Culture isolation and molecular identification of Blastocystis sp. in Brazilian human isolates: preliminary results.

Blastocystis sp. is a protist commonly found in stool samples of humans and animals. Biological and genetic factors of this organism remain controversial. The present study aimed to develop and implement the Blastocystis in vitro culture of Brazilian human isolates for routine use. The fecal isolates (n = 20) were maintained in our laboratory by several passages in Pavlova's medium. Cultures were monitored every 72 h by light microscopy. Genomic DNA was extracted to identify the subtypes (STs). In most isolates, the vacuolar form was prevalent. The amoeboid, granular and cystic forms were observed during in vitro cultivation. STs 1, 2, 3, 4 and 7 were identified. Our preliminary results show the generation time and forms present in the in vitro culture of Blastocystis subtypes isolated from Brazilian human isolates. Therefore, we emphasize the use of in vitro culture as a tool in future studies for the better understanding of the biological aspects of Blastocystis sp.

Mitochondrial Glycolysis in a Major Lineage of Eukaryotes.

The establishment of the mitochondrion is seen as a transformational step in the origin of eukaryotes. With the mitochondrion came bioenergetic freedom to explore novel evolutionary space leading to the eukaryotic radiation known today. The tight integration of the bacterial endosymbiont with its archaeal host was accompanied by a massive endosymbiotic gene transfer resulting in a small mitochondrial genome which is just a ghost of the original incoming bacterial genome. This endosymbiotic gene transfer resulted in the loss of many genes, both from the bacterial symbiont as well the archaeal host. Loss of genes encoding redundant functions resulted in a replacement of the bulk of the host's metabolism for those originating from the endosymbiont. Glycolysis is one such metabolic pathway in which the original archaeal enzymes have been replaced by bacterial enzymes from the endosymbiont. Glycolysis is a major catabolic pathway that provides cellular energy from the breakdown of glucose. The glycolytic pathway of eukaryotes appears to be bacterial in origin, and in well-studied model eukaryotes it takes place in the cytosol. In contrast, here we demonstrate that the latter stages of glycolysis take place in the mitochondria of stramenopiles, a diverse and ecologically important lineage of eukaryotes. Although our work is based on a limited sample of stramenopiles, it leaves open the possibility that the mitochondrial targeting of glycolytic enzymes in stramenopiles might represent the ancestral state for eukaryotes.

Seeing the Whole Elephant: Imaging Flow Cytometry Reveals Extensive Morphological Diversity within Blastocystis Isolates.

Blastocystis is a common protist isolated in humans and many animals. The parasite is a species complex composed of 19 subtypes, 9 of which have been found in humans. There are biological and molecular differences between Blastocystis subtypes although microscopy alone is unable to distinguish between these subtypes. Blastocystis isolates also display various morphological forms. Several of these forms, however, have not been properly evaluated on whether or not these play significant functions in the organism's biology. In this study, we used imaging flow cytometry to analyze morphological features of Blastocystis isolates representing 3 subtypes (ST1, ST4 and ST7). We also employed fluorescence dyes to discover new cellular features. The profiles from each of the subtypes exhibit considerable differences with the others in terms of shape, size and granularity. We confirmed that the classical vacuolar form comprises the majority in all three subtypes. We have also evaluated other morphotypes on whether these represent distinct life stages in the parasite. Irregularly-shaped cells were identified but all of them were found to be dying cells in one isolate. Granular forms were present as a continuum in both viable and non-viable populations, with non-viable forms displaying higher granularity. By analyzing the images, rare morphotypes such as multinucleated cells could be easily observed and quantified. These cells had low granularity and lower DNA content. Small structures containing nucleic acid were also identified. We discuss the possible biological implications of these unusual forms.

Features of Blastocystis spp. in xenic culture revealed by deconvolutional microscopy.

Blastocystis spp. are common human enteric parasites with complex morphology and have been reported to cause irritable bowel syndrome (IBS). Deconvolutional microscopy with time-lapse imaging and fluorescent spectroscopy of xenic cultures of Blastocystis spp. from stool samples of IBS patients and from asymptomatic, healthy pigs allowed observations of living organisms in their natural microbial environment. Blastocystis organisms of the vacuolated, granular, amoebic and cystic forms were observed to autofluorescence in the 557/576 emission spectra. Autofluorescence could be distinguished from fluorescein-conjugated Blastocystis-specific antibody labelling in vacuolated and granular forms. This antibody labelled Blastocystis subtypes 1, 3 and 4 but not 5. Surface pores of 1 µm in diameter were observed cyclically opening and closing over 24 h. Vacuolated forms extruded a viscous material from a single surface point with coincident deflation that may demonstrate osmoregulation. Tear-shaped granules were observed exiting from the surface of an amoebic form, but their origin and identity remain unknown.

Modified fields' stain: ideal to differentiate Dientamoeba fragilis and Blastocystis sp.

Dientamoeba fragilis, a trichomonad parasite is usually found in the gastrointestinal tract of human, and it is known to be the cause for gastrointestinal disease. The parasite is globally distributed and mostly found in rural and urban areas. The parasite is found in humans and nonhuman primates such as the macaques, baboons, and gorillas. Often, the parasite is confused with another largely found organism in stools called Blastocystis sp. especially when seen directly under light microscopy on culture samples containing both parasites. Both sometimes are seen with two nuclei with sizes tending to be similar which complicates identification. Stools were collected fresh from nine previously diagnosed persons infected with D. fragilis who also were found to be positive for Blastocystis sp. Samples were then cultured in Loeffler's medium and were stained with Giemsa, iron hematoxylin, and modified Fields' (MF) stain, respectively. D. fragilis was differentiated from Blastocystis sp. when stained with MF stain by the presence of a thinner outer membrane with clearly demarcated nuclei in the center of the cell whilst Blastocystis sp. had a darker and thicker stained outer membrane with the presence of two nuclei. The staining contrast was more evident with modified Fields' stain when compared with the other two. The simplicity in preparing the stain as well as the speed of the staining procedure make MF stain an ideal alternate. The modified Fields' stain is faster and easier to prepare when compared to the other two stains. MF stain provides a better contrast differentiating the two organisms and therefore provides a more reliable diagnostic method to precisely identify one from the other especially when cultures show mixed infections.

Comparison of direct fecal smear microscopy, culture, and polymerase chain reaction for the detection of Blastocystis sp. in human stool samples.

OBJECTIVE: To compare the sensitivity and specificity of direct fecal smear microscopy, culture, and polymerase chain reaction in the detection of Blastocystis sp. in human stool. METHODS: Human stool samples were collected from a community in San Isidro, Rodriguez, Rizal, Philippines. These samples were subjected to direct fecal smear microscopy, culture and polymerase chain reaction to detect the presence of Blastocystis sp. RESULTS: Of the 110 stool samples collected, 28 (25%) were detected positive for the presence of Blastocystis sp. by two or more tests. Culture method detected the highest number of Blastocystis-positive stool samples (n=36), followed by PCR of DNA extracted from culture (n=26), PCR of DNA extracted from stool (n=10), and direct fecal smear (n=9). Compared to culture, the sensitivity of the other detection methods were 66.7% for PCR from culture and 19.4% for both PCR from stool and direct fecal smear. Specificity of the methods was high, with PCR from culture and direct fecal smear having 97.3%, while PCR from stool at 95.9%. CONCLUSIONS: In this study, in vitro culture is the best method for detecting Blastocystis sp. in human stool samples.

Amoebic forms of Blastocystis spp. - evidence for a pathogenic role.

BACKGROUND: Blastocystis spp. are one of the most prevalent parasites isolated from patients suffering from diarrhea, flatulence, constipation and vomiting. It's pathogenicity and pathophysiology remains controversial to date. Protease activity and amoebic forms have been reported previously in symptomatic isolates but there has been no conclusive evidence provided to correlate the protease activity and any specific life cycle stage of the parasite thus far. METHODS: Symptomatic isolates with amoebic form were tested for protease activity and compared with symptomatic and asymptomatic isolates without amoebic form for 10 days culture period. RESULTS: The present study demonstrates an elevated protease activity in cultures having a higher percentage of amoebic forms seen in symptomatic isolates. The growth curve demonstrated a significantly (p < 0.05) higher average number of parasite counts in asymptomatic compared to symptomatic isolates. Symptomatic isolates showed amoebic forms with percentages ranging from 5% to 17%. Elevated protease activity was demonstrated in isolates that had higher percentages of amoebic forms with intense bands at higher molecular weight proteases (60 - 100 kDa). As days of culture proceeded, the protease quantification also showed a steady increase. CONCLUSION: This study elucidates a correlation between protease activity and percentage of amoebic forms. The finding implies that these forms could play a role in exacerbation of intestinal symptoms during Blastocystis spp. infection.

Apoptosis in Blastocystis spp. is related to subtype.

Previous studies have shown that apoptosis-like features are observed in Blastocystis spp., an intestinal protozoan parasite, when exposed to the cytotoxic drug metronidazole (MTZ). This study reports that among the four subtypes of Blastocystis spp. investigated for rate of apoptosis when treated with MTZ, subtype 3 showed the highest significant increase after 72h of in vitro culture when treated with MTZ at 0.1mg/ml (79%; p<0.01) and 0.0001mg/ml (89%; p<0.001). The close correlation between viable cells and apoptotic cells for both dosages implies that the pathogenic potential of these isolates has been enhanced when treated with MTZ. This suggests that there is a mechanism in Blastocystis spp. that actually regulates the apoptotic process to produce higher number of viable cells when treated. Apoptosis may not just be programmed cell death but instead a mechanism to increase the number of viable cells to ensure survival during stressed conditions. The findings of the present study have an important contribution to influence chemotherapeutic approaches when developing drugs against the emerging Blastocystis spp. infections.

Characterization of two cysteine proteases secreted by Blastocystis ST7, a human intestinal parasite.

Blastocystis spp. are unicellular anaerobic intestinal parasites of both humans and animals and the most prevalent ones found in human stool samples. Their association with various gastrointestinal disorders raises the questions of its pathogenicity and of the molecular mechanisms involved. Since secreted proteases are well-known to be implicated in intestinal parasite virulence, we intended to determine whether Blastocystis spp. possess such pathogenic factors. In silico analysis of the Blastocystis subtype 7 (ST7) genome sequence highlighted 22 genes coding proteases which were predicted to be secreted. We characterized the proteolytic activities in the secretory products of Blastocystis ST7 using specific protease inhibitors. Two cysteine proteases, a cathepsin B and a legumain, were identified in the parasite culture supernatant by gelatin zymographic SDS-PAGE gel and MS/MS analysis. These proteases might act on intestinal cells and disturb gut function. This work provides serious molecular candidates to link Blastocystis spp. and intestinal disorders.

Algunas consideraciones acerca de Blastocystissp, un parásito controversial / Some considerations about Blastocystis sp, a controversial parasite / Algumas considerações sobre Blastocystis sp, Um parasita controverso

Blastocystis es un organismo unicelular, anaerobio y uno de los parásitos intestinales de mayor prevalencia a nivel mundial. Durante mucho tiempo su ubicación taxonómica fue difícil de definir. Actualmente es considerado el único parásito humano del Reino Chromista. Se le reconocen numerosas formas evolutivas (vacuolares, granulares, ameboidales, avacuolares, multivacuolares y quísticas) que conforman un ciclo vital aún en estudio. Los quistes son las formas de resistencia y transmisión. Se lo considera un parásito zoonótico con 9 subtipos que pueden tener diferentes especificidades entre hospedadores humanos y animales. Su carácter patógeno ha sido motivo también de controversia dado que puede presentar infecciones asintomáticas o sintomáticas con manifestaciones digestivas, y dérmicas, entre otras. El diagnóstico de laboratorio puede ser microscópico o mediante cultivos, serología y técnicas moleculares.

Blastocystis is an anaerobic, unicellular organism and it is one of the most prevalent among intestinal parasites. It has a worldwide distribution. Nowadays, it is considered the only human parasite that belongs to the kingdom Chromista. Many morphological forms of the parasite are known: vacuolar, granular, amoeboid, avacuolar, multivacuolar and cyst. Its life cycle is not completely understood. Cysts are the forms of resistance and transmission. It is considered a zoonotic parasite with 9 subtypes, with different specificities for human and animal hosts. Its pathogenesis is controversial because it can cause asymptomatic and symptomatic infections, with gastrointestinal and cutaneous manifestations. Laboratory diagnosis may be performed by microscopic, cultural, serological and molecular techniques.

Blastocystis legumain is localized on the cell surface, and specific inhibition of its activity implicates a pro-survival role for the enzyme.

Programmed cell death (PCD) is crucial for cellular growth and development in multicellular organisms. Although distinct PCD features have been described for unicellular eukaryotes, homology searches have failed to reveal clear PCD-related orthologues among these organisms. Our previous studies revealed that a surface-reactive monoclonal antibody (mAb) 1D5 could induce multiple PCD pathways in the protozoan Blastocystis. In this study, we identified, by two-dimensional gel electrophoresis and mass spectrometry, the target of mAb 1D5 as a surface-localized legumain, an asparagine endopeptidase that is usually found in lysosomal/acidic compartments of other organisms. Recombinant Blastocystis legumain displayed biphasic pH optima in substrate assays, with peaks at pH 4 and 7.5. Activity of Blastocystis legumain was greatly inhibited by the legumain-specific inhibitor carbobenzyloxy-Ala-Ala-AAsn-epoxycarboxylate ethyl ester (APE-RR) (where AAsn is aza-asparagine) and moderately inhibited by mAb 1D5, cystatin, and caspase-1 inhibitor. Interestingly, inhibition of legumain activity induced PCD in Blastocystis, observed by increased externalization of phosphatidylserine residues and in situ DNA fragmentation. In contrast to plants, in which legumains have been shown to play a pro-death role, legumain appears to display a pro-survival role in Blastocystis.

Blastocystis exhibits inter- and intra-subtype variation in cysteine protease activity.

Blastocystis is an enteric protistan parasite of zoonotic potential and poorly understood pathogenesis. We have previously reported that Blastocystis cysteine proteases can degrade human secretory IgA and are also responsible for the induction of IL-8 response in colonic epithelial cells in vitro. Differences in virulence between Blastocystis subtypes have been reported recently in both animal models and clinical studies, although cellular mechanisms for these differences are currently unknown. Parasites such as Giardia intestinalis and Entamoeba histolytica have distinct virulent and non-virulent strains which may be attributable to variations in their cysteine proteases. In the present study, variations in cysteine protease activity was observed between avian (subtype 7) and rodent (subtype 4) isolates of Blastocystis with avian isolates exhibiting approximately two times higher peak cysteine protease activity than rodent isolates. Cysteine protease activity and parasite cell size varied over time within cultures of the same isolate. An association between parasite cell size and protease activity was observed.

Localization and nucleotide specificity of Blastocystis succinyl-CoA synthetase.

The anaerobic lifestyle of the intestinal parasite Blastocystis raises questions about the biochemistry and function of its mitochondria-like organelles. We have characterized the Blastocystis succinyl-CoA synthetase (SCS), a tricarboxylic acid cycle enzyme that conserves energy by substrate-level phosphorylation. We show that SCS localizes to the enigmatic Blastocystis organelles, indicating that these organelles might play a similar role in energy metabolism as classic mitochondria. Although analysis of residues inside the nucleotide-binding site suggests that Blastocystis SCS is GTP-specific, we demonstrate that it is ATP-specific. Homology modelling, followed by flexible docking and molecular dynamics simulations, indicates that while both ATP and GTP fit into the Blastocystis SCS active site, GTP is destabilized by electrostatic dipole interactions with Lys 42 and Lys 110, the side-chains of which lie outside the nucleotide-binding cavity. It has been proposed that residues in direct contact with the substrate determine nucleotide specificity in SCS. However, our results indicate that, in Blastocystis, an electrostatic gatekeeper controls which ligands can enter the binding site.

Cellular evolution: what's in a mitochondrion?

Mitochondria and their relatives constitute a wide range of organelles, only some of which function in aerobic respiration. Mitochondrial remnants from different anaerobic lineages show a striking degree of functional convergence.

Blastocystis ratti contains cysteine proteases that mediate interleukin-8 response from human intestinal epithelial cells in an NF-kappaB-dependent manner.

Blastocystis is a ubiquitous enteric protozoan found in the intestinal tracts of humans and a wide range of animals. Evidence accumulated over the last decade suggests association of Blastocystis with gastrointestinal disorders involving diarrhea, abdominal pain, constipation, nausea, and fatigue. Clinical and experimental studies have associated Blastocystis with intestinal inflammation, and it has been shown that Blastocystis has potential to modulate the host immune response. Blastocystis is also reported to be an opportunistic pathogen in immunosuppressed patients, especially those suffering from AIDS. However, nothing is known about the parasitic virulence factors and early events following host-parasite interactions. In the present study, we investigated the molecular mechanism by which Blastocystis activates interleukin-8 (IL-8) gene expression in human colonic epithelial T84 cells. We demonstrate for the first time that cysteine proteases of Blastocystis ratti WR1, a zoonotic isolate, can activate IL-8 gene expression in human colonic epithelial cells. Furthermore, we show that NF-kappaB activation is involved in the production of IL-8. In addition, our findings show that treatment with the antiprotozoal drug metronidazole can avert IL-8 production induced by B. ratti WR1. We also show for the first time that the central vacuole of Blastocystis may function as a reservoir for cysteine proteases. Our findings will contribute to an understanding of the pathobiology of a poorly studied parasite whose public health importance is increasingly recognized.

Colony growth of Philippine isolates of Blastocystis hominis in simplified, soft agar medium.

The agar-cloning technique of Blastocystis hominis has been observed in both solid and semisolid agar using Iscove's modified Dulbecco's medium. In this study, Philippine isolates of B. hominis were grown by pour-plate method in semisolid agar using Locke's solution. Inoculated plates contained 0.7% agar, 10% horse serum, and 0.1% sodium thioglycollate. Plates were incubated at 37 degrees C in a microaerophilic jar for 7-10 days. Biconvex disk-shaped colonies were seen abound at the bottom half of the medium. Colonies growing at the agar-glass interface were flat and consisted of thin layers of cells. From these colonies, large amoeboid cells were frequently seen on the periphery, whereas smaller cells were concentrated at the core. Analysis of the SSU rDNA genetically established the identity of the clones to be B. hominis. This is the first report on agar cloning of Blastocystis in a compound medium.

Freeze-fracture and cytochemical studies on the in vitro cyst form of reptilian Blastocystis pythoni.

After cultured cysts are osmotically shocked by treating with distilled water, there is an exponential increase in the cyst form of Blastocystis pythoni; this was demonstrated by an immunofluorescence antibody assay against the culture organisms. In 11-day-old cultures of B. pythoni, 68.8% of the organisms (= 2.2 x 10(8) cysts/ml) were in the cyst form. Examination of thin sections of cysts revealed many similarities to the cyst forms of Blastocystis obtained from fecal samples in previous investigations. Freeze-fracture images of the plasma membrane of non-cyst cells also revealed a similar distribution of the intramembrane particles (IMP) when compared to non-cysts of B. hominis, while the plasma membrane of the cyst form showed practically no IMP. The size and morphology of particle-rich small depressions and smooth small protrusions observed on the P face and E face of non-cyst cells, respectively, were similar to endocytic sites reported for B. hominis. In the present study glycogen was cytochemically demonstrated at the ultrastructural level by an alkaline bismuth staining method in both cyst and non-cyst cells.

A survey of Blastocystis in domestic chickens.

The prevalence and morphology of Blastocystis in fresh faecal material from 227 domestic chickens was investigated. A very high prevalence of infection (approximately 95%) was found in chickens from four of the five commercial farms studied. Extremely high numbers of Blastocystis were found in the majority of samples. Blastocystis cells showed considerable variation in size, ranging from approx. 3 microm to approx. 120 microm in diameter. This size range is more extreme than those previously recognised for the organism from chickens. All chickens from one farm appeared free of Blastocystis infection. Most Blastocystis cells appeared to be the vacuolar form, although the shape of the cells and the appearance of the central vacuole contents varied considerably within and among faecal samples. Nuclei showed "spots" of electron-opaque material, generally arranged as a band within the nuclei. Multiple individual cysts within a single outer fibrillar layer were found in addition to single cysts without an encompassing fibrillar layer.