Nearly two months have passed since state and federal health authorities first became aware on June 28 of two related cases of Cyclospora infection in Iowa residents. Considering Iowa typically sees one or two such cases a year, the small irregularity immediately caught the attention of state health officials. Within weeks, authorities had identified several hundred cases across a dozen states, with the bulk in Iowa, Nebraska and Texas. Patients’ illness onset dates spread from June 1 to the middle of July. On Aug. 1, investigators in Iowa and Nebraska concluded that the majority of cases in those two states were connected to lettuce grown by Taylor Farms de Mexico and in salads served at Olive Gardens and Red Lobsters, chains both owned by Darden Restaurants of Orlando, FL. As of Aug. 22, the foodborne parasite has infected at least 625 people in 22 states, making it the largest Cyclospora outbreak in the U.S. since 1997. Of those cases, 517 (83 percent) are concentrated in Iowa, Nebraska and Texas. But, as time goes on, the investigations in Texas and the remaining states have still not identified an outbreak source. The U.S. Food and Drug Administration has a team of investigators at Taylor Farms in Mexico performing environmental assessments of the facilities and traceback investigations to determine where else – if anywhere – contaminated lettuce might have been sold, according to FDA spokesman David Steigman. A number of cases in Texas are not easily connected to Darden Restaurants and Taylor Farms, said Dave Theno, Ph.D., CEO of Gray Dog Partners, a technical food safety consulting agency. But he said that the evidence remains “pretty compelling” that most of the illnesses are part of one event until an FDA investigation proves otherwise. “A lot of things are possible, but what appears to be most likely is likely to be the answer,” Theno said. “If you look at the map, you could make a case that this thing went right through the heart of the country.” Because fresh produce has a relatively short shelf-life, distribution channels typically run up north from Mexico, not east to west. Based on the geography of the outbreak, it would make sense to assume that contaminated produce was sent north through Texas and ended up in Iowa and Nebraska. Theno said the most important question for investigators in Mexico to answer is whether Taylor Farms has crop contamination problems that moved Cyclospora through their facilities or whether it’s an area-wide contamination issue – possibly from a water source – that might mean nearby crops were also affected. Investigation complications A number of factors complicate the investigation as a whole. First, a representative from Darden Restaurants told NBC News that they do not use Taylor Farms salad in their Texas restaurants. It is not clear if the restaurant sources Taylor Farms salad in any states besides Iowa and Nebraska. A Florida woman who tested positive for Cyclospora in July told Food Safety News that she ate salad at Olive Garden several days before falling ill. This may suggest that Darden uses Taylor Farms salad in Florida, which has 31 confirmed illnesses, the fourth-highest number of the states involved. Representatives for Darden, Olive Garden and Red Lobster have not responded to numerous requests for comment from Food Safety News. Further complicating the matter, not even all of the cases in Iowa and Nebraska have a clear connection to Darden. Roughly 80 percent of Iowa cases and 75 percent of Nebraska cases appear connected to Darden, leaving the remaining 20 to 25 percent more of a mystery, according to Iowa state epidemiologist Dr. Patricia Quinlisk. To complicate the investigation even more, the salad came into the U.S. pre-mixed with Taylor lettuce and other ingredients. Perhaps the biggest issue distancing the Texas illnesses is that they began, on average, a week later than those in Iowa and Nebraska. In Texas, where illnesses appear to have less of a connection to Darden and Taylor, patients are being interviewed and re-interviewed about all the places they may have dined. Texas state health department spokeswoman Christine Mann told Food Safety News that illnesses in Texas generally fall into smaller clusters than those in Iowa and Nebraska, making the investigation in Texas more difficult. The biggest roadblock, however, has been the relatively long incubation period of Cyclospora parasites. Patients may not experience symptoms such as diarrhea and nausea for several days – or even weeks – after exposure. “It’s difficult to explain to people why it’s taking so long,” Mann said. “By the time the cases are finally reported to the CDC, several weeks have gone by. When an epidemiologist finally interviews patients, they might not remember eating the food that got them sick.” Some cases, Quinlisk said, may be isolated Cyclospora illnesses that have gotten swept up in the outbreak investigation. A few hundred cases get reported in the U.S. each year on average. Lessons for the future Quinlisk described the investigation into the Iowa and Nebraska illnesses as “very detailed.” The classic food history interview involves an 18-page questionnaire that takes more than an hour for each patient to complete. Patients may then receive additional calls from the environmental health team looking for more information. The state health departments in those states performed targeted cluster investigations using case-control studies. In short, they interviewed patients who got sick, as well as fellow diners who didn’t get sick, to tease out any statistical differences in their meals. They even retrieved credit-card records from patients to coordinate dates of exposure. Another issue that makes these investigations tricky is that not everyone who eats the contaminated food ends up sick. The parasite is not evenly dispersed across all the salad, and so some diners may ingest a large number of organisms, while others may eat from the same salad bowl and ingest only a few or none at all. The general rule of thumb, Quinlisk said, is to expect about 40 percent of those who ate a contaminated food to actually end up with symptoms. One issue this outbreak raises is the need for more coordination between state health departments during large, interstate outbreaks, said Craig Hedberg, Ph.D., environmental health professor at the University of Minnesota. Hedberg compared the national jurisdiction of food regulation from FDA to the patchwork of state health departments that make up the majority of environmental health investigation resources. “Our public-health system is really based on individual state authorities, and we don’t have a national framework for conducting outbreak investigations in parallel with the kind of regulation we have with respect to the food itself,” Hedberg said. “The CDC attempts to help guide those investigations, but because of limited resources and different priorities across different states, that coordination doesn’t always occur as seamlessly as we’d like to see it.” Theno said he felt this current outbreak demonstrated the need for better cooperation and communication between federal investigators at FDA and the companies potentially involved in the outbreak. “I think we need to find a way for companies and regulatory agencies to work much more closely together on these outbreaks,” Theno said. “It takes a long time for the agency to reach back into the supply chain. The faster we can get to the source of an outbreak, the sooner we can stop it and the better we can prevent it in the future.” (Cyclospora is a single-celled food- or waterborne parasite that may cause diarrhea, nausea and vomiting. In the U.S., it is often associated with imported fresh produce. In 1996, at least 1,465 people were infected with Cyclospora in an outbreak linked to raspberries grown in Guatemala. Another 804 people were sickened by Guatemalan raspberries the next year. In 2005, 592 contracted Cyclospora infections after eating basil imported from Peru.)