Final Report for ONE14-201
Project Information
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. 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 Vibrio parahaemolyticus (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.
Introduction:
Illnesses associated with the consumption of raw and undercooked shellfish have increased over the past decade, presenting serious concerns to shellfish farmers, resource managers, and public health officials (Newton et al. 2012). The majority of seafood-associated illnesses, both in the mid-Atlantic and worldwide, are caused by infection with Vibrio parahaemolyticus (Vp), bacteria that occur naturally throughout estuaries and marine environments. Filter-feeding bivalves such as oysters accumulate these naturally occurring bacteria in their tissues through regular feeding processes, posing health risks to individuals consuming these shellfish raw. Both regulator and consumer concerns over such illnesses threaten the sustainability and growth of the shellfish aquaculture industry. Oyster aquaculture has emerged as a significant industry in New Jersey with an anticipated harvest of 3 million oysters in 2015. 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 provide ideal conditions for Vp to proliferate in oyster tissues (Nordstrom et al. 2004). 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. The objective of this work is to answer three basic questions: 1) do levels of Vp in oyster tissues increase when intertidal oysters are exposed 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.
- Determine if levels of Vp increase in oyster tissues when intertidal oysters are exposed during low tides.
- Determine if Vp levels rapidly decline to pre-low tide levels upon immersion with the incoming tide.
- Determine whether virulent strains of Vp are present at the intertidal Delaware Bay area oyster farms.
Cooperators
Research
We conducted field surveys during the summer 2014 to address these objectives specifically testing the three following hypotheses: 1) total Vp burdens increase during intertidal aerial exposure, 2) total Vp burdens decrease following submersion after aerial exposure, and 3) markers associated with Vp pathogenicity 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. We placed near-market to market-size oysters into six mesh bags at typical grow-out densities (150 per bag) in early June 2014. Additional bags containing surplus 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).
We continuously monitored water temperatures at each rack system using HOBO® data loggers (Onset Computers Corp., Bourne, MA USA) to assure homogeneity in the environmental conditions among the intertidal and subtidal grow-out systems. Oysters were collected across a tidal cycle, once monthly in June, July, and August. Specific collection dates each month 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 the following day’s low tide aerial 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 incorporating a multiplex real-time PCR assay to enable the detection of the Vp species-specific thermolabile hemolysin (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) were emptied into sterile vessels and homogenized. We performed a three-tube MPN, as described in the FDA Bacteriological Analytical Manual (DePaola and Kaysner 2004), on triplicate samples taken from each homogenate. After incubation, a 1 ml sample from each tube was removed and boiled for 10 min for real-time qPCR. The real-time qPCR 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 estimated as the most probable number (MPN) from the three-tube assay and log10 transformed to be analyzed by ANOVA. We also tested for differences in the presence of the trh and tdh markers over the tidal cycle and over the time course of sampling using logistic regression.
Recorded water temperatures ranged from 14.33°C to 41.34°C in subtidal bags and 11.82°C to 42.40°C in intertidal bags (Fig. 1). We did not see a significant difference in mean temperatures between the subtidal and intertidal bags (ANOVA, F1,10654 = 3.14, P = 0.08), but we did see a greater daily range of temperatures recorded between the subtidal and intertidal bags (ANOVA, F1,220 = 57.95, P < 0.001). The daily temperature range, measured as the difference between the minimum and maximum temperatures recorded daily, varied from 0.96°C to 23.11°C in subtidal bags and 1.14°C to 24.36°C in intertidal bags.
Levels of total Vp (95%CI) ranged from 19 (5 - 68) to 1,100 (260 – 4,700) CFU/g (Fig. 2). We did not find a significant effect of intertidal exposure on levels of total Vp (ANOVA, F1,40 = 0.07, P = 0.79), nor did we observes significant differences between Vp densities in oysters tissues along the tidal cycle (ANOVA, F2,40 = 0.60, P = 0.55). This result does not lend evidence for our hypotheses that total Vp burdens increase during intertidal aerial exposure. Densities of total Vp in oyster tissues after re-immersion with the incoming tide were similar to the densities we observed before aerial exposure. This pattern can result from either or both declining densities of Vp after re-immersion or limited, if any, increases in Vp density with aerial exposure. We also did not observe any significant difference in densities of total Vp within oyster tissues over the months sampled (ANOVA, F2,40 = 0.25, P = 0.78). These preliminary results suggest that that the aerial exposure experienced by oysters grown in the intertidal has little effect on densities of Vp in oyster tissues.
Patterns of the tdh and trh markers associated with Vp pathogenicity mirrored the patterns we saw in total Vp, however the levels of these markers observed in oyster tissues were much lower then the levels observed for total Vp (Fig. 2). We did not observe significant impacts of intertidal exposure on densities of tdh (ANOVA, F1,40 = 1.05, P = 0.31) and trh (ANOVA, F1,40 = 0.28, P = 0.59), nor did we see differences in tdh (ANOVA, F2,40 = 0.37, P = 0.69) and trh (ANOVA, F2,40 = 1.03, P = 0.37) over the tidal cycle. Densities of these markers were low in most of our samples and we did not observe tdh in any samples in July. This latter observation led to a significant effect of sampling month on the presence (Fig. 3; Analysis of Deviance, χ2 = 18.47, df = 2, P < 0.001) and density (ANOVA, F2,40 = 4.91, P = 0.01) of tdh. It remains open whether this result is due to limited sampling of a rare phenomenon (i.e. the presence of tdh genes in vibrios associated with Delaware Bay oysters) or a true effect of sampling month on the density tdh. Collectively, these results suggest that Vp levels remain at ambient levels when intertidal oysters are exposed during low tides, and are maintained at such levels upon immersion with the incoming tide. We observed the presence of markers that have been associated with Vp pathogenicity, suggesting that virulent strains of Vp are likely present in Delaware Bay. Levels of these markers were approximately two orders of magnitude lower than the levels observed for total Vp, implying that virulent strains of Vp are likely to be only a small fraction of the total Vp present. The U.S. Food and Drug Aministration recommends that the levels of Vibrio parahaemolyticus in oysters not exceed 10,000 cells/gram. Observed Vp levels in this experiment were well below this threshold.
- Figure 1: Temperatures of subtidal and intertidal bags, recorded 15 minute intervals
- Figure 2:Vibrio parahaemolyticus density in intertidal and subtidal oysters sampled in June, July, and August
- Figure 3: Proportion of samples containing the tdh and trh markers associated with V. parahaemolyticus pathogenicity
This study provides critically needed information on the interaction of Vibrio parahaemolyticus and oyster farm practices in the lower Delaware Bay. It is the first to compare Vp densities in intertidal and subtidal conditions at the site in the Bay environment and to apply molecular tools to evaluate the relative densities of markers associated with Vp pathogenicity. The results suggest that under conditions present in the lower Delaware Bay, intertidal oyster culture methods pose no higher risk for Vp than subtidal grow-out methods. At the present time New Jersey state regulations present more stringent harvesting requirements (time-temperature controls) for intertidally-harvested oysters than for oysters harvested from subtidal areas. However, given the importance of maintaining the safest product possible, and the limited scope of our work (only single year and single site) additional investigations should be conducted to confirm these results. Should additional investigations support these results, more relaxed restrictions may prove acceptable.
These results are preliminary and our understanding of the risk of human illness associated with oyster aquaculture would certainly benefit from a broader survey of Vp in oysters grown in mid-Atlantic estuaries. This preliminary study motivated a larger study, recently funded by New Jersey Sea Grant and in partnership with the Virginia Institute of Marine Science (VIMS), to assess the effects of common oyster aquaculture practices on levels of harmful vibrios in harvested oysters. This additional, broader study is further addressing regional patterns in Vp risk by sampling throughout the estuary salinity gradient of both the Chesapeake and Delaware Bays over a multi-year time horizon. Broadening our sampling will allow us to refine our understanding of farm practices associated with elevated levels of harmful vibrios in oysters.
Education & Outreach Activities and Participation Summary
Participation Summary:
The results of this work have been presented at regional and national meetings, respectively at the Northeast Aquaculture Conference and Exposition in Portland, ME January 14-16, 2015 and the Annual Meeting of the National Shellfisheries Association in Monterey, CA March 22-26, 2015. Additionally, the results will be presented to our local shellfish growers via the Rutgers University hosted Shellfish Growers Forum Series (scheduled for May 18, 2015). The report has also been shared with our local State authorities at the NJ Department of Environmental Protection Bureau of Marine Water Monitoring and NJ Department of Health. The attached project summary has been distributed to all the oyster growers presently working in the Delaware Bay, NJ and is available on the Haskin Shellfish Research Laboratory website.
Project Outcomes
Farmer Adoption
The results suggest that current culture practices involving rack and bag grow out systems located in intertidal Delaware Bay environments do not increase the risk of Vp. Oyster growers have been advised to continue to follow best management practices and New Jersey Vp Management Plan guidelines for harvesting oysters during seasons of elevated bay wide levels of Vp. However, no additional measures need to be implemented above and beyond current practice. Confirmation of these results through similar studies to be conducted this summer would provide a sound scientific basis for relaxing stringent time and temperature regulations that are presently imposed on oyster harvests from intertidal sites in New Jersey.
Areas needing additional study
We have very little understanding of the risks to human health from oyster consumption, and its relation to oyster farming practices. Our preliminary study found that levels of Vp, and markers associated with Vp pathogenicity in humans, change little when oysters emerge from the water and are exposed to aerial conditions over a normal tidal cycle, which generally occurs over a period of three to four hours in the mid-Atlantic region. Would the patterns we observed remain consistent if oysters were exposed to aerial conditions for a longer duration, such as the five to six hours seen in many oyster farms located in northern New England. And, how do the low levels of markers associated with Vp pathogenicity we observed relate to the risk of human illness? Our ability to predict where, when, and under what conditions the consumption of raw, half-shell oysters presents a risk to human health is expanding, but remains in its early stages. This present study and those following will help ensure the safety and sustainability of the emerging US oyster aquaculture industry.