In vitro development of exoerythrocytic forms of Plasmodium gallinaceum sporozoites in avian macrophages

Exoerythrocytic forms of Plasmodium gallinaceum were cultured in vitro using salivary gland sporozoites extracted from experimentally infected Aedes fluviatilis mosquitoes. The host cells were macrophage precursors from chicken bone marrow. At various times after introduction of sporozoites, the cultures were stained by Giemsa or by immunofluorescence assay (IFA) using anti-sporozoite-specific monoclonal antibodies (MAb). The time to complete parasite development in vitro was 50-70 h. By 70 h, ruptured segmenters and free merozoites were visible within the cells. Inoculation of normal chickens with infected cultures induced parasitemia after a pre-patent period of 10-11 days. In vitro young exoerythrocytic forms, late schizonts that include the matured segmenters, and free merozoites shared common antigens with the sporozoites as revealed by IFA using anti-sporozoite-specific MAbs. Our data indicate that macrophages support development of P. gallinaceum sporozoites and that the circumsporozoite proteins are present until the end of the primary exoerythrocytic schizogony.     

Fine Structure of Exoerythrocytic Merozoite Formation of Plasmodium berghei in Rat Liver1

The fine structure of exoerythrocytic merogony of Plasmodium berghei was studied after perfusion-fixation of rat livers from 51 h post-inoculation onwards. Meroblast formation was effected by clefts originating from the parasite plasmalemma and by fusion of vacuoles with each other. Invaginations at the periphery resulted in labyrinthine structures providing the parasites with an enormous increase in surface area, which might facilitate exchange of metabolites. When the parasitophorous vacuole membrane collapsed, the newly formed merozoites were lying free in the hepatocytic cytoplasm, which degenerated until the merozoites were sticking together by a stroma, obviously a remnant of the host hepatocyte. Groups of merozoites, still kept together by the spongy stroma, were subsequently released in the bloodstream. At 53 h most of the developmental stages leading to the release of merozoites could be found and thereafter parasite numbers decreased while large granulomas became apparent.     

In Vitro Development of Exoerythrocytic Forms of Plasmodium Gallinaceum Sporozoites In Avian Macrophages

ABSTRACT Exoerythrocytic forms of Plasmodium gallinaceum were cultured in vitro using salivary gland sporozoites extracted from experimentally infected Aedes fluviatilis mosquitoes. the host cells were macrophage precursors from chicken bone marrow. At various times after introduction of Sporozoites, the cultures were stained by Giemsa or by immunofluorescence assay (IFA) using anti-sporozoite-specific monoclonal antibodies (MAb). the time to complete parasite development in vitro was 50-70 h. By 70 h, ruptured segmenters and free merozoites were visible within the cells. Inoculation of normal chickens with infected cultures induced parasitemia after a pre-patent period of 10-11 days. In vitro young exoerythrocytic forms, late schizonts that include the matured segmenters, and free merozoites shared common antigens with the sporozoites as revealed by IFA using anti-sporozoite-specific MAbs. Our data indicate that macrophages support development of P. gallinaceum sporozoites and that the circumsporozoite proteins are present until Ac end of the primary exoerythrocytic schizogony.     

Fine structure of exoerythrocytic merozoite formation of Plasmodium berghei in rat liver

The fine structure of exoerythrocytic merogony of Plasmodium berghei was studied after perfusion-fixation of rat livers from 51 h post-inoculation onwards. Meroblast formation was effected by clefts originating from the parasite plasmalemma and by fusion of vacuoles with each other. Invaginations at the periphery resulted in labyrinthine structures providing the parasites with an enormous increase in surface area, which might facilitate exchange of metabolites. When the parasitophorous vacuole membrane collapsed, the newly formed merozoites were lying free in the hepatocytic cytoplasm, which degenerated until the merozoites were sticking together by a stroma, obviously a remnant of the host hepatocyte. Groups of merozoites, still kept together by the spongy stroma, were subsequently released in the bloodstream. At 53 h most of the developmental stages leading to the release of merozoites could be found and thereafter parasite numbers decreased while large granulomas became apparent.     

Comparative studies of antigens of erythrocytic and exoerythrocytic merozoites of Plasmodium lophurae: characterization of a surface glycoprotein from exoerythrocytic merozoites

Rabbits were immunized with merozoite-enriched preparations of erythrocytic and exoerythrocytic Plasmodium lophurae. The antisera were used to compare antigens of the two types of merozoites. The indirect immunofluorescent antibody test showed the presence of common antigens. The growth of exoerythrocytic parasites was inhibited by the homologous antiserum and to a lesser extent by the antiserum prepared against erythrocytic forms. Cultures of exoerythrocytic parasites as well as their normal host cells were labeled metabolically with 35S-methionine, tritiated proline and glucosamine. Nonidet P-40 extracts of labeled merozoite-enriched preparations, infected cells, and normal cells were immunoprecipitated with the two types of antisera and the immunoprecipitates were analyzed on polyacrylamide gels. The results showed that erythrocytic and exoerythrocytic merozoites have several common proteins. A major difference was a glycoprotein with an approximate molecular weight of 110,000 daltons. This glycoprotein was associated with the surface of exoerythrocytic merozoites and was not recognized by antibodies prepared against erythrocytic forms.     

Hepatocyte CD81 is required for Plasmodium falciparum and Plasmodium yoelii sporozoite infectivity

Plasmodium sporozoites are transmitted through the bite of infected mosquitoes and first invade the liver of the mammalian host, as an obligatory step of the life cycle of the malaria parasite. Within hepatocytes, Plasmodium sporozoites reside in a membrane-bound vacuole, where they differentiate into exoerythrocytic forms and merozoites that subsequently infect erythrocytes and cause the malaria disease. Plasmodium sporozoite targeting to the liver is mediated by the specific binding of major sporozoite surface proteins, the circumsporozoite protein and the thrombospondin-related anonymous protein, to glycosaminoglycans on the hepatocyte surface. Still, the molecular mechanisms underlying sporozoite entry and differentiation within hepatocytes are largely unknown. Here we show that the tetraspanin CD81, a putative receptor for hepatitis C virus, is required on hepatocytes for human Plasmodium falciparum and rodent Plasmodium yoelii sporozoite infectivity. P. yoelii sporozoites fail to infect CD81-deficient mouse hepatocytes, in vivo and in vitro, and antibodies against mouse and human CD81 inhibit in vitro the hepatic development of P. yoelii and P. falciparum, respectively. We further demonstrate that the requirement for CD81 is linked to sporozoite entry into hepatocytes by formation of a parasitophorous vacuole, which is essential for parasite differentiation into exoerythrocytic forms.     

Plasmodium vivax: exoerythrocytic schizonts recognized by monoclonal antibodies against blood-stage schizonts

Exoerythrocytic parasites of Plasmodium vivax grown in human hepatoma cells in vitro were probed with monoclonal antibodies raised against other stages of P. vivax. Monoclonal antibodies specific for four independent antigens on blood-stage merozoites all reacted with exoerythrocytic schizonts and merozoites by immunostaining. The characteristic staining pattern of each monoclonal antibody was similar on both blood- and exoerythrocytic-stage parasites and appeared only in mature schizont segmenters. In contrast, a monoclonal antibody specific for the caveolar-vesicle complex of the infected host cell membrane and a second monoclonal antibody reacting with an unknown internal antigen did not appear to react with exoerythrocytic parasites. We confirm prior reports that monoclonal antibodies against the sporozoite immunodominant repeat antigen react with all exoerythrocytic-stage parasites, but note that as the exoerythrocytic parasite matures the immunostaining is concentrated in plaques reminiscent of germinal centers and apparently distinct from mature merozoites. These results indicate that mature merozoites from either exoerythrocytic or blood-stage parasites are antigenically very similar, but that stage-specific antigens may be found in specialized structures present only in a specific host cell type.     

Elongate and Round Gametocytes of Leucocytozoon simondi (Mathis & Léger) in Ducks Inoculated with Megaloschizonts

SYNOPSIS. Single megaloschizonts give rise to elongate and round gametocytes, the former outnumbering the latter. Male and female elongate gametocytes develop from merozoites of a single megaloschizont. Elongate gametocytes were seen 2–7 days and round gametocytes 6–11 days after megaloschizonts had been inoculated into ducklings. Experimental evidence indicates that merozoites of megaloschizonts invade blood cells and develop into elongate gametocytes. Other merozoites infect tissue cells and develop into secondary exoerythrocytic schizonts which give rise to round gametocytes. Relapse in Leucocytozoon simondi infections is discussed in relation to megaloschizont-induced exoerythrocytic schizogony.     

The Morphology and Behavior of Living Exoerythrocytic Stages of Plasmodium gallinaceum and P. fallax and Their Host Cells

The morphology and behavior of living exoerythrocytic stages of Plasmodium gallinaceum and P. fallax were studied by the use of tissue cultures, phase contrast microscopy, and time-lapse cinephotomicrography. The morphology of exoerythrocytic stages of these two species was essentially that previously observed in fixed, stained material, with the following exceptions: (1) the presence of a filament on one end of the merozoite, (2) the absence of clefts in the cytoplasm of the large schizonts, and (3) the absence of a vacuole-like space around the parasite. The following behavior was observed either directly or in time-lapse sequences: (1) emergence of merozoites from mature schizonts, (2) progressive motility of free merozoites, (3) entry of merozoites, both actively and passively, into host cells, (4) nuclear division in the parasite, (5) the various stages of schizogony, including final production of merozoites, (6) massive infection of host cells, and (7) phagocytosis of merozoites and attempted phagocytosis of mature schizonts by macrophages. Exoerythrocytic stages of P. fallax differed from those of P. gallinaceum in that the merozoites of the former were (1) somewhat more curved in shape and (2) present in fewer numbers in mature schizonts. The use of tissue culture, phase contrast microscopy, and time-lapse cinephotomicrography promises to solve many of the remaining problems concerning exoerythrocytic stages of malarial parasites and their interrelationships with host cells.     

A circumsporozoite-like protein is present in micronemes of mature blood stages of malaria parasites

We demonstrate for the first time the presence of a circumsporozoite (CS)-like protein in invasive blood stages of malaria parasites. Immunogold electron microscopy using antisporozoite monoclonal antibodies localized these antigens in the micronemes of merozoites. Western immunoblot and two-dimensional gel electrophoresis of mature blood stage extracts of Plasmodium falciparum, P. berghei, P. cynomolgi, and P. brasilianum identified polypeptides having the same apparent molecular mass and isoelectric points as the corresponding sporozoite (CS) proteins. The CS-like protein of merozoites is present in relatively minor amounts, compared to the CS protein of sporozoites. Mice with long-term P. berghei blood-induced infections develop antibodies which react with sporozoites.     

The Fine Structure of Differentiating Merozoites of Haemoproteus columbae Kruse*

SYNOPSIS. The first sign of merozoite formation in schizonts of Haemoproteus columbae is the accumulation of dense material at intervals beneath the plasma membrane of the schizont. The schizont's membrane then invaginates in deep furrows cleaving the parasite into pseudo-cytomeres. thereby increasing the area of membrane available for differentiation. Signs of differentiation appear under this membrane as soon as it is formed. Rhoptries and polar rings develop in the region of the dense accumulations, the cytoplasm containing these structures begins to elevate, and each evagination differentiates into a merozoite. When the merozoite is half-formed, the cytostome appears, then dense bodies at the apex of the organism, and finally a spherical body intimately associated with a mitochondrion. These merozoites of Haemoproteus are assumed to be the forms that penetrate erythrocytes and become gametocytes. They contain the same organelles as merozoites of Plasmodium. However, the merozoites of Haemoproteus are oval like the erythrocytic merozoites of Plasmodium rather than elongate like the exoerythrocytic merozoites. This body shape may be a generic characteristic or it may indicate a structural difference between exoerythrocytic merozoites and merozoites that infect erythrocytes. When the merozoites of Plasmodium, Haemoproteus and Leucocytozoon are compared, the first 2 genera appear closely related, but Leucocytozoon seems very different. Perhaps it should not be included within the Haemoproteidae.     

Immunity to exoerythrocytic forms of malaria. II. Passive transfer of immunity to exoerythrocytic forms

The passive transfer of heat-inactivated or nonheat-inactivated convalescent serum, from turkeys inoculated with Plasmodium fallax exoerythrocytic forms and treated with chloroquine to suppress the development of erythrocytic forms and the development of immunity to them, delayed the exoerythrocytic-form infection in turkeys inoculated intravenously with exoerythrocytic forms. The degree of exoerythrocyticform parasitization in cerebral tissue was significantly less in the experimental groups than the degree of parasitization in control groups, and the experimental birds continued gaining weight for a longer period than the control birds. The passive transfer of immune serum had an effect on the course of the exoerythrocytic-form infection equivalent to killing 90% of the exoerythrocytic-form inoculum. The immunity to exoerythrocytic forms is form-specific, since the infected, chloroquine-treated, serum donors were just as susceptible to an erythrocytic-form challenge infection as normal turkeys.In vitro studies demonstrated that heat-inactivated serum from turkeys immune to exoerythrocytic-form infections caused a precipitate to form at the small end of exoerythrocytic merozoites. This precipitate was not observed on merozoites mixed with control serum.     

Plasmodium fallax: phagocytosis of exoerythrocytic stages in tissue culture

Wandering phagocytes in tissue cultures were attracted to the exoerythrocytic stages, both intracellular and extracellular, of Plasmodium fallax. They phagocytized free merozoites or schizonts that had been freed from host cells. They attempted to phagocytize large intracellular parasites.     

FINE STRUCTURE OF THE ASEXUAL STAGES OF PLASMODIUM ELONGATUM

Plasmodium elongatum, an avian malarial parasite, differs from other such parasites by infecting both the circulating red blood cells and the hematopoietic cells. The exoerythrocytic development of P. elongatum occurs mainly in these red cell precursors. The fine structure of the asexual stages of P. elongatum has been studied in the bone marrow and peripheral blood of canaries and compared with that of the asexual stages of other avian malarial parasites. With minor differences, the merozoites of P. elongatum possess the same organelles as those in the exoerythrocytic merozoites of P. fallax and the erythrocytic stages of P. cathemerium, P. lophurae, P. fallax, and P. gallinaceum. The developmental sequence is also essentially similar to that of other avian malarial parasites, in that upon entry into a new host cell, the dedifferentiation, growth, and redifferentiation phases take place. However, we have found some important differences in the feeding mechanism of P. elongatum. The cytostome is involved in the ingestion of host cell cytoplasm in both exoerythrocytic and erythrocytic stages, in contrast to P. fallax, in which the cytostome is inactive in the exoerythrocytic stages. In P. elongatum, host cell cytoplasm is ingested through the cytostome, and "boluses" are formed and incorporated into a large digestive vacuole. Subsequently, the digestion of the boluses takes place in this digestive vacuole. Thus, in regard to the function of the cytostome, the exoerythrocytic stages of P. elongatum appear to be closely related to the erythrocytic stage which has a feeding mechanism similar to that of the erythrocytic stage of other avian malarial parasites.     

Exoerythrocytic Development of Plasmodium Gallinaceum Sporozoites In A Chicken Fibroblast Cell Line and Inhibition of the Cell Invasion By Specific Anti-Sporozoite Monoclonal Antibodies

ABSTRACT. Cultivation of the Plasmodium gallinaceum exoerythrocytic forms from sporozoites was attempted in three diferent cell lines: HEPG2-A16 (from a human hepatoma), VERO (monkey kidney epithelial cells) and SL-29 (chicken embryo fibroblast cells). the sporozoites in vaded all three cell types but their development into exoerythrocytic forms ocurred only in the SL-29 cells. In the presence of specific monoclonal antibodies against the major circumsporozoite protein, there were varying degrees of inhibition of parasite invasion of the SL-29 cells. of seven monoclonal antibodies tested, two completely inhibited cell invasion at high concentrations and caused intense inhibition at concentrations as low as 2.5 μg/ml, four caused intense inhibition at these various concentrations, and one had no effect on sporozoite invasion.     

The influence of cell type and culture medium on the in vitro cultivation of exoerythrocytic stages of Plasmodium berghei

Plasmodium berghei sporozoites successfully entered and developed into exoerythrocytic schizonts in a variety of cell types cultured in vitro, but segmentation and release of merozoites was only observed in human embryonic lung cells. Exoerythrocytic development was generally not influenced by the culture medium, and NCTC-135 was used routinely. In vitro infectivity of P. berghei sporozoites was unaffected by the serum type used for isolation.     

约氏疟原虫红外期成熟裂殖体的超微结构研究

用细胞色素氧化酶组织化学方法处理感染了约工疟原虫子孢子的大鼠肝脏,通过透射电镜研究红外期裂殖体的超微结构。在接种子孢子后４８小时的标本中发现一成熟裂殖体,外周仍由一寄生虫质膜包裹,膜下有许多小泡,粗面内质肉、圆形或蚕豆形具明显嵴的线粒体,以及大量成熟裂殖子。     

The cultivation of the exoerythrocytic stages ofPlasmodium berghei from sporozoites

Summary Cultures of embryonic rat brain and liver, and embryonic turkey brain were inoculated with sporozoites ofPlasmodium berghei. Sporozoites succeeded in establishing exoerythrocytic infections in approximately 10% of the cultures. The exoerythrocytic parasites developed to a late schizont stage with some showing early segmentation although free merozoites were not observed. The morphology and rate of development of the exoerythrocytic parasites in culture appear similar to that seen in vivo. This work was supported by ONR Contract No. N00014-76-C-1132 and Naval Medical Research and Development Command, Research Work Unit No. M0095PN.002.5058. the opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. The experiments reported herein were conducted according to the principles set forth in theGuide for the Care and Use of Laboratory Animals, Institute of Laboratory Resources, National Research Council, DHEW, Pub. No. (NIH) 74-23.     

Immunoelectron microscopic localization of circumsporozoite antigen in the differentiating exoerythrocytic trophozoite of Plasmodium berghei

The distribution of the circumsporozoite (CS) antigens in the 24 hour exoerythrocytic trophozoite of P. berghei was studied using Lowicryl immunogold electron microscopy. These antigens were present on the plasmalemma of the parasite, in disrupted areas of the host cell cytoplasm adjacent to the trophozoite and around inclusions of the host cell cytoplasm. There was evidence of a redistribution of the CS antigens away from the pellicular region of the sporozoite.     

Ultrastructure of the pellicular complex of Plasmodium fallax

The exoerythrocytic merozoites of Plasmodium fallax grown in a tissue-culture system have been investigated by negative staining and thin-sectioning techniques, and the respective results have been compared. Negative staining provided additional information, corroborated findings obtained with thin sectioning, and contributed particularly to the study of the pellicular complex of the merozoites which has been demonstrated as being composed of three layers: a thin outer membrane, a thick interrupted inner membrane, and a partial layer of microtubules. Observations made of negatively stained parasites revealed that the thick, interrupted inner membrane in thin sections is actually a labyrinthine structure and covers the entire surface of the merozoite, except at the regions of the conoid and the cytostome. The microtubules which radiate from the conoid to the posterior end demonstrated a transverse periodicity and filamental subunits parallel to the axis of the microtubule. The detailed structure of the conoid and the cytostome is also described.