Observation of morphological changes of female osmiophilic bodies prior to Plasmodium gametocyte egress from erythrocytes.

Plasmodium parasites cause malaria in mammalian hosts and are transmitted by Anopheles mosquitoes. Gametocytes, which differentiate from asexual-stage parasites, are activated by environmental changes when ingested into the mosquito midgut, and are rapidly released from erythrocytes prior to fertilization. Secretory proteins localized to osmiophilic bodies (OBs), organelles unique to gametocytes, have been reported to be involved in female gametocyte egress. In this study, we investigate the dynamics of OBs in activated gametocytes of Plasmodium falciparum and Plasmodium yoelii using the female OB-specific marker protein, G377. After activation, female gametocyte OBs migrate to the parasite surface and fuse to form large vesicles beneath the parasite plasma membrane. At the marginal region of female gametocytes, fused vesicles secrete contents by exocytosis into the parasitophorous vacuole space, prior to parasite egress via the break-down of the erythrocyte membrane. This is the first detailed description of how proteins are transported through osmiophilic bodies.

Vancomycin improves Plasmodium yoelii malaria parasite in vitro liver stage cultures by controlling Elizabethkingia anophelis, a bacterium in the microbiome of lab-reared Anopheles mosquitoes.

Studies of Plasmodium sporozoites and liver stages require dissection of Anopheles mosquitoes to obtain sporozoites for experiments. Sporozoites from the rodent parasite P. yoelii are routinely used to infect hepatocytes for liver stage culture, but sometimes these cultures become contaminated. Using standard microbiological techniques, a single colony type of Gram-negative rod-shaped bacteria was isolated from contaminated cultures. Mass spectrometry and sequencing of the bacterial 16S ribosomal RNA gene identified the contaminant as Elizabethkingia spp. Based on sequence comparison and published studies of the Anopheles microbiome, the best match was E. anophelis. Culture contamination was not ameliorated by density gradient purification of sporozoites. However, the addition of vancomycin to the culture media consistently reduced contamination and improved culture outcomes as measured by liver stage parasite size. Thus, mosquito salivary gland-derived E. anophelis is identified a potential contaminant of Plasmodium liver stage cultures that can be mitigated by the addition of antibiotics.

Plasmodium yoelii: novel rhoptry proteins identified within the body of merozoite rhoptries in rodent Plasmodium malaria.

The biogenesis, organization and function of the rhoptries are not well understood. Antisera were prepared to synthetic peptides prepared as multiple antigenic peptides (MAPs) obtained from a Plasmodium yoelii merozoite rhoptry proteome analysis. The antisera were used in immunofluorescence and immunoelectron microscopy of schizont-infected erythrocytes. Twenty-seven novel rhoptry proteins representing proteases, metabolic enzymes, secreted proteins and hypothetical proteins, were identified in the body of the rhoptries by immunoelectron microscopy. The merozoite rhoptries contain a heterogeneous mixture of proteins that may initiate host cell invasion and establish intracellular parasite development.

Polychromatic polarization microscope: bringing colors to a colorless world.

Interference of two combined white light beams produces Newton colors if one of the beams is retarded relative to the other by from 400 nm to 2000 nm. In this case the corresponding interfering spectral components are added as two scalars at the beam combination. If the retardance is below 400 nm the two-beam interference produces grey shades only. The interference colors are widely used for analyzing birefringent samples in mineralogy. However, many of biological structures have retardance <100 nm. Therefore, cells and tissues under a regular polarization microscope are seen as grey image, which contrast disappears at certain orientations. Here we are proposing for the first time using vector interference of polarized light in which the full spectrum colors are created at retardance of several nanometers, with the hue determined by orientation of the birefringent structure. The previously colorless birefringent images of organelles, cells, and tissues become vividly colored. This approach can open up new possibilities for the study of biological specimens with weak birefringent structures, diagnosing various diseases, imaging low birefringent crystals, and creating new methods for controlling colors of the light beam.

Malaria Sporozoites Traverse Host Cells within Transient Vacuoles.

Plasmodium sporozoites are deposited in the host skin by Anopheles mosquitoes. The parasites migrate from the dermis to the liver, where they invade hepatocytes through a moving junction (MJ) to form a replicative parasitophorous vacuole (PV). Malaria sporozoites need to traverse cells during progression through host tissues, a process requiring parasite perforin-like protein 1 (PLP1). We find that sporozoites traverse cells inside transient vacuoles that precede PV formation. Sporozoites initially invade cells inside transient vacuoles by an active MJ-independent process that does not require vacuole membrane remodeling or release of parasite secretory organelles typically involved in invasion. Sporozoites use pH sensing and PLP1 to exit these vacuoles and avoid degradation by host lysosomes. Next, parasites enter the MJ-dependent PV, which has a different membrane composition, precluding lysosome fusion. The malaria parasite has thus evolved different strategies to evade host cell defense and establish an intracellular niche for replication.

Identification of the gene for a Plasmodium yoelii rhoptry protein. Multiple copies in the parasite genome.

Serum from mice hyperimmune to Plasmodium yoelii was used to screen a P. yoelii genomic DNA library. Antibodies selected from hyperimmune serum by lambda gt11 clone J7 or raised against a specific fusion protein or peptide produced a punctate pattern of immunofluorescence on fixed smears of parasitised erythrocytes and immunoprecipitated a 235-kDa protein apparently identical to a rhoptry protein previously implicated in red cell invasion. The cloned DNA hybridised to at least seven RsaI fragments of P. yoelii genomic DNA and to three DraI fragments of similar but not identical sequence. These results suggest that the gene encoding the 235-kDa rhoptry protein may be represented more than once in the P. yoelii genome.

A member of a conserved Plasmodium protein family with membrane-attack complex/perforin (MACPF)-like domains localizes to the micronemes of sporozoites.

Pore-forming proteins are employed by many pathogens to achieve successful host colonization. Intracellular pathogens use pore-forming proteins to invade host cells, survive within and productively interact with host cells, and finally egress from host cells to infect new ones. The malaria-causing parasites of the genus Plasmodium evolved a number of life cycle stages that enter and replicate in distinct cell types within the mosquito vector and vertebrate host. Despite the fact that interaction with host-cell membranes is a central theme in the Plasmodium life cycle, little is known about parasite proteins that mediate such interactions. We identified a family of five related genes in the genome of the rodent malaria parasite Plasmodium yoelii encoding secreted proteins all bearing a single membrane-attack complex/perforin (MACPF)-like domain. Each protein is highly conserved among Plasmodium species. Gene expression analysis in P. yoelii and the human malaria parasite Plasmodium falciparum indicated that the family is not expressed in the parasites blood stages. However, one of the genes was significantly expressed in P. yoelii sporozoites, the stage transmitted by mosquito bite. The protein localized to the micronemes of sporozoites, organelles of the secretory invasion apparatus intimately involved in host-cell infection. MACPF-like proteins may play important roles in parasite interactions with the mosquito vector and transmission to the vertebrate host.

Plasmodium sporozoites possess both positively and negatively charged sites accessible on their surface.

Studies were conducted on the distribution of anionic and cationic sites on the surface of intact Plasmodium berghei and P. yoelii sporozoites. Anionic and cationic ferritins were used as probes for electron microscope studies and fluorescein conjugates of the same charged ferritins were used for correlative studies by fluorescence light microscopy. We found that the surfaces of mature sporozoites from mosquito salivary glands possess both negatively and positively charged sites that are accessible to charged ferritin molecules. Sporozoites obtained from oocysts expressed negatively charged sites but were inconsistent in their expression of positively charged sites. The charged nature of the ferritin probes was confirmed by their binding to cellulose particles or cellular components with known negative or positive charges. The electrostatic nature of the binding of ferritin to sporozoites was established by showing that binding could be blocked by competition with high concentrations of NaCl. The most likely source of these surface charges is circumsporozoite (CS) protein, the major protein covering the surface of sporozoites. Circumsporozoite proteins contain a cluster of positively charged amino acids, which may play a role as components of a surface ligand capable of binding to a liver receptor.

Effect of ketotifen on the ultrastructure of the erythrocytic stages of Plasmodium yoelii.

The effect on the morphology of Plasmodium yoelii of ketotifen treatment was different from that of chloroquine and other antimalarial drugs. Following administration of a low dosage of ketotifen, the parasite first developed a multilamellate pellicular complex which resembled a medullary sheath and then developed vacuoles and cavitations. The development of large multilamellate whorls consisting of a pellicular structure enclosing cytoplasm was the typical change which occurred.

[Morphological characteristics of Plasmodium yoelii schizonts in ghost erythrocytes].

OBJECTIVE: To observe the morphological characteristics of Plasmodium yoelii schizogony in their ghost erythrocytes. METHODS: Saponify, hypotonic shock, and electron microscopy were used to observe the different fashions of erythrocytic parasites and their characteristic organellae in ghost erythrocytes. RESULTS: The malarial parasites and their fine structures were dramatically well preserved in the ghost erythrocytes, such as the ring-like early trophozoites, the brassiere-like early schizonts, the emerging buds on the surface of late schizonts, and the grape-cluster like late schizonts. The cytostome, food vacuole, and crystallized malarial pigments were found in the early trophozoites. The proliferations of nucleoplasma and nuclear membrane as well as and the clot-like nuclear division were followed by the budding during the schizogony. CONCLUSION: The saponify technique that makes the erythrocytic malaria parasites and their fine organellae to be dramatically revealed in their ghost erythrocytes, may be a useful method in the Plasmodium biological research and anti-malaria immunological researches.

[Immunity of peritoneal monocytes against Plasmodium yoelii infected erythrocytes].

OBJECTIVE: To test the immunity of peritoneal monocytes against Plasmodium yoelii infected red blood cells (target cells). METHODS: Saponinized Plasmodium yoelii infected red blood cells (SPRBC, Ghost erythrocyte) were used to immunize mice i.p twice. Three weeks later, the infected red blood cells were injected i.p.; 90 min later, the total peritoneal cells were isolated and washed for scanning electromicroscopy to observe the effects of the peritoneal monocyte to the target cell. RESULTS: The peritoneal cells of the immunized mice were activated after 90 min of the challenge of target cells. The size of the cell was not even and the pili on the cell surface turned to be long and densed. Cell interconnections were found among the cells. In some peritoneal monocytes, their cell plasma were scattered (omlette-like) or with the shape as "cellular bomb". The scattered or the sheeted pili and spredding cell plasma could adhere to the target cells which were perforated densely and damaged. CONCLUSION: The protective adaptive immunity exists in the peritoneal monocytes of immunized mice.

[Scanning electron microscopic observations of ookinete formation of Plasmodium yoelii yoelii in vitro].

The ookinete formation and mature ookinete of Plasmodium yoelii yoelii were described. During the development from zygote to ookinete, at first a finger-shaped or a stick-shaped projection protruded from one side of zygote. The anterior end of the projection assumed a truncated cone shape. Following enlargement of the projection, the body of zygote gradually wrinkled and shrank and became smaller. An annular structure developed between the newly formed ookinete and the remaining body of zygote, which ultimately separated from the ookinete. The ookinete was banana-shaped. The surface of mature ookinetes was smooth. On the tip there was an apical complex showing truncated cone shape. Behind the apical end there were a few pellicular folds. Through the course of development of the ookinete the parasite constantly showed obvious movement. As the parasites protruding forward, longitudinal and spiral wrinkles appeared on their body surface.

[Early ultrastructural evolution of murine malaria merozoites after entering red cells].

A TEM study of murine malaria parasites, Plasmodium berghei and P. yoelii was performed by consecutive sampling in vivo to look into the early sequential changes in the ultrastructure of the merozoites after entering red cells. The results showed that once finishing invasion, the merozoite resided in the peripheral cytoplasm of the red cell, creating a bulge at the invasion site, with an additional unit membrane around it (parasitophorous vacuole); apical structures disappeared; the spherical body was degenerative or atrophic and separated from the mitochondrion and nucleus. The mitochondrion became more extended and the nucleus elongated and curved. There were more Er vesicles in the cytoplasm, taking a dilated polyangular shape. The inner double membrane was separated from the outer membrane and got into incomplete, winding, finally disappeared. Sometimes multimembranous bodies could be seen in the peripheral spaces. Once the dedifferentiation process was over, the merozoite was transformed into an early trophozoite, with a single plasma membrane and decreased density. Individual large Er vesicle with acute angles was found in the cytoplasm, and small food pills appeared beneath the plasma membrane; then the shape of the parasite changed from a ball-like one to a pie-like one, gradually the flat cell body rolled up, with its edges met and fused, resulting in the formation of a large food vacuole, with digestive vacuoles and pigment granules around it. Thus, it grew into a middle-aged trophozoite.

[Study on the exoerythrocytic forms of Plasmodium yoelii yoelii in rats and mice].

SD rats and three strains of mice were infected with Plasmodium yoelii yoelii By265 strain by intravenous inoculation of sporozoites via tail vein. Liver tissues were taken 42 hours after infection and serial sections were made and stained by Colophonium-Giemsa method for microscopic examination. The ratio of the average value of major diameter/minor diameter of exoerythrocytic(EE)schizonts of Plasmodium yoelii yoelii in rats and mice of ICR/JCL, C57BL, KM strains was 35.81 +/- 4.56 microns/29.72 +/- 4.08 microns, 28.08 +/- 4.66 microns/23.66 +/- 4.44 microns, 28.14 +/- 4.16 microns/23.63 +/- 3.77 microns, 23.80 +/- 2.42 microns/21 +/- 0 microns, respectively. The results showed that the development of EE schizonts of Plasmodium yoelii yoelii was not synchronous. The EE schizonts in the rat liver were surrounded by Kupffer cells, monocytes and monocyte-derived macrophages. Since the parasitemia disappeared rapidly in rats, and EE schizonts were not well developed in KM strain, it may be considered that ICR/JCL and C57BL strains are more suitable as vertebrate host in Plasmodium yoelii yoelii-Anopheles stephensi system model (Figs. 1-9).

[Localization of the antigen in erythrocytic stages of Plasmodium yoelii by immuno-electron microscopy].

In this study, the antigen recognized by the protective McAb M26-32 in erythrocytic stages of P. yoelii was localized by immuno-electron microscopy with LR Whithe resin embedding and colloidal gold probe cytochemical techniques. The results indicated that the antigen which reacts specifically to McAb M26-32 was mainly localized within the cytoplasm of early and late trophozoites, schizonts and merozoites, being the common antigen of asexual blood stages of the plasmodium. The amount of the antigen was on the increase during the development of trophozoite, while a portion of the antigen might be transported outward by exocytosis of the parasites and then be localized in the cytoplasm of the infected erythrocytes adjacent to the parasites.

Plasmodium yoelii: contribution of oocysts melanization to natural refractoriness in Anopheles dirus.

It is well known that Anopheles dirus is naturally refractory to rodent malaria parasite, Plasmodium yoelii, but the mechanism is still largely unknown. Here, we found that some P. yoelii taken into An. dirus could develop into oocysts, but oocysts were partially melanized at 7 days and completely melanized at 15 days post-infectious blood meal. Transmission electronic microscopy could find the melanized P. yoelii oocysts in An. dirus as early as 5 days post-infection, with a few haemocytes attaching to the melanized oocysts, indicating a typical humoral melanization reaction. Although the change of protein pattern at 24h post-infection suggested that other unknown mechanisms and/or factors might be involved in killing ookinetes, our data implied that oocysts melanization was one of the mechanisms of An. dirus to block P. yoelii development. In addition, activity of phenoloxidase, such as monophenol oxidase and o-diphenoloxidase, in haemolymph of An. dirus fed on infectious blood meal was much higher than that of mosquitoes fed on 5% glucose or normal mouse blood (p<0.05), implying the possible role of PO in oocysts melanization by An. dirus.

[Ultrastructural study on effect of primaquine on sporogonic stage of Plasmodium yoelii nigeriensis].

Ultrastructural changes of oocysts and sporozoites of Plasmodium yoelii nigeriensis was observed. Anopheles stephensi were allowed to obtain blood meal from the mice which had been administered with primaquine diphosphate at different doses and times. Mosquitoes were directed and prepared 6-13 d following infection. Electron microscopy showed that the development and morphology of a number of oocysts and sporozoites in infected mosquitoes after treatment became abnormal. The cytoplasma of the oocysts were partially or totally dissolved and formed vacuoles. The walls of oocysts were thicken. The nuclei and organelles of oocysts and sporozoites were destroyed or damaged. The nuclei and organelles of oocysts and sporozoites were destroyed or damaged. The extent of damage of the oocysts and sporozoites related to the dose of primaquine and the time after the drug administration.

A cytochemical ultrastructural study of the lysosomal system of different species of malaria parasites.

We have used ultrastructural techniques in different malarial species to demonstrate a lysosomal system. First, we have tried to localize acid phosphatase, a typical lysosomal label. Its activity was localized in the endoplasmic reticulum and in endocytic vesicles, and in dense-cored vesicles near the digestive vacuoles, especially in Plasmodium falciparum (FCR3 strain). Then, we have studied the different cellular compartments of the malarial parasite by the zinc iodide-osmium tetroxide technique that heavily contrasted the cellular compartments of the parasite. This experiment led to the observation of a profound rearrangement of the endoplasmic reticulum, especially in P. berghei. A very atypical but functional Golgi apparatus was demonstrated in all the growing stages of the parasite and lysosome-like vesicles were observed, showing a structure very similar to those of the coated vesicles of a true Golgi complex. The presence of these organelles are in favor of the existence of a lysosomal system and of the endogenicity of some enzymes involved hemoglobin degradation.

The chemotherapy of rodent malaria XLI. Causal prophylaxis. Part V. Effect of mefloquine on exoerythrocytic schizogony in Plasmodium yoelii yoelii.

Previous studies using mefloquine in rodents have suggested that this compound has no effect on pre-erythrocytic schizogony. In the present study, direct observations on 46-hour-old pre-erythrocytic schizonts of P. yoelii in the liver of rats have shown that mefloquine does induce recognizable changes in the peripheral, enzyme-containing vesicles but that these are insufficient to hinder the maturation of the parasites, thus confirming that this compound has no causal prophylactic value.