Fine structure of entamoeba histolytica in culture and in intestinal mucosa
[영문]Although several investigations were reported on the ultrastructure of Entamoeba histolytica trophozoites (Osada, 1959; Miller et al., 1961; F letcher et al., 1962), no attempt has yet been made to characterize micromorphology of the trophozoite in the tissue invading phase. Recently, El-Hashimi and Pittman (1970) reported that the structural difference, between the trophozoites obtained from the human colon and those cultivated in vitro, was the presence of a fuzzy coat in the amoebae from colon.
This study was designed to observe the structural transformation of trophozoites after invasion of YS 27-strain E.histolytica to intestinal mucosa of rabbit.
The YS 27-strain was isolated from liver abscess of 72 year-old man at Severance Hospital in September 1969. The amoeba was cultured on a diphasic medium with calf serum and penicillin G added (Cho, 1968)-associated with unidentified intestinal
bacterial flora-and was subcultured every other day.
Young rabbits were inoculated intracecally following the technique described by Cho (1968). The sediments from several media of 48 hour-old amebae were pooled and spun at 500 rpm for 5 minutes. This sediment was washed twice in warmed normal saline (37℃) by low speed centrifugation. The amebae were counted using Spencer bright-line Neubauer counting chamber and were diluted with warmed normal saline for inoculation. The animals were anesthetized with ether, and the caecum was exposed by incision. the amebae were inoculated by means of 5 ml syring fitted with
a 20-gauge needle toward the blind end of the caecum from a distal margin to junction of caecum and colon. The incision was closed with #1 surgical silk. Twelve days after the inoculation, the rabbits were sacrificed by air injection to the auricular vein.
For electron microscopy, the rabbit caecum was removed and portions of the lesion were fixed with 3% glutaraldehyde in 0.1M phosphate buffer (pH 7.4) for 2 hours (4℃). They were washed and left overnight in 0.1M phosphate buffer (4℃), and post-fixed with 1% osmium tetroxide in phosphate buffer solution for 2 hours. Then they were dehydrated in an ascending series of ethanol from 60%. After embedding in Epon 812 (Luft, 1961), they were cut into sections 500A thick with glass knives using Sowall MT2 Porter Blum ultramicrotome. The sections were mounted on copper grids, stained with saturated uranyl acetate and lead citrate, and examined in a Hitachi HU-11E-1 electron microscope. Alternate sections 1μ thick were stained with basic fuchsin and examined with a light microscope.
In culture form E.histolytica, several 48-hour cultures of the parasite were spun at 500 rpm for 5 minutes and the supernate was discarded. 2 ml of 3% glutaraldehyde in 0.1M phosphate buffer (pH 7.4) was added tothe sediment. In order to increase the concentration of the fixative, 1ml-portion of the mixture of fixative was repeatedly withdrawn and replaced with flesh fixative, and fixed for 2 hours (4℃). The amebae were washed with 0.1M phosphate buffer (pH 7.4) containing 10% sucrose,
and left in the buffer solution overnight. They were post-fixed with 1% osmium tetroxide in phosphate buffer for 1 hour, and washed with 70% ethanol, then concentrated by low speed centrifugation. The ethanol was aspirated and replaced by a few drops of 1% warmed agar. After the agar solidified, it was cut into 1mm cubes, dehydrated, embedded, cut into sections, mounted, stained and examined in the manner as described in the intestinal phase.
By electron microscopy, trophozoites of E.histolytica from in vitro cultures were more or less round or oval in shape and measured 12 to 17 μ in diameter. Each ameba was bounded by a limiting unit membrane approximately 150 A in thickness. The
plasma membrane was irregular in appearance and two or three small nodular cytoplasmic process approximately 0.15x0.3μ in size were observed from the cell surface. The cytoplasm in the tip of the projections was less electron-dense than that in the central portion of the cell. The ground substance of the cytoplasm contained widely-scattered fine and coarse electron-dense granules, and some of those structures were similar to α-glycogen particles reported by Revel(1964). A number of vacuoles of different size and shape were present in the cytoplasm, and
were lined along with plasmalemma-like membrane. Vacuoles in central portion of the cell usually contained particulate matters, and those in peripheral portion were almost empty. The particulate matters probably represented ingested bacteria,
starch grains under digestion and some sort of membrane whorls. The endoplasmic reticulum was scarce and consisted of fine, thin tubules sometimes without constituent ribosomes, but sometimes surrounded by small ribosomes. It was regarded as rudimentary type. Neither mitochondria nor Goligi complex was observed. The
nucleus varied from oval to irregular in shape. The nuclear membrane was composed of a double membrane and distinct nuclear pores were seldom observed. The nuclear matrix contained many very fine or coarse electron-dense granules. Medium-sized,
electron-dense granules were aggregated beneath the nuclear membrane. Small vesicular inclusions of various sizes, some of which contained dense granules or concentric rings, were found in the peripheral region of the nucleus.
Ultrastructure of precyst-like from E.histolytica from in vitro culture were more or less round and measured approximately 12 μ in diameter. Each ameba was completely enclosed by a thin limiting unit membrane coated on the outside by a pale grey homogenous layer about 0.17 μ wide. Small electron-dense particles dotted evenly on the cytoplasmic surface of the membrane. Vacuoles present in the cytoplasm were surrounded by a double membrane, which were dissimilar to the plasma membrane, and varied greatly in size. Large vacuoles usually contained some sort of membranous matters, and small or minute vaculoes were almost empty. Short strands of endoplasmic reticulum were scattered through the cytoplasm. They usually appeared as fine smooth tubules, or sometimes lined with fine ribosome granules.
Fine and coarse electron-dense granules resembled those observed in the trophozoite from. Highly electron-dense cylindrical rods were distributed throughout the cytoplasm, and they were sometimes arranged in "rosette" patterns. The centers of the radiated rods were spherical clusters of very fine and highly electron-dense particles, and were seperated by a clear space. Moderately electron-dense filamentous structures of various length approximately 300A width were abundantly distributed through the cytoplasm. Each filament consisted of small particles
stacked along its length in a helical pattern. In some areas they were aggregated and formed a lamellae or crystalloid structure.
The lamellae consisted of 9-32 fibrils which ran parallel to one another, and the number of crystalloids varied from 33 to 50. The cystalloids were regarded as the cross-sectional profile of lamellae, and some showed the mixed pattern of longitudinal and cross-sectional profiles. These correspond to the helical
structure in ribonucleoprotein bodies-chromatoid bodies-described by Siddiqui and Rudzinska(1963) and Morgan and Uzman(1966). Nucleus was round shape approximately 4.5μ in diameter. Nuclear membrane and pore resembled to those in the trophozoite form. The background of the nucleus contained many fine or coarse electron-dense granules. The medium sized electron-dense granules were mostly pushed toward the corner of the nucleus and accumulated. Some of them were studded underneath nuclear membrane. In the accumulation of dense granules, 6-7 highly electron-dense nuclear
inclusions were present. They were irregular in size and shape, and comprised a agregation of heavy granular meterials.
Ultrastructure of amebae in intestinal mucosa were surrounded by a plenty of destroyed cellular contents of intestinal mucosa. The trophozoites were oval shape, measuring 15.5-18.0μx18.5-220.0μ in size. Each ameba was bounded by a limiting unit membrane approximately 150A in thickness, and no extra-membranous layer was observed. A large number of vacuoles of various sizes and shapes were present through the cytoplasm. The membrane usually resembled to the plasmalemma, but the membrane in some vacuole was thickened and less electron-dense than ordinary unit
membrane. In the region close to the plasma membrane, a portion of vacuole membrane was flattened and fused into the plasmalemma, and moderately electron-dense gluey masses were clung onto both inner and out surface of the membrane. It suggests the ejecting process of ingested food residuals from the vacuoles. Some vacuoles contained nuclear substances of surrounding cells in various stages of digestion. The ground substance of the cytoplasm consisted mainly of interspersed fine and coarse electron-dense granules, and short and spiral filaments dotted with granules. The latter resembled to the spiral polyribosome discribed by Lowe and Maegraith (1970b). Glycogen particles were sparse. Other electron-dense granules observed in the cytoplasm were tentatively classified into four different groups;
G1, G2, G3 and G4. G1 designated irregular shaped dense pigment body 0.5-1.0μ in width. G2 indicated highly electron-dense and spherical smaller granules measuring 0.1-0.2μ in diameter. Sometimes they were found 2-5 granules in a group. G3 was pointed to the moderately electron-dense ellipsoidal rods 0.4x0.2μ in size. G4 was the moderately electron-dense, spherical granule approximately 0.2μ in diameter. Another structure in the cytoplasm was the spherical aggregation of dense-granules
approximately 1.7μ in diameter. Endoplasmic reticulum was prominent as wavy strand with ribosomes along their length. Neither Golgi apparatus nor mitochondrium was seen in sections. Nuclei were oval and 3-3.5μx3.5μ in size. The nuclear membrane
was composed of a double membrane similar to that seen in the trophozoites from culture. There were regularly-spaced nuclear pores. The nucleoplasm showed a greater electron density than the cytoplasm. The background contained many fine or coarse and electron-dense granules. In some amoebae, the central part was more electron-dense than the other part of nucleoplasm. This was indicated as karyosome. Other electron-dense granules were aggregated beneath the nuclear membrane. Much larger round and very electron-dense bodies approximately 0.25μ in diameter were
found in the peripheral region of the nucleus.
The results were summarized as follows;
A. Trophozoite of E.histolytica in culture.
1. The trophozoites were irregular in shpae. Small cytoplasmic processes were observed on the cell surface, which were speculated as origin of pseudopodia.
2. Food vacuoles varied greatly in size and usually contained ingested bacteria, starch grains and some sorts of membraned whorl.
3. Nucleus was irregular in shape, and it contained several, slightly-dense, round vesicular inclusions beneath the nuclear membrane.
4. Some amoeba was "rounded-off", and plasmalemma was smoothly surfaced and coated on the outside by a pale grey homogenous layer. Food vacuoles were almost emptied. Electron-dense hollow rods were radiated in rosette from a central mass of a fine granules, and some were distributed in cluster irregularly in the cytoplasm. Bundles of the crystalloid aggregation were prominently observed in the cytoplasm. Nucleus was round in shape, and nuclear chromatins were densely pushed toward one
corner of the periphery of the nucleus, where highly electron-dense heavy granular inclusions were observed. This was considered as precystic trophozoite of E.histolytica.
B. Trophozoite of E.histolytica in intestinal mucosa.
1. The trophozoites were oval in shape and plasmalemma was rather smooth than the former. There was no obvious difference of endoplasm and ectoplasm.
2. Most of food vacuoles contained ingested nuclear contents of tissue cells which were digested in various stages. A considerable numbers of small empty vacuoles were also scattered at the periphery of cytoplasm. The membrane of some vacuole was thickly layered.
3. In the cytoplasm, electron-dense materials were observed in addition to fine dense granules, short filaments and rosette-like α-glycogen particles. They were grouped into four different catogories: a) irregular shaped dense pigment bodies, b) spherical smaller granules, c) moderatley electron-dense ellipsoidal rods, and d) moderately electron-dense spherical granules.
4. Nucleus was oval in shape. There were regularly spaced nuclear pores and patches of peripheral chromatin lining the nuclear membrane. Much larger round and very electron-dense bodies were found in the periphery of the nucleus.