Ecology and Epidemiology of Escherichia Coli O157:H7 in Fresh Produce Production Regions of Salinas, California

NON-TECHNICAL SUMMARY: Consumption of fresh fruits and vegetables is growing in the United States, however, this trend coincides with produce outbreaks. Since 1995, 16 outbreaks of Escherichia coli O157:H7 (EcO157) associated with fresh lettuce or spinach have occurred; 7 outbreaks were traced to the Salinas, California. Intensive investigation of 3 recent outbreaks implicated a single farm as a supplier of contaminated lettuce, indicating pre-harvest contamination. Our hypothesis is that key biotic and abiotic processes hydrologically link primary reservoirs of EcO157, resulting in bacterial contamination of produce. The goal is to develop and implement science-based strategies to prevent on-farm contamination of produce. We propose to sample extensively vertebrate animal feces, creek/ditch and irrigation water, soil and produce for commensal and EcO157, and use epidemiological approaches to determine if: (1) vertebrate populations are sources of EcO157 contamination of watersheds; (2) climate, landscape attributes, and irrigation management practices correlate with increased risk of contamination; and (3) in-field contamination of lettuce with EcO157 is associated with management production practices and environmental risk factors in the Salinas. The information obtained from this study will be used to inform produce growers about strategies to prevent pre-harvest microbial contamination, to educate the livestock community about potential impacts of rangeland runoff on watersheds and down-stream stakeholders, and to develop effective management practices for improving water quality.

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APPROACH: Our overall study design combines epidemiological and microbiological methods followed by outreach and education activities to disseminate prevention and control information. We will conduct an in-depth longitudinal study that identifies the key biotic and abiotic processes that sufficiently load, then hydrologically link and disseminate, primary environmental reservoirs of EcO157 within and between lettuce fields, resulting in bacterial contamination of this raw agricultural commodity. For each node (vertebrate sources, water, soil, lettuce) of the system, we will collect a detailed set of covariates that will be used to identify critical control points, points of environmental amplification, and management practices that either elevate or decrease the risk of in-field contamination and dissemination of EcO157 on lettuce. Samples obtained as part of the study will be analyzed by ARS and strains isolated will be analyzed by ARS by Multi-Locus Variable number tandem repeat Analysis (MLVA) and Pulsed Field Gel Electrophorsis (PFGE) to characterize isolates for genetic differences, determine molecular epidemiologic linkages among samples isolated or obtained from public health collaborators, and thus, pinpoint the mechanisms that link and disseminate vertebrate sources of EcO157 within and between fields of lettuce. Hierarchical regression models will identify significant covariates for our outcomes of interest. The information obtained will be disseminated to stakeholders by a variety of mechanisms. Documents Assistance Type Cooperative Agreement(ATCA) with UC Davis. <P>

PROGRESS: 2006/10 TO 2007/09<br>
Progress Report Objectives (from AD-416) It is hypothesized that vertebrate populations function as a key source of E. coli O157:H7 (EcO157) contamination of watersheds where lettuce and other leafy vegetables are grown; that climate, landscape attributes, and irrigation practices correlate with increased risks of EcO157 and commensal E. coli contamination; and in-field contamination of lettuce plants with EcO157 relates to combinations of production practices and environmental risk factors in the Salinas Valley. The major objectives of this project are the design of a sampling strategy and to sample the environment of leafy vegetable production to (1) quantify environmental loading by vertebrate sources, (2) characterize predisposing conditions for hydrological transport of EcO157 and E. coli to lettuce fields, (3) identify the in-field mechanism(s) of contamination of lettuce, and to (4) develop and disseminate educational materials for growers of fresh produce and the livestock community about microbial water quality, potential impacts on down-stream stakeholders, and effective livestock production practices for improving water quality. These objectives will be completed in coordination and collaboration with the USDA, Agricultural Research Service (ARS). Approach (from AD-416) Our overall study design combines epidemiological and microbiological methods followed by outreach and education activities to disseminate prevention and control information. We will conduct an in-depth longitudinal study that identifies the key biotic and abiotic processes that sufficiently load, then hydrologically link and disseminate, primary environmental reservoirs of EcO157 within and between lettuce fields, resulting in bacterial contamination of this raw agricultural commodity. For each node (vertebrate sources, water, soil, lettuce) of the system, we will collect a detailed set of covariates that will be used to identify critical control points, points of environmental amplification, and management practices that either elevate or decrease the risk of in- field contamination and dissemination of EcO157 on lettuce. Samples obtained as part of the study will be analyzed by ARS and strains isolated will be analyzed by ARS by Multi-Locus Variable number tandem repeat Analysis (MLVA) and Pulsed Field Gel Electrophorsis (PFGE) to characterize isolates for genetic differences, determine molecular epidemiologic linkages among samples isolated or obtained from public health collaborators, and thus, pinpoint the mechanisms that link and disseminate vertebrate sources of EcO157 within and between fields of lettuce. Hierarchical regression models will identify significant covariates for our outcomes of interest. The information obtained will be disseminated to stakeholders by a variety of mechanisms. Documents Assistance Type Cooperative Agreement(ATCA) with UC Davis. Significant Activities that Support Special Target Populations This report serves to document research conducted under a Grant (Assistance Type Cooperative Agreement) between ARS and University of California at Davis. Additional details of research can be found in the report for the parent project, 5325-42000-044-00D, Molecular Microbiology and Control of Enteric Pathogens that Contaminate Fresh Produce. The collaborators of PSMRU at UC Davis have met with PSMRU and multiple industry groups to explain the objectives of the project and to identify farms that are candidates for enrollment as study sites. Communications and meetings with other appropriate federal and state agencies expressing interest in collaboration are continuing. Research conducted with the support of this grant is monitored by the ADODR, by email and phone with the cooperators at UC Davis.

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PROGRESS: 2007/09 TO 2008/09<BR>
OUTPUTS: The start date for this proposal was Oct. 1, 2007. In year 2, the PD, Co-PD and collaborators continued to meet with multiple potential cooperators to explain the objectives of the project and to enroll study sites. Multiple trips to the central coast region of California were required to inform potential collaborators of the exact objectives of the studies and issues relevant to confidentiality. More than five sites appropriate for studies of risk factors were identified and sampling and testing was initiated in April, 2008. The multi-state spinach outbreak of Sept-2006 provided an opportunity to test and modify methods to be used in years 2 and 3 of the project. A PCR method for measurement of the presence of shigatoxin in enrichment broths was developed and tested for sensitivity with isolates of O157 and non-O157 E. coli spiked into broths cultured with environmental samples (water, lettuce, soil). Soil, leafy vegetable, water samples and deer fecal samples collected during the 2007 and 2008 deer hunting seasons, and feral pig, wild bird and small wild mammal samples collected by USDA Wildlife Services personnel, were provided to the ARS lab for testing for O157. More than 2500 samples have been processed to date for E. coli O157:H7, generic E. coli and non-O157 STECs. The deer sample results have been of interest to the CA Dept of Fish and Game, growers and produce representatives as a result of deer being considered a "significant risk" in the Leafy Greens Marketing Agreement. Clinical strains of E. coli O157:H7 from multiple states (CA, HI, OR, ID, WA, MN), cattle isolates, and meat isolates have been obtained also and genotyped. A genotyping database was created as one of the objectives of the project. The database contains genotyping (multilocus variable number tandem repeat analysis, MLVA) and source data results for >1100 strains as of October-2008. The ARS lab has become a member of CDC PulsNet and will be submitting profiles as they are obtained. Sampling and testing will continue into the rainy season with an emphasis on livestock, wild animal and watershed sampling. Communications and meetings with other appropriate federal and state agencies expressing interest in collaboration are continuing. <BR>PARTICIPANTS: The PI (RM) and Co-PI (RA) continued meeting in Year 2 with growers and ranchers to identify farms and ranches to enroll. Two people were hired to sample farms and ranches twice and sometimes three times each week. The PI has organized two meetings of the project collaborators. A collaborator, M. Jay-Russell, moved to the Western Institute for Food Safety and Security at UC Davis, and leads the sampling team and coordinates interactions with cooperators. All laboratory analyses are being done at the ARS laboratory (M. Cooley); three technicians were hired to assist. The PI communicated extensively with other public health agencies to obtain epidemiological information and relevant strains (outbreak, sporadic, produce-related) for genotyping. The Co-PI has consulted with K. Tate and R. Larsen on hydrology- and cattle-related issues relevant to the project. CDF&G has requested the PI's lab continue testing deer samples for E. coli O157:H7. Two USDA-APHIS wildlife staff members provide samples through an interagency agreement, in place of staff to be hired under the UC Davis specific cooperative agreement for this purpose. <BR>TARGET AUDIENCES: Outreach activities include numerous contacts of the PI and Co-PI with growers for enrollment of fields for study. Additional outreach activities include numerous meetings to explain the objectives and needs of the project with, for example, the CA Lettuce Research Board, CA Strawberry Commission, CA Dept. Fish and Game, Monterey County Resource Conservation District, USDA National Resource Conservation District, local and national cattlemen associations. The preliminary studies leading to the project, and project objectives and rationale, have been presented at numerous national and international meetings including IAFP, IFT, UK-Society for General Microbiology, FoodMicro-2008 and other meetings. There is broad awareness of the project by the major produce processors, trade organizations and many growers. <BR>PROJECT MODIFICATIONS: The objectives of the proposal were developed around the concept that flooding of fields during heavy rain events may explain some of the frequent outbreaks of E. coli O157. However, recognition of these results by processors and buyers has resulted in restrictions on purchase of produce from these types of fields. Therefore, our objectives have been modified slightly to minimize testing of the flooded field hypothesis, and concentrate more on testing wildlife near and on fields, as they may transport pathogens to fields directly. Also, testing for other STECs has been added, but may be limited considering it is resource intensive and was not funded in this grant. The other sampling objectives proposed remain the same.
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IMPACT: 2007/09 TO 2008/09 <BR>
Methods for measuring MPN of generic E. coli and methods for automation have been evaluated for sensitivity and ease of use for high-throughput testing. A PCR method for screening an enrichment broth for presence of potential STEC isolates was developed based on different primers to increase the sensitivity of detection. This method proved to be useful, but further development will be needed to increase sensitivity. A new commercial master mix for environmental samples was tested and increased detection of STECs by >50%. Results with >2000 samples have revealed that the Colilert and EC Chromagar methods each have strengths and weaknesses relative to the large number of samples that must be processed during this project. Additional methods developed for detecting STECs during this project are: (1) a novel shigatoxin (stx) capture method and detection of stx with anti-stx monoclonal antibody; (2) a 12 antisera immunochemical screening assay for detecting strain differences among multiple stx-positive isolates; this assay identifies rapidly isolates to retain for further analysis. Our method of non-selective enrichment of fecal/water/plant/soil samples, stx-PCR of enrichment sample, plating on selective media, picking of multiple suspect colonies, and immunochemical and PCR analysis of isolates has resulted in isolation of >200 strains of STEC to date. A method of genotyping STECs with higher resolution than PFGE is being developed for purposes of source tracking. Genotyping of strains isolated from watersheds, wildlife and livestock indicates transport of pathogens may be occurring during heavy rain events, through roaming wildlife and indicates persistent incidence of pathogen in specific locations. Sampling and testing will continue for 12-18 months.