In vitro and in vivo thermal stress induces proliferation of Blastocystis sp.

Blastocystis sp. is known to be the most commonly found intestinal protozoan parasite in human fecal surveys and has been incriminated to cause diarrhea and abdominal bloating. Binary fission has been widely accepted as the plausible mode of reproduction for this parasite. The present study demonstrates that subjecting the parasites in vitro to higher temperature shows the proliferation of parasite numbers in cultures. Transmission electron microscopy was used to compare the morphology of Blastocystis sp. subtype 3 isolated from a dengue patient having high fever (in vivo thermal stress) and Blastocystis sp. 3 maintained at 41 °C (in vitro thermal stress) and 37 °C (control). Fluorescence stains like acridine orange (AO) and 4',6'-diamino-2-phenylindole (DAPI) were used to demonstrate the viability and nuclear content of the parasite for both the in vitro and in vivo thermal stress groups of parasites. Blastocystis sp. at 37 °C was found to be mostly vacuolar whereas the in vitro thermal stressed isolates at 41 °C were granular with electron dense material seen to protect the granules within the central body. Parasites of the in vivo thermal stressed group showed similar ultrastructure as the in vitro ones. AO and DAPI staining provided evidence that these granules are viable which develop into progenies of Blastocystis sp. These granular forms were then observed to rupture and release progenies from the mother cells whilst the peripheral cytoplasmic walls were seen to degrade. Upon exposure to high temperature both in vitro and in vivo, Blastocystis sp. in cultures show higher number of granular forms seen to be protected by the electron dense material within the central body possibly acting as a protective mechanism. This is possibly to ensure the ability to survive for the granules to be developed as viable progenies for release into the host system.

Prevalence and staining characteristics of Blastocystis isolates from food animals in Tamil Nadu.

The present study was undertaken to investigate the prevalence and staining characteristics of Blastocystis isolated from food animals. Smears of the duodenal and caecal mucosal scrapings, collected from food animals, were stained with Giemsa, Gram's, modified acid-fast and acridine orange. Blastocystis was identified in 295 samples, including faeces and intestinal contents of animals like small ruminants (95), poultry (170) and pigs (30). The prevalence in pigs was found to be high (94.4%) followed by poultry (29.4%) and small ruminants (14%). Various forms of Blastocystis such as vacuolar, granular and amoeboid forms were identified by using different stains. The parasites stained with Giemsa were identified by the presence of eosinophilic nucleus and basophilic cytoplasm. In organisms stained with Gram's stain, the cytoplasm of the vacuolar forms took up the counter stain safranine. Blastocystis appeared as a pink colored cyst against bluish green background with modified acid-fast staining. The study shows that there is a very high prevalence of Blastocystis among the food animals investigated. Simple parasitological procedures, including direct microscopical examination and staining with agents like Giemsa, Gram's and acridine orange can assist identification of the parasites from intestinal contents and faecal material.

Blastocystis Isolate B Exhibits Multiple Modes of Resistance against Antimicrobial Peptide LL-37.

Blastocystis is one of the most common eukaryotic organisms found in humans and many types of animals. Several reports have identified its role in gastrointestinal disorders, although its pathogenicity is yet to be clarified. Blastocystis is transmitted via the fecal-to-oral route and colonizes the large intestines. Epithelial cells lining the intestine secrete antimicrobial peptides (AMPs), including beta-defensins and cathelicidin, as a response to infection. This study explores the effects of host colonic antimicrobial peptides, particularly LL-37, a fragment of cathelicidin, on different Blastocystis subtypes. Blastocystis is composed of several subtypes that have genetic, metabolic, and biological differences. These subtypes also have various outcomes in terms of drug treatment and immune response. In this study, Blastocystis isolates from three different subtypes were found to induce intestinal epithelial cells to secrete LL-37. We also show that among the antimicrobial peptides tested, only LL-37 has broad activity on all the subtypes. LL-37 causes membrane disruption and causes Blastocystis to change shape. Blastocystis subtype 7 (ST7), however, showed relative resistance to LL-37. An isolate, ST7 isolate B (ST7-B), from this subtype releases proteases that can degrade the peptide. It also makes the environment acidic, which causes attenuation of LL-37 activity. The Blastocystis ST7-B isolate was also observed to have a thicker surface coat, which may protect the parasite from direct killing by LL-37. This study determined the effects of LL-37 on different Blastocystis isolates and indicates that AMPs have significant roles in Blastocystis infections.

Increase number of mitochondrion-like organelle in symptomatic Blastocystis subtype 3 due to metronidazole treatment.

Blastocystis sp., an intestinal organism is known to cause diarrhea with metronidazole regarded as the first line of treatment despite reports of its resistance. The conflicting reports of variation in drug treatment have been ascribed to subtype differences. The present study evaluated in vitro responses due to metronidazole on ST3 isolated from three symptomatic and asymptomatic patients, respectively. Symptomatic isolates were obtained from clinical patients who showed symptoms such as diarrhea and abdominal bloating. Asymptomatic isolates from a stool survey carried out in a rural area. These patients had no other pathogens other than Blastocystis. Ultrastructural studies using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) revealed drug-treated ST3 from symptomatic patients were irregular and amoebic with surface showing high-convoluted folding when treated with metronidazole. These organisms had higher number of mitochondrion-like organelle (MLO) with prominent cristae. However, the drug-treated ST3 from asymptomatic persons remained spherical in shape. Asymptomatic ST3 showed increase in the size of its central body with the MLO located at the periphery.

Phenotypic variation in Blastocystis sp. ST3.

BACKGROUND: Blastocystis, is one of the most common human intestinal protozoan, which has many conflicting reports on its pathogenic role. Gut conditions which obviously varies in asymptomatic individuals, symptomatic and irritable bowel syndrome (IBS) patients in terms of gut flora, pH, osmotic pressure and water potentials could play an important role in its pathogenicity. The present study is the first study to investigate phenotypic characteristics of Blastocystis sp. ST3 isolated from asymptomatic, symptomatic and IBS isolates. METHODS: A total of 8 Blastocystis isolates were obtained from four IBS patients (IBS1-4) and four symptomatic patients (S1-4) at a local gastroenterology clinic. Asymptomatic isolates (A1-4) were obtained from a field survey at a local village. RESULTS: All 12 isolates were determined as subtype 3 (ST3). A1-4 isolates showed the highest peak growth followed by IBS1-4 isolates and S1-4 isolates for the growth profile. Parasites from IBS isolates (IBS1-4) showed the largest diameter with a mean 18.43 ± 2.22 µm compared to parasites of symptomatic isolates (isolates S1-4) 15.54 ± 3.02 µm and asymptomatic isolates (isolates A1-4) 11.76 ± 0.82 µm. The symptomatic isolates (average generation time: 9.87 ± 2.97 h) grew faster than the IBS isolates (average generation time: 7.56 ± 1.06 h) and asymptomatic isolates (average generation time: 5.97 ± 1.52 h). The parasites isolated from IBS isolates showed strong aggregation and clumping, which had seen reduced in parasites of isolates S1-4. No clumping was seen in parasites from A1-4. The outer surface of parasites in IBS isolates showed greater binding affinities towards FITC-labelled Concanavalin A (Con A) than symptomatic isolates and asymptomatic isolates. Scanning electron microscopy showed that in IBS isolates, the surface of Blastocystis showed a very coarse and intensely folded surface. The IBS isolates also exhibited a dense material and a thicker layer of surface coat can be seen compared to asymptomatic and symptomatic isolates. CONCLUSION: There have been no studies thus far providing evidence for phenotypic variation within a particular subtype. The present study is the first to demonstrate the phenomenon of gut environment facilitating adaptation of parasites possibly for survival leading to phenotypic differences for Blastocystis.

[Morphological identification of blastocystis].

The authors have attempted to systematize the currently known specific morphological features of the composition of Blastocystis spp. existing in different forms and to present this material as a reference table, by understanding the need for further data clarification. In addition, the paper describes observations of variations in the forms of human blastocysts. In particular, it depicts the species of multinucleated cysts, the identification of which may cause difficulties in diagnosing and differentiating these forms with some representative species of the genus Entamoeba.

Establishing mono-eukaryotic Histomonas meleagridis cultures from in vivo infection contaminated with Tetratrichomonas gallinarum and Blastocystis spp.

SUMMARY The necessity to easily establish Histomonas meleagridis cultures has been underlined extensively by many researchers in order to gain more insights in the biology of H. meleagridis. In addition the occurrence of different protozoa in the caeca of birds impedes, however, the isolation and propagation of H. meleagridis from field outbreaks. Therefore, in a kinetic study using transmission electron microscopy the deleterious effects of adventitious protozoa including Tetratrichomonas gallinarum and Blastocystis spp. on cultured H. meleagridis were examined. To overcome this issue, an easy and successful approach to establish the mono-eukaryotic H. meleagridis culture free of other host's protozoa is proposed. At 10 days post infection, liver lesions of H. meleagridis-infected birds were isolated and inoculated into culture media pre-incubated with caecal bacteria. After 48 h of incubation, presence of H. meleagridis in the cultures was confirmed through morphological evaluation. Additionally, TEM examination and analysis by PCR amplification of the small subunit rRNA gene could exclude the co-cultivation of T. gallinarum and Blastocystis spp. Furthermore, after successful propagation and maintenance of the cultured H. meleagridis, its pathogenicity was affirmed in an infection experiment in turkeys.

[Isolation of Blastocystis spp. from human hosts and in vitro determination of different morphological forms]. / Blastocystis spp.'nin Insandan Izolasyonu ve Besiyerinde Farkli Evrim Sekillerinin Izlenmesi.

OBJECTIVE: Blastocystis is a highly common parasite that infects the gastrointestinal tract of many different organisms. Morphology and the appropriate classification of Blastocystis spp. has only recently been resolved with molecular biological studies. This study was performed to determine the prevalence of Blastocystis spp. among humans and to isolate the parasite from clinical specimens. METHODS: Blastocystis spp. was detected in 0.48% of the stool samples and the positive samples were cultivated in Locke-Egg Serum (LES). During passages inoculums were investigated by direct microscopy and stained with trichrome and iron hematoxylene. RESULTS: Vacuolar and granular forms were the most common in cultures and also the amoeboid form was observed. CONCLUSION: LES medium may be a suitable selection for studies aiming to determine the frequency of Blastocystis spp. and for the diagnosis in routine laboratories.

Comparison of microscopy, culture, and conventional polymerase chain reaction for detection of blastocystis sp. in clinical stool samples.

We tested 513 stool samples from patients in Sydney, Australia for Blastocystis by using five diagnostic techniques: microscopy of a permanently stained smear using a modified iron-hematoxylin stain, two xenic culture systems (a modified Boeck and Drbohlav's medium and tryptone, yeast extract, glucose, methionine-9 medium), and two published conventional polymerase chain reaction methods specific for the small subunit ribosomal DNA. Ninety-eight (19%) samples were positive for Blastocystis in one or more of the diagnostic techniques. The PCR 2 method was the most sensitive at detecting Blastocystis with a sensitivity of 94%, and the least sensitive was microscopy of the permanent stain (48%). Subtype 3 was the most predominant subtype (present in 43% of samples assigned to this group). This study highlights the low sensitivity of microscopy when used as the sole diagnostic modality for detection of Blastocystis sp.

A functional Tom70 in the human parasite Blastocystis sp.: implications for the evolution of the mitochondrial import apparatus.

Core proteins of mitochondrial protein import are found in all mitochondria, suggesting a common origin of this import machinery. Despite the presence of a universal core import mechanism, there are specific proteins found only in a few groups of organisms. One of these proteins is the translocase of outer membrane 70 (Tom70), a protein that is essential for the import of preproteins with internal targeting sequences into the mitochondrion. Until now, Tom70 has only been found in animals and Fungi. We have identified a tom70 gene in the human parasitic anaerobic stramenopile Blastocystis sp. that is neither an animal nor a fungus. Using a combination of bioinformatics, genetic complementation, and immunofluorescence microscopy analyses, we demonstrate that this protein functions as a typical Tom70 in Blastocystis mitochondrion-related organelles. Additionally, we identified putative tom70 genes in the genomes of other stramenopiles and a haptophyte, that, in phylogenies, form a monophyletic group distinct from the animal and the fungal homologues. The presence of Tom70 in these lineages significantly expands the evolutionary spectrum of eukaryotes that contain this protein and suggests that it may have been part of the core mitochondrial protein import apparatus of the last common ancestral eukaryote.

Autophagy is involved in starvation response and cell death in Blastocystis.

Previous studies have demonstrated that colony forms of Blastocystis undergo cell death with numerous membrane-bound vesicles containing organelles located within the central vacuole, resembling morphological features of autophagy. In this study, we investigated whether Blastocystis underwent autophagy upon amino acid starvation and rapamycin treatment. Concurrently, we provide new insight into a possible function of the central vacuole. The use of the autophagy marker monodansylcadaverine, and the autophagy inhibitors 3-methyladenine and wortmannin, showed the existence of autophagy in amino-acid-starved and rapamycin-treated Blastocystis. Confocal microscopy and transmission electron microscopy studies also showed morphological changes that were suggestive of autophagy. The unusually large size of the autophagic compartments within the parasite central vacuole was found to be unique in Blastocystis. In addition, autophagy was found to be triggered when cells were exposed to the cytotoxic antibody mAb 1D5, and autophagy was intensified in the presence of the caspase inhibitor zVAD.fmk. Taken together, our results suggest that the core machinery for autophagy is conserved in Blastocystis, and that it plays an important role in the starvation response and cell death of the parasite.

Biochemical characterization of a mitochondrial-like organelle from Blastocystis sp. subtype 7.

A mitochondrion-like organelle (MLO) was isolated from isotonic homogenates of Blastocystis. The organelle sedimented at 5000 g for 10 min, and had an isopycnic density in sucrose of 1.2 g ml(-1). Biochemical characterization enabled the demonstration of several key enzymes that allowed the construction of a metabolic pathway consisting of an incomplete Krebs cycle linked to the oxygen-sensitive enzymes pyruvate : NADP(+) oxidoreductase (PNO), acetate : succinate CoA transferase (ASCT) and succinate thiokinase (STK), which cumulatively are responsible for recycling CoA and generating ATP. The organelle differs from typical aerobic mitochondria in possessing an oxygen-sensitive PNO that can use FAD(+) or FMN(+) as electron acceptor but is inactive with NAD(+), Spinacia oleracea ferredoxin or Clostridium pasteurianum ferredoxin. A gene with 77 % sequence similarity to the PNO mitochondrion precursor cluster from Euglena gracilis sp[Q941N5] was identified in the Blastocystis genome database. A second cluster with 56 % sequence similarity to the pyruvate : ferredoxin oxidoreductase (PFOR) from Trichomonas vaginalis was also identified, which is in agreement with the concept that the PNO gene arose through the fusion of a eubacterial gene for PFOR with the gene for NADPH : cytochrome p450 reductase. Hydrogenase activity was not detected under the conditions used in this study. The Blastocystis oranelle therefore demonstrates significant biochemical differences from traditional mitochondria and hydrogenosomes, but possesses features of both. Based upon the results of this study, the Blastocystis organelle falls into the category of a MLO.

[The morphological study of the cysts Pelomyxa palustris Greeff, 1874].

Pelomyxa palustris Greeff, 1874, is the only species of pelomixoid amoebas with the rest cysts in its life cycle. The morphology of the P. palustris has been studied by the light and electronic microscopy. Encystation of P. palustris under climatic conditions of North-West of Russia occurs within August-September. Rest cysts have a complex, trilaminar wall. Two inner lamina are the dense endocyst and the laminated mesocyst, thickness of each layer runs up to 0.6-0.7 microm. Thickness of the electron-dense ectocyst usually does not exceed 0.1-0.2 microm. The encystated cell of P. palustris has the unique structure. About 60 % of the cell volume are occupied by a huge vacuole placed in the center and filled up with the prokaryotic cytobionts. Different vacuoles, small vesicles of various nature, autophagosomes and lipid drops could be found inside that huge vacuole. The amoebae cytoplasm occupies the space in between endocyst's inner surface and the central vacuole. No any inclusions, prokaryotic cytobionts and most of cell organelles are absent in the cytoplasm. There are 4 large nuclei filled with relatively homogeneous karyoplasm lying in the cytoplasm. Nuclear envelope forms a lot of long tubular channels, running through the cytoplasm and lining the membrane of the central vacuole. Encysted pelomixoid stay in this state up until the beginning of excystation. Excystation of P. palustris in the studied region occurs in spring, during the latter half of April and the beginning of May. Cysts undergo complex morphofunctional changes, related to the reorganization of the wall and formation of young multinucleate amoebas. Only one wall lamina of the 3 initial ones is left up to the moment of excystation. The central vacuole endures ruination and its content penetrates into the cytoplasm. Pelomixoid nuclei divide twice. Prokaryotic cytobionts are localized in cytoplasm and in the perinuclear area. Young multinuclear species of P. palustris coming out of the cysts do not differ in their structure from the adult forms.

Ultrastructural and phylogenetic studies on Blastocystis isolates from cockroaches.

Four Blastocystis isolates from cockroaches were established and these isolates were morphologically confirmed as Blastocystis organisms by light and/or electron microscopy. As these isolates were morphologically indistinguishable from Blastocystis isolated from other animals, phylogenetic analyses were conducted using their small subunit ribosomal RNA genes. A analyses of these sequences with previously reported ones that had been classified into nine Blastocystis clades indicated the presence of a new clade that comprised only Blastocystis organisms from cockroaches (clade X). A clade comprised of amphibian and reptilian Blastocystis organisms (clade IX) was located at the basal position of the Blastocystis tree together with the common ancestor of Proteromonas and Protoopalina, clade X emerged after the divergences of these two basal clades and its branching position was clearly supported by bootstrap analysis.

A survey of Blastocystis infection in anuran and urodele amphibians.

Blastocystis infection in amphibians was surveyed in three species of anuran and one species of urodele amphibians captured at two distinct locations in Japan. All three species of frogs were highly infected with Blastocystis, while 69 individual urodele newts, Cynopus pyrrhogaster, were negative for infection. Eleven Blastocystis isolates (47.8%) were recovered from 23 Rana nigromaculata leopard frogs. Twenty-three (92%) of 25 Rana catesbeiana bullfrogs and all (100%) of 24 Bufo japonicus japonicus toads were positive for Blastocystis. Two distinct populations of the toad and bullfrog showed a high prevalence (100 or 84.6%) of Blastocystis infection, while in two populations of the leopard frog only one population was positive for Blastocystis (84.6%). Three Blastocystis isolates from different species of the frogs were established. Since none of the three isolates could survive at 37 degrees C, a temperature tolerance assay was performed to assess the optimal growth temperature and to determine the range of non-lethal temperatures. During the exponential growth phase of 3- or 4-day cultures at 25 degrees C, three isolates were exposed to 4, 28, 31, or 34 degrees C for 3 days and then returned to 25 degrees C to monitor the cell growth. Based on the optimal growth temperatures and different ranges of temperature tolerance among the three new isolates from frogs and two known species, Blastocystis hominis and Blastocystis lapemi, it was established that the three isolates recovered from different species of frogs had different physiological features from B. hominis and B. lapemi.

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.

Blastocystis sp. from pigs: ultrastructural changes occurring during polyxenic cultivation in Iscove's modified Dulbecco's medium.

A porcine strain of Blastocystis sp. grown in LES medium was transferred to Iscove's modified Dulbecco's medium (IMDM) and encystation medium (EM). In comparison with the cells maintained in LES medium, the cells cultivated for several days in both IMDM and EM exhibited considerable differences in their morphology and ultrastructure. The central vacuole was mostly electron-opaque, usually with electron-lucent granules in its center. Mitochondrial matrix became less electron-dense and cristae were shortened and reduced in number. Two membranes of the nuclear envelope were dilated and the intramembranous space was filled with intermediately electron-dense bodies. Two or three nuclei surrounded by one joint outer membrane were often seen. In the old cultures the surface coat often disappeared and electron-dense pits on the cell surface were much more numerous than in young cultures or in the cells grown in LES medium. The possible role of the cells with peculiar ultrastructural features in the Blastocystis life cycle is discussed.