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

View the project final report

• 2014 annual report

Commodities

• Animals: fish, shellfish

Practices

• Animal Production: preventive practices
• Education and Training: extension, on-farm/ranch research, workshop

Illnesses associated with the bacteria Vibrio parahaemolyticus (Vp) have increased during the last
five years presenting a serious concern to shellfish farmers. Oysters accumulate the naturally
occurring bacteria, posing health risks to individuals consuming raw shellfish. 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 2 million oysters in 2013. 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. 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.

Objectives:
(1) To determine if Vps spike when farmed oysters in the intertidal are
exposed to sunlight and air during low tides; and if so,
(2) to determine if Vp levels rapidly decline to pre-low
tide levels upon immersion with the next tide; and
(3) to determine whether 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.

Methods:
Three hypotheses will be tested:

1. Total Vp burdens increase during intertidal aerial exposure
2. Total Vp burdens decrease following submersion after aerial exposure
3. Pathogenic strains of Vp represent a small fraction of total Vp in intertidal oysters The study will be 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 (2.75-3.0 inch shell height) will be placed into six bags at typical grow-out densities (150 per bag) at least two weeks prior to each trial. Three bags will be 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 will be assigned to an adjacent floating cage grow-out system (subtidal rack). Height of the intertidal rack relative to tidal levels and distance from the bottom will be documented.

Temperature and salinity of the six bags will be monitored continuously using HOBO data loggers. Samples will be taken across a tidal cycle, once monthly in June, July, and August. Specific collection dates will target the Spring tide. On each sample date, 12 oysters will be randomly sampled from each bag along a time course spanning from
(1) first emergence on the ebbing tide,
(2) two hours after low tide air exposure and
(3) approximately 24 hours later just prior to low tide air exposure. Samples will be placed on ice and immediately transported to the laboratory where they will be processed for Vp analysis. The standard qualitative procedure method for quantifying Vp is the most probable number (MPN) technique on a pooled sample of 12 oysters (FDA, 2007). This technique is presently used by most states for coarse monitoring of Vp in oyster populations. The more recent multiplex real-time PCR assay (Nordstrom et al. 2007), has demonstrated higher sensitivity and specificity than the MPN technique, and allows the simultaneous 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). Using individual oysters as a template for the assay, the multiplex PCR allows for a more rigorous assessment of Vp burden at the individual level and variation in Vp burden at the population level. Our approach therefore employs the multiplex real-time PCR assay on individual oysters. Oysters will be cleaned and shucked, and entire shell contents (animal and liquor) will be emptied into a sterile tube and homogenized.

We will perform the three-tube MPN method as described in the FDA Bacteriological Analytical Manual (DePaola and Kaysner 2004). After incubation, a 1 ml sample from each tube will be removed and boiled for 10 min for PCR. Our PCR analysis will incorporate 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 will be 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 will test 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) will be treated as categorical fixed effects. The goal of the project is to assess and minimize human health risks associated with farmed-raised intertidal oysters.

We believe the study design enables a robust evaluation of Vp response to intertidal conditions providing measurable answers to the key questions posed. By answering these questions the study will present scientific evidence on which best management practices can be developed. Project results will be presented to both industry and state policy makers informing best management practices and regulations. Acceptance and implementation of the recommendations will serve to support the production of safe, sustainable, and profitable cultured oysters. The degree to which the project outcomes guide practice and policy decisions will be documented through direct discussion with industry and regulatory stakeholders. The percent of intertidal oyster farms adopting proposed recommendations will be determined and will serve as a measure of project success and goal achievement.

Timeline:
1. Research project is conducted on-farm, directly linking industry with researchers (Jun-Aug 2014, monthly sampling).
2. Laboratory analysis conducted to determine Vp abundances (Sept-Dec 2014).
3. Project results are presented to shellfish farmers, scientists, and extension agents attending regional and local aquaculture meeting (Winter 2015).
4. Project results are presented to State regulators to help inform Vp Control Plans (Winter 2015).
5. Shellfish Growers Forum involving roundtable discussion between scientists, State shellfish related regulatory agents, and industry regarding best practices for minimizing Vp in farmed oysters in intertidal environments of NJ offered (Winter 2015). 6. Project results and recommendations are posted on University and Sea Grant extension (including eXtension.org) and disseminated through printed copy (Winter 2015).
7. State regulators use study results to inform Vp Control Plans (Winter 2015). 8. All NJ intertidal farms implement recommended BMPs to reduce the risk of Vp on their farms (Summer 2015).

References:
Cox, A.M., M. Gomez-Chiarri. 2012. Vibrio parahaemolyticus in Rhode Island Coastal Ponds and the Estuarine Environment of Narragansett Bay. Appl. Environ. Microbiol. April 2012 vol. 78 no. 8 2996-2999

DePaola, A., Jr., and C. A. Kaysner.2004. Vibrio, Chapter 9. In Bacteriological Analytical Manual. U.S. Food and Drug Administration, Washington, DC.

FDA. 2007. Naturally occurring pathogens. National Shellfish Sanitation Program guide for the control of molluscan shellfish. FDA, Washington, DC.

Jones, J. Y. Hara-Kudo, J.A. Krantz, R.A. Benner, A.B. Smith, T.R.Dambaugh, J.C. Bowers, and A. DePaola. 2012. Comparison of molecular detection methods for Vibrio parahaemolyticus and Vibrio vulnificus. Food Microbiology 30 105-111.

Nordstrom, J. L., M. C. L. Vickery, G. M. Blackstone, S. L. Murray, and A. DePaola. 2007. Development of a multiplex real-time PCR assay with an internal amplification control for the detection of total and pathogenic Vibrio parahaemolyticus bacteria in oysters. Appl. Environ. Microbiol. 73:5840–5847.