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The overall objectives are to study E. coli O157:H7 and Salmonella enterica transmission by filth flies to understand routes of contamination and those processes which mediate fly infestations of leafy greens and other unprocessed fresh foods with the ultimate goal of reducing fly-borne bacterial contamination of fresh produce. <P>Specifically, we will compare transmission efficiencies of house flies and blow flies transferring E. coli O157:H7 to leafy greens or other unprocessed fresh foods, determine dispersal distances for flies bearing transmissible bacteria, determine the retention time of transmissible bacteria in and on flies, and document the fate of bacteria that are deposited on leaf surfaces via fly regurgitation or defecation. One of our goals is also to use fly microbial profiles as a forensic tool to locate the source of bacteria on field grown leafy greens. Filth flies harbor many species of bacteria, including several human pathogens. Using molecular tools, we will compare the bacterial species compositions of flies to those of known bacterial sources to see if origin of flies, and possible contamination sources, can be determined. Finally, filth flies are not normally associated with food plants, but can be attracted to volatiles associated with plants. We will test attractiveness of volatiles from homopteran-infested leafy greens and determine if honeydew-fly-plant interactions impact the probability of pathogen contamination by bacteria-bearing filth flies. We anticipate learning more about how filth flies interact and behave in agricultural ecosystems where fly breeding areas are in close proximity to fresh produce production areas and how these behaviors might increase risk for contamination of produce. <P>Our results will be used to develop fly management and fresh produce cropping strategies that reduce the risk of fly-borne pathogen transmission to pre-harvest fresh produce that will be communicated to the livestock and fresh produce industries through targeted extension and outreach programs.
NON-TECHNICAL SUMMARY: Filth flies are insects such as houseflies, blowflies, and/or flesh flies that develop in fecal material, decomposing animals or rotting plants. In addition to necrotic tissue, these flies must feed upon bacteria in order to complete their life cycle. As a result, filth flies are well-known vectors of human pathogens in situations where they can come in contact with prepared foods or hospital surfaces. However, the role of filth flies as carriers of pathogens to food plants is not well understood. In cropping systems where fly breeding areas such as animal production facilities are in close proximity to fresh produce production areas it is important to understand when and under which conditions flies will move to, and make contact with, plants intended for human consumption. The work outlined in this project will determine if flies are efficient vectors of two foodborne disease pathogens, Salmonella spp. and E. coli O157:H7 to pre-harvest leafy greens. Our work has several goals: We will determine 1. if some species of flies are better enteric bacteria vectors than others 2. the optimal dispersal distance of flies carrying transmissible pathogenic bacteria 3. if bacterial profiling can be used to trace the origin of flies 4. whether bacteria regurgitated by flies onto plant surfaces can move into the plant (internalize) and 5. identify odors emanating from plants infested with aphids or whiteflies that are attractive to flies. This information will be used to develop and implement foodborne pathogen risk-reduction strategies in fresh produce cropping systems.
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APPROACH: To define pathogen transmission parameters and compare vector competence of fly species we will use green fluorescent protein (GFP)-tagged E. coli O157:H7 and S. enterica strains. Flies will be exposed to bacteria in cages containing manure-bacteria mixtures and then exposed to plants under various conditions and constraints designed to tell us how long viable bacteria remains associated with flies and how easily flies transfer bacteria to plants over time. Flies will also be tested under field conditions for dispersal distances combined with vector competence (of naturally occurring pathogens) determination by capturing flies at 100m, 400m, 800m, and greater distances from a release source. Vector competence comparisons will be statistically analyzed using paired t-tests. For multiple treatment comparisons, data will be subjected to analysis of variance and Tukey's mean separation test when appropriate. The ability of E. coli and Salmonella to penetrate below a fly-regurgitation spot into leaf tissue will be assessed by transmission electron microscopy. Bacterial profiling of flies and fly developmental sources (such as animal manures, compost, decaying vegetation) will be done using a metagenomic approach. Bacteria obtained from these sources will be subjected to T-RFLP (Terminal Restricted Fragment Length Polymorphism) analysis which profiles the structure and relative contribution of all bacterial species that are present in a fly as well as the source environment using 16s rDNA. We will compare the bacterial profiles of the flies and source environments to ascertain the origin of the flies. Identification of volatiles emanating from plant/fungi/bacteria/honeydew combinations will be done by capturing volatiles on solid phase microencapsulation fibers and analysis by gas chromatography - mass spectrometry. We will also determine if fly abundance /pathogen load is dependent upon the presence/absence of homopterans in field-grown leafy greens by passive (yellow sticky cards) and active (sweep netting) capture of flies attracted to test fields. Collected flies will be tested for presence of gut sugars (an indication of the fly food source) and for the presence of E. coli O157:H7. Data will be analyzed by multivariate analysis of variance with appropriate mean separation tests. The final component of our project is the transfer of information we obtain to both leafy greens/produce growers and livestock facility managers. We will accomplish this by using both direct communication via face-to-face extension meetings that bring both production systems together and by passive dissemination via extension publications and a public website. We will assess current knowledge of fly biology, pathogen contamination routes via insects, and fly control measures during year one and again at the end of the project. Our results will be used to develop and implement fly control and fly mitigation strategies that result in lower risk of contamination of fly-borne pathogens. Finally, we will present our research findings at professional meetings and publish results in peer-reviewed journals.