Exoerythrocytic Schizogony Research Articles

A Plasmodium falciparum ATP-binding cassette transporter is essential for liver stage entry into schizogony

SummaryPlasmodium sporozoites invade hepatocytes and transform into liver stages within a parasitophorous vacuole (PV). The parasites then grow and replicate their genome to form exoerythrocytic merozoites that infect red blood cells. We report that the human malaria parasite Plasmodium falciparum (Pf) expresses a C-type ATP-binding cassette transporter, Pf ABCC2, which marks the transition from invasive sporozoite to intrahepatocytic early liver stage. Using a humanized mouse infection model, we show that Pf ABCC2 localizes to the parasite plasma membrane in early and mid-liver stage parasites but is not detectable in late liver stages. Pf abcc2— sporozoites invade hepatocytes, form a PV, and transform into liver stage trophozoites but cannot transition to exoerythrocytic schizogony and fail to transition to blood stage infection. Thus, Pf ABCC2 is an expression marker for early phases of parasite liver infection and plays an essential role in the successful initiation of liver stage replication.

A replication-competent late liver stage-attenuated human malaria parasite.

Whole-sporozoite vaccines engender sterilizing immunity against malaria in animal models and importantly, in humans. Gene editing allows for the removal of specific parasite genes, enabling generation of genetically attenuated parasite (GAP) strains for vaccination. Using rodent malaria parasites, we have previously shown that late liver stage-arresting replication-competent (LARC) GAPs confer superior protection when compared with early liver stage-arresting replication-deficient GAPs and radiation-attenuated sporozoites. However, generating a LARC GAP in the human malaria parasite Plasmodium falciparum (P. falciparum) has been challenging. Here, we report the generation and characterization of a likely unprecedented P. falciparum LARC GAP generated by targeted gene deletion of the Mei2 gene: P. falciparum mei2-. Robust exoerythrocytic schizogony with extensive cell growth and DNA replication was observed for P. falciparum mei2- liver stages in human liver-chimeric mice. However, P. falciparum mei2- liver stages failed to complete development and did not form infectious exoerythrocytic merozoites, thereby preventing their transition to asexual blood stage infection. Therefore, P. falciparum mei2- is a replication-competent, attenuated human malaria parasite strain with potentially increased potency, useful for vaccination to protect against P. falciparum malaria infection.

A Plasmodium falciparum Strain Expressing GFP throughout the Parasite's Life-Cycle

The human malaria parasite Plasmodium falciparum is responsible for the majority of malaria-related deaths. Tools allowing the study of the basic biology of P. falciparum throughout the life cycle are critical to the development of new strategies to target the parasite within both human and mosquito hosts. We here present 3D7HT-GFP, a strain of P. falciparum constitutively expressing the Green Fluorescent Protein (GFP) throughout the life cycle, which has retained its capacity to complete sporogonic development. The GFP expressing cassette was inserted in the Pf47 locus. Using this transgenic strain, parasite tracking and population dynamics studies in mosquito stages and exo-erythrocytic schizogony is greatly facilitated. The development of 3D7HT-GFP will permit a deeper understanding of the biology of parasite-host vector interactions, and facilitate the development of high-throughput malaria transmission assays and thus aid development of new intervention strategies against both parasite and mosquito.

GFP‐targeting allows visualization of the apicoplast throughout the life cycle of live malaria parasites

The Plasmodium parasite, during its life cycle, undergoes three phases of asexual reproduction, these being repeated rounds of erythrocytic schizogony, sporogony within oocysts on the mosquito midgut wall and exo-erythrocytic schizogony within the hepatocyte. During each phase of asexual reproduction, the parasite must ensure that every new daughter cell contains an apicoplast, as this organelle cannot be formed de novo and is essential for parasite survival. To date, studies visualizing the apicoplast in live Plasmodium parasites have been restricted to the blood stages of Plasmodium falciparum. In the present study, we have generated Plasmodium berghei parasites in which GFP (green fluorescent protein) is targeted to the apicoplast using the specific targeting sequence of ACP (acyl carrier protein), which has allowed us to visualize this organelle in live Plasmodium parasites. During each phase of asexual reproduction, the apicoplast becomes highly branched, but remains as a single organelle until the completion of nuclear division, whereupon it divides and is rapidly segregated into newly forming daughter cells. We have shown that the antimicrobial agents azithromycin, clindamycin and doxycycline block development of the apicoplast during exo-erythrocytic schizogony in vitro, leading to impaired parasite maturation. Using a range of powerful live microscopy techniques, we show for the first time the development of a Plasmodium organelle through the entire life cycle of the parasite. Evidence is provided that interference with the development of the Plasmodium apicoplast results in the failure to produce red-blood-cell-infective merozoites.

Structure and life cycle.

Plasmodium species, as members of Apicomplexa, share many common morphological features. Each of the developmental stages in the life cycle of malaria parasites exhibits a remarkable conservation and distinct patterns of structural organization [1, 2]. These conservations are supposed to have originated in the special adaptation to the tissues and/or cells in the different hosts of malaria parasites. As the technology of electron microscopy has improved, more detailed electron microscopic observations of the various stages of malaria parasites have been carried out and greatly advanced our knowledge of the life cycle and the fine structure of malaria parasites. Although the significance of the morphological changes is not fully understood, the introduction of the techniques of immunoelectron microscopy to the field of malaria parasites [3] has helped us in the meaningful and dynamic analysis of parasite morphology and cell biology, and our knowledge of the subcellular localization of malaria antigens and their functions in specific parasite organelles has been accumulated. Structural, biochemical and molecular biological aspects are different among the complex cycle comprising the erythrocytic schizogony, mosquito stages, and preerythrocytic (exoerythrocytic) schizogony. In this chapter, we will describe the ultrastructure of each specific stage, and the morphological and functional changes of the host cells induced by malaria parasites.

A study of selected Plasmodium yoelii messenger RNAs during hepatocyte infection

We investigated the expression of several mRNAs in exoerythrocytic and erythrocytic stages of Plasmodium yoelii in infected mice, focusing our attention on genes thought to be involved in signal transduction (like pypka and pymap- 1, encoding homologues of cAMP-dependent and mitogen-activated protein kinases, respectively) and cell cycle progression (those encoding the cdc2-related kinases Pycrk-1, Pycrk-3 and Pymrk). Messengers coding for enzymes involved in general processes such as DNA replication and RNA transcription (both subunits of the ribonucleotide reductase (Pyrnr1, Pyrnr2) and RNA polymerase II) as well as a messenger coding for Pys21, a sexual stage-specific protein, were also investigated. Total RNA was prepared from livers of infected mice at different times post sporozoite inoculation. In contrast to the pys21 transcript, which was observed only in infected erythrocytes, all messenger species could be detected in the liver by RT-PCR, peaking at 43 h post infection, a time when parasite burden was maximum, and decreasing markedly thereafter to become hardly visible at 168 h. Some transcripts ( pypka, pymap-1, pyrnr1 and pyrnr2) could be detected 12 h after infection, while others ( pymrk and pyrnapolII) did not become detectable until 24 h. In addition, we characterised all these messengers by Northern blot of total RNAs extracted from infected erythrocytes. Taken together, these data suggest that a similar set of regulatory genes is expressed during both exoerythrocytic and erythrocytic schizogony.

Plasmodium vinckei petteri: algunos aspectos de su esporogonia y esquizogonia exoeritrocitaria

A study of Plasmodium vinckei peterri sporogony was performed by experimental infection of Anopheles stephensi with gametocytes from infected mice. The study includes the description of the ookinete, complete evolution of oocysts and their final transformation to sporozoites. These were later used for intravenous infection of new mice, in order to study the exoerythrocytic schizogony. The morphology of exoerythrocytic schizonts was similar to that of other species of the same group. The minimal duration of the hepatic cycle was found to be of 61 hours. These data, along with other related to the hematic cycle characteristics, help to complete the information on the species, which can be recommended as a model for the study of human malaria.

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.

Interactions of Plasmodium berghei sporozoites and murine Kupffer cells in vitro.

Malaria sporozoites must leave the bloodstream and cross a layer of sinusoidal lining cells in order to infect hepatocytes and undergo exoerythrocytic schizogony. To determine whether Kupffer cells (KC) derived from this layer interact with sporozoites, murine KC were isolated from perfused livers of BALB/cJ mice and incubated in vitro with Plasmodium berghei sporozoites. Isolated KC had characteristic macrophage surface Ag and were phagocytic, ingesting both latex particles and Leishmania major amastigotes. In the absence of immune serum, sporozoites associated with fewer than 10% of these KC. By 30 min, 10% of the cell-associated sporozoites were completely ingested, 30% were in the process of being ingested, and 60% were attached to the surface of the cells. Opsonization of sporozoites with monoclonal or polyclonal antibodies directed against P. berghei circumsporozoite protein markedly enhanced sporozoite association with KC. Up to 40% of cells exposed to opsonized sporozoites had parasites inside or attached to their surfaces. Sporozoites attached to or ingested by KC were uniformly destroyed within 240 min in all cultures; there was no evidence of conversion of sporozoites to the exoerythrocytic stage within KC by light microscopy, and there was no evidence of residual sporozoites, either inside or outside of cells, by either light or electron microscopy. These data suggest that under nonimmune conditions, KC play a minor role in resistance to infection by malaria sporozoites. However, when sporozoites are opsonized by circumsporozoite antibodies, phagocytosis by KC may be an important immune mechanism that prevents parasitization of hepatocytes.

Histopathology of hepatocystosis in cynomolgus monkeys (Macaca fascicularis) imported from Southeast Asia.

Histopathological examination was carried out on the hepatic lesions of hepatocystosis in eighteen cynomolgus monkeys (Macaca fascicularis) from Southeast Asia, and the pathogenesis was discussed. The lesions caused by the protozoa were found only in the liver, showing the stages of exoerythrocytic schizogony of Hepatocystis, i.e., formation of merocysts and release of merozoites, with degenerative and/or progressive changes of the hepatic tissue. In these lesions, immature or mature merocysts unreleasing merozoite were destroyed and replaced by the granulation tissue. Early stage of the development of schizont and the necrotic lesions caused of release of merozoites from mature merocysts with the host reaction were found in a few monkeys within 20 days after imported into Japan. The disease without rupture of merocysts was not so harmful to the host animal, and the monkeys who passed a sufficient quarantine period could be utilized for experimental purposes. The parasite of the present cases was identified to be Hepatocystis semnopitheci by its characteristic structure of merocyst and the geographical localization of the host animals.

Membrane structure and function of malaria parasites and the infected erythrocyte.

SUMMARYAccording to the World Health Organization the global estimate of malaria is over 200 million infections, the majority of which are caused by the most life-threatening species,Plasmodium falciparum(Report of the Steering Committees of the Scientific Working Groups on Malaria, World Health Organization, June 1983). The causative agent of the disease, the malarial parasite, requires two hosts: a blood-sucking mosquito and a blood-containing vertebrate. Commonly, infection of the vertebrate begins when an infected mosquito bites a suitable vertebrate and injects minute sporozoites into the bloodstream. Within 30 mm the introduced sporozoites leave the bloodstream and enter parenchymal cells of the liver (mammals) or endothelial cells (birds). In these sites the parasite undergoes asexual multiplication (= exo-erythrocytic schizogony) producing daughter progeny called merozoites. The exo-erythrocytic merozoites are released from the tissues into the circulation where they invade red blood cells. Within an erythrocyte the merozoite undergoes asexual multiplication (= erythrocytic schizogony) producing a substantial number of merozoites. The erythrocyte lyses, merozoites are released, and invasion of another erythrocyte may then take place. The synchronous rupture of the red cell and merozoite release is marked by the periodic fever–chill cycles so characteristic of the malarial infection. Some merozoites continue to reinvade other erythrocytes and multiply by asexual means, whereas others enter erythrocytes and differentiate into sexual stages, male or female gametocytes. When a suitable mosquito feeds on an infected vertebrate gametocytes are ingested and the sexual cycle of development is initiated. In the mosquito stomach the gametocytes transform into gametes, fertilization takes place, the resultant worm-like zygote penetrates the cells of the mosquito gut and comes to lie on the outer surface of the stomach. Here each zygote forms a cyst-like body, the oocyst, within which thousands of sporozoites are produced by asexual multiplication. When the swollen oocysts burst, sporozoites are freed and these make their way to the salivary gland. At the next blood feeding the mosquito injects the infective sporozoites and the life-cycle is completed.

Plasmodium berghei: Inhibitors of ornithine decarboxylase block exoerythrocytic schizogony

DL-alpha-difluoromethylornithine and DL-alpha-monofluoromethyldehydroornithine methyl ester, inhibitors of ornithine decarboxylase, blocked exoerythrocytic schizogony of Plasmodium berghei in mice and in cultured human hepatoma cells. These effects were reversed by exogenous administration of the polyamine, spermidine. The antimalarial drug, primaquine, the side chain of which is structurally analogous to a natural polyamine, did not enhance the activity of alpha-difluoromethylornithine or alpha-monofluoromethyldehydroornithine methyl ester. These results extend previous observations that polyamines influence the malaria parasite's schizogony outside the red blood cell but not within it.

An outbreak of malaria in penguins at the Farm-land Zoo.

In June of 1983 authors had investigated the occurrence of the avian malaria in the Humboldt penguins imported from Japan to the Farm-land Zoo in central district of Korea. The disease was characterized by acute course and high mortality. The peripheral blood smears from the affected penguins demonstrated different developmental stages of Plasmodium sp. in the mature erythrocytes. The predominant gross lesions noticed were pulmonary and epicardial edema and hepato-splenomegaly. Microscopically the lesions were characterized by extensive reticuloendothelial cell hyperplasia with striking feature of exoerythrocytic schizogony affecting a variety of tissues. The report also signifies the first description of a disastrous epizootic of avian malaria in Korea.

Fine Structure of Babesia equi Laveran, 1901 within Lymphocytes and Erythrocytes of Horses: An In vivo and In vitro Study

The development of Babesia equi (Piroplasmia) in the vertebrate host was studied by electron microscopy. The tick-borne sporozoites initiated an exoerythrocytic schizogony in lymphocytes in vivo and in vitro. In lymphocyte cultures the schizonts of B. equi lay as fissured bodies directly within the host cell forming numerous cytomeres. These cytomeres were bordered by a single membrane and contained polymorphous nuclei. Merozoite differentiation was initiated at several places by protrusions appearing at the periphery of the cytomeres. The cytoplasm of the schizont merged progressively into the young merozoites. The mature merozoites were pear-shaped (2.0 X 1.5 microns), bounded by a pellicle and had an apical complex without conoid. Numerous merozoites were observed entering erythrocytes in vitro. In vivo, lymphocytes containing B. equi schizonts were seen in capillaries within lymph nodes from the 12th day onwards after attachment of infected ticks. At the same time some erythrocytes were found that contained B. equi merozoites. Development of B. equi in the vertebrate host shows many similarities to the corresponding development of Theileria parasites. This makes a new discussion of the taxonomic position of B. equi necessary.

Studies on Plasmodium atheruri

Plasmodium atheruri, a parasite of the African porcupine Atherurus africanus, is infective to splenectomised laboratory Rats and Mice, to Hamsters and to 2 exotic rodents which are easily bred in captivity: Calomys callosus and Meriones unguiculatus. Sporogony develops in Anopheles stephensi fed on Atherurus and on laboratory rodents; it is similar to that of the Rodent Plasmodia. Exoerythrocytic schizogony usually lasts 4 to 6 days but schizonts have been found in the liver of a porcupine at day 8. Schizogony in the blood (induced by inoculation of infected blood or by injections of sporozoites) follows two stages which are morphologically distinct: a) acute infections developing in clean Atherurus, following splenectomy of infected Atherurus or in laboratory rodents; it is characterized by large trophozoïtes, schizonts with 8 to 16 merozoites, gametocytes and infectivity to Anopheles. b) chronic infection which only occurs in the blood of Atherurus and follows the acute stage 15 to 21 days after its onset; it is characterized by small trophozoites and schizonts producing 4 merozoites. Thus it is thought that the chronic stage only occurs in the natural host when it is immunized. We think that, in nature, chronic schizogony maintains the parasitaemia through periods of low transmission and is followed by "recrudescences". The mechanisms that trigger the "recrudescences" are unknown but are probably connected with a decrease in the immune status of the host.

Inhibitory action of α-difluoromethylornithine on rodent malaria ( Plasmodium berghei)

The effect of α-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase was tested on rodent malaria. It inhibited the exoerythrocytic schizogony of Plasmodium berghei in mice but has little or no action on the erythrocytic schizogony. These results are examined in relation to the reduction in polyamines concentration and the retardation of cell division observed with this compound.

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.

Rodent plasmodia of the République Centrafricaine: The sporogony and tissue stages of Plasmodium chabaudi and P. berghei yoelii

The sporogony and primary exo-erythrocytic schizogony of two rodent plasmodia recently discovered in the République Centrafricaine were studied in Anopheles stephensi, and in laboratory rats and mice given single intravenous inoculations of sporozoites. Oöcysts of both Plasmodium berghei yoelii and P. chabaudi grow to a diameter of 80 μ at a temperature of 25°C. Sporozoites of the former parasite enter the glands of A. stephensi on the 9th day, and of the latter on the 11th day. The mean length of sporozoites of both parasites is between 13 and 14 μ. Descriptions are given of schizonts of P.b. yoelii in the parenchyma cells of livers of rats and mice 25, 30, 36, 45 and 50 hours after inoculation, and of P. chabaudi at 28, 42, 50, 53 and 54 hours. The inoculation of blood from sporozoite-infected rodents shows that the first schizonts of P.b. yoelii mature at 43 hours and those of P. chabaudi at 53 hours. Mature schizonts of both parasites grow to a mean diameter of 37 μ. Presumed secondary exo-erythrocytic schizonts found in the livers of two Thamnomys rutilans naturally infected with both P.b. yoelii and P. chabaudi are described; these forms differ morphologically from the primary exo-erythrocytic schizonts seen in experimentally infected rodents. The differential characters of P.b. yoelii and P.b. berghei are reviewed, and the relationships between P.b. berghei and P. vinckei from Katanga, and P.b. yoelii and P. chabaudi from the République Centrafricaine, are discussed. P.b. yoelii and P. chabaudi are easily maintained cyclically in the laboratory, and are likely to be extremely useful in studies on immunity, chemoprophylaxis and chemotherapy.

Studies on Malaria in Chimpanzees. V. The Sporogonous Cycle and Mosquito Transmission of Plasmodium vivax schwetzi

Plasm odium vivax schwietzi, the natural benign tertian malaria parasite of chimpanzees, is known by its erythrocytic schizogony and gametogony and by its sporogony in A4nopheles maculipennis atroparvus (Rodhain and Lassman 1940 and Rodhain 1955). There is no record of transmission of the parasite by the mosquito, nor is its exo-erythrocytic schizogony known. The present investigation was undertaken to elucidate these 2 points and to determine if any characteristics existed in the life cycle of the parasite which would constitute specific differences to the similar stages in the life cycle of P. ziva.r zivax. of man.

Studies on malaria in chimpanzees. VI. Laverania falciparum.

SummaryLaverania falciparum will grow in the liver of a chimpanzee and in the blood of a splenectomized chimpanzee. The tissue phase in the chimpanzee accords with previous descriptions in man, but the time of pre-erythrocytic development is a little longer in the chimpanzee. In the peripheral blood of a splenectomized chimpanzee L. falciparum was seen at all stages except the mature gametocytes. No second generation of exoerythrocytic schizogony or persisting exo-erythrocytic schizonts could be found in the liver 12 days after infection. On the basis of this and other evidence the choice is made to assign the parasite to the redefined genus Laverania.