific for PrPsc. We will then choose MS or MAb and validate our novel diagnostic for preclinical diagnosis of scrapie in sheep blood. Whereas MS and MAb methods rely on dissolved samples, contamination of agricultural products and environmental surfaces is associated with solid samples. So we will produce a cell culture based assay for TSE infectivity that is surface-adsorbed. After using the relatively convenient hamster model for early development, we will validate this technology for detection of scrapie in sheep brain on meat-and-bone meal and stainless steel. Transmissible prions that infect animals and their byproducts represent a human health threat through the consumption of contaminated food. The unusual biochemical properties of infectious prion proteins have presented a difficult diagnostic target and new methods are needed to improve their detection in livestock. We have demonstrated the effectiveness of novel biochemical separation techniques for the isolation, enrichment and detection of prions from biological samples. Application of these prion enrichment strategies have resulted in the generation of specific monoclonal antibodies that bind with high-affinity to prions from multiple species with improved sensitivity. We have demonstrated the utility of these anti-prion monoclonal antibodies for sensitive detection of prions in multiple immunoassay formats and have identified the exact amino acid sequence in the prion protein to which they bind. Using our prion enrichment methods along with a modified enzyme-linked immunosorbent assay (ELISA) we have established a selective and sensitive high-throughput prion immunoassay capable of identifying prion disease in asymptomatic animals. We have filed patents and have transferred this technology to our USDA, Animal and Plant Health Inspection Services (APHIS)partners. The use of this ARS technology as part of the ongoing national prion surveillance efforts by the National Veterinary Logistic System (NVLS) will improve early detection of prion infected livestock effectively containing disease transmission and ensuring the eradication of prion from food and agricultural byproducts. Significant Activities that Support Special Target Populations In support of our USDA-APHIS partners we have transferred our anti-prion monoclonal antibodies, techniques for prion enrichment of biological samples, and modified ELISA prion detection assay to NVLS for integration into their national prion surveillance program. Accomplishments 01 Major improvement in sensitivity allows early detection of �mad cow� and related diseases in livestock. Surveillance for "mad cow" and related prion diseases, such as scrapie in sheep, requires early diagnosis of disease. ARS scientists in Albany, California, have generated a significant advance in test sensitivity to produce a rapid, sensitive, cost-effective prion diagnostic assay. The advances include an improved sample enrichment method that concentrates prions into a smaller volume, and new antibodies that bind to prions under test conditions more tightl than previos antibodies. While the US patent for this technology is pending, it has been transferred to the National Veterinary Logistic System in Ames, Iowa, where higher test sensitivity improves monitoring US livestock to help secure our food supply and export markets. 02 Determination of protein shape involved in prion disease. Prion disease such as "mad cow" and scrapie, are associated with changes in shape of a specific protein in the victim's brain- the prion protein. A lack of information regarding these shape changes hampers diagnosis and control prion disease. ARS scientists in Albany, California, developed and used new approach to study this shape change through modification of prion protein by simple chemical addition reactions. Diseased proteins underwe significantly different modifications than normal proteins, and these differences were easily detected by changes in antibody binding. A US patent is pending for this new approach to protein based disease diagnos which is important in detection and control of prion disease.
Impacts
(N/A)
Publications
- Silva, C.J., Onisko, B.C., Dynin, I.A., Erickson, M.L., Requena, J.J., Carter, J.M. 2011. Utility of mass spectrometry in the diagnosis of prion diseases. Analytical Chemistry. (83)1609-1615. DOI: 10.1021/ac102527w.
- Gong, B., Ramos, A., Vazquez-Fernandez, E., Silva, C.J., Alonso, J., Requena, J.R. 2011. Probing structural differences between PrPC and PrPSc by surface nitration and acetylation: evidence of conformational change in the C-terminus. Biochemistry. 50:4963-4972.
- Silva, C.J. 2010. Mass spectrometry and prions: The need to simplify and remove oil from the system. International News on Fats, Oils and Related Materials.21(8):517-520.
- Hnasko, R.M., Lin, A.V., Mcgarvey, J.A., Stanker, L.H. 2011. A Rapid Method to Improve Protein Detection by Indirect ELISA. Biochemical and Biophysical Research Communications. DOI 10.1016/j.bbrc.2011.06.005.
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) We will develop highly sensitive diagnostic tests to detect transmissible spongiform encephalopathy (TSE) in livestock and wildlife animal species prior to the onset of clinical disease. We will also develop tests to confirm the presence or absence of TSE disease agents in ingredients of animal origin and decontaminated environments. Approach (from AD-416) Transmissible spongiform encephalopathies (TSEs) are a family of fatal diseases caused by exposure of animals to infectious prions. The zoonotic prion disease in cattle (Bovine Spongiform Encephalopathy [BSE]) represents an economic threat to the U.S. beef industry through deleterious changes in meat export, herd management practices, consumer perception and safety. Other TSEs, such as Scrapie in sheep and Chronic Wasting Disease (CWD) in cervids, directly impact the economics and management of those commodities and represent reservoirs of infectious prions with the potential for vertical transmission of disease to other agriculturally important animal species. Consequently, U.S. animal industry stakeholders have identified detection of infectious prions as a priority bio-security research issue essential for prevention of TSE diseases. Our expertise in pathogen diagnostics will be used to develop the methodology and molecular tools necessary for the detection of infectious prions (PrPSc) from biological tissues and environmental substrates. (1) We will generate new high-affinity monoclonal antibodies (MAb) against PrPSc for the development of sensitive immunoassays. (2) We will define new methods for the isolation and enrichment of PrPSc to promote improved detection of diseased animals or contaminated substrates. (3) We will develop a method for the detection of PrPSc by mass spectrometry. Anti-PrPSc immunoassays and analytical MS methods will be validated with prion contaminated samples and detection sensitivity determined. Improved sensitivity of PrPSc detection will be achieved by assay refinement and sample enrichment strategies toward predictive diagnostics of TSE in asymptomatic animals. Replacing 5325-32000-007-00D (3/19/2008). We have developed a method to enrich prions from biological tissues resulting in a significant increase in sensitivity of prion detection by immunoassay. This enrichment strategy is being applied for the detection of prions in biological samples with low-level contamination from asymptomatic animals. To overcome the challenges in making anti-PrPSc antibodies we, in collaboration with our academic partners, generated a transgenic mouse that lacks endogenous PrPC (Prnp0/0 Balbc/J). We demonstrated that wild- type mice fail to respond to a purified PrPSc immunogen whereas Prnp0/0 Balbc/J mice elicit a robust and specific immune response producing anti- prion antisera. This model is currently being used to generate hybridoma cells for the production of monoclonal antibodies (MAb) used in prion immunoassays. The identification of hybridoma cells producing anti-PrPSc MAbs requires a screening method that can distinguish PrPSc from normal PrPC. Current screening methods utilized recombinant PrPC or crude homogenates and lack the capacity to discriminate. Consequently, these methods favor identification of antibodies that recognize both PrPC and PrPSc. We have developed a screen that exploits our prion enrichment method to generate sufficient native PrPC and PrPSc for use as an antigen. By using a comparative ELISA as screen our method favors identification of hybridomas producing PrPSc selective MAbs. We have used this method to successfully identify hybridomas making high-affinity anti-prion MAbs useful for PrPSc detection. Together with our academic partners, we have generated eight new anti- prion MAbs and determined their binding properties to prions from different species. Five antibodies exhibit conformational dependent binding to discontinuous PrP epitopes. The high-affinity F4-31 anti-prion MAb has increased the sensitivity of prion detection and will be useful in a wide range of immunoassays. Mammalian cell lines were positively evaluated for PrPC expression by immunoassay. Prion challenge with infectious hamster, ovine, or deer brain extract was ineffective in converting PrPC to PrPSc in any cell line tested. However, PrPSc challenge (RML strain) of mouse cell lines resulted in time-dependent increase in detection of proteinase-K (PK) resistant PrPSc. Yet, the efficiency of conversion was low, the biochemical profile of cellular PrPSc by Western blot was variable, and infectivity of cellular PK-resistant material was indeterminate. Over- expression of recombinant mouse and ovine PrP in cell lines was ineffectual in promoting conversion of PrPC to PK-resistant PrPSc. We conclude that a cell model for the amplification of low-level prion contaminant is currently impractical for reliable prion detection. Blood plasma from hamsters infected with PrPSc was fractionated to enrich prions and shown to be infectious. Efforts to detect prions from plasma of infected animals by mass spectrometry have been unsuccessful, but we can capture and detect prion with our MAbs by immunoassay. We are currently refining our enrichment of plasma to evaluate prion detection in blood of asymptomatic animals. Accomplishments 01 Prion sample concentration. Early detection of prion diseases such as sheep scrapie requires concentration of the prion proteins that serve as disease markers from dilute biological samples. Researchers at the Foodborne Contaminants Research Unit in Albany, California, in collaboration with the University of California San Francisco, developed new method for concentrating prion proteins from animal tissues. We foun this method results in a significant increase in prion concentration, allowing more sensitive prion detection. This year we filed a patent application and published a manuscript on this new method, showing how i provides more sensitive and early detection of disease in infected anima 02 New strain of mouse for anti-prion antibody production. Production of antibodies for detection of prion proteins associated with diseases such as sheep scrapie is limited by the resistance of normal mice to making a immune response against their own proteins. Researchers at the Foodborne Contaminants Research Unit in Albany, California, in collaboration with our partners at the University of California San Francisco, have made a new strain of mouse that lacks the prion protein. Unlike normal mice, th new mice are highly sensitive to immunization with prions. This year we filed a patent application and published a manuscript showing the use of these mice in making new antibodies that can bind and detect prions. Suc antibodies may be used for more sensitive and early detection of disease in infected animals. 03 New method for identification of antibodies that detect prion proteins. Making new monoclonal antibodies for detection of prion proteins in diseases such as sheep scrapie requires selection of the best performing cells from among thousands of candidates. Researchers at the Foodborne Contaminants Research Unit in Albany, California have developed a fast a sensitive method to identify such cells, which are taken from mice that have been immunized to produce antibodies that strongly bind to prion protein. This method was used to identify improved antibodies which are now available for use in detection of disease. Furthermore, the new screening method may be used by other scientists for research to make additional new antibodies for prions. 04 New antibodies for detection of prion disease. Sensitive detection of prion proteins for early diagnosis of disease requires antibodies that a capable of strong binding to prions. Researchers at the Foodborne Contaminants Research Unit in Albany, California, in collaboration with our partners at the University of California San Francisco, generated eight new antibodies that detect prion disease in many different animals We have published a manuscript that describes these anti-prion antibodie and shows their value in improving detection of prions. Improved detecti methods will help in herd management and control of the spread of prion diseases. 05 Prion protein modification. Changes in the prion protein that may lead disease are poorly understood. Researchers at the Foodborne Contaminants Research Unit in Albany, California used mass spectrometry to detect and measure a specific chemical modification to the prion protein. In contra to results published by other scientists, we found no difference in this chemical modification between normal and infected forms of the prion protein. We have published a manuscript describing this work, which help scientists and veterinarians focus on other changes in the prion protein that might be involved in prion disease.
Impacts
(N/A)
Publications
- Silva, C.J., Onisko, B.C., Dynin, I.A., Erickson, M.L., Vensel, W.H., Requena, J., Antaki, E.M., Carter, J.M. 2010. Assessing the Role of Oxidized Methionine at Position 213 in the Formation of Prions in Hamsters. Journal of Biochemistry. (49):1854-1861.
- Hnasko, R.M., Serban, A.V., Carlson, G.A., Prusiner, S.B., Stanker, L.H. 2010. Generation of Antisera to Purified Prions in Lipid Rafts. Prion. 4:294-104.
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) We will develop highly sensitive diagnostic tests to detect transmissible spongiform encephalopathy (TSE) in livestock and wildlife animal species prior to the onset of clinical disease. We will also develop tests to confirm the presence or absence of TSE disease agents in ingredients of animal origin and decontaminated environments. Approach (from AD-416) The threat of Bovine Spongiform Encephalopathy(BSE) continues to affect export economics for US meat. Meanwhile scrapie continues to influence sheep profits and herd biosecurity, and CWD is spreading throughout North America. Thus U.S. animal industry stakeholders have identified detection of the TSE infectious agent (prions) as a priority biosecurity research issue essential for prevention of TSE diseases. We will build on our previous successes using mass spectrometry (MS) for high-sensitivity and specificity in detection of PrPsc as a marker for TSE infectivity in blood using a hamster scrapie model. We will also develop a novel PrP-null mouse strain and related myeloma cell culture system for production of monoclonal antibodies (MAb), which may be specific for PrPsc. We will then choose MS or MAb and validate our novel diagnostic for preclinical diagnosis of scrapie in sheep blood. Whereas MS and MAb methods rely on dissolved samples, contamination of agricultural products and environmental surfaces is associated with solid samples. So we will produce a cell culture based assay for TSE infectivity that is surface-adsorbed. After using the relatively convenient hamster model for early development, we will validate this technology for detection of scrapie in sheep brain on meat-and-bone meal and stainless steel. Replacing 5325-32000-007-00D (3/19/2008). Significant Activities that Support Special Target Populations In FY09 we continued to experiment with new combinations of detergents, temperature, and centrifugation conditions, to refine our method for enzyme-free isolation of prions from animal tissues. An enzyme-free method is required, because prions in blood are not resistant to the proteinase K enzyme cocktail that is typically used to isolate prions from brain. We have now demonstrated that our purified prion material remains infectious. Getting our method for hamster brain to work on sheep brain, this year, required a surprisingly large amount of work. We found that sheep brains contain significantly more fibrous tissue. This feature apparently evolved to meet the needs of a species remarkable for head-butting behavior. We are studying plasma and buffy coat as blood fractions to develop for prion diagnosis. Each of these fractions contains about half the infectivity present in blood. Buffy coat is more concentrated, while plasma may prove more robust for handling and storage. We have completed our evaluation of cell culture for detection of prions in commodities and environmental samples. This work involved collaboration with the world�s leading prion cell biologists. We found this method gave very poor agreement between experiments due to instability of the cell lines. We have continued to pursue a Contingency on covalent modification of PrP to generate disease-specific molecular differences that subsequently can be recognized by antibodies. This work attracted interest and collaboration with the Canadian company Amorfix. Technology Transfer Number of New Patent Applications filed: 2
Impacts
(N/A)
Publications
- Harman, J.L., Silva, C.J. 2009. Bovine Spongiform Encephalopathy. Journal of the American Veterinary Medical Association. (2009)234(1):59-72
- Hnasko, R.M., Bruederle, C.E. 2009. Inoculation of Scrapie with the Self- Assembling RADA-Peptide Disrupts Prion Accumulation and Extends Hamster Survival. PLoS ONE. 4(2): http://www.plosone.org/article/info:doi/10. 1371/journal.pone.0004440
- Silva, C.J. 2009.Prion Diseases:Sequelae and Long Term Consequences of Infectious Diseases. District of Columbia:ASM Press. 442p.
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report Objectives (from AD-416) We will develop highly sensitive diagnostic tests to detect transmissible spongiform encephalopathy (TSE) in livestock and wildlife animal species prior to the onset of clinical disease. We will also develop tests to confirm the presence or absence of TSE disease agents in ingredients of animal origin and decontaminated environments. Approach (from AD-416) The threat of BSE continues to affect export economics for US meat. Meanwhile scrapie continues to influence sheep profits and herd biosecurity, and CWD is spreading throughout North America. Thus U.S. animal industry stakeholders have identified detection of the TSE infectious agent (prions) as a priority biosecurity research issue essential for prevention of TSE diseases. We will build on our previous successes using mass spectrometry (MS) for high-sensitivity and specificity in detection of PrPsc as a marker for TSE infectivity in blood using a hamster scrapie model. We will also develop a novel PrP-null mouse strain and related myeloma cell culture system for production of monoclonal antibodies (MAb), which may be specific for PrPsc. We will then choose MS or MAb and validate our novel diagnostic for preclinical diagnosis of scrapie in sheep blood. Whereas MS and MAb methods rely on dissolved samples, contamination of agricultural products and environmental surfaces is associated with solid samples. So we will produce a cell culture based assay for TSE infectivity that is surface-adsorbed. After using the relatively convenient hamster model for early development, we will validate this technology for detection of scrapie in sheep brain on meat-and-bone meal and stainless steel. Replacing 5325-32000-007-00D (3/19/2008). Significant Activities that Support Special Target Populations At this point in the Project, in general, we are completing preliminary studies using our relatively convenient hamster and mouse models, and are starting to work with more agriculturally relevant sheep and deer tissues. We are finding the cervid tissues quite different from rodent tissues, in their requirements for sample workup (e.g., amount and quality of lipid and fiber) and in their expression of TSE infectivity and presence of markers. OSQR required us to establish a new collaboration with a reputable cell biologist, to assist with our cell-based scrapie assay. We now have a new MTA with Dr. Charles Weissmann (Scripps), under which we are sharing cell lines and laboratory protocols. We have completed one part of our speed congenics project to develop PrP- null (disease-resistant) mice for use in antibody generation. After conceiving a new procedure for immunogen enrichment, we performed experimental vaccination of these animals in our facilities. This project relates to NP103 Component 8: Prevention and control of transmissible spongiform encephalopathies. Problem statement 9A: Scrapie; 9B Chronic Wasting Disease (CWD); and 9C: Bovine Spongiform Encephalopathy (BSE).
Impacts
(N/A)
Publications
- Bruederle, C.E., Hnasko, R.M., Kraemer, T., Garcia, R.A., Haas, M.J., Marmer, W.N., Carter, J.M. 2008. Prion infected Meat-and-Bone Meal is still Infectious After Biodiesel Production. PLoS One 3(8): e2969. doi:10. 1371/journal.pone.0002969.
- Onisko, B.C., Chen, N., Napoli, J. 2008. The Nuclear Transcription Factor RAR Associates with Neuronal RNA Granules and Suppresses Translation. Journal of Biological Chemistry. 283(30):20841-20847.
- Sajnani, G., Pastrana, M.A., Dynin, I.A., Onisko, B.C., Requena, J.R. 2008. Insights on scrapie prion protein (prpsc) structure obtained by limited proteolysis and mass spectrometry. Journal of Molecular Biology. 382(2008) :88-98.
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