Number Of Merozoites Research Articles

In vitro interaction of Perkinsus marinus merozoites with eastern and pacific oyster hemocytes

This study compared hemocyte responses of eastern and Pacific oysters to Perkinsus marinus, in vitro. Except for the percentage of hemocytes associated with P. marinus there was little or no significant difference between eastern and Pacific oysters with regard to their hemocytic response to P. marinus. In phagocytosis assays, merozoites were bound to all hemocyte types but in unequal proportions, unlike zymosan which was found predominantly associated with granulocytes. The number of merozoites enlarging in Ray's fluid thioglycollate medium after incubation with hemocytes in plasma for one day was significantly lower than after incubation in plasma alone in both oyster species. Electron microscopy or merozoites indicated that the parasites were rapidly phagocytosed and that some of the merozoites showed signs of degeneration in less than 12 h. The results suggest that limited intracellular killing of P. marinus had occurred, but was probably not mediated by oxygen metabolites, since no increase in chemiluminescence was observed when hemocytes of either eastern or Pacific oysters were exposed to merozoites.

Plasmodium spp. (Apicomplexa: Plasmodiidae) of the flying lizardDraco volans (Agamidae)

The agamid lizardDraco volans from Palawan, Republic of the Philippines, was found to be parasitised by threePlasmodium species:P. draconis n. sp.,P. volans n. sp. andP. vastator, Laird 1960. BothP. draconis andP. volans, but notP. vastator, were also present inD. volans from Sarawak. The species are readily distinguished by schizont size, merozoite number, and gametocyte size and shape.P. volans has schizonts approximately one-half the size of those ofP. draconis and produces 4–6 merozoites in comparison to 4–16 in the larger species. Gametocytes of both new species are predominantly oval in the Palawan sample and elongate in that from Sarawak.P. draconis gametocytes are approximately twice the size ofP. volans gametocytes, but on average no more than one-half the size of gametocytes ofP. vastator.

Improved Method for the Initiation of Continuous Cultures of the Oyster PathogenPerkinsus marinus(Apicomplexa)

Abstract A simple method for initiating continuous cultures of Perkinsus marinus with hypnospores is described. The visceral mass of an infected eastern oyster Crassostrea virginica was first incubated in Ray's fluid thioglycollate medium in order to produce large hypnospores from the smaller histozoic stages of the protozoan. Hypnospores were then purified and transferred into the culture medium JL-ODRP-1 where, within a relatively short period of time, they further divided by either progressive cleavage or successive bipartition. These divisions produced large numbers of merozoites and flagellated cells. This two-step procedure surpassed the previously described method of initiating cultures from hearts of infected eastern oysters because a much higher number of purified, larger P. marinas cells could be obtained from a single infected eastern oyster.

Schizogony of Plasmodium yoelii nigeriensis. Role of latent merozoites

Several procedures were employed to try to specify the schizogonic cycle of Plasmodium yoelii nigeriensis. The Percoll-glucose gradient technique for concentrating the very young stages (rings and young trophozoites), allowing a very precise follow up of the development of the parasitaemia during the first schizogonic cycles. A method for studying the prepatencies, providing an approximation of the number of merozoites inoculated. A comparison between the numbers of merozoites present in the blood, after--firstly simple dilutions in saline, revealing the total number of merozoites,--secondly dilutions in saline after a passage of a few hours in the organism of a mouse, revealing the number of latent merozoites. It was shown that the infection, during the first two cycles, varies according to the time of inoculation. In all cases the increase of the parasitaemia occurred mainly from 00:01 to 06:00. This increase of parasitaemia in mice inoculated with the Percoll concentrated parasites was significantly high during the first cycle in mice inoculated at 06:00 and 09:00 and during the second cycle in those inoculated at 12:00, 15:00 and 18:00. However, differences were rapidly compensated and parasitaemias became comparable at the 3rd or 4th cycle when they ceased to be dependent on the time of inoculation.

Invasion of hereditary ovalocytes by Plasmodium falciparum in vitro and its relation to intracellular ATP concentration

Hereditary ovalocytes (stomatocytic ovalocytes), when examined within 1–2 days from the time that the blood sample is drawn, are invaded by Plasmodium falciparum in culture to the extent of at least 55% of normal control cells. The ovalocytes have extremely rigid membranes, characterised by a shear elastic modulus some 3–4 times greater than that of normal cells. The extent of invasion falls off very much more rapidly than that into normal cells on storage, and we surmise that this is the reason for earlier reports of resistance of ovalocytes to malarial invasion in vitro. The initial loss of susceptibility to invasion with time is not accompanied by any change in membrane rigidity, but is primarily a consequence of a rapid decline in intracellular ATP concentration: this falls to below the threshold level required for invasion (approx. 0.1 mM) over a period in which the ATP in normal cells remains almost constant. Incubation in a metabolic regenerating medium leads to a rise in the intracellular ATP concentration and invasion by P. falciparum is recovered, though to a much lower extent than in normal cells. The resistance of ovalocytes to invasion becomes irreversible, due possibly to degradative processes in the membrane, on further storage. The developing parasites in ovalocytes have a reduced number of merozoites and show distinct morphological abnormalities.

Cryptosporidium parvum: investigation of sporozoite excystation in vivo and the association of merozoites with intestinal mucus

Enteric cryptosporidiosis was studied in the small intestine of five-day-old sucking mice after infection with 10 6 Cryptosporidium parvum oocysts. It was shown that excystation and the majority of subsequent endogenous stages occurred predominantly in the ileum. During the first three days of infection the number of merozoites collected in ileal washings increased over 100-fold to approximately 106 merozoites per mouse on the third day. In contrast to control mice, wash fluid from infected mice contained numerous strands of dislodged mucus. Estimates of mucus in the ilea] washings of infected mice were similar to those made in controls until day 4 after infection when they increased and remained high throughout the remainder of the experiment. This study describes a method whereby ileal mucus washings from C parvum infected infant mice could be used as a rich source of merozoites.

Les formes atypiques dePlasmodium vivax.A propos d’une observation

In one case of human malaria imported from Gabon, abnormal forms of Plasmodium vivax are described; severe multiple infections of the host erythrocytes are noticed (up to 6 amoeboid trophozoites in a single red blood cell). Attention is drawn to the numerous morphologically abnormal intra-erythrocytic stages of P. vivax: severe multiple infections of red cells, parasites "ressembling" P. ovale, mature schizonts of small size, with reduced number of merozoites... The geographical distribution of P. vivax in Central Africa is discussed, with reflections on the factors allowing the importation of few P. vivax cases from this area.

In Vitro Effects of Primaquine and Primaquine Metabolites on Exoerythrocytic Stages of Plasmodium berghei

The antimalarial activities of primaquine and its metabolites against exoerythrocytic (EE) stages of Plasmodium berghei in vitro were compared with their abilities to spontaneously generate activated oxygen. A quantitative relationship between the number of sporozoites and the number of EE merozoites produced was established. The reduction in the number of merozoites was used as an assay of drug activity. The ED50 of primaquine, 3.7-3.9 x 10(-6) M, was the concentration of drug that reduced the number of merozoites to 50% of controls. Several of the primaquine metabolites were much more potent than primaquine, with ED50s as low as 2 x 10(-7) M. Metabolites containing the 4-amino-1-methylbutyl side chain were most effective in vitro. Superoxide generation was measured for the various metabolites. In general, superoxide generation did not correlate with antimalarial activity. However, for the 3 metabolites with 4-amino-1-methylbutyl side chains, there was a correlation between superoxide generation and antimalarial activity.

Continuous In vitro Cultivation of Sarcocystis cruzi

Sporozoites of Sarcocystis cruzi were inoculated onto monolayer cultures of bovine pulmonary artery endothelial (CPA) cells. Sporozoites entered the cells, formed large and small multinucleate schizonts, and produced large numbers of merozoites. Continuous cultivation from the original sporozoite inoculum has been maintained for more than 1,320 days by subinoculating merozoites onto new cultures of CPA cells. During this time, the capacity to produce both types of schizonts was preserved, and schizogony was the only form of reproduction that was observed.

Eimeria tenella: Use of a monoclonal antibody in determining the intracellular fate of the refractile body organelles and the effect on in vitro development

A monoclonal antibody, which recognizes the refractile body of Eimeria sporozoites, was used to study the developmental fate of this organelle during asexual development of E. tenella and to determine the effect of this monoclonal antibody on in vitro development of the parasite. Through use of immunofluorescent antibody and gold-labeling techniques at the light and electron microscopy level, the refractile body at 48 to 96 hr postinoculation was found to separate into 6 to 10 small globules, then diffuse throughout the schizont cytoplasm, and eventually reconcentrate as a small dot of material in each of the mature first-generation merozoites. The schizont did not develop to maturity if diffusion of the refractile body did not occur. The refractile body material was quickly lost as the merozoite left the schizont and invaded new cells and was not detected in any later developmental stages. The in vitro development of first- and second-generation schizonts of E. tenella was greatly inhibited (up to 100%) with exposure to the monoclonal antibody. There was an increase in the number of schizonts with nondispersed refractile body in the monoclonal antibody-treated cells when compared to the untreated controls, and the few mature schizonts seen had up to a 50-fold decrease in the number of merozoites. Immunofluorescent antibody labeling of the refractile body of intracellular sporozoites and schizonts treated in vitro with the monoclonal antibody for 24–96 hr postinoculation indicated that the antibody had crossed the host cell and parasite plasma membrane during incubation.

A new Plasmodium species from a lemur: Plasmodium coulangesi n. sp

The splenectomy of a female Lemur macaco macaco, showing a scanty blood infection by Haemosporidians and a Trypanosome was followed by a dramatic increase of the rate of intra-erythrocytic parasitaemia. Three species of Plasmodium where then identified. The first species was unknown; it is described in this paper and named Plasmodium coulangesi n. sp. It is characterised by--the low (6). constant number of merozoites in mature schizonts,--the disposition of the pigment, well apart from the parasitic mass to which it is linked by a tiny wisp of cytoplasm,--the normal host erythrocyte, the shape, size and colour of which are unaltered. The second species was provisionaly designated as Plasmodium sp.; it was previously seen in the same host by Garnham and Uilenberg in 1975 and designated by these authors as Plasmodium girardi Buck, Coudurier et Quesnel, 1952. Only gametocytes of the last species were found; they are similar to those of Plasmodium lemuris Huff and Hoogstraal, 1963.

Hematozoan parasites of Rio Grande wild turkeys from southern Texas.

One hundred twenty-three of 300 blood samples (41%) taken from Rio Grande wild turkeys (Meleagris gallopavo intermedia) from three locations in southern Texas (Welder Wildlife Refuge, Chaparrosa Ranch, and Campo Alegre Ranch) and subinoculated into domestic broad-breasted white turkey poults were positive for a Plasmodium (Novyella) sp. Analysis of blood films from 350 turkeys revealed Haemoproteus meleagridis in 76% of the birds. A significantly greater mean parasite intensity was observed in birds from Welder Wildlife Refuge. Birds from the Campo Alegre Ranch exhibited a significantly higher prevalence of H. meleagridis than birds from Chaparrosa. The Plasmodium sp. was infective for canaries (Serinus canaria), bobwhites (Colinus virginianus), and ring-necked pheasants (Phasianus colchicus), but would not produce infection in white leghorn chickens (Gallus gallus) or Coturnix quail (Coturnix coturnix). Attempts to infect Culex tarsalis and C. pipiens were unsuccessful. Asexual erythrocytic synchrony was not observed when blood-induced infections were monitored in two domestic turkey poults every 4 hr for 72 hr. Exoerythrocytic stages were not found upon examination of impression smears and tissue samples taken from brain, liver, spleen, kidney, lung, and bone marrow. The Plasmodium sp. is most similar morphologically to three species in the subgenus Novyella, P. hexamerium, P. vaughani, and P. kempi. The most striking similarities are to P. hexamerium, and involve mean merozoite number, erythrocytic schizont location, and vertebrate host susceptibility. It differs from P. vaughani in being able to infect turkeys and in type of parasitized erythrocytes. Differences to P. kempi include mean merozoite number, and ability to infect pheasants, and its inability to develop in C. pipiens and C. tarsalis.

Antigens of In vitro-Produced First-Generation Merozoites of Eimeria bovis (Apicomplexa)

Production of mammalian coccidia in cell culture and subsequent use of in vitro-produced organisms as a source of antigen has been mainly restricted to Toxoplasma gondii (Chernin and Weller, 1957, Journal of Parasitology 43: 33-39; Krahenbuhl et al., 1971, Journal of Parasitology 57: 386-390; Braveny et al., 1978, Tropenmedizin Parasitologie 29: 432-434). We describe here a procedure for the production in vitro of large numbers of first-generation merozoites of Eimeria bovis. Also presented are data concerned with the antigenic similarities of merozoites of E. bovis obtained in vivo or in vitro. Oocysts of E. bovis were collected from the feces of experimentally infected Holstein-Friesian bull calves, sporulated, purified and cleaned as described previously (Fayer and Hammond, 1967, Journal of Protozoology 14: 764-772). Oocysts were stored at 4 C in aqueous 2.5% (w/ v) K2Cr207 for no more than 6 mo prior to use. Sporozoites were excysted from oocysts and separated from oocyst and sporocyst debris immediately before inoculation of cultured cells for in vitro production of first-generation merozoites (Fayer and Hammond, loc. cit.; Speer, 1983, The coccidia in in vitro cultivation of protozoan parasites, J. B. Jensen (ed.). CRC Press, Inc., Boca Raton, Florida, 297 p.; Larsen et al., 1984, Journal of Parasitology 70: 597-601). In vivo-produced first-generation merozoites were obtained from an experimentally infected calf as described previously (Reduker and Speer, 1985, Canadian Journal of Zoology 63: 2478-2480). An established cell line of bovine monocytes (BM) was used as a source of host cells for cultivation of first-generation merozoites. The source and characteristics of this cell line, culture medium (CM), and the ability of BM to support penetration and development of E. bovis have been described previously (Speer et al., 1985, Infection and Immunity 50: 566-571). Each of four 150-cm2 polystyrene Corning tissue culture flasks was inoculated with enough BM to produce a 75% confluent monolayer within 24 to 48 hr. Each flask was then inoculated with 40 ml of CM containing 7.5 x 105 sporozoites, which resulted in an inoculum density of 104 sporozoites/ cm2 BM. Culture flasks were incubated at 38 C in 5% CO2-95% air in a Forma Scientific continuous flow CO2 incubator. When mature meronts and extracellular merozoites were detected with phase-contrast microscopy, the CM containing free merozoites was removed daily and replaced with fresh CM. Merozoites were harvested from each flask at 1021 days after sporozoite inoculation (DAI). After the culture flask was gently rapped 20 x with the palm of the hand, the medium was rocked back and forth 20 x, then decanted into a sterile 50ml centrifuge tube. Ten ml Hanks' balanced salts solution (calcium and magnesium free, pH 7.4; HBSS) were added to each flask and the process was repeated, after which fresh CM was added to each flask. The harvested suspension, which contained merozoites and some host cells, was pelleted by centrifugation at 200 g for 10 min, resuspended in 2-3 ml HBSS, and then agitated with 8-10 strokes on a motor-driven teflon-coated tissue grinder in order to disrupt any intact mature meronts. Numbers of merozoites harvested each day from each flask were estimated by counting on a hemacytometer, and mean number of merozoites harvested (?+ 1 SD) was calculated. At 21 DAI, the width of each flask was examined by phase-contrast microscopy (x 200) for number of intracellular sporozoites, meronts (immature and mature) and degenerate or ruptured meronts. The relative frequency of each stage was calculated as a percentage of total parasite stages observed. After examination, BM and parasites were removed from each flask (0.35% EDTA in RPMI 1640, 38 C, 15 min), and numbers of merozoites were determined as outlined above. Merozoites obtained from cultures were combined, centrifuged, and disrupted in 2% sodium dodecyl sulfate (SDS), 10% glycerol, 6.25 x 10-2 M Tris (hydroxymethyl) aminomethane, 4%

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.

A new malaria parasite Plasmodium (Sauramoeba) heischi in skinks (Mabuya striata) from Nairobi, with a brief discussion of the distribution of malaria parasites in the family Scincidae.

A new species of malaria parasite, Plasmodium (Sauramoeba) heischi, is described from African skinks (Mabuya striata). Eleven individuals of 90 specimens collected in Nairobi were found to be infected. The new parasite is a large species, characterized by spindle-shaped gametocytes, the female often with a subterminal nucleus. The schizonts produce up to 65 nuclei and cause great hypertrophy and distortion of the host cell. Although similar to P. (Sauramoeba) giganteum in dimensions and merozoite numbers, P. heischi is easily distinguished by gametocyte and schizont shapes.

Plasmodium falciparum Merozoites: Isolation by Density Gradient Centrifugation Using Percoll and Antigenic Analysis

Merozoites of Plasmodium falciparum were isolated and immunocytochemically analyzed. Mature parasites from knobby (K+) and knobless (K-) strains were incubated for 4 to 5 hr in RPMI 1640 with 10% serum and 10% RBC extract. About 12 to 14% of the merozoites released were recovered by density gradient centrifugation using Percoll. From 1 to 3 X 10(9) merozoites were obtained per collection. The merozoite preparations were contaminated with 10% residual bodies, about 0.1% infected and uninfected erythrocytes, about 0.1% RBC-free trophozoites and schizonts, and numerous small (less than 0.5 microns) membrane vesicles. Merozoites from the K+ and K- strains were morphologically and, by an indirect, ferritin-labeled antibody assay using serum from immune Aotus, antigenically indistinguishable. Although the residual body coats reacted with the immune Aotus serum, the membrane vesicles, some of which were seen to be blebbing from merozoites, did not react with this serum or a serum against erythrocytes. This paper describes a procedure that can be used to obtain large numbers of merozoites with little contamination by host erythrocytes.

Protection against plasmodiu chabaudi malaria I. — Vaccination of mice with merozoites and Freund's adjuvants

Rodent malaria can be a useful experimental model for preliminary studies of some of the variables of a future malaria vaccine ( e.g. optimal doses of merozoites to be used for immunization, nature of adjuvant, etc.). The present study shows that a partial protection can be achieved in outbred OF-1 mice after vaccination with isolated Plasmodium chabaudi merozoites and Freund's complete adjuvant. These results show the relative importance of parasite virulence, number of merozoites used for immunization and size of challenge inoculum. The optimal conditions for using such a model in vaccination experiments are discussed. In these conditions the major effect of immunization is to transform an acute lethal infection into an acute self-curing infection, which is subsequently followed by a solid state of protection against a secondary challenge.

Artificial infection of Rhipicephalm appendiculatus with Theileria parva by percutaneous injection

Rhipicephalus appendiculatus nymphs were inoculated with fresh or cryopreserved blood containing Theileria parva piroplasms, or with cell culture grown stages of T parva. The use of fresh blood was successful. Cryopreserved blood containing dimethylsulphoxide (DMSO), killed most nymphs after inoculation: DMSO could be removed by slow dialysis, without destroying the infectivity of the blood. Attempts to infect ticks by inoculating cell culture grown stages of T parva failed, even when large numbers of merozoites were present in the inoculum.

Factors affecting the ability of isolated Plasmodium knowlesi merozoites to attach to and invade erythrocytes.

Plasmodium knowlesi merozoites were prepared by the polycarbonate sieving method of Dennis, Mitchell, Butcher & Cohen (1975). Merozoite function was assayed by their attachment to and invasion of rhesus erythrocytes at 37 degrees C. The early merozoites from the culture chamber were the most invasive, although maximum numbers of merozoites appeared later. Merozoites were most stable when incubated at room temperature (23 degrees C). At 37 and 0 degrees C invasiveness rapidly declined to zero. Attachment was rapidly lost at 37 degrees C but was retained at 0 degrees C. Attachment was unchanged in the pH range 6.8--7.9 but invasion was reduced at pH 7.9. The presence of L-fucose, D-galactose, D-glucose, D-mannose, N-acetyl-D-galactosamine or N-acetyl-D-glucosamine did not reduce invasion. Attachment and invasion were greatly reduced or abolished by the presence of 2.5 mM EDTA or EGTA, by lactoperoxidase-catalysed iodination of the merozoites, or by treatment of the merozoites with trypsin at a concentration of 1 micrograms/ml or greater for 10 min at 23 degrees C.

The Continuous Cultivation of Plasmodium Fragile by the Method of Trager-Jensen

Using the Trager-Jensen method, a second malaria species, Plasmodium fragile, a simian counterpart to the human malaria P. falciparum, has been cultivated successfully. The average growth rate every 3--4 days was 5-fold and the average number of merozoites observed was 14. To date, only rhesus monkey red blood cells (RBC) would support the long-term cultivation of this parasite. Short-term observations indicate that RBC from the squirrel monkey (Saimiri sciureus) may support growth but human RBC of each of the four major AB-O types failed to support growth of the parasite. Availability of the P. fragile-rhesus monkey model would allow for a second parasite-host system for the in vitro and in vivo study of the immunologic responses of the falciparum-like parasite in a more natural host.