Table_2_Exploring Micro-Eukaryotic Diversity in the Gut: Co-occurrence of Blastocystis Subtypes and Other Protists in Zoo Animals.DOCX

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 co-occurrence 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.</p

Image_1_Exploring Micro-Eukaryotic Diversity in the Gut: Co-occurrence of Blastocystis Subtypes and Other Protists in Zoo Animals.tif

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 co-occurrence 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.</p

Table_1_Exploring Micro-Eukaryotic Diversity in the Gut: Co-occurrence of Blastocystis Subtypes and Other Protists in Zoo Animals.DOCX

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 co-occurrence 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.</p

Genetic Differentiation of the Mitochondrial Cytochrome Oxidase <i>c</i> Subunit I Gene in Genus <i>Paramecium</i> (Protista, Ciliophora)

<div><p>Background</p><p>The mitochondrial cytochrome <i>c</i> oxidase subunit I (<i>COI</i>) gene is being used increasingly for evaluating inter- and intra-specific genetic diversity of ciliated protists. However, very few studies focus on assessing genetic divergence of the <i>COI</i> gene within individuals and how its presence might affect species identification and population structure analyses.</p><p>Methodology/Principal findings</p><p>We evaluated the genetic variation of the <i>COI</i> gene in five <i>Paramecium</i> species for a total of 147 clones derived from 21 individuals and 7 populations. We identified a total of 90 haplotypes with several individuals carrying more than one haplotype. Parsimony network and phylogenetic tree analyses revealed that intra-individual diversity had no effect in species identification and only a minor effect on population structure.</p><p>Conclusions</p><p>Our results suggest that the <i>COI</i> gene is a suitable marker for resolving inter- and intra-specific relationships of <i>Paramecium</i> spp.</p></div

Phylogenetic tree of the barcoding region of 263 cytochrome <i>c</i> oxidase subunit I (<i>COI</i>) gene sequences of the genus <i>Paramecium</i> and genera <i>Lembadion</i> and, <i>Tetrahymena</i> inferred by Bayesian Inference (BI) analysis based on dataset <i>COI</i>-f.

<p>The branches are shaded according to subgenera <i>Chloroparamecium</i>, <i>Helianter</i>, <i>Cypriostomum</i>, <i>Paramecium</i>, proposed by Fokin <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077044#pone.0077044-Fokin1" target="_blank">[28]</a>. The scale bar corresponds to 30 substitutions per 100 nucleotide positions. For <i>P. bursaria</i>, Clade H includes populations sampled from Australia, Germany, and Poland; Clade I and J include populations sampled from Russia and Poland, Germany, Ukraine, and Canada; Clade K includes populations sampled from China (Pb1C1-4 & Pb2C &Pb3C2-3), Austria, Japan, and Italy; Clade L includes populations sampled from China (Pb3C1), Russia, and Japan (see details in Fig. S2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077044#pone.0077044.s003" target="_blank">file S3</a>). For <i>P.caudatum</i>, Clade A includes populations sampled from China (PcC1-4 and AM072774), Australia, USA, and Brazil while members of Clade B were sampled from Germany, Italy, Russia, UK, Norway, Hungary, Slovenia, and Austria (see details in Fig. S3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077044#pone.0077044.s003" target="_blank">file S3</a>). Inconsistent sequences (FJ905146, FJ905147, EU056259, EU056258, DQ837977, DQ837982, JF741258, JF304183) are marked in red <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077044#pone.0077044-Tarcz1" target="_blank">[14]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077044#pone.0077044-Barth3" target="_blank">[42]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077044#pone.0077044-StrderKypke2" target="_blank">[47]</a>.</p

Haplotype network of <i>Paramecium bursaria</i> generated on the basis of the maximum-likelihood tree.

<p>Black circles indicate intermediate or unsampled haplotypes, while lines between points represent nucleotide substitutions. Wherever there are more than four substitutions, they are indicated by numbers. Clades K, L, H, I, J are marked to match the corresponding clades in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077044#pone-0077044-g004" target="_blank">Figure 4</a>. Colored circles and squares indicate haplotypes whose size is proportional to the number of individuals showing that haplotype. Haplotype_7 is represented by 4 clones of Pb1C1; haplotype_14 is represented by 4 clones of Pb1C2 and 7 clones of Pb1C3; haplotype_22 is represented by 5 clones of Pb1C4 and 3 clones of Pb2C1; haplotype_25 is represented by 3 clones of Pb3C1.</p

Haplotype network of <i>Paramecium</i> sp. (A) based on the dataset <i>COI</i>_nw, <i>P. nephridiatum</i> (B) based on dataset <i>COI</i>_nn, <i>P. duboscqui</i> (C), based on dataset <i>COI</i>_nd and <i>P. caudatum</i> (D) based on dataset <i>COI</i>_nc generated on the basis of the maximum-likelihood tree.

<p>Each line between points represents a single mutational step. A haplotype is represented by a circle whose size is proportional to the number of individuals showing that haplotype. Haplotypes are colored to match the respective population in the map. A) Haplotype_1 is represented by 2 clones of PwC1; Haplotype_3 is represented by 4 clones of PwC1 and 2 clones of PwC2; B) Haplotype_4 is represented by 1 clone of PdC1, 1 clone of PdC2, 1 clone of PdC3 and 2 clones of PdC4; C) Haplotype_2 is represented by 4 clones of PnC1 and 1 clone of PnC3; D) Haplotype_6 is represented by 4 clones of PcC1 and 5 clones of PcC3; Haplotype_9 is represented by 6 clones of PcC2 and 5 clones of PcC4; Haplotype_13 is represented by 3 clones of PcC3.</p

Variable site details of <i>Paramecium bursaria</i> and <i>Paramecium caudatum</i>.

<p>Alignment of amplified <i>COI</i> sequences (primer binding regions excluded) based on datasets <i>COI</i>_nb and <i>COI</i>_nc. The nucleotides shaded with rectangles and circles are used to illustrate the levels of diversity found among different clones.</p

Data_Sheet_1_Blastocystis One Health Approach in a Rural Community of Northern Thailand: Prevalence, Subtypes and Novel Transmission Routes.pdf

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.</p

Table_1_Blastocystis One Health Approach in a Rural Community of Northern Thailand: Prevalence, Subtypes and Novel Transmission Routes.XLSX

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.</p