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Managing Risk to the Public and the Environment from Poultry Litter Application and from Food Borne Human Pathogens


Three risk management projects are contained in this proposal: <P>Project 1 - Risk Management for Maryland Grown Fresh Vegetables and Fruits has two objectives: a) to compare IPM & organic inputs of fertilizer & pesticides in the field using a replicated factorial study to test for the effects of these treatments on the microbial communities present on the leaf and fruit surface of four high-risk fresh-market crops and b) to conduct in vitro assays on the development of Salmonella biofilms, to test whether the development of these biofilms is affected by naturally-occurring plant extracts, and by commonly-used broad spectrum organic fungicides and inscecticides. <P>Project 2 - Assessment of Public Risk from Application of Antibiotics in Land Applied Poultry Litter and Biosolids as a Potential for Inducing Microbial Antibiotic Resistance - has 6 objectives: a) collect samples from many avian and biosolid sources & analyze for a broad suite of antibiotics & presence of antibiotic resistant bacteria, b) determine in the lab whether antibiotic resistant bacteria can persist in aqueous solition over time & can induce antibiotic resistance, c) determine in the lab the ability of various sources to generate resistant bacteria in natural receiving waters, d) measure the suite of residues in a range of streams impacted by land application of manure and biosolids, e) confirm the occurrence by isolating biotypes and f) determine antibiotic resistance patterns associated with human vs. avian fecal sources. <P>Project 3 - Sustainable Use of Pelletized Broiler Litter in the Bay Watershed has several objectives: a)to establish total and plant available nutrient content and nutrient mineralization rates of pelletized litter, b) to determine realistic methods of application, and c) to develop effective recommendations that promote environmentally and economically sustainable use in commercial grain production.

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NON-TECHNICAL SUMMARY: Project 1 addresses a situation that scientific & technical gaps exist in our understanding of the ways human pathogens survive in the environment & the role of routine agricultural practices on this survival. Farmers rely on Good Agricultural Practices to reduce risk of microbial contamination of crops & potential food-borne illnesses stemming from consumption of fresh fruits & vegetables. <P>Project 2 investigates a problem recently identified that interactions between bacterial organisms and antibiotics in the environment contribute to the development of antimicrobial-resistant bacterial strains & that resistance to animal use antibiotics can result in resistance to human use antibiotics. Do antibiotics from poultry litter and biosolids application confer resistance to human use antibiotics? That is what the project seeks to answer. <P>Project 3 - Looks into the problem of Non-point nutrient losses from agriculture, both P and N, continuing to be of environmental concern in Maryland and the Chsapeake Bay Watershed because of anthropogenic eutrophication due to nitruent enrichment from agricultural runoff.Pelletized broiler litter is often pointed to as having less environmental impact than applied fresh litter. This project will assess whether that is the case and if so, could it be a practice that is also economical to the farmer. The purpose of this proposal is to investigate techniques and management approaches that will reduce environmental risk and impact from poultry litter and reduce human risk from food -borne pathogens.

<P>APPROACH: Project 1 - Replicated field plots containing melon, tomato, spinach & strawberry will be established in Spring 2007. Small plots will be established in a 2 x 2 factorial study. Factors will be pesticide (organic schedule vs. industry-standard IPM)& fertilizer (organic provided by sterilized composted poultry litter vs. industry-standard chemical). All crops except sponach will be grown using plastic mulch and trickle irrigation. In each of 16 whole plots, one row of the four crops will be planted each year: melon, tomato, strawberry, spinach. 16 replicate plots will be required. Rows will be 20 ft. long and planted 6 ft. on center. Separation will be 50 feet to reduce cross-contamination. Total space will be 1.3 acres. Supplies and infrastructure are available on site.Yields will be hand harvested and recorded (measured). Standard microbiological assays will be conducted. Results will be compared to DNA washes taken from same tissues and crops to test for presence of enterobacteriaceae & natural microbial diversity of species. Using Salmonella standards, commonly used pesticides and plant extracts will be assayed in the lab to test for their effects on biofilm development in vitro (Prouty and Gunn standard assay). After incubation, biofilm quantified using spectrophotometer. Using this model system, the role of conventional pesticides & naturally occurring plant extracts will be tested. Pesticides will be added singly to the model systems along with plant extracts and compared to the controls. <P>Project 2 - Samples wil be collected from a number of waste sources (e.g. brioler, turkey, WWTPs). In Year 1, a representaative sample of litter and biosolids from Objective 1 will be introduced to diluent freshwater to determine presence & resistance. Lab exposures will investigate the ability of litter and biosolids to generate resistant bacteria in natural receiving waters. In Year 2, surface water sampes will be collected from 15 sites and analyzed for residues and resistance. Data will be analyzed using patterns identified in Yr 1lab exposures and Yr 2 antibiotic resistance patterns around STP effluents as a means of fecal source tracking. <P>Project 3-A 1 yr. lab incubation study will be done to determine and compare N and P mineralization rates and mechanisms for FPL, PPL and commercially available inorganic N and P fertilizers. A 1 yr. field study to compare these nutrient sources in commercial row crop production systems under no-till and conventional tillage practices will be conducted as well. Specific analytical methods will be used for soil samples, e.g. determination of N concentrations (Markus 1985). Total N and carbon concentrations will be determined by high temperature combustion using LECO CN analyzer.

PROGRESS: 2007/06 TO 2008/06

OUTPUTS: Three projects are funded pertaining to managing risk to the public and environment from poultry litter application and food-borne human pathogens. Project 1 addresses scientific & technical gaps existing in our understanding of the ways human pathogens survive in the environment & how routine practices affect this survival. At the end of 2007 growing season, replicated soil cores were taken from fields which had been used to grow certified organic crops or crops under conventional management systems. Using standard soil extraction kits and PCR, 16S rDNA was isolated from 400 bacterial isolates from certified organic and conventional soils. DNA from both soils was sequenced and species were identified using NCBI database BLAST. Sequences are being compared from both soils to estimate the number of sample sequences required. The project has also tested the role of various production inputs on the microbial load measured from leaves of high risk fresh market fruit & vegetable crops. From 03/2008-05/2008 leaves were assayed and sampled from 3 direct strawberry market locations. Two of the sites utilized perrennial matted-row plantings with the 3rd site planted in an annual system using raised beds mulched with plastic. In leafy greens, 2 pilot studies testing production practices on mircobial load were also conducted. Spinach and kale were used to examine the incidence of Enterobacteriaceae species & the impact of commercial type washing practices on surface microflora. Project 2 is looking into antibbiotic resistance from land applied poultry litter and biosolids. Poultry litter samples were provided after much difficulty in trying to obtain them. Biosolids that were lime stabilized and unstabilized were obtained from two treatment plants. Samples were taken from 3 lakes on the Eastern Shore which drain large watersheds where poultry litter is routinely applied as fertilizer. 160 tons of broiler litter were applied to two 33-acre research fields to monitor the effects of tillage practices on antibiotics in run-off following rain events. No till and Turbo Till were the practices used on the fields. Sediment and water samples were taken. Samples from each source mentioned above were sent to USGS for antibiotics analysis and to NOAA for antibiotic resistant bacteria analysis. Results are forthcoming. A new issue involving the detection of ionophores in the samples has bu USGS at no charge. Project 3 evaluates the sustainable use of pelletized brouler litter. To date replicated field studies have been established to evaluate pelletized poultry litter in field settings. A one year soil incubation study has been initiated in the lab. When the results from the laboratory and field studies are available, they will be used to develop the recommendations. <BR>PARTICIPANTS: For Project 1, participants include the Wye Research and Education Center in Queenstown, Maryland where the replicated plots for fruits and vegetables have been established. Ms. Zhou Huiling was awarded a State Scholarship from the China Scholarship Council to participate on this project. The CSC is a non-profit institution affiliated with the Ministry of Education of the P.R. China. For Project 2, sample analyses for biosolids, poultry litter, sediments and water samples from the 3 lakes and samples from the two fields are being analyzed by USGS (Mr. Dana Kolpin, Dr. Michael Meyer) and by NOAA (Mr. A.K. Leight). Dr. Meyer is also analyzing the ionophores that may also be detected in the samples. Mr. Al Razik, Manager of the Biosolids Program from Maryland Environmental Services obtained the biosolids from two treatment plants and Perdue through fresh broiler litter samples and samples of the pellets produced by the facility. For Project 3, participants include the University of Delaware, College of Agriculture and Natural Resources (Dr. J. Thomas Sims), the E.I. duPont de Nemours and Company (Dr. Mark C. Conner as Manager of Chesapeake Farms), the St. Andrew's Center for the 21st Century Farm and USDA-ARS Animal and Natural Resource Institute in Beltsville (Dr. Jack Meisinger). <BR>TARGET AUDIENCES: For Project 1, the central target audience are the local farmers who use GAPs in order to raise and market fresh fruits and vegetables. For Project 2, the central target audience is the Executive Council for the Chsapeake Bay who make decisions as to how to reduce nutrient pollution coming into the Chesapeake Bay and deal with other issues that can affect the Bay waters and the people and aquatic/wildlife that use those waters. Other target audiences would be State enforcing and managing agencies(Agriculture, Environment, Health and Mental Hygiene, Natural Resources, Maryland Environmental Service) as well as various stakeholders in the agricultural and environmental sectors and municipal treatment systems. Further audiences would be at the Federal level - EPA and USDA. For Project 3, the target audiences will be the farmers who attend field days and workshops. These will be hosted by the St. Andrew's Center, fields days hosted by the Dupont Company at Chesapeake Farms, and extension activities of the Soil Fertility and Nutrient Management Program at the University of Maryland. The Chesapeake Farms Field Day has been held annually since 1996 and is attended by 300-400 agricultural stakeholders. In addition findings will be shared through print and web publications. Guidance will also be provided on how individuals could conduct evaluations comparing PPL with current practices on their farms. <BR>PROJECT MODIFICATIONS: For Project 2, A TurboTill was used on one of the fields which incorporates the litter into the top few inches of the soil in an effort to stop run-off of nutrients and other water soluble contaminants while still maintaining some of the benefits of reduced sediment loss obtained from not tilling. This was not originally part of the Hatch antibiotics proposal but the litter and tillage was paid for from another contract to look at nutrient and steroid losses from poultry litter amended fields. Since the start of the Hatch project, a new issue concerning antibiotics developed. This concerns ionophores which are lipid-soluble molecules usually synthesized by microorganisms. Ionophores disrupt transmembrane ion concentration gradients required for the proper functioning and survival of microorganisms, and thus have antibiotic properties. Some of the known ionophores are monensin, lasalocid, laidlomycin, salinomycin and narasin. There are human health issues associated with ionophores with regard to direct exposures to the drugs that may be experienced by farm workers. In addition, there are also issues with regard to the development of ionophore resistance. Different resistance genes can be linked together on bacterial plasmids or chromosomes and the same is true for ionophore resistance genes. There is some concern that the use of an ionophore could select for not only ionophore resistance but also resistance to important human antibiotics such as the tetracyclines and fluroquinolones (due to genetic linkages). Because of this concern, USGS (Dr. Meyer) is in the process of developing methods for detection of ionophores in the samples that are sent to him under this Hatch funded project. The USGS feels that this is an emerging concern and will add these extra analyses at no charge for the Hatch samples.

<P>IMPACT: 2007/06 TO 2008/06 <BR>For project 1, preliminary results with strawberry showed that even during early spring, bacteria could be identified, enumerated and effectively compared between treatments. Leaves taken from strawberry grown with plastic mulch appeared to have lower counts than leaves taken from matted-row plants on the same date. This project will contribute to increased commercial awareness by growers of the need to implement good agricultural practices to minimize the risks of foodborne illnesses associated with fresh-market crops. <P>Project 2 is quite significant because it will be the first effort to systematically examine antibiotics in poultry litter and biosolids & the antibiotic resistance potential from runoff from fields amended with these fertilizers. When project 3 is completed, the results will be used to develop effective recommendations for pelletized poultry litter use in commercial row crop production that are environmentally and agronomically responsible.

Walsh, Christopher; Brinsfield, Russ
University of Maryland - College Park
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