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Food Safety Risks Within Diversified Farming Systems: Risk Assessment, Pathogen Movement and Detection in Fresh Produce.

Gutierrez-Rodriguez, Eduardo; Gunter, Christopher; Thakur, Siddhartha; Chapman, Benjamin
North Carolina State University
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1) Conduct controlled studies in reserach stations to determine the impact of buffer zone distances, temporal factors, air and insect on the movement of indicator (fecal coliforms, E. coli and Enterococcus sp.) and pathogenic (Salmonella, STEC O157:H7 and non- O157:H7 STEC) organisms at the animal: produce interface on research stations across the state of North Carolina.

2) Validate the outcome of the first objective by studying the movement of pathogenic microorganisms from known animal reservoirs and potential environmental sources into fresh produce field in commercial diversified farms in the State of North Carolina.

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The emergence and growth of the "Eat Local" movement has contributed directly to the growth of diversified farms which promotes rearing livestock and growing fresh produce within the same agricultural system. The interface of food animals and fresh produce in agricultural production is an area in need of information that could potentially reduce the risk of pathogen transmission and fresh produce contamination. The primary goal of this project will be to determine the potential transmission of indicator and pathogenic bacterial from animal operations that are in close proximity to vegetable production systems on an experimental research station and commercial diversified farming operations. Information collected from these two approaches will help identify the key sources, track movement of pathogens, narrow the 'how-to' information gap, and help the produce industry to strategize control measures to improve food safety.We anticipate that the information generated from this proposal will be directly applicable to the reassessment of microbiological standards and matrices for the produce industry. This information will provide practical evidence of produce contamination from nearby animal operations and the potential mitigation steps to reduce contamination across growing seasons. Furthermore, it will provide insight into pathogen testing regimes linked to potential sources of natural human pathogen contamination critical to strengthen the safety of the fresh produce industry and reduce foodborne outbreaks.

Methods Objective 1.1.1 Work PlanThe Piedmont and Clayton Research Station will be used as our control study locations. This layout would allow us to compare the prevalence of indicator and pathogenic microorganisms between cropping systems and to determine overall environmental differences within them that can potentially increase contamination of fresh produce with pathogenic microorganisms. Sampling will initiate in the month of February and continue until the end of September where most of the targeted crops (Spinach, Lettuce, Brassica cultivars, Tomato and Melon) at least will have gone through one rotation. Our focus will be on the detection and isolation of Salmonella, STEC O157:H7 and non-O157:H7 STEC and indicator microorganisms (fecal coliforms, E. coli and Enterococcus sp.) within the selected diversified farm.i) Sampling animals at livestock operations: Sampling will include manure solids, lagoon effluents and bio-aerosols from the stall, pen, Dairy lagoon and Broiler houses. The presence of Salmonella, STEC O157:H7 and non-O157:H7 STEC from these samples will be determined following the procedure described in section 2.3.ii) Environmental Sampling (soil, plant and irrigation water): Diversified fields will be placed at 50, 200, 400 and 800ft downwind from each animal operation; in addition to control plots located 1 mile away from animal operations as described previously. At each location, soil will be collected from top 6cm of the soil horizon and 10L of irrigation water will be processed following the procedure described by (Sbodio et al. 2013).iii) Indicator microorganism and pathogen isolation from crops: Leafy greens (Spinach/Lettuce): Twelve heads (lettuce) or bags (spinach, each 250g) per location (50, 200, 400 and 800ft) will be collected and combined into 4 replicates. From each replicate 150 g will be rinsed with 0.01M potassium phosphate buffer (PPB) supplemented with 0.05% Tween 20 (PPBT) at a 2: 1 ratio (buffer ? leaves). Each bag will then be pulsified for 30s at medium speed and the entire supernatant will be extracted and used for the determination of indicator microorganisms (total coliforms (TC), non-pathogenic E. coli and Enterococcus sp.), STEC and Salmonella spp. A portion of the supernatant will be used for DNA extraction that will allow us to describe the microbial communities within each diversified plot. Quantification of Enterococcus spp., will follow the procedure by (Haugland et al. 2005) using probe based real-time PCR. Non-pathogenic E. coli and TC will be isolated from the supernatant using a tenfold dilution series prepared with 9 ml PPB tubes and plated on Chrom-ECC. After plating, the supernatant will be used to determine the presence of STEC and Salmonella.Salmonella spp. isolation: after UPB enrichment 10ml will be transferred in to 90ml Tetrathionate Bile Broth (TBB) + iodine and incubated for 6h at 42 °C. After enrichment 10ml will be transferred into 90ml mBroth bags and incubated for 18h at 37 °C. mBroth enrichment will be streaked on Xylose Lactose Tergitol 4 agar (XLT4) plates. Black colonies present in the XLT4 will be further isolated and molecular identification will be done using probe-based PCR as described elsewhere by (McEgan et al. 2013). STEC isolation: 10ml from UPB enrichment will be transferred into 90ml of mEHEC (2x concentrations) and the enrichment will be incubated at 42 °C for 18h. After incubation the enrichment will be streaked on Chrom-STEC and incubated at 37 °C for 24h. Mauve colonies present on Chrom-STEC plates will be further isolated and characterized through probe-base PCR. A similar approach for further microbial community characterization and detection of indicator and pathogenic microorganisms will be followed for Brassica cultivars, Tomato and Melons.Tomatoes: Twenty fruit per location (50, 200, 400 and 800ft) will be collected and combined into 4 replicates each consisting of 5 fruit. Each replicate will be rinsed with 100 ml of PPBT and vigorously rubbed by hand for 1 min, followed by sonication for 10min to remove attached bacteria from the tomato surface (Tomas-Callejas et al. 2012). Fruit stages may include 1- mature green, 2- "turning" and 3-"red". Melons: Sixteen melons per location (50, 200, 400 and 800ft) will be collected and combined into 4 replicates each consisting of 4 fruit. For each replicate the rind of all 4 melons will be peeled using a sterilize knife, pooled and rinsed with PPBT at a 2:1 ratio.1.2 Microbial Community Analysis: A mall subset of samples will be used for microbial community analysis using Next Generation Sequencing (NGS) technologies to describe how the microbiome within the animal: crop interface varies when compared to operations away from this type of interface. DNA from soil, air, water and manure solids samples will be strategically selected based on the presence of indicator and pathogenic microorganism within the systems and analyzed using the Illumina MiHiSeq platform as described elsewhere (Bulgarelli et al. 2012, Caporaso et al. 2010a, Caporaso et al. 2010b).1.3 Bioaerosol Sampling: Samples will include aerosol capturing at 50, 200, 400 and 800 ft within the diversified fields located next to animal operations, in the control plots and in and around the dairy and poultry operations. Aerosol sampling in and around the animal: crop interface and was developed based on previous recommendations by Millner and Suslow 2007.Methods Objective 22.1 Sampling Design: We aim to collect livestock (swine, poultry, dairy) fecal samples, fresh produce (leafy greens, tomatoes, water melons) and the environment (soil, irrigation water, air and manure) in three different diversified farms in NC. Buffer distance between the animal and fresh produce fields will be identified and tagged at every farm. All samples will be georeferenced on the farm to determine the exact distance between each collection and ultimately to illustrate buffers and mechanisms of pathogen dispersal. All the samples will be processed for Salmonella, STEC O157:H7 and non-O157:H7 STEC detection as described previously. Phenotypic and genotypic characterization will be performed simultaneously.i) Farm livestock sample collection: The fecal samples will be collected from a freshly excreted pile with sterile tongue depressors (soft pats), plastic scoops (firm scat), or tweezers (dry pellets) and care will be taken to avoid the interface with the ground.ii) Farm produce and environmental sample collection: will follow the procedures described in section 1.1. 2.2 Microbiological Analysis: Pathogen isolation from livestock, produce and environmental samples: Salmonella, STEC O157:H7 and non-O157:H7 STEC from fecal will be isolated following standard protocols (Olsen et al. 2000, Thakur et al. 2006, Hoefer et al. 2011). Enteric pathogen and indicator microorganism isolation and detection from produce (Lettuce, spinach, tomato and melons) will follow the same procedure described in section 1.1. Environmental sample analysis including water, air, and soil samples will be analyzed as described in sections 1.2 and 1.3. Phenotypic characteristics of Salmonella serotypes will be performed at the National Veterinary Services Laboratory (Ames, Iowa). Genotypic Characterizations will be achieved through Pulsed field gel electrophoresis (PFGE) using the PulseNet protocol (Ribot et al. 2006).2.3 Data Analysis: Pathogen from the different sources will be compared using the likelihood ratio ?2 test, adjusting for dependencies within the sampling scheme (e.g., within farm, plot) using multi-level mixed logistic regression models (Stata 12.0, College Station, TX) and exact methods wherever applicable. Significant associations of the pathogen presence with a particular risk factor will be assessed via the odds ratio.

Funding Source
Nat'l. Inst. of Food and Agriculture
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Risk Assessment, Management, and Communication
Escherichia coli
Bacterial Pathogens
Sanitation and Quality Standards
Meat, Poultry, Game