Fine Structure of Schizonts and Merozoites of Eimeria nieschulzi*

SYNOPSIS. Schizonts of E. nieschulzi lie in a vacuole within the host cell. After nuclear division the cell membrane invaginates forming merozoites. Differentiation of the pellicle and other organelles occurs while merozoites are still attached to the schizont cytoplasm. Merozoites have a pellicle thickened at the anterior end to form a polar ring. Radiating posteriorly from the ring, directly beneath the pellicle, are about 25 microtubules. Within the polar ring is a dense conoid. Extending posteriorly from within the conoid is a paired organelle. The paired organelle varies in size and shape in each generation of merozoites. Numerous toxonemes occupy the anterior half of the merozoites. Two paranuclear bodies are present in 1st generation merozoites. One or 2 granular bodies were seen in the anterior end of 2nd generation merozoites. In 3rd generation merozoites 6 or more granular bodies were seen anterior to the nucleus. Each merozoite has a single nucleus containing diffuse chromatin material. Elongate mitochondria and glycogen granules are present. The vacuole surrounding mature merozoites contains residual cytoplasm of the schizont and some granular material. Microvilli project into the vacuole from the host cell membrane.     

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts.  Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies.       

Cryptosporidium parvum merozoites share neutralization-sensitive epitopes with sporozoites

Sporozoites and merozoites are stages in the life cycle of Cryptosporidium parvum that can cyclically infect intestinal cells, causing persistent infection and severe diarrhea in immunodeficient patients. Infection by sporozoites can be neutralized by surface-reactive mAb. We show that merozoite infectivity can also be neutralized by surface-reactive mAb. To do this, viable C. parvum merozoites were isolated by differential and isopycnic. centrifugation, and distinguished from sporozoites by transmission electron microscopy. Differential reactivity with a panel of seven mAb was used to determine the amount of sporozoite contamination in isolated merozoite preparations. The isolated merozoites were distinguished from sporozoites (p less than 0.0001) by four sporozoite-specific mAb (16.332, 16.502, 17.25, and 18.357) in an indirect immunofluorescence assay. Three mAb (16.29, 17.41, and 18.44) consistently reacted with both merozoites and sporozoites. Isolated merozoites were infectious for neonatal mice when administered by intraintestinal injection. Infectivity for mice was significantly neutralized (p less than 0.05) when 1 to 2 x 10(5) merozoites were incubated with sporozoite-neutralizing mAb 17.41 or 18.44, before inoculation. Merozoites incubated with an isotype control mAb remained infectious for neonatal mice. We conclude that C. parvum merozoites share neutralization-sensitive epitopes with sporozoites.     

Final stage of maturation of the erythrocytic schizonts of rodent Plasmodium in the lungs

Schizonts of all rodent Plasmodium studied (Plasmodium yoelii, P. chabaudi, P. vinckei) show a characteristic morphology when they are completely mature: rounded or slightly elongate merozoites, completely detached from the pigment mass. At this stage, they are localized principally in the spleen and the lungs but, in impression smears of these organs they show two different aspects. In the spleen, schizonts are either inside the host erythrocyte or extraglobular but still close to a pigment mass; free merozoites are rare. In the lungs, on the contrary, merozoites are often free and dispersed; electron microscopy showed them to lie against the endothelium. Work by physiologists has shown the blood circulation in the alveoli to be much slowed down. Free merozoites, lined against the endothelium of relatively rigid capillaries, are in the best possible conditions to make contact with the intact red blood cells. Lungs appear to be the privileged site for the invasion of erythrocytes by the merozoites.     

Preparation and purification of merozoites of Eimeria tenella.

Second-generation merozoites of Eimeria tenella were obtained from both infected cecal tissue and infected chorioallantoic membranes of embryonated eggs. The merozoites were harvested from the tissue by incubation with hyaluronidase, yielding approximately 4 times 10(7) merozoites per cecum and 3 times 10(6) merozoites per chorioallantoic membrane. Subsequent purification of the merozoites by density centrifugation and glass bead filtration resulted in a 54% overall yield and a final preparation of approximately 95% purity. The viability of such preparations was established by inoculation of the merozoites to the ceca of chickens, resulting in oocyst production by 48 hr. This purification procedure allows for a rapid preparation of E. tenella during its second asexual stage in sufficient quantity and purity for biochemical study.     

Further Studies on in vitro Development of Eimeria bovis and Attempts to Obtain Second-Generation Schizonts*

SYNOPSIS. Eimeria bovis merozoites occurred in tissue culture medium removed from Leighton tube cultures of embryonic bovine tracheal cells beginning 12-14 days after inoculation with 270,000-369,000 sporozoites per tube. The number of merozoites produced in these cultures increased daily until a peak was reached 18-21 days after inoculation. In 3 experiments an average of 2.0–15.6 million merozoites per tube was produced during the 20-day observation period. When such merozoites were frozen in liquid nitrogen and stored 26–42 days, some were motile upon thawing. These merozoites as well as others freshly obtained from cell cultures and from calves were inoculated into 11 different types of cultured mammalian cells including primary, cell line and established cell line cultures. Some merozoites were exposed to substances normally found in the lumen of the gut, before or at the time of inoculation. Altho small numbers of intracellular merozoites were found, no further development was observed. Gametocytes were observed in the cecum of a calf 4 days after merozoites from cell cultures were introduced into a ligated cecum of the calf.     

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.     

Development of Eimeria meleagrimitis Tyzzer from sporozoites and merozoites in turkey kidney cell cultures

Sporozoites and 1st-, 2nd-, and 3rd-generation merozoites of Eimeria meleagrimitis were inoculated into primary cultures of turkey kidney cells. In vitro-excysted sporozoites developed into mature macrogamonts in 8 days; in vivo-excysted sporozoites developed into 2nd- or 3rd-generation schizonts within 5 to 7 days. First-generation merozoites obtained from infected turkeys produced mature 2nd-generation schizonts within 24 h. Second-generation merozoites from turkeys produced mature macrogamonts and oocysts within 72 h, whereas 3rd-generation merozoites produced these stages within 48 h. The oocysts that developed from 3rd-generation merozoites sporulated at 25 C and were infective for turkeys. The timing of the early stages and the intervals between schizogonic generations in cultures were comparable with those in turkeys. Morphologic parameters, however, indicated that some differences existed between in vitro and in vivo development. Second- and 3rd-generation schizonts and gamonts that developed after inoculation of cultures with merozoites were similar to stages in turkeys. Oocysts, however, were significantly smaller (P less than 0.05) in cultures. All stages that developed after inoculation of cultures with sporozoites were smaller (P less than 0.05) than their in vivo counter parts.     

Timing niches of 3 species of Plasmodium coexisting in a rodent in Central Africa

Freeze-thawing of blood infected with malaria parasites is a technique which brings about the destruction of all stages except the merozoites and makes possible investigations on the behaviour of these merozoites and the schizogonic rhythm of each species. Merozoites of Plasmodium y. yoelii remain in the blood during the 24 hrs. following inoculation; it is concluded that their penetration in the erythrocytes occurs gradually during this time. Synchronism is poor. Merozoites of P. vinckei petteri penetrate rapidly inside the erythrocytes independently of the time of inoculation. Infection is therefore synchronous and does not follow the circadian rhythm of the host. Penetration of merozoites of P. c. chabaudi is predominant at midnight when rodents are maintained with a normal circadian rhythm (light from 8 am to 8 pm) and predominant at noon when the rhythm of the host is inverted (light from 8 pm to 8 am). Infection is therefore synchronous and follows the host rhythm. The three species of plasmodia coexisting in Thamnomys rutilans from CAR show the same periodicity of 24 hrs. but, because of differences in the biology of the merozoites, they occupy three distinct niches. These notions have great practical implications in chronotherapy, as many data lead to the idea that merozoites are drug resistant.     

Protein p126: a parasitophorous vacuole antigen associated with the release of Plasmodium falciparum merozoites

The p126 protein is synthesized by P. falciparum between the 32nd and the 36th hour of the erythrocytic cycle, and is localized in the parasitophorous vacuole. It is processed when schizonts rupture and the major fragments (50, 47 and 18 kDa), which are released into culture supernatant, have been characterized using monoclonal antibodies. The 47 kDa fragment has been mapped at the N-terminus of the molecule. The portion of the protein p126 gene coding for this fragment contains 3 introns and is characterized by a sequence coding for 6 repeats of 8 aminoacids and by repeats of TCA/T-AGT coding for a polyserine sequence of 37 serines in a row for the FCR-3 strain. The 50 kDa fragment is also found in culture supernatant when merozoites are released from mature schizonts. The incubation of mature schizonts with leupeptin inhibits the release of merozoites and, in this case, a 56 kDa intermediate product is found. In those conditions, merozoites were observed free in the erythrocyte cytoplasm, the membrane of the parasitophorous vacuole being destroyed. The 50 kDa fragment can be obtained from the 56 kDa fragment by treatment with trypsin (a protease inhibited by leupeptin). Our results suggest that the processing of the 56 kDa fragment: 1) is protease-dependent, and could depend on a trypsin-like activity; 2) cannot occur after the release of merozoites because of the protease inhibitors contained in the serum; 3) does not occur before the release of merozoites, since no processed products of the protein p126 are observed in unruptured schizonts.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Plasmodium fallax: the invasion of host cells by exoerythrocytic merozoites in tissue culture

Exoerythrocytic merozoites of Plasmodium fallax in tissue culture have been observed and photographed in time-lapse cinemicrography as they invaded new host cells. Entry into the host cells was rapid and frequently several merozoites invaded the cells in rapid succession at or near the same site. The invasion proceeded at the broad (posterior) area of the merozoites, away from a filamentous process that usually held clusters of merozoites together in rosette form.     

Etude Ultrastructurale des Mérozoites et de la Schizogonie des Coccidies (Eimeriina): Eimeria magna (Perard 1925) de I'Intestin des Lapins et E. tenella (Railliet et Lucet, 1891) des Coecums des Poulets

SYNOPSIS. An electron microscopic study is made of merozoites and schizogony of Eimeria magna and Eimeria tenella from rabbits and chickens infected 5 days before fixation.
The merozoite outer layer is formed by a unit membrane lined by a dense osmiophilic layer. A micropyle is present. The apical complex of the cell is constituted by a conoid surmounted by 2 rings and surrounded by another from which about 26 subpellicular, tubular fibrils start. Two "rhoptries" (= toxonemes) go thru the conoid to the apex of cell. Rare sarconemes (= convoluted tubes) are disseminated in the anterior part of merozoites. A nucleus with nucleolus, Golgi apparatus, mitochondria, endoplasmic reticulum, lipid globules and glucidic grains were observed.
Schizogony starts by the formation of a multinucleated schizont which has a centriolar structure. The new merozoites appear as evaginations of the schizont's membrane. Cellular organelles (conoid, rhoptries, micropyle, sarconemes) differentiate and the nuclei enter the diverticula of the schizont. Then the development of merozoites proceeds by "external budding".
The ultrastructural similarities between the merozoites of Eimeria and the endodyocytes of Toxoplasmea, appear to us to be extremely interesting and indicate a close relationship between the Toxoplasmea and the Coccidia.     

Identification of Surface Proteins on Viable Plasmodium knowlesi Merozoites

Viable merozoites of Plasmodium knowlesi were isolated and the proteins that were labeled on intact merozoites by lactoperoxidase-catalyzed radioiodination were identified. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography of Triton soluble extracts of labeled merozoites demonstrated eight major bands ranging in apparent molecular weight from 150,000 D to 22,000 D. Exposure of intact merozoites to trypsin (10 μg/ml) for 10 min resulted in the loss of the two highest molecular weight proteins (150,000 D and 105,000 D) and the appearance of two new bands at 70,000 D and 62,000 D. Trypsin treatment under these conditions also removed the receptor(s) for merozoite attachment to erythrocytes. Therefore, these high molecular weight proteins are candidates for the merozoite component that attaches to erythrocytes. There was no evidence that the labeled membrane components were serum or erythrocyte membrane components, two potential contaminants in the preparation. Anti-rhesus erythrocyte antibody did not precipitate labeled merozoite proteins. Furthermore, the immunoprecipitation of labeled merozoite proteins by rhesus anti-merozoite serum was not inhibited by erythrocyte ghosts.     

The ultrastructure of the intraerythrocytic stages of Babesia herpailuri-trophozoites and merozoites after treatment with imidocarb (3,3'-bis-2-imidazolin-2-yl)-carbanilide (author's transl)]

The fine structure of trophozoites and especially of merozoites of Babesia herpailuri is described before and after treatment with Imidocarb (Wellcome). The mostly piriform to oval merozoites possess an outer membrane and a supporting membrane below. The intratorium consists of a polar ring, rhoptries micronemes and the sperical body which lies beside the big nucleus and next to mitochondria. The endoplasmic reticulum and invaginations are not clearly formed. The cellular changes of Babesia herpailuri, observed one hour after drug treatment in trophozoites and six hours later in merozoites, concern the form and function of the parasite: widening of the subpellicular endoplasmic reticulum and of the perinuclear space; sporadic dilatation of the endoplasmic reticulum of the merozoites (9 fig.). Damaged membranes, dissolution of the cellular membrane, disintegration of the nuclei as are known effects of the Berenil treatment to Babesia herpailuri, are not noted results after the Imidocarb treatment. The original membrane systems of trophozoites as well as of merozoites, remain unaffected by the drug as long as investigations were carried on (24 h). The satisfying prophylactic effect of Imidocarb as well as the insignificant cellular damages on merozoites may be due to the small feeding of hemoglobin.     

The gamma interferon knockout mouse model for sarcocystis neurona: comparison of infectivity of sporocysts and merozoites and routes of inoculation.

The dose-related infectivity of Sarcocystis neurona sporocysts and merozoites of 2 recent isolates of S. neurona was compared in gamma interferon knockout (KO) mice. Tenfold dilutions of sporocysts or merozoites were bioassayed in mice, cell culture, or both. All 8 mice, fed 1,000 sporocysts, developed neurological signs with demonstrable S. neurona in their tissues. Of 24 mice fed low numbers of sporocysts (100, 10, 1), 18 became ill by 4 wk postinoculation, and S. neurona was demonstrated in their brains; antibodies (S. neurona agglutination test) to S. neurona and S. neurona parasites were not found in tissues of the 6 mice that were fed sporocysts and survived for >39 days. One thousand culture-derived merozoites of these 2 isolates were pathogenic to all 8 mice inoculated subcutaneously (s.c.). Of the 24 mice inoculated s.c. with merozoites numbering 100, 10, or 1, only 3 mice had demonstrable S. neurona infection; antibodies to S. neurona were not found in the 21 mice that had no demonstrable organisms. As few as 10 merozoites were infective for cell cultures. These results demonstrate that at least 1,000 merozoites are needed to cause disease in KO mice. Sarcocystis neurona sporocysts were infective to mice by the s.c. route.     

Development of Eimeria meleagrimitis Tyzzer from Sporozoites and Merozoites in Turkey Kidney Cell Cultures*

SYNOPSIS. Sporozoites and 1st-, 2nd-, and 3rd-generation merozoites of Eimeria meleagrimitis were inoculated into primary cultures of turkey kidney cells. In vitro-excysted sporozoites developed into mature macrogamonts in 8 days; in vivo-excysted sporozoites developed into 2nd- or 3rd-generation schizonts within 5 to 7 days. First-generation merozoites obtained from infected turkeys produced mature 2nd-generation schizonts within 24 h. Second-generation merozoites from turkeys produced mature macrogamonts and oocysts within 72 h, whereas 3rd-generation merozoites produced these stages within 48 h. The oocysts that developed from 3rd-generation merozoites sporulated at 25 C and were infective for turkeys. The timing of the early stages and the intervals between schizogonic generations in cultures were comparable with those in turkeys. Morphologic parameters, however, indicated that some differences existed between in vitro and in vivo development. Second- and 3rd-generation schizonts and gamonts that developed after inoculation of cultures with merozoites were similar to stages in turkeys. Oocysts, however, were significantly smaller (P < 0.05) in cultures. All stages that developed after inoculation of cultures with sporozoites were smaller (P < 0.05) than their in vivo counter parts.     

Plasmodium falciparum: isolation and purification of spontaneously released merozoites by nylon membrane sieves

A new procedure for isolating spontaneously released merozoites from in vitro cultures of Plasmodium falciparum (FVO and FCB strains) is described. The mature forms of relatively synchronous cultures containing predominantly trophozoites and few schizonts were concentrated with Plasmagel and then incubated at 37 C, without adding fresh red blood cells, until trophozoites matured into schizonts. Merozoites which were subsequently released were harvested and freed from host red blood cell material by low-speed centrifugations and nylon membrane sieves (3- and 1.2-μm pore size). From a culture containing about 5.2 × 10⁹ mature-form parasites, a total of about 10.7 × 10⁹ merozoites were released during three consecutive harvests and about 69% of these merozoites were recovered after the isolation and purification procedures. As demonstrated by both light and electron microscopy, most merozoites were morphologically intact and the merozoite preparations were free of host cell constituents. SDS-acrylamide gel electrophoresis confirmed the absence of host cell material and also showed that merozoites had a complex protein pattern of apparent molecular weights between 225 and 15 kdaltons. Such purified merozoite preparations will be invaluable for malaria immunization studies, for identification of protective antigens of P. falciparum, and for other immunological and biochemical studies.     

Monoclonal antibody characterization of Plasmodium falciparum antigens in immune complexes formed when schizonts rupture in the presence of immune serum

When Plasmodium falciparum parasites are cultured with some immune sera, merozoites are agglutinated by antibodies to form immune clusters of merozoites and prevent their invasion into erythrocytes. Within these immune clusters of merozoites, several antigens that are normally found in the soluble fraction after detergent extraction accumulate in relatively insoluble immune complexes. From mice immunized with these immune complexes, we obtained hybridomas secreting monoclonal antibodies (mAb) that react with various immune clusters of merozoites antigens, including mAb 3D5, which recognizes a 101-kDa antigen (p101) and mAb, 5E3, which recognizes a 113-kDa antigen (p113). Both mAb reacted with antigens at the surface of schizonts, in the vacuolar space, and at the surface of merozoites before their release from schizont-infected cells. Both p101 and p113 were synthesized by mature trophozoites and young schizonts. In pulse-chase experiments, p113 was processed to 100-, 70-, 55-, and 50-kDa products. Both p101 and p113 appeared in the culture medium when schizont rupture occurred in normal culture medium but were found in immune complexes when schizont rupture occurred in the presence of immune serum. Antibodies in immune complexes, when dissociated with acid and used to probe immunoblots, reacted with affinity-purified p101 and p113. Antigens such as these, which are accessible at the parasite surface and react with antibodies present in immune serum that inhibits parasite invasion, are logical candidates to study in the search for a vaccine against the erythrocytic stages of malaria.