The evaluation of a semiautomated computer method to determine the effects of DMSO on Giardia lamblia-intestinal cell interaction.

In this work, we describe a semiautomated computer method to evaluate the activity of a common drug solvent, dimethyl sulfoxide (DMSO), on in vitro Giardia lamblia-host cell interaction. To compare the number of intestinal cells (IEC-6) and the adhered trophozoites over a specific area in control and treated coculture, a computer routine was created. Using video-light microscopy and digital image-processing tools, the operator was able to count the number of epithelial cells or parasites when they were still lying on the slide surface and without the need to detach them from the substrate for counting with a hemocytometer or other counting devices. Using this strategy, we calculated the total cell number per area and verified the effects of different concentrations of DMSO on G. lamblia-intestinal cell interaction and on the IEC-6 culture. At concentrations of 0.2% and 1%, this solvent produced a fragmentation on the monolayer of epithelial cells. However, DMSO did not affect the attachment of G. lamblia. In the course of these experiments, we compared the semiautomated method to the manual counting method and found that the first one generated smaller standard deviations (SD) than the second.

Giardia lamblia: the effects of extracts and fractions from Mentha x piperita Lin. (Lamiaceae) on trophozoites.

Giardia lamblia is a parasite that causes giardiasis in humans and other mammals. The common treatment includes different classes of drugs, which were described to produce unpleasant side effects. Mentha x piperita, popularly known as peppermint, is a plant that is frequently used in the popular medicine to treat gastrointestinal symptoms. We examined the effects of crude extracts and fractions from peppermint against G. lamblia (ATCC 30888) on the basis of trophozoite growth, morphology and adherence studies. The methanolic, dichloromethane and hexanic extracts presented IC(50) values of 0.8, 2.5 and 9.0microg/ml after 48h of incubation, respectively. The aqueous extract showed no effect against the trophozoites with an IC(50)>100microg/ml. The aqueous fraction presented a moderate activity with an IC(50) of 45.5microg/ml. The dichloromethane fraction showed the best antigiardial activity, with an IC(50) of 0.75microg/ml after 48h of incubation. The morphological and adhesion assays showed that this fraction caused several alterations on plasma membrane surface of the parasite and inhibited the adhesion of G. lamblia trophozoites. Cytotoxic assays showed that Mentha x piperita presented no toxic effects on the intestinal cell line IEC-6. Our results demonstrated antigiardial activity of Mentha x piperita, indicating its potential value as therapeutic agent against G. lamblia infections.

Susceptibility of Giardia lamblia to Hovenia dulcis extracts.

Giardia lamblia is the causative agent of giardiasis, a common parasitic infection of the human and animal digestive tract. Although several drugs have been available to treat this infection, they present unpleasant side effects or cytotoxicity. In order to find a more natural treatment for the disease, we analyzed the effects of the methanolic extract and three fractions obtained from Hovenia dulcis Thunb. (Rhamnaceae) leaves on G. lamblia. Comparing all fractions, dichloromethane was more efficient in reducing Giardia growth. The exposition of G. lamblia to this fraction lead to degenerations in the surface, modifications in the cell shape and alterations in the localization of nuclei. Besides that, the adhesion of G. lamblia was also altered. Experiments revealed that the obtained fraction did not present cytotoxic effects in mammalian cells. In summary, dichloromethane fraction has strong antigiardial effects and could become an important new substance for the treatment of giardiasis.

Identification of a 43-kDa outer-membrane protein as an adhesin in Aeromonas caviae.

Aeromonas spp. are associated with intestinal and extra-intestinal infections. However, the virulence factors of A. caviae remain, for the most part, poorly known. This study examined the interactions involved in the adherence of A. caviae isolates Ae56, Ae391 and Ae398 to HEp-2 cells. All strains expressed high levels of aggregative adherence. Maximum adhesion occurred with bacteria grown at 22 degrees C, but transmission electron microscopy did not reveal the presence of fimbrial structures on the bacterial cell surface. Outer-membrane proteins (OMPs) extracted from isolate Ae398, grown at 22 degrees C and 37 degrees C, showed similar SDS-PAGE protein profiles. Most proteins were < 60 kDa. A major 43-kDa protein was seen only in the boiled OMP extract. The biotinylated 43-kDa protein bound specifically to HEp-2 cells. Microbeads coated with the 43-kDa protein were also adherent to HEp-2 cells, and anti-43-kDa protein antibody blocked adherence of 43-kDa protein-coated latex beads. These data suggest that the 43-kDa OMP functions as an adhesin in A. caviae.

Corynebacterium diphtheriae surface proteins as adhesins to human erythrocytes.

Corynebacterium diphtheriae strains express non-fimbrial surface proteins able to recognize and bind to specific host cells receptors. Protein extracts were obtained from bacterial cells by mechanical process and ammonium sulfate precipitation at 25 and 45% (w/v) saturation. SDS-PAGE analysis of the extracts detected two polypeptide bands of 67 and 72 kDa, named 67-72 p. The 67-72 p, rabbit anti-67-72 p IgG antibodies as well as human gastric mucin, N-acetylneuraminic acid and N-acetyl D-glucosamine molecules were able to inhibit bacterial hemagglutination. Hemagglutination assays using 67-72 p-coated latex beads and Western blot analysis of biotin-labeled 67-72 p and erythrocyte receptors demonstrated the binding of 67-72 p to human erythrocyte membranes. Immunolabeled colloidal gold-A protein transmission electron microscopy using anti-67-72 p revealed a diffuse distribution of non-fimbrial 67-72 p on the surface of C. diphtheriae strains of both sucrose-fermenting and non-fermenting biotypes. Non-fimbrial lectin-like surface 67-72 p may play a role as adhesins in bacterial attachment thereby facilitating the early steps in pathogenesis of both toxigenic and non-toxigenic C. diphtheriae.

Electron and video-light microscopy analysis of the in vitro effects of pyrantel pamoate on Giardia lamblia.

Electron and video-light microscopy analysis of the in vitro effects of pyrantel pamoate on Giardia lamblia. Experimental Parasitology 97, 9-14. Giardia infection is predominant in the small intestine of vertebrates, where the trophozoites attach to epithelial cells and adversely affect the microvilli and other epithelial cell structures. Giardiasis, the disease caused by this protozoan, is very common in developing countries and mainly affects children. Drugs currently used to treat Giardia infection, such as some benzimidazole derivatives, were originally designed to treat helminthic infections. Many of the drugs are known to cause severe side effects and disturbances to the patient. Using transmission electron microscopy and video-light microscopy, we studied the effects of pyrantel pamoate, a drug commonly used in the treatment of helminthic infections in horses and ruminants, on Giardia lamblia trophozoites. Pyrantel pamoate was administered to Giardia cells in four different concentrations. Using video-light microscopy, we observed the decrease in flagella beating frequency and severe changes in the lateral flange and in the general aspect of the cell. Using transmission electron microscopy, we observed changes in the cytoplasm and peripheral vesicles. The flagella and adhesive disk structure were not affected. Apparently, the effects of pyrantel pamoate are irreversible.

Do microtubules around the Toxoplasma gondii-containing parasitophorous vacuole in skeletal muscle cells form a barrier for the phagolysosomal fusion?

The intracellular fate of Toxoplasma gondii was studied in primary cultures of skeletal muscle cells (SMC). The labelling of secondary lysosomes with BSA-Au particles showed no phagolysosomal fusion with the vacuole containing the parasite. After internalization of the parasites, the parasitophorous vacuole became involved by closely apposed endoplasmic reticulum (ER) and mitochondria; within 18 h of interaction, microtubules were visualized in association with the parasitophorous vacuole, suggesting that they could form a barrier for the phagolysosomal fusion.

Contributions of the axostyle and flagella to closed mitosis in the protists Tritrichomonas foetus and Trichomonas vaginalis.

Tritrichomonas foetus and Trichomonas vaginalis are protists that undergo closed mitosis: the nuclear envelope remains intact and the spindle remains extranuclear. Here we show, in disagreement with previous studies, that the axostyle does not disappear during mitosis but rather actively participates in it. We document the main structural modifications of the cell during its cell cycle using video enhanced microscopy and computer animation, bright field light microscopy, confocal laser scanning microscopy, and scanning and transmission electron microscopy. We propose six phases in the trichomonad's cell cycle: an orthodox interphase, a pre-mitotic phase, and four stages during the cell division process. We report that in T. foetus and T. vaginalis: a) all skeletal structures such as the costa, pelta-axostyle system, basal bodies, flagella, and associated filaments of the mastigont system are duplicated in a pre-mitotic phase; b) the axostyle does not disappear during mitosis, otherwise playing a fundamental role in this process; c) axostyles participate in the changes in the cell shape, contortion of the anterior region of the cell, and karyokinesis; d) flagella are not under assembly during mitosis, as previously stated by others, but completely formed before it; and e) cytokinesis is powered in part by cell locomotion.

Tritrichomonas foetus: isolation and characterization of the Golgi complex.

An effective methodology to isolate and characterize the Golgi complex of Tritrichomonas foetus is described in this work. Using sucrose density gradient centrifugation, two highly enriched Golgi fractions (GF1 and GF2) were obtained. Enzymatic assays of GF1 and GF2 showed a strong enrichment in galactosyltransferase activity (20- and 7-fold, respectively), with minimal contamination with other organelles. The GF fraction was further subfractionated by alkaline treatment, which resulted in the production of Golgi content and membrane subfractions. Electron microscopic observations of intact cells or Golgi fractions fixed in solutions containing glutaraldehyde and tannic acid, as well as of deep-etched replicas of isolated fractions, revealed the presence of discrete bridges only between closely apposed cisternae.

Isolation and biochemical characterization of the plasma membrane of Tritrichomonas foetus.

A fraction containing plasma membrane-enriched vesicles has been prepared from Tritrichomonas foetus. Cells were ruptured using a Potter type homogenizer, under well controlled conditions, and membranes were isolated by differential centrifugation and in discontinuous sucrose gradient. This fraction was enriched 8 and 10-fold in the plasma membrane marker enzymes 5'-nucleotidase and (Na+ + K+)-dependent, ouabain-sensitive ATPase, respectively. Determination of Glucose-6-phosphatase and NADPH cytochrome c reductase activities in this fraction, indicates a minimal contamination with endoplasmic reticulum membranes. Analysis by Sodium Dodecyl Sulfate-Polyacrylamide (SDS-PAGE) gradient gel showed that the plasma membrane fraction contains several proteins with major bands corresponding to apparent molecular weights of 48, 45, 39, 37, 32, 30, 27, 23, 20, 19, 17, and 15 kDa.

Free movement of Tritrichomonas foetus in a liquid medium: a video-microscopy study.

The present paper describes in detail the complex movement of the protozoon Tritrichomonas foetus. By the use of analogue and digital video techniques, we were able to analyze frame by frame the beatings of the anterior flagella and discuss their role in the movement of the cell. We also measured the productive displacement of the cell during one flagellar beating cycle. The obtained data were digitally improved and compared to analogue quantifications. It is shown that during 1 s of recorded movement, T. foetus performs 4 complete anterior flagella beating cycles (with active-like and recovery-like beatings). In each cycle the cell swims +/- 6.5 microns forwards, after the recovery of +/- 1.5 microns of receded movement. These observations led us to conclude that the estimated average speed of T. foetus is 25 microns/s, and that all flagella participate in the cell movement. The recurrent flagellum continuously contribute to the forward movement of the protozoon. The cell also performs rotational movements. The obtained results led us to suggest a model for the movement of T.foetus.

Coordinated flagellar and ciliary beating in the protozoon Tritrichomonas foetus.

Tritrichomonas foetus is a flagellated protozoon found in urogenital tract of cattle. Its free movement in liquid medium is powered by the coordinated movement of three flagella projecting towards the anterior region of the cell, and one recurrent flagellum that forms a junction with the cell body and ends as a free projection in the posterior region of the cell. We have used video microscopy and digital image processing to analyze the relationships between the movements of these flagella. The anterior flagella beat in a ciliary type pattern displaying effective and recovery strokes, while the recurrent flagellum beats in a typical flagellar wave form. One of the three anterior flagella has a distinctive pattern of beating. It beats straight in its forward direction as opposed to the ample beats performed by the others. Frequency measurements obtained from cells swimming in a viscous medium shows that the beating frequency of the recurrent flagellum is approximate twice the frequency for the three anterior flagella. We also observed that the costa and the axostyle do not show any active motion. On the contrary, they form a cytoskeletal base for the anchoring and orientation of the flagella.

Isolation and biochemical characterization of the costa of Tritrichomonas foetus.

The costae are cytoskeletal structures found in Trichomonadidae. Both the structural organization and composition of this organelle are still unknown. In the present work we have introduced a new methodology for the costa isolation. Using sucrose density-gradient centrifugation an enriched costa fraction was obtained. Analyses by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the costa contains several proteins, with major bands corresponding to apparent molecular masses of 122, 115, 112, 93, 87, 82, 59, 52, 44, 41, 32, and 26 kDa. No significant amount of carbohydrates was detected in the costa fraction. The fractionation methodology described here has the advantage of using normal centrifugation methods and is being applied to trichomonas in the size range of Tritrichomonas foetus and Trichomonas vaginalis.