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Based on the following hypotheses, the study includes five objectives: E. coli 0157:H7 colonizes different tissues of the spinach plant, and achieves different titers, depending on the site and method of entry. Phytopathogenic bacteria enter plants through natural openings, wounds, or deposition by insects. Depending on the mode of entry, bacteria encounter different plant micro-environments and develop different interactions with the host.
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Objective 1: Assess whether a green fluorescent protein-tagged E. coli 0157:H7, introduced into different plant tissues, colonizes and moves through intercellular spaces, the vascular system, or both, using confocal and fluorescence microscopy of roots, stems and leaves. Spinach cultivars will be inoculated by root drench, leaf surface contamination, needle injection, vacuum infiltration, and an insect vector. Plate counts and quantitative (Q-PCR) will be used to assess bacterial titers reached after internalization.
Spinach cultivar and plant age at the time of inoculation affect susceptibility to E. coli O157:H7 internalization, colonization, and titer. Different plant cultivars often differ in susceptibility to bacterial invasion.
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Objective 2: Test susceptibility to E. coli 0157:H7 for a variety of spinach cultivars. Plant tissues (mature and developing leaves and roots) will be assessed by quantitative polymerase chain reaction (Q-PCR) and culturing. The effect of plant age on colonization will be tested using 1, 2, 3, and 4 week-old plants.
Insect species that are known phytopathogen vectors inhabit livestock pasture areas in close proximity to leafy greens production areas.
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Objective 3: Survey cattle pastures and nearby lettuce/spinach growing areas for insects. Insect sticky traps will be placed in fencerows bordering pastured cattle at three to four locations in Southern California and the Salinas Valley. Sweep samples will be collected from each pasture and insects identified. Pasture grasses will be sampled and tested for E. coli O157:H7.
Insects common to livestock and field production areas (muscid flies, cabbage loopers, leafhoppers, and aphids) are capable of acquiring E. coli from contaminated livestock sources.
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Objective 4a: Confirm that houseflies acquire bacteria. Flies will be exposed to E. coli by caging with open agar cultures, dishes of E. coli suspension, fecal material containing E. coli, or potted grasses sprayed with 105 cells/ml bacteria, and flies will be held in mesh cages and tested for E. coli by polymerase chain reaction (PCR) methodology;<p>
Objective 4b: Test a butterfly model for its capacity to drink and acquire from above sources. Cabbage looper adults will be enclosed with E. coli-contaminated water or fresh fecal material and then tested along with liquid inoculation sources by PCR and cell plating; <p>
Objective 4c: Test phytopathogen vectors for their capacity to acquire pathogens from the above sources and from traditional artificial feeding sachets. Circulifer tenellus (a vector of spinach virus), and Acyrthosiphon pisum or Myzus persicae (aphid vectors of plant viruses) will be caged with E. coli contaminated water, fecal material, and plant material, or placed in feeding sachets containing E. coli and insects will be tested after 1, 2 or 3 weeks by PCR and culturing.
Flies, butterflies, leafhoppers, and aphids are competent vectors of E. coli transmitted to leafy greens.
Objective 5a: Determine dispersal flight distances of flies and cabbage looper butterflies marked with fluorescent powder, released in cattle production area, and recaptured at distances of 0.8 km, 1.6 km, and 2.4 km using fly-bait traps and sugar-water traps set in concentric circles around the release point; <p>
Objective 5b: Assess propensity of contaminated flies, butterflies to alight on and contaminate greens. Houseflies or butterflies exposed to E. coli will be released into a chamber containing lettuce or spinach plants, and plants will be tested for E. coli by PCR and cell plating, at 1, 3 and 5 days; <p>
Objective 5c: Evaluate E. coli vector competence of leafhoppers and aphids; C. tenellus, A. pisum, M. persicae and Bemesia tabaci will acquire E. coli from feeding sachets and caged with lettuce or spinach and plants will be tested for E. coli by PCR or culturing. If any experimental transmissions are successful, transmission of E. coli O157:H7 by those insect species will be tested.
The role of insects and other arthropods as potential vectors for E. coli O157:H7 dissemination resulting in contamination of produce is examined in this study. The likelihood of insect transmission of E. coli O157:H7 from animal areas to leafy green produce is not known, although flies have been previously implicated in the transmission of E. coli O157:H7 to cattle, feed, and water. This project will evaluate E. coli O157:H7 internalization patterns and titers in spinach plants, based on the route of internalization, the type of cultivar, and the age of the plant. It will also determine whether key insect species are capable of transmitting or disseminating E. coli to leafy greens. The results will inform the development and prioritization (based on bacterial internalization sites, titer and translocation propensity) of strategies used to prevent or interrupt bacterial dissemination and/or invasion of plant tissues, including choice of cultivar, pest management practices, and bacterial removal or inactivation treatments.
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