K. T. Moe

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.

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.

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).

Production and characterization of murine monoclonal antibodies to Blastocystis hominis

Several hybridomas producing antibodies detected by enzyme-linked immunosorbent assay (ELISA) were established by fusions of mouse myeloma P3.X63.Ag8.U1 with spleen cells from BALB/c mice immunized against an isolate of Blastocystis hominis. Five strongly positive hybrids (6B6, 1D5, 1E7, 4F7 and 4G11) were cloned and all were found to secrete IgM monoclonal antibodies. Four MAbs (6B6, 1E7, 4F7 and 4G11) reacted in immunoblots with a number of B. hominis antigens (mol. wt ranging from 25,000 to 220,000) which were likely to be repeating oligosaccharide epitopes located on glycoproteins, as indicated by pronase and periodate treatment. Another MAb (1D5) reacted with a single antigenic band (mol. wt 30,5000). Similar results were obtained in immunoblots using 4 other B. hominis isolates. Indirect fluorescent-antibody assay (IFA) using MAbs showed 3 patterns of reactivity. 1D5 showed patchy fluorescence, 4F7 showed peripheral fluorescence and 6B6, 1E7 and 4G11 showed bright diffuse fluorescence. These patterns were observed for all 5 human Blastocystis isolates. The MAbs exhibited some cross-reactivity with 2 reptilian Blastocystis isolates but not with Giardia intestinalis, Trichomonas vaginalis or Entamoeba histolytica.

Clonal growth of Blastocystis hominis in soft agar with sodium thioglycollate

Abstract The present report describes a method for establishment of colonies of Blastocystis hominis from single cells in soft agar. The percentage of colony-forming efficiency (% CFE = number of colonies grown / number of cells inoculated × 100) for the cultures was greatly improved by the addition of sodium thioglycollate. Five human Blastocystis isolates chosen for this study showed no apparent variation in colonial morphology. Isolated colonies were also successfully grown in liquid medium, providing a means of obtaining large numbers of B. hominis cells that had arisen from a single clone.

Cytopathic effect of Blastocystis hominis after intramuscular inoculation into laboratory mice

Abstract The present study investigated the pathogenesis of Blastocystis hominis by intramuscular injection of the organism into experimental mice. A total of 27 naïve BALB/c mice aged 6–8 weeks were injected in the leg muscle with axenic culture isolate B of B. hominis. Histological examination at different times revealed that B. hominis could produce a severe inflammatory reaction and myonecrosis. Most changes were observed at 6 h after injection and for up to 2–3 days. By 2 weeks the muscle had regained normal histology. There was infiltration of polymorphonuclear leukocytes (PML) into the injection site, indicating that B. hominis had a strong chemoattractant activity for PML.

Survival of Blastocystis hominis clones after exposure to a cytotoxic monoclonal antibody.

Our previous studies have shown that monoclonal antibodies (MAbs) to Blastocystis hominis react mainly with carbohydrate epitopes, while 1 MAb (1D5) reacts specifically with a protein of 30.5 kDa. In the present study, 3 monoclonal antibodies (1D5, 1E7 and 4F7) were used in immunogold localization. 1E7 and 4F7 were found to react primarily with the surface coat, while 1D5 was plasma membrane-specific. In the presence of complement, only 1D5 exhibited a cytotoxic effect on B. hominis whereas 1E7 and 4F7 did not, suggesting that the surface coat of B. hominis could serve as an immunological barrier against host antibodies. Using a recently described agar plating method, only 1D5 exhibited significant (P < 0.01) complement-independent cytotoxicity to B. hominis, inhibiting colony growth at low concentrations. Parasites that had been exposed to 1D5 were morphologically smaller than those that were not exposed to this MAb. Colonies that grew in the presence of 1D5 were isolated and grown in liquid medium containing increasing amounts of the cytotoxic MAb. Two clones that grew well in liquid medium containing 1D5 were also able to develop into colonies in soft agar. This study has shown that the 30.5 kDa protein found on the plasma membrane of B. hominis is a functionally important protein and that not all cells within a certain population would be susceptible to the cytotoxic effects of 1D5. These findings suggest that a heterogenous population exists in continuously maintained cultures of B. hominis.

Colony formation of Blastocystis hominis in soft agar.

Abstract This is the first description of a method for growing axenized Blastocystis hominis as colonies in petri dishes containing soft agar. Blastocystis cells cultured in two types of agar appeared to show different colonial morphologies as well as differing colony yields. Microscopic examination of the colonies revealed many amoeboid and giant cells. Many cells were also shown to possess thin filament-like structures that appeared to stretch across the central vacuole.