2005 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? What does it matter? The food and water supply of the United States is 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 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 suggests that drinking water supplies may be vulnerable to pathogen contamination from animal production operations. 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 Component. Problem Area 1. Methods Assessment and Development and Problem Area 2. Fate and Transport of Pathogens. 2.List the milestones (indicators of progress) from your Project Plan. Milestones - Year 1 (2005) Objective 1. Detection, quantification and characterization of pathogen survivalin 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 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 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 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.What was the single most significant accomplishment this past year? Although most waterborne viruses are of fecal origin, knowledge of the processes that control the transport and fate of viruses in the presence of manure contaminated water is still incomplete. Laboratory and modeling studies were undertaken at the ARS scientists at the USDA George E. Brown, Jr. Salinity Lab, in collaboration with Dr. Jan Yin of the University of Delaware, to quantify mechanisms of virus transport and fate in the presence and absence of manure suspension. Results from this study indicate that the survival of viruses may be enhanced in the presence of manure suspension, and that virus transport studies conducted with pure solutions may significantly underestimate the migration potential of viruses in manure-contaminated environments. Conversely, manure contamination was also found to increase the retention of viruses in the soil by filling and/or clogging small soil pores. Provides guidelines and information necessary to assess the risk and vulnerability of water resources to virus contamination, and to develop cost-effective treatment strategies to minimize human and animal exposure. 4b.List other significant accomplishments, if any. During on-farm contamination of fresh produce by pathogens from contaminated water or manure used for fertilization, pathogenic bacterial strains compete with other microorganisms for nutrients. However, information concerning the rate of pathogen persistence in that environment is limited. Growth chamber and microcosm studies were conducted by ARS scientists at the USDA George E. Brown Jr. Salinity Laboratory to determine the persistence of Escherichia coli O157:H7 in two soils that may provide different nutrient levels for bacterial survival. Bacteria were tagged with green fluorescent protein to quantify persistence of pathogens in soil, roots and leaf surfaces. Persistence of pathogen was consistently higher in sandy soil than clay soil and results showed that E. coli O157:H7 can persist in the environment for extended periods of time, and under favorable conditions the pathogen can regrow. Provides information on a very significant pathway for pathogen recontamination in the environment. Although disease causing microorganisms are commonly found in animal manure, the potential implications of manure particles on microbe transport are not yet known. ARS scientists, in collaboration with Dr. Yakov Pachepsky of USDA-ARS, Beltsville Maryland, conducted a study at the USDA George E.Brown Jr. Salinity Laboratory to investigate the transport behavior of manure particles and cysts of Giardia in several sands. Results indicate that manure particles and Giardia were retained in the small pore spaces of the sand, and that the migration potential of larger sized particles and microbes was enhanced over time due to filling of these sites with manure particles. Therefore, transport studies conducted in the absence of manure particles may underestimate the transport potential of microbes in manure-contaminated environments. Treatment techniques to remove microorganisms from ground water and/or surface water by soil passage (riverbank filtration, infiltration trenches, and sand filters) may potentially be compromised due to filling of staining sites. Surface and groundwater quality in the Chino-Santa Ana River Basin, California, a major source of drinking water for the Los Angeles metropolitan area, has been affected significantly by intensive dairy operations and the disposal of untreated wastewater into the Basin. Development of treatment options are needed to prevent contamination of these waters by mineral ions that may be hazardous to human health. ARS scientists from the USDA George E. Brown Salinity Laboratory conducted a study to determine the effectiveness of surface flow wetlands in combination with wetland plant species for the removal of nitrate from the Santa Ana River. Wetland cells with 100% plant cover were the least efficient cells for nitrate removal, whereas cells with 75% plant cover were most efficient due to higher microbial diversity at these sites. Results demonstrate that wetland systems depend on the development and maintenance of healthy, diverse microbial communities for optimal reduction of nitrate from dairy wastewaters. Provides a treatment option for removal of a major contaminant from agricultural wastewater. Microorganism deposition in porous media is frequently not consistent with traditional attachment theory, and various competing explanations have been proposed in the literature to account for these deviations. To resolve this issue, researchers at the GEBJr. Salinity Lab constructed and used a micromodel chamber (2x7x0.2cm) to microscopically examine and video record the deposition behavior of colloids and microbes in porous media. Visual observations demonstrate that straining of colloids and microbes occurred at grain-grain junctions. This tool also allows researchers to visually examine the attachment of colloids and microbes to grain surfaces as a function of solution chemistry and mineralogy. An accurate description of microorganism deposition in porous media will facilitate the development of models to predict pathogen fate and improved treatment technologies. 4c.List any significant activities that support special target populations. None 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. 5a.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 a quality, 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. 5b.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 only use less reliable indicator organisms. 5c. 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 the contamination of ground water with nitrate. 5d. Accurate information on pathogen transport processes 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. The following accomplishments contribute to defining such processes: Recognition that straining is an important mechanism of pathogen deposition that is dependent on pathogen and porous media size, on colloid concentration, and the magnitude and distribution of manure particles in suspension; n 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; eecognition that the survival of viruses may be enhanced in the presence of manure suspension, and that virus transport studies conducted with pure solutions may significantly underestimate the migration potential of viruses in manure-contaminated environments. 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 pasture. 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 United States, the Netherlands, Germany, and New Zealand. Review Publications Pachepsky, Y.A., Bradford, S.A., Sadeghi, A.M., Guber, A.K., Shelton, D.R. 2004. Modeling fate and transport of manure-borne pathogens in the environment. In: Proceedings of the Pathogens in the Environment Workshop, February 23-25, 2004, Kansas City, Missouri. p. 34-38. O'Carroll, D.M., Bradford, S.A., Abriola, L.M. Infiltration of PCE in a system containing spatial wettability variations. Journal of Contaminant Hydrology. 2004. 73:39-63. Bradford, S.A., Simunek, J., Van Genuchten, M.T., Bettahar, M. 2004. Straining of colloids at textural interfaces. ASA-CSSA-SSSA Annual Meeting Abstracts. CD-ROM, Seattle, WA. O'Carroll, D.M., Abriola, L.M., Polityka, C.A., Bradford, S.A., Demond, A.H. 2005. Prediction of two-phase capillary pressure-saturation relationships in fractional wettability systems. Journal of Contaminant Hydrology. 77:247-270. Ibekwe, A.M., Lyon, S. 2005. Impact of dairy production on microbial characteristics through drinking water aquifer material. In: Proceedings of First International Conference on Environmental Science and Technology, January 23-26, 2005, New Orleans, Louisiana. 1:215-221. Ibekwe, A.M., Watt, P.M., Shouse, P.J., Grieve, C.M. 2005. Fate of Escherichia coli O157:H7 in irrigation water on soils and plants as validated by culture method and real-time PCR. Canadian Journal of Microbiology. 50:1007-1014. Ibekwe, A.M., Papiernik, S.K., Yang, C. 2004. Enrichment and molecular characterization of chloropicrin- and metam-sodium-degrading microbial communities. Applied Microbiology and Biotechnology. 66:325-332. Yang, S., Perna, N.T., Coksey, D.A., Okinaka, Y., Lindow, S.E., Ibekwe, A.M., Keen, N.T., Yang, C.H. 2004. Genome-wide identification of plant-up-regulated genes of erwinia chrysanthemi 3937 using a gfp based ivet leaf array. Molecular Plant Microbe Interactions. 2004. 17(9):999-1008. Ibekwe, A.M., Leddy, M., Lyon, S.R., Jacobson, M. 2005. Impact of plant species and density on microbial community composition in a free water surface constructed wetland. American Society for Microbiology Annual Meeting. CD-ROM, Atlanta, GA. Ibekwe, A.M., Watt, P.M., Shouse, P.J., Grieve, C.M. 2005. Fate of escherichia coli o157:h7 in irrigation water on soils and plants. In: First International Conference on Environmental Science and Technology, January 23-26, 2005, New Orleans, LA. 2:11-17. Bradford, S.A. 2005. Concentration dependent colloid transport in saturated porous media. American Chemical Society Abstracts, San Diego, CA. 229:U646-U647. Bradford, S.A., Tadassa, Y.F., Bettahar, M. 2004. Transport of microorganisms in the presence and absence of manure suspensions. American Geophysical Union, Abstracts of Fall Meeting, San Francisco, CA. 85:47. Bradford, S.A., Bettahar, M. 2005. Straining, attachment, and detachment of Cryptosporidium oocysts in saturated porous media. Journal of Environmental Quality. 34:469-478. Ibekwe, A.M., Grieve, C.M., Poss, J.A., Grattan, S., Suarez, D.L. 2005. Determining rate of change in cucumber rhizosphere microbial community composition in response to soil pH, salinity, and boron. In: Proceedings of the International Salinity Forum, Managing Saline Soils and Water: Science, Technology, and Soil Issues. April 25-27, 2005. Riverside, CA pp:77-80.