Molecular Identification and Subtype Analysis of Blastocystis.

Several typing methods have been used in studies aiming to unravel the molecular epidemiology of Blastocystis, which is one of the most common intestinal parasites in human and many non-human hosts. Such studies have the potential to add to knowledge on Blastocystis transmission, host specificity, phylogeography, and clinical and public health significance, but rely on robust, standardized methods by which data can be generated and compared directly between studies. One of the most used methods is "barcoding,", which involves single-round PCR amplification and sequencing of partial small subunit ribosomal RNA genes of the parasites. Recently, a publicly available online facility was developed for quick and standardized identification of subtypes (ribosomal lineages) and subtype alleles (variation within subtypes) based on sequence data obtained by barcoding PCR. Moreover, a modified barcoding approach is now available using nested PCR, which enables detection of mixed subtype infections. © 2016 by John Wiley & Sons, Inc

Variable geographic distribution of Blastocystis subtypes and its potential implications.

Blastocystis is a common intestinal micro-eukaryote found in both humans and non-human hosts and known to be genetically very diverse. It has been divided into numerous subtypes (STs), nine of which have been identified in humans to date. Surveys of ST prevalence have started to emerge over the past few years but to date no data are available for any African country except Egypt and Tanzania. In this study, we determined the prevalence of Blastocystis STs in populations from Libya, Liberia and Nigeria, as well as expanding the dataset available for the UK. A total of 356 Blastocystis STs were identified in this study, 271 from the UK, 38 from Libya, 25 from Liberia and 22 from Nigeria. SSU rRNA gene sequences revealed the presence of eight of the nine STs known from humans but at varying frequencies between countries. ST1 was the most common ST in Libya and Nigeria whereas ST3 showed the highest frequency in the other two countries, as indeed is the case in most populations around the world. ST4 was absent in Libya and ST2 in Nigeria, while no ST5, ST6, ST8 or ST9 infections were detected in any of the three African populations. The picture emerging from this and other surveys suggests that there is significant variation in ST prevalence between populations. Some of the possible reasons for and implications of this diversity are discussed

Genetic diversity of blastocystis in livestock and zoo animals.

Blastocystis is a common unicellular anaerobic eukaryote that inhabits the large intestine of many animals worldwide, including humans. The finding of Blastocystis in faeces in mammals and birds has led to proposals of zoonotic potential and that these hosts may be the source of many human infections. Blastocystis is, however, a genetically diverse complex of many distinct organisms (termed subtypes; STs), and sampling to date has been limited, both geographically and in the range of hosts studied. In order to expand our understanding of host specificity of Blastocystis STs, 557 samples were examined from various non-primate animal hosts and from a variety of different countries in Africa, Asia and Europe. STs were identified using 'barcoding' of the small subunit rRNA gene using DNA extracted either from culture or directly from faeces. The host and geographic range of several STs has thereby been greatly expanded and the evidence suggests that livestock is not a major contributor to human infection. Two new STs were detected among the barcode sequences obtained; for these, and for three others where the data were incomplete, the corresponding genes were fully sequenced and phylogenetic analysis was undertaken

Are molecular tools clarifying or confusing our understanding of the public health threat from zoonotic enteric protozoa in wildlife?

Emerging infectious diseases are frequently zoonotic, often originating in wildlife, but enteric protozoa are considered relatively minor contributors. Opinions regarding whether pathogenic enteric protozoa may be transmitted between wildlife and humans have been shaped by our investigation tools, and has led to oscillations regarding whether particular species are zoonotic or have host-adapted life cycles. When the only approach for identifying enteric protozoa was morphology, it was assumed that many enteric protozoa colonized multiple hosts and were probably zoonotic. When molecular tools revealed genetic differences in morphologically identical species colonizing humans and other animals, host specificity seemed more likely. Parasites from animals found to be genetically identical - at the few genes investigated - to morphologically indistinguishable parasites from human hosts, were described as having zoonotic potential. More discriminatory molecular tools have now sub-divided some protozoa again. Meanwhile, some infection events indicate that, circumstances permitting, some “host-specific” protozoa, can actually infect various hosts. These repeated changes in our understanding are linked intrinsically to the investigative tools available. Here we review how molecular tools have assisted, or sometimes confused, our understanding of the public health threat from nine enteric protozoa and example wildlife hosts (Balantoides coli - wild boar; Blastocystis sp. - wild rodents; Cryptosporidium spp. - wild fish; Encephalitozoon spp. - wild birds; Entamoeba spp. - non-human primates; Enterocytozoon bieneusi - wild cervids; Giardia duodenalis - red foxes; Sarcocystis nesbitti - snakes; Toxoplasma gondii - bobcats). Molecular tools have provided evidence that some enteric protozoa in wildlife may infect humans, but due to limited discriminatory power, often only the zoonotic potential of the parasite is indicated. Molecular analyses, which should be as discriminatory as possible, are one, but not the only, component of the toolbox for investigating potential public health impacts from pathogenic enteric protozoa in wildlife

Extreme genome diversity in the hyper-prevalent parasitic eukaryote Blastocystis

Blastocystis is the most prevalent eukaryotic microbe colonizing the human gut, infecting approximately 1 billion individuals worldwide. Although Blastocystis has been linked to intestinal disorders, its pathogenicity remains controversial because most carriers are asymptomatic. Here, the genome sequence of Blastocystis subtype (ST) 1 is presented and compared to previously published sequences for ST4 and ST7. Despite a conserved core of genes, there is unexpected diversity between these STs in terms of their genome sizes, guanine-cytosine (GC) content, intron numbers, and gene content. ST1 has 6,544 protein-coding genes, which is several hundred more than reported for ST4 and ST7. The percentage of proteins unique to each ST ranges from 6.2% to 20.5%, greatly exceeding the differences observed within parasite genera. Orthologous proteins also display extreme divergence in amino acid sequence identity between STs (i.e., 59%–61%median identity), on par with observations of the most distantly related species pairs of parasite genera. The STs also display substantial variation in gene family distributions and sizes, especially for protein kinase and protease gene families, which could reflect differences in virulence. It remains to be seen to what extent these inter-ST differences persist at the intra-ST level. A full 26% of genes in ST1 have stop codons that are created on the mRNA level by a novel polyadenylation mechanism found only in Blastocystis. Reconstructions of pathways and organellar systems revealed that ST1 has a relatively complete membrane-trafficking system and a near-complete meiotic toolkit, possibly indicating a sexual cycle. Unlike some intestinal protistan parasites, Blastocystis ST1 has near-complete de novo pyrimidine, purine, and thiamine biosynthesis pathways and is unique amongst studied stramenopiles in being able to metabolize ?-glucans rather than ?-glucans. It lacks all genes encoding heme-containing cytochrome P450 proteins. Predictions of the mitochondrion-related organelle (MRO) proteome reveal an expanded repertoire of functions, including lipid, cofactor, and vitamin biosynthesis, as well as proteins that may be involved in regulating mitochondrial morphology and MRO/endoplasmic reticulum (ER) interactions. In sharp contrast, genes for peroxisome-associated functions are absent, suggesting Blastocystis STs lack this organelle. Overall, this study provides an important window into the biology of Blastocystis, showcasing significant differences between STs that can guide future experimental investigations into differences in their virulence and clarifying the roles of these organisms in gut health and disease

Levels of genetic diversity vary dramatically between Blastocystis subtypes.

Blastocystis is a common single-celled parasite of humans and other animals comprising at least 13 genetically distinct small subunit ribosomal RNA lineages (subtypes (STs)). In this study we investigated intra-subtype genetic diversity and host specificity of two of the most common subtypes in humans, namely ST3 and ST4, by analysing and comparing over 400 complete and partial nuclear SSU-rDNAs and data from multilocus sequence typing (MLST) of the mitochondrion-like organelle (MLO) genome of 132 samples. Inferences from phylogenetic analyses of nuclear SSU-rDNA and concatenated MLST sequences were compatible. Human ST3 infections were restricted to one of four identified MLO clades except where exposure to non-human primates had occurred. This suggests relatively high host specificity within ST3, that human ST3 infections are caused predominantly by human-to-human transmission, and that human strains falling into other clades are almost certainly the result of zoonotic transmission. ST4 from humans belonged almost exclusively to one of two SSU-rDNA clades, and only five MLST sequence types were found among 50 ST4s belonging to Clade 1 (discriminatory index: 0.41) compared to 58 MLST sequence types among 81 ST3s (discriminatory index: 0.99). The remarkable differences in intra-subtype genetic variability suggest that ST4 has a more recent history of colonising humans than ST3. This is congruent with the apparently restricted geographical distribution of ST4 relative to ST3. The implications of this observation are unclear, however, and the population structure and distribution of ST4 should be subject to further scrutiny in view of the fact ST4 is being increasingly linked with intestinal disease

The significance of 'Blastocystis' in different hosts

Blastocystis is an obligate anaerobic, protistan parasite found in the intestinal tract of human and various other hosts. Blastocystis is placed within the stramenopiles. This diverse group also includes slime nets, water moulds and brown algae. The transmission of Blastocystis is believed to take place through the faecal-oral route. Waterborne transmission of Blastocystis through the use of untreated water or poor sanitary conditions has also been reported. Numerous epidemiological surveys have shown prevalence up to 10% of the population in developed countries and as much as 50-60% in developing countries. Differences in virulence among Blastocystis subtypes have been reported in a recent animal infection study. Blastocystis shows extensive genetic diversity and is divided into numerous genetic subtypes The parasite is commonly associated with gastrointestinal symptoms such as watery and mucous diarrhea, vomiting, and abdominal cramps and bloating. Epidemiological studies suggest an association between Blastocystis infection and irritable bowel syndrome. Irritable bowel syndrome (IBS) is identified as a functional bowel disorder in which abdominal pain or discomfort is associated with a defect or alteration in the consistency or frequency of stools. Diagnosis of IBS by physicians is carried out using symptom -based criteria known as the Rome criteria. To see whether there is any link between Blastocystis infection and irritable bowel syndrome (IBS), I have compared the frequency of subtypes of Blastocystis in IBS patients with those in the general population. In human population both UK and Libya showed similar distribution of Blastocystis subtypes 2 ST I, 2 and 3 are common in the two populations and ST3 has the highest frequency in UK while STI was the most common in Libya. Epidemiology studies on Blastocystis infection in animals have revealed high frequency of occurrence in cattle, pigs, primates and birds and it has often been suggested that Blastocystis infection is a zoonosis. In Libya, Blastocystis subtypes were detected from humans, birds and numerous mammals' hosts (camel, cow, sheep, goat, gazelle, Barbary sheep and gundi). Ten subtypes were detected (1, 2, 3, 5, 7,10,14, IS, 16, 17) and four new subtypes were found in cow, camel and gundi. Subtype I, 3, and 7 were in common between animal and human but subtypes 5, 10 and the four new subtypes were found only in animals. ST 2 was found in human only. Also I discovered four new hosts for Blastocystis from mouse deer, gundi, gazelle and barbary sheep. Both human and animal showed diversity in Blastocystis sUbtypes. Both human and animals become infected with same Blastocystis subtype and for this reason we need to find refined tool to differentiate between them, so I have developed MLST for Blastocystis subtype I based on mitochondrial DNA. Application ofMLST to 39 isolates from different host and different geographic place showed variation in the sequence of related isolates. Over all MLST proved to be a highly discriminatory and stable method for unambiguous characterization of Blastocystis