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.

Marked increase in cryptosporidiosis cases, Spain, 2023.

BackgroundBy mid-September 2023, several event notifications related to cryptosporidiosis had been identified from different regions in Spain. Therefore, a request for urgent notification of cryptosporidiosis cases to the National Surveillance Network was launched.AimWe aimed at assessing the extent of the increase in cases, the epidemiological characteristics and the transmission modes and compared to previous years.MethodsWe analysed data on case notifications, outbreak reports and genotypes focusing on June-October 2023 and compared the results to 2016-2022.ResultsIn 2023, 4,061 cryptosporidiosis cases were notified in Spain, which is an increase compared to 2016-2022. The cumulative incidence was 8.3 cases per 100,000 inhabitants in 2023, sixfold higher than the median of 1.4 cases per 100,000 inhabitants 2016-2022. Almost 80% of the cases were notified between June and October. The largest outbreaks were related to contaminated drinking water or swimming pools. Cryptosporidium hominis was the most common species in the characterised samples (115/122), and the C. hominis IfA12G1R5 subtype, previously unusual in Spain, was detected from 76 (62.3%) of the 122 characterised samples.ConclusionsA substantial increase in cryptosporidiosis cases was observed in 2023. Strengthening surveillance of Cryptosporidium is essential for prevention of cases, to better understand trends and subtypes circulating and the impact of adverse meteorological events.

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.

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.

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.

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.

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.

Molecular detection and characterization of Blastocystis in herbivore livestock species in Portugal.

Blastocystis is a ubiquitous intestinal protist in humans and animals worldwide. The traditional livestock free-roaming raising system in rural communities increases the risk of infection with contact with a wider range of pathogens transmitted via the faecal-oral route associated with that wildlife-livestock-human interface. However, no studies have been conducted to determine the occurrence and subtype distribution of Blastocystis in livestock in Portugal. Here, we collected 180 faecal samples from herbivore livestock (cattle, goats, horses, and sheep) in different regions of the country to investigate Blastocystis prevalence and subtype diversity using PCR and next-generation amplicon sequencing. Blastocystis was present in 40.6% (73/180; 95% CI: 33.31-48.11) of the samples (goats, 81.0%; sheep, 60.9%; cattle, 32.2%). None of the horse samples were Blastocystis-positive. Eighteen subtypes were detected (ST1-ST3, ST5-ST7, ST10, ST13, ST14, ST21, ST23-ST26, ST30, ST42-ST44). Mixed infections were detected in 97.3% of the Blastocystis-positive samples. Potentially zoonotic subtypes were identified in 75.0%, 96.4%, and 100% of the Blastocystis-positive specimens collected from cattle, sheep, and goats, respectively. These results demonstrate that cattle, sheep, and goats harbour a high diversity of Blastocystis subtypes in the study regions. Importantly, our data provide novel molecular evidence strongly suggesting that some Blastocystis STs/ST subgroups may have differential host specificity.

Microscopy detection and molecular characterisation of Giardia duodenalis infection in outpatients seeking medical care in Egypt.

Introduction: Giardiosis remains one of the most prevalent enteric parasitic infections globally. Earlier molecular-based studies conducted in Egypt have primarily focused on paediatric clinical populations and most were based on single genotyping markers. As a result, there is limited information on the frequency and genetic diversity of G. duodenalis infections in individuals of all age groups. Methods: Individual stool samples (n = 460) from outpatients seeking medical care were collected during January-December 2021 in Kafr El-Sheikh governorate, northern Egypt. Initial screening for the presence of G. duodenalis was conducted by coprological examination. Microscopy-positive samples were further confirmed by real-time PCR. A multilocus sequence typing approach targeted amplification of the glutamate dehydrogenase (gdh), beta-giardin (bg), and triose phosphate isomerase (tpi) genes was used for genotyping purposes. A standardised epidemiological questionnaire was used to gather basic sociodemographic and clinical features of the recruited patients. Results: Giardia duodenalis cysts were observed in 5.4% (25/460, 95% CI: 3.6-7.9) of the stool samples examined by conventional microscopy. The infection was more frequent in children under the age of 10 years and in individuals presenting with diarrhoea but without reaching statistical significance. Stool samples collected during the winter period were more likely to harbour G. duodenalis. All 25 microscopy-positive samples were confirmed by real-time PCR, but genotyping data was only available for 56.0% (14/25) of the isolates. Sequence analyses revealed the presence of assemblages A (78.6%, 11/14) and B (21.4%, 3/14). All assemblage A isolates were identified as sub-assemblage AII, whereas the three assemblage B sequences belonged to the sub-assemblage BIII. Patients with giardiosis presenting with diarrhoea were more frequently infected by the assemblage A of the parasite. Conclusion: This is one of the largest epidemiological studies evaluating G. duodenalis infection in individuals of all age groups in Egypt. Our molecular data suggest that G. duodenalis infections in the surveyed population are primarily of anthropic origin. However, because assemblages A and B are zoonotic, some of the infections identified can have an animal origin. Additional investigations targeting animal (domestic and free-living) and environmental (water) samples are warranted to better understand the epidemiology of giardiosis in Egypt.

Detection and Molecular Diversity of Cryptosporidium spp. and Giardia duodenalis in the Endangered Iberian Lynx (Lynx pardinus), Spain.

Cryptosporidium spp. and Giardia duodenalis are the main non-viral causes of diarrhoea in humans and domestic animals globally. Comparatively, much less information is currently available in free-ranging carnivore species in general and in the endangered Iberian lynx (Lynx pardinus) in particular. Cryptosporidium spp. and G. duodenalis were investigated with molecular (PCR and Sanger sequencing) methods in individual faecal DNA samples of free-ranging and captive Iberian lynxes from the main population nuclei in Spain. Overall, Cryptosporidium spp. and G. duodenalis were detected in 2.4% (6/251) and 27.9% (70/251) of the animals examined, respectively. Positive animals to at least one of them were detected in each of the analysed population nuclei. The analysis of partial ssu rRNA gene sequences revealed the presence of rodent-adapted C. alticolis (n = 1) and C. occultus (n = 1), leporid-adapted C. cuniculus (n = 2), and zoonotic C. parvum (n = 2) within Cryptosporidium, and zoonotic assemblages A (n = 5) and B (n = 3) within G. duodenalis. Subgenotyping analyses allowed for the identification of genotype VaA19 in C. cuniculus (gp60 locus) and sub-assemblages AI and BIII/BIV in G. duodenalis (gdh, bg, and tpi loci). This study represents the first molecular description of Cryptosporidium spp. and G. duodenalis in the Iberian lynx in Spain. The presence of rodent/leporid-adapted Cryptosporidium species in the surveyed animals suggests spurious infections associated to the Iberian lynx's diet. The Iberian lynx seems a suitable host for zoonotic genetic variants of Cryptosporidium (C. parvum) and G. duodenalis (assemblages A and B), although the potential risk of human transmission is regarded as limited due to light parasite burdens and suspected low excretion of infective (oo)cysts to the environment by infected animals. More research should be conducted to ascertain the true impact of these protozoan parasites in the health status of the endangered Iberian lynx.

Prevalence and associated risk factors of intestinal parasites among schoolchildren in Ecuador, with emphasis on the molecular diversity of Giardia duodenalis, Blastocystis sp. and Enterocytozoon bieneusi.

BACKGROUND: Intestinal helminths, including Soil-Transmitted Helminth (STH), and Gastrointestinal Protist (GP) infections are major contributors to the global burden of disease, particularly in low-income countries such Ecuador. Their epidemiology in these settings is largely unknown. METHODOLOGY: This prospective cross-sectional study investigates the carriage of intestinal helminths, including STH, and GP in asymptomatic schoolchildren (3-11 years) in the Chimborazo and Guayas provinces, Ecuador. Single stool samples (n = 372) and epidemiological questionnaires on demographics and potential risk factors were collected from participating schoolchildren. Conventional microscopy examination was used as screening method, and molecular (PCR and Sanger sequencing) assays were used to further investigate the epidemiology of some GP. A multivariate logistic regression analysis was used to evaluate the strength of the association of suspected risk factors with the presence of helminths and GP. PRINCIPAL FINDINGS: At least one intestinal parasite species was observed by microscopy in 63.2% (235/372) of the participating schoolchildren. Enterobius vermicularis (16.7%, 62/372; 95% CI: 13.0-20.9) and Blastocystis sp. (39.2%, 146/372; 95% CI: 34.2-44.2) were the most prevalent among helminths and GP, respectively. Assemblages A (50.0%), B (37.5%) and A+B (12.5%) were detected within Giardia duodenalis and ST3 (28.6%), ST1 and ST2 (26.2% each), and ST4 (14.3%) within Blastocystis sp. Three genotypes, two known (A: 66.7%; KB-1: 16.7%) and a novel (HhEcEb1, 16.7%) were identified within Enterocytozoon bieneusi. Municipality of origin, household overcrowding, and poor sanitation and personal hygiene habits were risk factors for childhood intestinal parasites colonization. CONCLUSIONS/SIGNIFICANCE: Despite massive government drug administration programs, STH and GP infection remain a public health concern in paediatric populations living in poor-resource settings. Molecular analytical methods are required to better understand the epidemiology of these intestinal parasites. This study provides novel information on the occurrence of Blastocystis sp. and E. bieneusi genetic variants circulating in Ecuadorian human populations.

Prevalence and public health relevance of enteric parasites in domestic dogs and cats in the region of Madrid (Spain) with an emphasis on Giardia duodenalis and Cryptosporidium sp.

BACKGROUND: Pet dogs and cats exert an unquestionable beneficial effect in the well-being of their owners, but can also act as a source of zoonotic infections if improperly cared. OBJECTIVES: We investigated the occurrence, risk factors, genetic variability and zoonotic potential of intestinal parasites in dogs and cats attended in a clinical veterinary setting in Spain. METHODS: Canine (n = 252) and feline (n = 35) faecal samples were collected during 2017-2019 and analysed by coproparasitological methods. A rapid lateral immunochromatographic test (ICT) was used for detecting Giardia duodenalis and Cryptosporidium sp. Samples positive at microscopy examination and/or ICT were reassessed by molecular methods. RESULTS: Overall, 48.8% (123/252) of dogs and 48.6% (17/35) of cats were infected by enteric parasites. In dogs, G. duodenalis was the most prevalent species (40.9%), followed by Cystoisospora sp. (7.1%), and Toxocara canis (5.2%). In cats, Joyeuxiella sp. and Toxocara cati were the dominant species (20.0% each), followed by G. duodenalis (14.3%), D. caninum (5.7%) and Cystoisospora felis and Toxascaris leonina (2.9% each). Pups and kittens were more likely to harbour intestinal parasites and develop clinical signs. Sequence analyses of dog isolates revealed the presence of assemblages A (n = 1), C (n = 4), D (n = 4) and C+D (n = 1) within G. duodenalis; C. parvum (n = 1) and C. canis (n = 4) within Cryptosporidium and PtEb IX (n = 1) in Enterocytozoon bieneusi. A novel C. canis subtype family, named XXi, is reported. CONCLUSIONS: Our results highlight that (i) well-cared dogs carry zoonotic enteric protozoan parasites of public health relevance, (ii) proper hygiene practices and routine veterinary treatment are essential to prevent zoonotic infections, (iii) vulnerable populations should avoid contact with pups/kittens with diarrhoea and (iv) infected dogs might be major contributors to the environmental contamination with soil-transmitted helminths (STHs) eggs.

Molecular detection and characterization of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi infections in dromedary camels (Camelus dromedaries) in Egypt.

Introduction: Few studies have investigated the occurrence of microeukaryotic gut parasites in dromedary camels in Egypt, and the majority of these investigations are based on microscopic analysis of fecal material. Methods: Herein, we assessed the occurrence, molecular diversity, and zoonotic potential of protozoan (Cryptosporidium spp. and Giardia duodenalis) and microsporidian (Enterocytozoon bieneusi) pathogens in individual fecal samples (n = 102) of dromedary camels with (n = 26) and without (n = 76) diarrhea from Aswan Governorate, Upper Egypt. Other factors possibly associated with an increased risk of infection (geographical origin, sex, age, and physical condition) were also analyzed. The SSU rRNA or ITS genes were targeted by molecular (PCR and Sanger sequencing) techniques for pathogen detection and species identification. Results and discussion: The most abundant species detected was G. duodenalis (3.9%, 4/102; 95% CI: 1.1-9.7), followed by Cryptosporidium spp. (2.9%, 3/102; 95% CI: 0.6-8.4). All samples tested negative for the presence of E. bieneusi. Sequence analysis data confirmed the presence of zoonotic C. parvum (66.7%, 2/3) and cattle-adapted C. bovis (33.3%, 1/3). These Cryptosporidium isolates, as well as the four Giardia-positive isolates, were unable to be amplified at adequate genotyping markers (Cryptosporidium: gp60; Giardia: gdh, bg, and tpi). Camels younger than 2 years old were significantly more likely to harbor Cryptosporidium infections. This connection was not statistically significant, although two of the three cryptosporidiosis cases were detected in camels with diarrhea. The spread of G. duodenalis infections was unaffected by any risk variables studied. This is the first report of C. parvum and C. bovis in Egyptian camels. The finding of zoonotic C. parvum has public health implications since camels may function as sources of oocyst pollution in the environment and potentially infect livestock and humans. Although preliminary, this study provides useful baseline data on the epidemiology of diarrhea-causing microeukaryotic parasites in Egypt. Further research is required to confirm and expand our findings in other animal populations and geographical regions of the country.

High Diversity of Giardia duodenalis Assemblages and Sub-Assemblages in Asymptomatic School Children in Ibadan, Nigeria.

Giardia duodenalis is a significant contributor to the burden of diarrheal disease in sub-Saharan Africa. This study assesses the occurrence and molecular diversity of G. duodenalis and other intestinal parasites in apparently healthy children (n = 311) in Ibadan, Nigeria. Microscopy was used as a screening method and PCR and Sanger sequencing as confirmatory and genotyping methods, respectively. Haplotype analyses were performed to examine associations between genetic variants and epidemiological variables. At microscopy examination, G. duodenalis was the most prevalent parasite found (29.3%, 91/311; 95% CI: 24.3-34.7), followed by Entamoeba spp. (18.7%, 58/311; 14.5-23.4), Ascaris lumbricoides (1.3%, 4/311; 0.4-3.3), and Taenia sp. (0.3%, 1/311; 0.01-1.8). qPCR confirmed the presence of G. duodenalis in 76.9% (70/91) of the microscopy-positive samples. Of them, 65.9% (60/91) were successfully genotyped. Assemblage B (68.3%, 41/60) was more prevalent than assemblage A (28.3%, 17/60). Mixed A + B infections were identified in two samples (3.3%, 2/60). These facts, together with the absence of animal-adapted assemblages, suggest that human transmission of giardiasis was primarily anthroponotic. Efforts to control G. duodenalis (and other fecal-orally transmitted pathogens) should focus on providing safe drinking water and improving sanitation and personal hygiene practices.

Molecular characteristics and zoonotic potential of enteric protists in domestic dogs and cats in Egypt.

Introduction: Domestic dogs and cats can be a source of human infection by a wide diversity of zoonotic pathogens including parasites. Genotyping and subtyping tools are useful in assessing the true public health relevance of canine and feline infections by these pathogens. This study investigated the occurrence, genetic diversity, and zoonotic potential of common diarrhea-causing enteric protist parasites in household dogs and cats in Egypt, a country where this information is particularly scarce. Methods: In this prospective, cross-sectional study a total of 352 individual fecal samples were collected from dogs (n = 218) and cats (n = 134) in three Egyptian governorates (Dakahlia, Gharbeya, and Giza) during July-December 2021. Detection and identification of Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, and Blastocystis sp. were carried out by PCR and Sanger sequencing. Basic epidemiological variables (geographical origin, sex, age, and breed) were examined for association with occurrence of infection by enteric protists. Results and discussion: The overall prevalence rates of Cryptosporidium spp. and G. duodenalis were 1.8% (95% CI: 0.5-4.6) and 38.5% (95% CI: 32.0-45.3), respectively, in dogs, and 6.0% (95% CI: 2.6-11.4) and 32.1% (95% CI: 24.3-40.7), respectively, in cats. All canine and feline fecal samples analyzed tested negative for E. bieneusi and Blastocystis sp. Dogs from Giza governorate and cats from Dakahlia governorate were at higher risk of infection by Cryptosporidium spp. (p = 0.0006) and G. duodenalis (p = 0.00001), respectively. Sequence analyses identified host-adapted Cryptosporidium canis (n = 4, one of them belonging to novel subtype XXe2) and G. duodenalis assemblages C (n = 1) and D (n = 3) in dogs. In cats the zoonotic C. parvum (n = 5) was more prevalent than host-adapted C. felis (n = 1). Household dogs had a limited (but not negligible) role as source of human giardiasis and cryptosporidiosis, but the unexpected high frequency of zoonotic C. parvum in domestic cats might be a public health concern. This is the first molecular-based description of Cryptosporidium spp. infections in cats in the African continent to date. Molecular epidemiological data provided here can assist health authorities and policy makers in designing and implementing effective campaigns to minimize the transmission of enteric protists in Egypt.

Iberian wild leporidae as hosts of zoonotic enteroparasites in Mediterranean ecosystems of Southern Spain.

Wild lagomorphs including rabbits and hares can act as natural carriers or reservoirs of bacterial and parasitic zoonotic diseases. However, little is known on the epidemiology and potential public health significance of intestinal eukaryotes in wild leporids. We examined faecal samples from European wild rabbits (Oryctolagus cuniculus, n = 438) and Iberian hares (Lepus granatensis, n = 111) collected in the Autonomous Region of Andalusia in southern Spain during 2012-2021. We searched for the presence of DNA from the main intestinal protist and microsporidial pathogens of veterinary and public health concerns using molecular methods (PCR followed by Sanger and next-generation sequencing). Giardia duodenalis was the most prevalent species found (27.8%, 153/550; 95% CI: 24.1-31.8), followed by Cryptosporidium spp. (1.3%, 7/550; 95% CI: 0.5-2.6), Blastocystis sp. (1.1%, 6/550; 95% CI: 0.4-2.4) and Encephalitozoon intestinalis (0.2%, 1/550; 95% CI: 0.0-10.1). All samples tested negative for Enterocytozoon bieneusi. Sequence analyses revealed the presence of sub-assemblage BIV (n = 1) within G. duodenalis, and Cryptosporidium cuniculus (n = 6) and Cryptosporidium andersoni (n = 1) within Cryptosporidium. The presence of ruminant-adapted C. andersoni is indicative of a potential cross-species transmission event, although a spurious infection (mechanical carriage) cannot be ruled out. Samples assigned to C. cuniculus belonged to the gp60 subtype families Va (n = 3) and Vb (n = 2). The six Blastocystis-positive samples were identified as ST2 (n = 3) and ST1 + ST2 (n = 3). Our molecular results suggest that wild rabbits and hares were primarily infected by leporid-adapted species of eukaryotic pathogens. However, the occasional findings of zoonotic G. duodenalis sub-assemblage BIV, Blastocystis sp. ST1 and ST2, and Encephalitozoon intestinalis could be of public health relevance.