Structure and invasive behaviour of Plasmodium knowlesi merozoites in vitro.

The structure and invasive behaviour of extracellular erythrocytic merozoites prepared by a cell sieving method have been studied with the electron microscope. Free merozoites contain organelles similar to those described in late schizonts of Plasmodium knowlesi. Their surface is lined by a coat of short filaments. On mixing with fresh red cells, merozoites at first adhere, then cause the red cell surface to invaginate rapidly, often with the formation of narrow membranous channels in the red cell interior. As the merozoite enters the invagination it forms an attachment by its cell coat to the rim of the pit, and finally leaves this coat behind as it is enclosed in a red cell vacuole. Dense, rounded intracellular bodies then move to the merozoite periphery, and apparently rupture to cause further localized invagination of the red cell vacuole. The merozoite finally loses its rhoptries, the pellicle is reduced to a single membrane and the parasite becomes a trophozoite. Invasion is complete by 1 min after adhesion, and the trophozoite is formed by 10 min.     

Ultrastructure of Cryptosporidium wrairi from the Guinea Pig

SYNOPSIS. The ultrastructure of the known tissue stages of Cryptosporidium wrairi Vetterling, Jervis, Merrill, and Sprinz, 1971 parasitizing the ileum of guinea pigs is described. Young trophozoites are surrounded by 4 unit membranes, the outer 2 of host origin, the inner 2 the pellicle of the parasite. Each trophozoite contains a vesicular nucleus with a large nucleolus. Its cytoplasm contains ribosomes, but eventually fills with cisternae of the rough endoplasmic reticulum. As the trophozoite matures the area of attachment of the parasite to the host cell becomes vacuolated, with vertical membranous folds. It is apparent that the parasite acquires nourishment from the host cell thru this area of attachment. As schizonts develop, (a) multiple nuclei appear, (b) the endoplasmic reticulum enlarges, (c) the attachment zone increases in area, (d) large vacuoles, which develop as endocytotic vesicles in the attachment area, are found in the cytoplasm and (e) the inner unit membrane of the parasite pellicle is resorbed around the sides of the developing schizont. Following nuclear division, merozoites develop from the schizont by budding. Merozoites have an ultrastructure similar to that described for other coccidia except that no mitochondria, micropores, or subpellicular tubules were observed. Merozoites penetrate the epithelial cell causing invagination of the microvillar membrane and lysing it. No unit membrane is formed between the parasite and the host cell. However, the cell produces one or 2 dense bands adjacent to the parasite attachment area. The macrogamete contains a nucleus, endoplasmic reticulum, attachment zone, and large vacuoles. It also contains a variety of granules, some of which are polysaccharide. The immature microgametocyte contains multiple compact nuclei. No mature microgametocytes or zygotes were found.     

THE FINE STRUCTURE OF THE EXOERYTHROCYTIC STAGES OF PLASMODIUM FALLAX

The fine structure of the exoerythrocytic cycle of an avian malarial parasite, Plasmodium fallax, has been analyzed using preparations grown in a tissue culture system derived from embryonic turkey brain cells which were fixed in glutaraldehyde-OsO₄. The mature merozoite, an elongated cell 3- to 4-µ long and 1- to 2-µ wide, is ensheathed in a complex double-layered pellicle. The anterior end consists of a conoid, from which emanate two lobed paired organelles and several closely associated dense bodies. A nucleus is situated in the mid portion of the cell, while a single mitochondrion wrapped around a spherical body is found in the posterior end. On the pellicle of the merozoite near the nucleus a cytostomal cavity, 80 to 100 mµ in diameter, is located. Based on changes in fine structure, the subsequent sequence of development is divided into three phases: first, the dedifferentiation phase, in which the merozoite loses many complex structures, i.e. the conoid, paired organelles, dense bodies, spherical body, and the thick inner layers of the pellicle, and transforms into a trophozoite; second, the growth phase, which consists of many nuclear divisions as well as parallel increases in mitochondria, endoplasmic reticulum, and ribosomes; and third, the redifferentiation and cytoplasmic schizogony phase, in which the specialized organelles reappear as the new merozoites bud off from the mother schizont.     

An electron microscopic study of Cryptosporidium. II. The stages of gametogenesis and sporogony in Cryptosporidium parvum

The ultrastructure of stages of gametogony and sporogony of C. parvum from the intestine of experimentally infected suckling rats was studied by transmission electron microscope. Unlike merogony, in which the whole cytoplasm of the mother meront is used up for the merozoite formation, during microgametogony the large residual mass of gamonts remains in contact with the feeder organelle even after microgamete outbudding. Unlike other coccidia, during the microgametogenesis in C. parvum, the nuclear substance of the daughter nuclei is not separated into osmiophilic (containing the condensed chromatin) and achromatinic parts. The gamete outbudding in C. parvum is accompanied by evagination of the pellicle of the mother gamont whose cytoplasm displays some slit-like canals that seem to sequester the daughter nuclei with some portion of the surrounding cytoplasm. The flagella-free microgametes of C. parvum resemble somatic cells, rather than male sexual cells of other coccidia. The study of thick-walled oocysts of C. parvum made it possible to suggest that the fragile wall of the oocyst proper may be easily destroyed in the course of processing of the material to look eventually as a ghost of electron lucent substance in the parasitophorous vacuole, whereas the structures revealed on the electronograms may presumably represent the outer and inner layers of the sporocyst. If so, the suture described elsewhere in the cryptosporidial oocysts, is to be considered as belonging to the sporocyst wall rather than to the oocyst wall, i.e. likely as in other investigated coccidia. However, the question on the mode of sporozoite excystment in the thin-walled oocysts of C. parvum still remains obscure.     

Stage-dependent processing and localization of a Plasmodium falciparum protein of 130,000 molecular weight

A Plasmodium falciparum protein of 130,000 molecular weight (m.w.) has been identified, cloned in Escherichia coli, and completely sequenced (Kochan et al. 1986). The protein appeared to bind to soluble glycophorin, a host erythrocyte surface protein. In the present study, extracts of parasites from different intraerythrocytic stages were immunoblotted with antibodies, raised against a 30,000 m.w. fusion protein corresponding to the 3' end of the 130,000 m.w. protein. It was demonstrated that the protein is synthesized at the trophozoite stage, accumulates at the schizont stage, and is processed at the merozoite stage to a triplet of three polypeptides. The processed proteins are present in the culture supernatant at the time of merozoite burst from the red cell. Immunofluorescent staining of the parasite at different intracellular stages indicates that the protein is localized on the parasite at the trophozoite stage. At late trophozoite stage, it appears to be transported to the erythrocyte cytoplasm, where it is present in small vesicles or inclusions. In mature schizonts the protein accumulates around the plasma membrane of the erythrocyte. At the segmenter stage, just prior to merozoite release, it appears also to surround the intracellular merozoite, as well as the erythrocyte plasma membrane. The soluble 130,000 m.w. protein binds to erythrocytes but binds significantly greater to erythrocyte membranes, suggesting it binds to an internal domain of glycophorin rather than the domain exposed on the surface. The 130,000 m.w. protein is present in 11 different geographic isolates of P. falciparum from diverse geographic origins. Its molecular weight is similar in all isolates.     

Secretion of Plasmodium falciparum rhoptry protein into the plasma membrane of host erythrocytes

The rhoptry is an organelle of the malarial merozoite which has been suggested to play a role in parasite invasion of its host cell, the erythrocyte. A monoclonal antibody selected for reactivity with this organelle identifies a parasite synthesized protein of 110 kD. From biosynthetic labeling experiments it was demonstrated that the protein is synthesized midway through the erythrocytic cycle (the trophozoite stage) but immunofluorescence indicates the protein is not localized in the organelle until the final stage (segmenter stage) of intraerythrocytic development. Immunoelectron microscopy shows that the protein is localized in the matrix of the rhoptry organelle and on membranous whorls secreted from the merozoite. mAb recognition of the protein is dithiothreitol (DTT) labile, indicating that the conformation of the epitope is dependent on a disulfide linkage. During erythrocyte reinvasion by the extracellular merozoite, immunofluorescence shows the rhoptry protein discharging from the merozoite and spreading around the surface of the erythrocyte. The protein is located in the plasma membrane of the newly invaded erythrocyte. These studies suggest that the 110-kD rhoptry protein is inserted into the membrane of the host erythrocyte during merozoite invasion.     

Plasmodium lophurae: the ultrastructure of the exoerythrocytic stages

The fine structure of the exoerythrocytic stages of Plasmodium lophurae was studied. in specimens grown in tissue cultures of avian cells. Specimens were prepared for sectioning by a method which minimizes disturbance and permits precise selection and orientation specimens.Plasmodium lophurae is similar in many aspects to P. fallax. Merozoites are highly specialized and differentiated. Analysis of their ultrastructure revealed the polar complex to be a specialization of the pellicular envelope and its associated underlying microtubules. The polar rings may simply be a modification of the inner membrane of the pellicle and not discrete structures as previously reported. The electron-dense polar organelles are separated on morphological grounds into three groups: the large paired organelles and the small dense bodies which are both linked to microducts, and the transitional bodies, a third organelle being reported for the first time. Transitional bodies are without microducts, occur in fully mature merozoites and persist only for a short period. All three of these organelles appear to be related to and possibly even derived from internal membrane systems and ribosomes. The apolar end of the merozoite contains the mitochondrion and its associated spherical body. Detailed study of the latter shows it to be cylindrical.Upon entering the host cell, the parasite adds a third membrane at the interface between it and the cell. The merozoite becomes spherical and undergoes transformation into a trophozoite. During this reorganization phase, dedifferentiation occurs and is followed by a rapid growth phase. The end of the growth phase is signaled by the appearance of germinal clefts and nuclear division. The entire process of schizogony culminates in a highly synchronized formation of merozoites.Processes of the limiting membrane forming the host parasite interface were observed extending deply into the cytoplasm of the host cell and often appeared to form bridges between two or more parasites. The significance of this new observation is not yet established.     

Plasmodium falciparum-infected erythrocytes: qualitative and quantitative analyses of parasite-induced knobs by atomic force microscopy

We used the combination of an atomic force microscope and a light microscope equipped with epifluorescence to serially image Plasmodium falciparum-infected erythrocytes. This procedure allowed us to determine unambiguously the presence and developmental stage of the malaria parasite as well as the number and size of knobs in singly, doubly, and triply infected erythrocytes. Knobs are not present during the ring stage of a malaria infection but a lesion resulting from invasion by a merozoite is clearly visible on the erythrocyte surface. This lesion is visible into the late trophozoite stage of infection. Knobs begin to form during the early trophozoite stage of infection and have a single-unit structure. Our data suggest the possibility that a two-unit structure of knobs, which was reported by Aikawa et al. (1996, Exp. Parasitol. 84, 339-343) using atomic force microscopy, appears to be a double-tipped image. The number of knobs per unit of host cell surface area is directly proportional to parasite number in both early and late trophozoite stages. These results indicate that knob formation by one parasite does not influence knob formation by other parasites in a multiply infected erythrocyte. In addition, knob volume is not influenced by either parasite stage or number at the late trophozoite stage, indicating that the number of component molecules per knob is constant throughout the parasite maturation process.     

Electron microscopic observation of the invasion process of Cryptosporidium parvum in severe combined immunodeficiency mice

Cryptosporidium parvum mainly invades the intestinal epithelium and causes watery diarrhea in humans and calves. However, the invasion process has not yet been clarified. In the present study, the invasion process of C. parvum in severe combined immunodeficiency (SCID) mice was examined. Infected mice were necropsied; the ilea were double-fixed routinely and observed by scanning and transmission electron microscopy. In addition, the microvillus membrane was observed by ruthenium red staining. Scanning electron micrographs showed elongation of the microvilli at the periphery of the parasite. The microvilli were shown to be along the surface of the parasite in higher magnification. Transmission electron microscopy confirmed that the invading parasites were located among microvilli. Parasites existed in the parasitophorous vacuole formed by the microvillus membrane. The parasite pellicle attached to the host cell membrane at the bottom of the parasite, and then the pellicle and host cell membrane became unclear. Subsequently, the pellicle became complicated and formed a feeder organelle. In addition, invasion of the parasite was not observed in either a microvillus or the cytoplasm of the host cell. Therefore, C. parvum invades among microvilli, is covered with membranes derived from numerous microvilli, and develops within the host cell.     

An ultrastructural comparison of the attachment sites between Gregarina steini and Cryptosporidium muris

Early developmental stages of Gregarina steini Berndt, 1902 from the intestine of Tenebrio molitor larvae were studied by transmission electron microscopy. The formation and structure of the eugregarine attachment site were compared with comparable features found on the feeder organelle of Cryptosporidium muris Tyzzer, 1907, from the stomach of experimentally infected rodents. The similarity of the attachment strategy between both organisms was revealed. The membrane fusion site in G. steini, formed by the trophozoite plasma membrane, host cell plasma membrane and a membrane-like structure limiting the cortical zone of the epimerite, resembles the Y-shaped membrane junction between the host cell plasma membrane, the trophozoite plasma membrane and membrane surrounding the anterior vacuole in C. muris. The anterior vacuole of C. muris appears to be the precursor of the feeder organelle and its structure is very similar to the epimeritic bud and the cortical zone of G. steini trophozoites. In both investigated organisms, the apical complex disappears early during cell invasion. The possibility of the epicellular location of Cryptosporidium on the surface of host cells is discussed.     

Freeze fracture studies on the interaction between the malaria parasite and the host erythrocyte in Plasmodium knowlesi infections.

The freeze fracture technique has been used to study the internal cyto-architecture of the surface membranes of the parasite and erythrocyte in Plasmodium knowlesi infections. Six fracture faces, derived from the plasma membrane and 2 pellicular membranes, have been identified at the surface of the free merozoite. The apposed leaflets of the 2 pellicular membranes show the characteristic features of E fracture faces, a result compatible with the view that the pellicular membranes line a potential cisterna. There is evidence to suggest that there may be changes in the distribution and density of the integral proteins in the merozoite plasma membrane at invasion. Furthermore, vesicles consisting of stacked membranes occur within and around the erythrocyte invagination at invasion; it is suggested that these vesicles are released from the merozoite rhoptries. Formation of the parasitophorous vacuole is accompanied by dramatic changes in the density and distribution of intra-membraneous particles (IMP) in the vacuolar membrane. Initially there is a great reduction in particle numbers, but subsequently the particles reappear and show reversed polarity. The possible causes and implications of these changes are discussed. The intra-erythrocytic parasite synthesizes new transmembrane proteins as development proceeds, and the trophozoite and schizont stages of development are characterized by the appearance of circular, particle-free regions in the parasite plasmalemma. There is a decrease in the density of transmembrane proteins in the erythrocyte plasma membrane during parasite maturation, and the P face IMP show the characteristic features of aggregation.     

Pellicle complex ofEuglena gracilis: Characterization by disruptive treatments

Summary Euglena gracilis was treated with 4% colchicine, 0.5% -mercaptoethanol, 0.5% Triton X-100, and 8% tannic acid in attempting to characterize its pellicle complex. Colchicine had no visible effects on the microtubules of the pellicle, canal or reservoir. Colchicine in dimethyl sulfoxide disorganized the reservoir region. Colchicine was shown to enter the cell by its ability to inhibit flagellar regeneration. Mercaptoethanol destroyed most of the organelles in the cell. Only the tripartite plasmalemma of the pellicle complex remained normal. The microtubules of the cell and the protein layer underlying the plasmalemma were disrupted by mercaptoethanol treatment. Triton X-100 caused membranes of internal organelles to round, up become distended and swollen, but had no morphological effect on the plasmalemma. The ridge-groove shape of the pellicle remained intact after every treatment. Mercaptoethanol caused an indentation of the ridges over the position of a disrupted microtubule, suggesting a supporting function of the microtubules. Distension of endoplasmic reticulum by Triton X-100 revealed more clearly its association with the pellicle complex. The ability of the pellicle ofEuglena gracilis to maintain its integrity under a number of disruptive treatments was demonstrated.     

Dynamic subcellular localization of isoforms of the folate pathway enzyme serine hydroxymethyltransferase (SHMT) through the erythrocytic cycle of Plasmodium falciparum

Background

The folate pathway enzyme serine hydroxymethyltransferase (SHMT) converts serine to glycine and 5,10-methylenetetrahydrofolate and is essential for the acquisition of one-carbon units for subsequent transfer reactions. 5,10-methylenetetrahydrofolate is used by thymidylate synthase to convert dUMP to dTMP for DNA synthesis. In Plasmodium falciparum an enzymatically functional SHMT (PfSHMTc) and a related, apparently inactive isoform (PfSHMTm) are found, encoded by different genes. Here, patterns of localization of the two isoforms during the parasite erythrocytic cycle are investigated.

Methods

Polyclonal antibodies were raised to PfSHMTc and PfSHMTm, and, together with specific markers for the mitochondrion and apicoplast, were employed in quantitative confocal fluorescence microscopy of blood-stage parasites.

Results

As well as the expected cytoplasmic occupancy of PfSHMTc during all stages, localization into the mitochondrion and apicoplast occurred in a stage-specific manner. Although early trophozoites lacked visible organellar PfSHMTc, a significant percentage of parasites showed such fluorescence during the mid-to-late trophozoite and schizont stages. In the case of the mitochondrion, the majority of parasites in these stages at any given time showed no marked PfSHMTc fluorescence, suggesting that its occupancy of this organelle is of limited duration. PfSHMTm showed a distinctly more pronounced mitochondrial location through most of the erythrocytic cycle and GFP-tagging of its N-terminal region confirmed the predicted presence of a mitochondrial signal sequence. Within the apicoplast, a majority of mitotic schizonts showed a marked concentration of PfSHMTc, whose localization in this organelle was less restricted than for the mitochondrion and persisted from the late trophozoite to the post-mitotic stages. PfSHMTm showed a broadly similar distribution across the cycle, but with a distinctive punctate accumulation towards the ends of elongating apicoplasts. In very late post-mitotic schizonts, both PfSHMTc and PfSHMTm were concentrated in the central region of the parasite that becomes the residual body on erythrocyte lysis and merozoite release.

Conclusions

Both PfSHMTc and PfSHMTm show dynamic, stage-dependent localization among the different compartments of the parasite and sequence analysis suggests they may also reversibly associate with each other, a factor that may be critical to folate cofactor function, given the apparent lack of enzymic activity of PfSHMTm.     

毁灭泰泽球虫(Tyzzeria perniciosa)内生发育的超微结构研究——Ⅰ.裂殖生殖(Schizogony)

利用透射电镜对寄生于北京鸭小肠的毁灭泰泽球虫的裂殖生殖过程进行了观察。滋养体内未见多糖颗粒、脂肪体和致密体,在细胞质的被膜空泡内发现退化的微线和棒状体。在裂殖体核分裂过程中,出现典型的球虫型有丝分裂装置(如中心粒、中心锥、纺锤体)。裂殖子的发生是外瓣生方式,裂殖子在裂殖体的表面形成,并以母细胞的限制膜为外膜。     

Electron microscopic observation of the early stages of Cryptosporidium parvum asexual multiplication and development in in vitro axenic culture

The stages of Cryptosporidium parvum asexual exogenous development were investigated at high ultra-structural resolution in cell-free culture using transmission electron microscopy (TEM). Early C. parvum trophozoites were ovoid in shape, 1.07 × 1.47 μm² in size, and contained a large nucleus and adjacent Golgi complex. Dividing and mature meronts containing four to eight developing merozoites, 2.34 × 2.7 μm² in size, were observed within the first 24 h of cultivation. An obvious peculiarity was found within the merozoite pellicle, as it was composed of the outer plasma membrane with underlying middle and inner membrane complexes. Further novel findings were vacuolization of the meront's residuum and extension of its outer pellicle, as parasitophorous vacuole-like membranes were also evident. The asexual reproduction of C. parvum was consistent with the developmental pattern of both eimerian coccidia and Arthrogregarinida (formerly Neogregarinida). The unique cell-free development of C. parvum described here, along with the establishment of meronts and merozoite formation, is the first such evidence obtained from in vitro cell-free culture at the ultrastructural level.     

贝氏隐孢子虫在北京鸭体内发育的超微结构研究

贝氏隐孢子虫各期虫体均位于宿主粘膜上皮细胞的带虫空泡中。在虫体与上皮细胞接触处,虫体表膜反复折迭形成营养器。子孢子或裂殖子与粘膜上皮细胞接触后,逐步过渡为球形的滋养体;滋养体经2—3次核分裂、产生含4或8个裂殖子的两代裂殖体,裂殖体以外出芽方式产生裂殖子;裂殖子无微孔,顶端表皮形成3—4个环嵴,裂殖子进一步发育成为配子体;大配子体含有两种类型的成囊体。小配子呈楔形,无鞭毛和顶体,有一个致密的长椭圆形细胞核,小配子表膜内侧有9根膜下微管;孢子化卵囊内含四个裸露的子孢子和一个大残体。本文是有关鸭体内隐孢子虫超微结构的首次报导。     

Investigations into the fire structure of the asexual developmental stages of Frenkelia in the liver of the bank vole (author's transl)]

Bank voles (Clethrionomys glareolus) were infected by stomach tube with Frenkelia sporocysts from the faeces of buzzards (Buteo buteo). The voles were sacrificed at regular intervals and their livers examined electronmicroscopically. Seven days p.i. developmental stages of Frenkelia could be detected in liver parenchymal cells. The youngest schizonts detected are enveloped by a pellicle consisting of two membranes. This pellicle, which is in direct contact with the host cell mitochondria, shows marked invaginations which increase with the development of the schizont. A parasitophorous vacuole is not detectable. In developing schizonts numerous sections through nuclei with nucleic spindles and merozoite anlagen (dome-shaped) structures) are visible. It is not clear whether there are several nuclei or a section through one large and lobed nucleus. Within the merozoite anlagen the conoid and the subpellicular microtubules are formed first. By the prolongation of the dome-shaped structures towards the posterior pole, the nucleus and the other newly formed cell organelles are incorporated into the forming merozoite. The posterior pole of the merozoite still remains open at this stage of development. With increasing differentiation the merozoites become lancet-shaped, their apical poles bing always directed towards the periphery of the schizont. The outer membrane of the pellicle of the schizont forms the outer part of the pellicle of the merozoites by invaginating around them. At this stage of development the inner membrane of the pellicle of the schizont is no longer detectable. Thus the typical pellicle of the motile stages of sporozoaonsisting of three membranes is formed. In the centre of the merozoites which lie freely in the liver cell a residual body is present. The host cell reacts against the parasites by forming a thick border of mitochondria and distinct endoplasmic reticulum.     

An Ultrastructural Study of Eimeria iroquoina Molnar & Fernando, 1974 in Experimentally Infected Fathead Minnows (Pimephales promelas,Cyprinidae) 3. Merogony1

Fathead minnows, Pimephales promelas, raised from eggs in the laboratory, were experimentally infected with oocysts of Eimeria iroquoina from either P. promelas or the common shiner, Notropis cornutus. Within intestinal epithelial cells, trophozoites thought to be derived from the sporozoites contained a prominent electron-dense refractile body. Merozoites dedifferentiated into trophic forms by losing components of their apical complex and pellicle. The inner membrane components of the pellicle appeared discontinuous, and the micronemes became enclosed within vacuoles. Prior to merozoite formation, multinucleate meronts were limited by a single membrane. Golgi complexes were associated with the nuclei of this stage. Merozoites were formed by ectomerogony in one generation and by endomerogony in the final generation. In both forms of merogony the final nuclear division was coupled with the onset of differentiation of the merozoites and featured eccentric mitotic spindles associated with centrocones located within the nuclear envelope and with the precursors of the apical complex. A Golgi complex was closely associated with the nucleus and apical tip of the forming merozoite. Unlike other Eimeria species, the complete pellicle of the merozoites of the final asexual generation of E. iroquoina was formed within the cytoplasm of the meront, without association with the limiting membrane, thus, all pellicular components are synthesized de novo. The inner membranes of the pellicle initially appeared as longitudinal strips, each of which was associated with a pair of the 22–24 subpellicular microtubules. Mature meronts of the final asexual generation averaged 9 μm in diameter and produced 13–16 merozoites. With the exception of the internal completion of the pellicle of the final generation merozoites, the basic processes of merogony in fish Eimeria species are similar to those recorded in terrestrial hosts.     

Transformation of sporozoites of Plasmodium berghei into exoerythrocytic forms in the liver of its mammalian host

Summary Intrahepatocytic transformation in vivo of the rodent malaria sporozoite of Plasmodium berghei, into the young trophic exoerythrocytic tissue stage was studied by immunofluorescence, light- and electron microscopy. The first 20 h of intracellular life were involved entirely in dedifferentiation with limited proliferation of organelles. From about 20 h onwards nuclear division commenced, rough endoplasmic reticulum became markedly expanded, and mitochondria increased in numbers. However, remains of the sporozoite pellicle (i.e., inner membranes and subpellicular microtubules) persisted for at least 28 h, which correlates with the persisting reaction of young exoerythrocytic forms with antisporozoite antibodies. In general, the basic mechanism of transformation resembles that of the ookinete into oocyst and that of the merozoite into erythrocytic trophozoite.     

Ultrastructural study of asexual development of Cryptosporidium parvum in a human intestinal cell line.

The lack of a well-defined in vitro model of Cryptosporidium infection has severely hampered research on the biology of parasitic invasion of the host cell and on intracellular development of the parasite. In vitro infection of the differentiated human enterocyte cell line HT29.74 was studied by electron microscopy to detect changes in parasite and host cell morphology. Cryptosporidium oocysts obtained from AIDS patients were applied to a monolayer of cloned differentiated HT29.74 cells. Parasites and infected cells were evaluated by transmission electron microscopy at 20 min, 1 h, 6 h, 24 h and 7 days. Sporozoite invagination within the epithelial cell microvilli and subsequent penetration was evident at 1 h. At 6 h, the development of a dense band and feeder layer was visible. Development of the trophozoite into a schizont occurred over 24 h. Micronemes and dense granules were clearly visible within sporozoites and merozoites. Organization of vacuoles within the cytoplasm of the host cell was evident below the dense band. A sexual Cryptosporidium development in vitro was morphologically no different from initial development in vivo. In vitro infection of HT29.74 cells provides an excellent model to study parasite-host cell interaction and asexual parasite development.