Historically, Cyclospora infection in the United States has been associated with imported fresh produce. However, in 2018, the U.S. saw two significant outbreaks associated with vegetables grown in the United States.
“We had more than 2,000 non-travel associates cyclosporiasis cases,” said Ynes Ortega, member of the Center for Tropical and Emerging Global Diseases and associate professor in the Department of Food Science and Technology’s Center for Food Safety.
Fresh produce vegetable trays containing broccoli, cauliflower, carrots, and dill dip, which are not often associated with Cyclospora outbreaks, were implicated in the first outbreak affecting 250 people and lettuce from a salad mix used by a fast food chain was the source of the second outbreak with 511 cases.
“Cyclospora has previously been detected in salad greens produced in the US and lettuce implicated in the second outbreak was produced in the U.S.,” said Ortega. “Clearly, we need to determine if Cyclospora is not only a parasite present in other countries but also in the U.S.”
Cyclospora cayetanensis, the single-cell parasite that causes cyclosporiasis, was first described by Ortega in the 1990s. A person becomes infected with the parasite by consuming contaminated food, mostly fresh fruits and vegetables, and water. Infection results in gastrointestinal illness characterized primarily by diarrhea. Cyclosporiasis is treated with sulfa drugs, fluids, and rest. If left untreated, the symptoms can persist for up to a month and can be recurring. Prevention of infection is accomplished by frequent hand washing by those who process fruits and vegetables, thoroughly rinsing fruits and vegetables with water prior to consumption, and avoiding potentially contaminated water while traveling in countries where C. cayetanensis is endemic.
Ortega has been awarded a 2-year grant from the Center for Produce Safety, a non-profit organization committed to addressing issues faced by the produce industry, to investigate C. cayetanensis presence in the United States.
“We will be testing surface water for the presence of Cyclospora cayetanensis, improving sample collection methods, and genotyping of the parasite,” said Ortega.
Up until 2018, it was believed that Cyclospora was not present in the United States. Therefore, one of the main goals of this grant is to determine how widely distributed this parasite is within the United States. To aid in this determination, a simpler and more sensitive method of detection is needed which Ortega’s laboratory is already working on.
“These two objectives are critical to implement monitoring and intervention strategies not only in the U.S. but also in endemic locations, with the ultimate goal of reducing the number of domestic cases of cyclosporiasis,” said Ortega.
BACKGROUND: Cyclospora cayetanensis is an important enteric pathogen, causing diarrhea and food-borne cyclosporiasis outbreaks. For effective outbreak identification and investigation, it is essential to rapidly assess the genetic heterogeneity of C. cayetanensis specimens from cluster cases and identify the likely occurrence of outbreaks.
METHODS: In this study, we developed a quantitative PCR (qPCR) targeting the polymorphic link region between copies of the mitochondrial genome of C. cayetanensis, and evaluated the genetic heterogeneity among 36 specimens from six countries using melt curve, gel electrophoresis, and sequence analyses of the qPCR products.
RESULTS: All specimens were amplified successfully in the qPCR and produced melt peaks with different Tm values in the melt curve analysis. In gel electrophoresis of the qPCR products, the specimens yielded bands of variable sizes. Nine genotypes were identified by DNA sequencing of the qPCR products. Geographical segregation of genotypes was observed among specimens analyzed, which could be useful in geographical source-tracking.
CONCLUSIONS: The length and nucleotide sequence variations in the mitochondrial genome marker allow rapid assessment of the genetic heterogeneity among C. cayetanensis specimens by melt curve, gel electrophoresis, or DNA sequence analysis of qPCR products. The sequence data generated could be helpful in the initial source-tracking of the pathogen.
KEYWORDS: Cyclospora cayetanensis; Genotyping; Mitochondrion; Source-tracking; qPCR