On Oct. 5, 2018, in a seemingly ordinary transaction, Eddie and Patti Clinton, retired Wake Co., North Carolina educators/administrators, purchased two 10-pound bags of raw whole fresh shrimp. The original source of the crustaceans was a fisherman who reportedly harvested the shrimp in or near the New River in the southeastern area of the Tarheel State.
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Eddie placed the shrimp on ice in a cooler overnight. The next day he scooped the shrimp with his bare hands into smaller bags, then put them in his freezer. And, yes, he washed his hands after handling the raw shrimp.
Within about 24 hours, Eddie began experiencing soreness in his legs, shaking, feeling simultaneously hot and cold, loss of appetite, and slurred speech. By October 8, he was on life support at a Raleigh hospital in a medically-induced coma. The next day, doctors determined Eddie was infected with Vibrio vulnificus.
Eddie’s heart, liver, and kidneys were affected by the V. vulnificus, and his left leg was amputated below the knee on November 6. “This has been a horrific journey and it will be a long road to recovery,” Patti relates. “But Eddie is alive and that’s what matters most.”
“The doctors suspect Eddie may have wiped his mouth with his hand or that he had a small cut on his hand when handling the shrimp, but they never found any open wound on him,” says Patti. “Every doctor that saw Eddie said they had never seen a case like this before, where a person was infected with Vibrio without ingesting it in food.”
According to the CDC, people become infected with vibriosis typically “by eating raw or undercooked shellfish, particularly oysters.” Certain Vibrio species can also cause a skin infection when an open wound, which could be a cut or scrape, is exposed to raw seafood, raw seafood juices, or brackish or salt water, CDC says. Brackish water is a mixture of fresh and sea water, often found where rivers meet the sea.
CDC estimates that 80,000 people in the U.S. become sick with vibriosis each year, and 100 people die from their infection. About 52,000 of these illnesses are estimated to be the result of eating contaminated food. The most commonly reported Vibrio species, Vibrio parahaemolyticus, is estimated to cause 45,000 cases of vibriosis each year in the U.S.
When ingested, Vibrio bacteria can cause watery diarrhea, often accompanied by abdominal cramping, nausea, vomiting, fever, and chills, CDC says. Usually these symptoms occur within 24 hours of ingestion and last about three days. Severe illness is rare and typically occurs in people with a weakened immune system.
Most people with a mild case of vibriosis recover after about three days with no lasting effects, CDC points out. However, people with a V. vulnificus infection can get seriously ill and need intensive care or limb amputation. About one in four people with this type of infection die, sometimes within a day or two of becoming ill, CDC says.
Patti Clinton says her husband has diabetes, congestive heart failure, and chronic obstructive pulmonary disorder, plus he’s a smoker. “The doctors said most anyone else could have handled those shrimp we purchased with no negative health impacts, but since Eddie is an immuno-compromised senior male, he got life-threatening vibriosis,” she points out.
Postharvest Processing Pearls
Postharvest processing (PHP) methods can be used to reduce human-pathogenic Vibrio bacteria, such as V. vulnificus, from oysters intended for the raw, half-shell market, most especially those that are harvested from the Gulf of Mexico during warmer months when the organism is most prolific.
With oysters, PHP is any process that has been validated using National Shellfish Sanitation Program validation procedures, according to Corinne Audemard, PhD, an associate research scientist with the Virginia Institute of Marine Science, College of William & Mary. “PHP aims to reduce the levels of pathogenic hazards to below the appropriate FDA action level or, in the absence of such a level, below the appropriate level as determined by the Interstate Shellfish Sanitation Conference (ISSC),” Dr. Audemard relates.
Along with reducing Vibrio and other bacteria to non-detectable levels, Dr. Audemard says PHP kills spoilage bacteria, thus extending shelf life.
Four PHP technologies are currently utilized by ISSC-approved firms for PHP: 1.) Individual quick freezing, which involves rapid freezing of half shell oysters on trays, then adding a thin glaze of ice to seal in the natural juices before storing them frozen; 2.) Heat-cool pasteurization, a process whereby live oysters are placed in warm water for a certain time period and then immediately dipped in cold water to stop the cooking process; 3.) High hydrostatic pressure, that subjects oysters to high pressures (35,000 to 40,000 pounds per square inch) for three to five minutes; and 4.) Low-dose gamma irradiation.
High Salinity Relay
A relatively unexplored PHP method called relaying holds promise as an alternative strategy for reducing V. vulnificus levels, Dr. Audemard says.
“High salinity relaying involves transferring oysters from salinity waters, 8 to 15 psu (practical salinity units), to higher salinity waters, 30 to 35 psu, to achieve a reduction in pathogenic bacteria, to less than 30 V. vulnificus per gram in as little as 14 days,” Dr. Audemard explains. “High salinity waters appear to negatively affect the survival of V. vulnificus.”
High salinity relay differs from previously approved PHP methods in that it is not a controlled process, Dr. Audemard points out. “That’s because the procedure typically relies on the exposure of oysters to natural high salinity waters for several weeks,” she says. “However, high salinity relaying is also used in molluscan shellfish transfer to more controlled environments, such as land-based tanks with similar results.”
In research published in August 2018, Dr. Audemard and several colleagues evaluated high salinity relay as a PHP for reducing V. vulnificus.
Dr. Audemard says the study was based on FDA validation guidelines, which specify, among other things, the initial Vibrio density before the process, the number of samples to be analyzed, the analytical methods to be used, and the endpoint concentration criteria to be reached for process validation, 30 per gram (g). (See page 203 of the National Shellfish Sanitation Program Guide for the Control of Molluscan Shellfish 2017 Revision.)
During each of three relay experiments, oysters cultured from three different Chesapeake Bay sites of contrasting salinities (10 to 21 psu) were relayed without acclimation to high salinity waters (31 to 33 psu) for up to 28 days. Overall, nine lots of oysters were relayed with six exhibiting initial V. vulnificus greater than 10,000 per g.
“As recommended by the FDA PHP validation guidelines, these lots reached both the 3.52 log reduction and the less than 30 perg densities requirements for V. vulnificus after 14 to 28 days of relay,” Dr. Audemard relates. “Densities of total and pathogenic V. parahaemolyticus in relayed oysters were significantly lower than densities at the sites of origin, suggesting an additional benefit associated with high salinity relay. While relay did not have a detrimental effect on oyster condition, oyster mortality levels ranged from 2 percent to 61 percent after 28 days of relay. Although the identification of the factors implicated in oyster mortality will require further examination, this study strongly supports the validation of high salinity relay as an effective PHP method to reduce levels of V. vulnificus in oysters to endpoint levels approved for human consumption.”
Relative to the 61 percent mortality, Dr. Audemard believes the oysters were either stressed by the temperature abuse they underwent before relay to increase the Vibrio levels in these oysters, or the relay gear was not optimal, or some other factors. “We actually do not think high salinity relay in itself caused the mortality based on this study and previous ones by us and others,” she mentions.
Indicator for Pathogenic Vibrios
In 2005, under the leadership of Gary Richards, PhD, a USDA Agricultural Research Service laboratory team in Dover, Del., developed and published a simple and rapid procedure called the colony overlay procedure for peptidases (COPP) assay to quantify total vibrios (TV) in oysters and seawater.
Salina Parveen, PhD, a professor in the Food Science and Technology Program at the University of Maryland Eastern Shore, and several collaborators sought to validate the use of the COPP assay. “Regulatory agencies and industry currently use comparable testing of fecal coliform bacteria as an indicator of fecal pollution,” Dr. Parveen relates. “We thought the use of the COPP assay might serve a similar role as an indicator of pathogenic vibrios.”
Dr. Parveen and her colleagues collected oyster and seawater samples from the Delaware Inland Bays and the Maryland Chesapeake Bay and analyzed for TV, as well as pathogenic strains of V. vulnificus and V. parahaemolyticus.
They compared the COPP assay with direct plating and a molecular method that detects TV and pathogenic vibrios.“The results of the study indicate that the COPP assay is a viable alternative to other, more complicated methods, such as the most probable number (MPN) method, for the detection of V. vulnificus in oysters and seawater and it is currently under further evaluation for its ability to serve as an indicator for V. parahaemolyticus,” Dr. Parveen explains.
Dr. Parveen points out that the COPP assay reduces analysis time from three days (using the standard FDA-approved MPN-based method) to 24 hours, and it provides a practical method for monitoring pathogenic Vibrio species in shellfish and the aquatic environment.
Farm-Raised Oyster Project
Just underway is a three-year study to determine whether an oyster farm’s geographic location, handling practices, and choice of equipment affect Vibrio levels in farm-raised oysters.
William Walton, PhD, an associate professor in Auburn University’s School of Fisheries, Aquaculture and Aquatic Sciences, landed a $456,646 USDA NIFA grant in August 2018 to fund the project.
Dr. Walton and his collaborators, FDA microbiologist Jessica Jones, PhD, and Auburn doctoral student Victoria Pruente, are focusing on a management system called off-bottom oyster farming, where oysters are maintained in floating cages or suspended baskets above the ocean floor in food-rich coastal waters.
“The off-bottom industry has boomed over the last two decades and continues to expand,” Dr. Walton relates. “Oysters grown this way are typically hatchery-reared single set oysters instead of clumps of oysters normally found in the wild. When properly operated, the containers provide protection from predators and eliminate burial in the sediment, allowing oysters to be cultured in areas where they would not survive on the bottom. Adverse bottom environments include high salinity areas with high predation rates or areas where the substrate is too soft.”
Once a week, off-bottom farmers raise the baskets out of the water and allow the oysters to air-dry. “This practice prevents barnacles, seaweed, and other undesirable organisms from attaching to and marring the oysters,” Dr. Walton notes.
“Though the air-drying is process is crucial to product quality, it is not risk-free,” Pruente interjects. “The frequent handling exposes the oysters to elevated air temperatures and also interrupts filter feeding, and those conditions cause Vibrio levels to rise.”
Once the baskets are lowered back into the ocean, Vibrio levels gradually subside, but questions remain. “In our trials, we will look at how long after the oysters are resubmerged the Vibrio levels return to naturally occurring levels,” Dr. Walton says.
To achieve the shell shape, meat quality, and cleanliness that high-end market demands, farmers routinely handle their oysters for grading, splitting, control of biofouling, and many other reasons. “Routine handling techniques include tumbling and desiccation to prevent biofouling from barnacles, mud worms, and other organisms that clog the mesh in the containers, thus reducing the flow of water, food, and oxygen, and also to eliminate predators such as oyster drills,” Dr. Walton explains.
In contrast to any elective handling techniques, farmers could just leave their oysters alone and let them grow, Dr. Walton mentions. “When they do, with minimal intervention/care/handling, the oysters tend to grow long, skinny, and flat, which diminishes their market value,” he relates.
Dr. Walton says the increased demand for farm-raised oysters, combined with improved handling techniques for farm-raised oysters, has raised concerns among public health agencies and scientists about how routine handling on the farm can elevate the risk to humans posed by Vibrio, which are concentrated within the oyster tissue.
“The routine handling exposes oysters to elevated air temperatures for extended periods of time, permitting increased growth of Vibrio within the oysters,” Dr. Walton points out. “Fortunately, re-submersing the oysters allows them to begin filtering again and reduce the Vibrio levels back down to the background levels, which can vary and depend on environmental conditions, including water temperature and salinity. Our prior work determined the time required for the reduction of the elevated risk to background levels is seven days when using suspended gear.”
Dr. Walton’s team will now determine the re-submersion period required for elevated levels of Vibrio to return to ambient levels in oysters grown in a different type of gear that is commonly used and using various handling treatments.
New Vibrio Test Kit
On Oct. 1, 2018, Bio-Rad Laboratories, Inc., Hercules, Calif., launched its iQ-Check Vibrio PCR (polymerase chain reaction) Detection Kit, which provides qualitative detection of the three main Vibrio species that cause vibriosis, V. cholerae, V. parahaemolyticus, and V. vulnificus, according to Frédéric Pastori, the company’s international product manager.
With its main targeted matrices being seafood, including fish and shellfish (mollusks and crustaceans), the kit is a multiplex test based on gene amplification and detection by real-time PCR, Pastori says, noting that one kit can be used for up to 94 samples.
“This kit uses Bio-Rad’s Vibrio Enrichment Broth that promotes the growth of Vibrio while inhibiting associated competitive flora,” he relates. “The broth shortens culture from 18 hours to eight hours incubation time, and the entire detection process can be completed within 11 hours.”
Pastori says the iQ-Check Vibrio Kit reduces false positive results that can sometimes occur with real-time PCR methods when Bio-Rad’s Free DNA Removal Solution is incorporated into the testing process.
“The solution is easy to use, non-toxic, and optimizes the extraction process by reducing free DNA in the sample that can lead to false positive results,” Pastori explains. “Additionally, in an effort to manage PCR inhibition, an internal amplification control is included in the reaction mix to validate negative results.”