Genetic identification of the ciliates from greater rheas (Rhea americana) and lesser rheas (Rhea pennata) as Balantioides coli.

The ciliate species Balantioides coli can be cross-transmitted between humans and several animal species. Usually harmless, sometimes it can be pathogenic and cause the death of the host. In birds, B. coli has been confirmed in ostriches by genetic analysis, but the identification from South American greater rheas (Rhea americana) and lesser rheas (Rhea pennata pennata) is tentative. Since these species are reared for commercial purposes and for reintroduction into the wild, it is necessary to elucidate whether the ciliate from rheas is B. coli to minimize health risks for humans and for other domestic and wild animals. Individual parasite cells are collected from Argentinean isolates of reared greater rheas and of wild and reared lesser rheas, and their ITS region was PCR amplified; the cloning products were sequenced and compared with sequences available in public databases. The results have shown that several sequence types are expressed at the same time in the parasite cells, and all correspond to B. coli, confirming the possibility of cross-transmission of the parasite between wild and reared South American rheas and several mammal species and humans.

Morphological Redescription of Opalina undulata Nie 1932 from Fejervarya limnocharis with Molecular Phylogenetic Study of Opalinids (Heterokonta, Opalinea).

The redescription of Opalina undulata Nie 1932, collected from the rectum of the frog Fejervarya limnocharis, is presented in this paper based on detailed morphological information and molecular data. Our results revealed that specimens collected from Diaocha Lake in late August were larger and had more nuclei than those collected from the same site in early May. We sequenced their SSU rDNA-ITS1-5.8S rDNA-ITS2-LSU rDNA (5' end) and found that they were completely identical, which means that the two populations belonged to the same species. These facts gave us a hint that body dimension and number of nuclei are not reliable taxonomic parameters for opalinids during their life cycle. Therefore, we recommended that the specific identification of opalinids based on morphological features should be carried out during seasons except spring. Meanwhile, our molecular phylogenetic analysis confirmed the monophyly of Opalinata. Within Opalinata, Opalinea were monophyletic with all opalinid species grouping together. Karotomorpha and Proteromonas did not group together confirming the paraphyly of Proteromonadea.

Molecular phylogeny based on six nuclear genes suggests that Echinococcus granulosus sensu lato genotypes G6/G7 and G8/G10 can be regarded as two distinct species.

Tapeworms of the species complex of Echinococcus granulosus sensu lato (s. l.) are the cause of a severe zoonotic disease - cystic echinococcosis, which is listed among the most severe parasitic diseases in humans and is prioritized by the World Health Organization. A stable taxonomy of E. granulosus s. l. is essential to the medical and veterinary communities for accurate and effective communication of the role of different species in this complex on human and animal health. E. granulosus s. l. displays high genetic diversity and has been divided into different species and genotypes. Despite several decades of research, the taxonomy of E. granulosus s. l. has remained controversial, especially the species status of genotypes G6-G10. Here the Bayesian phylogeny based on six nuclear loci (7387 bp in total) demonstrated, with very high support, the clustering of G6/G7 and G8/G10 into two separate clades. According to the evolutionary species concept, G6/G7 and G8/G10 can be regarded as two distinct species. Species differentiation can be attributed to the association with distinct host species, largely separate geographical distribution and low level of cross-fertilization. These factors have limited the gene flow between genotypic groups G6/G7 and G8/G10, resulting in the formation of distinct species. We discuss ecological and epidemiological differences that support the validity of these species.

Genetic diversity and phylogeography of the elusive, but epidemiologically important Echinococcus granulosus sensu stricto genotype G3.

Cystic echinococcosis (CE) is a severe parasitic disease caused by the species complex Echinococcus granulosus sensu lato. Human infections are most commonly associated with E. granulosus sensu stricto (s.s.), comprising genotypes G1 and G3. The objective of the current study was to provide first insight into the genetic diversity and phylogeography of genotype G3. Despite the epidemiological importance of the genotype, it has remained poorly explored due to the ambiguity in the definition of the genotype. However, it was recently demonstrated that long sequences of mitochondrial DNA (mtDNA) provide a reliable method to discriminate G1 and G3 from each other. Therefore, we sequenced near-complete mtDNA of 39 G3 samples, covering most of the known distribution range and host spectra of the genotype. The phylogenetic network revealed high genetic variation within E. granulosus s.s. G3 and while G3 is significantly less prevalent worldwide than G1, the genetic diversity of both of the genotypes is equally high. We also present the results of the Bayesian phylogeographic analysis, which yielded several well-supported diffusion routes of genotype G3 originating from Turkey and Iran, suggesting the Middle East as the origin of the genotype.

Morphology of Nyctotheroides hubeiensis Li et al. 1998 from Frog Hosts with Molecular Phylogenetic Study of Clevelandellid Ciliates (Armophorea, Clevelandellida).

The morphology of Nyctotheroides hubeiensis (Acta Hydrobiol. Sin. 1998, 22(suppl.):187), collected from the rectum of Phelophylax nigromaculatus, is presented in this paper based on detailed morphological information and molecular data. Our phylogenetic analysis showed that N. hubeiensis fell into the Nyctotheroides clade, which was strongly supported as monophyletic and clustered as basal to the genera Nyctotherus and Clevelandella. Also, the monophyly of the Order Clevelandellida and the affinity of parasitic nyctotherids and free-living metopids were indicated in our work. The origin of clevelandellid ciliates as well as their possible evolutionary history was also discussed here; however, the analysis of more species from other vertebrate hosts (fish, reptiles) should be made before a well-supported conclusion can be drawn.

High-resolution phylogeography of zoonotic tapeworm Echinococcus granulosus sensu stricto genotype G1 with an emphasis on its distribution in Turkey, Italy and Spain.

Echinococcus granulosus is the causative agent of cystic echinococcosis. The disease is a significant global public health concern and human infections are most commonly associated with E. granulosus sensu stricto (s. s.) genotype G1. The objectives of this study were to: (i) analyse the genetic variation and phylogeography of E. granulosus s. s. G1 in part of its main distribution range in Europe using 8274 bp of mtDNA; (ii) compare the results with those derived from previously used shorter mtDNA sequences and highlight the major differences. We sequenced a total of 91 E. granulosus s. s. G1 isolates from six different intermediate host species, including humans. The isolates originated from seven countries representing primarily Turkey, Italy and Spain. Few samples were also from Albania, Greece, Romania and from a patient originating from Algeria, but diagnosed in Finland. The analysed 91 sequences were divided into 83 haplotypes, revealing complex phylogeography and high genetic variation of E. granulosus s. s. G1 in Europe, particularly in the high-diversity domestication centre of western Asia. Comparisons with shorter mtDNA datasets revealed that 8274 bp sequences provided significantly higher phylogenetic resolution and thus more power to reveal the genetic relations between different haplotypes.

Trichomonas gypaetinii n. sp., a new trichomonad from the upper gastrointestinal tract of scavenging birds of prey.

In the context of an epidemiological study carried out by several wildlife recovery centers in Spain, trichomonads resembling Trichomonas gallinae were found in the oropharyngeal cavity of 2 Egyptian vultures (Neophron percnopterus) and 14 cinereous vultures (Aegypius monachus) which did not show any symptoms of trichomonosis. In order to characterize them, these isolates along with seven other T. gallinae isolates obtained from different hosts and from different geographical origin were analyzed. Genetic analyses were performed by sequencing the small subunit ribosomal RNA (SSU-rRNA) and the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA regions. The morphological study of the isolates in both light and scanning electron microscopy was also performed. The sequences obtained in the genetic analysis coincide with previously published sequences of an isolate named as Trichomonas sp., obtained from a bearded vulture (Gypaetus barbatus), and showed clear differences to the T. gallinae sequences (97 and 90-91% homology, respectively, for SSU-rRNA and ITS regions) and display higher similarity with Trichomonas vaginalis and Trichomonas stableri than with T. gallinae. Multivariate statistical analysis of the morphometric study also reveals significant differences between the trichomonads of vultures and the isolates of T. gallinae. The isolates from vultures presented smaller values for each variable except for the length of axostyle projection, which was higher. These results together with the different nature of their hosts suggest the possibility of a new species of trichomonad which we hereby name Trichomonas gypaetinii, whose main host are birds of the subfamily Gypaetinae.

New insights into the molecular phylogeny of Balantidium (Ciliophora, Vetibuliferida) based on the analysis of new sequences of species from fish hosts.

We obtained sequences of the small subunit ribosomal RNA (rRNA) for two new isolates of Balantidium from fishes, Balantidium polyvacuolum and Balantidium ctenopharingodoni. This is the first introduction of molecular data of Balantidium species from fish hosts in the phylogenetic analyses of the ciliate subclass Trichostomatia. Despite the fact that these species share morphological characteristics common to other species of Balantidium, the phylogenetic analysis of their sequences has shown that they are to be placed in a different branch closely related to the so-called Australian clade. Thus, our results indicate that the genus Balantidium is polyphyletic and possibly should be represented by two different genera; however, the analysis of more species from other poikilothermic hosts (amphibians, reptiles) should be made before a revised taxonomical proposal could be made.

Wild Animals in Captivity: An Analysis of Parasite Biodiversity and Transmission among Animals at Two Zoological Institutions with Different Typologies.

We have conducted a 10-year-long coprological study of the animals housed in two zoological institutions (ZooAquarium and Faunia, Madrid, Spain) to assess the parasite biodiversity, prevalence, and their relation with host class, diet, and enclosure type (soil type and level of isolation from wild fauna). A total of 4476 faecal samples from 132 mammal species and 951 samples from 86 avian species were examined. The results indicated that only 12.8% of avian species had parasites at least once during the study period, whereas 62.1% of mammal species tested positive. Predominantly, protists (Entamoeba, flagellates, and ciliates) and nematodes (mainly Trichuris) were identified in the findings. Carnivorous species were primarily infected by nematodes, while herbivorous and omnivorous species were mainly infected by protists. The number of infected herbivorous and omnivorous species was significantly greater than carnivorous species. Differences were observed based on soil type (artificial, natural, mixed) and isolation level (isolated/accessible), but these differences were not statistically significant. Several parasites (Entamoeba spp., Giardia spp., Balantidoides coli, Trichuris spp.) could potentially be transmitted between humans and some mammals and birds. Regular animal analyses and a personnel health program in the institutions would minimise transmission risks between zoo animals, wildlife, and humans.

Prevalence of Toxoplasma gondii in Endangered Wild Felines (Felis silvestris and Lynx pardinus) in Spain.

The wildcat (Felis silvestris) and the Iberian lynx (Lynx pardinus) are important species in Spain, considered as near-threatened and endangered, respectively. Both can be infected by Toxoplasma gondii, a parasite that can cause morbidity and mortality in transplacentally-infected or immunocompromised mammals. The data on the prevalence of this parasite in wild populations of these species in Spain are outdated. The objective of this study was to update information and evaluate the role of these felines in parasite epidemiology and the potential impact of the parasite on their conservation. Blood and fecal samples were collected from captured animals, as well as the tongue, diaphragm, and spleen, from animals killed in road accidents in central Spain. An indirect fluorescent antibody test (IFAT) was used to detect parasite antibodies in serum, microscopy and molecular analysis were used to detect oocysts in feces, and molecular analysis was used to determine the existence of tissue cysts. Seroprevalence was 85% in wildcats and 45% in lynx, and parasite DNA was detected in the feces of one wildcat and in tissue samples from 10 wildcats and 11 Iberian lynxes. These results highlight the epidemiological importance and high risk of T. gondii infection in animals and humans in the studied areas. Considering feline susceptibility to infection, monitoring programs are needed to assess the health status of wild felines.

Development of a specific polymerase chain reaction assay for the detection of Basidiobolus.

The etiology of chronic diarrhea is complex in humans and animals. It is always necessary to evaluate a list of differential diagnosis, including bacteria, protozoa and fungi. Basidiobolomycosis is a fungal disease reported sporadically worldwide, mainly caused by B. ranarum, a frequent organism found in soil or in the intestine and skin of lizards and frogs. It is an opportunistic pathogen that causes infections characterized by granulomatous lesions in the subcutaneous tissues as well as in the intestinal wall in humans and animals. In this work we have developed a PCR technique to differentiate Basidiobolus from other causes of intestinal disease in dogs and humans. To test the specificity of the PCR assay we included closely related organisms, common intestinal microbiota and pathogenic organisms, such as Aspergillus, Candida, Cryptosporidium, Escherichia, Giardia, Mucor, Proteus, Rhizopus and Salmonella. Pythium insidiosum, which cause clinically similar disease in dogs but require a different treatment. Only Basidiobolus was positive to the PCR assay.

Cyst detection and viability assessment of Balantioides coli in environmental samples: Current status and future needs.

The ciliate Balantioides coli is a human enteric parasite that can cause life-threatening infections. It is a food- and waterborne parasite, with cysts being the infective stage. Despite its importance as a potential pathogen, few reports have investigated its presence in environmental samples, and some issues need attention including i) The accuracy of B. coli identification. In most cases, the protozoa is identified only by its morphological traits, which can be identical to those from other parasitic ciliates of animals. Genetic analysis of cysts recovered from environmental samples is necessary for species confirmation. In addition, genetic methods used with faecal samples need to be adequately validated with environmental matrices. ii) The methodology for searching this parasite in environmental samples. The protocols include an initial phase to isolate the cysts from the matrix followed by a second phase in which concentration procedures are usually applied. The methods may be valid but are not standardised and differences between studies could affect the results obtained. iii) The areas that needs further research. The development of genetic identification methods and standardised analytical protocols in environmental samples are required, as well as the assessment of viability and infectivity of B. coli cysts. The development of axenic culture systems will boost research on this parasite.

Human-Borne Pathogens: Are They Threatening Wild Great Ape Populations?

Climate change and anthropic activities are the two main factors explaining wild great ape habitat reduction and population decline. The extent to which human-borne infectious diseases are contributing to this trend is still poorly understood. This is due to insufficient or fragmented knowledge on the abundance and distribution of current wild great ape populations, the difficulty obtaining optimal biological samples for diagnostic testing, and the scarcity of pathogen typing data of sufficient quality. This review summarises current information on the most clinically relevant pathogens of viral, bacterial, parasitic, and fungal nature for which transmission from humans to wild great apes is suspected. After appraising the robustness of available epidemiological and/or molecular typing evidence, we attempt to categorise each pathogen according to its likelihood of truly being of human origin. We further discuss those agents for which anthroponotic transmission is more likely. These include two viral (Human Metapneumovirus and Respiratory Syncytial Virus), one bacterial (diarrhoeagenic Escherichia coli), and two parasitic (Cryptosporidium spp. and Giardia duodenalis) pathogens. Finally, we identify the main drawbacks impairing research on anthroponotic pathogen transmission in wild great apes and propose research lines that may contribute to bridging current knowledge gaps.

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.

Chromosome-scale Echinococcus granulosus (genotype G1) genome reveals the Eg95 gene family and conservation of the EG95-vaccine molecule.

Cystic echinococcosis is a socioeconomically important parasitic disease caused by the larval stage of the canid tapeworm Echinococcus granulosus, afflicting millions of humans and animals worldwide. The development of a vaccine (called EG95) has been the most notable translational advance in the fight against this disease in animals. However, almost nothing is known about the genomic organisation/location of the family of genes encoding EG95 and related molecules, the extent of their conservation or their functions. The lack of a complete reference genome for E. granulosus genotype G1 has been a major obstacle to addressing these areas. Here, we assembled a chromosomal-scale genome for this genotype by scaffolding to a high quality genome for the congener E. multilocularis, localised Eg95 gene family members in this genome, and evaluated the conservation of the EG95 vaccine molecule. These results have marked implications for future explorations of aspects such as developmentally-regulated gene transcription/expression (using replicate samples) for all E. granulosus stages; structural and functional roles of non-coding genome regions; molecular 'cross-talk' between oncosphere and the immune system; and defining the precise function(s) of EG95. Applied aspects should include developing improved tools for the diagnosis and chemotherapy of cystic echinococcosis of humans.

Occurrence and genetic characterization of Giardia duodenalis from captive nonhuman primates by multi-locus sequence analysis.

Giardia is the most common enteric protozoan that can be pathogenic to both humans and animals. Transmission can be direct through the faecal-oral route, or through ingestion of contaminated water or food. Genetic characterization of Giardia duodenalis isolates has demonstrated the existence of seven groups (assemblages A to G) which differ in their host distribution. Assemblages A and B are present in humans and other primates, dogs, cats, rodents, and other species of wild mammals, but the role of the different host animals in the epidemiology of human infection remains unclear. With this preliminary data, we can infer that nonhuman primates (NHP) might be a potential reservoir for zoonotic transmission. This research paper discusses the presence of Giardia in nonhuman primates housed in two Spanish zoological gardens (located in Valencia and Madrid). Twenty faecal samples obtained from 16 different species of NHP were studied; 70% were positives to Giardia, and genetic analyses were performed by sequencing of four genes (SSrRNA, glutamate dehydrogenase, triose phosphate isomerase, and beta-giardin). The assemblage A was the most frequent (63.4%) in the species studied. A sequence from a red ruffed lemur (corresponding to genotype AI) was obtained, and this is the first reported sequence of a gdh gene obtained from this species. The multi-locus sequence analysis was also performed on the samples positive to nested PCR belonging to assemblage B. After amplification using the GDHeF, GDHiF, and GDHiR gdh primers; AL3543, AL3546, AL3544, and AL3545 tpi primers; G7, G759, GBF, and GBR bg primers, amplicons of 432, 500, and 511 bp respectively were obtained. Amplification products were sequenced and the sequence and phylogenetic analyses showed that genotype IV like was the most frequent in the samples belonging to this assemblage.

Balantioides coli.

Balantioides coli (=Balantidium coli) is the only ciliate that parasitizes humans. Pigs are the main reservoir. Other species, as camels, cattle, donkey, sheep and goat have been also proposed as reservoirs for human infections. The parasite has a direct life cycle, being transmitted by the faecal-oral route. This type of cycle and the large number of host species imply an important potential for zoonotic transmission of the parasite. Infections are most commonly found in tropical and temperate regions, with prevalence up to 100% in pigs; high prevalence values have been also recorded in some non-human primates and camels. In humans, prevalence is usually under 10% in the population at risk. The main epidemiological factors involved in the transmission of this parasite include close contact with pigs, lack of basic sanitation infrastructures (water supply, wastewater disposal) and hygiene. Individual health status, intestinal microbiota and diet are also important for the onset of the infection. Outbreaks caused by this parasite are rare; those reported to date were related to poor hygienic conditions or to catastrophic natural disasters. Balantioides coli infections can be asymptomatic and symptomatic, which can be chronic (with intermittent diarrhoea), or acute (a dysenteric form which can be life-threatening). Efective treatments include tetracycline, iodoquinol and 5-nitroimidazole compounds (metronidazole, secnidazole). The main effective individual preventive measure is the use of disinfected water for drinking and other uses. Adequate water supply infrastructures, proper disposal of wastewater and animal faeces, and regular monitoring programs on farms will help limit transmission.

Long-Term Preservation and Storage of Faecal Samples in Whatman® Cards for PCR Detection and Genotyping of Giardia duodenalis and Cryptosporidium hominis.

Preservation and conservation of biological specimens, including faecal samples, is a challenge in remote areas or poor-resource settings where the cold chain cannot be maintained. This study aims at evaluating the suitability of filter cards for long-term storage of faecal samples of animal and human origin positive to the diarrhoea-causing protozoan parasites, Giardia duodenalis and Cryptosporidium hominis. Three commercially available Whatman® Filter Cards were comparatively assessed: the FTA® Classic Card, the FTA® Elute Micro Card, and the 903 Protein Saver Card. Human faecal samples positive to G. duodenalis (n = 5) and C. hominis (n = 5) were used to impregnate the selected cards at given storage (1 month, 3 months, and 6 months) periods and temperature (-20 °C, 4 °C, and room temperature) conditions. Parasite DNA was detected by PCR-based methods. Sensitivity assays and quality control procedures to assess suitability for genotyping purposes were conducted. Overall, all three Whatman® cards were proven useful for the detection and molecular characterisation of G. duodenalis and C. hominis under the evaluated conditions. Whatman® cards represent a simple, safe, and cost-effective option for the transportation, preservation, and storage of faecal samples without the need of the cold chain.

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.