Objective:
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.

Approach:
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.