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. To 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. We 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. SNPs 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.

Cyclospora cayetanensis is a food-borne intestinal human parasite that causes outbreaks of diarrhea. There is a need for efficient laboratory methods for strain-level characterization to assist in outbreak investigations. By using next generation sequencing, genomic sequences can be obtained and compared to identify potential genotyping markers. However, there is no method available to propagate this parasite in the laboratory. Therefore, genomic DNA must be extracted from oocysts purified from human stool. The objective of this study was to apply optimized methods to purify C. cayetanensis oocysts and extract DNA in order to obtain high-quality whole genome sequences with minimum contamination of DNA from other organisms. Oocysts from 21 human stool specimens were separated from other stool components using discontinuous density gradient centrifugation and purified further by flow cytometry. Genomic DNA was used to construct Ovation Ultralow libraries for Illumina sequencing. MiSeq sequencing reads were taxonomically profiled for contamination, de novo assembled, and mapped to a draft genome available in GenBank to assess the quality of the resulting genomic sequences. Following all purification steps, the majority (81–99%) of sequencing reads were from C. cayetanensis. They could be assembled into draft genomes of around 45 MB in length with GC-content of 52%. Density gradients performed in the presence of a detergent followed by flow cytometry sorting of oocysts yielded sufficient genomic DNA largely free from contamination and suitable for whole genome sequencing of C. cayetanensis. The methods described here will facilitate the accumulation of genomic sequences from various samples, which is a prerequisite for the development of typing tools to aid in outbreak investigations.

Cryptosporidium parvum is a species of protozoa that causes cryptosporidiosis, an intestinal disease affecting many mammals including humans. Typically, in healthy individuals, cryptosporidiosis is a self-limiting disease. However, C. parvum can cause a severe and persistent infection that can be life-threatening for immunocompromised individuals, such as AIDS patients. As there are no available treatments for these patients that can cure the disease, there is an urgent need to identify treatment options. We tested the anti-parasitic activity of the alkylphosphocholine oleylphosphocholine (OlPC), an analog of miltefosine, against C. parvum in in vitro and in vivo studies. In vitro experiments using C. parvum infected human ileocecal adenocarcinoma cells (HCT-8 cells) showed that OlPC has an EC50 of 18.84 nM. Moreover, no cell toxicity has been seen at concentrations ≤50 µM. C57BL/6 interferon gamma receptor knock-out mice, were infected by gavage with 4000 C. parvum oocysts on Day 0. Oral treatments, with OlPC, miltefosine, paromomycin or PBS, began on Day 3 post-infection for 10 days. Treatment with OlPC, at 40 mg/kg/day resulted in 100% survival, complete clearance of parasite in stools and a 99.9% parasite burden reduction in the intestines at Day 30. Doses of 30 mg/kg/day and 20 mg/kg/day also demonstrated an increased survival rate and a dose-dependent parasite burden reduction. Mice treated with 10 mg/kg/day of miltefosine resulted in 50% survival at Day 30. In contrast, control mice, treated with PBS or 100 mg/kg/day of paromomycin, died or had to be euthanized between Days 6 and 13 due to severe illness. Results of parasite burden were obtained by qPCR and cross-validated by both flow cytometry of stool oocysts and histological sections of the ileum. Together, our results strongly support that OlPC represents a potential candidate for the treatment of C. parvum infections in immunocompromised patients.