Hostname: page-component-78c5997874-xbtfd Total loading time: 0 Render date: 2024-11-09T16:30:24.019Z Has data issue: false hasContentIssue false

Pinning down the role of common luminal intestinal parasitic protists in human health and disease – status and challenges

Published online by Cambridge University Press:  08 February 2019

Christen Rune Stensvold*
Affiliation:
Laboratory of Parasitology, Department of Bacteria, Parasites and Fungi, Statens Serum Institut, Artillerivej 5, DK–2300 Copenhagen S, Denmark
*
Author for correspondence: Christen Rune Stensvold, E-mail: run@ssi.dk
Get access
Rights & Permissions [Opens in a new window]

Abstract

While some single-celled intestinal parasites are direct causes of diarrhoea and other types of intestinal pathology, the impact of other gut micro-eukaryotes on human health remains elusive. The fact that some common luminal intestinal parasitic protists (CLIPPs) have lately been found more often in healthy than in diseased individuals has fuelled the hypothesis that some parasites might in fact be protective against disease. To this end, the use of new DNA technologies has helped us investigate trans-kingdom relationships in the gut. However, research into these relationships is currently hampered by the limited data available on the genetic diversity within the CLIPPs genera, which results in limited efficacy of publicly available DNA sequence databases for taxonomic annotation of sequences belonging to the eukaryotic component of the gut microbiota. In this paper, I give a brief overview of the status on CLIPPs in human health and disease and challenges related to the mapping of intestinal eukaryotic diversity of the human gut.

Type
Review Article
Copyright
Copyright © Cambridge University Press 2019 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

Introduction

Recent research into the human gut microbiome has vastly increased our understanding of the relationship between gut microbes and human health and disease. For instance, we now know that in adults, a low-diversity microbiota with increases in proportions of facultative anaerobes is linked to acute diarrhoea, inflammatory bowel disease, Clostridium difficile infection, metabolic syndrome and liver disease, just to mention a couple of conditions (Cani, Reference Cani2018; Kriss et al., Reference Kriss, Hazleton, Nusbacher, Martin and Lozupone2018). Although most gut microbiome research has focussed on prokaryotic diversity, we have also gained significant insight into the micro-eukaryotic diversity of the human gut. DNA-based methods have been instrumental to this advancement. Three important points have emerged: (1) For some common luminal intestinal parasitic protists (CLIPPs), genetic diversity is surprisingly high; still, DNA sequence data available in publicly available databases such as the NCBI database is rudimentary, hence not reflecting this amount of diversity (Stensvold et al., Reference Stensvold, Lebbad, Victory, Verweij, Tannich, Alfellani, Legarraga and Clark2011c, Reference Stensvold, Lebbad and Clark2012, Reference Stensvold, Winiecka-Krusnell, Lier and Lebbad2018; Royer et al., Reference Royer, Gilchrist, Kabir, Arju, Ralston, Haque, Clark and Petri2012; Poulsen and Stensvold, Reference Poulsen and Stensvold2016). (2) We have come to realize that some CLIPPs are very common and often more common in gut-healthy individuals than in those with functional and inflammatory bowel diseases, contrary to previous general belief (Petersen et al., Reference Petersen, Stensvold, Mirsepasi, Engberg, Friis-Møller, Porsbo, Hammerum, Nordgaard-Lassen, Nielsen and Krogfelt2013; Andersen et al., Reference Andersen, Bonde, Nielsen and Stensvold2015; Krogsgaard et al., Reference Krogsgaard, Engsbro, Stensvold, Nielsen and Bytzer2015, Reference Krogsgaard, Andersen, Johannesen, Engsbro, Stensvold, Nielsen and Bytzer2018; Rossen et al., Reference Rossen, Bart, Verhaar, van Nood, Kootte, de Groot, D'Haens, Ponsioen and van Gool2015; Beghini et al., Reference Beghini, Pasolli, Truong, Putignani, Cacciò and Segata2017; Jokelainen et al., Reference Jokelainen, Hebbelstrup Jensen, Andreassen, Petersen, Röser, Krogfelt, Nielsen and Stensvold2017; Mirjalali et al., Reference Mirjalali, Abbasi, Naderi, Hasani, Mirsamadi, Stensvold, Balaii, Asadzadeh Aghdaei and Zali2017). (3) Robust links between CLIPPs and gut bacteria have been identified by several research teams (Stensvold and van der Giezen, Reference Stensvold and van der Giezen2018).

These three points currently stimulate interdisciplinary research across the fields of parasitology, clinical microbiology, gastroenterology and ecology. Nevertheless, compared with advances within e.g. bacteriology and virology, progress in the research into CLIPPs and their role in human health and disease is still reflected mostly in simple stool microscopy-based surveys of parasites in selected populations, and is therefore still facing some major challenges. In the following, I will try to detail the status of the three above-mentioned points, and highlight some of the limitations and challenges to the work ahead that aims to identify the significance of CLIPPs in human health and disease.

Which are the most common luminal intestinal parasitic protists?

Intestinal eukaryotes that need a host to complete their life cycles (i.e. organisms that are referred to as ‘parasites’) include both helminths and protists. More typically, the distinction is made between ‘helminths’ and ‘protozoa’, but from a taxonomical point of view, the group of organisms referred to as protozoa does not include one of the most common micro-eukaryotes, namely Blastocystis, and so, the term ‘protists’ appears more relevant and applicable than ‘protozoa’ in this context. Moreover, while some parasitic intestinal protists are invasive (e.g. sporozoa) or adhere to the mucosal lining (e.g. Giardia), quite a few genera appear to be confined mainly to the gut lumen. These include Blastocystis, Dientamoeba, Endolimax, Iodamoeba and most species of Entamoeba, and given their prevalence, they could be referred to as CLIPPs.

Contrary to the situation in developing countries, the number of carriers of helminth infestations other than those attributable to pinworm (Enterobius vermicularis) appears to be rapidly plummeting in human populations in the Western world (Verweij and van Lieshout, Reference Verweij and van Lieshout2011; Verweij, Reference Verweij2014), and also in some parts of the developing world, which would probably reflect improved hygienic standards. Still, and for incompletely known reasons, a substantial proportion of the population is colonized by CLIPPs, especially Blastocystis and Dientamoeba (Verweij and van Lieshout, Reference Verweij and van Lieshout2011; Roser et al., Reference Roser, Simonsen, Nielsen, Stensvold and Molbak2013; Krogsgaard et al., Reference Krogsgaard, Engsbro, Stensvold, Nielsen and Bytzer2015, Reference Krogsgaard, Andersen, Johannesen, Engsbro, Stensvold, Nielsen and Bytzer2018; Jokelainen et al., Reference Jokelainen, Hebbelstrup Jensen, Andreassen, Petersen, Röser, Krogfelt, Nielsen and Stensvold2017) and, to a lesser extent, by one or more of the Amoebozoa, e.g. Entamoeba coli (Bruijnesteijn van Coppenraet et al., Reference Bruijnesteijn van Coppenraet, Wallinga, Ruijs, Bruins and Verweij2009; Krogsgaard et al., Reference Krogsgaard, Andersen, Johannesen, Engsbro, Stensvold, Nielsen and Bytzer2018; Stensvold and Nielsen, Reference Stensvold and Nielsen2012; ten Hove et al., Reference ten Hove, Schuurman, Kooistra, Möller, van Lieshout and Verweij2007); these organisms will be introduced briefly below.

Blastocystis

Blastocystis is a genus comprising a perplexing variety of ribosomal lineages that are arguably separate species, judging from the amount of genetic diversity across complete nuclear ribosomal genes. So far, at least 17 ribosomal lineages, the so-called ‘subtypes’, have been acknowledged in humans, non-human primates, other mammals and birds (Alfellani et al., Reference Alfellani, Taner-Mulla, Jacob, Imeede, Yoshikawa, Stensvold and Clark2013b; Clark et al., Reference Clark, van der Giezen, Alfellani and Stensvold2013; Stensvold and Clark, Reference Stensvold and Clark2016). Also reptiles, amphibia and insects have been identified as hosts for various species of Blastocystis (Yoshikawa et al., Reference Yoshikawa, Koyama, Tsuchiya and Takami2016). While this parasite appears to be a rare or at least not so common finding in strict or moderately strict carnivores such as cats, dogs and hyenas (Ruaux and Stang, Reference Ruaux and Stang2014; Wang et al., Reference Wang, Owen, Traub, Cuttell, Inpankaew and Bielefeldt-Ohmann2014; Heitlinger et al., Reference Heitlinger, Ferreira, Thierer, Hofer and East2017; Cociancic et al., Reference Cociancic, Zonta and Navone2018; Moura et al., Reference Moura, Oliveira-Silva, Pedrosa, Nascentes and Cabrine-Santos2018; Udonsom et al., Reference Udonsom, Prasertbun, Mahittikorn, Mori, Changbunjong, Komalamisra, Pintong, Sukthana and Popruk2018), it may be more common in omni- and herbivores, including pigs, cows and sheep (Pakandl, Reference Pakandl1991; Navarro et al., Reference Navarro, Domínguez-Márquez, Garijo-Toledo, Vega-García, Fernández-Barredo, Pérez-Gracia, García, Borrás and Gómez-Muñoz2008; Ramirez et al., Reference Ramirez, Sanchez, Bautista, Corredor, Florez and Stensvold2014; Masuda et al., Reference Masuda, Sumiyoshi, Ohtaki and Matsumoto2018; Moura et al., Reference Moura, Oliveira-Silva, Pedrosa, Nascentes and Cabrine-Santos2018; Udonsom et al., Reference Udonsom, Prasertbun, Mahittikorn, Mori, Changbunjong, Komalamisra, Pintong, Sukthana and Popruk2018). Nine distinct ribosomal lineages, the so-called ‘subtypes’, have been isolated from humans, with subtypes 1–4 predominating. Some subtypes even exhibit extensive within-subtype diversity that to some degree is host-specific; e.g. ST3 (Alfellani et al., Reference Alfellani, Jacob, Perea, Krecek, Taner-Mulla, Verweij, Levecke, Tannich, Clark and Stensvold2013a). Colonization is common in older children and adults than in infants and young children (El Safadi et al., Reference El Safadi, Gaayeb, Meloni, Cian, Poirier, Wawrzyniak, Delbac, Dabboussi, Delhaes, Seck, Hamze, Riveau and Viscogliosi2014; Scanlan et al., Reference Scanlan, Stensvold, Rajilić-Stojanović, Heilig, De Vos, O'Toole and Cotter2014; Poulsen et al., Reference Poulsen, Efunshile, Nelson and Stensvold2016; Salehi et al., Reference Salehi, Haghighi, Stensvold, Kheirandish, Azargashb, Raeghi, Kohansal and Bahrami2017; Scanlan et al., Reference Scanlan, Hill, Ross, Ryan, Stanton and Cotter2018), with prevalence rates reaching 100% in developing countries (El Safadi et al., Reference El Safadi, Gaayeb, Meloni, Cian, Poirier, Wawrzyniak, Delbac, Dabboussi, Delhaes, Seck, Hamze, Riveau and Viscogliosi2014). Moreover, Blastocystis may colonize the human gut for several years (Scanlan et al., Reference Scanlan, Stensvold, Rajilić-Stojanović, Heilig, De Vos, O'Toole and Cotter2014).

Dientamoeba fragilis

DNA-based methods helped overcome the diagnostic challenges related to the detection of Dientamoeba fragilis, a non-flagellated flagellate for which a cyst stage was reported only very recently (Munasinghe et al., Reference Munasinghe, Vella, Ellis, Windsor and Stark2013; Stark et al., Reference Stark, Garcia, Barratt, Phillips, Roberts, Marriott, Harkness and Ellis2014). Dientamoeba fragilis is the only known species in the genus. The first DNA-based detection methods for D. fragilis appeared in the mid-00s (Peek et al., Reference Peek, Reedeker and van Gool2004; Stark et al., Reference Stark, Beebe, Marriott, Ellis and Harkness2005a, Reference Stark, Beebe, Marriott, Ellis and Harkness2005b, Reference Stark, Beebe, Marriott, Ellis and Harkness2006; Verweij et al., Reference Verweij, Mulder, Poell, van Middelkoop, Brienen and van Lieshout2007). Since then, such methods have helped us to realize that this parasite is very common in some populations, especially in Northern Europe (Röser et al., Reference Roser, Simonsen, Nielsen, Stensvold and Molbak2013; de Jong et al., Reference de Jong, Korterink, Benninga, Hilbink, Widdershoven and Deckers-Kocken2014; Ögren et al., Reference Ögren, Dienus, Löfgren, Einemo, Iveroth and Matussek2015; Holtman et al., Reference Holtman, Kranenberg, Blanker, Ott, Lisman-van Leeuwen and Berger2017; Jokelainen et al., Reference Jokelainen, Hebbelstrup Jensen, Andreassen, Petersen, Röser, Krogfelt, Nielsen and Stensvold2017). In Denmark, D. fragilis is almost an obligate finding in children (Röser et al., Reference Roser, Simonsen, Nielsen, Stensvold and Molbak2013; Jokelainen et al., Reference Jokelainen, Hebbelstrup Jensen, Andreassen, Petersen, Röser, Krogfelt, Nielsen and Stensvold2017). In other regions where methods of high sensitivity are also used, such as Australia, the parasite appears to be a lot less common (Stark et al., Reference Stark, Barratt, Chan and Ellis2016); however, studies involving screening of asymptomatic individuals for D. fragilis are very rare, and so the prevalence of the parasite in individuals without symptoms in most parts of the world remains largely unknown. Apart from humans, D. fragilis has been found in non-human primates and pigs (Stark et al., Reference Stark, Phillips, Peckett, Munro, Marriott, Harkness and Ellis2008; Cacciò et al., Reference Cacciò, Sannella, Manuali, Tosini, Sensi, Crotti and Pozio2012). The diversity within the species appears very limited, and most cases of D. fragilis colonization are attributable to one of only two acknowledged genotypes (Genotype 1), no matter where sampling is performed (Stark et al., Reference Stark, Beebe, Marriott, Ellis and Harkness2005a, Reference Stark, Beebe, Marriott, Ellis and Harkness2005b; Stensvold et al., Reference Stensvold, Clark and Röser2013; Cacciò et al., Reference Cacciò, Sannella, Bruno, Stensvold, David, Guimarães, Manuali, Magistrali, Mahdad, Beaman, Maserati, Tosini and Pozio2016; Greigert et al., Reference Greigert, Abou-Bacar, Brunet, Nourrisson, Pfaff, Benarbia, Pereira, Randrianarivelojosia, Razafindrakoto, Solotiana Rakotomalala, Morel, Candolfi and Poirier2018).

Entamoeba

A number of Entamoeba species can colonize the human intestine. Infections due to the potentially highly pathogenic Entamoeba histolytica are relatively rare compared with colonization by Entamoeba dispar, Entamoeba hartmanni, and, especially, Entamoeba coli, which has been found to colonise between 20 and 30% of individuals in surveyed populations in Brazil (Aguiar et al., Reference Aguiar, Gonçalves, Sodré, Pereira, Bóia, de Lemos and Daher2007; Neres-Norberg et al., Reference Neres-Norberg, Guerra-Sanches, Blanco Moreira-Norberg, Madeira-Oliveira, Santa-Helena and Serra-Freire2014; Higa et al., Reference Higa, Cardoso, Weis, França, Pontes, Silva, Oliveira and Dorval2017; Jeske et al., Reference Jeske, Bianchi, Moura, Baccega, Pinto, Berne and Villela2018). Substantial genetic variation has been detected within E. coli, with E. coli subtype 1 and subtype 2 differing by 13% (Stensvold et al., Reference Stensvold, Lebbad, Victory, Verweij, Tannich, Alfellani, Legarraga and Clark2011c). Overall, the genetic diversity within octo-nucleated Entamoebas appears vast and still largely unaccounted for (Jacob et al., Reference Jacob, Busby, Levy, Komm and Clark2016; Elsheikha et al., Reference Elsheikha, Regan and Clark2018). Entamoeba polecki rarely infects humans; nevertheless, four subtypes have been detected with quite varying geographical distribution and host reservoirs (Stensvold et al., Reference Stensvold, Lebbad, Victory, Verweij, Tannich, Alfellani, Legarraga and Clark2011c, Reference Stensvold, Winiecka-Krusnell, Lier and Lebbad2018); all four subtypes have been found in humans (Verweij et al., Reference Verweij, Polderman and Clark2001; Stensvold et al., Reference Stensvold, Winiecka-Krusnell, Lier and Lebbad2018).

It is currently unclear to which extent non-histolytica Entamoebas contribute to the development of intestinal symptoms.

Some other protists show up in stool every now and then (often accompanied by other CLIPPs) and these include parasites belonging to ciliates and the Amoebozoa. Although the amount of documentation is scarce, it is clear that for some of these parasites, especially Iodamoeba and Endolimax, the intra-generic diversity is vast, with a maximum genetic divergence of at least 31% (Stensvold et al., Reference Stensvold, Lebbad and Clark2012; Constenla et al., Reference Constenla, Padrós and Palenzuela2014; Poulsen and Stensvold, Reference Poulsen and Stensvold2016). Endolimax nana was recently shown to colonize 28.8% of 3245 individuals attending the Evandro Chagas National Institute of Infectious Diseases, Rio de Janeiro, Brazil (Faria et al., Reference Faria, Zanini, Dias, da Silva, de Freitas, Almendra, Santana and Sousa2017). To date, no complete, annotated nuclear genome sequences have been published for CLIPPs other than Blastocystis and Entamoeba dispar.

The extensive genetic diversity documented so far within these CLIPPs has informed the taxonomic terminology, and so, depending on the availability of morphology data and genetic diversity and SSU rDNA sequence coverage, sequences are annotated to species, subtypes, ribosomal lineage, or conditional lineage (Jacob et al., Reference Jacob, Busby, Levy, Komm and Clark2016). Importantly, it appears that specific taxonomic terminologies are being developed for individual genera; these are based first and foremost on a pragmatic basis.

CLIPPs in a gut ecology setting

In addition to exploring parasite diversity in the gut, it could be important to try and look to gut microbial and ecological relationships in non-human hosts and in the environment, respectively, to better understand the role of CLIPPs in human health and disease. In the field of ecology, protists have been identified as important components of terrestrial and aquatic environments where they are integral constituents of trophic chains and nutrient cycles (Bates et al., Reference Bates, Clemente, Flores, Walters, Parfrey, Knight and Fierer2013; Maritz et al., Reference Maritz, Rogers, Rock, Liu, Joseph, Land and Carlton2017). In geothermal springs, protist diversity appears to rely on pH and temperature (Oliverio et al., Reference Oliverio, Power, Washburne, Cary, Stott and Fierer2018). The introduction of Acanthamoeba into the rhizosphere of Arabidopsis thaliana leads to rapid changes in associated bacterial communities due to the grazing of the amoeba (Rosenberg et al., Reference Rosenberg, Bertaux, Krome, Hartmann, Scheu and Bonkowski2009). Gut flagellates and ciliates assist termites and ruminants in metabolizing/fermenting carbohydrates (Veira, Reference Veira1986; Ohkuma, Reference Ohkuma2008; Moon-van der Staay et al., Reference Moon-van der Staay, van der Staay, Michalowski, Jouany, Pristas, Javorský, Kišidayová, Varadyova, McEwan, Newbold, van Alen, de Graaf, Schmid, Huynen and Hackstein2014); examples are endless. The presence of protists in various niches therefore appears to be driven by a variety of host- and environment-derived factors and may in turn have a number of vital or less vital consequences for the associated microbiome, be it the host-associated gut microbiome, plant rhizosphere or terrestrial and aquatic biomes. This understanding has to a large extent failed to resonate with professionals in clinical microbiology and related medical fields, where CLIPPs are generally seen as ‘intruders’ and (potential) pathogens, despite the fact that most of these are most probably non-invasive and may have unknown functions of potential benefit (Parfrey et al., Reference Parfrey, Walters and Knight2011; Lukeš et al., Reference Lukeš, Stensvold, Jirků-Pomajbíková and Wegener Parfrey2015; Andersen and Stensvold, Reference Andersen and Stensvold2016).

Nevertheless, the concept of certain gut parasitic protists as ‘ecosystem engineers’ also in humans is sinking in, and studies on trans-kingdom relationships are emerging. For instance, Laforest-Lapointe and Arriet (Reference Laforest-Lapointe and Arrieta2018) recently proposed a model for the ecological role of Blastocystis in the human gut microbiota. They suggested that Blastocystis by predation on abundant bacterial taxa lowers the competition for nutrients and space, leading to an increase in bacterial richness and community evenness. And indeed, carriers of Blastocysts and other CLIPPs have been shown to have gut bacterial microbiomes that differ significantly from those who do not carry these parasites in several recent studies, the findings of which were recently summarized by Stensvold and van der Giezen (Reference Stensvold and van der Giezen2018). In fact, higher diversity and higher richness are typically observed in CLIPPs-positive individuals than in those who are negative. What is more is the fact that observations from a recent meta-analysis of metagenomics data indicated that Blastocystis carriage is linked to low body mass index (Andersen et al., Reference Andersen, Bonde, Nielsen and Stensvold2015; Beghini et al., Reference Beghini, Pasolli, Truong, Putignani, Cacciò and Segata2017), which again lends support to specific links to gut bacterial diversity. However, it remains to be identified, to which extent Blastocystis is actively driving this difference as proposed by Laforest-Lapointe and Arrieta, or whether Blastocystis is merely an indicator or specific bacterial community patterns. Stensvold and van der Giezen (Reference Stensvold and van der Giezen2018) recently hypothesized that the increased intestinal oxygen concentrations observed during gut dysbiosis may prevent Blastocystis from establishing in the gut, which would suggest a role for Blastocystis as an indicator organism.

Experimental models, such as that recently proposed by Pomajbikova and colleagues (Růžková et al., Reference Růžková, Květoňová, Jirků, Lhotská, Stensvold, Parfrey and Jirků Pomajbíková2018), could be used to develop longitudinal studies on bacterial community changes after the establishment of Blastocystis colonization. Blastocystis is one the few parasites that is readily established in culture (Clark and Stensvold, Reference Clark and Stensvold2016), and cysts induced in cultures or obtained from donor material, isolated from stool by gradient centrifugation, can be used for inoculation in order not to co-introduce bacteria that would lead to experimental bias (Rene et al., Reference Rene, Stensvold, Badsberg and Nielsen2009). Here, the use of both eubiotic and dysbiotic animals could be used to study potential differences in colonization success rate.

The fact that some hosts (e.g. cats and dogs) are not so prone to harbouring a parasite such like Blastocystis while others (e.g. humans and artiodactyls) in the same habitat are much more likely hosts, should also be explored in detail, to identify whether this boils down to diet, behaviour (exposure), and/or other factors. If all the subtypes of the parasite are globally pervasive and the overall colonization pressure of Blastocystis strong, differences in intestinal colonization between hosts may rely – at least in part – on differences in gut microbiota composition.

It is intriguing that not only Blastocystis, but also other CLIPPs have been shown to be linked to specific microbiota patterns (Stensvold and van der Giezen, Reference Stensvold and van der Giezen2018). Studying gut microbiomes of rural Africans, Morton et al. (Reference Morton, Lynch, Froment, Lafosse, Heyer, Przeworski, Blekhman and Ségurel2015) could predict the presence/absence of Entamoeba by 79% accuracy, based on the composition of any individual's gut microbiota. To this end, Xiong et al. (Reference Xiong, Yu, Dai, Zhang, Qiu and Ou2018) identified that shrimp health status could be predicted with 92.4% accuracy based on eukaryotic taxon profiling.

Nucleated life within the human intestine also include fungi. Common genera found in stool include Candida, Saccharomyces, Malassezia, Pichia and Aspergillus (Laforest-Lapointe and Arrieta, Reference Laforest-Lapointe and Arrieta2018); however, our understanding of the extent to which these genera in fact colonize the human intestinal tract or merely reflect dietary components is incomplete, and recent evidence appears to suggest that fungal colonization of the intestinal tract of healthy individuals is minimal (Auchtung et al., Reference Auchtung, Fofanova, Stewart, Nash, Wong, Gesell, Auchtung, Ajami and Petrosino2018).

The faecal eukaryome – mapping of eukaryotic diversity in vertebrate stool

As observed by e.g. Hamad et al. (Reference Hamad, Abou Abdallah, Ravaux, Mokhtari, Tissot-Dupont, Michelle, Stein, Lagier, Raoult and Bittar2018), differences in observed microbiome profiles may reflect differences in DNA extraction protocols, DNA amplification and sequencing technologies, plus queried databases (SILVA, Greengenes, RDP, NCBI, self-curated databases, etc.). So far, mapping of eukaryotic diversity in human and non-human stool samples has used mainly one of two approaches: Shotgun sequencing or amplicon-based sequencing of genomic DNA extracted from stool (Cristescu, Reference Cristescu2014). The applicability of shotgun sequencing in terms of detecting and differentiating CLIPPs is hampered by the fact that relatively few CLIPPs genomes are available for reference. Amplicon-based sequencing has typically used nuclear small subunit ribosomal DNA (18S) as the target. However, some variation in amplicon-based approaches is seen, mostly in terms of the choice of target(s) and DNA sequence data processing. The most informative regions of the 18S appear to be the V3, V4, V5 and the V9 regions (Maritz et al., Reference Maritz, Rogers, Rock, Liu, Joseph, Land and Carlton2017; Krogsgaard et al., Reference Krogsgaard, Andersen, Johannesen, Engsbro, Stensvold, Nielsen and Bytzer2018). As an example, Krogsgaard and colleagues used three different primer sets for eukaryotic DNA (G3F1/G3R1 and G6F1/G6R1 targeting the V3–V4 region of the 18S rRNA gene and G4F1/G4R1 targeting the V3–V5 region) and one set of primers for prokaryotic DNA [341F/806R (Yu et al., Reference Yu, Lee, Kim and Hwang2005)] (Krogsgaard et al., Reference Krogsgaard, Andersen, Johannesen, Engsbro, Stensvold, Nielsen and Bytzer2018). Sequences were mapped using BION (http://box.com/bion), a newly developed k-mer-based analytical semi-commercial open-source package which allows annotation to species level. Prokaryotic DNA sequences were mapped against the RDP 11.04 reference database, while eukaryotic DNA sequences were mapped using SILVA version 123 reference database with an improved in-house seven-tier taxonomy for eukaryotes, similar to the tiers defined for prokaryotes (phylum, class, order, family, genus, species and sequence levels).

Published data on differences in the eukaryome across vertebrate populations and links between bactieral and eukaryotic signatures are still scarce.

Krogsgaard et al. (Reference Krogsgaard, Engsbro, Stensvold, Nielsen and Bytzer2015) found that CLIPPs diversity was higher in healthy individuals compared with patients with irritable bowel syndrome and also observed that individuals colonized by CLIPPs typically had a higher bacterial richness and diversity than those without (Krogsgaard et al., Reference Krogsgaard, Andersen, Johannesen, Engsbro, Stensvold, Nielsen and Bytzer2018).

Heitlinger et al. (Reference Heitlinger, Ferreira, Thierer, Hofer and East2017) used 4 16S and 44 18S primers in a Fluidigm-based approach, followed by taxonomic analysis using dada2 to map eukaryotic diversity in spotted hyenas. While no differences were found in eukaryome richness, diversity, evenness or genus abundance across age groups in a population of spotted hyenas, a more diverse eukaryome was identified in high-ranking than in low-ranking animals (Heitlinger et al., Reference Heitlinger, Ferreira, Thierer, Hofer and East2017).

Maritz et al. (Reference Maritz, Rogers, Rock, Liu, Joseph, Land and Carlton2017) recently developed and evaluated an 18S rRNA assay employing ILLUMINA-based sequencing and annotation of sequence data using locally curated as well as QIIME formatted SILVA databases with a view to detecting and differentiating protists in sewage with special emphasis on trichomonads. The team used vertebrate blocking primers to increase protist data yield (Maritz et al., Reference Maritz, Rogers, Rock, Liu, Joseph, Land and Carlton2017). Choice of primers is critical too as evidenced by the differing outcomes in terms of e.g. Amoebozoan data obtained by Moreno et al., Reference Moreno, Matz, Kjelleberg and Manefield2010 and Matsunaga et al. (Reference Matsunaga, Kubota and Harada2014), who both aimed at mapping eukaryotic diversity in wastewater/sludge.

The extent to which primate gut eukaryotic diversity is only rudimentarily reflected in reference databases can be exemplified by the following: In a metabarcoding study of non-human primate gut eukaryomes, only 0.01% of all SSU rDNA reads matched sequences in the Silva 123 database at a 100% threshold (Wilcox and Hollocher, Reference Wilcox and Hollocher2018). In that study, de novo operational taxonomic unit (OTU) assignment revealed 4293 eukaryotic OTUs at a 97%-identity level, and reference-based taxonomy assignment matched sequences to 2021 unique eukaryotic genera. Investigating the sewage eukaryome of sludge digesters in Japan, Matsubayashi et al. (Reference Matsubayashi, Shimada, Li, Harada and Kubota2017) found that 85% of the clones obtained by 18S rRNA gene clone library construction showed less than 97.0% sequence identity to what they termed as ‘described eukaryotes’, indicating most of the eukaryotes in anaerobic sludge digesters are largely unknown.

Advancing the mapping of intestinal eukaryotic diversity: Wastewater and new sequencing technologies–the way forward?

In summary, the characterization of nuclear small subunit (SSU) ribosomal RNA genes has been the backbone of DNA-mapping the tree of life. In the field of clinical microbiology, taxon-specific genetic variation across nuclear SSU ribosomal RNA genes has been instrumental to the development of a vast variety of targeted DNA-based diagnostic methods over the past few decades (Verweij and Stensvold, Reference Verweij and Stensvold2014); however, the development and use of such diagnostics are limited by the DNA sequence data available in NCBI (Stensvold et al., Reference Stensvold, Lebbad and Verweij2011b).

The SSU rRNA gene has proved useful for the detection and differentiation of several species of parasites. For helminths, however, this gene generally appears very conserved, and mitochondrial genes or ITS data are taxonomically more informative. Likewise, ITS data appear more relevant for differentiating between non-parasitic eukaryotic organisms often found in the gut, such as yeasts and molds, and so the genes providing most taxonomic resolution differ and depend on the type of organism.

The presence of large intra-generic diversity in some parasites has spurred hypotheses on differences in pathogenicity being associated with species/subtype/genotype, and so our ability to detect and differentiate not only genera and species but also subtypes, ribosomal lineages, etc., is important. Again, while the 18S has proved particularly useful in differentiating between Blastocystis subtypes and even subtype alleles (Stensvold et al., Reference Stensvold, Alfellani and Clark2011a), this marker provides very little resolution within the species of for instance D. fragilis. For other parasites, such as a couple of genera belonging to the Amoebozoa, namely Entamoeba, Endolimax and Iodamoeba, we are only beginning to appreciate the vast extent of genetic diversity (Silberman et al., Reference Silberman, Clark, Diamond and Sogin1999; Clark, Reference Clark2000; Verweij et al., Reference Verweij, Polderman and Clark2001; Stensvold et al., Reference Stensvold, Lebbad and Clark2010, Reference Stensvold, Lebbad, Victory, Verweij, Tannich, Alfellani, Legarraga and Clark2011c; Royer et al., Reference Royer, Gilchrist, Kabir, Arju, Ralston, Haque, Clark and Petri2012; Jacob et al., Reference Jacob, Busby, Levy, Komm and Clark2016; Elsheikha et al., Reference Elsheikha, Regan and Clark2018). The work and methodological limitations involved in mapping the intra-generic diversity in these organisms have led to issues related to resolving the phylogeny among this group of organisms and left some ‘dark holes’ in publicly available databases. Briefly, the largest limitations here are as follows: although hypervariable regions within 18S, ITS or 28S may prove useful for studies into eukaryotic diversity, robust analysis of phylogenetic relationships, including the very delineation of novel ribosomal lineages, and optimal yield of analysis of sequence data from metagenomics or other amplicon-based sequencing studies requires sequencing of complete, or near-complete ribosomal genes. When genomic DNA extracted directly from e.g. stool is used, the application of general primers with a view to amplifying near-complete ribosomal genes often results in preferential amplification of some organisms over other. As an example, individuals colonized by Iodamoeba and/or Endolimax are typically co-colonised with Blastocystis, and because the length of the SSU rRNA gene is only 1.8 kbp in Blastocystis while 2.5 kbp or more in Iodamoeba and Endolimax, Blastocystis ribosomal genes are more likely to be amplified from faecal genomic DNA due to the shorter DNA sequence. Another limitation is related to intra-cellular variation (hypervariable regions), which makes Sanger sequencing of polymerase chain reaction (PCR) products of some sequence stretches unsuitable, e.g. due to the presence of sequence variation within homo-polymers. TA cloning of PCR products has been tried with some success, but this is relatively expensive, time-consuming and laborious (Stensvold et al., Reference Stensvold, Lebbad and Clark2012). Even next-generation sequencing methods such as ILLUMINA do not provide much better solutions to overcoming this issue. Clearly, alternative ways to effectively obtain data are needed.

Meanwhile, Pacific Biosciences (PacBio) RS II, considered a third-generation sequencer, uses single-molecule real-time technology and can be used for sequencing of single DNA molecules in real-time without prior amplification steps, enabling direct observation of DNA synthesis by DNA polymerase (Nakano et al., Reference Nakano, Shiroma, Shimoji, Tamotsu, Ashimine, Ohki, Shinzato, Minami, Nakanishi, Teruya, Satou and Hirano2017). Importantly, this technology enables the production of long reads (typically >20 kbp with a maximum of 60 kbp) at relatively low costs (Nakano et al., Reference Nakano, Shiroma, Shimoji, Tamotsu, Ashimine, Ohki, Shinzato, Minami, Nakanishi, Teruya, Satou and Hirano2017). Orr and colleagues used culturing and targeted PacBio RS II amplicon sequencing to expand on data on the diversity within the class of Diphyllatea, a group of protists that may represent one of the earliest diverging eukaryotic lineages (Orr et al., Reference Orr, Zhao, Klaveness, Yabuki, Ikeda, Makoto and Shalchian-Tabrizi2018). By obtaining near full-length 18S rRNA sequences in addition to mining publicly available databases, they were able to resolve the phylogeny within the class and better map the distribution of members of the class. The technology was also recently used for characterizing and quantifying protistan sequences from environmental samples (Jones and Kustka, Reference Jones and Kustka2017), and in terms of gut microbial diversity, one of the few studies using it so far is that by Myer et al. (Reference Myer, Kim, Freetly and Smith2016) to generate data for phylogenetic analysis of rumen bacterial communities.

A limit to this technology is the relatively high rate of sequencing-related introduced errors; however, there are several ways to reduce or completely eliminate these errors using software tools and by decreasing the time the machine is used. Moreover, PacBio appears to be better at overcoming the issues related to the sequencing of hypervariable regions that e.g. ILLUMINA sequencing may have problems with. Critics of PacBio might argue that the use of this technology should rather be seen as an adjunctive, supportive and possibly exploratory tool that may provide a scaffold that could inform and guide more sophisticated and precise analyses. Such analyses could include Illumina-based sequencing of overlapping 300–400-bp amplicons using sequence-specific primers. Nevertheless, complete and accurate de novo assemblies of Escherichia coli strains could be accomplished using data generated solely from the PacBio RS II (Powers et al., Reference Powers, Weigman, Shu, Pufky, Cox and Hurban2013). The team found that addition of other sequencing technology data obtained by Ion Torrent and MiSeq offered no improvements over the use of PacBio data alone (Powers et al., Reference Powers, Weigman, Shu, Pufky, Cox and Hurban2013).

Apart from identifying the best possible technological and data processing pipelines, it is also worthwhile considering types of material for studying diversity. For instance, untreated sewage may be particularly useful in terms of detecting and mapping micro-eukaryotic diversity, since this material reflects pooled faecal samples from a large population of humans with some spill-over of material from non-human sources.

Chouari et al. (Reference Chouari, Leonard, Bouali, Guermazi, Rahli, Zrafi, Morin and Sghir2017) investigated eukaryotic diversity in wastewater using 18S sequencing, and of 1519 analysed sequences, 160 operational taxonomic units were identified. No less than 56.9% of the phylotypes were assigned to novel phylogenetic molecular species, showing <97% sequence similarity with their nearest affiliated representative within public databases. Similarly, Matsunaga et al. (Reference Matsunaga, Kubota and Harada2014) observed that 60% of their 18S rRNA gene clones obtained from DNA extracted from municipal wastewater had <97% sequence identity to described eukaryotes. In both studies, data on Blastocystis and Amoebozoa were observed. These studies highlight not only the vast DNA data gap in the eukaryotic tree of life, but also the relevance of using sewage as study material for investigations into eukaryotic diversity.

In conclusion, DNA mapping of nucleated life within the intestine and exploring it in ecological contexts are critical to further our understanding of gut microbial diversity and its role in health and disease. Application of more detailed reference data will allow for subtle and robust trans-kingdom analyses of gut microbes and will moreover expand our knowledge on host specificity, transmission patterns and links to clinical phenotypes. The use of genomic DNA from the pooled stool, as e.g. represented by sewage and amplicon-based third-generation sequencing may be a way to ensure the acquisition of quick and robust data to uncover the missing branches of the gut microbial eukaryotic tree.

Author ORCIDs

Christen Rune Stensvold, 0000-0002-1417-7048.

Financial support

This research received no specific grant from any funding agency, commercial or not-for-profit sectors.

Conflict of interest

None.

Ethical standards

Not applicable.

References

Aguiar, JI, Gonçalves, AQ, Sodré, FC, Pereira, SOR, Bóia, MN, de Lemos, ER and Daher, RR (2007) Intestinal protozoa and helminths among Terena Indians in the State of Mato Grosso do Sul: high prevalence of Blastocystis hominis. Revista da Sociedade Brasileira de Medicina Tropical 40, 631634.Google Scholar
Alfellani, MA, Jacob, AS, Perea, NO, Krecek, RC, Taner-Mulla, D, Verweij, JJ, Levecke, B, Tannich, E, Clark, CG and Stensvold, CR (2013 a) Diversity and distribution of Blastocystis sp. subtypes in non-human primates. Parasitology 140, 966971.Google Scholar
Alfellani, MA, Taner-Mulla, D, Jacob, AS, Imeede, CA, Yoshikawa, H, Stensvold, CR and Clark, CG (2013 b) Genetic diversity of Blastocystis in livestock and zoo animals. Protist 164, 497509.Google Scholar
Andersen, LO and Stensvold, CR (2016) Blastocystis in health and disease: are we moving from a clinical to a public health perspective? Journal of Clinical Microbiology 54, 524528.Google Scholar
Andersen, LO, Bonde, I, Nielsen, HB and Stensvold, CR (2015) A retrospective metagenomics approach to studying Blastocystis. FEMS Microbiology Ecology 91, PubMed PMID: 26130823. doi: 10.1093/femsec/fiv072.Google Scholar
Auchtung, TA, Fofanova, TY, Stewart, CJ, Nash, AK, Wong, MC, Gesell, JR, Auchtung, JM, Ajami, NJ and Petrosino, JF (2018) Investigating colonization of the healthy adult gastrointestinal tract by fungi. mSphere 3, pii: e00092–18. doi: 10.1128/mSphere.00092-18.Google Scholar
Bates, ST, Clemente, JC, Flores, GE, Walters, WA, Parfrey, LW, Knight, R and Fierer, N (2013) Global biogeography of highly diverse protistan communities in soil. The ISME Journal 7, 652659.Google Scholar
Beghini, F, Pasolli, E, Truong, TD, Putignani, L, Cacciò, SM and Segata, N (2017) Large-scale comparative metagenomics of Blastocystis, a common member of the human gut microbiome. The ISME Journal 11, 28482863.Google Scholar
Bruijnesteijn van Coppenraet, LE, Wallinga, JA, Ruijs, GJ, Bruins, MJ and Verweij, JJ (2009) Parasitological diagnosis combining an internally controlled real-time PCR assay for the detection of four protozoa in stool samples with a testing algorithm for microscopy. Clinical Microbiology and Infection 15, 869874.Google Scholar
Cacciò, SM, Sannella, AR, Manuali, E, Tosini, F, Sensi, M, Crotti, D and Pozio, E (2012) Pigs as natural hosts of Dientamoeba fragilis genotypes found in humans. Emerging Infectious Diseases 18, 838841.Google Scholar
Cacciò, SM, Sannella, AR, Bruno, A, Stensvold, CR, David, EB, Guimarães, S, Manuali, E, Magistrali, C, Mahdad, K, Beaman, M, Maserati, R, Tosini, F and Pozio, E (2016) Multilocus sequence typing of Dientamoeba fragilis identified a major clone with widespread geographical distribution. International Journal for Parasitology 46, 793798.Google Scholar
Cani, PD (2018) Human gut microbiome: hopes, threats and promises. Gut 67, 17161725.Google Scholar
Chouari, R, Leonard, M, Bouali, M, Guermazi, S, Rahli, N, Zrafi, I, Morin, L and Sghir, A (2017) Eukaryotic molecular diversity at different steps of the wastewater treatment plant process reveals more phylogenetic novel lineages. World Journal of Microbiology and Biotechnology 33, 44.Google Scholar
Clark, CG (2000) Cryptic genetic variation in parasitic protozoa. Journal of Medical Microbiology 49, 489491.Google Scholar
Clark, CG and Stensvold, CR (2016) Blastocystis: isolation, xenic cultivation, and cryopreservation. Current Protocols in Microbiology 43, 20A.21.2120A.21.28.Google Scholar
Clark, CG, van der Giezen, M, Alfellani, MA and Stensvold, CR (2013) Recent developments in blastocystis research. Advances in Parasitology 82, 132.Google Scholar
Cociancic, P, Zonta, ML and Navone, GT (2018) A cross-sectional study of intestinal parasitoses in dogs and children of the periurban area of La Plata (Buenos Aires, Argentina): zoonotic importance and implications in public health. Zoonoses and Public Health 65, e44e53.Google Scholar
Constenla, M, Padrós, F and Palenzuela, O (2014) Endolimax piscium sp. nov. (Amoebozoa), causative agent of systemic granulomatous disease of cultured sole, Solea senegalensis Kaup. Journal of Fish Diseases 37, 229240.Google Scholar
Cristescu, ME (2014) From barcoding single individuals to metabarcoding biological communities: towards an integrative approach to the study of global biodiversity. Trends in Ecology & Evolution 29, 566571.Google Scholar
de Jong, MJ, Korterink, JJ, Benninga, MA, Hilbink, M, Widdershoven, J and Deckers-Kocken, JM (2014) Dientamoeba fragilis and chronic abdominal pain in children: a case-control study. Archives of Disease in Childhood 99, 11091113.Google Scholar
El Safadi, D, Gaayeb, L, Meloni, D, Cian, A, Poirier, P, Wawrzyniak, I, Delbac, F, Dabboussi, F, Delhaes, L, Seck, M, Hamze, M, Riveau, G and Viscogliosi, E (2014) Children of Senegal River Basin show the highest prevalence of Blastocystis sp. ever observed worldwide. BMC Infectious Diseases 14, 164.Google Scholar
Elsheikha, HM, Regan, CS and Clark, CG (2018) Novel entamoeba findings in nonhuman primates. Trends in Parasitology 34, 283294.Google Scholar
Faria, CP, Zanini, GM, Dias, GS, da Silva, S, de Freitas, MB, Almendra, R, Santana, P and Sousa, MD (2017) Geospatial distribution of intestinal parasitic infections in Rio de Janeiro (Brazil) and its association with social determinants. PLoS Neglected Tropical Diseases 11, e0005445.Google Scholar
Greigert, V, Abou-Bacar, A, Brunet, J, Nourrisson, C, Pfaff, AW, Benarbia, L, Pereira, B, Randrianarivelojosia, M, Razafindrakoto, JL, Solotiana Rakotomalala, R, Morel, E, Candolfi, E and Poirier, P (2018) Human intestinal parasites in Mahajanga, Madagascar: the kingdom of the protozoa. PLoS ONE 13, e0204576.Google Scholar
Hamad, I, Abou Abdallah, R, Ravaux, I, Mokhtari, S, Tissot-Dupont, H, Michelle, C, Stein, A, Lagier, JC, Raoult, D and Bittar, F (2018) Metabarcoding analysis of eukaryotic microbiota in the gut of HIV-infected patients. PLoS ONE 13, e0191913.Google Scholar
Heitlinger, E, Ferreira, SCM, Thierer, D, Hofer, H and East, ML (2017) The intestinal eukaryotic and bacterial biome of spotted hyenas: the impact of social status and age on diversity and composition. Frontiers in Cellular and Infection Microbiology 7, 262.Google Scholar
Higa, MG, Cardoso, WM, Weis, SMDS, França, AO, Pontes, ERJC, Silva, PVD, Oliveira, MP and Dorval, MEMC (2017) Intestinal parasitism among waste pickers in Mato Grosso do Sul, Midwest Brazil. Revista do Instituto de Medicina Tropical de Sao Paulo 59, e87.Google Scholar
Holtman, GA, Kranenberg, JJ, Blanker, MH, Ott, A, Lisman-van Leeuwen, Y and Berger, MY (2017) Dientamoeba fragilis colonization is not associated with gastrointestinal symptoms in children at primary care level. Family Practice 34, 2529.Google Scholar
Jacob, AS, Busby, EJ, Levy, AD, Komm, N and Clark, CG (2016) Expanding the entamoeba universe: new hosts yield novel ribosomal lineages. Journal of Eukaryotic Microbiology 63, 6978.Google Scholar
Jeske, S, Bianchi, TF, Moura, MQ, Baccega, B, Pinto, NB, Berne, MEA and Villela, MM (2018) Intestinal parasites in cancer patients in the South of Brazil. Brazilian Journal of Biology 78, 574578.Google Scholar
Jokelainen, P, Hebbelstrup Jensen, B, Andreassen, BU, Petersen, AM, Röser, D, Krogfelt, KA, Nielsen, HV and Stensvold, CR (2017) Dientamoeba fragilis – a commensal in children in Danish day care centers. Journal of Clinical Microbiology 55, 17071713.Google Scholar
Jones, BM and Kustka, AB (2017) A quantitative SMRT cell sequencing method for ribosomal amplicons. Journal of Microbiological Methods 135, 7784.Google Scholar
Kriss, M, Hazleton, KZ, Nusbacher, NM, Martin, CG and Lozupone, CA (2018) Low diversity gut microbiota dysbiosis: drivers, functional implications and recovery. Current Opinion in Microbiology 44, 3440.Google Scholar
Krogsgaard, LR, Engsbro, AL, Stensvold, CR, Nielsen, HV and Bytzer, P (2015) The prevalence of intestinal parasites is not greater among individuals with irritable bowel syndrome: a population-based case-control study. Clinical Gastroenterology and Hepatology 13, 507513.e502Google Scholar
Krogsgaard, LR, Andersen, LO, Johannesen, TB, Engsbro, AL, Stensvold, CR, Nielsen, HV and Bytzer, P (2018) Characteristics of the bacterial microbiome in association with common intestinal parasites in irritable bowel syndrome. Clinical and Translational Gastroenterology 9, 161.Google Scholar
Laforest-Lapointe, I and Arrieta, MC (2018) Microbial eukaryotes: a missing link in gut microbiome studies. mSystems 3, pii: e00201–17.Google Scholar
Lukeš, J, Stensvold, CR, Jirků-Pomajbíková, K and Wegener Parfrey, L (2015) Are human intestinal eukaryotes beneficial or commensals? PLoS Pathogens 11, e1005039.Google Scholar
Maritz, JM, Rogers, KH, Rock, TM, Liu, N, Joseph, S, Land, KM and Carlton, JM (2017) An 18S rRNA workflow for characterizing protists in sewage, with a focus on zoonotic trichomonads. Microbial Ecology 74, 923936.Google Scholar
Masuda, A, Sumiyoshi, T, Ohtaki, T and Matsumoto, J (2018) Prevalence and molecular subtyping of Blastocystis from dairy cattle in Kanagawa, Japan. Parasitology International 67, 702705.Google Scholar
Matsubayashi, M, Shimada, Y, Li, YY, Harada, H and Kubota, K (2017) Phylogenetic diversity and in situ detection of eukaryotes in anaerobic sludge digesters. PLoS ONE 12, e0172888.Google Scholar
Matsunaga, K, Kubota, K and Harada, H (2014) Molecular diversity of eukaryotes in municipal wastewater treatment processes as revealed by 18S rRNA gene analysis. Microbes and Environments 29, 401407.Google Scholar
Mirjalali, H, Abbasi, MR, Naderi, N, Hasani, Z, Mirsamadi, ES, Stensvold, CR, Balaii, H, Asadzadeh Aghdaei, H and Zali, MR (2017) Distribution and phylogenetic analysis of Blastocystis sp. subtypes isolated from IBD patients and healthy individuals in Iran. European Journal of Clinical Microbiology & Infectious Diseases 36, 23352342.Google Scholar
Moon-van der Staay, SY, van der Staay, GW, Michalowski, T, Jouany, JP, Pristas, P, Javorský, P, Kišidayová, S, Varadyova, Z, McEwan, NR, Newbold, CJ, van Alen, T, de Graaf, R, Schmid, M, Huynen, MA and Hackstein, JH (2014) The symbiotic intestinal ciliates and the evolution of their hosts. European Journal of Protistology 50, 166173.Google Scholar
Moreno, AM, Matz, C, Kjelleberg, S and Manefield, M (2010) Identification of ciliate grazers of autotrophic bacteria in ammonia-oxidizing activated sludge by RNA stable isotope probing. Appl Environ Microbiol 76, 22032211.Google Scholar
Morton, ER, Lynch, J, Froment, A, Lafosse, S, Heyer, E, Przeworski, M, Blekhman, R and Ségurel, L (2015) Variation in Rural African gut microbiota is strongly correlated with colonization by entamoeba and subsistence. PLoS Genetics 11, e1005658.Google Scholar
Moura, RGF, Oliveira-Silva, MB, Pedrosa, AL, Nascentes, GAN and Cabrine-Santos, M (2018) Occurrence of Blastocystis spp. in domestic animals in Triângulo Mineiro area of Brazil. Revista da Sociedade Brasileira de Medicina Tropical 51, 240243.Google Scholar
Munasinghe, VS, Vella, NG, Ellis, JT, Windsor, PA and Stark, D (2013) Cyst formation and faecal-oral transmission of Dientamoeba fragilis – the missing link of the life cycle of an emering pathogen. International Journal for Parasitology 43, 879883.Google Scholar
Myer, PR, Kim, M, Freetly, HC and Smith, TP (2016) Metagenomic and near full-length 16S rRNA sequence data in support of the phylogenetic analysis of the rumen bacterial community in steers. Data in Brief 8, 10481053.Google Scholar
Nakano, K, Shiroma, A, Shimoji, M, Tamotsu, H, Ashimine, N, Ohki, S, Shinzato, M, Minami, M, Nakanishi, T, Teruya, K, Satou, K and Hirano, T (2017) Advantages of genome sequencing by long-read sequencer using SMRT technology in medical area. Human Cell 30, 149161.Google Scholar
Navarro, C, Domínguez-Márquez, MV, Garijo-Toledo, MM, Vega-García, S, Fernández-Barredo, S, Pérez-Gracia, MT, García, A, Borrás, R and Gómez-Muñoz, MT (2008) High prevalence of Blastocystis sp. in pigs reared under intensive growing systems: frequency of ribotypes and associated risk factors. Veterinary Parasitology 153, 347358.Google Scholar
Neres-Norberg, A, Guerra-Sanches, F, Blanco Moreira-Norberg, PR, Madeira-Oliveira, JT, Santa-Helena, AA and Serra-Freire, NM (2014) [Intestinal Parasitism in Terena Indigenous People of the Province of Mato Grosso do Sul, Brazil]. Revista de Salud Publica (Bogota) 16, 859870.Google Scholar
Ögren, J, Dienus, O, Löfgren, S, Einemo, IM, Iveroth, P and Matussek, A (2015) Dientamoeba fragilis prevalence coincides with gastrointestinal symptoms in children less than 11 years old in Sweden. European Journal of Clinical Microbiology & Infectious Diseases 34, 19951998.Google Scholar
Ohkuma, M (2008) Symbioses of flagellates and prokaryotes in the gut of lower termites. Trends in Microbiology 16, 345352.Google Scholar
Oliverio, AM, Power, JF, Washburne, A, Cary, SC, Stott, MB and Fierer, N (2018) The ecology and diversity of microbial eukaryotes in geothermal springs. The ISME Journal 12, 19181928.Google Scholar
Orr, RJS, Zhao, S, Klaveness, D, Yabuki, A, Ikeda, K, Makoto, WM and Shalchian-Tabrizi, K (2018) Enigmatic Diphyllatea eukaryotes: culturing and targeted PacBio RS amplicon sequencing reveals a higher order taxonomic diversity and global distribution. BMC Evolutionary Biology 18, 115.Google Scholar
Pakandl, M (1991) Occurrence of Blastocystis sp. in pigs. Folia Parasitologica (Praha) 38, 297301.Google Scholar
Parfrey, LW, Walters, WA and Knight, R (2011) Microbial eukaryotes in the human microbiome: ecology, evolution, and future directions. Frontiers in Microbiology 2, 153.Google Scholar
Peek, R, Reedeker, FR and van Gool, T (2004) Direct amplification and genotyping of Dientamoeba fragilis from human stool specimens. Journal of Clinical Microbiology 42, 631635.Google Scholar
Petersen, AM, Stensvold, CR, Mirsepasi, H, Engberg, J, Friis-Møller, A, Porsbo, LJ, Hammerum, AM, Nordgaard-Lassen, I, Nielsen, HV and Krogfelt, KA (2013) Active ulcerative colitis associated with low prevalence of Blastocystis and Dientamoeba fragilis infection. Scandinavian Journal of Gastroenterology 48, 638639.Google Scholar
Poulsen, CS and Stensvold, CR (2016) Systematic review on Endolimax nana: a less well studied intestinal ameba. Tropical Parasitology 6, 829.Google Scholar
Poulsen, CS, Efunshile, AM, Nelson, JA and Stensvold, CR (2016) Epidemiological aspects of Blastocystis colonization in children in Ilero, Nigeria. American Journal of Tropical Medicine and Hygiene 95, 175179.Google Scholar
Powers, JG, Weigman, VJ, Shu, J, Pufky, JM, Cox, D and Hurban, P (2013) Efficient and accurate whole genome assembly and methylome profiling of E. coli. BMC Genomics 14, 675.Google Scholar
Ramirez, JD, Sanchez, LV, Bautista, DC, Corredor, AF, Florez, AC and Stensvold, CR (2014) Blastocystis subtypes detected in humans and animals from Colombia. Infection Genetics and Evolution 22, 223228.Google Scholar
Rene, BA, Stensvold, CR, Badsberg, JH and Nielsen, HV (2009) Subtype analysis of Blastocystis isolates from Blastocystis cyst excreting patients. American Journal of Tropical Medicine and Hygiene 80, 588592.Google Scholar
Rosenberg, K, Bertaux, J, Krome, K, Hartmann, A, Scheu, S and Bonkowski, M (2009) Soil amoebae rapidly change bacterial community composition in the rhizosphere of Arabidopsis thaliana. The ISME Journal 3, 675684.Google Scholar
Roser, D, Simonsen, J, Nielsen, HV, Stensvold, CR and Molbak, K (2013) Dientamoeba fragilis in Denmark: epidemiological experience derived from four years of routine real-time PCR. European Journal of Clinical Microbiology & Infectious Diseases 32, 13031310.Google Scholar
Rossen, NG, Bart, A, Verhaar, N, van Nood, E, Kootte, R, de Groot, PF, D'Haens, GR, Ponsioen, CY and van Gool, T (2015) Low prevalence of Blastocystis sp. in active ulcerative colitis patients. European Journal of Clinical Microbiology & Infectious Diseases 34, 10391044.Google Scholar
Royer, TL, Gilchrist, C, Kabir, M, Arju, T, Ralston, KS, Haque, R, Clark, CG and Petri, WA (2012) Entamoeba bangladeshi nov. sp., Bangladesh. Emerging Infectious Diseases 18, 15431545.Google Scholar
Ruaux, CG and Stang, BV (2014) Prevalence of blastocystis in shelter-resident and client-owned companion animals in the US Pacific Northwest. PLoS ONE 9, e107496.Google Scholar
Růžková, J, Květoňová, D, Jirků, M, Lhotská, Z, Stensvold, CR, Parfrey, LW and Jirků Pomajbíková, K (2018) Evaluating rodent experimental models for studies of Blastocystis ST1. Experimental Parasitology 191, 5561.Google Scholar
Salehi, R, Haghighi, A, Stensvold, CR, Kheirandish, F, Azargashb, E, Raeghi, S, Kohansal, C and Bahrami, F (2017) Prevalence and subtype identification of. Gastroenterology and Hepatology From Bed To Bench 10, 235241.Google Scholar
Scanlan, PD, Stensvold, CR, Rajilić-Stojanović, M, Heilig, HG, De Vos, WM, O'Toole, PW and Cotter, PD (2014) The microbial eukaryote Blastocystis is a prevalent and diverse member of the healthy human gut microbiota. FEMS Microbiology Ecology 90, 326330.Google Scholar
Scanlan, PD, Hill, CJ, Ross, RP, Ryan, CA, Stanton, C and Cotter, PD (2018) The intestinal protist Blastocystis is not a common member of the healthy infant gut microbiota in a Westernized country (Ireland). Parasitology 145, 12741278.Google Scholar
Silberman, JD, Clark, CG, Diamond, LS and Sogin, ML (1999) Phylogeny of the genera Entamoeba and Endolimax as deduced from small-subunit ribosomal RNA sequences. Molecular Biology and Evolution 16, 17401751.Google Scholar
Stark, D, Beebe, N, Marriott, D, Ellis, J and Harkness, J (2005 a) Detection of Dientamoeba fragilis in fresh stool specimens using PCR. International Journal for Parasitology 35, 5762.Google Scholar
Stark, D, Beebe, N, Marriott, D, Ellis, J and Harkness, J (2005 b) Prospective study of the prevalence, genotyping, and clinical relevance of Dientamoeba fragilis infections in an Australian population. Journal of Clinical Microbiology 43, 27182723.Google Scholar
Stark, D, Beebe, N, Marriott, D, Ellis, J and Harkness, J (2006) Evaluation of three diagnostic methods, including real-time PCR, for detection of Dientamoeba fragilis in stool specimens. Journal of Clinical Microbiology 44, 232235.Google Scholar
Stark, D, Phillips, O, Peckett, D, Munro, U, Marriott, D, Harkness, J and Ellis, J (2008) Gorillas are a host for Dientamoeba fragilis: an update on the life cycle and host distribution. Veterinary Parasitology 151, 2126.Google Scholar
Stark, D, Garcia, LS, Barratt, JL, Phillips, O, Roberts, T, Marriott, D, Harkness, J and Ellis, JT (2014) Description of Dientamoeba fragilis cyst and precystic forms from human samples. Journal of Clinical Microbiology 52, 26802683.Google Scholar
Stark, D, Barratt, J, Chan, D and Ellis, JT (2016) Dientamoeba fragilis, the neglected trichomonad of the human bowel. Clinical Microbiology Reviews 29, 553580.Google Scholar
Stensvold, CR and Clark, CG (2016) Current status of Blastocystis: a personal view. Parasitology International 65, 763771.Google Scholar
Stensvold, CR and Nielsen, HV (2012) Comparison of microscopy and PCR for detection of intestinal parasites in Danish patients supports an incentive for molecular screening platforms. Journal of Clinical Microbiology 50, 540541.Google Scholar
Stensvold, CR and van der Giezen, M (2018) Associations between Gut Microbiota and common luminal intestinal parasites. Trends in Parasitology 34, 369377.Google Scholar
Stensvold, CR, Lebbad, M and Clark, CG (2010) Genetic characterisation of uninucleated cyst-producing Entamoeba spp. from ruminants. International Journal for Parasitology 40, 775778.Google Scholar
Stensvold, CR, Alfellani, M and Clark, CG (2011a) Levels of genetic diversity vary dramatically between Blastocystis subtypes. Infection Genetics and Evolution 12, 263273.Google Scholar
Stensvold, CR, Lebbad, M and Verweij, JJ (2011b) The impact of genetic diversity in protozoa on molecular diagnostics. Trends in Parasitology 27, 5358.Google Scholar
Stensvold, CR, Lebbad, M, Victory, EL, Verweij, JJ, Tannich, E, Alfellani, M, Legarraga, P and Clark, CG (2011c) Increased sampling reveals novel lineages of Entamoeba: consequences of genetic diversity and host specificity for taxonomy and molecular detection. Protist 162, 525541.Google Scholar
Stensvold, CR, Lebbad, M and Clark, CG (2012) Last of the human protists: the phylogeny and genetic diversity of iodamoeba. Molecular Biology and Evolution 29, 3942.Google Scholar
Stensvold, CR, Clark, CG and Röser, D (2013) Limited intra-genetic diversity in Dientamoeba fragilis housekeeping genes. Infection Genetics and Evolution 18, 284286.Google Scholar
Stensvold, CR, Winiecka-Krusnell, J, Lier, T and Lebbad, M (2018) Evaluation of a PCR method for detection of Entamoeba polecki, with an overview of Its molecular epidemiology. Journal of Clinical Microbiology 56, pii: e00154–18.Google Scholar
ten Hove, R, Schuurman, T, Kooistra, M, Möller, L, van Lieshout, L and Verweij, JJ (2007) Detection of diarrhoea-causing protozoa in general practice patients in The Netherlands by multiplex real-time PCR. Clinical Microbiology and Infection 13, 10011007.Google Scholar
Udonsom, R, Prasertbun, R, Mahittikorn, A, Mori, H, Changbunjong, T, Komalamisra, C, Pintong, AR, Sukthana, Y and Popruk, S (2018) Blastocystis infection and subtype distribution in humans, cattle, goats, and pigs in central and western Thailand. Infection Genetics and Evolution 65, 107111.Google Scholar
Veira, DM (1986) The role of ciliate protozoa in nutrition of the ruminant. Journal of Animal Science 63, 15471560.Google Scholar
Verweij, JJ (2014) Application of PCR-based methods for diagnosis of intestinal parasitic infections in the clinical laboratory. Parasitology 141, 18631872.Google Scholar
Verweij, JJ and Stensvold, CR (2014) Molecular testing for clinical diagnosis and epidemiological investigations of intestinal parasitic infections. Clinical Microbiology Reviews 27, 371418.Google Scholar
Verweij, JJ and van Lieshout, L (2011) Intestinal parasitic infections in an industrialized country; a new focus on children with better DNA-based diagnostics. Parasitology 138, 14921498.Google Scholar
Verweij, JJ, Polderman, AM and Clark, CG (2001) Genetic variation among human isolates of uninucleated cyst-producing entamoeba species. Journal of Clinical Microbiology 39, 16441646.Google Scholar
Verweij, JJ, Mulder, B, Poell, B, van Middelkoop, D, Brienen, EA and van Lieshout, L (2007) Real-time PCR for the detection of Dientamoeba fragilis in fecal samples. Molecular and Cellular Probes 21, 400404.Google Scholar
Wang, W, Owen, H, Traub, RJ, Cuttell, L, Inpankaew, T and Bielefeldt-Ohmann, H (2014) Molecular epidemiology of Blastocystis in pigs and their in-contact humans in Southeast Queensland, Australia, and Cambodia. Veterinary Parasitology 203, 264269.Google Scholar
Wilcox, JJS and Hollocher, H (2018) Unprecedented symbiont eukaryote diversity Is governed by internal trophic webs in a wild Non-human primate. Protist 169, 307320.Google Scholar
Xiong, J, Yu, W, Dai, W, Zhang, J, Qiu, Q and Ou, C (2018) Quantitative prediction of shrimp disease incidence via the profiles of gut eukaryotic microbiota. Applied Microbiology and Biotechnology 102, 33153326.Google Scholar
Yoshikawa, H, Koyama, Y, Tsuchiya, E and Takami, K (2016) Blastocystis phylogeny among various isolates from humans to insects. Parasitology International 65, 750759.Google Scholar
Yu, Y, Lee, C, Kim, J and Hwang, S (2005) Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction. Biotechnology and Bioengineering 89, 670679.Google Scholar