Enteric protists in wild western chimpanzees (Pan troglodytes verus) and humans in Comoé National Park, Côte d'Ivoire.

The western chimpanzee (Pan troglodytes verus), a subspecies of the common chimpanzee, is currently listed as Critically Endangered. Human-driven habitat loss and infectious diseases are causing dramatic chimpanzee population declines and range contractions that are bringing these primates to the brink of extinction. Little information is currently available on the occurrence of diarrhoea-causing enteric protist species in chimpanzees in general, and in western chimpanzees in particular, or on the role of humans as a potential source of these infections. In this prospective molecular epidemiological study, we investigated the presence, genetic variability, and zoonotic potential of enteric protists in faecal samples from western chimpanzees (n = 124) and humans (n = 9) in Comoé National Park, Côte d'Ivoire. Parasite detection and genotyping were conducted by using polymerase chain reaction (PCR) and Sanger sequencing. The protist species found in the chimpanzee samples were Entamoeba dispar (14.5%), Blastocystis sp. (11.3%), Giardia duodenalis (5.8%), Troglodytella abrassarti (2.5%) and Cryptosporidium hominis (0.8%). The protist species found in the human samples were G. duodenalis (22.2%) and Blastocystis sp. (11.1%). Entamoeba histolytica, Enterocytozoon bieneusi, and Balantioides coli were undetected in both chimpanzee and human samples. Sequence analyses revealed the presence of Blastocystis subtype (ST) 1 (alleles 4 and 8) and ST3 (allele 24) in chimpanzees, and ST3 (allele 52) in humans. ST1 allele 8 represents a chimpanzee-adapted Blastocystis genetic variant. Cross-species transmission of pathogenic enteric protists between chimpanzees and humans might be possible in Comoé National Park, although the frequency and extent of zoonotic events remain to be fully elucidated.

Presence and genetic diversity of enteric protists in captive and semi-captive non-human primates in côte d'Ivoire, Sierra Leone, and Peru.

Little information is currently available on the occurrence and genetic diversity of pathogenic and commensal protist species in captive and semi-captive non-human primates (NHP) resident in zoological gardens or sanctuaries in low- and medium-income countries. In this molecular-based study, we prospectively collected individual faecal samples from apparently healthy NHP at the Abidjan Zoological Garden (AZG) in Côte d'Ivoire, the Tacugama Sanctuary (TS) in Sierra Leone, and the Quistococha Zoological Garden (QZG) in Peru between November 2018 and February 2020. We evaluated for the presence of pathogenic (Cryptosporidium spp., Entamoeba histolytica, Giardia duodenalis, Blastocystis sp., Enterocytozoon bieneusi, Balantioides coli) and commensal (Entamoeba dispar, Troglodytella abrassarti) protist species using PCR methods and Sanger sequencing. Giardia duodenalis was the most prevalent species found (25.9%, 30/116), followed by Blastocystis sp. (22.4%, 26/116), and E. dispar (18.1%, 21/116). We detected E. bieneusi (4.2%, 1/24) and T. abrassarti (12.5%, 3/24) only on NHP from AZG. Cryptosporidium spp., E. histolytica, and B. coli were undetected at the three sampling sites investigated here. Sequence analyses revealed the presence of zoonotic sub-assemblages BIII (n = 1) in AZG and BIV (n = 1) in TS within G. duodenalis. We identified Blastocystis subtype ST3 (100%, 6/6) in AZG, ST1 (80.0%, 12/15), ST2 (6.7%, 1/15), and ST3 (13.3%, 2/15) in TS, and ST2 (80.0%, 4/5) and ST3 (20.0%, 1/5) in QZG. The only E. bieneusi isolate detected here was identified as zoonotic genotype CAF4. Our PCR-based data indicate that potentially pathogenic protist species including G. duodenalis, Blastocystis sp., E. bieneusi, and B. coli are present at variable rates in the three NHP populations investigated here. The identification of zoonotic genotypes within these species indicates that human-NHP transmission is possible, although the extent and directionality of these events need to be elucidated in future molecular surveys.

Molecular Detection and Characterization of Intestinal and Blood Parasites in Wild Chimpanzees (Pan troglodytes verus) in Senegal.

Wild chimpanzee populations in West Africa (Pan troglodytes verus) have dramatically decreased as a direct consequence of anthropogenic activities and infectious diseases. Little information is currently available on the epidemiology, pathogenic significance, and zoonotic potential of protist species in wild chimpanzees. This study investigates the occurrence and genetic diversity of intestinal and blood protists as well as filariae in faecal samples (n = 234) from wild chimpanzees in the Dindefelo Community Nature Reserve, Senegal. PCR-based results revealed the presence of intestinal potential pathogens (Sarcocystis spp.: 11.5%; Giardia duodenalis: 2.1%; Cryptosporidium hominis: 0.9%), protist of uncertain pathogenicity (Blastocystis sp.: 5.6%), and commensal species (Entamoeba dispar: 18.4%; Troglodytella abrassarti: 5.6%). Entamoeba histolytica, Enterocytozoon bieneusi, and Balantioides coli were undetected. Blood protists including Plasmodium malariae (0.4%), Trypanosoma brucei (1.3%), and Mansonella perstans (9.8%) were also identified. Sanger sequencing analyses revealed host-adapted genetic variants within Blastocystis, but other parasitic pathogens (C. hominis, P. malariae, T. brucei, M. perstans) have zoonotic potential, suggesting that cross-species transmission between wild chimpanzees and humans is possible in areas where both species overlap. Additionally, we explored potential interactions between intestinal/blood protist species and seasonality and climate variables. Chimpanzees seem to play a more complex role on the epidemiology of pathogenic and commensal protist and nematode species than initially anticipated.

Molecular Detection and Characterization of Blastocystis sp. and Enterocytozoon bieneusi in Cattle in Northern Spain.

Some enteric parasites causing zoonotic diseases in livestock have been poorly studied or even neglected. This is the case in stramenopile Blastocystis sp. and the microsporidia Enterocytozoon bieneusi in Spain. This transversal molecular epidemiological survey aims to estimate the prevalence and molecular diversity of Blastocystis sp. and E. bieneusi in cattle faecal samples (n = 336) in the province of Álava, Northern Spain. Initial detection of Blastocystis and E. bieneusi was carried out by polymerase chain reaction (PCR) and Sanger sequencing of the small subunit (ssu) rRNA gene and internal transcribed spacer (ITS) region, respectively. Intra-host Blastocystis subtype diversity was further investigated by next generation amplicon sequencing (NGS) of the ssu rRNA gene in those samples that tested positive by conventional PCR. Amplicons compatible with Blastocystis sp. and E. bieneusi were observed in 32.1% (108/336, 95% CI: 27.2-37.4%) and 0.6% (2/336, 95% CI: 0.0-1.4%) of the cattle faecal samples examined, respectively. Sanger sequencing produced ambiguous/unreadable sequence data for most of the Blastocystis isolates sequenced. NGS allowed the identification of 10 Blastocystis subtypes including ST1, ST3, ST5, ST10, ST14, ST21, ST23, ST24, ST25, and ST26. All Blastocystis-positive isolates involved mixed infections of 2-8 STs in a total of 31 different combinations. The two E. bieneusi sequences were confirmed as potentially zoonotic genotype BEB4. Our data demonstrate that Blastocystis mixed subtype infections are extremely frequent in cattle in the study area. NGS was particularly suited to discern underrepresented subtypes or mixed subtype infections that were undetectable or unreadable by Sanger sequencing. The presence of zoonotic Blastocystis ST1, ST3, and ST5, and E. bieneusi BEB4 suggest cross-species transmission and a potential risk of human infection/colonization.

Occurrence and Genetic Diversity of Protist Parasites in Captive Non-Human Primates, Zookeepers, and Free-Living Sympatric Rats in the Córdoba Zoo Conservation Centre, Southern Spain.

Little information is currently available on the epidemiology of parasitic and commensal protist species in captive non-human primates (NHP) and their zoonotic potential. This study investigates the occurrence, molecular diversity, and potential transmission dynamics of parasitic and commensal protist species in a zoological garden in southern Spain. The prevalence and genotypes of the main enteric protist species were investigated in faecal samples from NHP (n = 51), zookeepers (n = 19) and free-living rats (n = 64) by molecular (PCR and sequencing) methods between 2018 and 2019. The presence of Leishmania spp. was also investigated in tissues from sympatric rats using PCR. Blastocystis sp. (45.1%), Entamoeba dispar (27.5%), Giardia duodenalis (21.6%), Balantioides coli (3.9%), and Enterocytozoon bieneusi (2.0%) (but not Troglodytella spp.) were detected in NHP. Giardia duodenalis (10.5%) and Blastocystis sp. (10.5%) were identified in zookeepers, while Cryptosporidium spp. (45.3%), G. duodenalis (14.1%), and Blastocystis sp. (6.25%) (but not Leishmania spp.) were detected in rats. Blastocystis ST1, ST3, and ST8 and G. duodenalis sub-assemblage AII were identified in NHP, and Blastocystis ST1 in zookeepers. Giardia duodenalis isolates failed to be genotyped in human samples. In rats, four Cryptosporidium (C. muris, C. ratti, and rat genotypes IV and V), one G. duodenalis (assemblage G), and three Blastocystis (ST4) genetic variants were detected. Our results indicate high exposure of NHP to zoonotic protist species. Zoonotic transmission of Blastocysts ST1 was highly suspected between captive NHP and zookeepers.

Molecular Diversity of Giardia duodenalis, Cryptosporidium spp., and Blastocystis sp. in Symptomatic and Asymptomatic Schoolchildren in Zambézia Province (Mozambique).

Infections by the protist enteroparasites Giardia duodenalis, Cryptosporidium spp., and, to a much lesser extent, Blastocystis sp. are common causes of childhood diarrhoea in low-income countries. This molecular epidemiological study assesses the frequency and molecular diversity of these pathogens in faecal samples from asymptomatic schoolchildren (n = 807) and symptomatic children seeking medical attention (n = 286) in Zambézia province, Mozambique. Detection and molecular characterisation of pathogens was conducted by polymerase chain reaction (PCR)-based methods coupled with Sanger sequencing. Giardia duodenalis was the most prevalent enteric parasite found [41.7%, 95% confidence interval (CI): 38.8‒44.7%], followed by Blastocystis sp. (14.1%, 95% CI: 12.1‒16.3%), and Cryptosporidium spp. (1.6%, 95% CI: 0.9‒2.5%). Sequence analyses revealed the presence of assemblages A (7.0%, 3/43) and B (88.4%, 38/43) within G. duodenalis-positive children. Four Cryptosporidium species were detected, including C. hominis (30.8%; 4/13), C. parvum (30.8%, 4/13), C. felis (30.8%, 4/13), and C. viatorum (7.6%, 1/13). Four Blastocystis subtypes were also identified including ST1 (22.7%; 35/154), ST2 (22.7%; 35/154), ST3 (45.5%; 70/154), and ST4 (9.1%; 14/154). Most of the genotyped samples were from asymptomatic children. This is the first report of C. viatorum and Blastocystis ST4 in Mozambique. Molecular data indicate that anthropic and zoonotic transmission (the latter at an unknown rate) are important spread pathways of diarrhoea-causing pathogens in Mozambique.

Intestinal Protists in Captive Non-human Primates and Their Handlers in Six European Zoological Gardens. Molecular Evidence of Zoonotic Transmission.

We assessed the occurrence, genetic diversity, and zoonotic potential of four protozoan (Cryptosporidium spp., Entamoeba histolytica, Entamoeba dispar, Giardia duodenalis), one stramenopile (Blastocystis sp.), one microsporidia (Enterocytozoon bieneusi), and two ciliate (Balantioides coli, Troglodytella abrassarti) intestinal parasite or commensal protist species in captive non-human primates (NHP) and their zookeepers from six European zoological gardens in France (n = 1), Germany (n = 1), and Spain (n = 4). Faecal samples from NHP (n = 454) belonging to 63 species within 35 genera and humans (n = 70) were collected at two sampling periods in each participating institution between October 2018-August 2021. Detection and species identification was accomplished by PCR and Sanger sequencing of the ssu rRNA and/or ITS genes. Sub-genotyping analyses using specific markers were conducted on isolates positive for G. duodenalis (gdh, bg, tpi) and Cryptosporidium spp. (gp60). Overall, 41.0% (186/454) and 30.0% (21/70) of the faecal samples of NHP and human origin tested positive for at least one intestinal protist species, respectively. In NHP, Blastocystis sp. was the most prevalent protist species found (20.3%), followed by G. duodenalis (18.1%), E. dispar (7.9%), B. coli and T. abrassarti (1.5% each), and Cryptosporidium spp. and E. bieneusi (0.9% each). Occurrence rates varied largely among NHP host species, sampling periods, and zoological institutions. The predominant protist species found in humans was Blastocystis sp. (25.7%), followed by Cryptosporidium spp. (2.9%), E. dispar (1.4%), and G. duodenalis (1.4%). Sequencing of PCR-positive amplicons in human and/or NHP confirmed the presence of Cryptosporidium in six isolates (C. hominis: 66.7%, C. parvum: 33.3%), G. duodenalis in 18 isolates (assemblage A: 16.7%, assemblage B: 83.3%), Blastocystis in 110 isolates (ST1:38.2%, ST2:11.8%, ST3: 18.2%, ST4: 9.1%, ST5: 17.3%, ST8: 2.7%, ST13: 0.9%), and E. bieneusi in four isolates (CM18: 75.0%, Type IV: 25.0%). Zoonotic transmission events involving Blastocystis ST1-ST4 were identified in four zoological institutions. Zoonotic transmission of C. hominis was highly suspected, but not fully demonstrated, in one of them. Monitoring of intestinal protist species might be useful for assessing health status of captive NHP and their zookeepers, and to identify transmission pathways of faecal-orally transmitted pathogens.

First identification of genotypes of Enterocytozoon bieneusi (Microsporidia) among symptomatic and asymptomatic children in Mozambique.

Enterocytozoon bieneusi is a human pathogen with a broad range of animal hosts. Initially, E. bieneusi was considered an emerging opportunistic pathogen in immunocompromised, mainly HIV-infected patients, but it has been increasingly reported in apparently healthy individuals globally. As in other African countries, the molecular epidemiology of E. bieneusi in Mozambique remains completely unknown. Therefore, we undertook a study to investigate the occurrence and genetic diversity of E. bieneusi infections in children with gastrointestinal symptoms as well as in asymptomatic children in Mozambique. Individual stool specimens were collected from 1,247 children aged between 0 and 14 years-old living in urban and rural settings in Zambézia (n = 1,097) and Maputo (n = 150) provinces between 2016 and 2019. Samples were analysed for E. bieneusi by nested-PCR targeting the internal transcribed spacer (ITS) region of the rRNA gene. All positive amplicons were confirmed and genotyped. Penalised logistic regression (Firth) was used to evaluate risk associations. The overall prevalence of E. bieneusi in this children population was 0.7% (9/1,247). A 10-fold higher prevalence was found in Maputo (4.0%; 6/150) than in Zambézia (0.3%; 3/1,097). All E. bieneusi-positive samples were from children older than 1-year of age, and most (8/9) from asymptomatic children. Nucleotide sequence analysis of the ITS region revealed the presence of four genotypes, three previously reported (Peru11, n = 1; Type IV, n = 2, and S2, n = 2) and a novel genotype (named HhMzEb1, n = 4). Novel genotype HhMzEb1 was identified in both asymptomatic (75%, 3/4) and symptomatic (25%, 1/4) children from a rural area in Maputo province in southern Mozambique. Genotypes HhMzEb1, Peru11, S2, and Type IV belonged to the Group 1 that includes genotypes with low host specificity and the potential for zoonotic and cross-species transmission. Being infected by enteric protozoan parasites and no handwashing were identified as risk associations for E. bieneusi infection. This study reports the first investigation of E. bieneusi genotypes in Mozambique with the identification of three previously reported genotypes in humans as well as a novel genotype (HhMzEb1). Findings highlight the need to conduct additional research to elucidate the epidemiology of E. bieneusi in the country, especially in rural areas where poor hygiene conditions still prevail. Special attention should be paid to the identification of suitable animal and environmental reservoirs of this parasite and to the characterization of transmission pathways.