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.

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.

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.

Single-strand conformation polymorphism-based genetic characterization of the Cyclospora cayetanensis strains collected from different provinces in Turkey.

INTRODUCTION AND OBJECTIVE: Cyclospora cayetanensis, a coccidian protozoan species, has been recently found to cause diarrhea in all age groups in immunocompetent and immunocompromised individuals in most regions of the world. This study aimed to conduct the molecular detection of C. cayetanensis and to determine the genetic diversity of the 18S ribosomal RNA (rRNA) gene sequence of C. cayetanensis isolated from individuals living in different provinces in Turkey by using PCR-single-strand conformation polymorphism (SSCP). MATERIAL AND METHODS: A total of 22 subjects were included in the study. Fourteen of the subjects were female and eight were male, with ages ranging between 7-65 years. Stool specimens were examined using wet mount and modified acid-fast staining methods, which revealed the presence of oocysts in the samples. The 18S rRNA ITS-1 Ccits37f-GCTTGCTATGTTTTAGCATGTGG and Ccits501r-GCACAATGAATGCACACACA gene regions were used as primers. The PCR products were analyzed by agarose gel electrophoresis and visualized on a UV transilluminator. For the SSCP, the PCR products were denatured with formamide, run for 16 h in 6% (49:1) polyacrylamide gel, and then imaged with silver staining. RESULTS: SSCP assay was performed given that the DNA strands demonstrated different folds; the DNA strands contain different nucleotides based on the PCR-SSCP results for the Cyclospora strains collected in 4 provinces. Moreover, 3 different band profiles were observed in the investigated samples. A slight mutation difference was observed among the strains collected. CONCLUSIONS: Further comprehensive studies involving more C. cayetanensis-positive specimens and utilizing different mutation screening methods are warranted to demonstrate mutation differences in Cyclopora strains in Turkey.

Molecular characterization of the waterborne pathogens Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, Cyclospora cayetanensis and Eimeria spp. in wastewater and sewage in Guangzhou, China.

BACKGROUND: The waterborne pathogens Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi and Cyclospora cayetanensis can cause intestinal diseases in humans. An understanding of their occurrence and transport in the environment is essential for accurate quantitative microbial risk assessment. METHODS: A total of 238 influent samples were collected from four wastewater treatment plants (WWTPs) and 88 samples from eight sewer locations in Guangzhou, China. PCR-based tools were used to detect and genetically characterize Cryptosporidium spp., G. duodenalis and E. bieneusi. Eimeria spp. and Cyclospora spp. were also analyzed to assess the sources of Cryptosporidium spp., G. duodenalis and E. bieneusi in wastewater. RESULTS: The overall occurrence rates in the WWTP and sewer samples were 14.3% (34/238) and 13.6% (12/88) for Cryptosporidium spp., 55.5% (132/238) and 33.0% (29/88) for G. duodenalis, 56.3% (134/238) and 26.1% (23/88) for E. bieneusi and 45.4% (108/238) and 47.7% (42/88) for Eimeria spp., respectively. Altogether, 11 Cryptosporidium species and genotypes, six G. duodenalis genotypes, 11 E. bieneusi genotypes and four C. cayetanensis were found, together with the presence of nine Eimeria species. The common occurrence of Cryptosporidium rat genotype IV, C. muris and Eimeria papillata and E. nieschulzi suggested that rodents were significant sources of the enteric pathogens detected in the wastewater samples. CONCLUSIONS: While the dominant Cryptosporidium spp. detected in the raw wastewater sampled in this study are not pathogenic to humans, the widely detected G. duodenalis assemblage A and E. bieneusi genotypes D and Type IV are well-known zoonotic pathogens. Further studies are needed to monitor the occurrence of these waterborne pathogens in WWTPs to better understand their transmission and environmental transport in China.

Modifications of the U.S. food and drug administration validated method for detection of Cyclospora cayetanensis oocysts in prepared dishes: Mexican-style salsas and guacamole.

Although multiple outbreak clusters of Cyclospora cayetanensis have been traced back to consumption of dishes in Mexican-style restaurants, the FDA Bacteriological Analytical Manual (BAM) does not currently provide methods to detect C. cayetanensis in dishes that contain multiple produce ingredients, such as salsas and guacamole. These complex food matrices also may contain high levels of fats, which can interfere with the detection. Several modifications to the BAM Chapter 19b method (washing produce, DNA extraction, and a TaqMan real-time PCR assay targeting the 18S rRNA gene of C. cayetanensis) were assessed with the goal to detect as few as 5 oocysts of C. cayetanensis in 25 g samples of commercial salsa/pico de gallo, guacamole, and salsa verde. Both freshly prepared and frozen versions of these foods were seeded with 5, 10 and 200 oocysts. For salsa samples, using a gentler washing step than recommended by BAM, we achieved detection of 5 oocysts in the samples (81.8%, n = 11). Increasing the amount of Alconox® in the wash solution to 1%, rather than the 0.1% used in BAM, and adjusting the DNA extraction protocol to process large wash pellets, enabled detection of 5 oocysts in guacamole. To reach the desired level of detection in salsa verde, two types of modifications were necessary: gentler washing and DNA extraction modifications. The use of these same method modifications on previously frozen food samples, provided levels of detection similar to those achieved with fresh dishes. Our modifications enabled robust and reproducible detection of C. cayetanensis in multi-ingredient Mexican dishes, detecting as few as 5 oocysts in 25 g samples. Validating and deploying effective methods to detect C. cayetanensis in high risk fresh produce and prepared dishes are critically important for prevalence studies and outbreak investigations of this parasite.

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.

Sporadic cyclosporiasis in symptomatic Cuban patients: Confirmation of positive results from conventional diagnostic methods by molecular assay.

In Cuba, there are few studies on cyclosporiasis. Here, we report results from 1247 stool samples from symptomatic patients that were examined by microscopy methods and positive cases confirmed by nested PCR targeting the 18S rRNA gene, followed by sequencing. Seven positive samples, all diagnosed during May-June, were confirmed by the molecular method, indicating an occurrence in this patient cohort of 0.56%.

Molecular typing of Cyclospora cayetanensis in produce and clinical samples using targeted enrichment of complete mitochondrial genomes and next-generation sequencing.

BACKGROUND: Outbreaks of cyclosporiasis, a diarrheal illness caused by Cyclospora cayetanensis, have been a public health issue in the USA since the mid 1990's. In 2018, 2299 domestically acquired cases of cyclosporiasis were reported in the USA as a result of multiple large outbreaks linked to different fresh produce commodities. Outbreak investigations are hindered by the absence of standardized molecular epidemiological tools for C. cayetanensis. For other apicomplexan coccidian parasites, multicopy organellar DNA such as mitochondrial genomes have been used for detection and molecular typing. METHODS: We developed a workflow to obtain complete mitochondrial genome sequences from cilantro samples and clinical samples for typing of C. cayetanensis isolates. The 6.3 kb long C. cayetanensis mitochondrial genome was amplified by PCR in four overlapping amplicons from genomic DNA extracted from cilantro, seeded with oocysts, and from stool samples positive for C. cayetanensis by diagnostic methods. DNA sequence libraries of pooled amplicons were prepared and sequenced via next-generation sequencing (NGS). Sequence reads were assembled using a custom bioinformatics pipeline. RESULTS: This approach allowed us to sequence complete mitochondrial genomes from the samples studied. Sequence alterations, such as single nucleotide polymorphism (SNP) profiles and insertion and deletions (InDels), in mitochondrial genomes of 24 stool samples from patients with cyclosporiasis diagnosed in 2014, exhibited discriminatory power. The cluster dendrogram that was created based on distance matrices of the complete mitochondrial genome sequences, indicated distinct strain-level diversity among the 2014 C. cayetanensis outbreak isolates analyzed in this study. CONCLUSIONS: Our results suggest that genomic analyses of mitochondrial genome sequences may help to link outbreak cases to the source.

Cyclospora cayetanensis infection in humans: biological characteristics, clinical features, epidemiology, detection method and treatment.

Cyclospora cayetanensis, a coccidian parasite that causes protracted and relapsing gastroenteritis, has a short recorded history. At least 54 countries have documented C. cayetanensis infections and 13 of them have recorded cyclosporiasis outbreaks. Cyclospora cayetanensis infections are commonly reported in developing countries with low-socioeconomic levels or in endemic areas, although large outbreaks have also been documented in developed countries. The overall C. cayetanensis prevalence in humans worldwide is 3.55%. Among susceptible populations, the highest prevalence has been documented in immunocompetent individuals with diarrhea. Infections are markedly seasonal, occurring in the rainy season or summer. Cyclospora cayetanensis or Cyclospora-like organisms have also been detected in food, water, soil and some other animals. Detection methods based on oocyst morphology, staining and molecular testing have been developed. Treatment with trimethoprim-sulfamethoxazole (TMP-SMX) effectively cures C. cayetanensis infection, whereas ciprofloxacin is less effective than TMP-SMX, but is suitable for patients who cannot tolerate co-trimoxazole. Here, we review the biological characteristics, clinical features, epidemiology, detection methods and treatment of C. cayetanensis in humans, and assess some risk factors for infection with this pathogen.

Identification of human pathogenic Enterocytozoon bieneusi, Cyclospora cayetanensis, and Cryptosporidium parvum on the surfaces of vegetables and fruits in Henan, China.

Cryptosporidium spp., Giardia duodenalis, Cyclospora cayetanensis, and Enterocytozoon bieneusi are known etiological agents of self-limiting diarrhea, chronic disorders, and severe debilitating illnesses in humans, particularly children and patients with immunodeficiency diseases. To assess the pathogen carriage status of raw vegetables and fruits and the potential transmission routes of the aforementioned parasites in Henan province, China, a total of 1099 vegetables and fruits samples (21 items) were purchased and collected from agricultural farms or open markets. Cryptosporidium spp., E. bieneusi, C. cayetanensis and G. duodenalis were screened by employing polymerase chain reaction (PCR) amplification of species-specific genes. Three kinds of human pathogenic agent (E. bieneusi, C. cayetanensis and C. parvum) were identified on the surfaces of the vegetables and fruits (3.7%, 41/1099). E. bieneusi was found in 3.5% (38/1099) of the samples, whereas C. cayetanensis and C. parvum were only identified in two (0.2%) and one (0.1%) of the vegetable and fruit samples, respectively. No G. duodenalis contamination was detected in the present study. In total, 12 different E. bieneusi ITS genotypes (eight known and four novel) were detected, of which the ten (EbpA, CM8, CHG19, EbpC, CTS3, Henan-IV, and CHV1 to CHV4) that occurred in 20 samples (20/38, 52.6%) clustered into the previously described high potential zoonotic group 1 in the phylogenetic analysis. The remaining two known genotypes (BEB8 and CD6) detected in 18 samples (18/38, 47.4%) belonged to group 2. That C. cayetanensis, C. parvum and some E. bieneusi genotypes have been reported in humans, highlights the possible risk of foodborne related disease outbreaks.

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.

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.

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.

Molecular detection and genotyping of pathogenic protozoan parasites in raw and treated water samples from southwest Colombia.

BACKGROUND: Protozoan parasites such as Giardia duodenalis, Cryptosporidium spp., Cyclospora cayetanensis, Toxoplasma gondii and Entamoeba histolytica represent a great challenge to the systems producing water for human consumption because their cystic forms are persistent in the environment and resist to the disinfection methods conventionally used for their control. In this study, we investigated the presence of these protozoan pathogens in both raw and treated water samples used for the production of drinking water in Nariño Department, southwest Colombia. We collected 110 water samples (10 lof each sample) and analyzed them with real-time PCR (qPCR). qPCR-positive samples were genotyped with PCR and DNA sequencing. RESULTS: Giardia duodenalis was detected in 35/110 (31.8%) of the samples and Cryptosporidium spp. in 9/110 (8.2%) of the samples; no sample was positive for T. gondii, E. histolytica or C. cayetanensis. Giardia duodenalis was detected in samples of both raw water (Drinking Water Treatment Plants (DWTP): 47.83%;Drinking Water Rural Plants (DWRP): 18.42%) and water collected either after conventional physicochemical treatment (26.09%) or after disinfection by chlorine (50%), whereas Cryptosporidium spp. were only detected in raw waters (DWTP: 17.39%; DWRP: 13.16%). The two pathogens were detected in both types of treatment plants supplying water to urban areas and to rural zones. Analysis of gdh and tpi markers identified assemblages AI, AII and H of G. duodenalis, while analysis of the small subunit rRNA and gp60 markers of Cryptosporidium-positive samples identified C. parvum (Subtype IIcA5G3c), C. galli, C. molnari, Cryptosporidium sp. genotype II of bats and Cryptosporidium sp. genotype VIII of birds. CONCLUSIONS: The results obtained demonstrate the presence of protozoan parasites in the water of the study region, and the need to improve the surveillance systems for these pathogens and identify the corresponding sources of contamination.

Population genetic characterization of Cyclospora cayetanensis from discrete geographical regions.

Cyclospora cayetanensis is an emerging pathogen that is endemic in developing countries and responsible for many large foodborne cyclosporiasis outbreaks in North America since 1990s. Because of the lack of typing targets, the genetic diversity and population genetics of C. cayetanensis have not been investigated. In this study, we undertook a population genetic analysis of multilocus sequence typing data we recently collected from 64 C. cayetanensis specimens. Despite the extensive genetic heterogeneity in the overall C. cayetanensis population, there were significant intra- and inter-genic linkage disequilibria (LD). A disappearance of LD was observed when only multilocus genotypes were included in the population genetic analysis, indicative of an epidemic nature of C. cayetanensis. Geographical segregation-associated sub-structuring was observed between specimens from China and those from Peru and the United States. The two subpopulations had reduced LD, indicating the likely occurrence of genetic exchange among isolates in endemic areas. Further analyses of specimens from other geographical regions are necessary to fully understand the population genetics of C. cayetanensis.

Pathogenic characteristics of a novel intranuclear coccidia in Japanese black calves and its genetic identification as Eimeria subspherica.

Bovine intranuclear coccidiosis is caused by the protozoans Eimeria alabamensis and Cyclospora spp. Here, we characterized the disease and genetically identified the causative species in Japanese black calves with chronic and refractory watery diarrhea. Histologic examinations revealed atrophy of the jejunal villi and numerous parasites in the nucleus of epithelial cells in the jejunum. Based on molecular analyses using 18S ribosomal RNA gene-specific primers that we designed, the parasites were found to be formed in the same cluster as Eimeria subspherica in the phylogenetic tree, which was separated from those of other related Eimeria spp. These results constitute the first report of E. subspherica as a cause of bovine intranuclear coccidiosis.

Multilocus sequence typing and clonal population genetic structure of Cyclospora cayetanensis in humans.

To investigate the prevalence of Cyclospora cayetanensis in a longitudinal study and to conduct a population genetic analysis, fecal specimens from 6579 patients were collected during the cyclosporiasis - prevalent seasons in two urban areas of central China in 2011-2015. The overall incidence of C. cayetanensis infection was 1·2% (76/6579): 1·6% (50/3173) in Zhengzhou and 0·8% (26/3406) in Kaifeng (P 0·05). All the isolates clustered in the C. cayetanensis clade based on the small subunit ribosomal RNA gene sequence phylogenetic analysis. There were 45 specimens positive for all the five C. cayetanensis microsatellite loci, and formed 29 multilocus genotypes (MLGs). The phylogenetic relationships of 54 distinct MLGs (including 25 known reference MLGs), based on the concatenated multilocus sequences, formed three main clusters. A population structure analysis showed that the 79 isolates (including 34 known reference isolates) of C. cayetanensis produced three distinct subpopulations based on allelic profile data. In conclusion, we determined the frequency of C. cayetanensis infection in humans in Henan Province. The clonal population structure of the human C. cayetanensis isolates showed linkage disequilibrium and three distinct subpopulations.

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.

Molecular detection of Cyclospora in water, soil, vegetables and humans in southern Italy signals a need for improved monitoring by health authorities.

To date, in Europe, there is scant information on the occurrence of Cyclospora in water from treatment plants and in humans, and no data are available on soil or fresh plant products. Here, we undertook the first molecular survey of Cyclospora in multiple biological matrices collected from the Apulia region of southern Italy. Samples of irrigation water from four municipal treatment plants, eight different types of vegetables or fruit (cucumber, lettuce, fennel, celery, tomato, melon, endive and chicory) and soil from the same farms on which these plants were grown, as well as faecal samples from humans living in the same region were tested by qPCR-coupled single-strand conformation polymorphism (SSCP) analysis and DNA sequencing. Cyclospora was detected in 15.5% of all 213 samples tested. Specifically, this protist was detected in (i) treated water (21.3% of 94 samples), well water (6.2% of 16), but not drinking water (0% of 3); (ii) soil (11.8% of 51 samples) and vegetables (12.2% of 49), with the highest prevalence (18.7%) on fennel; and (iii) human stools (27.5% of 40 samples). In environmental and food samples, Cyclospora was detected mainly in autumn and was significantly more prevalent in the faeces from humans of 40-50 years of age. This is the first comprehensive molecular survey of Cyclospora in environmental, food and human faecal samples in Europe. These data suggest that irrigation water, soil and vegetables might be contaminated by Cyclospora cayetanensis, which might represent a source of infection to humans in the study area and calls for monitoring by health authorities.