Sequence introgression from exogenous lineages underlies genomic and biological differences among Cryptosporidium parvum IOWA lines.

The IOWA strain of Cryptosporidium parvum is widely used in studies of the biology and detection of the waterborne pathogens Cryptosporidium spp. While several lines of the strain have been sequenced, IOWA-II, the only reference of the original subtype (IIaA15G2R1), exhibits significant assembly errors. Here we generated a fully assembled genome of IOWA-CDC of this subtype using PacBio and Illumina technologies. In comparative analyses of seven IOWA lines maintained in different laboratories (including two sequenced in this study) and 56 field isolates, IOWA lines (IIaA17G2R1) with less virulence had mixed genomes closely related to IOWA-CDC but with multiple sequence introgressions from IOWA-II and unknown lineages. In addition, the IOWA-IIaA17G2R1 lines showed unique nucleotide substitutions and loss of a gene associated with host infectivity, which were not observed in other isolates analyzed. These genomic differences among IOWA lines could be the genetic determinants of phenotypic traits in C. parvum. These data provide a new reference for comparative genomic analyses of Cryptosporidium spp. and rich targets for the development of advanced source tracking tools.

Cyclospora cayetanensis comprises at least 3 species that cause human cyclosporiasis.

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).

Investigation of US Cyclospora cayetanensis outbreaks in 2019 and evaluation of an improved Cyclospora genotyping system against 2019 cyclosporiasis outbreak clusters.

Cyclosporiasis is an illness characterised by watery diarrhoea caused by the food-borne parasite Cyclospora cayetanensis. The increase in annual US cyclosporiasis cases led public health agencies to develop genotyping tools that aid outbreak investigations. A team at the Centers for Disease Control and Prevention (CDC) developed a system based on deep amplicon sequencing and machine learning, for detecting genetically-related clusters of cyclosporiasis to aid epidemiologic investigations. An evaluation of this system during 2018 supported its robustness, indicating that it possessed sufficient utility to warrant further evaluation. However, the earliest version of CDC's system had some limitations from a bioinformatics standpoint. Namely, reliance on proprietary software, the inability to detect novel haplotypes and absence of a strategy to select an appropriate number of discrete genetic clusters would limit the system's future deployment potential. We recently introduced several improvements that address these limitations and the aim of this study was to reassess the system's performance to ensure that the changes introduced had no observable negative impacts. Comparison of epidemiologically-defined cyclosporiasis clusters from 2019 to analogous genetic clusters detected using CDC's improved system reaffirmed its excellent sensitivity (90%) and specificity (99%), and confirmed its high discriminatory power. This C. cayetanensis genotyping system is robust and with ongoing improvement will form the basis of a US-wide C. cayetanensis genotyping network for clinical specimens.

Evaluation of an ensemble-based distance statistic for clustering MLST datasets using epidemiologically defined clusters of cyclosporiasis.

Outbreaks of cyclosporiasis, a food-borne illness caused by the coccidian parasite Cyclospora cayetanensis have increased in the USA in recent years, with approximately 2300 laboratory-confirmed cases reported in 2018. Genotyping tools are needed to inform epidemiological investigations, yet genotyping Cyclospora has proven challenging due to its sexual reproductive cycle which produces complex infections characterized by high genetic heterogeneity. We used targeted amplicon deep sequencing and a recently described ensemble-based distance statistic that accommodates heterogeneous (mixed) genotypes and specimens with partial genotyping data, to genotype and cluster 648 C. cayetanensis samples submitted to CDC in 2018. The performance of the ensemble was assessed by comparing ensemble-identified genetic clusters to analogous clusters identified independently based on common food exposures. Using these epidemiologic clusters as a gold standard, the ensemble facilitated genetic clustering with 93.8% sensitivity and 99.7% specificity. Hence, we anticipate that this procedure will greatly complement epidemiologic investigations of cyclosporiasis.

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

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.

TITLE: Développement d'un flux de travail pour l'identification de marqueurs de génotypage nucléaire pour Cyclospora cayetanensis. ABSTRACT: Cyclospora cayetanensis est un parasite intestinal responsable de la cyclosporose, maladie diarrhéique. Le génotypage moléculaire, utilisant le séquençage ciblé des amplicons, fournit un outil complémentaire pour les enquêtes sur les épidémies, en particulier lorsque les données épidémiologiques sont insuffisantes pour relier les cas et identifier les grappes. Le but de cette étude était d'identifier des marqueurs candidats de génotypage à l'aide d'un nouveau flux de travail pour la détection des polymorphismes d'un seul nucléotide (SNP) différentiateurs dans les génomes de C. cayetanensis. Quatre génomes entiers de C. cayetanensis ont été comparés à l'aide de ce flux de travail et quatre marqueurs candidats ont été sélectionnés pour l'évaluation de leur utilité de génotypage par PCR et séquençage Sanger. Ces quatre marqueurs couvraient 13 SNP et ont résolu les parasites provenant de 57 spécimens de selles, différenciant C. cayetanensis en 19 nouveaux génotypes uniques.

Cryptosporidium Oocyst Purification Using Discontinuous Gradient Centrifugation.

Many laboratory studies in cryptosporidial research require a source of purified oocysts. Sources can include experimentally infected laboratory animals or from samples collected from naturally infected animals and from clinical cases of human cryptosporidiosis. Purification of oocysts can be accomplished with readily available laboratory equipment including tabletop centrifuges and microcentrifuges. Following purification, oocysts can be stored in antibiotic-supplemented buffers or in 2.5% aqueous potassium dichromate for over 6 months. Ultimately, oocyst viability and infectivity decline to less than 10% after 1 year, so if isolates are expected to be maintained, serial passage in a suitable host at ≤6-month intervals is recommended. Oocysts purified as described in this chapter are suitable for animal infection studies, cell culture studies, and a wide range of molecular biological studies, environmental studies, drug testing, and disinfection studies.

Cell Culture Infectivity to Assess Chlorine Disinfection of Cryptosporidium Oocysts in Water.

This chapter provides a detailed protocol to assess disinfection efficacy of chlorine against Cryptosporidium oocysts including the core chlorine disinfection assay, the in vitro cell culture infectivity assay, and microscopy analysis and data interpretation.

Simultaneous detection of four protozoan parasites on leafy greens using a novel multiplex PCR assay.

Pathogen contamination of fresh produce presents a health risk for consumers; however, the produce industry still lacks adequate tools for simultaneous detection of protozoan parasites. Here, a simple multiplex PCR (mPCR) assay was developed for detection of protozoan (oo)cysts and compared with previously published real-time PCR assays and microscopy methods. The assay was evaluated for simultaneous detection of Cryptosporidium, Giardia, Cyclospora cayetanensis, and Toxoplasma gondii followed by parasite differentiation via either a nested specific PCR or a restriction fragment length polymorphism (RFLP) assay. Spiking experiments using spinach as a model leafy green were performed for assay validation. Leaf-washing yielded higher recoveries and more consistent detection of parasites as compared with stomacher processing. Lowest limits of detection using the nested mPCR assay were 1-10 (oo)cysts/g spinach (in 10 g samples processed), and this method proved more sensitive than qPCR for parasite detection. Microscopy methods were more reliable for visual detection of parasites in lower spiking concentrations, but are more costly and laborious, require additional expertise, and lack molecular confirmation essential for accurate risk assessment. Overall, the nested mPCR assay provides a rapid (<24 h), inexpensive ($10 USD/sample), and simple approach for simultaneous detection of protozoan pathogens on fresh produce.

Genotyping genetically heterogeneous Cyclospora cayetanensis infections to complement epidemiological case linkage.

Sexually reproducing pathogens such as Cyclospora cayetanensis often produce genetically heterogeneous infections where the number of unique sequence types detected at any given locus varies depending on which locus is sequenced. The genotypes assigned to these infections quickly become complex when additional loci are analysed. This genetic heterogeneity confounds the utility of traditional sequence-typing and phylogenetic approaches for aiding epidemiological trace-back, and requires new methods to address this complexity. Here, we describe an ensemble of two similarity-based classification algorithms, including a Bayesian and heuristic component that infer the relatedness of C. cayetanensis infections. The ensemble requires a set of haplotypes as input and assigns arbitrary distances to specimen pairs reflecting their most likely relationships. The approach was applied to data generated from a test cohort of 88 human fecal specimens containing C. cayetanensis, including 30 from patients whose infections were associated with epidemiologically defined outbreak clusters of cyclosporiasis. The ensemble assigned specimens to plausible clusters of genetically related infections despite their complex haplotype composition. These relationships were corroborated by a significant number of epidemiological linkages (P < 0.0001) suggesting the ensemble's utility for aiding epidemiological trace-back investigations of cyclosporiasis.

Mitochondrial Junction Region as Genotyping Marker for Cyclospora cayetanensis.

Cyclosporiasis is an infection caused by Cyclospora cayetanensis, which is acquired by consumption of contaminated fresh food or water. In the United States, cases of cyclosporiasis are often associated with foodborne outbreaks linked to imported fresh produce or travel to disease-endemic countries. Epidemiologic investigation has been the primary method for linking outbreak cases. A molecular typing marker that can identify genetically related samples would be helpful in tracking outbreaks. We evaluated the mitochondrial junction region as a potential genotyping marker. We tested stool samples from 134 laboratory-confirmed cases in the United States by using PCR and Sanger sequencing. All but 2 samples were successfully typed and divided into 14 sequence types. Typing results were identical among samples within each epidemiologically defined case cluster for 7 of 10 clusters. These findings suggest that this marker can distinguish between distinct case clusters and might be helpful during cyclosporiasis outbreak investigations.

Evaluation of Multilocus Sequence Typing of Cyclospora cayetanensis based on microsatellite markers.

Cyclospora cayetanensis is a human parasite transmitted via ingestion of contaminated food or water. Cases of C. cayetanensis infection acquired in the United States often go unexplained, partly because of the difficulties associated with epidemiologic investigations of such cases and the lack of genotyping methods. A Multilocus Sequence Typing (MLST) method for C. cayetanensis based on five microsatellite loci amplified by nested PCR was described in 2016. The MLST loci had high variability, but many specimens could not be assigned a type because of poor DNA sequencing quality at one or more loci. We analyzed Cyclospora-positive stool specimens collected during 1997-2016 from 54 patients, including 51 from the United States. We noted limited inter-specimen variability for one locus (CYC15) and the frequent occurrence of unreadable DNA sequences for two loci (CYC3 and CYC13). Overall, using the remaining two loci (CYC21 and CYC22), we detected 17 different concatenated sequence types. For four of five clusters of epidemiologically linked cases for which we had specimens from >1 case-patient, the specimens associated with the same cluster had the same type. However, we also noted the same type for specimens that were geographically and temporally unrelated, indicating poor discriminatory power. Furthermore, many specimens had what appeared to be a mixture of sequence types at locus CYC22. We conclude that it may be difficult to substantially improve the performance of the MLST method because of the nucleotide repeat features of the markers, along with the frequent occurrence of mixed genotypes in Cyclospora infections.

Purification of Cyclospora cayetanensis oocysts obtained from human stool specimens for whole genome sequencing.

BACKGROUND: 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. RESULTS: 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%. CONCLUSIONS: 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.

Inflammasome components caspase-1 and adaptor protein apoptosis-associated speck-like proteins are important in resistance to Cryptosporidium parvum.

Cryptosporidium spp. are opportunistic protozoan parasites that infect epithelial cells in the intestinal tract and cause a flu-like diarrheal illness. Innate immunity is key to limiting the expansion of parasitic stages early in infection. One mechanism in which it does this is through the generation of early cytokines, such as IL-18. The processing and secretion of mature IL-18 (and IL-1ß) is mediated by caspase-1 which is activated within an inflammasome following the engagement of inflammasome-initiating sensors. We examined how the absence of caspase-1 and caspase-11, the adapter protein Asc, and other inflammasome components affects susceptibility to cryptosporidial infection by these and other key cytokines in the gut. We found that Casp-11-/-Casp-1-/- knockout mice have increased susceptibility to Cryptosporidium parvum infection as demonstrated by the 35-fold higher oocyst production (at peak infection) compared to wild-type mice. Susceptibility correlated with a lack of IL-18 in caspase-1 and caspase1/11 knockout mice, whereas IL-18 is significantly elevated in wildtype mice. IL-1ß was not generated in any significant amount following infection nor was any increased susceptibility observed in IL-1ß knockout mice. We also show that the adapter protein Asc is important to susceptibility, and that the caspase-1 canonical inflammasome signaling pathway is the dominant pathway in C. parvum resistance.

Removals of cryptosporidium parvum oocysts and cryptosporidium-sized polystyrene microspheres from swimming pool water by diatomaceous earth filtration and perlite-sand filtration.

Removal of Cryptosporidium-sized microspheres and Cryptosporidium parvum oocysts from swimming pools was investigated using diatomaceous earth (DE) precoat filtration and perlite-sand filtration. In pilot-scale experiments, microsphere removals of up to 2 log were obtained with 0.7 kg·DE/m2 at a filtration rate of 5 m/h. A slightly higher microsphere removal (2.3 log) was obtained for these DE-precoated filters when the filtration rate was 3.6 m/h. Additionally, pilot-scale perlite-sand filters achieved greater than 2 log removal when at least 0.37 kg/m2 of perlite was used compared to 0.1-0.4 log removal without perlite both at a surface loading rate of 37 m/h. Full-scale testing achieved 2.7 log of microspheres and oocysts removal when 0.7 kg·DE/m2 was used at 3.6 m/h. Removals were significantly decreased by a 15-minute interruption of the flow (without any mechanical agitation) to the DE filter in pilot-scale studies, which was not observed in full-scale filters. Microsphere removals were 2.7 log by perlite-sand filtration in a full-scale swimming pool filter operated at 34 m/h with 0.5 kg/m2 of perlite. The results demonstrate that either a DE precoat filter or a perlite-sand filter can improve the efficiency of removal of microspheres and oocysts from swimming pools over a standard sand filter under the conditions studied.

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

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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Evaluation of library preparation methods for Illumina next generation sequencing of small amounts of DNA from foodborne parasites.

Illumina library preparation methods for ultra-low input amounts were compared using genomic DNA from two foodborne parasites (Angiostrongylus cantonensis and Cyclospora cayetanensis) as examples. The Ovation Ultralow method resulted in libraries with the highest concentration and produced quality sequencing data, even when the input DNA was in the picogram range.

Multilocus Sequence Typing Tool for Cyclospora cayetanensis.

Because the lack of typing tools for Cyclospora cayetanensis has hampered outbreak investigations, we sequenced its genome and developed a genotyping tool. We observed 2 to 10 geographically segregated sequence types at each of 5 selected loci. This new tool could be useful for case linkage and infection/contamination source tracking.

Comparative genomics reveals Cyclospora cayetanensis possesses coccidia-like metabolism and invasion components but unique surface antigens.

BACKGROUND: Cyclospora cayetanensis is an apicomplexan that causes diarrhea in humans. The investigation of foodborne outbreaks of cyclosporiasis has been hampered by a lack of genetic data and poor understanding of pathogen biology. In this study we sequenced the genome of C. cayetanensis and inferred its metabolism and invasion components based on comparative genomic analysis. RESULTS: The genome organization, metabolic capabilities and potential invasion mechanism of C. cayetanensis are very similar to those of Eimeria tenella. Propanoyl-CoA degradation, GPI anchor biosynthesis, and N-glycosylation are some apparent metabolic differences between C. cayetanensis and E. tenella. Unlike Eimeria spp., there are no active LTR-retrotransposons identified in C. cayetanensis. The similar repertoire of host cell invasion-related proteins possessed by all coccidia suggests that C. cayetanensis has an invasion process similar to the one in T. gondii and E. tenella. However, the significant reduction in the number of identifiable rhoptry protein kinases, phosphatases and serine protease inhibitors indicates that monoxenous coccidia, especially C. cayetanensis, have limited capabilities or use a different system to regulate host cell nuclear activities. C. cayetanensis does not possess any cluster of genes encoding the TA4-type SAG surface antigens seen in E. tenella, and may use a different family of surface antigens in initial host cell interactions. CONCLUSIONS: Our findings indicate that C. cayetanensis possesses coccidia-like metabolism and invasion components but unique surface antigens. Amino acid metabolism and post-translation modifications of proteins are some major differences between C. cayetanensis and other apicomplexans. The whole genome sequence data of C. cayetanensis improve our understanding of the biology and evolution of this major foodborne pathogen and facilitate the development of intervention measures and advanced diagnostic tools.

Draft Genome Sequences from Cyclospora cayetanensis Oocysts Purified from a Human Stool Sample.

The parasite Cyclospora cayetanensis causes foodborne diarrheal illness. Here, we report draft genome sequences obtained from C. cayetanensis oocysts purified from a human stool sample. The genome assembly consists of 865 contigs with a total length of 44,563,857 bases. These sequences can facilitate the development of subtyping tools to aid outbreak investigations.

Oleylphosphocholine (OlPC) arrests Cryptosporidium parvum growth in vitro and prevents lethal infection in interferon gamma receptor knock-out mice.

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 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.