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? The food and water supply of the United States are among the safest in the world. An increasing number of outbreaks of human disease are, however, occurring as a result of pathogen contamination of fruits, vegetables, and water resources. Liquid and solid animal wastes originating from Concentrated Animal Feeding Operations are believed to be a major source of pathogenic microorganisms. At present, our ability to detect and quantify pathogenic microorganisms, and predict their fate and transport in the environment is not completely understood. Hence, the two primary objectives of this research are to: (I) develop methods for the detection, quantification, and source identification of pathogenic microorganisms; and (II) quantify mechanisms and processes affecting the transport and survival of pathogenic microorganisms. The development of economically viable methods to treat and /or manage animal wastes is a secondary objective of this project. To this end, an interdisciplinary team of research scientists is conducting laboratory, growth chamber, field, and numerical experiments to develop novel detection techniques and to explore the influence of microbe, water, manure, and soil characteristics on pathogen fate and transport in the environment. Pathogens are considered by most consumers, food processors, food safety researchers and regulatory agencies to be the most important food safety problem today. It is estimated that there are 10 million to 100 million cases of food-borne illness each year. Investigations of water-borne disease outbreaks also suggest that drinking water supplies may be vulnerable to pathogen contamination from animal production operations. Pathogenic microorganisms in groundwater are estimated to cause between 750,000 and 5 million illnesses per year in the United States. Research that helps to detect, and minimize the transport and survival of pathogens from animal manure to the environment is important for maintaining confidence in the food and water supply. This research also aids in the development of control strategies to prevent the transmission of pathogenic microorganisms to food-producing animals, agricultural crops, and the environment in general. This research falls under National Program 206 Manure and Byproduct Utilization: Pathogen and Pharmaceutically Active Compounds Components. Problem Area 1. Methods Assessment and Development and Problem Area 2. Fate and Transport of Pathogens. 2.List by year the currently approved milestones (indicators of research progress) Milestones - Year 1 (2005) Objective 1. Detection, quantification and characterization of pathogen survival in different environmental matrices. Initiate study of primers and probes for E. coli and enterococci quantification. Objective 2. Determine inactivation/survival rates and transport of pathogens from manure sources within a watershed. Complete instrumentation of collection sites for establishing nonpoint fecal pollution at sites along the San Antonio Creek. Objective 3. Determine sources of nonpoint fecal pollution at the Santa Ana River Watershed by bacterial source tracking at the ecosystem level. Complete instrumentation of sampling sites in the watershed. Objective 4. Quantify important mechanisms influencing the transport and retention of pathogens in subsurface environments. Continue study of the release behavior of variously sized pathogens from dairy manures. Initiate study of pathogen transport at various measurement scales. Initiate study to determine the influence of colloid input and duration on straining behavior. Objective 5. Model development of pathogen transport in saturated and unsaturated zones. Develop conceptual model for pathogen transport across textural interfaces; transfer technology Milestones -Year 2 (2006) Objective 1. Detection, quantification and characterization of pathogen survival in different environmental matrices. Evaluation of primer and probe sensitivity. Initiate testing of primers and probes with environmental samples using community DNA/RNA. Objective 2. Determine inactivation/survival rates and transport of pathogens from manure sources within a watershed. Complete annual analysis of water and sediment samples. Objective 3. Determine sources of nonpoint fecal pollution at the Santa Ana River Watershed by bacterial source tracking at the ecosystem level. Complete annual collection and analysis of water and sediment samples for genetic profiling and source tracking of pathogens in the Santa Ana River Watershed. Objective 4. Quantify important mechanisms influencing the transport and retention of pathogens in subsurface environments. Initiate study of mechanisms affecting the unsaturated transport of pathogens. Initiate study of the role of solution salinity and composition on pathogen transport. Continue study on transport of pathogens at various measurement scales. Continue study of the release behavior of variously sized pathogens from dairy manures. Objective 5. Model development of pathogen transport in saturated and unsaturated zones. Develop conceptual model for pathogen transport in the presence/absence of manure suspensions; transfer technology. Milestones - Year 3 (2007) Objective 1. Detection, quantification and characterization of pathogen survival in different environmental matrices. Continue testing primers and probes with environmental samples using community DNA/RNA. Transfer technology on primers and probes for assessment of viable pathogens. Objective 2. Determine inactivation/survival rates and transport of pathogens from manure sources within a watershed. Continue analysis of inactivation/survival rates and transport of pathogens in the watershed. Objective 3. Determine sources of nonpoint fecal pollution at the Santa Ana River Watershed by bacterial source tracking at the ecosystem level. Continue collection and analysis of microbial transport data. Obtain isolates from other locations and assay for geographic variability. Objective 4. Quantify important mechanisms influencing the transport and retention of pathogens in subsurface environments. Continue study of the role of solution salinity and composition on pathogen transport. Continue study of mechanisms affecting the unsaturated transport of pathogens. Objective 5. Model development of pathogen transport in saturated and unsaturated zones. Develop conceptual model of release behavior of variously sized pathogens from dairy manures; transfer technology. Develop conceptual model of influence of colloid input and duration on straining behavior; transfer technology. Year 4 (2008) Objective 1. Detection, quantification and characterization of pathogen survival in different environmental matrices. Determine best method for quantifying viable cells in environmental samples; transfer technology; prepare reports/manuscripts. Objective 2. Determine inactivation/survival rates and transport of pathogens from manure sources within a watershed. Complete analysis of microbial transport and inactivation rate. Objective 3. Determine sources of nonpoint fecal pollution at the Santa Ana River Watershed by bacterial source tracking at the ecosystem level. Transfer technology concerning inactivation/survival rates and transport of pathogens in the Santa Ana River Watershed. Objective 4. Quantify important mechanisms influencing the transport and retention of pathogens in subsurface environments. Complete study of mechanisms affecting the unsaturated transport of pathogens. Complete study of the role of solution salinity and composition on pathogen transport. Objective 5. Model development of pathogen transport in saturated and unsaturated zones. Develop conceptual model of the role of aqueous solution salinity and composition on colloid/pathogen transport; transfer technology. Year 5 (2009) Objective 1. Detection, quantification and characterization of pathogen survival in different environmental matrices. Research completed and technology transferred. Objective 2. Determine inactivation/survival rates and transport of pathogens from manure sources within a watershed Transfer technology concerning inactivation/survival rates and transport of pathogens; prepare reports/manuscripts. Objective 3. Determine sources of nonpoint fecal pollution at the Santa Ana River Watershed by bacterial source tracking at the ecosystem level. Contribute isolates and microbial fingerprints to the national database; Transfer technology. Objective 4. Quantify important mechanisms influencing the transport and retention of pathogens in subsurface environments. Continue study of pathogen transport at various measurement scales. Objective 5. Model development of pathogen transport in saturated and unsaturated zones. Develop conceptual model of unsaturated transport of pathogens. 4a.List the single most significant research accomplishment during FY 2006. Monitoring pollutants at the watershed level - The middle Santa Ana River (San Antonio and Chino Creek) is listed as an impaired water body with main pollutants, nutrients, and pathogens which originate from densely populated areas, agricultural activities, and urban and storm-water runoff. Field studies were undertaken by scientists at the USDA George E. Brown, Jr. Salinity Lab, in collaboration with scientists at the Orange County Water District, Inland Empire Utility Commission, Santa Ana River Water Board, and USGS to quantify the main sources of fecal pollution to Santa Ana River using specific gene markers. Results from this study showed that the Middle Santa Ana River watershed is affected by multiple sources of non-point microbial pollution. The spatial and temporal distributions of these markers showed that human-specific Bacteroides species was detected in all the samples except the control sites and in a few other sites while Enterococcus species showed unique genetic profiling based on land use. This study demonstrates the usefulness of gene markers of non-culture-based microbial source tracking approaches for determining sources of fecal pollution. When fully developed, this technology will contribute significantly to best management method for water quality improvement in the watershed. Accomplishments are aligned with National Program 206 Manure and Byproduct Utilization: Pathogen and Pharmaceutically Active Compounds Component. Problem Area 1. Methods Assessment and Development and Problem Area 2. Fate and Transport of Pathogens. 4b.List other significant research accomplishment(s), if any. Transport and deposition of E. coli O157:H7 - Water contamination by various strains of bacteria is becoming common in rural areas of the United States, and may pose a risk to drinking water resources. Laboratory and modeling studies were undertaken by ARS scientists at the Salinity Lab, in collaboration with Drs. Sharon Walker and Jirka Simunek of the University of California, Riverside, to quantify the influence of grain size distribution and flow rate on the transport and deposition behavior of pathogenic E. coli O157:H7. Results suggest that straining and aggregation of E. coli O157:H7 can occur in fine sand, and that increasing the water velocity minimizes straining. An improved understanding of the processes that control the subsurface transport and survival of pathogenic E. coli O157:H7 will aid in the assessment of drinking water contamination and in the development of efficient water treatment techniques. Accomplishments are aligned with National Program 206 Manure and Byproduct Utilization: Pathogen and Pharmaceutically Active Compounds Component. Problem Area 1. Methods Assessment and Development and Problem Area 2. Fate and Transport of Pathogens. Coupling of physical and chemical mechanisms of colloid deposition – Little research has examined the influence of solution chemistry on straining deposition in small soil pores. Experimental and theoretical studies were undertaken by ARS scientists at Salinity Lab, in collaboration with Dr. Sharon Walker and Saeed Torkzaban of U.C. Riverside, to quantify the coupling of physical and chemical mechanisms of colloids deposition under unfavorable attachment conditions (pH=10). Results indicate that the transport and deposition behavior of negatively charged colloid particles were strongly dependent on the solution chemistry, system hydrodynamics, the colloid size, and grain size distribution characteristics. An improved understanding of the role of physical and chemical factors on colloid deposition is needed to predict the transport and fate of pathogenic microorganisms. Accomplishments are aligned with National Program 206 Manure and Byproduct Utilization: Pathogen and Pharmaceutically Active Compounds Component. Problem Area 1. Methods Assessment and Development and Problem Area 2. Fate and Transport of Pathogens. Unsaturated colloid and bacteria transport – An understanding of colloid and bacteria transport in unsaturated soils is important to protect groundwater resources from microorganism contamination and to develop efficient bioremediation strategies for recalcitrant chemicals. Experimental and modeling studies were conducted in collaboration with researchers in Julich Germany, U.C. Riverside, and ARS scientists at the Salinity Lab to examine the influence of soil grain size, water content, bacteria hydrophobicity, the metabolic state of bacteria, and the solution chemistry on bacteria and colloid transport in unsaturated sands. Results indicate that colloid surface chemistry and aqueous phase solution chemistry has a big impact on the magnitudes of attachment and straining deposition; with straining tending to increase with decreasing sand size and water content, and increasing cell hydrophobicity and solution ionic strength. The role of attachment increased for more hydrophilic bacteria, for coarser textured sand, and increasing solution ionic strength. This information has improved our ability to quantify and predict bacteria transport in unsaturated soils. Accomplishments are aligned with National Program 206 Manure and Byproduct Utilization: Pathogen and Pharmaceutically Active Compounds Component. Problem Area 1. Methods Assessment and Development and Problem Area 2. Fate and Transport of Pathogens. Methodology for microbial source tracking- Bacterial pollution has been a frequent concern of many agencies and localities where water bodies are used for recreational or commercial purposes. Detection of fecal pollution indicators have been limited by methodologies that are too intensive, involves building of an extensive library, and watershed specific. Field studies were undertaken at the USDA George E. Brown, Jr. Salinity Lab, in collaboration with Orange County Water District, Inland Empire Utility Commission, Santa Ana River Water Board, and USGS to quantify the major genes that are targets for detection of E. coli as tools for non-library-based bacterial source tracking. Water and sediment samples were collected quarterly for 18 months from different points along the Santa Ana River that has received input from human or agricultural activities. Fingerprinting analysis of E. coli isolates using uidA 1939 gene marker showed several clusters of E. coli relating to land use, flow pattern, weather conditions, and if the sample site was lined or natural. The uidA1939 marker was a good marker for microbial source tracking due to its sensitivity and discriminatory power and needs. Accomplishments are aligned with National Program 206 Manure and Byproduct Utilization: Pathogen and Pharmaceutically Active Compounds Component. Problem Area 1. Methods Assessment and Development and Problem Area 2. Fate and Transport of Pathogens. 4c.List significant activities that support special target populations. None. 4d.Progress report. None. 5.Describe the major accomplishments to date and their predicted or actual impact. A quantitative real-time PCR methodology was developed to quantify E. coli O157:H7 in manure, soil, and waste and irrigation water. This method enables the collection of a large data set with reliable and high through-put technology. These assays were optimized to specifically quantify very low levels; >100 bacterial cells per gram of soil without enrichment or 1 to 10 bacterial cells per gram of soil with enrichment. Impact: This work represents a considerable advancement in pathogen quantification in different ecosystems. These methods will allow for routine monitoring of surface and ground water soil and food for the occurrence/prevalence of pathogenic E. coli. Survival of pathogens: E. coli O157:H7 survived more than 45 days in rhizosphere soil, which was determined with a newly developed real time PCR method. Impact: A sensitive method to measure the actual fate of Escherichia coli 0157:H7 is available so that researchers and regulators no longer need to use less reliable indicator organisms. Constructed wetlands for removal of contaminants: The removal of the main pollutants from the dairy washwater has had a beneficial impact on the surface and groundwater in the Chino Basin, and, in turn, has improved the quality of water leading into the Santa Ana River and the Orange County groundwater basin. Impact: The wetlands provided a cost-effective, low-maintenance process that can be independently built and managed. This project has already benefited the water district by reducing cost of contaminant removal down stream and prevented the contamination of ground water by nitrate. Transport and fate of microorganisms – Experimental and modeling studies have been conducted to improve our understanding of the mechanisms that control the transport and fate of microorganisms in the environment. Specific research findings include: Recognition that pathogen transport is highly dependent on pathogen and porous media size, on colloid concentration, and the magnitude and distribution of manure particles in suspension; an improved understanding of pathogen transport and deposition behavior across soil textural interfaces and in heterogeneous aquifer systems; a mathematical model has been developed to describe pathogen attachment, detachment, straining, blocking and size exclusion; quantification of physical and chemical factors that influence pathogen release and loading rates from animal waste to water, and the development of a conceptual model to describe and predict this process; an improved understanding and ability to simulate the coupling of solution chemistry and hydrodynamics on straining deposition; and quantification of factors that influence the transport and fate of colloid in unsaturated porous media. The development of a stochastic stream tube model to examine the influence of heterogeneity of colloid and porous media characteristics on transport and fate. Impact – This accurate information on pathogen transport processes discussed above is needed to predict and numerical model pathogen transport in subsurface environments, to assess the vulnerability and risk of resource contamination, and to develop cost effective remediation technologies and best management practices to protect water supplies. Accomplishments are aligned with National Program 206 Manure and Byproduct Utilization: Pathogen and Pharmaceutically Active Compounds Component. Problem Area 1. Methods Assessment and Development and Problem Area 2. Fate and Transport of Pathogens. 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? Quantitative Real-time PCR methods for quantitative detection of E. coli O157:H7 in environmental samples has been transferred to other scientists and commercial water testing laboratories. The major constrain is that this is a PCR based assay and may be subjected to PCR artifacts. Constructed wetland technology has been transfer to some local farmers in the Chino area. This may be a possible alternative to direct spreading of wash water on their pastures. Research findings on pathogen transport and release will continue to be transferred to other scientists via peer-reviewed publications and presentations at national and international meetings. The developed models of pathogen attachment, straining, and exclusion have been incorporated into the HYDRUS 1D computer program. These modifications have been disseminated to several user of this software in the scientific community. Review Publications Bradford, S.A., Simunek, J., Bettahar, M., Tadassa, Y.F., Van Genuchten, M.T., Yates, S.R. 2005. Straining of colloids at textural interfaces. Water Resources Research. 41:W10404, doi:10.1029/2004WR003675. Bettahar, M., Bradford, S.A. 2005. Concentration dependent transport of colloids in saturated porous media. Journal of Contaminant Hydrology. 82:99-117. Bradford, S.A., Tadassa, Y.F., Pachepsky, Y.A. 2006. Transport of giardia and manure suspensions in saturated porous media. Journal of Environmental Quality. 35:749-757. Bradford, S.A., Torkzaban, S., Walker, S. 2006. Coupling of physical and chemical mechanisms of colloid deposition. Annual Colloid and Surface Science Symposium. Paper No. 63. Bradford, S.A., Van Genuchten, M.T., Simunek, J. 2005. Modeling of colloid transport and deposition in porous media. Proceedings of the workshop on HYDRUS Applications, Oct. 19, 2005, Utrecht Univ., The Netherlands. p. 1-5. He, C., Simunek, J., Pang, L., Bradford, S.A. 2005. Colloid-facilitated transport in variably saturated porous media: numerical modeling and experimental verification. [CD-ROM] Agronomy Abstracts. SSSA Annual Meeting. Salt Lake City, UT. Ibekwe, A.M. 2005. Persistence of Escherichia coli O157:H7 in rhizosphere and non-rhizosphere soils after fumigation. International Union of Microbiological Societies Proceedings/Abstracts. Page 18. Torkzaban, S., Bradford, S.A., Walker, S. 2006. Colloid transport in unsaturated porous media: the role of water content and ionic strength on particle straining. Annual Colloid and Surface Science Symposium. Paper No. 21. Walker, S., Bradford, S.A. 2006. A new paradigm for pathogen transport and deposition in porous media: the role of pore structure and colloid-colloid interactions. Annual Colloid and Surface Science Symposium. Paper No. 14. Wildenschild, D., Bradford, S.A. 2005. Interfacial area estimates for a napl-water system and their effect on predicting groundwater remediation efficiency. EOS Transactions, Fall Meeting. 86:H33A-1372. American Geophysical Union. Simunek, J., Van Genuchten, M.T., Bradford, S.A., Lenhard, R.J. 2005. New Features of HYDRUS-1D, Version 3.0. In: S. Torkzaban and S. Majid Hassanizadeh (eds.), Proceedings of Workshop on HYDRUS Applications, p. 86-89, October 19, 2005, ISBN 90-39341125, Department of Earth Sciences, Utrecht University, Utrecht, The Netherlands. Ibekwe, A.M. 2006. Impact of plant density and microbial composition on water quality from a free water surface constructed wetland. American Society of Agronomy Abstracts, annual meeting, Salt Lake City, UT. [CD-ROM] Ibekwe, A.M., Grieve, C.M., Yang, C.-. 2006. Persistence of escherichia coli O157:H7 in soil after fumigations. [CD-ROM] American Society for Microbiology Annual Meeting. Orlando, FL.