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Meteorological and Environmental Conditions Leading to Increases in Virulent Vibrio vulnificus Abundances in Shellfish


The pathogenic bacterium,Vibrio vulnificus,is ubiquitous to estuarine and coastal environments and can be concentrated in in seafood, especially filter-feeding animals such as oysters. Ingestion of seafood contaminated with this organism can lead to infections resulting in gastroenteritis with associated abdominal pain, diarrhea, and vomiting, but have the potential to quickly progress to lethal septicemia. This bacterium is the cause of 95% of seafood-borne deaths in the United States, and is the leading cause of all foodborne deaths in Florida. V. vulnificus exhibits a high degree of variation, both genotypically and phenotypically and strains are divided into genotypes, with an E-genotype that predominantly recovered from environmental samples, and a C-genotype that is most frequently isolated from clinical sources. Water column data suggests that the deadlier C-type strains respond differently to environmental conditions than the closely related E-type strains. In particular, they behave differently to factors such as pH and dissolved oxygen and this can lead to an increase in the C-type to E-type ratio. We intend to quantify the abundance of these bacteria in shellfish meats while concurrently recording data on the environmental and meteorological conditions at the time of shellfish harvesting. By identifying the relationships between external factors shifts in the ratio of virulent strains, we will generate predictive models that can be used to forecast times when public health risk of foodborne illness is elevated when consuming or purchasing oysters or other harvested shellfish, potentially reducing the number of infections.

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<p>NON-TECHNICAL SUMMARY:<br/> Approximately 95% of seafood related deaths in the United States are caused by a single bacterium known as Vibrio vulnificus. Commonly found in coastal waters, these harmful bacteria are concentrated by shellfish such as oysters and have the potential to cause disease when oysters are eaten raw or undercooked. The severity of the disease can range from stomach upset, including vomiting and diarrhea, to a severe blood infection that can result in death in as little as 24 hours. One method of looking at the genetics of these bacteria separates them into two types, with one of these types, known as C-type, more able to cause food-borne infections. While there has been a great deal of research on how the species as a whole reacts to environmental conditions, very few studies exist that look at what conditions might trigger a rapid short-term
increase in the deadly C-types. Preliminary research suggests that while salinity and temperature are the drivers of the total abundance of these bacteria in the water, these have no relevance on the concentration of the C-type strains. Other factors such as pH and dissolved oxygen, however, correlate with the ratio of the C-type strains. These factors are particularly interesting as anthropogenic effects upstream of the oyster beds can affect these specific environmental conditions. For example, eutrophication stemming from nutrient runoff from upstream agriculture operations causing depressed dissolved oxygen in by increasing biological oxygen demand and encouraging phytoplankton blooms. This research linking the bacterial dynamics with meteorological and environmental drivers is a vital step in developing accurate predictions for the deadly forms of V. vulnificus bacteria. This will
ultimately reduce the number of illnesses and deaths caused this emerging pathogen in the United States.
<p>APPROACH:<br/> Objective 1). Quantify V. vulnificus total abundance in shellfish meats and the water column using both culture and quantitative PCR (qPCR)-based methods that specifically target or identify the more virulent strains of this species. The project will be focused in Carteret County, North Carolina, a location that has been well studied by Project Director Froelich and Primary Mentor Noble for many years. The study area includes the Croatan-Albemarle-Pamlico estuary system, the second largest in the United States. Oysters will be collected three times per month, from four sites that are open for shellfish harvesting . Two sites have higher average salinities than the other two, and these four sites together allow for a range of environmental parameters, even when collected on the same day. Sampling will be conducted year-round and will be conducted over 18
months of the two-year project. Water will also be concurrently collected from each oyster harvest site in sterile 1-liter bottles. Water and oysters will be transported to the laboratory for analysis immediately after collection. Water samples will be filtered and the filters placed onto media selective for V. vulnificus, while oysters will be aseptically shucked and homogenized before being plated on selective media. PCR analysis will be be utilized to confirm presumptive bacterial isolates as V. vulnificus while simultaneously determining their genotype (C-type or E-type). For sample collection and DNA extraction to be used in qPCR based quantification of pathogenic V. vulnificus from water and oysters100 ml of water samples will be filtered onto a polycarbonate filter and bead beating used to extract the DNA. For oyster samples, shucked oyster meats will be homogenized and treated
with proteinase K, and then extracted via NucliSens magnetic extraction technology. After extraction virulent V. vulnificus will be quantified using qPCR and previously tested primers, probes, and protocols designed to detect polymorphism in the pilF gene. The culture-based data generation will occur concurrently with the oyster harvesting. The samples collected for qPCR analysis will be flash frozen and stored at -80?C until after the Vibrio bacterial population decline in the winter. At this time, the frozen samples will be processed and analyzed. An electronic database will be maintained that contains all collected data. Objective 2). Collect data on environmental conditions in the water column of oyster and shellfish harvesting areas including parameters such as temperature, dissolved oxygen, turbidity, pH, and chlorophyll a and simultaneously collect meteorological parameters
during, and antecedent to, harvesting of shellfish including precipitation, wind speed, and wind direction. Simultaneous to the oyster collection, water will be measured using a handheld, calibrated, YSI 6000 multiprobe Sonde that collects data on the above-mentioned factors. These data will be collected from water immediately above or adjacent to harvested oysters and be logged in the above-mentioned database for correlation analysis. Meteorological data, including precipitation, wind speed (average and maximum), wind direction, and air temperature, will be collected for each sampling date, as well as the two days antecedent to the sampling date, using local weather stations accessible via the internet. Objective 3). Identify the relationships between measured environmental parameters and "blooms" of the pathogenic strains of V. vulnificus in both the water column and shellfish meats.
Each individual environmental condition will be compared with each other in a correlation matrix to identify significant correlations and remove potentially interdependent factors. Multiple linear regression or Spearman Rank correlation analysis will be performed on the remaining factors including microbial concentrations and ratios of virulent sub-populations, with log10-transformation as necessary to reduce skewness. The results of these analyses will be used in the final objective. Objective 4). Use the identified relationships to generate new improved predictive models that that can be used to forecast times when public health risk of foodborne illness is elevated when consuming or purchasing oysters or other harvested shellfish. Regression models will be created and compared using R2 and root mean squared error statistics using SPSS statistical analysis software (SPSS Inc.,
Chicago, IL). We expect to generate a database containing at least 54 individual sampling events each of water and oysters. This database will generate a predictive model that will estimate the quantity of virulent forms of V. vulnificus in shellfish based on current or recent environmental and meteorological conditions. The success of this model will be measured by a hindcasting study in which prior data on V. vulnificus bacteria isolated from oysters in similar locations in previous years will be compared to the values predicted by the model. Additionally, the interim success of will be evaluated by generating a model from the data generated in the first year of the study and using that model to forecast the V. vulnificus abundance that will be measured in the second year of the study.

Froelich, Brett
University of North Carolina
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