G. C. Ng

Genome sequence of the stramenopile Blastocystis, a human anaerobic parasite

BackgroundBlastocystis is a highly prevalent anaerobic eukaryotic parasite of humans and animals that is associated with various gastrointestinal and extraintestinal disorders. Epidemiological studies have identified different subtypes but no one subtype has been definitively correlated with disease.ResultsHere we report the 18.8 Mb genome sequence of a Blastocystis subtype 7 isolate, which is the smallest stramenopile genome sequenced to date. The genome is highly compact and contains intriguing rearrangements. Comparisons with other available stramenopile genomes (plant pathogenic oomycete and diatom genomes) revealed effector proteins potentially involved in the adaptation to the intestinal environment, which were likely acquired via horizontal gene transfer. Moreover, Blastocystis living in anaerobic conditions harbors mitochondria-like organelles. An incomplete oxidative phosphorylation chain, a partial Krebs cycle, amino acid and fatty acid metabolisms and an iron-sulfur cluster assembly are all predicted to occur in these organelles. Predicted secretory proteins possess putative activities that may alter host physiology, such as proteases, protease-inhibitors, immunophilins and glycosyltransferases. This parasite also possesses the enzymatic machinery to tolerate oxidative bursts resulting from its own metabolism or induced by the host immune system.ConclusionsThis study provides insights into the genome architecture of this unusual stramenopile. It also proposes candidate genes with which to study the physiopathology of this parasite and thus may lead to further investigations into Blastocystis-host interactions.

Predominance of subtype 3 among Blastocystis isolates from a major hospital in Singapore

Blastocystis is an enteric protozoan parasite commonly found in humans and animals. Phylogenetic and genotypic analyses have shown that Blastocystis exhibits extreme genetic diversity, and humans are host to a number of zoonotic isolates. In the present study, the prevalence of Blastocystis in 276 stool samples from a hospital in Singapore was examined, and for the first time, riboprinting using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to determine the genetic diversity of the Blastocystis isolated from the Singapore population. The prevalence rate was determined to be 3.3% (9/276), and Blastocystis displaying two main ribotypes were isolated. As a comparison, we performed PCR-RFLP using two different published methodologies, and both methods allowed the isolates to be divided into two distinct groups based on their riboprint patterns. According to a recently proposed classification scheme, 78% (7/9) of the isolates were of subtype 3, while 22% (2/9) were subtype 1. The predominance of subtype 3 in an urbanized city state such as Singapore is in agreement with the idea that subtype 3 is a genotype of human origin.

Experimental Blastocystis hominis infection in laboratory mice

Abstract Young (less than 8 weeks old) immunocompetent BALB/c mice became infected with Blastocystis hominis after inoculation of fecal cysts orally and of in vitro axenic-culture forms intracecally. This study confirmed that the fecal cyst was the form responsible for external transmission and that the mode of transmission was by the fecal-oral route. The infection was self-limiting and the infected BALB/c mice appeared normal except that some of them showed weight loss and lethargy. Both vacuolar and granular forms were found in the cecum, but only cyst forms were observed in the colon. Histological examination of the cecum and colon showed intense inflammatory-cell infiltration, edematous lamina propria, and mucosal sloughing. It is apparent that although B. hominis is not invasive, it is capable of causing pathogenesis in BALB/c mice.

Elucidation of the life cycle of the intestinal protozoan Blastocystis hominis.

This paper elucidates the status of the different morphological forms ofBlastocystis and reports the existence of thin-and thick-walled cysts inB. hominis on the basis of current experimental evidence. It is suggested that the thin-walled cysts are autoinfectious, leading to multiplication of the organism in the intestinal tract. The thick-walled cysts are responsible for external transmission via the faecal-oral route. A life cycle forB. hominis is postulated on the basis of these findings.

Observations on the ultrastructure and viability of the cystic stage of Blastocystis hominis from human feces

This report describes the ultrastructure and viability of cysts of Blastocystis hominis from feces of infected patients. The cysts were round to ovoid, measured 2-5 μm in size, and contained a condensed cytoplasm that had vacuoles of varying sizes, four nuclei, and as many as six cristate mitochondria. The cell wall was rather electron-lucent. Surprisingly, chromatoid-like structures were found in the cytoplasm and nucleus of some of the cysts. These have not previously been reported in Blastocystis. The cysts can survive in water for up to 19 days at normal temperatures but are fragile at extreme temperatures and in common disinfectants.

In vitro encystment and experimental infections of Blastocystis hominis

Cultures ofBlastocystis hominis were induced to encyst using three encystation media: (a) an encystation medium (EM) comprising yeast extract in buffered saline containing 50% horse serum, (b) an encystation medium (CEM) comprising EM conditioned with bacterial soluble products and (c) an encystation medium (TEM) containing 0.5% trypticase in EM. Two isolates ofB. hominis were studied, an axenized isolate C and a non-axenized isolate MS. In EM, isolate C did not encyst, whereas 6.1% of isolate MS had encysted by day 1. However, in CEM and TEM, 17.4% and 25.7% of isolate C, respectively, had encysted by day 5. In all three media, isolate MS encysted more readily than isolate C, with as much as 91.7% of the former encysting in TEM. As viewed by phase-contrast microscopy, cyst-like stages appeared highly refractile. Direct stool examination of juvenile Wistar rats infected with 10000 cyst-like stages of both C and MS isolates showedBlastocystis at day 2 post-infection. At autopsy on day 7, large numbers ofBlastocystis were seen in the cecum, with smaller numbers being observed in the large intestine. In contrast, rats fed with various inocula of the vacuolar stages of isolates C and MS did not become infected, indicating the importance of the encysted stages in the transmission of the parasite.

A survey of Blastocystis in reptiles.

A total of 28 species of reptiles were investigated forBlastocystis using light microscopy and in vitro culture in biphasic egg slant medium.Blastocystis species were detected in 8 (28.6%) of these 28 species in 3 tortoises (Geochelone elephantopus, G. elegans andG. carbonaria), 3 snakes (Boiga dendrophilla, Python reticulatus andElaphe radiata), 1 crocodile (Crocodylus porosus) and 1 iguana lizard (Cyclura cornuta). The reptilianBlastocystis appeared to be morphologically similar toB. hominis.

Description of a Blastocystis species from Rattus norvegicus

Abstract Two isolates (WR1 and WR2) of Blastocystis from laboratory-bred Wistar rats were axenized by a method of colony growth in soft agar combined with antibiotic treatment. The colonies were cultured in Iscoves modified Dulbeccos medium (IMDM) and Bacto agar mixture supplemented with 10% horse serum in the presence of thioglycollate. The cells from the colonies had an ameboid outline with a central body. Large inclusions were seen in the central body of some cells. Some granular forms were also found. In the axenic culture of isolate WR2, about one-third of the organisms were granular forms. Cysts were found in the axenic culture of both isolates. This is the first report of such cyst formation in in vitro culture. The karyotypic patterns of both isolates of the rat Blastocystis were analyzed by pulsed-field gel electrophoresis (PFGE). A total of 13 chromosomal bands were separated, ranging from 1.86 Mb to 295 kb. The karyotypic patterns of the rat Blastocystis were different from those of B. hominis and reptilian Blastocystis. On the basis of the above-mentioned differences, the rat Blastocystis is assigned as B.␣ratti sp. nov.

Axenic culture of reptilian Blastocystis isolates in monophasic medium and speciation by karyotypic typing

Abstract The growth of axenic reptilian isolates of Blastocystis in Iscove’s modified Dulbecco’s medium (IMDM) was studied and the morphology of the parasite was examined by phase-contrast microscopy. The chromosomal patterns of these reptilian isolates of Blastocystis were examined by pulsed-field gel electrophoresis (PFGE) and compared with those of B. hominis and B. lapemi, a sea snake Blastocystis. IMDM with 10% horse serum supported excellent growth of the reptilian Blastocystis isolates. The parasites from all the isolates were predominantly vacuolar, but multivacuolar and amoeboid forms were also seen. Amoeboid forms with rather elongate pseudopodia were also observed. There were some differences in size, morphology, and growth characteristics in the different reptilian isolates. The karyotypic patterns of the Blastocystis isolates from tortoise, iguana, and python were distinctly different from one another and from those obtained with B. hominis and B. lapemi. On the basis of the above-mentioned differences in chromosomal patterns, the tortoise, iguana, and python isolates are described as new species, viz., B. geocheloni sp. nov. from Geochelone carbonaria (red-footed tortoise), B. cycluri sp. nov. from Cyclura cornuta (rhino iguana), and B. pythoni sp. nov. from Python reticulatus (reticulated python).

In vitro response of Blastocystis hominis against traditional Chinese medicine.

This is the first inn vitro study on the activity of 20 kinds of crude extracts of traditional Chinese medicine (TCM) on the intestinal parasite, Blastocystis hominis using the criteria of living cell count (LCC) and living cell rate (LCR). LCC and LCR were applied as observation indicators, the former as a fixed-quantity and the latter as a fixed-quality method. LCR calculated percentage rate of living cells using eosin-brilliant cresyl blue staining which could differentiate between living cells and dying or dead cells. There were five extracts with no inhibitory activity, thirteen with moderate inhibition and two with high inhibition. The crude extracts of Coptis chinensis (CC) and Brucea javanica (BJ) were found to be most active against B. hominis. The active concentration of CC was 100 micrograms/ml. The active concentration of BJ was 500 micrograms/ml. The active concentration of metronidazole (MD) was 10 micrograms/ml and this was taken as an active standard drug for B. hominis.