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? This project is aligned to NP-108, Food Safety Program and addresses Strategic Plan Performance Measure 3.1.2. Incidental contamination of foods by pathogenic bacteria and/or their toxins is a serious threat to public health and the economy. Intentional contamination of the food supply (bioterrorism) may pose even greater threats. The presence of food-borne pathogens and toxins must be rapidly determined at various stages of food production, processing and distribution. Producers, processors, retailers, researchers and public health professionals need simple and cost-effective biosensor-based methods to detect multiple pathogens and toxins in foods. Thus, we will develop rapid, specific, and sensitive biosensor-based assays for diverse pathogenic bacteria which can be widely adopted in applications ranging from simple field tests to high speed, high throughput laboratory screening assays. Specific objectives of the research are to develop (1) specific, high affinity biorecognition reagents for food-borne pathogens and toxins, (2) rapid and effective means to separate and concentrate targeted pathogens without carryover of background organisms and (3) integrated assay systems based on multiple target biosensor platforms. The proposed research addresses the need for development or modification (and validation) of biosensor-based diagnostic tools for the detection of pathogenic microorganisms and their products as identified in the Problem areas 1.2.1 (Detection) and 1.2.9 (Food Security). The research also addresses Agency Performance Measure 3.1.2: Develop and transfer to Federal agencies and the private sector systems that rapidly and accurately recognize, detect, and differentiate the most critical and economically important foodborne microbial pathogens. The proposed biosensor research will produce reliable, cost-effective, quantitative measurements of microbial pathogens and/or toxins in food products. These multi-sample, multi-analyte detection tools are needed by food producers and regulators to ensure the safety of the food supply. In addition, these detection methodologies will provide data to carry out risk assessment, to develop and validate predictive microbial models, and to identify where intervention is most needed. This information will assist the implementation of Hazard Analysis and Critical Control Point (HACCP) programs by FSIS, FDA, and their regulated industries. With respect to food security, the developed biosensor detection technologies will allow ARS to strengthen and expand laboratory preparedness and to develop rapid laboratory methods for the detection of select agents (microbial pathogens, toxins and chemical contaminants) in adulterated foods. 2.List by year the currently approved milestones (indicators of research progress) Approved yearly milestones associated research objectives are: FY 2007: Objective 1. Develop specific, high affinity biorecognition reagents for food-borne pathogens and toxins. Milestone 1. Select and develop immuno reagents for Y. pestis. Obtain non-immuno DNA targeting technique. Objective 2. Develop rapid and effective means to separate and concentrate targeted pathogens without carryover of background organisms. Milestone 1. Synthesize and screen tagged microbial binding agents. Characterize matrix effects for large-volume filtration Objective 3. Develop integrated assay systems based on multiple target biosensor platforms. Milestone 1. Fabricate miniturized multi-target platform using immuno and DNA detection reagents. Characterize L. mono. detection using BIACORE insturment. Synthesize La-immuno reagents against pathogens and toxins. FY 2008: Objective 1. Develop specific, high affinity biorecognition reagents for food-borne pathogens and toxins. Milestone 2. Identify antibodies against F. tularensis. Identify effective DNA target reagents against L. monocytogenes. Objective 2. Develop rapid and effective means to separate and concentrate targeted pathogens without carryover of background organisms. Milestone 2. Establish efficacy of tagged binding agents. Synthesize magnetic reagents for detection of pathogens and their toxins. Develop methods for pathogen screening in large volumes. Objective 3. Develop integrated assay systems based on multiple target biosensor platform Milestone 2. Select and optimize target DNA sequences. Prepare immunomagnetic reagents for F. tularensis and L. monocytogenes identification. Generate La-labeled antibodies against L. monocytogenes and Y. pestis. FY 2009: Objective 1. Develop specific, high affinity biorecognition reagents for food-borne pathogens and toxins. Milestone 3. Complete development of immuno reagents for F. tularensis. Characterize DNA target reagents against L. monocytogenes. Objective 2. Develop rapid and effective means to separate and concentrate targeted pathogens without carryover of background organisms. Milestone 3. Optimize magnetic reagents for pathogen and toxin capture. Develop multi-pathogen concentration columns. Objective 3. Develop integrated assay systems based on multiple target biosensor platforms. Milestone 3. Establish the molecular basis for the capture efficiency of pathogens and their toxins. Investigate fluidic manifolds. FY 2010. Objective 1. Develop specific, high affinity biorecognition reagents for food-borne pathogens and toxins. Milestone 4. Complete immuno reagents selection for E.coli O-157:H7 toxin. Develop L. monocytogenes DNA target magnetic beads. Objective 2. Develop rapid and effective means to separate and concentrate targeted pathogens without carryover of background organisms. Milestone 4. Identify new material(s) to minimize background in pathogen detection. Optimize multi-pathogen concentration columns Objective 3. Develop integrated assay systems based on multiple target biosensor platforms. Milestone 4. Develop multi-target pathogen enrichment methods for foods. Capture and detect bacteria on antibody platforms. Fabricate filters and affinity columns. FY 2011: Objective 1. Develop specific, high affinity biorecognition reagents for food-borne pathogens and toxins. Milestone 5. Complete immuno reagents selection for E.coli O-157:H7 toxin. Develop L. monocytogenes DNA target magnetic beads. Objective 2. Develop rapid and effective means to separate and concentrate targeted pathogens without carryover of background organisms. Milestone 5. Prepare and characterize magnetic reagents. Determine capture efficiency of mixed and bifunctional magnetic reagents. Detect foodborne pathogens with multi-pathogen filtration and concentration system Objective 3. Develop integrated assay systems based on multiple target biosensor platforms. Milestone 5. Detect multiple pathogens in foods using DNA and immuno methods on miniaturized plate formats. Establish L. monocytogenes and Y. pestis detection methods using IMB-TRF. Process food samples with integrated fluidic modules for pathogen detection. 4a.List the single most significant research accomplishment during FY 2006. This project was developed to replace 1935-42000-046-00D and was approved on 5-9-2006. Related accomplishments are reported for -46. 4b.List other significant research accomplishment(s), if any. None. 4c.List significant activities that support special target populations. None. 4d.Progress report. 1935-42000-058-01R. This report serves to document reserch conducted under reimbursable cooperative agreement between ARS and Polysciences. Additional details of the reserch can be found in the parent report for project 1935-42000-058-00D, entitled "Integrated Biosensor-Based Processes for Multipathogenic Analyte Detection". The objective of this cooperative research project is to determine the effects of various particle characteristics on the overall efficiency of capturing pathogenic bacteria, e.g., Escherichia coli O157:H7, Salmonella, in different foods. The use of immunomagnetic beads (IMB) to capture targeted pathogenic bacteria in foods has been applied to many rapid biosensor technologies. In addition of being a simple and effective mean of separating pathogens from other food components, the application of IMB also can substantially increased the concentration of pathogen in the detection volume. However, the controlling factors of the capture are yet to be established. In this agreement, systematical identification of those factors, e.g., sizes and densities were conducted. The results indicated that the bacterial capture efficiency of the beads was enhanced by larger sizes and heavier densities. This conclusion was supported by a through hydrodynamic analysis. In addition, the agreement also supported a project to reduce the costs of detection process. IMB-captured pathogens beads were further labeled by immuno fluorescent beads (FB) for capture signal amplification. FB was normally widely used for particle size in industry. All those research progresses were published and were reported in annual reports associated with the in-house project. The agreement was terminated on 9/30/2006 and new agreement is being developed with Polysciences. 1935-42000-058-02R. This report serves to document research conducted under a reimbursable cooperative agreement between ARS and FSIS. Additional details of this research can be found in the parent project report for 1935-42000-058-00D, entitled "Integrated Biosensor-Based Processes for Multipathogenic Analyte Detection". Selection of Antibody Fragments for Detection of Y. pestis. Yersinia pestis is the causative agent of plague and a recent concern with respect to food security. Commercially available antibodies and detection methods for Y. pestis rely on the detection of one unique protein present on the surface of the bacterium, the F1-capsular antigen. These detection methods were developed for the detection of Y. pestis from clinical samples and are unlikely to be applicable for the detection of Y. pestis in foods. At the request of FSIS, we have begun to develop methods for the detection of Y. pestis from foods. Using antibody phage display method, we have isolated several antibody fragments that detected Y. pestis. Although these antibodies did not react with the Y. pestis F1-capsular antigens they did exhibit cross-reactivity with some other bacterial species necessitating further antibody selection and screening. The success selection of specific antibodies will allow further development of methods for detecting Y. pestis in foods. Development of Immunomagentic Beads for the capture of Y. pestis. Yersinia pestis is the causative agent of plague and an potential threat to food security. Methods for the detection of Y. pestis from clinical samples have been developed, but these methods are not directly applicable to the detection of the pathogen from food. At the request of FSIS, we have generated immunomagnetic beads (IMBs) for the capture of Y. pestis using commercially available streptavidin coated magnetic particles and monoclonal antibodies directed against the Y. pestis F1-capsular antigen. The IMBs were evaluated and found to capture Y. pestis. We also observed and characterized the cross-reactivity of the antibody with a few other bacterial species. The cross-reactivity will necessitate the development of new antibodies for more specific detection of Y. pestis from foods. The success selection of specific antibodies will allow further development of methods for detecting Y. pestis in foods. 5.Describe the major accomplishments to date and their predicted or actual impact. The project was approved in May of 2006. There is no documented accomplishment yet. 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 collaboration with Polysciences is expected to continue during the life of this project to advance the immunomagnetic bead technology. 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). Gehring, A.G., Albin, D.M., Irwin, P.I., Tu, S-I. Detection and Potential Identification of Escherichia Coli O157:H7 using a Microparticle-based Chemiluminescent Immunoassay, The Ninth World Congress on Biosensor, May 10-12, 2006, Toronto Canada Tu, S-I., Cooke, P., Paoli, G., Gehring, A. Detection of Escherichia coli O157:H7 by the Use of Immunomagnetic and Fluorescent Beads. The Ninth World Congress on Biosensor, May 10-12, 2006, Sheraton Centre, Toronto Canada Paoli, G.C., L.G. Kleina, S.-I. Tu. 2006. Immunomagnetic Capture and Detection of Yersinia pestis from Milk. 93rd Annual Meeting of the International Association for Food Protection. Calgary, Alberta, Canada. Poster No. P3-18. Paoli, G.C. Food borne Pathogen Detection Reagents from Phage Displayed Antibodies. 26th Annual International Workshop on Rapid Methods and Automation in Microbiology. Kansas State University. Manhattan, KS, July 20, 2006. Tu, S-I. Immuno magnetic capture and biosensor amplification for pathogen detection in foods.