Effect and Localization of Trifluralin in Plasmodium falciparum Gametocytes: An Electron Microscopic Study1

Trifluralin, a herbicide which is known to bind to plant and algal tubulin, induced ultrastructural changes in the microtubules of the mature Plasmodium falciparum gametocytes in vitro. Trifluralin treatment led to disassembly of the well ordered subpellicular microtubules, whereas it had no effect on microtubules of human platelets or of rat neuronal cells in vitro. The disassembled microtubules showed fragmented large tubular structures, which frequently were associated with the pellicular membranes. Electron microscopic autoradiography showed radioactive trifluralin associated with the microtubule fragments. These results provide evidence that trifluralin selectively binds to microtubules in malaria parasites and causes disruption of their structure.     

Ultrastructure of Mitosis and Cytokinesis In the Colorless Flagellate Katablepharis Ovalis Skuja

ABSTRACT. Mitosis and cytokinesis in Katablepharis ovalis , a colorless flagellate, was investigated. Two new flagella are produced prior to prophase, resulting in a motile quadriflagellate cell during mitosis. the inner array of microtubules of the feeding apparatus disappears before prophase begins. the nuclear envelope disperses during prophase, apparently being converted into rough endoplasmic reticulum. the chromatin condenses and the nucleolus disperses with spindle microtubules appearing oriented perpendicular to the longitudinal axis of the cell. At metaphase, the chromatin is condensed as a single disc-shaped mass and rough endoplasmic reticulum flanks the chromatin mass on each side. Groups of spindle microtubules pass through tunnels in the rough endoplasmic reticulum and through electron-translucent areas of the chromatin. the spindle microtubules end at a number of minipoles in the cytoplasm. Vesicles, ribosomes, mitochondria and endoplasmic reticulum migrate among the spindle microtubules. There is no polar body or any electrondense area associated with the spindle poles. the basal bodies of the flagella remain attached to the axonemes and do not participate in mitosis. In anaphase, the chromatin separates and migrates to the poles. During telophase, the nuclear envelope reforms from the rough endoplasmic reticulum and the nucleoli reappear. the spindle microtubules are persistent during telophase. Cytokinesis occurs by longitudinal fission, starting at the anterior end and progressing posteriorly. Cytokinesis may be driven by elongation of the spindle microtubules since there is no visible structure associated with the furrowing.     

Gametogenesis and Sporogony of Hepatozoon Mocassini (Apicomplexa: Adeleina: Hepatozoidae) In an Experimental Mosquito Host, Aedes Aegypti

ABSTRACT. The sexual life cycle of the hemogregarine Hepatozoon mocassini was studied in Aedes aegypti , an experimental mosquito host, using transmission electron microscopy. Gamonts were observed leaving the host snake erythrocyte as early as 30 min after mosquitoes ingested infected blood, and some gamonts had penetrated the gut epithelial cells by this time. Six hours post-feeding, gamonts were identified within cells of the abdominal fat body. Twenty-four hours post-feeding, gamonts were often entrapped within the peritrophic membrane, but were no longer observed within the gut wall. Parasites pairing up in syzygy and undergoing sexual differentiatioe were observed within fat cells at this time, and by 48 hours post-feeding, well-developed macro- and microgametocytes as well as microgametes were discernible. Developing zygotes observed 3 days post-feeding were enclosed within a panoitophorous vacuole. By day 6, multinucleate oocysts with crystalloid bodies in the cytoplasm were seen. Sporazoites developing within sporocysts appeared by day 12. Seventeen days post-feeding, mature oocysts with sporocysts containing approximately 16 sporozoites were observed upon dissection of mosquitoes. Large crystalloid bodies no longer bound by rough endopbsmic reticulum were located anterior and posterior to the sporozoite nucleus. Free sporozoites were not observed.     

Effects of Mesoionic Xanthine Analogs On Trypanosoma Musculi Development In Mice

ABSTRACT. Two derivatives of the mesoionic thiazolo[3,2-a]pyrimidine-5,7-diones 1 were prepared and examined for in vivo antiprotozoan activity to study structure-activity relationships that might lead us to more active derivatives. Mesoionic compounds 1A and 1B were inoculated into Swiss Webster male mice with Trypanosoma musculi infection. the effects were measured by studying parasite populations during the course of patent period (days 9 through 15 post-infection).
The injection of 200 μg of compound 1A along with 5 times 10⁴ trypanosomes affected the level of parasitemia at both the peak and during days 9 to 13 post infection. Experimental animals that received drug 1A prior to and after infection with T. musculi developed significantly lower parasitemia as compared to the control animals at the height of parasite populations (day 11 of observation). the group that received the drug simultaneously with trypanosome infection had significantly lower parasitemias on day 11 and 13 of infection compared to the controls.     

In Vitro Propagation of the Protozoan Perkinsus Marinus, A Pathogen of the Eastern Oyster, Crassostrea Virginica

ABSTRACT. Perkinsus marinus , a pathogen of eastern oysters ( Crassostrea virginica ), has been successfully propagated in vitro. Cultures of the parasite were initiated from heart fragments of an infected oyster. the cultured protozoan (designated Parkinsus -1) was similar in morphology at both the light and transmission electron microscopy levels to histozoic stages of P. marinus in naturally infected oysters. In addition, cultured cells incubated in fluid thioglycollate medium produced enlarged cells (prezoosporangia) that stained blue-black in Lugol's solution, a response characteristic to Perkinsus spp. and used in routine diagnosis. Polyclonal antibodies raised against P. marinus prezoosporangia reacted positively to Perkinsus -1. Finally, the cultured cells infected susceptible oysters and reisolation of Perkinsus -1 cells was possible from the hearts of experimentally infected oysters. the culture medium contained most of the known constituents of cell-free hemolymph of oysters. the success achieved in culturing P. marinus will allow further investigations aimed at reducing mortalities caused by this important oyster pathogen and at addressing many unanswered questions about its biology and pathobiology.     

Oxygen Uptake In Cysts and Trophozoites of Giardia Lamblia

ABSTRACT. Oxygen uptake in cysts and trophozoites of the parasitic protozoan Giardia lamblia was examined. Both showed oxygen uptake activity, but that of cysts was only 10% to 20% that of trophozoites. Oxygen dependence of oxygen uptake in cysts and trophozoites showed oxygen maxima above which oxygen uptake decreased. the oxygen concentration at which the oxygen uptake rate was greatest was higher for trophozoites than for cysts. the effect of various inhibitors on cyst and trophozoithe oxygen uptake suggested that flavoproteins and quinones play some role in oxygen uptake. the substrate specificities and the effect of inhibitors on G. lamblia trophozoites were similar to those observed for G. muris. Metronidazole, the drug most commonly used in treatment of giardiasis, inhibited oxygen uptake and motility in trophozoites; however, it had no obvious effect on either oxygen uptake or excystation in cysts. Menadione, a redox cycling naphthaquinone, first stimulated, then completely inhibited, oxygen uptake in cysts and trophozoites; a complete loss of cyst viability and trophozoite motility was also observed. the effect of menadione on G. Iamblia may indicate that redox cycling compounds have potential as chemotherapeutic agents for the treatment of giardiasis.     

Evaluation of Rhodamine 123 As A Probe For Monitoring Mitochondrial Function In Trypanosoma Brucei Spp.

ABSTRACT. Rhodamine 123, a membrane potential-specific dye, has been evaluated as a probe to monitor the function of the mitochondrion in long slender bloodstream and procyclic trypomastigotes of several Trypanosoma brucei spp. By epifluorescence microscopy, mitochondrial development has been followed in long slender bloodstream and procyclic organisms stained with rhodamine 123. to photograph stained long slender bloodstream forms, it was necessary to develop a method to completely immobilize viable organisms. In both parasite forms, as the cell cycle progressed, the mitochondrion developed from a thread-like structure to a highly branched organelle. A dramatic reorganization occurred preceding cytokinesis to produce two progeny thread-like structures which were partitioned into newly formed daughter cells. the organelle within the long slender trypomastigote was found to stain optimally at 0.3 μ/ml of rhodamine 123, while the procyclic form required 3.0 μ/ml. the results suggest that the plasma membrane potential is higher in the long slender parasite than in the procyclic form. the effects of inhibitors that disrupt mitochondrial function were examined in long slender and procyclic parasites, and some of these agents were shown to affect rhodamine 123 accumulation and retention. In long slender trypomastigotes the trypanosome alternative oxidase does not appear to be coupled to proton pumping, whereas in procyclic organisms the effects of inhibitors indicate that this oxidase may be coupled to a pathway that is branched preceding an antimycin A₁-sensitive site.     

An Improved Medium For Plasmodium chabaudi In Vitro Erythrocyte Invasion Assays

ABSTRACT. RPMI-1640 is routinely used as the basal medium for the in vitro maintenance of malaria parasites. In this study we tested several commercially available nutritional media in a Plasmodium chabaudi chabaudi erythrocyte invasion assay and showed that three media, BME Basal Medium—modified, Dulbecco's Modified Eagle Medium, and William's Medium E, improved the level of merozoite invasion when compared with RPMI-1640. These media improve the rate of maturation of newly invaded rings to young trophozoites. Radioisotope incorporation by trophozoites maintained in these three media was also improved when compared to trophozoites maintained in RPMI-1640. BME Basal Medium—modified, or a combination of three parts BME Basal Medium—modified with one part William's Medium E, supported higher levels of erythrocyte invasion by merozoites. We suggest that either of these media replace the currently used RPMI-1640 for in vitro studies on P. c. chabaudi.     

Myosin-Like Protein (MR 175,000) In Gregarina Blaberae

ABSTRACT. A myosin-like protein (M_r 175,000) was detected in the parasitic protozoan Gregarina blaberae , by both immunofluorescence and immunoblotting of one- and two-dimensional electrophoresis gels using anti-myosin antibodies. This protein was present in the trophozoite ghost but not in the cytoplasmic extract, nor in extract from the sexual stage, suggesting a protein-stage-dependent expression. the protein tightly bound to the cortical membranes was insoluble at low ionic strength, or in detergent solutions, but could be extracted from Gregarina ghosts by 6 M urea in high ionic strength solution (0.5 M NaCI) and in the presence of reducing agents (20 mM DTT). the protein was localized by indirect immunofluorescence in the cortex of the epimerite, in the fibrillar disc (the socalled septum) separating the proto- and the deutomerite segments, in the contractile ring or sphincter at the top of the protomerite, and as longitudinal lines underlying the G. blaberae epicyte folds. the presence of both actin-like and myosin-like proteins would be consistent with a role in gliding and other cell motility processes of this parasite.     

Disruption of Ca2+ Homeostasis In Trypanosoma Cruzi By Crystal Violet

ABSTRACT. We have demonstrated previously that crystal violet induces a rapid, dose-related collapse of the inner mitochondrial membrane potential of Trypanosoma cruzi epimastigotes. In this work, we show that crystal violet-induced dissipation of the membrane potential was accompanied by an efflux of Ca²⁺ from the mitochondria. In addition, crystal violet inhibited the ATP-dependent, oligomycin-, and antimycin A-insensitive Ca²⁺ uptake by digitonin-permeabilired epimastigotes. Crystal violet also induced Ca²⁺ release from the mitochondria and endoplasmic reticulum of digitonin-permeabilized trypomastigotes. Furthermore, crystal violet inhibited Ca²⁺ uptake and the (Ca²⁺-Mg²⁺)ATPase of a highly enriched plasma membrane fraction of epimastigotes, thus indicating an inhibition of other calcium transport mechanisms of the cells. Disruption of Ca²⁺ homeostasis by crystal violet may be a key process leading to trypanosome cell injury by this drug.     

Oral Morphogenesis of the Microstome to Macrostome Transformation in Miamiensis avidus Strain Ma/21

ABSTRACT. Miamiensis avidus, a marine scuticociliate, undergoes microstome to macrostome transformation. This process is induced by prey ciliates. the morphogenesis of this process was examined using scanning electron microscopy and light microscopy of quantitative protargol-stained specimens. Several stages are identified and described.     

Low Genetic Diversity In Willaertia Magna From Wide Geographical Sources, and Characterisation of Willaertia Minor N. Sp. (Heterolobosea, Vahlkampfidae)

ABSTRACT. Twelve isolates assigned to Willaertia magna by morphological and physiological criteria, including the type material, were characterised at 23 enzyme-encoding loci. Isolates from Australia, Bangladesh, Belgium, England, France, Indonesia, Japan, Korea, Madagascar and Papua New Guinea showed fixed alleles at 18 loci and polymorphism at Acp, Lap, Me, 6Pgd and Pgm 1. the maximum difference in pairwise comparisons was 8.7%, consistent with intra-specific variation in the related genus Naegleria. an additional isolate, assigned to Willaertia by features of its flagellate and cyst stages, represents a new species. Willaertia minor n. sp. shares alleles with W. magna at only three loci, has a maximum growth temperature of 38°C and is significantly smaller in all stages than W. magna. Flagellates appear to divide only once and fail to replace the lost flagella, producing biflagellate daughter cells. Cysts and trophozoites are more readily mistaken for Naegleria , but lack perinuclear granules, as do the mature cysts of W. magna.     

Antagonistic Action of the Bacterium Bacillus Licheniformis M-4 Toward the Amoeba Naegleria Fowleri

ABSTRACT. Free-living amoebae belonging to the species Naegleria fowleri are known to be the etiological agents for a form of fulminant meningoencephalitis that is generally fatal (primary amoebic meningoencephalitis). In a broad bacterial screening from soil and water we have isolated three strains (M-4, D-13 and A-12) belonging to the species Bacillus licheniformis that have remarkable amoebicidal activity against Naegleria sp. and also against different Gram-positive and Gram-negative bacteria. Physical-chemical characteristics, partial purification and biological activities of a substance produced by the M-4 strain have been investigated. This substance (m-4) is stable at high temperature (up to 100°C) and extremes of pH (2.5-9.5) and also at -20°C for months. Its production is greatly influenced by oxygenation of the cultures and is probably related to the sporulation process of the bacterium. Scanning electron microscope observations reveal that amoebae are lysed after a few minutes contact with m-4.     

In Vitro Induced Anaerobic Resistance to Metronidazole In Trichomonas Vaginalis

ABSTRACT. Resitance to metronidazole detectable under anaerobic conditions was induced in two Trichomonas vaginalis strains (TV 10-02 and MRP-2) by cultivation at gradually increasing pressure of the drug (1-100 μ/ml) for 12 to 21 months. the resistant derivatives reproduced in anaerobic trypticase-yeast-extract-maltose medium at 100 μ/ml metronidazole and showed very high values of minimal lethal concentration for metronidazole in anaerobic in vitro assays (556-1,600 μ/ml at 48-h exposure to the drug). Stepwise selection was necessary to develop the resistance in either strain. Attempts to induce resistance by prolonged maintenance of trichomonads with constant, low or moderate drug concentrations (3-10 μ/ml) were unsuccessful. Freshly developed resistance to high concentrations of metronidazole was unstable in absence of drug pressure as well as after cryopreservation. Development of stable resistance required further cultivation at 100 μ/ml metronidazole. Unstable substrains did not revert to original susceptibility. They retained a moderate level of resistance, being able to grow at 10 μ/ml metronidazole. the strains with fully developed resistance had no activity of the hydrogenosomal enzymes pyruvate: ferredoxin oxidoreductase and hydrogenase and ceased uptake of [¹⁴C]-metronidazole. These findings indicate that the pyruvate oxidizing pathway responsible for metronidazole activation was inactivated and metabolism of the drug stopped.     

Phylogeny of Trichomonads Based On Partial Sequences of Large Subunit Rrna and On Cladistic Analysis of Morphological Data

ABSTRACT. Several domains of large subunit rRNA from nine trichomonad species have been sequenced. Molecular phylogenies obtained with parsimony and distance methods demonstrate the trichomonads are a monophyletic group which branches very early in the eukaryotic tree. the topology of the trees is in general agreement with traditional views on evolutionary and systematic relationships of trichomonads. A clear dichotomy is noted between the subfamily Trichomonadinae and the subfamily Tritrichomonadinae. In the latter subfamily, a second division separates the " Tritrichomonas muris -type" species from the " Tritrichomonas augusta -type" ones. Previous evolutionary schemes in which the Monocercomonadidae were regarded as the most "primitive" and the Trichomonadidae as more "evolved" are not in agreement with our molecular data. the emergence of Monocercomonas and Hypotrichomonas at the base of the Tritrichomonas lineage suggests a secondary loss of some cytoskeletal structures, the costa and undulating membrane in these genera. This is corroborated by the early branching position of Trichomitus. which possesses a costa and an undulating membrane and has usually been placed among the Trichomonadidae on the basis of cytological characters. A cladistic analysis was applied to the available morphological characters in order to produce a hierarchical grouping of the taxa reflecting their morphological diversity. Supplementary key words. Evolution, molecular phylogeny, morphological cladistic analysis.     

Freeze-Fracture Study of Malaria Sporozoites: Antibody-Induced Changes of the Pellicular Membrane*

Plasmodium cynomolgi, Plasmodium knowlesi, and Plasmodium berghei sporozoites, before and after incubation with immune serum, were studied after freeze-fracture by electron microscopy. There were evenly distributed numerous intramembranous particles (IMP) on the P face of the outer membrane. The E face of the plasma membrane had fewer IMP than its P face. The E face of the intermediate membrane had few IMP and also linear arrays of slightly raised ridges running the length of the parasite. The P face of the intermediate membrane had many IMP aligned along the long axis of the sporozoite. On the P face of the inner membrane. IMP were arranged in very distinct rows conforming to the long axis of the parasite; the E face of this membrane had a few randomly distributed IMP. A prominent change in the sporozoite incubated in immune serum was the appearance of a layer of aggregated particles around the parasite. The P face of the plasma membrane had several clear areas devoid of IMP and IMP aggregates. No changes were seen in the other fractured faces of the pellicle. These observations suggest that immune serum acts only on the P face of the plasma membrane.     

The Effect of Free Radicals Induced by Paraquat and Copper on the In Vitro Development of Plasmodium falciparum

The role of transition metals in paraquat toxicity was studied in cultures of Plasmodium falciparum. We showed that addition of copper led to an enhancement of the plasmodium killing, whereas addition of chelating agents. such as desferrioxamine and diethylenetriamine pentaacetic acid markedly reduced the toxic effects. Parsitized G6PD deficient erythrocytes were more sensitive than parasitized normal eryth-rocytes to copper and to the combination of copper and paraquat.