Development of a workflow for identification of nuclear genotyping markers for Cyclospora cayetanensis.

Katelyn A Houghton,Joel Barratt,Mark Borodovsky,Eldin Talundzic,Fernanda S Nascimento,Yvonne Qvarnstrom,Erik Vanroey,Subin Park,Alexandre Lomsadze,Michael J Arrowood

doi:10.1051/parasite/2020022

Abstract

Cyclospora cayetanensis is an intestinal parasite responsible for the diarrheal illness, cyclosporiasis. Molecular genotyping, using targeted amplicon sequencing, provides a complementary tool for outbreak investigations, especially when epidemiological data are insufficient for linking cases and identifying clusters. The goal of this study was to identify candidate genotyping markers using a novel workflow for detection of segregating single nucleotide polymorphisms (SNPs) in C. cayetanensis genomes. Four whole C. cayetanensis genomes were compared using this workflow and four candidate markers were selected for evaluation of their genotyping utility by PCR and Sanger sequencing. These four markers covered 13 SNPs and resolved parasites from 57 stool specimens, differentiating C. cayetanensis into 19 new unique genotypes.

Highlights

The coccidian parasite Cyclospora cayetanensis, identified as a cause of food-borne diarrheal illness in the early 1990s, is routinely linked to sporadic cases and annual, seasonal outbreaks of cyclosporiasis [27]
Three studies described the use of a multilocus sequence typing (MLST) method based on microsatellites [10, 13, 16], an approach successfully applied to other parasites [29]
The set of C. cayetanensis specific gene prediction parameters was determined by training of the GeneMark-EP+ gene prediction algorithm on the Centers for Disease Control and Prevention (CDC):HCNY16:01 isolate genome

Summary

Introduction

The coccidian parasite Cyclospora cayetanensis, identified as a cause of food-borne diarrheal illness in the early 1990s, is routinely linked to sporadic cases and annual, seasonal outbreaks of cyclosporiasis [27]. Epidemiologic investigations are the primary method for identifying clusters of cases in food-borne illness; they are the only tool available for cyclosporiasis. Three studies described the use of a multilocus sequence typing (MLST) method based on microsatellites [10, 13, 16], an approach successfully applied to other parasites [29]. A third approach targeting three genomic regions of high entropy, possessing several single nucleotide polymorphisms (SNPs) and an algorithm to predict sample relatedness, resolved four of eight epidemiologically linked outbreak clusters [2]

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