2010 Annual Report 1a.Objectives (from AD-416) Our overall goals are 1. to understand better the survival kinetics and hydrologic transport characteristics of manure pathogens and their associated fecal indicator bacteria in agricultural watersheds, and 2. to improve our understanding of the emissions of ammonia and greenhouse gases from manures and manure applications. To accomplish goal 1., we propose three interrelated objectives: A) develop a method detection limit for Salmonella in environmental soil, poultry litter, and water samples with quantitative, real-time polymerase chain reaction (qPCR); B) determine inactivation or die off rates of Salmonella and the fecal indicator bacteria Escherichia coli and fecal Enterococcus spp. In field soils under controlled and field conditions; and C: determine the hydrologic transport characteristics of manure pathogens and fecal indicator bacteria at the field plot and small zero-order watershed scales. To accomplish goal 2., we propose to pursue the following two objectives: D) to measure ammonia and global-change trace gas emissions from land application of poultry litter under different tillage management, and E) from wetlands receiving nitrogen inputs from pastures. 1b.Approach (from AD-416) Goal 1.: A real-time, quantitative polymerase chain reaction (qPCR) method will be developed to quantity the concentrations of Salmonella in poultry litter, and in environmental soil and water samples exposed to field applied poultry litter. With sentinel chambers, the aforementioned qPCR method, and defined cultural methods inactivation rates of Salmonella, and fecal indicator bacteria in agricultural soils will be determined under laboratory and field conditions. In collaboration with the Poultry Microbiological Safety Unit at the Russell Research Center, Athens,GA, and Southeast Watershed Research Laboratory, Tifton, GA overland transport of manure pathogens and fecal indicator bacteria from field applied poultry litter will be characterized at the field-plot level under various slope and aspect and under conditions of simulated rain, and at the small watershed-scale level that will depend on natural weather conditions. Goal 2.: Soil chambers will be used to measure gaseous emissions from poultry litter after soil application. Emissions will be evaluated under different tillage management systems. Small riparian/wetland areas will be studied to determine the proportion of hydrologic input nitrogen is transformed into gaseous nitrogenous emissions. 3.Progress Report Using modifications of our previously published method of enumerating E. coli 0157:H7, we completed in situ and in vitro experiments to determine comparative die-off rates of E. coli 0157:H7, generic E. coli, and fecal enterococci. Anticipating rain events with runoff, loads of fecal of indicator bacteria were determined for our four cropped and instrumented watersheds that receive poultry litter and are periodically grazed by cattle. Seven runoff events were measured and data analyzed. 4.Accomplishments 1. Significance of Wall Structure, Macromolecular Composition, and Surface Polymers to the Survival and Transport of Cryptosporidium parvum Oocysts. Because the pathogenic parasite Cryptosporidium parvum does not reproduce outside of its animal host, the parasite’s microscopic eggs depend on their protective shell to survival in soil and water environments after being shed in feces. To better understand survival and hydrologic transport of this parasite in agricultural environments, researchers at Cornell University and Agriculture Research Service (ARS) researchers at Watkinsville, Georgia determined the macromolecular structure and chemical composition of the shell. This new information will enhance the effectiveness of mathematical models designed to predict die-off and movement of this pathogen in agricultural landscapes and aid efforts to protect public health served by surface water supplies. 2. Performance Assessment of Polymerase Chain Reaction (PCR)-Based Assays Targeting Bacteroidales Genetic Markers of Bovine Fecal Pollutions—Tools for Microbial Source Tracking. It is necessary to discriminate between cattle and other (such as human) sources of fecal contamination, and evaluate human health risks associated with agricultural runoff and management of water for human use. Testing genetic-based methods for identifying cattle fecal sources from cattle herds across the Umnited States, United States Environmental Protection Agency (USEPA) researchers at Cincinnati, Ohio, and Agriculture Research Service (ARS) researchers at Watkinsville, Georgia, found that variability in assay performance existed between different herds and suggested that various methods of microbial source tracking could be tailored for each watershed of interest. This is important information for researchers involved in microbial source tracking research and environmental protection agencies mandated to monitor surface water for health risks across the nation. Review Publications Shanks, O.C., White, K., Kelty, C.A., Hayes, S., Sivaganesan, M., Jenkins, M., Varma, M., Haugland, R.A. 2010. Performance assessment PCR-based assays targeting Bacteroidales genetic markers of bovine fecal pollution. Applied and Environmental Microbiology. 76:1359-1366. Jenkins, M., Eaglesham, B., Anthony, L., Kachlany, S., Bowman, D., Ghiorse, W. 2010. Significance of wall structure, macromolecular composition, and surface polymers to the survival and transport of Cryptosporidium parvum Oocysts. Applied and Environmental Microbiology. 76:1926-1934.