Cyclospora cayetanensis is a coccidian parasite of humans, with a direct fecal–oral transmission cycle. It is globally distributed and an important cause of foodborne outbreaks of enteric disease in many developed countries, mostly associated with the consumption of contaminated fresh produce. Because oocysts are excreted unsporulated and need to sporulate in the environment, direct person-to-person transmission is unlikely. Infection by C. cayetanensis is remarkably seasonal worldwide, although it varies by geographical regions. Most susceptible populations are children, foreigners, and immunocompromised patients in endemic countries, while in industrialized countries, C. cayetanensis affects people of any age. The risk of infection in developed countries is associated with travel to endemic areas and the domestic consumption of contaminated food, mainly fresh produce imported from endemic regions. Water and soil contaminated with fecal matter may act as a vehicle of transmission for C. cayetanensis infection. The disease is self-limiting in most immunocompetent patients, but it may present as a severe, protracted or chronic diarrhea in some cases, and may colonize extra-intestinal organs in immunocompromised patients. Trimetoprim-sulfamethoxazole is the antibiotic of choice for the treatment of cyclosporiasis, but relapses may occur. Further research is needed to understand many unknown epidemiological aspects of this parasitic disease. Here, we summarize the biology, epidemiology, outbreaks, clinical symptoms, diagnosis, treatment, control and prevention of C. cayetanensis; additionally, we outline future research needs for this parasite.

Cholera toxin (CT) and toxin-co-regulated pili (TCP) are the major virulence factors of Vibrio cholerae O1 and O139 strains that contribute to the pathogenesis of disease during devastating cholera pandemics. However, CT and TCP negative V. cholerae strains are still able to cause severe diarrheal disease in humans through mechanisms that are not well understood. To determine the role of other virulence factors in V. cholerae pathogenesis, we used a CT and TCP independent infection model in the nematode Caenorhabditis elegans and identified the hemolysin A (hlyA) gene as a factor responsible for animal death and developmental delay. We demonstrated a correlation between the severity of infection in the nematode and the level of hemolytic activity in the V. cholerae biotypes. At the cellular level, V. cholerae infection induces formation of vacuoles in the intestinal cells in a hlyA dependent manner, consistent with the previous in vitro observations. Our data strongly suggest that HlyA is a virulence factor in C. elegans infection leading to lethality and developmental delay presumably through intestinal cytopathic changes.

Vibrio cholerae cytolysin (VCC) is among the accessory V. cholerae virulence factors that may contribute to disease pathogenesis in humans. VCC, encoded by hlyA gene, belongs to the most common class of bacterial toxins, known as pore-forming toxins (PFTs). V. cholerae infects and kills Caenorhabditis elegans via cholerae toxin independent manner. VCC is required for the lethality, growth retardation and intestinal cell vacuolation during the infection. However, little is known about the host gene expression responses against VCC. To address this question we performed a microarray study in C. elegans exposed to V. cholerae strains with intact and deleted hlyA genes. Many of the VCC regulated genes identified, including C-type lectins, Prion-like (glutamine [Q]/asparagine [N]-rich)-domain containing genes, genes regulated by insulin/IGF-1-mediated signaling (IIS) pathway, were previously reported as mediators of innate immune response against other bacteria in C. elegans. Protective function of the subset of the genes up-regulated by VCC was confirmed using RNAi. By means of a machine learning algorithm called FastMEDUSA, we identified several putative VCC induced immune regulatory transcriptional factors and transcription factor binding motifs. Our results suggest that VCC is a major virulence factor, which induces a wide variety of immune response- related genes during V. cholerae infection in C. elegans.

Cyclospora cayetanensis is a human-specific coccidian parasite responsible for several food and water-related outbreaks around the world, including the most recent ones involving over 900 persons in 2013 and 2014 outbreaks in the USA. Multicopy organellar DNA such as mitochondrion genomes have been particularly informative for detection and genetic traceback analysis in other parasites. We sequenced the C. cayetanensis genomic DNA obtained from stool samples from patients infected with Cyclospora in Nepal using the Illumina MiSeq platform. By bioinformatically filtering out the metagenomic reads of non-coccidian origin sequences and concentrating the reads by targeted alignment, we were able to obtain contigs containing Eimeria-like mitochondrial, apicoplastic and some chromosomal genomic fragments. A mitochondrial genomic sequence was assembled and confirmed by cloning and sequencing targeted PCR products amplified from Cyclospora DNA using primers based on our draft assembly sequence. The results show that the C. cayetanensis mitochondrion genome is 6274 bp in length, with 33% GC content, and likely exists in concatemeric arrays as in Eimeria mitochondrial genomes. Phylogenetic analysis of the C. cayetanensis mitochondrial genome places this organism in a tight cluster with Eimeria species. The mitochondrial genome of C. cayetanensis contains three protein coding genes, cytochrome (cytb), cytochrome C oxidase subunit 1 (cox1), and cytochrome C oxidase subunit 3 (cox3), in addition to 14 large subunit (LSU) and nine small subunit (SSU) fragmented rRNA genes.

Arsenic, a known human carcinogen, is widely distributed around the world and found in particularly high concentrations in certain regions including Southwestern US, Eastern Europe, India, China, Taiwan and Mexico. Chronic arsenic poisoning affects millions of people worldwide and is associated with increased risk of many diseases including arthrosclerosis, diabetes and cancer. In this study, we explored genome level global responses to high and low levels of arsenic exposure in Caenorhabditis elegans using Affymetrix expression microarrays. This experimental design allows us to do microarray analysis of dose-response relationships of global gene expression patterns. High dose (0.03%) exposure caused stronger global gene expression changes in comparison with low dose (0.003%) exposure, suggesting a positive dose-response correlation. Biological processes such as oxidative stress, and iron metabolism, which were previously reported to be involved in arsenic toxicity studies using cultured cells, experimental animals, and humans, were found to be affected in C. elegans. We performed genome-wide gene expression comparisons between our microarray data and publicly available C. elegans microarray datasets of cadmium, and sediment exposure samples of German rivers Rhine and Elbe. Bioinformatics analysis of arsenic-responsive regulatory networks were done using FastMEDUSA program. FastMEDUSA analysis identified cancer-related genes, particularly genes associated with leukemia, such as dnj-11, which encodes a protein orthologous to the mammalian ZRF1/MIDA1/MPP11/DNAJC2 family of ribosome-associated molecular chaperones. We analyzed the protective functions of several of the identified genes using RNAi. Our study indicates that C. elegans could be a substitute model to study the mechanism of metal toxicity using high-throughput expression data and bioinformatics tools such as FastMEDUSA.

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.