Blastocystis is an anaerobic protist, commonly inhabiting the intestinal tract of both humans and other animals. Blastocystis is extremely diverse comprising 17 genetically distinct subtypes in mammals and birds. Pathogenicity of this enteric microbe is currently disputed and knowledge regarding its distribution, diversity and zoonotic potential is fragmentary. Most research has focused on Blastocystis from primates, while sampling from other animals remains limited. Herein, we investigated the prevalence and distribution of Blastocystis in animals held within a conservation park in South East England. A total of 118 samples were collected from 27 vertebrate species. The barcoding region of the small-subunit ribosomal RNA was used for molecular identification and subtyping. Forty one per cent of the species were sequence positive for Blastocystis indicating a high prevalence and wide distribution among the animals in the park. Six subtypes were identified, one of which is potentially novel. Moreover, the majority of animals were asymptomatic carriers, suggesting that Blastocystis is not pathogenic in animals. This study provides a thorough investigation of Blastocystis prevalence within a wildlife park in the UK and can be used as a platform for further investigations on the distribution of other eukaryotic gut microbes.

Gregarine apicomplexans are closely related to parasites such as Plasmodium, Toxoplasma, and Cryptosporidium, which are causing severe health and economic burdens. Colonizing only invertebrates and having no obvious medical relevance, they are mostly ignored in 'omics' studies, although gregarines are the most basal apicomplexans and therefore key players in the understanding of the evolution of parasitism in the Apicomplexa from free-living ancestors. They belong to the largest exclusively parasitic phylum, but is this perception actually true? The effects of gregarines on their hosts seem to cover the whole spectrum of symbiosis from mutualistic to parasitic. We suggest future research directions to understand the evolutionary role of gregarines, by elucidating their biology and interaction with their hosts and the hosts' microbiota. Parasitism in the Apicomplexa The phylum Apicomplexa contains unicellular parasites (currently more than 6000 named species) and is well known for its notorious pathogens of humans and livestock, such as Plasmodium (causative agent of malaria; mainly infecting humans, humanoids), Toxoplasma (toxoplasmosis; humans, cats), Eimeria (eimeriosis; poultry, cattle, ruminants), Theileria (theileriosis; cattle), Babesia (babesiosis; cattle, humans); Isospora (isosporiasis; humans), Cyclospora (cyclosporiasis; dogs, humans), and Cryptosporidium (cryptosporidiosis; humans, most livestock). These pathogens are of great public health concern and economic relevance, and they affect millions of people each year [1]. They all have intracellular life stages with the exception of Cryptosporidium [2], which has intracellular and extracytoplasmic stages [3,4]. Apicomplexans infect both invertebrates and vertebrates and have complex life cycles that differ considerably between the abovementioned groups [5] (Figure 1). Most of these life cycles involve at least two host species (i.e., a heteroxenous life cycle) (see Glossary). The apicomplexan clade is referred to in publications and textbooks as the largest phylum of eukaryotes that consists of obligate parasitic (Box 1) species only; but is this assumption really true for all apicomplexan species? The Gregarines Within the apicomplexans, gregarines are a unique subgroup that infects a wide range of freshwater, marine, and terrestrial invertebrates (almost exclusively). Different views concerning the taxonomy of the gregarines are emerging [6,7], but comprehensive evidence for a reliable overall taxonomic review is still missing. The latest review of eukaryotes still refers to the historic major groups Archigregarinorida, Eugregarinorida, and Neogregarinorida, mainly based on habitat, host range, and trophozoite morphology, to which is added the Cryptogregarinorida to accommodate Cryptosporidium [8] (Figure 1). Archigregarines are the most ancestral group, with a mix of ancestral and derived features, occurring in marine habitats only. Eugregarines can be found in marine, freshwater, and terrestrial habitats with large trophozoites that are morphologic...

Blastocystis is a genetically diverse microbial eukaryote thriving in the gut of humans and other animals. While Blastocystis has been linked with gastrointestinal disorders, its pathogenicity remains controversial. Previous reports have suggested that one out of six humans could be carrying Blastocystis in their gut, while the numbers could be even higher in animals. Most studies on Blastocystis are either exclusively targeting the organism itself and/or the associated prokaryotic microbiome, while cooccurrence of other microbial eukaryotes has been mainly ignored. Herein, we aimed to explore presence and genetic diversity of Blastocystis along with the commonly occurring eukaryotes Cryptosporidium, Eimeria, Entamoeba and Giardia in the gut of asymptomatic animals from two conservation parks in the United Kingdom. Building upon a previous study, a total of 231 fecal samples were collected from 38 vertebrates, which included 12 carnivorous and 26 non-carnivorous species. None of the animals examined herein showed gastrointestinal symptoms. The barcoding region of the small subunit ribosomal RNA was used for subtyping of Blastocystis. Overall, 47% of animal species were positive for Blastocystis. Twenty six percent of samples carried more than one subtypes, including the newly identified hosts Scottish wildcat, bongo and lynx. Fifty three percent of samples carried at least another microbial eukaryote. Herewith, we discuss potential implications of these findings and the increasingly blurred definition of microbial parasites.

(1) Background: Blastocystis is a microbial eukaryote inhabiting the gastrointestinal tract of a broad range of animals including humans. Several studies have shown that the organism is associated with specific microbial profiles and bacterial taxa that have been deemed beneficial to intestinal and overall health. Nonetheless, these studies are focused almost exclusively on humans, while there is no similar information on other animals. (2) Methods: Using a combination of conventional PCR, cloning and sequencing, we investigated presence of Blastocystis along with Giardia and Cryptosporidium in 16 captive water voles sampled twice from a wildlife park. We also characterised their bacterial gut communities. (3) Results: Overall, alpha and beta diversities between water voles with and without Blastocystis did not differ significantly. Differences were noted only on individual taxa with Treponema and Kineothrix being significantly reduced in Blastocystis positive water voles. Grouping according to antiprotozoal treatment and presence of other protists did not reveal any differences in the bacterial community composition either. (4) Conclusion: Unlike human investigations, Blastocystis does not seem to be associated with specific gut microbial profiles in water voles.

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Blastocystis is an opportunistic parasite commonly found in the intestines of humans and other animals. Despite its high prevalence, knowledge regarding Blastocystis biology within and outside the host is limited. Analysis of the metabolites produced by this anaerobe could provide insights that can help map its metabolism and determine its role in both health and disease. Due to its controversial pathogenicity, these metabolites could define its deterministic role in microbiome’s “health” and/or subsequently resolve Blastocystis’ potential impact in gastrointestinal health. A common method for elucidating the presence of these metabolites is through 1H nuclear magnetic resonance (NMR). However, there are currently no described benchmarked methods available to extract metabolites from Blastocystis for 1H NMR analysis. Herein, several extraction solvents, lysis methods and incubation temperatures were compared for their usefulness as an extraction protocol for this protozoan. Following extraction, the samples were freeze-dried, re-solubilized and analysed with 1H NMR. The results demonstrate that carrying out the procedure at room temperature using methanol as an extraction solvent and bead bashing as a lysis technique provides a consistent, reproducible and efficient method to extract metabolites from Blastocystis for NMR.

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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.