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Attachment of Giardia lamblia trophozoites to enterocytes is essential for colonization of the small intestine and is considered a prerequisite for parasite-induced enterocyte dysfunction and clinical disease. In this work, coincubation of Giardia with Int-407 cells, was used as an in vitro model to study the role of cytoskeleton and surface lectins involved in the attachment of the parasite. This interaction was also studied by scanning and transmission electron microscopy. Adherence was dependent on temperature and was maximal at 37°C. It was reduced by 2.5 mM colchicine (57%), mebendazole (10 g/ml) (59%), 100 mM glucose (26%), 100 mM mannose (22%), 40 mM mannose-6-phosphate (18%), and concanavalin A (100 g/ml) (21%). No significant modification was observed when Giardia was pretreated with cytochalasins B and D and with EDTA. Giardia attachment was also diminished by preincubating Int-407 cells with cytochalasin B and D (5 g/ml) (16%) and by glutaraldehyde fixation of intestinal cells and of G. lamblia trophozoites (72 and 100%, respectively). Ultrastructural studies showed that Giardia attaches to the Int-407 monolayer predominantly by its ventral surface. Int-407 cells contact trophozoites with elongated microvilli, and both trophozoite imprints and interactions of Giardia flagella with intestinal cells were also observed. Transmission electron microscopy showed that Giardia lateral crest and ventrolateral flange were important structures in the adherence process. Our results suggest a combination of mechanical and hydrodynamic forces in trophozoite attachment; surface lectins also seem to mediate binding and may be involved in specific recognition of host cells.Giardia lamblia, a parasitic flagellated protozoan, is the most common causative agent of diarrheal illness worldwide. In spite of significant recent advances in the knowledge on the biochemistry and molecular biology of G. lamblia, little is known about the pathogenesis of symptomatic infections in humans and the factors that determine the variability of the clinical outcome. A combination of parasitic factors and host responses seems to be involved, but damage of the intestinal epithelium by adherent trophozoites of G. lamblia has been proposed as one important mechanism in the pathogenesis of the infection (21). The structural modifications produced by G. lamblia trophozoites on epithelial cells are the result of close attachment of a contractile region of the ventral disk (30).The mechanism of attachment of trophozoites to intestinal cells has not been established definitively. Evidence supports roles for the ventral disk, which is considered a specific attachment organelle (19), trophozoite contractile elements (12), hydrodynamic and mechanical forces (20), and lectin-mediated binding (8, 26). However, experimental verification has been hindered by the lack of a suitable model. Previous studies of adherence have used a variety of model systems, including synthetic surfaces such as plastic and glass, nonhuman cells such as isolated rat enterocyt...

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A quantitative, simple, and rapid assay has been developed to assess Giardia lamblia trophozoite sensitivity to metronidazole [1-(2-hydroxyetyl)-2-methyl-5-nitroimidazole] (MTZ). This new assay utilizes the ability of live (surviving) trophozoites to take up oxygen after have been exposed to MTZ. The effect of MTZ on oxygen uptake was compared with its effect on viability as evaluated by a culture method and morphological assays. Oxygen uptake rates decreased in trophozoites treated with MTZ, and this effect was drug concentration dependent: O2 uptake rates went from 3.04 μM O2 min−1 per 106 cells to 0.72 μM O2 min−1 per 106 cells with increasing drug concentration (0.15 to 0.6 mM) in the preincubation. Concentrations of the drug which inhibited oxygen uptake by 28 to 76% in trophozoites killed from 39 to 82% of trophozoites, as evaluated by the culture method, and altered the morphology of 21 to 86% of the trophozoites. Thus, the trophozoites killed by MTZ are nonmotile cells and do not take up oxygen. A good correlation was found between the inhibitory effects of MTZ, as evaluated by oxygen uptake, and cellular viability. Similar 50% inhibitory concentrations were obtained: 0.33 mM by oxygen uptake, 0.26 mM by the culture method, and 0.35 mM by morphological criteria. Oxygen uptake appears to be a good indicator of parasite viability. Therefore, this new method can provide a convenient means to assess MTZ susceptibility in G. lamblia and can be applied for screening potential antigiardial agents.

The cytotoxicity of ciprofloxacin was investigated in culture of Giardia lamblia (ATCC 30957), used as an in vitro cellular model, on the basis of cell growth, morphology, viability, adherence and metabolic studies. The effects on cell membrane integrity were evaluated by permeability of the cells to trypan blue, and the morphological alterations were studied by optical and transmission electronic microscopy. The metabolic studies were performed by measured oxygen uptake with a Clark-type oxygen electrode. Our data show that ciprofloxacin induces loss of viability with morphological alterations and loss of membrane integrity. Ultrastructural observations revealed that this drug promoted modifications on the cell shape, pronounced dorsal vesiculation, plasma membrane blebbing, disaggregation of ribosome, coagulation of cytoplasmic matrix, and heavy deposit of electron-dense precipitates on the cytoplasm and nucleus. Ciprofloxacin also inhibits parasite growth, adherence and O 2 uptake in a concentrationdependent manner. Some of these observations suggest that cytotoxicity of ciprofloxacin results in cell death by a necrosis process. We demonstrate that ciprofloxacin has a lethal effect in G. lamblia trophozoites and could be used as an alternative drug in giardiasis treatment, particularly in infections that are resistant to other antibiotics. #