Minimizing risks of Vibrio bacteria in farm-raised oysters grown in intertidal environments of the Delaware Bay

Minimizing risks of Vibrio bacteria in farm-raised oysters grown in intertidal environments of the Delaware Bay

Summary

Illnesses associated with the bacteria Vibrio parahaemolyticus (Vp) have increased during the last five years presenting a serious concern to shellfish farmers. The majority of New Jersey oyster farms are located on the intertidal shores of the lower Delaware Bay where growing conditions are ideal. However, some data suggest that the intertidal environment may also increase Vp. The ability to predict where, when and under what conditions Vp presents a health risk is limited by a poor understanding of its basic ecology and relationship with farm practices.

New Jersey’s oyster farms are concentrated on the extensive intertidal sand flats of the lower Delaware Bay where they are exposed twice daily during low tide. Previous studies from the Pacific Northwest indicate that intertidal exposure accelerates the proliferation of vibrios, increasing the risk to human health.

The objective of this work is to answer three basic questions—(1) Does Vp spike when farmed oysters in the intertidal are exposed to sunlight and air during low tides; and if so, (2) do Vp levels rapidly decline to pre-low tide levels upon immersion with the next tide; and (3) are virulent strains of Vp present at the intertidal Delaware Bay area oyster farms? Answers to these fundamental questions will be used to develop best management practices for the safe harvest of oysters, ensuring consumer health and a sustainable and profitable future for oyster farms in New Jersey and from Maine to Florida.

We conducted a preliminary study to test whether this result applies to mid-Atlantic intertidal environments. Oysters were collected from subtidal and intertidal rack and bag grow-out systems monthly from June through August 2014. Samples were collected at the initial exposure of intertidal oysters on the receding tide, and then at three and 24 hours following this initial exposure. Total and pathogenic Vp levels were enumerated using a most-probable number quantitative PCR assay with probes targeting the thermolabile direct hemolysin (tlh) and thermostable direct hemolysin (tdh) genes associated with pathogenicity. Observed Vp densities (± 95% CI) ranged from 19 (5 – 68) to 1,100 (260 – 4,700) CFU/g for total Vp, 0 to 11 (3 – 43) CFU/g for trh, and 0 to 459 (100 – 2,100) CFU/g for tdh. We did not see a significant difference between levels of total and pathogenic Vp between subtidal and intertidal oysters, nor was there a significant increase in vibrio burdens over the time course of low-tide exposure. This initial result suggests that the relationship between grow-out conditions and vibrio levels in oysters is not as straightforward as previously thought, and highlights the need for locally relevant aquaculture practices to minimize the risk of vibrio illness.

Objectives/Performance Targets

(1) To determine if Vp spikes when farmed oysters in the intertidal are exposed to sunlight and air during low tides.

(2) To determine if Vp levels rapidly decline to pre-low tide levels upon immersion with the next tide.

(3) To determine whether virulent strains of Vp present at the intertidal Delaware Bay area oyster farms.

Accomplishments/Milestones

Investigations were conducted during summer 2014 to test three hypotheses: (1) Total Vp burdens increase during intertidal aerial exposure, (2) Total Vp burdens decrease following submersion after aerial exposure, and (3) Pathogenic strains of Vp represent a small fraction of total Vp in intertidal oysters. 

The study was conducted in collaboration with oyster farmer Elizabeth Haskin, owner of Betsy’s Cape Shore Salts. Ms. Haskin employs rack and bag grow-out systems in the intertidal on a riparian lease located on the Cape Shore flats in the lower Delaware Bay. Near-market to market-size oysters were  placed into six bags at typical grow-out densities (150 per bag) in early June 2014. Additional bags containing extra oysters were deployed at the same time. These oysters were used for replacement of oysters sampled and lost to mortality. Three bags were randomly assigned to a location on the farm in an area having the longest air exposure during low tide (intertidal rack), and the remaining three bags were assigned to an adjacent offshore area, where oysters remained submerged at low tide (subtidal rack). 

Water temperature at each rack system was monitored continuously using HOBO data loggers. Oyster samples were taken across a tidal cycle, once monthly in June, July, and August. Specific collection dates targeted the Spring tide. On each sample date, 12 oysters were randomly sampled from each bag along a time course spanning from (1) first emergence on the ebbing tide, (2) three hours after low tide air exposure and (3) approximately 24 hours later just prior to low tide air exposure. Samples were placed in coolers on icepacks and immediately transported to the laboratory where they were processed for Vp analysis. Vibrio parahaemolyticus densities were enumerated by the most probable number (MPN) technique on a pooled sample of 12 oysters followed by multiplex real-time PCR assay enabling the  detection of the Vp species-specific tlh gene and the thermostable direct hemolysin (tdh) and thermostable-related hemolysin (trh) genes, which have been identified as key determinants of Vp pathogenicity (Nordstrom et al. 2007, Cox and Gomez-Chiarri 2012, Jones et al. 2013). Briefly, oysters were cleaned and shucked, and entire shell contents (animal and liquor) was emptied into a sterile vessels and homogenized. Three-tube MPN method as described in the FDA Bacteriological Analytical Manual (DePaola and Kaysner 2004) was performed. After incubation, a 1 ml sample from each tube was removed and boiled for 10 min for PCR. PCR analysis incorporated the internal amplification control (IAC) from Nordstrom et al. (2007) to ensure PCR integrity and eliminate false-negative reporting. Quantities of total Vp, trh+ Vp, and tdh+ Vp were log10 transformed and analyzed by repeated-measures ANOVA to test whether Vp burdens increase following intertidal aerial exposures and decrease with submersion. To assure homogeneity in the environmental conditions among the grow-out systems (intertidal and subtidal racks), we tested for within group correlations in temperature and salinity profiles recorded in each bag. The grow-out system (intertidal vs. subtidal racks) and time course (initial emergence on the ebbing tide vs. two hours following aerial exposure vs. 24 hours following initial emergence) were treated as categorical fixed effects. 

The project has moved forward along the proposed time course. We are presently finalizing our data analysis and preparing abstracts for regional and national meetings where the results will be presented. A meeting to discuss results with local shellfish growers is being planned for early February 2015.

Impacts and Contributions/Outcomes

Collaborators: