Performing Department
Food Science & Technology
Non Technical Summary
In the United States (US), contaminated produce is estimated to cause over 1.1 million illnesses, 7,125 hospitalizations, and 134 deaths annually, with associated costs of $1.4 billion. Should a producer be involved in litigation following an outbreak where negligence is directly pushed back onto the grower, costs are estimated to exceed $100,000 per case of foodborne illness (35, 36). Negative media and press from such an outbreak could render produce from Virginia non­ marketable and threaten the sustainability of a 100+ million-dollar industry (38). A recent US agricultural census in 2015, ranked Virginia 9th in direct vegetable sales, producing 217,343,742 on 3,415 farms (38). In addition to their substantial economic impact, foodborne illnesses also have an impact on human well-being in the US. When ranking the top 10 pathogen-food combinations in terms of annual disease burden, "Salmonellaproduce" ties for 8th; quality­ adjusted life year loss (2,781), cost of illness ($548,000,000); number of illnesses (170,264), hospitalizations (3,204), and deaths (1). Since 2002, produce grown in the Delmarva region (Delaware, Maryland Eastern Shore and Virginia Eastern Shore) have been linked to six foodborne disease outbreaks caused by Salmonella Newport. Specifically, produce contaminated with Salmonella Newport reportedly caused illness in 333, 72, 115, 65, 51 and 275 persons in 2002, 2005, 2006, 2007, 2010, and 2014 (21). This data highlights the impact of foodborne pathogen contamination on produce and the resulting impact to public health. Therefore, applied research is needed to assist the produce industry (e.g., growers, suppliers, etc.) in developing science-based recommendations to mitigate potential foodborne pathogen contamination events, and reduce the likelihood of foodborne outbreaks. Currently, the safety of produce is based on the implementation of Good Agricultural Practices (GAPs) to prevent microbial contamination during growing, harvesting, and postharvest handling. The goal of a GAPs program is to provide stakeholders with science-based recommendations on where microbial contamination may originate, survive, and transfer to fresh produce. A successfully implemented GAPs program should minimize the likelihood of contamination events and subsequent foodborne illnesses. While the research community and produce industry have gained a better understanding of how produce items may become contaminated (through water, soil amendments, sick workers), knowledge gaps remain including the risk of specific soil amendments or the rates specific pathogens need to die over time). Furthermore, the Food Safety Modernization Act's Produce Safety Rule has yet to set standards on raw manure application to produce fields until more research can be performed (defered action).Therefore,the purpose of this project is to determine the survival characteristics of selected foodborne pathogen strains, specifically Salmonella spp. (12 strains), Escherichia coli (E. coli) O157 (8 strains), and non-O157 Shiga toxin-producing E. coli (STEC) (7 strains) in raw manure-amended agricultural soils. We will use raw poultry manure to study the survival of Salmonella and we will use raw cattle manure for the study of E. coli O157 and non-O157 STEC. The raw biological soil amendment and pathogen pair was selected as prior studies, have shown those biological soil amendments may contain the respected pathogen. Additionally, the project will evaluate the survival of these pathogens in two different soil types commonly found in agricultural environments (sandy-loam andclay-loam soils). The data will also be used to develop models for use of raw manure in agricultural settings and provide recommendations (to growers, produce industry) for their application to fields where produce will be grown.
Animal Health Component
0%
Research Effort Categories
Basic
25%
Applied
75%
Developmental
0%
Goals / Objectives
Produce continues to be linked to foodborne outbreaks since many fruits and vegetables are consumed raw without a processing step that could inactivate microorganisms (2). Over a 15-year period beginning in 1990, produce was linked to 713 outbreaks and 34,049 cases of foodborne illnesses (3). Most recently in the US, outbreaks related to Escherichia coli O157:H7, O145, O26, Salmonella serovars, and Listeria monocytogenes have been linked to tomatoes, peppers, cantaloupe, mangoes, cucumbers, spinach, lettuce, and sprouts (4-19).The Food Safety Modernization Act (FSMA; which incorporates seven different food safety rules) was passed by Congress and signed into law by president Obama in 2011. FSMA emphasizes a preventive approach to food safety rather than responding and reacting to outbreaks (or contamination events). One of the seven rules in FSMA, the Produce Safety Rule (PSR), regulates the standards for the growing, harvesting, packing and holding of produce for human consumption (20). As of November 27, 2015, the final Produce Safety Rule was published in the federal registrar and provides standards for agricultural water, biological soil amendments, sprouts, worker health and hygiene, domestic and wild animals, equipment, building and tools, and other areas. In the final rule, the Food and Drug Administration (FDA) deferred action on the appropriate time interval or intervals between the application of untreated biological soil amendments of animal origin (including raw manure) and crop harvesting. While GAPs are voluntary, FSMA PSR compliance will be mandatory for many (as outlined by their size and profits, 20). Thus, the purpose of this project is to determine the survival characteristics of selected foodborne pathogen strains, specifically Salmonella spp. (12 strains), Escherichia coli (E. coli) O157 (8 strains), and non-O157 Shiga toxin-producing E. coli (STEC) (7 strains) in raw manure-amended agricultural soils. We will use raw poultry manure to study the survival of Salmonella and we will use raw cattle manure for the study of E. coli O157 and non-O157 STEC. The raw biological soil amendment and pathogen pair was selected, as prior studies have shown those biological soil amendments may contain the respected pathogen (25, 26). Additionally, the project will evaluate the survival of these pathogens in two different soil types commonly found on the Delmarva Peninsula (sandy-loam) and mainland Virginia (clay-loam) (37). The data will also be used to develop models for use of raw manure in agricultural settings and provide recommendations (to growers, produce industry) for their application to fields where produce will be grown. Specific objectives include:Salmonella survival "strain variability" (12 strains) in two types of soil (sandy and clay) with simulated soil moistureE. coli O157 survival "strain variability" (8 strains) in two types of soil (sandy and clay) with simulated soil moistureNon-O157 STEC survival "strain variability" (7 strains) in two types of soil (sandy and clay) with simulated soil moistureDevelopment of models to provide guidance on factors that influence the likelihood of pathogen survival in manure-amended soils
Project Methods
Research will be conducted at the Eastern Shore Agriculture Research and Extension Center in Painter, VA. The Biosafety Level, BSL-2 Greenhouse will be used for individual experiments. The approach for addressing each objective is described in greater detail below. Objectives 1-3. Investigate Salmonella (Y1), E. coli O157 (Y2) and non-O157 STEC (Y3) survival "strain variability" in two types of soil with simulated soil moisture.This set of experiments will be conducted at the Virginia Tech Eastern Shore AREC "Biosafety Level 2" Greenhouse. Whenever appropriate, standard methods such as those from the Compendium of Methods for the Microbiological Examination of Foods, the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM), or other applicable sources (27-31), will be used for the enumeration or identification of organisms.Experiments will be arranged in a randomized complete block design with three replications for a total of 72 planting pots (3 replicates × 12 strains × 2 soil types) for Salmonella, 48 planting pots (3 replicates × 8 strains × 2 soil types) for E. coli O157:H7 and 42 planting pots (3 replicates × 7 strains × 2 soil types) for non-O157 STEC. The experiments for all strains of a target organism will be performed simultaneously, with each replication occurring at different times. For each replication, positive (Green-Fluorescent Protein-tagged pathogen strain) and negative (no pathogen) control pots will also be included. See timeline for further details. Methods are detailed for the "strain variability" experiments. The methodology framework is similar, with minor details due to organism differences (e.g., Salmonella vs. E. coli O157:H7 selective media agars).Manure. Fresh poultry or cow manure will be obtained from local chicken or cow operations in Virginia. Key descriptive physical/chemical properties of manure samples will be collected including pH, moisture content, ammonia-N, nitrogen, and phosphorus. In order to mimic the real production environment, manure will not be autoclaved to kill resident microorganisms, but, if possible, the microflora will be characterized.Soil. Sandy-loam or clay-loam soil will be obtained from the 0-20 cm layer of agricultural farms located in Painter and Petersburg, Virginia, respectively. Soil will be collected prior to each replication. Soil samples will be tested for key descriptive physical/chemical properties including pH, moisture content, ammonia-N, nitrogen, nitrate, and carbon. Soil will be weighed into 2,000-g portions in new plastic pots and sterilized distilled water will be added to bring the soil moisture to levels typically observed in agricultural fields (approximately 12-15% sandy-loam and 22-25% clay-loam). Bacteria. We will select up to 12 Salmonella, 8 E. coli O157:H7 and 7 non-O157 STEC strains for use in experiments. Strains considered will include:Strains used in previous studies by USDA-ARS and FDA (21, 24)Strains that have been associated with outbreaks from produce commodities of interest (4-19, 22)Other significant strains (e.g., environmental strains), when outbreak strains are unavailable (23).Strains isolated by Virginia Tech Eastern Shore in previous studies (unpublished Strawn, unpublished Gu)Inoculation. Frozen stock cultures of bacterial strains are typically stored in glycerol stock solutions at -80°C. Prior to use, strains are streaked onto non-selective media supplemented with selective agents as appropriate. Inocula will be prepared from media or broth cultures and will be washed prior to use. Careful consideration will be taken to ensure inocula will be prepared similarly to the physiological state of the organism in manure and produce growing environment, by using appropriate growth media and adaptation procedures. Each inocula will be prepared at a level of approximately 7-8 log CFU/mL (24). Inocula will then be incorporated into the amended soil mixture and placed in pots. Briefly, soil will be amended with poultry litter or raw manure at a commercial agricultural standard rate of 4 kg/acre (M. Reiter, personal communication). This mixture (soil amended with poulty litter or raw cow manure) and inocula will be transferred to a sterile plastic bag, where it will be thoroughly homogenized (combined using a 30-s shake, 30-s rub, 30-s shake, repeat method). In total, 2000-g of inoculated manure-amended soil will be transferred to separate pots (one pot for each strain and soil type). The target inoculum in the amended soil (in each pot) will be approximately 4-5 log CFU/g. Test trial experiments will be performed prior to the study to determine the starting inoculum level that will yield a 4-5 log CFU/g in amended soil mixture. Throughout the incubation, the moisture content of the samples will be maintained by weekly weighing and addition of sterilized distilled water, as needed. Soil moisture levels will be targeted according to traditional precipitation data. Manure, soil, and manure-amended soil samples without inoculation will serve as controls.Sampling and enumeration. Organism counts will be determined at 0, 0.167, 1, 2, 4, 7, 14, 21, 28, 56, 84, 112, 168, 210, 252, and 336 days post-inoculation. If possible, more sampling points within the first 24 h will be added (e.g. 8 h time point), if necessary after test trials. Sampling and enumeration will be performed according to standard methods for Salmonella and STEC protocols by the Compendium of Methods for the Microbiological Examination of Foods and the U.S. Food and Drug Administration's Bacteriological Analytical Manual (27-31).Environmental Measurements. Select conditions within the greenhouse will be monitored during all trials. Air temperature and relative humidity will be monitored with measurements being recorded every 1 h. Soil samples (100 g) will be taken from control pots (no pathogens) and sent to the Waypoint Soil Testing Laboratory in Richmond, Virginia where they will determine soil pH, carbon content, amount and form of nitrogen present, among other soil variables.Objective 4. Development of models to provide guidance on factors that influence the likelihood of pathogen survival in manure-amended soil with different moisture conditions (Y4-5). In the first three years of this research a large amount of survival data will be collected and managed in excel spreadsheets. Each pathogen strain survival data (including pathogen enumeration, weather, soil data sets, etc.) will be used to support the development of risk models to determine the likelihood of produce contamination in fields with raw manure-amended soils. Once models have been developed, key recommendations will be derived for the produce industry.Data analysis. Statistics will be performed in appropriate software, such as SAS (SAS release 9.4, SAS Institute Inc., Cary, North Carolina). The survival of the strains will be compared by each soil type and over time for each replication, as well as overall (all three replications). Population data for each strain will be analyzed and fit into models to assess survival trends; for example, the analysis of variance with repeated measures across sampling times will be performed to determine whether general differences exist between the different strains (ANOVA using the PROC GLIMMIX procedure). Treatments (e.g., soil type or strain) will be treated as fixed factors, whereas location and replication will be treated as random. Means will be separated using Fisher's protected LSD at P=0.05 when appropriate.