Comparative sequence analysis of Cyclospora cayetanensis apicoplast genomes originating from diverse geographical regions

Abstract

Background Cyclospora cayetanensis is an emerging coccidian parasite that causes endemic and epidemic diarrheal disease called cyclosporiasis, and this infection is associated with consumption of contaminated produce or water in developed and developing regions. Food-borne outbreaks of cyclosporiasis have occurred almost every year in the USA since the 1990s. Investigations of these outbreaks are currently hampered due to lack of molecular epidemiological tools for trace back analysis. The apicoplast of C. cayetanensis, a relict non-photosynthetic plastid with an independent genome, provides an attractive target to discover sequence polymorphisms useful as genetic markers for detection and trace back analysis of the parasite. Distinct differences in the apicoplast genomes of C. cayetanensis could be useful in designing advanced molecular methods for rapid detection and, subtyping and geographical source attribution, which would aid outbreak investigations and surveillance studies.MethodsTo obtain the genome sequence of the C. cayetanensis apicoplast, we sequenced the C. cayetanensis genomic DNA extracted from clinical stool samples, assembled and annotated a 34,146 bp-long circular sequence, and used this sequence as a reference genome in this study. We compared the genome and the predicted proteome to the data available from other apicomplexan parasites. To initialize the search for genetic markers, we mapped the raw sequence reads from an additional 11 distinct clinical stool samples originating from Nepal, New York, Texas, and Indonesia to the apicoplast reference genome.ResultsWe identified several high quality single nucleotide polymorphisms (SNPs) and small insertion/deletions spanning the apicoplast genome supported by extensive sequencing reads data, and a 30 bp sequence repeat at the terminal spacer region in a Nepalese sample. The predicted proteome consists of 29 core apicomplexan peptides found in most of the apicomplexans. Cluster analysis of these C. cayetanensis apicoplast genomes revealed a familiar pattern of tight grouping with Eimeria and Toxoplasma, separated from distant species such as Plasmodium and Babesia.ConclusionsSNPs and sequence repeats identified in this study may be useful as genetic markers for identification and differentiation of C. cayetanensis isolates found and could facilitate outbreak investigations.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1896-4) contains supplementary material, which is available to authorized users.

Molecular typing of Cyclospora cayetanensis in produce and clinical samples using targeted enrichment of complete mitochondrial genomes and next-generation sequencing

“…Organelle genomes, such as genomes of the mitochondria and apicoplasts, are particularly informative in tracing population dynamics due to their non-recombining nature [20]. We reported the complete apicoplast genome of C. cayetanensis, and performed an intraspecies comparative sequence analysis of genomes from samples collected in Nepal, New York, Texas and Indonesia, and concluded that SNPs and sequence repeats identified in this study may be useful as genetic markers for geo-genomic identification and classification of C. cayetanensis [17]. Mitochondrial DNA has been widely used in phylogenetics [36][37][38], evolutionary biology [38], forensic sciences [39,40] and anthropology [41] due to its unique attributes, such as maternal inheritance without recombination events [42,43] and relatively higher mutation rate compared to nuclear DNA [44,45].…”

“…Despite the significant clinical and public health importance of C. cayetanensis, genetic information was sparse (limited to ribosomal complex and heat-shock protein sequences) due to technical difficulties and limited accessibility of C. cayetanensis samples. Since 2014, FDA and CDC in collaboration with academia developed methods and strategies to sequence C. cayetanensis genomes, including complete mitochondrial and apicoplast genomes, and high quality whole genome assemblies using NGS technologies [11][12][13][14][15][16][17][18]. With the advances in NGS technologies, whole genome sequencing (WGS)-based typing has become a preferred approach in clinical and public health microbiology in the recent years [28].…”

“…We obtained C. cayetanensis oocysts from a known clinical sample C5 by a purification method described previously [11,17]. Briefly, C. cayetanensis oocysts were recovered from sieved fecal samples by differential sucrose and cesium chloride gradient centrifugations.…”

“…Progress on the development of molecular epidemiologic tools to link cases with sources of infection has been hindered by the limited DNA sequence information. With the recent advances in sequencing technologies such as nextgeneration sequencing (NGS) and availability of efficient genome assembly programs, whole genome assemblies, complete mitochondrial and apicoplast genomes of C. cayetanensis have become available [11][12][13][14][15][16][17][18]. NGS is the preferred technology for generation of high-throughput high-resolution data in streamlined fashion for clinical diagnostics, forensic science, and public health research.…”

A Review of Cyclospora cayetanensis Transport in the Environment

“…Phylogenetically, Cyclospora is closest to Eimeria, given the 94% to 96% similarity in the 18S rRNA sequence (Relman et al, 1996). Cinar et al (2016) sequenced a variety of C. cayetanensis samples from around the world and found that they largely overlapped. However, additional sequencing of various geographically and temporally distinct organisms is needed in order to confidently define the genus.…”