The effects of metronidazole and furazolidone during Giardia differentiation into cysts.

The protozoon Giardia lamblia infects millions of people worldwide, most of them in underdeveloped countries, where it is frequently a hyperendemic disease. The search for an effective anti-Giardia treatment has been intense, but recurrent infections, virulence factors, and drug resistance imposed obstacles in the achievement of an efficient medication. Most papers about drug effects in Giardia are related to the trophozoite form, although viable cysts, the infective forms, are continuously eliminated in the stools during the treatment. Supported by this knowledge, we analyzed the inhibitory effects of metronidazole (MZ) and furazolidone (FZ) on the differentiation of Giardia into cysts and its viability. The presence of cavities, lamellar bodies and thread-like structures were the most frequent morphological alterations. The results showed also that FZ was more effective by 50% than MZ in inhibiting in vitro cyst differentiation.

The caudal complex of Giardia lamblia and its relation to motility.

This paper presents a detailed study of the caudal complex of Giardia lamblia and its relation to movements observed in this region. The caudal complex of Giardia, composed of axonemes from the caudal flagella plus associated microtubular sheets, was investigated by light, electron microscopy, and 3D reconstruction tools. By the use of video-microscopy and digital image processing techniques, we were able to visualize in detail the caudal movements. A non-ionic detergent, Triton X-100, was used to isolate the complex that was afterwards analyzed by video-microscopy and transmission electron microscopy (TEM). We showed for the first time, using video-microscopy, that the intracellular portion of the caudal flagella axonemes presented motility, even after the disrupture of the cell membrane, contrasting with the caudal flagella themselves, that do not show active beating pattern. To check if actin filaments play a role in the above described movements, as previously supposed, we incubated the cells with jasplakinolide, a drug that induces the disruption of actin filaments in living cells. The experiments demonstrated that the drug did not affect the caudal motility. The analysis of the caudal complex by transmission electron microscopy (TEM) revealed that, even after the exposure to higher detergent concentrations, the connections between their components remained intact. The information obtained by TEM and 3D reconstruction tools showed that the region between both nuclei marks the intracellular end of the caudal complex, which proceeds toward the caudal portion of the cell following its longitudinal axis, where the axonemes emerge as the caudal flagella. The results obtained from video-microscopy assays of the isolated beating complex together with the 3D reconstruction data indicated that the internal portion of the caudal flagella is the force-generator of the movements in this region.

Effects of iron limitation on adherence and cell surface carbohydrates of Corynebacterium diphtheriae strains.

Iron limitation may cause bacterial pathogens to grow more slowly; however, it may also stimulate these microorganisms to produce greater tissue damage, given that many virulence factors are controlled by the iron supply in the environment. The present study investigated the influence of low iron availability on the expression of proteins and surface sugar residues of two toxigenic strains of Corynebacterium diphtheriae subsp. mitis and evaluated their adherence to human group B erythrocytes and HEp-2 cells. A comparison was made between bacteria grown in (i) Trypticase soy broth (TSB), (ii) TSB treated with dipyridyl to deplete free iron, and (iii) TSB enriched with FeCl(3). The effects of iron concentration on adhesive properties were different for strains 241 and CDC-E8392, of the sucrose-fermenting and non-sucrose-fermenting biotypes, respectively. Iron-limited conditions enhanced interaction of strain 241 with erythrocytes and HEp-2 cells. Inhibition assays suggested the involvement of nonfimbrial protein combination 67-72p on hemagglutination of diphtheria bacilli grown under iron-limited conditions. Conversely, iron limitation inhibited adherence to glass and expression of electron-dense material on the bacterial surface. Lectin binding assays demonstrated a reduction in the number of sialic acid residues and an increase in D-mannose and D-galactose residues on the surfaces of both strains. Thus, iron exerts a regulatory role on adhesive properties of diphtheria bacilli, and low iron availability modulates the expression of C. diphtheriae surface carbohydrate moieties. The significant changes in the degree of lectin binding specific for D-mannose, D-galactose and sialic acid residues may have an effect on binding of host cells. The expression of dissimilar microbial virulence determinants may be coordinately controlled by common regulatory systems. For C. diphtheriae, the present results imply regulation of adherence and slime production as part of a global response to iron-limited environmental conditions that includes derepression of genes for the synthesis of cytotoxin and siderophores and for transport of the Fe(III)-siderophore complexes.

Comparison of three computer methods of sperm head analysis.

OBJECTIVE: Analysis of sperm heads using three different computer morphometrical tools and experimental conditions to find a more reliable and secure strategy among them. DESIGN: Controlled experiments on sperm morphology analysis from volunteers. SETTING: Laboratory of microscopy and imaging processing. PATIENT(S): Ten human semen samples donated by different zoospermic men. INTERVENTION(S): Semen samples were collected by masturbation after > or =72 hours of abstinence. MAIN OUTCOME MEASURE(S): Spermatozoon head morphology was compared by the use of different video-microscopy systems, three computer programs, and various staining conditions and manipulation by different operators. Nonbiological material in the form of latex beads was also used. RESULT(S): The data obtained suggest that the semiautomatic computer program is the most reliable and secure method for performing sperm analysis, besides the fact that it is a fast process compared with manual methods. CONCLUSION(S): Computer systems of sperm analysis should incorporate a step of interactive object identification to work properly, allowing the operator to confirm or correct possible computer misidentification. The latex beads were used to confirm the capability of all three computer programs to correctly evaluate nonbiological material.

Tubulin diversity in trophozoites of Giardia lamblia.

Giardia lamblia is a diplomonad that parasitizes the small intestine of vertebrates. The trophozoite is multiflagellar and its cytoskeleton presents a complex organization of microtubular structures. One of these, the adhesive disk, consists of a microtubular spiral. The median body, whose function is not yet determined, is also composed by microtubules. The cell has eight flagella and two microtubule sheets, known as the funis. In this study we used several antibodies and immunofluorescence microscopy to help in the characterization of these structures. We observed that Giardia tubulin reacts with antibodies raised against very distinct immunogens. The antibodies used were against: (1) alpha-tubulin from chicken embryo brain, Trypanosoma brucei, sea urchin sperm, Paramecium, acetylated alpha-tubulin from Paramecium, and tyrosinated alpha-tubulin, (2) beta-tubulin from chicken embryo brain and Physarum polycephalum, and (3) an antibody with specificity to beta-tubulin having as immunogen the FtsZ bacterial protein. Each cytoskeletal structure of Giardia presented a distinct pattern of labeling by the several antibodies used. These data indicate that even being considered one of the most ancient of organisms, Giardia shares similarities (at least in relation to tubulin) with other organisms. They also open some questions about the organization and composition of its microtubular structures.

Gold finder: a computer method for fast automatic double gold labeling detection, counting, and color overlay in electron microscopic images.

This work presents a computerized method to identify, detect, evaluate, and, by colored overlay, present gold particle pairs in electron microscopy (EM), even in wide-field views. Double gold immunolabeled specimens were analyzed in a LEO 912 electron microscope equipped with a 2k x 2k-pixel slow-scan cooled CCD camera connected to a computer with analySIS 3.1 PRO image processing software. The acquisition of a high-resolution and high-dynamic-range image by the camera allowed correct segmentation of the gold particles, separating them from other cell structures and from the substrate. Particle identification was performed by a classification module designed by us. Based on shape and size, the computer recognized the group of small particles and classified them as either singular or clustered and differentiated these from the single bigger type. The final image shows the particle types separated and colored, and indicates the total number of objects encountered in the specific region of interest. Moreover, a montage tool allowed us to obtain final representative images of large microscopic fields, which on analysis by the Gold Finder module provided information on the distribution and localization of antigens comparable to that provided by the wide-field light microscope images.

Interaction of Leptomonas wallacei with the intestinal tract of its natural host Oncopeltus fasciatus (Hemiptera: Lygaeidae).

While investigating the distribution of Leptomonas wallacei in the intestine of the insect host Oncopeltus fasciatus, promastigotes and cyst-like forms of L. wallacei were observed only in the midgut ventricles V(3) and V(4) and the hindgut. In video-microscopy, once contact had occurred, the parasites remained attached to the midgut epithelium. Scanning electron microscopy revealed the adhesion of flagellates and cyst-like forms to the midgut wall and to the rectal pads of the hindgut. Using transmission electron microscopy, we observed that adhesion occurred mainly between the flagellum and the perimicrovillar membranes secreted by the midgut epithelium. No modifications were observed either in the parasite or in the epithelial cells. In the hindgut, adhesion to the superficial wax layer of the epithelial cells of the rectal pads was via flagellum. Host cell morphology appeared unaffected by L. wallacei.

Intracellular viability of toxigenic Corynebacterium diphtheriae strains in HEp-2 cells.

Corynebacterium diphtheriae, generally considered an extracellular coloniser, was evaluated for its ability to enter and survive within HEp-2 monolayers by gentamicin protection assay. Intracellular viability of HC01 strain, isolated from endocarditis, was more expressive (2.59%) than observed in 241 (0.21%) and CDC-E8392 (1.93%) strains. Electron microscopy of C. diphtheriae-infected HEp-2 cells revealed intracellular bacteria inside membrane-bound vacuoles. Bacterial internalisation was totally inhibited by 5 microM cytochalasin E and significantly inhibited by 100 microM genistein (P<0.05). Therefore, C. diphtheriae presents the ability to survive within cultured epithelial cells and signalling cascade as well as actin polymerisation are required for entry of diphtheria bacilli into HEp-2 cells.

Video-microscopy observations of fast dynamic processes in the protozoon Giardia lamblia.

Video-microscopy in combination with digital image processing was used to analyze dynamic processes associated to the life cycle of Giardia lamblia trophozoites. These parasites swim and attach to the epithelial cells, producing the disease known as Giardiasis. Giardia is a multiflagellar cell, presenting 4 pairs of flagella. With the use of analogue and digital tools, we observed that in cells attached to glass slides only 2 of the 4 pairs present active beating (wave propagation). The frequency observed was 17-18 Hz to the anterior and 8-11 Hz to the ventral flagella. These data resulted from several hours of recording using both analogue video and high-speed digital camera. The caudal pair did not show active beating patterns and the same holds true for the posterior one. In this latter pair, oscillations were observed, but they were always associated to the transit of the wave produced by the ventral pair. The analysis performed with free moving cells showed that during its forward dislocation, Giardia lamblia presented either a lateral rocking or a complete rotational (tumbling) movement around its longitudinal axis. A dislocation of the caudal region of the cell both in the lateral and dorso-ventral direction was observed. This movement was completely independent from the flagellar beating and it is likely to be produced by a microtubular complex located in the caudal portion of the cell. The adhesion process of Giardia lamblia was also followed by video-microscopy and the data showed that the ventral disk had an active participation in this process.

The effects of the antiprotozoal drugs metronidazole and furazolidone on trophozoites of Giardia lamblia (P1 strain).

The effects of metronidazole and furazolidone on Giardia lamblia trophozoites were analyzed by video-light and transmission electron microscopy. In addition, growth curves were drawn based on four concentrations of the drugs. The IC50 was 4.6 microM for metronidazole and 2.9 microM for furazolidone. By light microscopy we observed that metronidazole-treated cells maintained the characteristic body shape, but many showed bubbles in the dorsal and ventral surfaces. The effects of furazolidone include changes in the morphology (the cells were roundish) and also cytoplasmic extrusions. When observed by transmission microscopy, cells treated with metronidazole appeared rounder than usual and membranous structures were observed in the cytoplasm. Cells treated with furazolidone showed the cytoplasm depleted of its contents and great changes in volume. Our results show that furazolidone was more effective than metronidazole and its effects were observed in cells treated with 1 microg/ml (the lowest concentration) as early as 6 h after the start of exposure.