P1-17 Molecular Characterization of Cyclospora cayetanensis in Produce and Clinical Samples Using Whole Mitochondrial Genome Sequencing

Monday, July 10, 2017
Exhibit Hall (Tampa Convention Center)
Hediye Cinar , U.S. Food and Drug Administration–CFSAN, Office of Applied Research and Safety Assessment , Laurel , MD
Gopal Gopinath , U.S. Food and Drug Administration , Laurel , MD
SeonJu Choi , U.S. Food and Drug Administration , Laurel , MD
Jeongu Lee , U.S. Food and Drug Administration , Laurel , MD
Sonia Almeria , U.S. Food and Drug Administration–CFSAN, Office of Applied Research and Safety Assessment , Laurel , MD
Mauricio Durigan , U.S. Food and Drug Administration–CFSAN, Office of Applied Research and Safety Assessment , Laurel , MD
Helen Murphy , U.S. Food and Drug Administration–CFSAN, Office of Applied Research and Safety Assessment , Laurel , MD
Alexandre da Silva , U.S. Food and Drug Administration–CFSAN, Office of Applied Research and Safety Assessment , Laurel , MD
Introduction:  Cyclospora cayetanensis is a coccidian parasite causing large outbreaks in different countries. In the United States, outbreaks affected 1,481 people from 2013 to 2015. Outbreak investigations are limited by the absence of molecular epidemiological tools for tracebacks. Because of difficulties in the recovery of the oocysts from produce, the unculturable nature of the organism, and limitations in efficient DNA extraction due to resistant oocyst wall structure, it is extremely challenging to generate sufficient DNA templates for robust amplification of large DNA fragments, used in traditional sequencing analysis.

Purpose: In different apicomplexan parasites, multicopy organellar DNA, such as mitochondrion genomes, have been used for detection and molecular epidemiology analysis. We developed a workflow, to obtain complete mitochondrial genome sequences from contaminated foods and clinical samples, for differentiation of C. cayetanensis isolates.

Methods:   The 6,274 bp long C. cayetanensis mitochondrial genome, in four overlapping amplicons from genomic DNA, extracted from cilantro and spiked with oocysts and extracted from clinical stool samples, was amplified by PCR. DNA sequence libraries of the gel-purified amplicons were prepared using the Ovation Ultralow System library kit (NuGEN Technologies) and sequenced using MiSeq. Sequence reads were assembled using CLC Genomics WorkBench, and Geneious programs, to map to a reference C. cayetanensis mitochondrial genome.

Results:   This approach allowed us to sequence complete mitochondria genomes from produce samples seeded with C. cayetanensis oocysts, and from stool samples. SNP profiles of 30 stool samples, from a 2014 C. cayetanensis outbreak, exhibited discriminatory power based on geographical metadata. Isolates from different states grouped together in an evolutionary tree, suggesting that genomic analyses of mitochondria sequences may help to link outbreak cases to the source.

Significance:  The described method will facilitate the application of genomics tools to epidemiologically link C. cayetanensis identified in clinical and food samples during outbreak investigations.