A 6-year-old outbreak of food poisoning linked to eating raw Chesapeake Bay oysters has left behind a lingering mystery. Scientists seeking to identify the water-borne pathogen that sickened a pair of Baltimore restaurant patrons have tracked the culprit to Asia.

How a potent strain of Vibrio bacteria seemingly from so far away wound up in the Bay continues to puzzle Maryland health officials, who worked with researchers at the U.S. Food and Drug Administration to investigate the 2010 cases.

The microorganism could have gotten here in the ballast water of the many oceangoing ships that ply the Chesapeake every year, state and federal scientists suggested in a recently published journal article. Or, they added, perhaps it came via the introduction of nonnative oysters or some exotic fish.

“It really is speculation,” acknowledged Dr. Clifford Mitchell, environmental health bureau director for the Maryland Department of Health and Mental Hygiene. “We didn’t sample ballast water. We didn’t take specimens that would lead us to know that we had fish coming over, or migration.”

But the case, published in the June issue of Applied and Environmental Microbiology, illustrates how disease-carrying organisms may travel around the world, researchers said. And while steps have been taken since 2010 to prevent the unintentional transport of pathogens, parasites and other potentially harmful organisms via ships’ ballast water, those safeguards still have significant gaps in them.

Vibrio bacteria are found naturally in brackish or salty water, and about a dozen species cause an estimated 80,000 illnesses, 500 hospitalizations and 100 deaths each year across the United States, according to the U.S. Centers for Disease Control and Prevention.

People can get infected from cuts or skin punctures while swimming, wading or fishing in bacteria-laden waters. Infections also can result from eating raw or undercooked seafood.

The bacterium involved in the 2010 food poisoning outbreak was Vibrio parahaemolyticus, strains of which are commonly found in coastal waters worldwide — including the Bay — though only some have been found to cause illness. When those are ingested, they can cause acute gastrointestinal distress, including diarrhea, stomach pain, nausea, fever and chills. It usually passes within a few days, but in rare cases can be more severe, especially in people with weakened immune systems.

There were 45 cases of Vibrio infections reported in Maryland in 2010, but it’s not that often, state health officials said, that they’re able to pinpoint the source of the bacteria that may have sickened a particular person. By the time laboratory tests identify Vibrio as the cause of someone’s GI distress and the information gets reported to the state, days or even weeks may have passed, and the food that person had eaten is long gone.

In this case, though, state health investigators got a lucky break. Two individuals who got sick said that shortly before they became ill that summer, they had eaten raw oysters at different Baltimore restaurants. They hadn’t traveled out of state or done anything else that likely could have exposed them.

When investigators visited the restaurants, they found the half-shells eaten by the two victims were from the Bay. And when they visited the Maryland aquaculture operation that supplied both eateries, investigators pulled some oysters from the water and discovered that they had Vibrio in them as well — 11 different potentially disease-causing strains, in fact. One of those appeared to match the Asian strain found in the two food poisoning victims.

The investigation ended there, for the time being. Even though the Vibrio involved were similar, researchers couldn’t positively identify them as the same, using the analytic techniques they had at the time. “The chromosome patterns matched, but we weren’t sure how common that pattern was in the environment,” explained Robert Myers, director of the state health department’s laboratory administration. “We hadn’t seen it before.”

A few years later, though, “whole genome sequencing” technology became available, Myers said, giving researchers the ability to draw a more detailed map of an organism’s genetic makeup.

With that new, more powerful analytical tool, FDA researchers re-examined the Vibrio strains involved in the 2010 outbreak and those from the oysters that state health investigators had sampled. They identified them as belonging to a family of strains known as “sequence type 8.” 

When researchers consulted a worldwide Vibrio database, they found that the Maryland strains were unlike any seen to date in the United States. Instead, they were closely related to strains reported only in Asia, most recently in Hong Kong about four years before the outbreak.

How they got here may never be known, as so much time had passed by the time the Asian angle turned up. Scientists at the Virginia Institute of Marine Science conducted research for several years before the outbreak on a species of oyster, Crassostrea ariakensis, that originally is from Asia. But Kimberly Reece, a marine scientist at VIMS, said it’s “extremely unlikely” that research could have been the source of the Vibrio in the outbreak.

The oysters used in the VIMS research were all spawned in the United States, Reece said, and all held under “very strict protocols” devised to avoid any unintentional release of nonnative pathogens or parasites.

A more likely source, Bay scientists say, is ballast water. Oceangoing ships carry large quantities of water in their hulls to help stabilize them against wind and waves. Water is pumped into and out of ballast tanks to balance shifting cargo loads and other circumstances. Parasites, pathogens, even fish can be transported from one coastal area to another, sometimes wreaking havoc on local ecosystems.

“There’s every possibility that a ship’s ballast water could result in transport of Vibrio parahaemolyustic,” said Fred Dobbs, a marine microbial ecologist at Old Dominion University, whose research has found a different Vibrio species — the one that can cause cholera — both in ships’ ballast water and in the coastal waters that the ships ply.

As a precaution, ships are now forbidden to discharge in port unless they have previously flushed out their ballast tanks at sea and refilled them with ocean water, or they have a system on board that can treat any discharge to kill off potential invaders.

The rationale for ballast exchange, as the former method is known, is that organisms capable of living in fresh or brackish waters can’t survive the saltier ocean water. If done properly, it has been shown to get rid of 95 percent of whatever microorganisms were in the tanks, according to Whitman Miller, director of the National Ballast Information Clearinghouse, which is operated by the Smithsonian Environmental Research Center in Edgewater, MD.

But that residue can still harbor potential invaders, Miller cautioned. And there are occasions when ships can’t do an exchange at sea and for safety reasons have to discharge in coastal waters, he said.

The U.S. Coast Guard has issued discharge standards for ballast water released in coastal waters. They require the discharge to be treated so that any Vibrio present will be reduced to extremely low concentrations, Miller said. A number of treatment systems have been developed, which use filters, ultraviolet radiation and chemicals to cut down pathogens and other organisms. But while such systems are in use on some vessels, the Coast Guard has yet to approve any, so they are not widespread.

“We’re getting there, but not there yet,” said Mario Tamburri, director of the Maritime Environmental Resource Center of the University of Maryland Center for Environmental Science. Tamburri’s center has been testing treatment systems.

“The rules are basically in place, or internationally almost in place,” he said, “but now we need to be sure the technologies that are available do meet those rules.”

Of 1,900 vessels calling in Baltimore in the previous year, according to the clearinghouse, 400 reported discharging ballast in local waters. Only 19 of those reported using any treatment system.

It’s also possible, said Reece at VIMS, that Asian Vibrio may have been introduced into the Bay by some “rogue introduction” of oysters from that continent. But she said she hadn’t heard of any.

Another possibility, some scientists suggested, is that the Vibrio strain involved in the Maryland outbreak isn’t really all that alien after all. Only a relatively small share of all the pathogens in the Bay have undergone this kind of detailed analysis to identify them, they noted.

“I think it’s very reasonable to entertain the idea that they’ve been here all the time,” ODU’s Dobbs said of the Vibrio strains in the 2010 outbreak. “Our environment is under-sampled in this regard. Most of the time, we’re not going out looking for these things in surveys. We only start looking when someone comes down with a bad case of shellfish poisoning.”

However the pathogen got here, state and federal researchers say the case demonstrates the potential for whole genome sequencing to help run down future outbreaks of Vibrio.

“We haven’t seen this strain in the Bay again since 2010,” said Julie Haendiges, a molecular biologist with the state health department who participated in the investigation. But with this new technology, she added, “if an outbreak occurs, we may be able to mobilize faster and find a better source for these infections — for all food-borne pathogens, not just Vibrio.”

State and federal health and environmental agencies have established strict protocols meant to ensure the safety of seafood produced and eaten in the United States. The health department’s Mitchell noted that their investigation of the outbreak found no problems with the aquaculture site

Changes were made to shellfish safety protocols after a larger outbreak in 2013 of Vibrio parahaemolyticus illnesses associated with eating raw oysters harvested along the Atlantic Coast. More than 100 people in 13 states, including Maryland and Virginia, became ill.  According to a spokesman for the Maryland Department of the Environment, which regulates shellfish harvest waters in the state, the Interstate Shellfish Sanitation Conference, a joint state-federal body, tightened its requirements for investigating such cases, closing implicated harvest areas and ordering a product recall when more than 10 cases are traced to a given area. 

But officials caution that the protocols are not foolproof, and cases like this are a reminder of the risk people run in consuming raw seafood, Mitchell said, especially if they have underlying health conditions.

The number of reported Vibrio infections in the state varies from year to year, but has been trending upward since 2005, according to state health data. Concentrations of the bacteria increase in warmer weather, and climate change could be a factor as Bay water temperatures tick upward. But Mitchell cautioned that the bacteria are present year-round.

“Given the number of people who eat oysters, certainly it’s a relatively small number of infections, but it can be a very significant one,” Mitchell said.