Parasitological loads estimated at the beginning of the study between cases and controls.

<p>Plots drew with all sample data according to the number of <i>Blastocystis</i> organisms/mg feces by microscopy (A) and number of <i>Blastocystis</i> DNA copies/mg feces by qPCR (B). The black bars mean the average in each group.</p

Population genetic indexes between different <i>Blastocystis</i> ST sequences.

<p>^a95%CI, 95% Confidence interval.</p><p>Population genetic indexes between different <i>Blastocystis</i> ST sequences.</p

Phylogenetic inference of <i>Blastocystis</i> spp.

<p>Bayesian phylogenetic tree using a fragment of SSUrDNA sequences; the values of the nodes indicate posterior probabilities values using 10 million generations. GenBank accession numbers are included, as well as if correspond to a case or a control; each ST clade is shown in different branch colors.</p

Generation times data in Blastocystis by qPCR and by microscopy

<p>Generation time (Tg) data of Blastocystis cultures in Barret’s and in Pavlova’s media during 48 h., from IBS patients and from asymptomatic carriers, measured by microscopy and by quantitative PCR are shown in present table. The generation times of Blastocystis isolates, were calculated according to Zhang et al. [17], with the following equation: Tg=(T2-T1)/(log2(n2/n1)), where Tg denotes the generation time, n1 represents the number of cultured parasitic organisms at the initial time (T1), and n2 represents the number of parasitic cells at subsequent time (T2). Thus, (T2-T1) = 48 hours of in vitro culture. Besides, for to absolute quantification by qPCR, it was necessary to consider i) the size of the Blastocystis genome ~ 18.8Mbp[36]; ii) 1pg of DNA ~ 978Mbp[37] and iii) the concentration of DNA control was 160ng/µL; thus, by cross multiplications, the number of copies of the genetic marker amplified were estimated.</p> <p> </p

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

Generation time (<i>T</i>_<i>g</i>) values of <i>Blastocystis</i> isolates between case and control groups.

<p>Average and standard deviation of <i>T</i>_{<b><i>g</i></b>} in Barret’s and Pavlova media, based on microscopic measures and qPCR assays.</p

Characteristics of the carriers and of <i>Blastocystis</i> ST identified in IBS patients and in the control group.

<p>^a<i>p</i> = 0.043,</p><p>OR(95%IC) = 0.43(0.17–1.06).</p><p>Characteristics of the carriers and of <i>Blastocystis</i> ST identified in IBS patients and in the control group.</p

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