Blastocystis is the most ubiquitous protist inhabiting the gastrointestinal tract of human and other animal hosts (Roberts et al., 2013; Beghini et al., 2017; Stensvold and van der Giezen, 2018). Historically, diagnosis of Blastocystis has been based on light microscopy of fecal smears or in vitro cultures. The organism has four morphological forms: vacuolar, granular, amoeboid, and cyst (Tan, 2008; Parija and Jeremiah, 2013). The lack of distinct morphological features had, in the past, blurred the extent of Blastocystis diversity. Based on the genetic heterogeneity of the small subunit ribosomal RNA (SSU rRNA), Blastocystis is currently divided into at least 28 subtypes (STs) consisting of ST1-ST17, ST21, and ST23-ST32, all of which have been found in mammalian and avian hosts and are likely separate species (Stensvold et al., 2012; Alfellani et al., 2013b; Zhao et al., 2017; Maloney et al., 2020, 2021a,b; Stensvold and Clark, 2020; Higuera et al., 2021). Several genetically distinct Blastocystis lineages have also been identified in amphibian, insect and reptilian hosts, however, these are not part of the subtyping nomenclature as yet (Yoshikawa et al., 2007, 2016).

Despite earlier assumptions, subtypes do not seem to be host specific. So far, ST1-ST9 and ST12 have been reported in humans along with a single instance of ST10, ST14, and ST16 (Stensvold and Clark, 2016; Khaled et al., 2020; Osorio-Pulgarin et al., 2021). The most frequently encountered subtypes in humans are ST1-ST4, with the latter being most often reported in Europe (Deng et al., 2019; Jiménez et al., 2019; Stensvold et al., 2020). Nonetheless, the subtypes reported in humans have also been found in non-human hosts. For example, ST1 and ST3 have been identified from pigs, while ST4 is dominant in rodents (Yoshikawa et al., 2004; Stensvold et al., 2009a; Alfellani et al., 2013a; Wang et al., 2018; Betts et al., 2021).

After more than a century of research, the pathogenicity of Blastocystis remains questionable. Its presence in sufferers of chronic gastrointestinal illnesses including irritable bowel syndrome and inflammatory bowel disease has led to speculations about possible links to these disease states (Dogruman-Al et al., 2009a; Tan et al., 2010; Poirier et al., 2012; Cifre et al., 2018; Kesuma et al., 2019; Peña et al., 2020; Shirvani et al., 2020). However, recent studies have increasingly shown that Blastocystis is a frequent and stable inhabitant in the gut of hosts without gastrointestinal symptoms (Scanlan et al., 2014; Mirjalali et al., 2017; Riabi et al., 2018; Yowang et al., 2018; Kataki et al., 2019; Lhotská et al., 2020; Padukone et al., 2020). In parallel, this protist has been linked with increased bacterial richness and diversity in the human gut (Audebert et al., 2016; Chabé et al., 2017; Laforest-Lapointe and Arrieta, 2018; Tito et al., 2019; Deng et al., 2021). Therefore, a plethora of researchers now consider Blastocystis as a commensal rather than a pathogen.

Understanding various aspects of Blastocystis epidemiology will contribute significantly toward determining its pathogenicity and/or virulence of the various subtypes. To that end, elucidating routes of transmission and contributions of various sources to these routes is essential. The human-to-human, zoonotic, and waterborne transmission routes have been explored in relation to Blastocystis prevalence (Eroglu and Koltas, 2010; Alfellani et al., 2013b; Maloney et al., 2019). Occurrence of certain subtypes in both human and other animal hosts has led to the hypothesis that these are subtypes of zoonotic potential. For instance, ST5, typically found in pigs, and ST6, ST7 typical subtypes of avian hosts, have also been found in humans that handle them extensively (Wang et al., 2014; Greige et al., 2018). Transmission of ST8 has also been noted between non-human primates and their human zookeepers (Stensvold et al., 2009a). Waterborne transmission of Blastocystis has been long recognized (Li et al., 2012; Andersen and Stensvold, 2016). For instance, ST1 was identified in the water supply of a rural community in central Thailand and schoolchildren that consumed it (Leelayoova et al., 2008) and in untreated drinking water in Peninsular Malaysia (Anuar et al., 2013). Nonetheless, only scant studies simultaneously consider the contribution of more than one source to Blastocystis transmission.

In general, investigating transmission dynamics requires conditions that allow for uninterrupted cycling of an organism in a community. As such, developing countries comprise ideal areas to undertake these types of approaches. Herein, we undertook a One Health approach to examine Blastocystis epidemiology in a rural community of northern Thailand. We collected samples from humans, other animals and the environment and screened them for presence of Blastocystis. Data were analyzed at singular and community levels. We identified water and soil as the primary contributing sources to Blastocystis transmission routes in this particular community. These findings provide a multi-layered understanding of the transmission dynamics (spreading and cycling) of this controversial protist.

The study took place in a century old isolated rural community in northern Thailand comprising approximately 500 people. Inhabitants live in very close proximity to their animals, primarily chickens and secondarily buffalo and pigs. Part of the community’s water supply comes from the river that runs through it. The river also provides a major food source for the villagers, as fish constitutes the primary protein source of the community, along with vegetables (which also grow inside the river and the river bank) and locally farmed sticky rice. The increased influence of westernized diet noted in urban centers of Thailand has a minor impact in this community. Collectively, the small population, distance from urban centers, unique gastronomy (minimal effect from westernization) and the general lifestyle make this particular community ideal for local One Health approaches. Herein, we used Blastocystis, a microbial eukaryote of controversial pathogenicity, to obtain a comprehensive view of its transmission dynamics.

Blastocystis is the most frequently encountered intestinal protist of metazoans with most studies focusing on either its prevalence in humans, other animals and/or the environment. Nonetheless, only very few investigations explore the organism’s transmission dynamics using a tripartite approach, whereby all of the aforementioned factors are considered collectively. In order to understand the role of this organism in health and disease it is essential to determine its occurrence simultaneously in human and non-human hosts and environments.

In humans, the prevalence of Blastocystis has been frequently reported in those with and without gastrointestinal symptoms (Dogruman-Al et al., 2009b; Scanlan et al., 2014; Yowang et al., 2018; Kataki et al., 2019; Lhotská et al., 2020; Padukone et al., 2020). Overall prevalence of Blastocystis might vary due to sampling population, region and detection method (Stensvold et al., 2009b; Tan et al., 2010; Alfellani et al., 2013a; Anuar et al., 2013; Clark et al., 2013). Herein, the prevalence of Blastocystis in asymptomatic human hosts was 73%, in asymptomatic non-human hosts 100% and in environmental samples 91%. We used microscopy and molecular methods to determine presence of Blastocystis. The most sensitive detection method was qPCR matching previous studies (Poirier et al., 2011; Stensvold and Nielsen, 2012; Stensvold et al., 2012). After sequencing all positive samples, a broad diversity of subtypes (STs) was detected: ST1, ST2, ST3, ST4, ST5, ST6, ST7, ST10, ST23, and a potential new subtype. Subtype 10 was detected in six human volunteers. The subtype has been previously found in two Senegalese children (Khaled et al., 2020), but it is a typical cattle subtype (Cian et al., 2017; Zhu et al., 2017; Masuda et al., 2018; Wang et al., 2018). To our great surprise, we found ST23 in 12 human samples making it the dominant subtype in this host. So far, ST23 has only been identified in ruminants. The occurrence of ST10 and ST23 in several adults in an Asian country raises questions regarding the host range and transmission dynamics of Blastocystis subtypes.

The following transmission routes have been widely discussed for Blastocystis: human-to-human, animal-to-human and environment-to-human. The former mode of transmission has been speculated to occur via the fecal-oral route much like other common gastrointestinal parasites. Herein, investigation of individuals within households showed no subtype sharing and there was even an instance of co-habiting individuals, whereby one was Blastocystis positive and another negative. This finding matches previous recent reports derived from family units elsewhere (Scanlan et al., 2016; Lhotská et al., 2020).

We also aimed to look at the animal-to-human transmission route. Previous studies have suggested that specific subtypes are zoonotic (Parkar et al., 2010; Alfellani et al., 2013b; Wang et al., 2014). For instance, ST5 has been proposed as potentially zoonotic from pigs (Yan et al., 2007; Wang et al., 2014) and Blastocystis ST6 and ST7 from poultry (Ramírez et al., 2014; Cian et al., 2017; Greige et al., 2018; Udonsom et al., 2018). Subtype 1, ST7, ST10, and ST23 were found in both human and animal hosts in the studied area giving the impression of zoonotic transmission. However, when looking at a fine-scale level there was no sharing of subtypes between animals and their respective owners. Collective consideration of the evidence points toward the source of Blastocystis in this specific community being elsewhere.

This prompted us to look at the two most commonly encountered environmental sources in the community: water and soil. Water contamination has been speculated as a risk factor to acquire Blastocystis. However, only few studies have looked at presence of Blastocystis in both water and humans that use it and even fewer have employed subtyping to examine overlap between the two (Leelayoova et al., 2008; Angelici et al., 2018; Pawestri et al., 2021). Blastocystis has been detected in drinking water (Leelayoova et al., 2008), tap water (Eroglu and Koltas, 2010), rain water tanks (Noradilah et al., 2017; Waters et al., 2019), bodies of freshwater (Ithoi et al., 2011; Khalifa et al., 2014), drinking water treatment facilities (Richard et al., 2016) and waste water (Suresh et al., 2005; Banaticla and Rivera, 2011; Stensvold et al., 2020). Herein, Blastocystis ST1 and ST3 were detected in community supply water, while ST1, ST3, ST5, ST6, ST7, ST10, ST23, and ST26 were found in rain collection vessels. Both these sources comprise the drinking water of this community. The rain collection vessels contain water that is filtered for large debris, but the water is untreated and is consumed unboiled (Li et al., 2007; Leelayoova et al., 2008; Anuar et al., 2013; Wongthamarin et al., 2018; Waters et al., 2019). The community supply water is filtered and occasionally treated. Given the exposed nature of the community water, various wildlife animal hosts harboring a range of subtypes (known and unknown) can easily access it. Thus, presence of the organism in these two sources could be due to a combination of factors including contamination by animal droppings and/or substandard management (i.e., filtration and chlorine usage). Water in the vessels is also used to wash vegetables and tubers hence transfer of cysts of a variety of subtypes could occur this way. Indeed Blastocystis has been previously found in vegetables (Al Nahhas and Aboualchamat, 2020; Li et al., 2020). Through fine-scale analysis we identified a case of ST3 in humans overlapping with the subtypes found in their rain collection vessels. Presence of Blastocystis in an environment, where there is continuous circulation of oxygen supported recently raised hypotheses that this previously considered strictly anaerobic organism tolerates oxygen (Tsaousis et al., 2012, 2018). Thus, future studies should aim toward investigating additional environments including extreme habitats for the presence of Blastocystis.

To that end, we broadened our approach and also explored occurrence of Blastocystis in soil. Most collected soil samples were positive for the organism, while ST1, ST3, ST7, ST23, and ST26 were identified. To our knowledge this is the first report of this protist being recorded in natural soil. The presence of Blastocystis in the soil could be due to extensive use of animal excrement and intestinal contents (especially from fish), which are typically utilized as garden fertilizer in the community. Nonetheless, while sampling, care was taken to collect from gardens that had not been recently fertilized. Moreover, wildlife hosts roaming the community could also shed Blastocystis. This finding suggests a new route of transmission that has been previously overlooked. In that vein, we propose that soil should not only be checked for presence of the organism in future studies, but that it should also be included along with water as a transmission route in the life cycle of Blastocystis (Figure 4).

Comparison of sequences found in different hosts and environments indicated that highly similar strains of ST1 and ST3 are circulating in the community. This suggests a shared Blastocystis transmission cycle among humans, animals and the environment for these subtypes. In contrast, ST7 showed an extraordinary amount of diversity with multiple strains distributed within and between hosts and the environment. This indicates that the full extent of ST7 genetic diversity and host range in the community has yet to be captured. Nonetheless a cluster of highly similar strains was found in chicken, pig and buffalo suggesting transmission among the three hosts.

This study has revealed no clear patterns of direct transmission between human-to-human or animal-to-human in this community (Figure 4). Instead, it points out to the existence of multiple independent routes of transmission. Previous efforts investigating Blastocystis sources of transmission have been geared toward dissecting dipartite relationships (i.e., animal-to-human or environment-to-human). Results from these studies have enhanced our understanding of the organism and its epidemiology. Nonetheless, they frequently only provide pieces of the overall picture, which remains fragmentary. Here, we have provided a step forward toward integrating a One Health approach to Blastocystis by considering both living and non-living sources. In this community, environmental sources comprise the reservoir of Blastocystis supplying a multitude of subtypes that circulate in both human and non-human hosts. Our study is pioneer in that we investigated a rural area, while taking into account the community structure and environmental factors toward understanding Blastocystis circulation.

Limited sample size does pose a limitation in our study. Specifically, the sample size was low, in particular samples from various animal hosts including stray animals and wildlife. Thus transmission cycles between and within hosts and the environment cannot be precisely deduced at this time.

Moving forward, additional communities both rural and urban should be explored under the One Health umbrella to determine whether similar patterns occur. Using the same approach in a temporal context, future studies should also investigate, whether Blastocystis and its various subtypes are true colonizers or passengers. Finally, supplementing One Health-based studies with culturomics and microbiome (pathogenic and non-pathogenic residents of the gut) and metabolome investigations will contribute significantly in uncovering the true roles of Blastocystis in gut health and disease.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

The studies involving human participants were reviewed and approved by the Ethics Committee of Mae Fah Luang University. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Ethics Committee of Mae Fah Luang University. Written informed consent for participation was not obtained from the owners because only fecal samples were obtained, there was no invasive procedure involved.

VJ: fieldwork, methodology, conceptualization, investigation, and draft writing. SM: methodology. SP: resources and methodology. AT: supervision, validation, and final draft. EG: funding acquisition, project administration, supervision, validation, data curation, and final draft. All authors have read and approved the submitted version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.