Abstract
BackgroundOutbreaks of cyclosporiasis, a diarrheal illness caused by Cyclospora cayetanensis, have been a public health issue in the USA since the mid 1990’s. In 2018, 2299 domestically acquired cases of cyclosporiasis were reported in the USA as a result of multiple large outbreaks linked to different fresh produce commodities. Outbreak investigations are hindered by the absence of standardized molecular epidemiological tools for C. cayetanensis. For other apicomplexan coccidian parasites, multicopy organellar DNA such as mitochondrial genomes have been used for detection and molecular typing.MethodsWe developed a workflow to obtain complete mitochondrial genome sequences from cilantro samples and clinical samples for typing of C. cayetanensis isolates. The 6.3 kb long C. cayetanensis mitochondrial genome was amplified by PCR in four overlapping amplicons from genomic DNA extracted from cilantro, seeded with oocysts, and from stool samples positive for C. cayetanensis by diagnostic methods. DNA sequence libraries of pooled amplicons were prepared and sequenced via next-generation sequencing (NGS). Sequence reads were assembled using a custom bioinformatics pipeline.ResultsThis approach allowed us to sequence complete mitochondrial genomes from the samples studied. Sequence alterations, such as single nucleotide polymorphism (SNP) profiles and insertion and deletions (InDels), in mitochondrial genomes of 24 stool samples from patients with cyclosporiasis diagnosed in 2014, exhibited discriminatory power. The cluster dendrogram that was created based on distance matrices of the complete mitochondrial genome sequences, indicated distinct strain-level diversity among the 2014 C. cayetanensis outbreak isolates analyzed in this study.ConclusionsOur results suggest that genomic analyses of mitochondrial genome sequences may help to link outbreak cases to the source.
Highlights
Outbreaks of cyclosporiasis, a diarrheal illness caused by Cyclospora cayetanensis, have been a public health issue in the Unites States of America (USA) since the mid 1990’s
PCR amplification of complete mitochondrial genomes from fresh produce and stool samples To identify and type the C. cayetanensis isolates based on the sequence diversity, we amplified the 6.3 kb long C. cayetanensis mitochondrial genome from genomic DNA extracted from cilantro spiked with oocysts and from stool samples
Stool samples were classified in levels of positivity for C. cayetanensis based on the oocyst load evaluated via microscopy and qPCR Cq values
Summary
A diarrheal illness caused by Cyclospora cayetanensis, have been a public health issue in the USA since the mid 1990’s. In 2018, 2299 domestically acquired cases of cyclosporiasis were reported in the USA as a result of multiple large outbreaks linked to different fresh produce commodities. Large cyclosporiasis outbreaks have been reported in the USA, since the mid-1990’s linked to various types of imported fresh produce (e.g. basil, cilantro, mesclun lettuce, raspberries and snow peas) [5, 6]. A major constraint in outbreak investigations is the lack of molecular epidemiology tools that would be useful in linking patients to the sources of infection. Progress on the development of molecular epidemiologic tools to link cases with sources of infection has been hindered by the limited DNA sequence information. Whole genome sequencing of bacterial species such as Listeria, Salmonella and E. coli is becoming a mainstream approach in public health and food safety research, targeted NGS approaches are needed for organisms with larger genomes, and/or for unculturable organisms
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