The epidemiology of intestinal protozoa in the Israeli population based on molecular stool test: a nationwide study.

Stool examination using microscopy was the traditional method for the diagnosis of intestinal parasites. Recently, the use of molecular tests to identify stool protozoa has become the main tool used in most clinical laboratories in Israel. This study aimed to evaluate the prevalence of intestinal parasites in Israel and to compare this prevalence in laboratories that use molecular tests vs a laboratory that uses microscopy. Samples collected from January to October 2021 at seven laboratories were analyzed by real-time PCR (RT-PCR) or by microscopy. The multiplex panel included the following pathogens: Giardia lamblia, Entamoeba histolytica, Cryptosporidium spp., Cyclospora, Dientamoeba fragilis, and Blastocystis spp. Overall, 138,415 stool samples were tested by RT-PCR and 6,444 by microscopy. At least one protozoa species was identified in 28.4% of the PCR-tested samples compared to 4.6% of the microscopy-tested samples. D. fragilis was the most common PCR-identified species (29%). D. fragilis, G. lamblia, and Cryptosporidium spp. were mainly found in pediatric population, while Blastocystis spp. was most prevalent among adults (P < 0.001). In a sub-cohort of 21,480 samples, co-infection was found in 4,113 (19.15%) samples, with Blastocystis spp. and D. fragilis being the most common (14.9%) pair. Molecular stool testing proved more sensitive compared to microscopy. D. fragilis was the most commonly detected pathogen. The above profile was identified during the COVID pandemic when traveling was highly restricted and most likely represents the locally circulating protozoa. IMPORTANCE: This study sheds light on the prevalence of stool parasites in Israel. Additionally, this study indicates that the shift from microscope analysis to molecular tests improved protozoa diagnosis.

Evaluation of coccidia DNA in irrigation pond water and wastewater sludge associated with Cyclospora cayetanensis 18S rRNA gene qPCR detections.

The coccidian parasite Cyclospora cayetanensis is the causative agent for foodborne outbreaks of cyclosporiasis disease and multiple annual fresh produce recalls. The aim of this study was to identify potential cross-reacting species for the C. cayetanensis 18S rRNA and MIT1C gene target real-time quantitative polymerase chain reaction (qPCR) assays. The environmental samples evaluated were irrigation pond water, produce wash water, and wastewater treatment sludge from a previous study with qPCR detections of C. cayetanensis by the 18S rRNA gene target qPCR. From these samples, longer regions of the 18S rRNA gene and the mitochondrial cytochrome c oxidase subunit III gene (cox3) were sequenced. Of 65 irrigation pond water samples with positive test results using the C. cayetanensis 18S rRNA gene qPCR assay, none had MIT1C qPCR assay detections or sequences that clustered with C. cayetanensis based on sequencing of the cox3 and 18S rRNA gene. Sequences from these samples clustered around coccidia sequences found in bird, fish, reptile, and amphibian hosts. Of 26 sludge samples showing detections by either qPCR assay, 14 (54%) could be confirmed as containing C. cayetanensis by sequencing of cox3 and 18S rRNA gene regions. In three of the remaining sludge samples, sequenced reads clustered with coccidia from rodents. This study demonstrated that caution should be taken when interpreting qPCR C. cayetanensis detection data in environmental samples and sequencing steps will likely be needed for confirmation. IMPORTANCE: Fresh produce is a leading transmission source in cyclosporiasis outbreaks. It is therefore essential to understand the role that produce-growing environments play in the spread of this disease. To accomplish this, sensitive and specific tests for environmental and irrigation waters must be developed. Potential cross-reactions of Cyclospora cayetanensis real-time quantitative polymerase chain reaction (qPCR) assays have been identified, hindering the ability to accurately identify this parasite in the environment. Amplicon sequencing of the cox3 and 18S rRNA genes revealed that all irrigation pond water and two sludge samples that initially detected C. cayetanensis by qPCR were most likely cross-reactions with related coccidian organisms shed from birds, fish, reptiles, amphibians, and rodents. These results support that a single testing method for environmental samples is likely not adequate for sensitive and specific detection of C. cayetanensis.

Detection of Cyclospora cayetanensis in Food and Water Samples: Optimized Protocols for Specific and Sensitive Molecular Methods from a Regulatory Agency Perspective.

Cyclospora cayetanensis is a coccidian parasite of the phylum Apicomplexa that causes cyclosporiasis, a human-specific gastrointestinal disease. Unlike most enteric pathogens, C. cayetanensis does not infect via direct fecal-oral transmission between humans because shed oocysts must be exposed to environmental triggers prior to becoming infectious. The development of specific and sensitive detection methods for C. cayetanensis is crucial to effectively address data gaps and provide regulatory support during outbreak investigations. In this study, new more specific molecular markers for the detection of C. cayetanensis were developed based on updated genomic databases of Apicomplexa mitochondrial sequences. Novel alternative reagents and supplies, as well as optimization protocols, were tested in spiked produce and agricultural water samples. The selected Mit1C primers and probe combined showed at least 13 mismatches to other related species. The new optimized qualitative real-time PCR assay with modifications to sample processing and replacement of discontinued items produced results comparable to the previously validated methods. In conclusion, the new optimized qualitative Mit1C real-time PCR assay demonstrated an increase in its specificity in comparison to other detection methods previously published, while it showed to be robust and as sensitive as the previously validated method at the FDA. This study has also expanded the array of PCR reagents that can be used to detect C. cayetanensis in produce and agricultural water samples and provided several improvements to the method for detection in agricultural water including replacements for discontinued items and a new dialysis filter for water filtration.

Enteric parasites Cyclospora cayetanensis and Cryptosporidium hominis in domestic and wildlife animals in Ghana.

BACKGROUND: Enteric parasitic infections remain a major public health problem globally. Cryptosporidium spp., Cyclospora spp. and Giardia spp. are parasites that cause diarrhea in the general populations of both developed and developing countries. Information from molecular genetic studies on the speciation of these parasites and on the role of animals as vectors in disease transmission is lacking in Ghana. This study therefore investigated these diarrhea-causing parasites in humans, domestic rats and wildlife animals in Ghana using molecular tools. METHODS: Fecal samples were collected from asymptomatic school children aged 9-12 years living around the Shai Hills Resource Reserve (tourist site), from wildlife (zebras, kobs, baboons, ostriches, bush rats and bush bucks) at the same site, from warthogs at the Mole National Park (tourist site) and from rats at the Madina Market (a popular vegetable market in Accra, Ghana. The 18S rRNA gene (18S rRNA) and 60-kDa glycoprotein gene (gp60) for Cryptosporidium spp., the glutamate dehydrogenase gene (gdh) for Giardia spp. and the 18S rDNA for Cyclospora spp. were analyzed in all samples by PCR and Sanger sequencing as markers of speciation and genetic diversity. RESULTS: The parasite species identified in the fecal samples collected from humans and animals included the Cryptosporidium species C. hominis, C. muris, C. parvum, C. tyzzeri, C. meleagridis and C. andersoni; the Cyclopora species C. cayetanensis; and the Gardia species, G. lamblia and G. muris. For Cryptosporidium, the presence of the gp60 gene confirmed the finding of C. parvum (41%, 35/85 samples) and C. hominis (29%, 27/85 samples) in animal samples. Cyclospora cayetanensis was found in animal samples for the first time in Ghana. Only one human sample (5%, 1/20) but the majority of animal samples (58%, 51/88) had all three parasite species in the samples tested. CONCLUSIONS: Based on these results of fecal sample testing for parasites, we conclude that animals and human share species of the three genera (Cryptosporidium, Cyclospora, Giardia), with the parasitic species mostly found in animals also found in human samples, and vice-versa. The presence of enteric parasites as mixed infections in asymptomatic humans and animal species indicates that they are reservoirs of infections. This is the first study to report the presence of C. cayetanensis and C. hominis in animals from Ghana. Our findings highlight the need for a detailed description of these parasites using high-throughput genetic tools to further understand these parasites and the neglected tropical diseases they cause in Ghana where such information is scanty.

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

BACKGROUND: Cyclospora cayetanensis is a protozoan parasite that causes intestinal illness in humans worldwide. Despite its global distribution, most genomic data for C. cayetanensis has been obtained from isolates collected in the United States, leaving genetic variability among globally distributed isolates underexplored. RESULTS: In the present study, the genome of an isolate of C. cayetanensis obtained from a child with diarrhea living in Mexico was sequenced and assembled. Evaluation of the assembly using a lineage typing system recently developed by the Centers for Disease Control and Prevention revealed that this isolate is lineage A. CONCLUSIONS: Given that the only other whole genome assembly available from Mexico was classified as lineage B, the data presented here represent an important step in expanding our knowledge of the diversity of C. cayetanensis isolates from Mexico at the genomic level.

Comparison of two novel one-tube nested real-time qPCR assays to detect human-infecting Cyclospora spp.

IMPORTANCE: Human-infecting Cyclospora spp. cause gastrointestinal distress among healthy individuals contributing to morbidity and putting stress on the economics of countries and companies in the form of produce recalls. Accessible and easy-to-use diagnostic tools available to a wide variety of laboratories would aid in the early detection of possible outbreaks of cyclosporiasis. This, in turn, will assist in the timely traceback investigation to the suspected source of an outbreak by informing the smallest possible recall and protecting consumers from contaminated produce. This manuscript describes two novel detection methods with improved performance for the causative agents of cyclosporiasis when compared to the currently used 18S assay.

Evaluation of nuclear and mitochondrial phylogenetics for the subtyping of Cyclospora cayetanensis.

Cyclospora cayetanensis is an enteric coccidian parasite responsible for gastrointestinal disease transmitted through contaminated food and water. It has been documented in several countries, mostly with low-socioeconomic levels, although major outbreaks have hit developed countries. Detection methods based on oocyst morphology, staining, and molecular testing have been developed. However, the current MLST panel offers an opportunity for enhancement, as amplification of all molecular markers remains unfeasible in the majority of samples. This study aims to address this challenge by evaluating two approaches for analyzing the genetic diversity of C. cayetanensis and identifying reliable markers for subtyping: core homologous genes and mitochondrial genome analysis. A pangenome was constructed using 36 complete genomes of C. cayetanensis, and a haplotype network and phylogenetic analysis were conducted using 33 mitochondrial genomes. Through the analysis of the pangenome, 47 potential markers were identified, emphasizing the need for more sequence data to achieve comprehensive characterization. Additionally, the analysis of mitochondrial genomes revealed 19 single-nucleotide variations that can serve as characteristic markers for subtyping this parasite. These findings not only contribute to the selection of molecular markers for C. cayetanensis subtyping, but they also drive the knowledge toward the potential development of a comprehensive genotyping method for this parasite.

The limit of detection of the BioFire® FilmArray® gastrointestinal panel for the foodborne parasite Cyclospora cayetanensis.

Cyclosporiasis is a foodborne diarrheal illness caused by the parasite Cyclospora cayetanensis. The BioFire® FilmArray® gastrointestinal (FilmArray GI) panel is a common method for diagnosing cyclosporiasis from clinical stool samples. The currently published limit of detection (LOD) of this panel is in genome equivalents; however, it is unclear how this relates to the number of C. cayetanensis oocysts in a clinical sample. In this study, we developed a technique to determine the LOD in terms of oocysts, using a cell sorter to sort 1 to 50 C. cayetanensis oocyst(s) previously purified from three human stool sources. We found the FilmArray GI panel detected samples with ≥20 C. cayetanensis oocysts in 100% of replicates, with varying detection among samples with 1, 5, or 10 C. cayetanensis oocysts. This method provides a parasitologically relevant LOD that should enable comparison among C. cayetanensis detection techniques, including the FilmArray GI panel.

Retrospective evaluation of an integrated molecular-epidemiological approach to cyclosporiasis outbreak investigations - United States, 2021.

Cyclosporiasis results from an infection of the small intestine by Cyclospora parasites after ingestion of contaminated food or water, often leading to gastrointestinal distress. Recent developments in temporally linking genetically related Cyclospora isolates demonstrated effectiveness in supporting epidemiological investigations. We used 'temporal-genetic clusters' (TGCs) to investigate reported cyclosporiasis cases in the United States during the 2021 peak-period (1 May - 31 August 2021). Our approach split 655 genotyped isolates into 55 genetic clusters and 31 TGCs. We linked two large multi-state epidemiological clusters (Epidemiologic Cluster 1 [n = 136 cases, 54 genotyped] and Epidemiologic Cluster 2 [n = 42 cases, 15 genotyped]) to consumption of lettuce varieties; however, product traceback did not identify a specific product for either cluster due to the lack of detailed product information. To evaluate the utility of TGCs, we performed a retrospective case study comparing investigation outcomes of outbreaks first detected using epidemiological methods with those of the same outbreaks had TGCs been used to first detect them. Our study results indicate that adjustments to routine epidemiological approaches could link additional cases to epidemiological clusters of cyclosporiasis. Overall, we show that CDC's integrated genotyping and epidemiological investigations provide valuable insights into cyclosporiasis outbreaks in the United States.

Development of a new multiplex PCR to detect fecal coccidian parasite.

BACKGROUND: Cryptosporidium spp., Cystoisospora belli and Cyclospora cayetanensis are common intestinal coccidian parasites causing gastroenteritis. The clinical presentation caused by each parasite is indistinguishable from each other. Uniplex polymerase chain reaction (PCR) for these three groups of intestinal coccidian parasites was developed by us in our laboratory. Thereafter, we planned to develop a single-run multiplex polymerase chain reaction (mPCR) assay to detect Cryptosporidium spp., C. belli and C. cayetanensis simultaneously from a stool sample and described it here as coccidian mPCR. METHODS: New primers for C. belli and C. cayetanensis were designed and uniplex PCRs were standardized. The coccidian mPCR was standardized with known positive DNA control isolates. It was validated with 58 known positive and 58 known negative stool samples, which were previously identified by uniplex PCR. RESULTS: The coccidian mPCR was standardized with earlier primers designed by us for Cryptosporidium spp. and C. cayetanensis, and a newly designed primer for the internal transcribed spacer-1 (ITS-1) gene for C. belli. The coccidian mPCR was 92.1% sensitive for Cryptosporidium spp., and 100% sensitive for C. belli and C. cayetanensis each, when tested on 116 known samples. It was 100% specific for all intestinal coccidian parasites. Two representative PCR products of the newly designed ITS-1 primer for C. belli were sequenced and submitted to the GenBank, which best match with the sequences of C. belli. CONCLUSION: A highly sensitive, specific, cost-effective, indigenous, single-run coccidian mPCR has been developed, which can simultaneously detect Cryptosporidium spp., C. belli and C. cayetanensis.

Immunogenic multi-epitope-based vaccine development to combat cyclosporiasis of immunocompromised patients applying computational biology method.

Cyclospora cayetanensis infections, also known as cyclosporiasis, persist to be the prevalent emerging protozoan parasite and an opportunist that causes digestive illness in immunocompromised individuals. In contrast, this causal agent can affect people of all ages, with children and foreigners being the most susceptible populations. For most immunocompetent patients, the disease is self-limiting; in extreme circumstances, this illness can manifest as severe or persistent diarrhea as well as colonize on secondary digestive organs leading to death. According to recent reports, worldwide 3.55% of people are infected by this pathogen, with Asia and Africa being more prevalent. For the treatment, trimethoprim-sulfamethoxazole is the only licensed drug and does not appear to work as well in some patient populations. Therefore, the much more effective strategy to avoid this illness is immunization through the vaccine. This present study uses immunoinformatics for identifying a computational multi-epitope-based peptide vaccine candidate for Cyclospora cayetanensis. Following the review of the literature, a highly efficient, secure, and vaccine complex based on multi-epitopes was designed by utilizing the identified proteins. These selected proteins were then used to predict non-toxic and antigenic HTL-epitopes, B-cell-epitopes, and CTL-epitopes. Ultimately, both a few linkers and an adjuvant were combined to create a vaccine candidate with superior immunological epitopes. Then, to establish the vaccine-TLR complex binding constancy, the TLR receptor and vaccine candidates were placed into the FireDock, PatchDock, and ClusPro servers for molecular docking and iMODS server for molecular-dynamic simulation. Finally, this selected vaccine construct was cloned into Escherichia coli strain-K12; thus, the constructed vaccines against Cyclospora cayetanensiscould improve the host immune response and can be produced experimentally.

Development and Single Laboratory Evaluation of a Refined and specific Real-time PCR Detection Method, Using Mitochondrial Primers (Mit1C), for the Detection of Cyclospora cayetanensis in Produce.

Regulatory methods for detection of the foodborne protozoan parasite Cyclospora cayetanensis must be specific and sensitive. To that end, we designed and evaluated (in a single laboratory validation) a novel and improved primer/probe combination (Mit1C) for real-time PCR detection of C. cayetanensis in produce. The newly developed primer/probe combination targets a conserved region of the mitochondrial genome of C. cayetanensis that varies in other closely related organisms. The primer/probe combination was evaluated both in silico and using several real-time PCR kits and polymerases against an inclusivity/exclusivity panel comprised of a variety of C. cayetanensis oocysts, as well as DNA from other related Cyclospora spp. and closely related parasites. The new primer/probe combination amplified only C. cayetanensis, thus demonstrating specificity. Sensitivity was evaluated by artificially contaminating cilantro, raspberries, and romaine lettuce with variable numbers (200 and 5) of C. cayetanensis oocysts. As few as 5 oocysts were detected in 75%, 67.7%, and 50% of the spiked produce samples (cilantro, raspberries, and romaine lettuce), respectively, all uninoculated samples and no-template real-time PCR controls were negative. The improved primer/probe combination should prove an effective analytical tool for the specific detection of C. cayetanensis in produce.

Epidemiologic Utility of a Framework for Partition Number Selection When Dissecting Hierarchically Clustered Genetic Data Evaluated on the Intestinal Parasite Cyclospora cayetanensis.

Comparing parasite genotypes to inform parasitic disease outbreak investigations involves computation of genetic distances that are typically analyzed by hierarchical clustering to identify related isolates, indicating a common source. A limitation of hierarchical clustering is that hierarchical clusters are not discrete; they are nested. Consequently, small groups of similar isolates exist within larger groups that get progressively larger as relationships become increasingly distant. Investigators must dissect hierarchical trees at a partition number ensuring grouped isolates belong to the same strain; a process typically performed subjectively, introducing bias into resultant groupings. We describe an unbiased, probabilistic framework for partition number selection that ensures partitions comprise isolates that are statistically likely to belong to the same strain. We computed distances and established a normalized distribution of background distances that we used to demarcate a threshold below which the closeness of relationships is unlikely to be random. Distances are hierarchically clustered and the dendrogram dissected at a partition number where most within-partition distances fall below the threshold. We evaluated this framework by partitioning 1,137 clustered Cyclospora cayetanensis genotypes, including 552 isolates epidemiologically linked to various outbreaks. The framework was 91% sensitive and 100% specific in assigning epidemiologically linked isolates to the same partition.

First report on occurrence and genotypes of Enterocytozoon bieneusi, Cryptosporidium spp. and Cyclospora cayetanensis from diarrheal outpatients in Ningbo, Southeast China.

Enterocytozoon bieneusi, Cryptosporidium spp. and Cyclospora cayetanensis are three important zoonotic pathogens which were a major cause of foodborne or waterborne intestinal diseases in humans and animals. However, very little data about occurrence and genotypes of the three parasites in Ningbo in the south wing of the Yangtze River Delta, China, which is important for a tourist city. In the present study, molecular characterization of E. bieneusi, C. cayetanensis and Cryptosporidium spp. in fecal samples from 489 diarrheal outpatients were carried out. As a result, a total of 35 (7.16%, 35/489) and three (0.61%, 3/489) samples were positive for E. bieneusi and C. cayetanensis respectively. No Cryptosporidium-positive sample or mixed-infections were detected. Four known E. bieneusi genotypes (Type IV, D, I and CHN4) and 8 novel genotypes (NBH1-NBH8) were identified with type IV was the dominant genotype (n = 14), followed by genotypes D (n = 5), NBH8 (n = 5) and NBH7 (n = 3). The remaining genotypes were found in one sample each, and these genotypes were belonged to the previously described high-potential zoonotic group 1. One novel sequence named NBC315, and the other two sequences (NBC30 and NBC370) identical with the reported sequence were detected. Therefore, the existence and importance of zoonotic potential of E. bieneusi and C. cayetanensis in diarrheal outpatients in Ningbo indicates the public health threats, and more investigations should be carried out in human populations, animals and other environmental sources from the One Health perspective.

Review on Cyclosporiasis Outbreaks and Potential Molecular Markers for Tracing Back Investigations.

Cyclosporiasis is an emerging disease caused by Cyclospora cayetanensis, which induces protracting and relapsing gastroenteritis and has been linked to huge and complicated travel- and food-related outbreaks worldwide. Cyclosporiasis has become more common in both developing and developed countries as a result of increased global travel and the globalization of the human food supply. It is not just a burden on individual human health but also a worldwide public health problem. As a pathogen of interest, the molecular biological characteristics of C. cayetanensis have advanced significantly over the last few decades. However, only one FDA-approved molecular platform has been commercially used in the investigation of cyclosporiasis outbreaks. More potential molecular markers and genotyping of C. cayetanensis in samples based on the polymorphic region of the whole genomes might differentiate between separate case clusters and would be useful in tracing back investigations, especially during cyclosporiasis outbreak investigations. Considering that there is no effective vaccine for cyclosporosis, epidemiological investigation using effective tools is crucial for controlling cyclosporiasis by source tracking. Therefore, more and more epidemiological investigative studies for human cyclosporiasis should be promoted around the world to get a deeper understanding of its characteristics as well as management. This review focuses on major cyclosporiasis outbreaks and potential molecular markers for tracing back investigations into cyclosporiasis outbreaks.

Evaluation of various distance computation methods for construction of haplotype-based phylogenies from large MLST datasets.

Multi-locus sequence typing (MLST) is widely used to investigate genetic relationships among eukaryotic taxa, including parasitic pathogens. MLST analysis workflows typically involve construction of alignment-based phylogenetic trees - i.e., where tree structures are computed from nucleotide differences observed in a multiple sequence alignment (MSA). Notably, alignment-based phylogenetic methods require that all isolates/taxa are represented by a single sequence. When multiple loci are sequenced these sequences may be concatenated to produce one tree that includes information from all loci. Alignment-based phylogenetic techniques are robust and widely used yet possess some shortcomings, including how heterozygous sites are handled, intolerance for missing data (i.e., partial genotypes), and differences in the way insertions-deletions (indels) are scored/treated during tree construction. In certain contexts, 'haplotype-based' methods may represent a viable alternative to alignment-based techniques, as they do not possess the aforementioned limitations. This is namely because haplotype-based methods assess genetic similarity based on numbers of shared (i.e., intersecting) haplotypes as opposed to similarities in nucleotide composition observed in an MSA. For haplotype-based comparisons, choosing an appropriate distance statistic is fundamental, and several statistics are available to choose from. However, a comprehensive assessment of various available statistics for their ability to produce a robust haplotype-based phylogenetic reconstruction has not yet been performed. We evaluated seven distance statistics by applying them to extant MLST datasets from the gastrointestinal parasite Cyclospora cayetanensis and two species of pathogenic nematode of the genus Strongyloides. We compare the genetic relationships identified using each statistic to epidemiologic, geographic, and host metadata. We show that Barratt's heuristic definition of genetic distance was the most robust among the statistics evaluated. Consequently, it is proposed that Barratt's heuristic represents a useful approach for use in the context of challenging MLST datasets possessing features (i.e., high heterozygosity, partial genotypes, and indel or repeat-based polymorphisms) that confound or preclude the use of alignment-based methods.

Surveillance of berries sold on the Norwegian market for parasite contamination using molecular methods.

The risk of foodborne parasite infection linked to the consumption of contaminated fresh produce has long been known. However, despite epidemiological links between the outbreaks and contaminated berries, few studies have assessed the magnitude of parasite contamination on fresh produce sold in Europe. The present study was aimed to address the knowledge gap on parasite contamination of berries sold in Norway. Samples of blueberries, strawberries, and raspberries were analysed by multiplex qPCR for detection of Echinococcus multilocularis, Toxoplasma gondii, and Cyclospora cayetanensis. In addition, a simplex qPCR method was employed for detecting contamination of the berries with Cryptosporidium spp. A total of 820 samples of berries, each of around 30 g (274 samples of blueberries, 276 samples of raspberries, and 270 samples of strawberries), were analysed. We found an overall occurrence of 2.9%, 6.6%, and 8.3% for T. gondii, C. cayetanensis, and Cryptosporidium spp., respectively, whereas E. multilocularis was not detected from any of the samples investigated. Strawberries and raspberries were most often contaminated with Cryptosporidium spp., whereas blueberries were contaminated mostly with C. cayetanensis. Detection of parasite contaminants on fresh berries indicates the need for a system to ensure the parasitological safety of fresh berries.

Genotyping Cyclospora cayetanensis From Multiple Outbreak Clusters With An Emphasis on a Cluster Linked to Bagged Salad Mix-United States, 2020.

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.

Dynamically expressed genes provide candidate viability biomarkers in a model coccidian.

Eimeria parasites cause enteric disease in livestock and the closely related Cyclospora cayetanensis causes human disease. Oocysts of these coccidian parasites undergo maturation (sporulation) before becoming infectious. Here, we assessed transcription in maturing oocysts of Eimeria acervulina, a widespread chicken parasite, predicted gene functions, and determined which of these genes also occur in C. cayetanensis. RNA-Sequencing yielded ~2 billion paired-end reads, 92% of which mapped to the E. acervulina genome. The ~6,900 annotated genes underwent temporally-coordinated patterns of gene expression. Fifty-three genes each contributed >1,000 transcripts per million (TPM) throughout the study interval, including cation-transporting ATPases, an oocyst wall protein, a palmitoyltransferase, membrane proteins, and hypothetical proteins. These genes were enriched for 285 gene ontology (GO) terms and 13 genes were ascribed to 17 KEGG pathways, defining housekeeping processes and functions important throughout sporulation. Expression differed in mature and immature oocysts for 40% (2,928) of all genes; of these, nearly two-thirds (1,843) increased their expression over time. Eight genes expressed most in immature oocysts, encoding proteins promoting oocyst maturation and development, were assigned to 37 GO terms and 5 KEGG pathways. Fifty-six genes underwent significant upregulation in mature oocysts, each contributing at least 1,000 TPM. Of these, 40 were annotated by 215 GO assignments and 9 were associated with 18 KEGG pathways, encoding products involved in respiration, carbon fixation, energy utilization, invasion, motility, and stress and detoxification responses. Sporulation orchestrates coordinated changes in the expression of many genes, most especially those governing metabolic activity. Establishing the long-term fate of these transcripts in sporulated oocysts and in senescent and deceased oocysts will further elucidate the biology of coccidian development, and may provide tools to assay infectiousness of parasite cohorts. Moreover, because many of these genes have homologues in C. cayetanensis, they may prove useful as biomarkers for risk.