2009 Annual Report 1a.Objectives (from AD-416) Develop or refine methodology and conduct analysis of bacterial, viral, and protozoan pathogens and nutrients in manure, agricultural runoff, soils, and forage materials. Collaborate with other PIs in interpretation of data and writing of manuscripts for journal publication. 1b.Approach (from AD-416) Improved management of dairy farms requires successfully managing forage production and associated nutrient flows, both to maximize nutrient use by animals and crops in order to optimize profit, and to minimize nutrient loss to the environment in order to optimize sustainability. While much of our research project focuses on improving forage and nutrient management, another critical area of concern is the transport of manure-derived pathogens from agricultural fields and their potential to contaminate surface waters. This SCA adds the pathogen component, as well as providing additional support for forage and nutrient aspects, of experiments designed to evaluate crop, soil, and manure management effects on nutrient use efficiency, soil and forage quality, and transport of contaminants in surface runoff from manured fields. This includes an ongoing experiment that employs a paired-watershed design to compare packages of practices, both conventional and improved nutrient/crop/soil systems at a landscape scale. Other planned replicated plot studies will examine the effect of manure application methods on nutrient and pathogen transport from alfalfa-grass mixtures, and assess the effects of timing of dairy slurry application on the forage quality, silage fermentation characteristics, and pathogen survival in growing and harvested forages. Water samples will be collected by one or both of the following methods, depending on the study design and availability of runoff:. 1)Unconcentrated ambient samples taken by an automated ISCO sampler (paired watershed) or other sampling techniques (plot studies);. 2)Novel glass wool filtration technology for simultaneously concentrating protozoa, bacteria, and virus pathogens, as well as indicators like E. coli. Forage and soil samples will be analyzed directly without concentration. E. coli will be quantified by the chromogenic substrate method in a most-probable-number format. Cryptosporidium species will be quantified by immunofluorescent microscopy, and Salmonella and Campylobacter species will be enumerated by standard culture techniques. One or more viruses (types to be determined from manure analyses) will be detected by quantitative real-time RT-PCR. We will also explore quantifying Cryptosporidium, Salmonella, and Campylobacter, in runoff and forage samples by qPCR. 3.Progress Report Significant progress has been accomplished on this Specific Cooperative Agreement. Progress has encompassed three components of the overall work plan.. 1)Pathogen enumeration. Real-time quantitative PCR (qPCR) assays were established for four pathogens common to both humans and bovines that may be present in dairy farm manure (Cryptosporidium parvum, E. coli O157:H7, Campylobacter jejuni, and Salmonella serotype Typhimurium). In addition, assays were developed for six groups of bovine pathogenic viruses that may be present in manure and could be transported to waterways from field runoff. Performance of these assays was excellent.. 2)Pathogen concentration. Before enumeration, pathogens must be concentrated from field runoff. The method selected for this study is glass wool filtration, which has been previously validated by the research team to be effective for concentrating waterborne human viruses. The effectiveness of glass wool filtration for recovering all ten pathogens indicated above was determined by seeding the pathogens into simulated runoff at three concentrations of suspended sediment and measuring pathogen recovery using the qPCR assays. Recovery efficiencies varied by pathogen type and ranged from 10 to 80%. Pathogen concentration was effective even at the highest sediment load tested.. 3)Pathogen analysis of run-off samples. Following development and validation of the qPCR assays and pathogen concentration method, analysis of run-off samples from the Nutrient Management Systems paired watershed field experiment for the 10 pathogen groups is in progress. Samples have also been analyzed for indicator E. coli by a standard color-based substrate assay. Other activities have included lab and field support for soil and nutrient assessment in this and other experiments. Monitoring of the activities and progress in this SCA were accomplished by frequent communication (one to four times per month) via e-mail, telephone, and field or office visits. The progress reported here contributes to the Pathogens and Pharmaceutically Active Compounds Component of the NP 206 Action Plan by development of methods of pathogen detection and use of those methods in studies of transport in surface runoff from agricultural fields.