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.

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.

Enterocytozoon bieneusi and Encephalitozoon intestinalis (microsporidia) in HIV-positive patients in central Spain.

Microsporidia are fungi-related eukaryotic intracellular parasites that opportunistically infect immunocompromised individuals such as those infected by the human immunodeficiency virus (HIV). Among them, Enterocytozoon bieneusi and Encephalitozoon spp. are the most clinically relevant species. We investigated the occurrence and genetic diversity of microsporidial and protist infections in mostly immunocompetent HIV-positive patients in Madrid, Spain. A structured questionnaire was used to retrieve data on factors potentially associated with an increased risk of infection, including sexual attitudes and sex-risk behaviour. Faecal samples (n = 96) from 81 HIV-positive patients were collected and analysed by molecular (PCR and Sanger sequencing) methods. Two microsporidial pathogens were detected: Ent. bieneusi (2.5%, 95% CI: 0.3-8.6) and Enc.intestinalis (4.9%, 95% CI: 1.4-12.2). The two Ent. bieneusi isolates were identified as zoonotic genotype A. Among protists, Entamoeba dispar was the species most prevalently found (33.3%, 95% CI: 23.2-44.7), followed by Blastocystis spp. (19.8%, 95% CI: 11.7-30.1), Giardia duodenalis (13.6%, 95% CI: 7.0-23.0), and Cryptosporidium spp. and Entamoeba histolytica (2.5%, 95% CI: 0.3-8.6 each). Cyclospora cayetanensis and Cystoisospora belli were not detected. Subtypes ST1 (70.6%, 12/17) and ST3 (29.4%, 5/17) were identified within Blastocystis sp., sub-assemblages AII and BIII (50%, 1/2 each) within G. duodenalis, and Cry. parvum and canine-adapted Cry. canis (50%, 1/2 each) within Cryptosporidium spp. Microsporidial and protist parasites were frequent in well-controlled, mostly immunocompetent HIV-positive patients and should be included in diagnostic algorithms when diarrhoea is present.

Opportunistic microsporidial and protist intestinal infections were relatively common in well-controlled HIV-positive patients in Madrid, Spain. These agents should be suspected and appropriately diagnosed in HIV-positive patients presenting with diarrhoea regardless of their immunological status.

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.

Prevalence and temporal dynamics of Cryptosporidium spp., Giardia duodenalis, and Blastocystis sp. among toddlers attending day-care centres in Spain. A prospective molecular-based longitudinal study.

Giardia duodenalis, Cryptosporidium spp., and Blastocystis sp. are common intestinal eukaryotic parasites affecting children in developed and resource-limited countries. Lack of information on the epidemiology and long-term stability in asymptomatic children complicates interpretation of transmission and pathogenesis. To assess the occurrence, genetic diversity, and temporal dynamics of intestinal eukaryotic parasites in young children, 679 stool samples from 125 toddlers attending six public day-care centres in Central Spain were collected bimonthly within a 1-year period. Detection and identification of species/genotypes were based on PCR and Sanger sequencing methods. Four eukaryotic species were identified: G. duodenalis (2.5‒31.6%), Cryptosporidium spp. (0.0‒2.4%), Blastocystis sp. (2.5‒6.4%), and Entamoeba dispar (0.0‒0.9%). Entamoeba histolytica and Enterocytozoon bieneusi were undetected. Sequence analyses identified assemblage A (63.6%) and B (36.4%) within G. duodenalis (n = 11), C. hominis (40%), C. parvum (40%), and C. wrairi (20%) within Cryptosporidium spp. (n = 5), and ST1 (3.8%), ST2 (46.2%), ST3 (15.4%), and ST4 (34.6%) within Blastocystis sp. (n = 26). Giardia duodenalis sub-assemblage AII/AIII was detected in a toddler for 10 consecutive months. Stable carriage of Blastocystis ST2 allele 9, ST3 allele 34, and ST4 allele 42 was demonstrated in five toddlers for up to 1 year.   Conclusions: Giardia duodenalis and Blastocystis sp. were common in toddlers attending day-care centres in Central Spain. Long-term infection/colonization periods by the same genetic variant were observed for G. duodenalis (up to 10 months) and Blastocystis sp. (up to 12 months). What is Known: • Asymptomatic carriage of G. duodenalis and Blastocystis sp. is frequent in toddlers. • The epidemiology and long-term stability of these eukaryotes in asymptomatic young children is poorly understood. What is New: • Long-term colonization/infection periods by the same genetic variant were described for Blastocystis sp. (up to 12 months) and G. duodenalis (up to 10 months).

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.

First report of rodent-adapted Cryptosporidium wrairi in an immunocompetent child, Spain.

Cryptosporidiosis is a leading cause of childhood diarrhoea. Two species, Cryptosporidium hominis and Cryptosporidium parvum, are responsible for most confirmed cases globally. Close contact with pet animals can be an unnoticed source of children infections. We describe a case of infection by rodent-adapted Cryptosporidium wrairi in a 22-month-old immunocompetent toddler with no clinical manifestations in close contact with a pet guinea pig and poor personal hygiene practices in Majadahonda (Madrid, Spain). Attempts to determine the C. wrairi genotype family at the 60-kDa glycoprotein marker failed repeatedly. This is the first description of C. wrairi in a human host. Although a spurious infection cannot be completely ruled out, data presented here suggest that C. wrairi can be transmitted zoonotically.

Detection of enteric parasites and molecular characterization of Giardia duodenalis and Blastocystis sp. in patients admitted to hospital in Ankara, Turkey.

This epidemiological study assesses the occurrence of enteric parasites in 4303 patients attended at two public hospitals in Ankara (Turkey) during 2018-2019. Microscopy was used as a screening test. Giardia duodenalis was also identified using a commercial ELISA for the detection of parasite-specific coproantigens. Giardia-positive samples by microscopy/ELISA were confirmed by real-time PCR and characterized using a multilocus genotyping scheme. Blastocystis sp. was genotyped in a sample subset. Blastocystis sp. (11.1%, 95% CI 11.4‒14.8%) and G. duodenalis (1.56%, 95% CI 1.22‒1.96) were the most prevalent pathogens found. Cryptosporidium spp., Entamoeba histolytica and intestinal helminths were only sporadically (<0.5%) found. For G. duodenalis, sequence (n = 30) analyses revealed the presence of sub-assemblages AII (23.3%), discordant AII/AIII (23.3%) and mixed AII + AIII (6.7%) within assemblage A, and BIII (10.0%), BIV (3.3%) and discordant BIII/BIV (23.3%) within assemblage B. Two additional sequences (6.7%) were assigned to the latter assemblage but sub-assemblage information was unknown. No associations between G. duodenalis assemblages/sub-assemblages and sociodemographic and clinical variables could be demonstrated. For Blastocystis sp., sequence (n = 6) analyses identified subtypes ST1, ST2 and ST3 at equal proportions. This is the first molecular characterization of G. duodenalis based on MLG conducted in Turkey to date.

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.

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.

Substantial prevalence of enteroparasites Cryptosporidium spp., Giardia duodenalis and Blastocystis sp. in asymptomatic schoolchildren in Madrid, Spain, November 2017 to June 2018.

BackgroundProtozoan enteroparasites Cryptosporidium species and Giardia duodenalis are major contributors to the burden of gastrointestinal illness in children globally, whereas the stramenopile Blastocystis species has been associated with irritable bowel syndrome and skin disorders.AimTo investigate the carriage of these parasites in voluntary asymptomatic schoolchildren (4‒14 years) in 12 different primary and secondary schools in Leganés (Madrid, Spain).MethodsIn a prospective cross-sectional study, stool samples and epidemiological questionnaires on demographics and potential risk factors were collected from participating schoolchildren. Detection of enteric parasites was conducted by PCR-based methods and confirmed by sequence analysis. We calculated prevalence and odds ratios (OR) with logistic regression.ResultsStool samples and questionnaires were provided by 1,359 schoolchildren from 12 schools. The individual prevalence for any parasite was 28%; Blastocystis sp.: 13%; G. duodenalis: 18%; Cryptosporidium spp.: 1%. Two schoolchildren were infected with all three species and 53 with two species. Multivariable risk factor analysis using logistic regression models indicated that an existing infection with one parasite increased the odds for an additional infection with another parasite. The odds of Blastocystis sp. carriage increased up to the age of 10 years and being female increased the odds of Cryptosporidium spp. infection. Washing vegetables before preparing a meal was protective for Blastocystis sp. infection.ConclusionWe detected a larger than expected proportion of asymptomatic cases in the participanting schoolchildren. Further investigation of asymptomatic children should be considered. Good hygiene measures should be encouraged for individuals of all ages to protect from protozoal infections.

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.

Occurrence and molecular epidemiology of Giardia duodenalis infection in dog populations in eastern Spain.

BACKGROUND: Giardia duodenalis is one of the most common enteric parasites in domestic animals including dogs. Young animals are more prone to the infection, with clinical manifestations ranging from asymptomatic to acute or chronic diarrhoea. Dogs are primarily infected by canine-specific (C-D) assemblages of G. duodenalis. However, zoonotic assemblages A and B have been increasingly documented in canine isolates, raising the question of whether and to which extent dogs can act as natural reservoirs of human giardiosis. METHODS: In this cross-sectional epidemiological survey we assessed the molecular diversity of G. duodenalis in dogs in the province of Castellón, Eastern Spain. A total of 348 individual faecal samples from sheltered (n = 218), breeding (n = 24), hunting (n = 68), shepherd (n = 24), and pet (n = 14) dogs were collected between 2014 and 2016. Detection of G. duodenalis cysts in faecal material was carried out by direct fluorescence microscopy as a screening test, whereas a qPCR targeting the small subunit ribosomal RNA gene of the parasite was subsequently used as a confirmatory method. RESULTS: Giardia duodenalis was detected in 36.5% (95% CI: 31.6-41.7%) of dogs. No significant differences in prevalence rates could be demonstrated among dogs according to their sex and geographical origin, but breeding (45.8%; 95% CI: 27.9-64.9%) and sheltered (40.4%; 95% CI: 34.1-47.0%) dogs harboured significantly higher proportions of G. duodenalis. Multi-locus sequence-based genotyping of the glutamate dehydrogenase and ß-giardin genes of G. duodenalis allowed the characterization of 35 canine isolates that were unambiguously assigned to assemblages A (14.3%), B (22.9%), C (5.7%), and D (37.1%). A number of inter-assemblage mixed infections including A + B (11.4%), A + D (2.9%), and A + B + D (5.7%) were also identified. CONCLUSIONS: Data presented here are strongly indicative of high infection pressures in kennelled animals. Zoonotic sub-assemblages AII, BIII, and BIV were responsible for a considerable proportion of the G. duodenalis infections detected, but very few of the genotypes identified have been previously documented in Spanish human populations. Although possible, zoonotic transmission between dogs and humans seems an infrequent event in this Spanish region.

Molecular genotyping of Giardia duodenalis in children from Behbahan, southwestern Iran.

Giardia duodenalis is an intestinal flagellated protozoan that infects humans and several animal species. Giardiasis causing more than 200 million symptomatic infections globally is one of the most common causes of diarrhea in developing countries. Based on molecular studies mainly targeting the small-subunit (SSU) rRNA gene locus of the parasite, eight assemblages (A to H) have been identified in humans and other animal species. The aim of the current study was to evaluate the frequency and molecular diversity of G. duodenalis in children from rural and urban day care centers from Behbahan, southwestern Iran. This cross-sectional study was based on a concentration method for the microscopic detection of G. duodenalis in stool samples of 450 children, aged 1-7 years, in Behbahan, southwestern Iran. The survey was conducted from December 2015 to May 2016. PCR methods targeting the SSU rRNA and triose phosphate isomerase (TPI) genes of G. duodenalis were used for the identification and genotyping of the parasite isolates. Based on sucrose flotation and microscopy techniques, 2.7% (12/450) of children were infected with G. duodenalis, of which six (50.0%) were males and the other six (50.0%) were females. Overall, 91.7% (11/12) of the infections were detected in children from rural areas. The SSU rRNA and TPI genes were amplified successfully in nine and eight, respectively, of the Giardia-positive samples at microscopy. Among the eight TPI sequences, assemblage A, sub-assemblage AII, was identified in five of the isolates. The sequences of the three remaining samples were untypable. Although no significantly statistical difference between genotype and clinical symptoms was found, five out of the eight isolates identified as assemblage A were obtained in asymptomatic children. Giardia duodenalis infections were more prevalent in children from rural day care schools, and the predominant assemblage was A, sub-assemblage AII. The higher prevalence of giardiasis in rural areas might be related to differences in personal hygiene habits, parents' education level, source of drinking water, and inadequate hygienic toilet facilities in rural areas.

Structure of mammalian AMPK and its regulation by ADP.

The heterotrimeric AMP-activated protein kinase (AMPK) has a key role in regulating cellular energy metabolism; in response to a fall in intracellular ATP levels it activates energy-producing pathways and inhibits energy-consuming processes. AMPK has been implicated in a number of diseases related to energy metabolism including type 2 diabetes, obesity and, most recently, cancer. AMPK is converted from an inactive form to a catalytically competent form by phosphorylation of the activation loop within the kinase domain: AMP binding to the γ-regulatory domain promotes phosphorylation by the upstream kinase, protects the enzyme against dephosphorylation, as well as causing allosteric activation. Here we show that ADP binding to just one of the two exchangeable AXP (AMP/ADP/ATP) binding sites on the regulatory domain protects the enzyme from dephosphorylation, although it does not lead to allosteric activation. Our studies show that active mammalian AMPK displays significantly tighter binding to ADP than to Mg-ATP, explaining how the enzyme is regulated under physiological conditions where the concentration of Mg-ATP is higher than that of ADP and much higher than that of AMP. We have determined the crystal structure of an active AMPK complex. The structure shows how the activation loop of the kinase domain is stabilized by the regulatory domain and how the kinase linker region interacts with the regulatory nucleotide-binding site that mediates protection against dephosphorylation. From our biochemical and structural data we develop a model for how the energy status of a cell regulates AMPK activity.

Sarcocystis arctica (Apicomplexa: Sarcocystidae): ultrastructural description and its new host record, the Alaskan wolf (Canis lupus).

Sarcocystis sarcocysts are common in muscles of herbivores but are rare in muscles of carnivores. Here, we report sarcocysts in the muscles of a gray wolf (Canis lupus) from Alaska, USA, for the first time. Sarcocysts extracted from the tongue of the wolf were up to 900 µm long and slender and appeared to have a relatively thin wall by light microscope. By transmission electron microscopy, the sarcocyst wall most closely resembled "type 9c," and had a wavy parasitophorous vacuolar membrane folded as pleomorphic villar protrusions (vp), with anastomoses of tips. The vp and the ground substance (gs) layer were smooth without tubules or granules. The gs was up to 2.0 µm thick. The total width of the wall including vp and the gs was 3.5 µm. The vp were up to 1.5 µm long. Mature sarcocysts contained numerous bradyzoites and few metrocytes. The bradyzoites were 9.5 µm long and 1.5 µm wide, and contained all organelles found in Sarcocystis bradyzoites with at least two rhoptries. Molecular characterization showed the highest identity for 18S rRNA, 28S rRNA, ITS-1, and cox1 sequences of Sarcocystis arctica of the Arctic fox (Vulpes lagopus) from Norway. The ultrastructure of S. arctica from the fox is unknown. Here, we provide ultrastructure of S. arctica from the Alaskan wolf for the first time. The definitive host of S. arctica remains unknown.

Detection and molecular characterisation of Giardia duodenalis, Cryptosporidium spp. and Entamoeba spp. among patients with gastrointestinal symptoms in Gambo Hospital, Oromia Region, southern Ethiopia.

OBJECTIVES: To assess the prevalence and genetic diversity of the enteric protozoa species G. duodenalis, Cryptosporidium spp. and Entamoeba histolytica in individuals with gastrointestinal symptoms compatible with infections by these pathogens seeking medical attention in a rural area in southern Ethiopia. METHODS: A total of 92 stool samples were initially screened by direct microscopy and immunochromatography and further confirmed by molecular methods. G. duodenalis-positive samples were molecularly characterised by multilocus genotyping of the glutamate dehydrogenase and ß-giardin genes of the parasite. PCR and DNA sequence analysis of the gene encoding the 60-kDa glycoprotein was used for the subtyping of Cryptosporidium isolates. Detection and differential diagnosis of E. histolytica/dispar were conducted by real-time PCR. RESULTS: PCR-based prevalences were 10.9% for G. duodenalis, 1.1% for Cryptosporidium spp. and 3.3% for Entamoeba spp. Seven (four novel and three known) subtypes of G. duodenalis assemblage B were identified at the GDH locus and 5 (one novel and four known) at the BG locus. A novel variant of C. hominis subtype IbA9G3 was also identified. Two Entamoeba isolates were assigned to E. dispar and an additional one to E. histolytica. CONCLUSION: Although preliminary, our results strongly suggest that giardiasis, cryptosporidiosis and amoebiasis represent a significant burden in Ethiopian rural population.

Cryptosporidium occultus in disguise.

As data accumulate in GenBank, the difficulties of delineating species of Cryptosporidium based on nuclear small subunit ribosomal RNA (ssu rRNA) gene information alone becomes increasingly evident. Here, we summarize currently available evidence suggesting that several ssu rDNA sequences primarily referred to as Cryptosporidium suis (some of them from non-suid hosts) should be considered Cryptosporidium occultus.

Evaluation of the Use of Singleplex and Duplex CerTest VIASURE Real-Time PCR Assays to Detect Common Intestinal Protist Parasites.

Cryptosporidium spp., Giardia duodenalis and Entamoeba histolytica are species of protozoa- causing diarrhoea that are common worldwide, while Entamoeba dispar, Dientamoeba fragilis and Blastocystis sp. appear to be commensal parasites whose role in pathogenicity remains controversial. We conducted the clinical evaluation of five singleplex and one duplex CerTest VIASURE Real-Time PCR Assays against a large panel of positive DNA samples (n = 358), and specifically to Cryptosporidium spp. (n = 96), G. duodenalis (n = 115), E. histolytica (n = 25) E. dispar (n = 11), Blastocystis sp. (n = 42), D. fragilis (n = 37), and related parasitic phylum species such as Apicomplexa, Euglenozoa, Microsporidia and Nematoda. DNA samples were obtained from clinical stool specimens or cultured isolates in a national reference centre. Estimated diagnostic sensitivity and specificity values were 0.94-1 for Cryptosporidium spp., 0.96-0.99 for G. duodenalis, 0.96-1 for E. histolytica, 1-1 for E. dispar, and 1-0.99 for D. fragilis in the evaluated singleplex assays. In the duplex assay for the simultaneous detection of Blastocystis sp. and D. fragilis these values were 1-0.98 and 1-0.99, respectively. Measures of diagnostic precision for repeatability and reproducibility were found to be under acceptable ranges. The assays identified six Cryptosporidium species (C. hominis, C. parvum, C. canis, C. felis, C. scrofarum, and C. ryanae), four G. duodenalis assemblages (A, B, C, and F), and six Blastocystis subtypes (ST1-ST5, and ST8). The evaluated singleplex and duplex VIASURE Real-Time PCR assays provide sensitive, practical, and cost-effective choices to the molecular diagnosis of the main diarrhoea-causing intestinal protists in clinical microbiology and research laboratories.