Cyclospora cayetanensis comprises at least 3 species that cause human cyclosporiasis

Abstract

The apicomplexan parasite Cyclospora cayetanensis causes seasonal foodborne outbreaks of the gastrointestinal illness cyclosporiasis. Prior to the coronavirus disease-2019 pandemic, annually reported cases were increasing in the USA, leading the US Centers for Disease Control and Prevention to develop a genotyping tool to complement cyclosporiasis outbreak investigations. Thousands of US isolates and 1 from China (strain CHN_HEN01) were genotyped by Illumina amplicon sequencing, revealing 2 lineages (A and B). The allelic composition of isolates was examined at each locus. Two nuclear loci (CDS3 and 360i2) distinguished lineages A and B. CDS3 had 2 major alleles: 1 almost exclusive to lineage A and the other to lineage B. Six 360i2 alleles were observed – 2 exclusive to lineage A (alleles A1 and A2), 2 to lineage B (B1 and B2) and 1 (B4) was exclusive to CHN_HEN01 which shared allele B3 with lineage B. Examination of heterozygous genotypes revealed that mixtures of A- and B-type 360i2 alleles occurred rarely, suggesting a lack of gene flow between lineages. Phylogenetic analysis of loci from whole-genome shotgun sequences, mitochondrial and apicoplast genomes, revealed that CHN_HEN01 represents a distinct lineage (C). Retrospective examination of epidemiologic data revealed associations between lineage and the geographical distribution of US infections plus strong temporal associations. Given the multiple lines of evidence for speciation within human-infecting Cyclospora, we provide an updated taxonomic description of C. cayetanensis, and describe 2 novel species as aetiological agents of human cyclosporiasis: Cyclospora ashfordi sp. nov. and Cyclospora henanensis sp. nov. (Apicomplexa: Eimeriidae).

The first Cyclospora cayetanensis lineage A genome from an isolate from Mexico

“…In this study, the genome of an isolate of C. cayetanensis obtained from a human patient in Mexico was sequenced via Illumina MiSeq and given the isolate name USDA_Mex32. To determine if the isolate USDA_Mex32 represents a clonal population, the methods proposed by Barratt et al, (2023) to assess the purity of a strain based on several genotyping loci was used [ 4 ]. Based on this analysis, if two or less haplotypes are observed for nuclear loci and only one haplotype is observed for mitochondrial loci, the assumption of a strain pure isolate can be made [ 4 ].…”

“…To determine if the isolate USDA_Mex32 represents a clonal population, the methods proposed by Barratt et al, (2023) to assess the purity of a strain based on several genotyping loci was used [ 4 ]. Based on this analysis, if two or less haplotypes are observed for nuclear loci and only one haplotype is observed for mitochondrial loci, the assumption of a strain pure isolate can be made [ 4 ]. Using these criteria, USDA_Mex32 represents a strain pure isolate with the patient sample likely representing a clonal population.…”

“…Gene marker phylogenetic analyses were performed by extracting and concatenating three loci from all available whole genome C. cayetanensis assemblies on NCBI in the following order: partial apicoplast genome (∼ 18 kb), putative cysteine proteinase (∼ 1.5 kb), partial polyamine-modulated factor 1-binding protein 1 (∼ 2.5 kb). These loci were selected because they have been previously shown to support the recently described lineage designations within C. cayetanensis [ 4 ]. The total concatenated length was ∼ 22 kb.…”

“…In the present study, the genome of a C. cayetanensis isolate, obtained from a child with diarrhea living in Mexico, was sequenced using Illumina MiSeq. The assembly from this isolate was assessed for lineage markers as recently described by the Centers for Disease Control and Prevention (CDC) to determine its lineage with a method that uses similarity scores between representative loci for lineage assignments [ 4 ]. Comparisons between this isolate and other available C. cayetanensis whole genome assemblies from National Center for Biotechnology Information (NCBI) were also made.…”

Optimizing hierarchical tree dissection parameters using historic epidemiologic data as ‘ground truth’

“…This is because historical genetic data can inform molecular epidemiologists of how genetically similar isolates of the same strain typically are; however, for many human parasites, including the foodborne parasite Cyclospora spp. [5], historic knowledge of circulating strains may be limited or absent, and the concept of what constitutes a strain may be complicated by sexual reproduction [6].…”