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.

Enterocytozoon bieneusi Infection after Hematopoietic Stem Cell Transplant in Child, Argentina.

We report a case of Enterocytozoon bieneusi infection in a pediatric hematopoietic stem cell transplant recipient in Argentina. Spores were visualized in feces using Calcofluor White and modified trichrome stainings. PCR and sequencing identified E. bieneusi genotype D in fecal samples and liver samples, confirming extraintestinal dissemination of the parasite.

Division of Blastocystis ST10 into three new subtypes: ST42-ST44.

The Blastocystis subtype ST10 has been recognized to contain a great deal of diversity at the sequence level, potentially indicating the presence of multiple new STs within the clade. However, the data needed to validate these new STs were not available. To help resolve this diversity, full-length small subunit (SSU) rRNA gene reference sequences were generated using Oxford Nanopore MinION long-read sequencing from 21 samples representing multiple domestic and wild hosts and geographic regions and covering the sequence diversity previously described using fragments of the SSU rRNA gene. Phylogenetic and pairwise distance analyses were used to compare full-length sequences of the SSU rRNA gene generated in this study with all other valid STs of Blastocystis. We present data supporting the division of ST10/ST23 cluster into five subtypes, ST10, ST23, and three new subtypes with the proposed ST designations of ST42, ST43, and ST44. As the host range of Blastocystis continues to expand with new subtypes and new hosts being frequently identified, the reference sequences provided in this study will assist in accurate sequence classification and help to clarify the epidemiology of this common intestinal microeukaryote.

Detection and genotyping of zoonotic microsporidia in the endangered Iberian lynx (Lynx pardinus).

Microsporidia is a diverse group of obligate, intracellular, and spore-forming parasites that infect a wide range of animals. Among them, Enterocytozoon bieneusi and Encephalitozoon spp. are the most frequently reported species in humans. Limited information is available about the presence and molecular diversity of microsporidian species in the endangered Iberian lynx (Lynx pardinus). Presence of Enterocytozoon bieneusi and Encephalitozoon spp. was investigated by molecular methods in wild and captive Iberian lynxes from Spain. Overall, E. bieneusi was detected in 3.2% (8/251) of the animals examined. None of the samples tested were positive for Encephalitozoon spp. Four known (D, EbfelA, PigEBITS7, and Type IV) and a novel (named as LynxSpEb1) E. bieneusi genotypes were identified. All the genotypes found belonged to the zoonotic Group 1 of E. bieneusi. This study provides the first genotyping data of E. bieneusi in Iberian lynx in Spain. Our result indicate that the Iberian lynx does not seem to play a relevant role in the epidemiology of Encephalitozoon spp., and that this endangered felid is likely acting as spillover host rather than a true reservoir of E. bieneusi. Additional studies should be conducted to assess the impact of this parasite in the health status of the endangered Iberian lynx.

Sympatric Recombination in Zoonotic Cryptosporidium Leads to Emergence of Populations with Modified Host Preference.

Genetic recombination plays a critical role in the emergence of pathogens with phenotypes such as drug resistance, virulence, and host adaptation. Here, we tested the hypothesis that recombination between sympatric ancestral populations leads to the emergence of divergent variants of the zoonotic parasite Cryptosporidium parvum with modified host ranges. Comparative genomic analyses of 101 isolates have identified seven subpopulations isolated by distance. They appear to be descendants of two ancestral populations, IIa in northwestern Europe and IId from southwestern Asia. Sympatric recombination in areas with both ancestral subtypes and subsequent selective sweeps have led to the emergence of new subpopulations with mosaic genomes and modified host preference. Subtelomeric genes could be involved in the adaptive selection of subpopulations, while copy number variations of genes encoding invasion-associated proteins are potentially associated with modified host ranges. These observations reveal ancestral origins of zoonotic C. parvum and suggest that pathogen import through modern animal farming might promote the emergence of divergent subpopulations of C. parvum with modified host preference.

Identification and validation of novel Blastocystis subtype ST41 in a Colombian patient undergoing colorectal cancer screening.

Blastocystis sp. is among the most frequent intestinal protists identified in humans globally. However, characterization of Blastocystis subtype diversity in humans is ongoing. We report here the identification of novel Blastocystis subtype ST41 in a Colombian patient undergoing colorectal cancer screening involving colonoscopy and fecal testing (microscopy, culture, PCR). The full-length ssu rRNA gene sequence of the protist was generated using MinION long-read sequencing technology. The validity of the novel subtype was confirmed via phylogenetic and pairwise distance analyses of the full-length ST41 sequence and all other valid subtypes. The study provides reference material essential for conducting subsequent experimental studies.

Occurrence and molecular characterization of Enterocytozoon bieneusi in wild and domestic animal species in Portugal.

The phylum Microsporidia encompasses a diverse group of obligate, intracellular, and spore-forming organisms able to infect a wide range of animal hosts. Among them, Enterocytozoon bieneusi is the most frequently reported species in humans and animals. Little is known about the presence and epidemiology of E. bieneusi in wildlife. We investigated E. bieneusi occurrence and genetic diversity in wild and domestic mammals, through molecular-detection methods, from different regions across Portugal. A total of 756 samples were collected from 288, 242, and 226 wild carnivores, wild ungulates, and domestic animals, respectively. Overall, eight specimens were E. bieneusi-positive (1.1%, 8/756) obtained from five wild (Iberian lynx, Iberian wolf, red fox, stone marten, and wild boar) and one domestic (sheep) host. Nucleotide sequence analysis identified four genotypes of E. bieneusi, Type IV, Wildboar3, BEB6, and PtEbIX. Three of those genotypes belong to Groups 1 (Type IV and Wildboar3) and 2 (BEB6), which are known to contain genotypes capable of infecting a variety of hosts, including humans, highlighting their public health importance. PtEbIX belongs to the dog-specific Group 11. This study represents the first, largest, and most comprehensive molecular-based epidemiology survey carried out in Portugal in wild and domestic animals to date and the first worldwide identification of E. bieneusi in wolf species. Our study showed that wild carnivores and ungulates may act as reservoirs of zoonotic genotypes of E. bieneusi, establishing their role in maintaining the sylvatic cycle of this parasite while representing a potential source of infection for humans and domestic animals.

The identification of human-pathogenic genotypes of fungi-related Enterocytozoon bieneusi in wild carnivores and ungulates in Portugal suggests cross-species infection events and overlapping of the sylvatic and domestic transmission cycles, demonstrating a potential transmission risk to humans.

Exploring Blastocystis genetic diversity in rural schoolchildren from Colombia using next-generation amplicon sequencing reveals significant associations between contact with animals and infection risk.

Blastocystis is a common intestinal protist with a global distribution in humans and many other animals. Yet, the status of Blastocystis as a pathogen, the risk factors associated with its transmission, and its zoonotic potential remain ill-defined. Here, we explored subtype (ST) diversity and potential risk factors for Blastocystis infection in 98 children from Apulo, Colombia. Samples were screened for Blastocystis via PCR, and ST identification was performed through next-generation amplicon sequencing (NGS). Associations between the presence of Blastocystis and individual STs and sociodemographic variables were assessed via logistic regression analyses. Seventy-one samples (72.4%) were Blastocystis-positive, and NGS revealed the presence of five STs (ST1-ST5). ST1, ST2, and ST3 were common and observed in nearly equal proportions (~ 40%), while samples with ST4 (1.4%) and ST5 (5.6%) were comparatively rare. The presence of mixed STs in the same sample was also common (28.2%). Comparisons among children within the same household identified that shared ST profiles were common, but diversity within family units was also observed. Logistic regression analyses returned significant associations between the presence of Blastocystis, individual subtypes, or mixed subtypes for several variables. Intriguingly, the presence of animals was one of the most common significant associations. Taken together, these data represent an important step forward in understanding both the potential routes and risk factors that may influence Blastocystis transmission and will be useful in shaping future studies which seek to clarify the relationships between STs, pathogenicity, and zoonotic transmission.

Zoonotic Enterocytozoon bieneusi genotypes in free-ranging and farmed wild ungulates in Spain.

Microsporidia comprises a diverse group of obligate, intracellular, and spore-forming parasites that infect a wide range of animals. Among them, Enterocytozoon bieneusi is the most frequently reported species in humans and other mammals and birds. Data on the epidemiology of E. bieneusi in wildlife are limited. Hence, E. bieneusi was investigated in eight wild ungulate species present in Spain (genera Ammotragus, Capra, Capreolus, Cervus, Dama, Ovis, Rupicapra, and Sus) by molecular methods. Faecal samples were collected from free-ranging (n = 1058) and farmed (n = 324) wild ungulates from five Spanish bioregions. The parasite was detected only in red deer (10.4%, 68/653) and wild boar (0.8%, 3/359). Enterocytozoon bieneusi infections were more common in farmed (19.4%, 63/324) than in wild (1.5%, 5/329) red deer. A total of 11 genotypes were identified in red deer, eight known (BEB6, BEB17, EbCar2, HLJD-V, MWC_d1, S5, Type IV, and Wildboar3) and three novel (DeerSpEb1, DeerSpEb2, and DeerSpEb3) genotypes. Mixed genotype infections were detected in 15.9% of farmed red deer. Two genotypes were identified in wild boar, a known (Wildboar3) and a novel (WildboarSpEb1) genotypes. All genotypes identified belonged to E. bieneusi zoonotic Groups 1 and 2. This study provides the most comprehensive epidemiological study of E. bieneusi in Spanish ungulates to date, representing the first evidence of the parasite in wild red deer populations worldwide. Spanish wild boars and red deer are reservoir of zoonotic genotypes of E. bieneusi and might play an underestimated role in the transmission of this microsporidian species to humans and other animals.

The fungal-related intracellular parasite Enterocytozoon bieneusi is a worldwide public health and veterinary problem. Here we demonstrated that it was present in wild boar, and wild and farmed red deer in Spain, with genotypes potentially capable of infecting humans, posing a public health risk.

Next-generation sequencing reveals wide genetic diversity of Blastocystis subtypes in chickens including potentially zoonotic subtypes.

Blastocystis sp. is an intestinal protist parasite commonly found in the feces of humans and animals worldwide. Blastocystis exhibits extensive genetic diversity and has been identified in humans and a variety of animals including other mammals and birds. Blastocystis subtypes do not exhibit strict host specificity which raises the possibility of zoonotic transmission through either direct contact or fecal contamination of food or water. However, reports detailing the subtypes and prevalence of Blastocystis in avian species are limited. Therefore, this study investigated the presence of Blastocystis in chickens by molecular characterization of the small subunit rRNA (SSU rRNA) gene. Fecal samples from 130 chickens were collected from local markets in Uberlândia and Belo Horizonte in the state of Minas Gerais, Brazil. To detect and identify subtypes of Blastocystis, a next-generation amplicon sequencing protocol was used. Forty-four of the 130 (33.8%) chickens examined were positive for Blastocystis. Blastocystis subtypes ST6 (23/130; 17.7%), ST7 (43/130; 33.1%), ST10 (1/130; 0.8%), ST14 (5/130; 3.8%), ST25 (1/130; 0.8%), and a novel subtype (ST29) (2/130; 1.5%) were observed. A nanopore sequencing strategy was used to obtain the near full-length SSU rRNA gene nucleotide sequence and validate novel subtype ST29. Mixed infections containing multiple subtypes were common and identified in 63.6% of Blastocystis-positive chickens. All positive samples contained one or both potentially zoonotic subtypes ST6 and ST7. The prevalence of Blastocystis in chickens was high, and molecular characterization mostly identified subtypes previously found in humans. Thus, chickens may be a source of human infection and environmental contamination.

Blastocystis sp. Subtype Diversity in Wild Carnivore Species from Spain.

The occurrence and molecular diversity of the stramenopile eukaryote Blastocystis sp. was investigated by PCR and sequencing (Sanger and NGS) methods in 380 faecal specimens of free-living carnivores in Spain. Blastocystis sp. was confirmed in 1.6% (6/380) of the specimens analysed. Two samples from a common genet and a fox were successfully subtyped as ST7 by Sanger. Using NGS, ST14 was found in a fox and a European polecat, ST7 in a fox, and two additional foxes presented mixed infections of ST1/ST2/ST4 and ST1/ST2/ST7, respectively. Wild carnivore species could act as carriers of zoonotic Blastocystis subtypes.

Zoonotic and genetically diverse subtypes of Blastocystis in US pre-weaned dairy heifer calves.

Blastocystis is an emerging zoonotic pathogen with global distribution. However, limited data exist on the prevalence and genetic diversity of Blastocystis in the USA and in food animals. We conducted the first large-scale molecular investigation of Blastocystis in the USA by testing 2539 fecal samples from dairy heifer calves from 13 states. Blastocystis was detected in 73 (2.9%) fecal samples and in 10 of the 13 tested states. Molecular characterization showed a wide diversity of subtypes. Eleven subtypes were identified, seven previously reported (ST-3, ST-4, ST-5, ST-10, ST-14, ST-17, and ST-21) and four potentially novel subtypes (named ST-23 to ST-26). Zoonotic subtypes 3, 4, and 5 were found in 67% (49) of the positive specimens in this population. Our results suggest that cattle could serve as reservoirs of infection for humans and other domestic animals highlighting the potential risk of zoonotic transmission for Blastocystis.

Occurrence and genetic diversity of Enterocytozoon bieneusi (Microsporidia) in owned and sheltered dogs and cats in Northern Spain.

Enterocytozoon bieneusi is an obligate intracellular protist-like fungi parasite that infects numerous mammal hosts including humans, raising concerns of zoonotic transmission. There is little information available on the presence and diversity of E. bieneusi genotypes in companion animals. Here, we determined the occurrence and genetic diversity of E. bieneusi in domestic dogs and cats from Northern Spain. A total of 336 genomic DNA samples extracted from canine (n = 237) and feline (n = 99) faecal specimens were retrospectively investigated. The presence of E. bieneusi was assessed by PCR of the rRNA internal transcribed spacer (ITS) gene. The parasite was detected in 3.0% (3/99) and 0.8% (2/237) of the cats and dogs examined, respectively. All three feline positive samples were from stray cats living in an urban setting, whereas the two canine samples were from owned dogs living in rural areas. Sequence analysis revealed the presence of two genotypes in dogs, BEB6 and PtEb IX, and two genotypes in cats, D and Peru11. The identification of Peru11 in a cat and BEB6 in a dog constitutes the first report of those genotypes in such hosts as well as first report in Spain. This is also the first evidence of genotype D in cats and PtEb IX in dogs in Spain. Three out of the four genotypes, BEB6, D and Peru11, have been previously reported as human pathogens and are potentially zoonotic indicating that dogs and cats need to be considered potential sources of human infection and environmental contamination.

Molecular Characterization of Enterocytozoon bieneusi in Wild Carnivores in Spain.

Microsporidia comprises a diverse group of obligate intracellular parasites that infect a broad range of invertebrates and vertebrates. Among Microsporidia, Enterocytozoon bieneusi is the most frequently detected species in humans and animals worldwide bringing into question the possible role of animal reservoirs in the epidemiology of this pathogen. Although E. bieneusi is an emerging zoonotic pathogen able to infect many domestic and wild mammals that could act as reservoir of infection for humans and other animals, only few studies have documented its occurrence in wild carnivores. To determine the occurrence of E. bieneusi in wild carnivores, we examined 190 wild carnivores collected from different locations in Spain. Twenty-five fecal samples (13.2%) from three host species (European badger, beech marten, and red fox) were E. bieneusi-positive by PCR. Nucleotide sequence analysis of the ITS region revealed a high degree of genetic diversity with a total of eight distinct genotypes including four known (PtEbIX, S5, S9, and WildBoar3) and four novel (EbCar1-EbCar4) genotypes identified. Phylogenetic analysis showed that the four novel genotypes (EbCar1-EbCar4), S5, S9, and WildBoar3 clustered within the previously designated zoonotic Group 1. Our results demonstrate that human-pathogenic genotypes are present in wild carnivores, corroborating their potential role as a source of human infection and environmental contamination.

Interlaboratory validation of an improved method for detection of Cyclospora cayetanensis in produce using a real-time PCR assay.

A collaborative validation study was performed to evaluate the performance of a new U.S. Food and Drug Administration method developed for detection of the protozoan parasite, Cyclospora cayetanensis, on cilantro and raspberries. The method includes a sample preparation step in which oocysts are recovered from produce using an enhanced produce washing solution containing 0.1% Alconox and a commercially available method to disrupt the C. cayetanensis oocysts and extract DNA. A real-time PCR assay targeting the C. cayetanensis 18S rDNA gene with an internal amplification control to monitor PCR inhibition provides species-specific identification. Five laboratories blindly analyzed a total of 319 samples consisting of 25 g of cilantro or 50 g of raspberries which were either uninoculated or artificially contaminated with C. cayetanensis oocysts. Detection rates for cilantro inoculated with 200, 10, and 5 oocysts, were 100%, 80%, and 31%, respectively. For raspberries, the detection rates for samples inoculated with 200, 10, and 5 oocysts were 100%, 90% and 50%, respectively. All uninoculated samples, DNA blank extracts, and no-template PCR controls were negative. Reproducibility between laboratories and analysts was high and the method was shown to be an effective analytical tool for detection of C. cayetanensis in produce.

Molecular identification of Enterocytozoon bieneusi, Cryptosporidium, and Giardia in Brazilian captive birds.

A total of 85 fecal samples from captive birds collected from October 2013 to September 2014 in Uberlândia and Belo Horizonte in the state of Minas Gerais (Brazil) were evaluated for the presence of Enterocytozoon bieneusi, Cryptosporidium, and Giardia by PCR. Of these, three birds were found positive for E. bieneusi (3.5%), two for Cryptosporidium (2.3%), and one for Giardia (1.2%). Two genotypes of E. bieneusi were detected by nucleotide sequence analysis of the ITS region, genotypes D and Peru 6 in a swan goose and in two rock pigeons, respectively. For Cryptosporidium and Giardia, nucleotide sequence analysis of the SSU rRNA identified Cryptosporidium baileyi and Duck genotype in a swan goose and a mandarin duck, respectively, and Giardia duodenalis assemblage A in a toco toucon. Our results demonstrate that human-pathogenic E. bieneusi genotypes D and Peru6 and G. duodenalis assemblage A are present in captive birds in Brazil, corroborating their potential role as a source of human infection and environmental contamination.

Evaluation of Fecal Indicators and Pathogens in a Beef Cattle Feedlot Vegetative Treatment System.

Runoff from open-lot animal feeding areas contains microorganisms that may adversely affect human and animal health if not properly managed. One alternative to full manure containment systems is a vegetative treatment system (VTS) that collects runoff in a sediment basin and then applies it to a perennial vegetation (grass) treatment area that is harvested for hay. Little is known regarding the efficacy of large-scale commercial VTSs for the removal of microbial contaminants. In this study, an active, pump-based VTS designed and built for a 1200-head beef cattle feedlot operation was examined to determine the effects of repeated feedlot runoff application on fecal indicator microorganisms and pathogens over short-term (2 wk) and long-term (3 yr) operations and whether fecal bacteria were infiltrating into deeper soils within the treatment area. In a short-term study, fecal bacteria and pathogen numbers declined over time in soil. Measurements of total coliforms and Enterococcus counts taken on control soils were not effective as fecal indicators. The repeated application of manure-impacted runoff as irrigation water did not enrich the pathogens or fecal indicators in the soil, and no evidence was seen to indicate that pathogens were moving into the deeper soil at this site. These results indicate that large-scale, active VTSs reduce the potential for environmental contamination by manure-associated bacteria. Also, this study has implications to full-containment systems that apply runoff water to land application areas (cropland) and the fate of pathogens in the soils of land application sites.

Effects of Enterococcus faecalis CECT 7121 on Cryptosporidium parvum infection in mice.

Cryptosporidium is an opportunistic protozoan parasite of humans and animals worldwide and causes diarrheal disease that is typically self-limiting in immunocompetent hosts but often life threatening to immunocompromised individuals. However, there is a lack of completely efficient therapy available. Probiotics have attracted the attention as potential antiparasite compounds against protozoa involved in intestinal infections. This study investigated the effects of administration of probiotic Enterococcus faecalis CECT 7121 on Cryptosporidium parvum infection in immunosuppressed mice. Effects on C. parvum infection at the intestinal mucosa were studied and scored at each portion of the gut. It was demonstrated that Ef CECT 7121 interfered with C. parvum infection when both probiotic and parasite were present in the same intestinal location suggesting that Ef CECT 7121 supplementation can alleviate the negative effects of C. parvum infection.

Enterocytozoon bieneusi, giardia, and Cryptosporidium infecting white-tailed deer.

Despite a white-tailed deer (WTD) population in the United States of approximately 32 million animals extremely little is known of the prevalence and species of the protists that infect these animals. This study was undertaken to determine the presence of potential human protist pathogens in culled WTD in central Maryland. Feces from fawns to adults were examined by molecular methods. The prevalence of Enterocytozoon bieneusi, Cryptosporidium, and Giardia was determined by PCR. All PCR-positive specimens were sequenced to determine the species and genotype(s). Of specimens from 80 WTD, 26 (32.5%) contained 17 genotypes of E. bieneusi. Four genotypes were previously reported (I, J, WL4, LW1) and 13 novel genotypes were identified and named DeerEb1-DeerEb13. Genotypes I, J, and LW1 are known to infect humans. Ten (12.5%) specimens contained the Cryptosporidium deer genotype, and one (1.25%) contained Giardia duodenalis Assemblage A. The identification zoonotic G. duodenalis Assemblage A as well as four E. bieneusi genotypes previously identified in humans suggest that WTD could play a role in the transmission of those parasites to humans.

Subtyping Cryptosporidium ubiquitum,a zoonotic pathogen emerging in humans.

Cryptosporidium ubiquitum is an emerging zoonotic pathogen. In the past, it was not possible to identify an association between cases of human and animal infection. We conducted a genomic survey of the species, developed a subtyping tool targeting the 60-kDa glycoprotein (gp60) gene, and identified 6 subtype families (XIIa-XIIf) of C. ubiquitum. Host adaptation was apparent at the gp60 locus; subtype XIIa was found in ruminants worldwide, subtype families XIIb-XIId were found in rodents in the United States, and XIIe and XIIf were found in rodents in the Slovak Republic. Humans in the United States were infected with isolates of subtypes XIIb-XIId, whereas those in other areas were infected primarily with subtype XIIa isolates. In addition, subtype families XIIb and XIId were detected in drinking source water in the United States. Contact with C. ubiquitum-infected sheep and drinking water contaminated by infected wildlife could be sources of human infections.