Blastocystis subtypes in raw vegetables from street markets in northern Thailand.

Blastocystis is a stramenopile protist of controversial pathogenicity. The organism colonizes a broad range of vertebrate and invertebrate hosts. Blastocystis has also been found in the environment both in water and soil. Several routes of transmission have been proposed including human to human, animal to human, and via contaminated food and water. In recent years, the presence of Blastocystis in vegetables has started to be explored. However, most studies have focused on microscopic detection. Moreover, works of this type from Asia are barely available. Hence, the aim of this preliminary study was to examine the occurrence of Blastocystis in raw vegetables sold in markets in northern Thailand. Fresh produce (n = 20) commonly used in Thai cuisine (Lanna) was purchased from two street markets and screened for Blastocystis using qPCR. Blastocystis was detected in 45% of the samples with the dominant subtype being ST3. Produce growing underground, such as galangal, carrot, and beetroot, were positive for the organism suggesting soil or inadequately composted manure as the source of contamination. To our knowledge, our study is the first to perform subtyping of Blastocystis in vegetables. Our results hint toward fresh produce being a, as yet, not widely explored, transmission route of Blastocystis in the studied community. Looking forward, large-scale investigations on the prevalence of this and other organisms under the One Health umbrella should be undertaken.

Blastocystis in tap water of a community in northern Thailand.

Blastocystis is the most common protist in the gut of humans and other animals having global distribution. Occasionally, this organism has also been reported in the environment. Transmission to humans occurs via the fecal-oral route, while water also comprises a transmission route. Blastocystis has been commonly found in rivers, lakes, and wells. Nonetheless, there is limited data about the prevalence and genetic diversity of Blastocystis in tap water. The main aim of this study was to examine the presence of Blastocystis subtypes in tap water (n=20) in a community in northern Thailand. Molecular characterization using the small subunit ribosomal RNA was used to screen for Blastocystis and identify the diversity of subtypes in samples. The overall prevalence was 30% with only subtype three (ST3) encountered in the tap water. These results indicate that tap water has a potential role in the transmission of this subtype in the studied community. Further investigations should focus on expanding sampling to include additional housing complexes and screening for Blastocystis in humans who are exposed to this water.

Metabolic Fluctuations in the Human Stool Obtained from Blastocystis Carriers and Non-Carriers.

Blastocystis is an obligate anaerobic microbial eukaryote that frequently inhabits the gastrointestinal tract. Despite this prevalence, very little is known about the extent of its genetic diversity, pathogenicity, and interaction with the rest of the microbiome and its host. Although the organism is morphologically static, it has no less than 28 genetically distinct subtypes (STs). Reports on the pathogenicity of Blastocystis are conflicting. The association between Blastocystis and intestinal bacterial communities is being increasingly explored. Nonetheless, similar investigations extending to the metabolome are non-existent.Using established NMR metabolomics protocols in 149 faecal samples from individuals from South Korea (n = 38), Thailand (n = 44) and Turkey (n = 69), we have provided a snapshot of the core metabolic compounds present in human stools with (B+) and without (B-) Blastocystis. Samples included hosts with gastrointestinal symptoms and asymptomatics. A total of nine, 62 and 98 significant metabolites were associated with Blastocystis carriage in the South Korean, Thai and Turkish sample sets respectively, with a number of metabolites increased in colonised groups. The metabolic profiles of B+ and B- samples from all countries were distinct and grouped separately in the partial least squares-discriminant analysis (PLS-DA). Typical inflammation-related metabolites negatively associated with Blastocystis positive samples. This data will assist in directing future studies underlying the involvement of Blastocystis in physiological processes of both the gut microbiome and the host. Future studies using metabolome and microbiome data along with host physiology and immune responses information will contribute significantly towards elucidating the role of Blastocystis in health and disease.

Blastocystis One Health Approach in a Rural Community of Northern Thailand: Prevalence, Subtypes and Novel Transmission Routes.

Blastocystis is the most commonly found eukaryote in the gut of humans and other animals. This protist is extremely heterogeneous genetically and is classified into 28 subtypes (STs) based on the small subunit ribosomal RNA (SSU rRNA) gene. Numerous studies exist on prevalence of the organism, which usually focus on either humans or animals or the environment, while only a handful investigates all three sources simultaneously. Consequently, understanding of Blastocystis transmission dynamics remains inadequate. Our aim was to explore Blastocystis under the One Health perspective using a rural community in northern Thailand as our study area. We surveyed human, other animal and environmental samples using both morphological and molecular approaches. Prevalence rates of Blastocystis were 73% in human hosts (n = 45), 100% in non-human hosts (n = 44) and 91% in environmental samples (n = 35). Overall, ten subtypes were identified (ST1, ST2, ST3, ST4 ST5, ST6, ST7, ST10, ST23, and ST26), eight of which were detected in humans (ST1, ST2, ST3, ST4, ST5, ST7, ST10, and ST23), three in other animals (ST6, ST7, and ST23), while seven (ST1, ST3, ST6, ST7, ST10, ST23, and ST26) were found in the environment. In our investigation of transmission dynamics, we assessed various groupings both at the household and community level. Given the overall high prevalence rate, transmission amongst humans and between animals and humans are not as frequent as expected with only two subtypes being shared. This raises questions on the role of the environment on transmission of Blastocystis. Water and soil comprise the main reservoirs of the various subtypes in this community. Five subtypes are shared between humans and the environment, while three overlap between the latter and animal hosts. We propose soil as a novel route of transmission, which should be considered in future investigations. This study provides a thorough One Health perspective on Blastocystis. Using this type of approach advances our understanding on occurrence, diversity, ecology and transmission dynamics of this poorly understood, yet frequent gut resident.

Prototheca bovis, a unicellular achlorophyllous trebouxiophyte green alga in the healthy human intestine.

Introduction. Prototheca species are non-photosynthetic trebouxiophyte algae ubiquitously distributed in nature and can be found in sewage and soil. This microbial eukaryote causes human protothecosis in immunocompromised individuals. Thus, Prototheca presence in the stool of individuals without gastrointestinal symptoms has been reported only rarely.Hypothesis/Gap statement. There is an absence of detailed characterization of human Prototheca isolates.Aim. The aim of this study was to perform morphological and molecular characterization of Prototheca isolates obtained from human stool.Methodology. Prototheca was isolated from faecal samples of four individuals living in a rural area in Thailand. A combination of bioimaging along with molecular and bioinformatics tools was used to characterize the four strains. The growth rate was tested using four media and three temperature conditions. Phylogenetic analysis using the small subunit ribosomal RNA (SSU rRNA) and cytochrome b (cytb) was also performed.Results. Static and live microscopy demonstrated the various life stages of Prototheca and its major defining cellular characteristics. An optimized DNA extraction methodology that improves DNA yield is provided. Partial fragments of the SSU rRNA and cytb genes were obtained. Phylogenetic analysis placed all four strains in the clade with Prototheca bovis. More broadly, Prototheca was not monophyletic but split into at least two distinct clades instead.Conclusion. The results represent the first molecular characterization of Prototheca in Thailand. The study provides insight into transmission dynamics of the organism and potential caveats in estimating the global prevalence of Prototheca. These will spearhead further investigations on Prototheca occurrence in rural areas of both industrialized and developing nations.

Entamoeba chiangraiensis n. sp. (Amoebozoa: Entamoebidae) isolated from the gut of Asian swamp eel (Monopterus albus) in northern Thailand.

The genus Entamoeba comprises mostly gut parasites and commensals of invertebrate and vertebrate animals including humans. Herein, we report a new species of Entamoeba isolated from the gut of Asian swamp eels (Monopterus albus) in northern Thailand. Morphologically, the trophozoite is elongated and has a single prominent pseudopodium with no clear uroid. The trophozoite is actively motile, 30-50 µm in length and 9-13 µm in width. Observed cysts were uninucleate, ranging in size from 10 to 17.5 µm in diameter. Chromatin forms a fine, even lining along the inner nuclear membrane. Fine radial spokes join the karyosome to peripheral chromatin. Size, host and nucleus morphology set our organism apart from other members of the genus reported from fish. The SSU rRNA gene sequences of the new isolates are the first molecular data of an Entamoeba species from fish. Phylogenetic analysis places the new organism as sister to Entamoeba invadens. Based on the distinct morphology and SSU rRNA gene sequence we describe it as a new species, Entamoeba chiangraiensis.