1a. Objectives (from AD-416):
Conduct controlled environmental studies to: 1) compare survival of manure-borne pathogens with surrogates; and 2) develop a baseline reference of pathogen and surrogate survival in manure-amended soils for comparison to field survival, accounting for environmental effects. Conduct field studies to evaluate survival of surrogates in raw manure-amended soils used for growing fresh produce. Contribute to development of a systematized protocol to determine safe practices for use of raw manure as a soil amendment in the production of fresh fruits and vegetables in a manner compliant with safety standards of the Food Drug and Cosmetics Act, and mandates put forth in the Food Safety Modernization Act of 2011.
1b. Approach (from AD-416):
For controlled environmental studies, USDA-ARS and UMES will collect raw manure from local animal production facilities, will characterize (and supplement, as necessary) the microbial load of the manure, will amend with appropriate pathogenic microorganisms, and determine survival as under different environmental conditions. For field studies, USDA-ARS and UMES will collect raw manure from local animal production facilities, will characterize (and supplement, as necessary) the microbial load of the manure, will apply manure to production fields consistent with current industry practices, and will monitor local weather and environmental impacts and the resultant survival and persistence of microorganisms of interest. USDA-ARS and UMES, in consultation with FDA, will determine selected manure types/categories for study, appropriate field application methods, sampling times, analytical protocols and methods. USDA-ARS and UMES will provide the FDA Project Officer, on at least a quarterly basis, a status report on the current state of the study, with information on data generated from completed milestones, status of current efforts, any technical difficulties encountered and plans to alleviate the issues while moving forward, and brief “next-step” discussions. No more than six months after the completion of field studies from all locations, USDA/ARS and UMES shall provide FDA/CFSAN with a final report on the project.
3. Progress Report:
Manure amendment of soils used to grow fresh produce can introduce pathogens which may persist and contaminate vulnerable market fresh commodities. Edaphic, environmental, and biological factors influence microbial survival differently. Reducing the risk of pathogen contamination on fresh produce is a high priority for food safety research. Field, growth chamber, and greenhouse studies were established and conducted at the University of Maryland Eastern Shore (UMES), which is located in the coastal plain, in Princess Anne. The purpose of the studies conducted in this project was to develop a model protocol to conduct field studies on the survival and persistence of manure pathogens in the field and compare those results to survival in pot studies in greenhouses using the same soils with the same rates of manure and microbial inoculated strains as used in the field studies. Results from these studies are also to be compared to study results using the same inputs except for soil and climate zone (Western Slope Upland (WSU) near the Piedmont Plateau Fall Line, Beltsville, MD). At both locations, the same non-pathogenic generic E. coli (Ec) and non-pathogenic strains of E. coli O157:H7 (attO157) were inoculated on the soils to which by broiler litter (BL) or dairy manure (DM) had been applied. A field experiment comprising eight treatments with four replications was conducted in Othello soil at the UMES Agricultural Research Farm. The BL and DM were applied to the soil plots at rates consistent with nutrient management requirements. After manure application, individual plots, except controls, received spray inoculum containing three rifampicin-resistant (RifR) strains of Ec and two RifR strains of attO157 grown in dairy manure extract were sprayed onto plots as a single inoculation event at either low, ~5x10^6 CFU/m^2, or high, 5x10^8 CFU/m^2 cell densities. Soil samples collected at 0, 1, 2, 3, 7, 14 days, and monthly for 4 months post-inoculation were analyzed for the viable inoculated strains of E. coli by direct plating or mini-MPN analysis. Results show that survival of Ec and attO157 was influenced by manure type and inoculum concentration. When soil was inoculated with high concentrations of Ec and amended with BL and DM bacterial counts declined to 4.6 and 2.43 log10 CFU/g, respectively, by 30-da. A similar trend, but to a lesser extent, was found for high inoculum concentrations of attO157. No Ec or attO157 bacteria were found in control soil. Data from remaining day of the study up through 4-months post-inoculation are being processed and analyzed. In addition, data collected from growth chamber study using a multi-strain mixture of generic and pathogenic E. coli O157:H7 strains and the UMES and WSU soils also are being analyzed. Companion studies in fields and greenhouses at the ES location are given in the progress report for Project 1265-32420-005-24R, titled: Development and Analysis of a Protocol to Assess Survival of Fecal Organisms in Agricultural Soils Amended with Raw Manure. Results from the first fall season application studies show that the protocol for conducting these studies allows for detection of early phase die-off and the slowly declining persistence generic E. coli and E. coli O157:H7 throughout the remaining 120 days post-inoculation. It also shows that manure and bacterial type, as well as bacterial inoculum concentration can have a substantial influence on survival and persistence of E. coli and E. coli O157:H7, and should be taken into account when setting guidelines for fresh produce safety. Non-pathogenic, field-isolated E. coli were more persistent than attO157 in manure-amended soils.