Can the pyruvate: ferredoxin oxidoreductase (PFOR) gene be used as an additional marker to discriminate among Blastocystis strains or subtypes?

Background Blastocystis spp. are the most prevalent intestinal eukaryotes identified in humans, with at least 17 genetic subtypes (ST) based on genes coding for the small-subunit ribosomal RNA (18S). It has been argued that the 18S gene should not be the marker of choice to discriminate between STs of these strains because this marker exhibits high intra-genomic polymorphism. By contrast, pyruvate:ferredoxin oxidoreductase (PFOR) is a relevant enzyme involved in the core energy metabolism of many anaerobic microorganisms such as Blastocystis, which, in other protozoa, shows more polymorphisms than the 18S gene and thus may offer finer discrimination when trying to identify Blastocystis ST. Therefore, the objective of the present study was to assess the suitability of the PFOR gene as an additional marker to discriminate among Blastocystis strains or subtypes from symptomatic carrier children. Methods Faecal samples from 192 children with gastrointestinal symptoms from the State of Mexico were submitted for coprological study. Twenty-one of these samples were positive only for Blastocystis spp.; these samples were analysed by PCR sequencing of regions of the 18S and PFOR genes. The amplicons were purified and sequenced; afterwards, both markers were assessed for genetic diversity. Results The 18S analysis showed the following frequencies of Blastocystis subtypes: ST3 = 43%; ST1 = 38%; ST2 = 14%; and ST7 = 5%. Additionally, using subtype-specific primer sets, two samples showed mixed Blastocystis ST1 and ST2 infection. For PFOR, Bayesian inference revealed the presence of three clades (I-III); two of them grouped different ST samples, and one grouped six samples of ST3 (III). Nucleotide diversity (π) and haplotype polymorphism (θ) for the 18S analysis were similar for ST1 and ST2 (π = ~0.025 and θ = ~0.036); remarkably, ST3 showed almost 10-fold lower values. For PFOR, a similar trend was found: clade I and II had π = ~0.05 and θ = ~0.05, whereas for clade III, the values were almost 6-fold lower. Conclusions Although the fragment of the PFOR gene analysed in the present study did not allow discrimination between Blastocystis STs, this marker grouped the samples in three clades with strengthened support, suggesting that PFOR may be under different selective pressures and evolutionary histories than the 18S gene. Interestingly, the ST3 sequences showed lower variability with probable purifying selection in both markers, meaning that evolutionary forces drive differential processes among Blastocystis STs

Clarifying the Cryptic Host Specificity of <i>Blastocystis</i> spp. Isolates from <i>Alouatta palliata</i> and <i>A</i>. <i>pigra</i> Howler Monkeys

<div><p>Although the presence of cryptic host specificity has been documented in <i>Blastocystis</i>, differences in infection rates and high genetic polymorphism within and between populations of some subtypes (ST) have impeded the clarification of the generalist or specialist specificity of this parasite. We assessed the genetic variability and host specificity of <i>Blastocystis</i> spp. in wild howler monkeys from two rainforest areas in the southeastern region of Mexico. Fecal samples of 225 <i>Alouatta palliata</i> (59) and <i>A</i>. <i>pigra</i> (166) monkeys, belonging to 16 sylvatic sites, were analyzed for infection with <i>Blastocystis</i> ST using a region of the small subunit rDNA (SSUrDNA) gene as a marker. Phylogenetic and genetic diversity analyses were performed according to the geographic areas where the monkeys were found. <i>Blastocystis</i> ST2 was the most abundant (91.9%), followed by ST1 and ST8 with 4.6% and 3.5%, respectively; no association between <i>Blastocystis</i> ST and <i>Alouatta</i> species was observed. SSUrDNA sequences in GenBank from human and non-human primates (NHP) were used as ST references and included in population analyses. The haplotype network trees exhibited different distributions: ST1 showed a generalist profile since several haplotypes from different animals were homogeneously distributed with few mutational changes. For ST2, a major dispersion center grouped the Mexican samples, and high mutational differences were observed between NHP. Furthermore, nucleotide and haplotype diversity values, as well as migration and genetic differentiation indexes, showed contrasting values for ST1 and ST2. These data suggest that ST1 populations are only minimally differentiated, while ST2 populations in humans are highly differentiated from those of NHP. The host generalist and specialist specificities exhibited by ST1 and ST2 <i>Blastocystis</i> populations indicate distinct adaptation processes. Because ST1 exhibits a generalist profile, this haplotype can be considered a metapopulation; in contrast, ST2 exists as a set of local populations with preferences for either humans or NHP.</p></div

Schematic representation of interactions among population indexes.

<p>The gene flow (Nm), genetic differentiation index (F_ST), and Tajima’s D values of <i>Blastocystis</i> ST by SSUrDNA analysis, according to different sampling sites; only those sites in which there were enough infected howlers to obtain the indexes are shown. The number together the sampling size circle, mean the Tajima’s D value. * <i>p</i><0.01</p

Haplotype networks for <i>Blastocystis</i>.

<p>Haplotype network trees using SSUrDNA sequences from different countries and hosts for ST1 (a) and ST2 (b). Numbers in branches refer to mutational changes; sizes of circles and colors are proportional to haplotype frequencies. For those animal haplotypes, an image and Roman reference numbers were included, while for human haplotypes, asterisks were added.</p

Infection rates of <i>Blastocystis</i> subtype (ST) for howler monkey populations.

<p>Infection rates of <i>Blastocystis</i> subtype (ST) for howler monkey populations.</p