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 table-top 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 declines 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.

Recent cyclosporiasis outbreaks associated with fresh produce grown in the United States highlight the need to better understand Cyclospora cayetanensis prevalence in U.S. agricultural environments. In this study, C. cayetanensis occurrence was assessed in municipal wastewater sludge, on-farm portable toilets, irrigation pond water, and spent packing house dump tank water in a Southeastern Georgia growing region over two years. Detection of the C. cayetanensis 18S rRNA qPCR gene target in pond samples was 0%, 28%, and 42% (N = 217) depending on the detection definition used, and ≤1% in dump tank samples (N = 46). However, no qPCR detections were confirmed by sequencing, suggesting false detection occurred due to cross-reactions. C. cayetanensis qPCR detections were confirmed in 9% of wastewater sludge samples (N = 76). The human-specific fecal markers HF183 and crAssphage were detected in 33% and 6% of pond samples, respectively, and 4% and 0% of dump tank samples, respectively. Despite community Cyclospora shedding and evidence of human fecal contamination in irrigation water, there was no correlation between C. cayetanensis and HF183 qPCR detections, further supporting that 18S gene target qPCR amplifications were due to cross-reactions. When evaluating C. cayetanensis qPCR environmental detection data, the impact of assay specificity and detection criteria should be considered. Moreover, additional sequence-based testing may be needed to appropriately interpret Cyclospora qPCR environmental data.

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.

Abstract The parasites traditionally referred to as coccidia that develop in the intestines of humans— Cystoisospora belli , Cyclospora cayetanensis , and Sarcocystis spp.—are in the Stramenopiles, Alveolates, Rhizaria supergroup in the current taxonomic scheme used by protozoologists. Oocysts of these coccidia are found in the feces of humans, and diagnosis is based ultimately on demonstrating oocysts in human stool samples. The use of molecular methods to evaluate the phylogenetics and genetic diversity of parasites has resulted in a revision of the coccidian parasites. In vitro culture of intestinal coccidian parasites is most often used as a tool to study developmental biology or to identify active chemotherapeutic agents. Differentiation of the intestinal coccidia to the subspecies/strain level—genotyping—can be important for epidemiologic reasons, for example to link cases in outbreak investigations or to better understand clinical outcome differences.

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).

Abstract Cyclosporiasis is a diarrheal illness caused by the foodborne parasite Cyclospora cayetanensis. Annually reported cases have been increasing in the United States prompting development of genotyping tools to aid cluster detection. A recently developed Cyclospora genotyping system based on 8 genetic markers was applied to clinical samples collected during the cyclosporiasis peak period of 2020, facilitating assessment of its epidemiologic utility. While the system performed well and helped inform epidemiologic investigations, inclusion of additional markers to improve cluster detection was supported. Consequently, investigations have commenced to identify additional markers to enhance performance.

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.

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.

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.

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.

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.

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.

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.

The functional mechanism of anti-merozoite antibodies.Antibodies to merozoite surface proteins can mediate several eff ector mechanisms, including complement fi xation due to cytophillic antibodies that result in merozoite lysis of C3b opsonization; inhibition of merozoite invasion into the RBC; phagocytosis of IgG-opsonized merozoites; production of reactive oxygen species (ROS) or Nitric oxide (NO) in response to opsonized parasites

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.

epithelium, we believed this organism to be a Cyclospora sp. rather than a Cystoisospora sp. We searched the serum bank of the Division of Parasitic Diseases, Centers for Disease Control and Prevention, and identified a serum sample from a person with a case of C. cayetanensis cyclosporiasis. An indirect immunofluorescence test was performed by using this serum on a deparaffinized section of the tissue biopsy specimen. Different stages of the coccidian organism were labeled brightly and produced apple-green fluorescence against a red counterstain (Eriochrome Black T), indicating that the parasite could possibly be a Cyclospora sp. (Figure, panels E, F). We considered that the Cyclospora-positive serum sample obtained from this particular patient may not be species-specific, since he might have also been infected with Cystoisospora. Therefore, we performed a realtime PCR assay that can distinguish C. cayetanensis from other coccidian parasites to identify the parasite definitively (3). DNA recovered from tissue in paraffin sections was successfully amplified and detected with this assay (data not shown), confirming the presence of C. cayetanensis. The patient’s illness was treated with albendazole for E. bieneusi infection and with trimethoprim and sulfamethoxazole for C. cayetanensis infection. The patient’s diarrhea subsided after 1 week, and several subsequent fecal samples were negative for microsporidia spores and Cyclospora oocysts. His immunosuppressive medications were reduced, and he remained diarrhea-free for the following 3-year period of April 2010 to April 2013.

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.

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-1 beta) 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(-/-) 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-1 beta was not generated in any significant amount following infection nor was any increased susceptibility observed in IL-1 beta 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. (C) 2018 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.