Intestinal protists in the gut microbiome are increasingly studied, but their basic epidemiology is not well understood. We explored the prevalence, genetic diversity, and potential zoonotic transmission of two protists colonizing the large intestine - Blastocystis sp. and Dientamoeba fragilis - in 37 species of non-human primates (NHPs) and their caregivers in six zoos in the Czech Republic. We analyzed 179 fecal samples (159 from NHPs, 20 from humans) by qPCR. Blastocystis sp. was detected in 54.7% (98/179) of samples, in 24 NHP species and in 57.2% of NHP samples (prevalence ranged between 36 and 80%), and in 35% of human samples (prevalence ranged between 0 and 67%). Using next generation amplicon sequencing, nine Blastocystis subtypes (ST1-ST5, ST7, ST8, and two novel subtypes) were identified. The two new Blastocystis subtypes (named ST47 and ST48) were described using Nanopore sequencing to produce full-length reference sequences of the small subunit ribosomal RNA gene. Some subtypes were shared between NHPs and their caregivers, suggesting potential zoonotic transmission. Mixed subtype colonization was frequently observed, with 52% of sequenced samples containing two or more subtypes. Dientamoeba was found only in NHPs with a prevalence of 6%. This study emphasizes the critical role of molecular diagnostics in epidemiological and transmission studies of these protists and calls for further research to better understand their impact on public health.

Department of Medical Microbiology Charles University and Motol University Hospital 5 Úvalu 84 150 06 Prague Czech Republic

Environmental Microbial and Food Safety Laboratory Agricultural Research Service United States Department of Agriculture 307 Center Road Beltsville MD 20705 USA

Faculty of Science University of South Bohemia Branišovská 1160 31 370 05 České Budějovice Czech Republic

Institute of Parasitology Biology Centre the Czech Academy of Sciences Branišovská 1160 31 370 05 České Budějovice Czech Republic

Institute of Vertebrate Biology Czech Academy of Sciences Květná 8 603 00 Brno Czech Republic

Zobrazit více v PubMed

Stensvold C.R., Clark C.G. Current status of Blastocystis: a personal view. Parasitol. Int. 2016;65:763–771. doi: 10.1016/j.parint.2016.05.015. PubMed DOI

Stark D., Barratt J., Chan D., Ellis J.T. Dientamoeba fragilis, the neglected trichomonad of the human bowel. Clin. Microbiol. Rev. 2016;29:553–580. doi: 10.1128/CMR.00076-15. PubMed DOI PMC

Clark C.G., van der Giezen M., Alfellani M.A., Stensvold C.R. Recent developments in Blastocystis research. Adv. Parasitol. 2013;82:1–32. doi: 10.1016/B978-0-12-407706-5.00001-0. PubMed DOI

Stensvold C.R., Tan K.S.W., Clark C.G. Blastocystis. Trends Parasitol. 2020;36:315–316. doi: 10.1016/j.pt.2019.12.008. PubMed DOI

Stensvold C.R., van der Giezen M. Associations between gut microbiota and common luminal intestinal parasites. Trends Parasitol. 2018;34:369–377. doi: 10.1016/j.pt.2018.02.004. PubMed DOI

Billy V., Lhotská Z., Jirků M., et al. Blastocystis colonization alters the gut microbiome and, in some cases, promotes faster recovery from induced colitis. Front. Microbiol. 2021;12 doi: 10.3389/fmicb.2021.641483. PubMed DOI PMC

Deng L., Wojciech L., Gascoigne N.R.J., et al. New insights into the interactions between Blastocystis, the gut microbiota, and host immunity. PLoS Pathog. 2021;17 doi: 10.1371/JOURNAL.PPAT.1009253. PubMed DOI PMC

Jokelainen P., Jensen B.H., Andreassen B.U., et al. Dientamoeba fragilis, a commensal in children in Danish day care centers. J. Clin. Microbiol. 2017;55:1707–1713. doi: 10.1128/JCM.00037-17. PubMed DOI PMC

Bart A., Wentink-Bonnema E.M.S., Gilis H., et al. Diagnosis and subtype analysis of Blastocystis sp. in 442 patients in a hospital setting in the Netherlands. BMC Infect. Dis. 2013;13:2–7. doi: 10.1186/1471-2334-13-389. PubMed DOI PMC

El Safadi D., Gaayeb L., Meloni D., et al. Children of Senegal River basin show the highest prevalence of Blastocystis sp. ever observed worldwide. BMC Infect. Dis. 2014;14:164. doi: 10.1186/1471-2334-14-164. PubMed DOI PMC

El Safadi D., Cian A., Nourrisson C., et al. Prevalence, risk factors for infection and subtype distribution of the intestinal parasite Blastocystis sp. from a large-scale multi-center study in France. BMC Infect. Dis. 2016;16:451. doi: 10.1186/s12879-016-1776-8. PubMed DOI PMC

Lhotská Z., Jirků M., Hložková O., et al. A study on the prevalence and subtype diversity of the intestinal protist Blastocystis sp. in a gut-healthy human population in the Czech Republic. Front cell infect Microbiol. 2020;10:544335. doi: 10.3389/fcimb.2020.544335. PubMed DOI PMC

Leelayoova S., Rangsin R., Taamasri P., et al. Evidence of waterborne transmission of Blastocystis hominis. Am. J. Trop. Med. Hyg. 2004;70:658–662. doi: 10.4269/ajtmh.2004.70.658. PubMed DOI

Leelayoova S., Siripattanapipong S., Thathaisong U., et al. Drinking water: a possible source of Blastocystis spp. subtype 1 infection in schoolchildren of a rural community in Central Thailand. Am. J. Trop. Med. Hyg. 2008;79:401–406. doi: 10.4269/ajtmh.2008.79.401. PubMed DOI

Garcia L.S. Dientamoeba fragilis, one of the neglected intestinal protozoa. J. Clin. Microbiol. 2016;54:2243–2250. doi: 10.1128/JCM.00400-16. PubMed DOI PMC

Clark C.G., Röser D., Stensvold C.R. Transmission of Dientamoeba fragilis: pinworm or cysts? Trends Parasitol. 2014;30:136–140. doi: 10.1016/j.pt.2014.01.005. PubMed DOI

Röser D., Nejsum P., Carlsgart A.J., et al. DNA of Dientamoeba fragilis detected within surface-sterilized eggs of Enterobius vermicularis. Exp. Parasitol. 2013;133:57–61. doi: 10.1016/j.exppara.2012.10.009. PubMed DOI

Ögren J., Dienus O., Löfgren S., et al. Dientamoeba fragilis DNA detection in Enterobius vermicularis eggs. Pathog Dis. 2013;69:157–158. doi: 10.1111/2049-632X.12071. PubMed DOI PMC

Munasinghe V.S., Vella N.G.F., Ellis J.T., et al. Cyst formation and faecal-oral transmission of Dientamoeba fragilis - the missing link in the life cycle of an emerging pathogen. Int. J. Parasitol. 2013;43:879–883. doi: 10.1016/j.ijpara.2013.06.003. PubMed DOI

Stark D., Garcia L.S., Barratt J.L.N., et al. Description of Dientamoeba fragilis cyst and precystic forms from human samples. J. Clin. Microbiol. 2014;52:2680–2683. doi: 10.1128/JCM.00813-14. PubMed DOI PMC

Jirků M., Kašparová A., Lhotská Z., et al. A cross-sectional study on the occurrence of the intestinal protist, Dientamoeba fragilis, in the gut-healthy volunteers and their animals. Int. J. Mol. Sci. 2022;23:15407. doi: 10.3390/ijms232315407. PubMed DOI PMC

Šloufová M., Lhotská Z., Jirků M., et al. Comparison of molecular diagnostic approaches for the detection and differentiation of the intestinal protist Blastocystis sp. in humans. Parasite. 2022;29:30. doi: 10.1051/parasite/2022029. PubMed DOI PMC

Maloney J.G., da Cunha M.J.R., Molokin A., et al. Next-generation sequencing reveals wide genetic diversity of Blastocystis subtypes in chickens including potentially zoonotic subtypes. Parasitol. Res. 2021;120:2219–2231. doi: 10.1007/s00436-021-07170-3. PubMed DOI

Hernández P.C., Maloney J.G., Molokin A., et al. Exploring Blastocystis genetic diversity in rural schoolchildren from Colombia using next-generation amplicon sequencing reveals significant associations between contact with animals and infection risk. Parasitol. Res. 2023;122:1451–1462. doi: 10.1007/s00436-023-07841-3. PubMed DOI PMC

Maloney J.G., Molokin A., Segu R., et al. Identification and molecular characterization of four new Blastocystis subtypes designated ST35-ST38. Microorganisms. 2023;11:46. doi: 10.3390/microorganisms11010046. PubMed DOI PMC

Santin M., Figueiredo A., Molokin A., et al. Division of Blastocystis ST10 into three new subtypes : ST42-ST44. J. Eukaryot. Microbiol. 2024;71 doi: 10.1111/jeu.12998. PubMed DOI

Koehler A.V., Herath H.M.P.D., Hall R.S., et al. Marked genetic diversity within Blastocystis in Australian wildlife revealed using a next generation sequencing–phylogenetic approach. Int J Parasitol Parasites Wildl. 2024;23 doi: 10.1016/j.ijppaw.2023.100902. PubMed DOI PMC

Stensvold C.R., Clark C.G., Röser D. Limited intra-genetic diversity in Dientamoeba fragilis housekeeping genes. Infect. Genet. Evol. 2013;18:284–286. doi: 10.1016/j.meegid.2013.05.003. PubMed DOI

Windsor J.J., Clark C.G., Macfarlane L. Molecular typing of Dientamoeba fragilis. Br. J. Biomed. Sci. 2004;61:153–155. doi: 10.1080/09674845.2004.11978138. PubMed DOI

Johnson J.A., Clark C.G. Cryptic genetic diversity in Dientamoeba fragilis. J. Clin. Microbiol. 2000;38:4653–4654. doi: 10.1128/jcm.38.12.4653-4654.2000. PubMed DOI PMC

Peek R., Reedeker F.R., Van Gool T. Direct amplification and genotyping of Dientamoeba fragilis from human stool specimens. J. Clin. Microbiol. 2004;42:631–635. doi: 10.1128/JCM.42.2.631-635.2004. PubMed DOI PMC

Stensvold C.R., Clark C.G., Röser D. Limited intra-genetic diversity in Dientamoeba fragilis housekeeping genes. Infect. Genet. Evol. 2013;18:284–286. doi: 10.1016/j.meegid.2013.05.003. PubMed DOI

Alfellani M.A., Stensvold C.R., Vidal-Lapiedra A., et al. Variable geographic distribution of Blastocystis subtypes and its potential implications. Acta Trop. 2013;126:11–18. doi: 10.1016/j.actatropica.2012.12.011. PubMed DOI

Hublin J.S.Y., Maloney J.G., Santin M. Blastocystis in domesticated and wild mammals and birds. Res. Vet. Sci. 2021;135:260–282. doi: 10.1016/j.rvsc.2020.09.031. PubMed DOI

Yoshikawa H., Wu Z., Pandey K., et al. Molecular characterization of Blastocystis isolates from children and rhesus monkeys in Kathmandu. Nepal. Vet Parasitol. 2009;160:295–300. doi: 10.1016/j.vetpar.2008.11.029. PubMed DOI

Betts E.L., Gentekaki E., Tsaousis A.D. Exploring micro-eukaryotic diversity in the gut: co-occurrence of Blastocystis subtypes and other protists in zoo animals. Front. Microbiol. 2020;11:288. doi: 10.3389/fmicb.2020.00288. PubMed DOI PMC

Köster P.C., Dashti A., Bailo B., et al. 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. Animals. 2021;11:700. doi: 10.3390/ani11030700. PubMed DOI PMC

Rudzińska M., Kowalewska B., Waleron M., et al. Molecular characterization of Blastocystis from animals and their caregivers at the gdańsk zoo (Poland) and the assessment of zoonotic transmission. Biology (Basel) 2021;10:984. doi: 10.3390/biology10100984. PubMed DOI PMC

Köster P.C., Martínez-Nevado E., González A., et al. Intestinal protists in captive non-human primates and their handlers in six European zoological gardens. Molecular Evidence of Zoonotic Transmission. Front Vet Sci. 2022;8 doi: 10.3389/fvets.2021.819887. PubMed DOI PMC

Helenbrook W.D., Wade S.E., Shields W.M., et al. Gastrointestinal parasites of ecuadorian mantled howler monkeys (Alouatta palliata aequatorialis) based on fecal analysis. J. Parasitol. 2015;101:341–350. doi: 10.1645/13-356.1. PubMed DOI

Menu E., Davoust B., Mediannikov O., et al. Occurrence of ten protozoan enteric pathogens in three non-human primate populations. Pathogens. 2021;10:280. doi: 10.3390/pathogens10030280. PubMed DOI PMC

Stark D., Phillips O., Peckett D., et al. Gorillas are a host for Dientamoeba fragilis: an update on the life cycle and host distribution. Vet. Parasitol. 2008;151:21–26. doi: 10.1016/j.vetpar.2007.10.002. PubMed DOI

Maloney J.G., Molokin A., Santin M. Next generation amplicon sequencing improves detection of Blastocystis mixed subtype infections. Infect. Genet. Evol. 2019;73:119–125. doi: 10.1016/j.meegid.2019.04.013. PubMed DOI

Edgar R.C. Search and clustering orders of magnitude faster than BLAST. Bioinformatics. 2010;26:2460–2461. doi: 10.1093/bioinformatics/btq461. PubMed DOI

Scicluna S.M., Tawari B., Clark C.G. DNA barcoding of Blastocystis. Protist. 2006;157:77–85. doi: 10.1016/j.protis.2005.12.001. PubMed DOI

Maloney J.G., Molokin A., Santin M. Use of Oxford Nanopore MinION to generate full - length sequences of the Blastocystis small subunit (SSU) rRNA gene. Parasit. Vectors. 2020;13:595. doi: 10.1186/s13071-020-04484-6. PubMed DOI PMC

Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 1980;16:111–120. doi: 10.1007/BF01731581. PubMed DOI

Tamura K., Stecher G., Kumar S. MEGA11: molecular evolutionary genetics analysis version 11. Mol. Biol. Evol. 2021;38:3022–3027. doi: 10.1093/molbev/msab120. PubMed DOI PMC

Stark D., Beebe N., Marriott D., et al. Detection of Dientamoeba fragilis in fresh stool specimens using PCR. Int. J. Parasitol. 2005;35:57–62. doi: 10.1016/j.ijpara.2004.09.003. PubMed DOI

De Quadros R.M., Weiss P.H.E., Marques S.M.T., Miletti L.C. Potential cross-contamination of similar Giardia duodenalis assemblage in children and pet dogs in southern Brazil, as determined by PCR-RFLP. Rev. Inst. Med. Trop. Sao Paulo. 2016;58:3–9. doi: 10.1590/S1678-9946201658066. PubMed DOI PMC

Cian A., El Safadi D., Osman M., et al. Molecular epidemiology of Blastocystis sp. in various animal groups from two French zoos and evaluation of potential zoonotic risk. PLoS One. 2017;12 doi: 10.1371/journal.pone.0169659. PubMed DOI PMC

Valença-Barbosa C., Do Bomfim T.C.B., Teixeira B.R., et al. Molecular epidemiology of Blastocystis isolated from animals in the state of Rio de Janeiro, Brazil. PLoS One. 2019;14 doi: 10.1371/journal.pone.0210740. PubMed DOI PMC

Ma L., Qiao H., Wang H., et al. Molecular prevalence and subtypes of Blastocystis sp. in primates in northern China. Transbound. Emerg. Dis. 2020;67:2789–2796. doi: 10.1111/tbed.13644. PubMed DOI

Deng L., Yao J., Chen S., et al. First identification and molecular subtyping of Blastocystis sp. in zoo animals in southwestern China. Parasit. Vectors. 2021;14:11. doi: 10.1186/s13071-020-04515-2. PubMed DOI PMC

Köster P.C., Lapuente J., Pizarro A., et al. Presence and genetic diversity of enteric protists in captive and semi-captive non-human primates in côte d’Ivoire, Sierra Leone, and Peru. Int J Parasitol Parasites Wildl. 2022;17:26–34. doi: 10.1016/j.ijppaw.2021.12.004. PubMed DOI PMC

Zanzani S.A., Gazzonis A.L., Epis S., Manfredi M.T. Study of the gastrointestinal parasitic fauna of captive non-human primates (Macaca fascicularis) Parasitol. Res. 2016;115:307–312. doi: 10.1007/s00436-015-4748-9. PubMed DOI

Lankester F., Kiyang J.A., Bailey W., Unwin S. Dientamoeba fragilis: initial evidence of pathogenicity in the western lowland gorilla (Gorilla gorilla gorilla) J. Zoo Wildl. Med. 2010;41:350–352. doi: 10.1638/2009-0190.1. PubMed DOI

Yu M., Yao Y., Xiao H., et al. Extensive prevalence and significant genetic differentiation of Blastocystis in high- and low-altitude populations of wild rhesus macaques in China. Parasit. Vectors. 2023;16:107. doi: 10.1186/s13071-023-05691-7. PubMed DOI PMC

Hernández-Castro C., Maloney J.G., Agudelo-lópez S.P., et al. Identification and validation of novel Blastocystis subtype ST41 in a Colombian patient undergoing colorectal cancer screening. J. Eukaryot. Microbiol. 2023;70:el2978. doi: 10.1111/jeu.12978. PubMed DOI

Oliveira-Arbex AP, David ÉB, Tenório M da S, et al (2020) Diversity of Blastocystis subtypes in wild mammals from a zoo and two conservation units in southeastern Brazil. Infect. Genet. Evol. 78:104053. doi:10.1016/j.meegid.2019.104053. PubMed DOI

Alfellani M.A., Jacob A.S., Perea N.O., et al. Diversity and distribution of Blastocystis sp. subtypes in non-human primates. Parasitology. 2013;140:966–971. doi: 10.1017/S0031182013000255. PubMed DOI

Stensvold C.R., Alfellani M.A., Nørskov-Lauritsen S., et al. Subtype distribution of Blastocystis isolates from synanthropic and zoo animals and identification of a new subtype. Int. J. Parasitol. 2009;39:473–479. doi: 10.1016/j.ijpara.2008.07.006. PubMed DOI

Rojas-Velázquez L., Maloney J.G., Molokin A., et al. Use of next-generation amplicon sequencing to study Blastocystis genetic diversity in a rural human population from Mexico. Parasit. Vectors. 2019;12:566. doi: 10.1186/s13071-019-3814-z. PubMed DOI PMC

Sarzhanov F., Dogruman-Al F., Santin M., et al. Investigation of neglected protists Blastocystis sp. and Dientamoeba fragilis in immunocompetent and immunodeficient diarrheal patients using both conventional and molecular methods. PLoS Negl. Trop. Dis. 2021;15 doi: 10.1371/JOURNAL.PNTD.0009779. PubMed DOI PMC