Assessing Risk Factors for the Persistence of Salmonella Enteritidis Phage Type 30 in Almond Orchards

The specific hypotheses and objectives of the proposed research are: Hypothesis I: Unique production management practices among the outbreak-associated orchards lead to the spread and persistence of SE PT30.
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Objective 1. To identify geographic, demographic, and management practices that may contribute to the persistence of SE PT30.
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Objective 2. To characterize the microbial communities, including populations of SE PT30, of soils in the almond production environment by soil type, age of the trees, season, and soil humidity.
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Objective 3. To study the interaction of SE PT30 with other members of soil communities in the soil, including protozoa and nematodes, and fungi and soft-rot bacteria on almonds and almond hulls and shells.
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Objective 4. To determine the localization and fitness of Salmonella Enteritidis PT30 on pre- and post-harvest almonds including almond tree flowers, leaves and soil, and almond hull, shell, and nut surface. Hypothesis II: S. Enteritidis PT30 is typical of other phage types of S. Enteritidis and other Salmonella serotypes. It has no unique physiological characteristics that make it capable of enhanced persistence in an almond production environment.
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Objective 5. To characterize and compare the physiology of SEPT30 with a variety of SE phage-type strains. Hypothesis III: Unique conditions existed during the 2000 harvest season that significantly amplified the population of S. Enteritidis PT30 on the hulls and shells of the harvested almonds leading to substantial contamination of the almonds during the hulling and shelling operation.
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Objective 6. To determine the environmental conditions that occurred during the production, and particularly harvesting of the outbreak-associated almonds and evaluate the effect of these conditions on the growth and survival of SE PT 30.
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Objective 7. To develop educational materials for growers of tree-nuts and fresh fruits and vegetables regarding specific practices that would reduce the liklihood for persistence of Salmonella in the production environment.
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Scientists with expertise related to almond production practices, microbial ecology of almond orchard soils and of plant surfaces, and growth and survival characteristics of Salmonella in produce systems, epidemiology and ecology of S. Enteritidis, and the molecular biology of Salmonella have joined to provide a multidisciplinary approach to address the research objectives.
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Epidemiological approach: A case control study will be conducted with positive farms and surrounding almond orchards. Statistical analysis will be used to try to pinpoint potential risk factors for the prevalence and persistance of SE PT30 in the environment. Production systems approach: Orchard floor microclimate of outbreak-associated almonds will be monitored by placing dataloggers with relative humidity, air temperature, soil temperature and soil moisture sensor in several locations in the blocks that have tested positive for SE PT30 as well as some of the younger blocks that have not tested positive. Based on the soil and plant water potential data as well as preliminary results on the characterization of microbial flora, a deficit irrigation trial will be set up in the second year. Altering soil moisture conditions near the soil surface should affect the survival of Salmonella.
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Molecular approach: This part of the study will characterize microbial communities in soils on outbreak-associated farms and will determine how these communities change during the growth cycle. Soil samples from the outbreak-associated farms and nearby SE PT30-negative farms will be collected. PCR-based approaches will be used to produce fingerprints of the microbial communities. Selected SEPT30 strains isolated from almonds and orchard soil will be made fluorescence by transforming them with a stable plasmid containing a green fluorescence protein (GFP) gene downstream of a constitutive promoter. This strain will be used to determine the localization of attachment and colonization by SE PT30 on almond hulls, shells, and nuts.
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Microbial physiology approach: Selected SEPT30 isolates and other phage type of SE will be characterized for carbon, nitrogen and phosphate utilization. We will also examine the surface structures of SEPT30 that might make this strain unique. The environmental conditions that occurred during the production, and particularly harvesting of the outbreak-associated almonds will be evaluated for their effect on the growth and survival of SE PT 30 in the almond environment.
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Ecological approach: Protozoa and nematodes that feed on bacteria have been recognized as potential reservoirs for bacteria, and an environment where bacteria may be protected from harsh conditions and evolve mechanisms for intracellular survival (e.g. Salmonella). To address the possibility that SEPT30 may have persisted in the orchard soil in such microbial reservoirs, protozoa and nematodes will be isolated from contaminated orchard soil and tested for their ability to feed on SEPT30 and host it internally.
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S. enteritidis was isolated from orchard soil a year after outbreak-associated almonds had been harvested indicating unexpected long-term persistence of this organism in the environment. Study of this almond-production environment provides an opportunity to learn more about the pre-harvest conditions that may be risk factors for contamination of tree nuts, and for raw produce in general.