Comparative studies of the anti-leishmanial activity of three <Emphasis Type="Italic">Crotalus durissus</Emphasis> ssp. venoms

In this study, we compared the anti-leishmanial activity of three crotalic venoms (Crotalus durissus terrificus–Cdt, Crotalus durissus cascavella–Cdca, and Crotalus durissus collilineatus–Cdcol). Different concentrations of each venom incubated with Leishmania (Leishmania) amazonensis promastigotes were used. Cdt venom exhibited a higher anti-leishmanial activity (Inhibitory concentration-IC50-value of 4.70 ± 1.72 μg/ml) in comparison with that of Cdca venom (IC50 value of 9.41 ± 1.21 μg/ml), while Cdcol venom increased parasite numbers in 50% at a concentration of 44.30 ± 2.18 μg/ml. In addition, this venom showed a low anti-leishmanial activity in higher concentrations (IC50 value of 281.00 ± 9.50 μg/ml). The main fractions of Cdca venom were isolated and assayed under similar conditions used for assessing crude venom. The most active fractions were gyroxin and crotamine that had IC50 values of 3.80 ± 0.52 μg/ml and 19.95 ± 4.21 μg/ml, respectively. Convulxin also inhibited parasite growth rate, although this effect was not dose-dependent. Crotoxin was the least effective fraction with an IC50 value of 99.80 ± 2.21 μg/ml. None of the protein fractions presented cytotoxic effects against J774 cells in culture. In vivo assays using BALB/c mice revealed that crotoxin and crotamine were the main toxic fractions. In conclusion, C. durissus cascavella venom has three main fractions with anti-leishmanial activity. These results open new possibilities to find proteins that might be used as possible agents against cutaneous leishmaniasis.     

Synergistic Effect of Febantel and Pyrantel Embonate in Elimination of Giardia in a Gerbil Model

The objectives of the study were to determine the optimal dose of febantel, pyrantel embonate and a combination of febantel/pyrantel embonate required to effectively treat Giardia in a gerbil model and to determine if there is a synergistic effect with the two drugs. SPF gerbils were infected by oral inoculation with 105 Giardia duodenalis trophozoites (day 0). On days 5 to 7, animals (n = 6) were treated once daily via oral gavage with febantel, pyrantel embonate, febantel and pyrantel embonate, metronidazole or placebo. Gerbils were euthanised 24 hours after last treatment and duodenal trophozoites were enumerated on a haemocytometer to obtain a concentration of trophozoites/ cm of gut. Febantel alone, effectively eliminated Giardia trophozoites at 160 and 80 mg/kg. Pyrantel embonate did not eliminate Giardia from the animals but significantly reduced parasite counts at all dosages. Febantel combined with pyrantel embonate effectively eliminated Giardia trophozoites at 160, 80 and 40 mg/kg. Metronidazole did not eliminate Giardia trophozoites from the gut. All placebo-treated animals were heavily infected with Giardia trophozoites. It can be concluded that febantel is more effective in elimination of Giardia infections when combined with pyrantel embonate compared to the agents used alone.     

Adherence and multiplication ofGiardia intestinalis on human enterocyte-like differentiated cells in vitro

This report describes a technique for studying the adherence and growth ofGiardia intestinalis trophozoites (strains PARIS/86/LCF/1, PARIS/86/LCF/2 and PARIS/88/LCF/8) using the human colon carcinoma cell line Caco2.Giardia trophozoites were cultured with Caco2 cells in a modified HSP₃ culture medium. The biochemical differentiation of Caco2 cells was established by an increase in sucrase isomaltase activities to values of 4.51±0.90 and 10.39±3.00 milliunits/mg protein for 8- and 12-day-old cultures, respectively.Giardia, adherence to 8- and 12-day-old Caco2 cells reached a value of >75% after 60 and 30 min, respectively. Adherence diminished significantly at 24° C and was almost undetectable at 4° C. Depletion of divalent cations reduced the proportion of adherent trophozoites by up to 46%. Adherence was pH-independent between pH 6.0 and 7.6. Parasite growth increased when Caco2 cell monolayers were used instead of exenic cultures. This in vitro human cell model may contribute to the study of the mechanisms and factors involved in the host-parasite interaction.     

The application of on-chip optofluidic microscopy for imaging Giardia lamblia trophozoites and cysts

The optofluidic microscope (OFM) is a lensless, low-cost and highly compact on-chip device that can enable high-resolution microscopy imaging. The OFM performs imaging by flowing/scanning the target objects across a slanted hole array; by measuring the time-varying light transmission changes through the holes, we can then render images of the target objects at a resolution that is comparable to the holes’ size. This paper reports the adaptation of the OFM for imaging Giardia lamblia trophozoites and cysts, a disease-causing parasite species that is commonly found in poor-quality water sources. We also describe our study of the impact of pressure-based flow and DC electrokinetic-based flow in controlling the flow motion of Giardia cysts—rotation-free translation of the parasite is important for good OFM image acquisition. Finally, we report the successful microscopy imaging of both Giardia trophozoites and cysts with an OFM that has a focal plane resolution of 0.8 microns.     

Adherence of Giardia lamblia Trophozoites to Int-407 Human Intestinal Cells

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 μg/ml) (59%), 100 mM glucose (26%), 100 mM mannose (22%), 40 mM mannose-6-phosphate (18%), and concanavalin A (100 μg/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 μg/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.     

Anti-Giardia activity of phenolic-rich essential oils: effects of Thymbra capitata, Origanum virens, Thymus zygis subsp. sylvestris, and Lippia graveolens on trophozoites growth, viability, adherence, and ultrastructure

The present work evaluates the anti-Giardia activity of phenolic-rich essential oils obtained from Thymbra capitata, Origanum virens, Thymus zygis subsp. sylvestris chemotype thymol, and Lippia graveolens aromatic plants. The effects were evaluated on parasite growth, cell viability adherence, and morphology. The tested essential oils inhibited the growth of Giardia lamblia. T. capitata essential oil is the most active followed by O. virens, T. zygis subsp. sylvestris, and L. graveolens oils. The tested essential oils at IC50 (71–257) μg/ml inhibited parasite adherence (p < 0.001) since the first hour of incubation and were able to kill almost 50% of the parasites population in a time-dependent manner. The main ultrastructural alterations promoted by essential oils were deformations in typical trophozoite appearance, often roundly shape, irregular dorsal and ventral surface, presence of membrane blebs, electrodense precipitates in cytoplasm and nuclei, and internalization of flagella and ventral disc. Our data suggest that essential oils induced cell death probably by processes associated to the loss of osmoregulation caused by plasmatic membrane alterations. Experiments revealed that the essential oils did not present cytotoxic effects in mammalian cells. In conclusion, T. capitata, O. virens, T. zygis subsp. sylvestris chemotype thymol, and L. graveolens essential oils have antigiardial activity in vitro and seem to have potential for the treatment of the parasitic disease caused by the protozoan G. lamblia.     

The effects of dihydroartemisinin on Giardia lamblia morphology and cell cycle in vitro

Several anti-Giardia drugs, such as metronidazole, tinidazole, mebendazole, albendazole and furazolidone, are usually effective but have severe side effects and potential toxicity. An urgent need exists for more effective and less toxic agents that can act against this protozoan. For this purpose, the effects of dihydroartemisinin (DHA) on Giardia lamblia were investigated in vitro. Axenically grown G. lamblia trophozoites were treated with DHA (LD₅₀ = 200 μg/mL) at different time intervals. The morphological and ultrastructural changes of the treated trophozoites were observed by both light and transmission electron microscopy (TEM). Changes in the cell cycle of the treated cells were observed by flow cytometry. By light microscopy, we observed that DHA-treated trophozoites were detached from the wall of the culture tube and shown bradypraxia and bubbles in the dorsal and ventral surfaces. Ultrastructural observations by TEM revealed that DHA promoted modifications of the cell shape, pronounced dorsal vesiculation, plasma membrane blebbing, disaggregation of ribosomes, depletion of cytoplasmic matrix and heavy deposition of electron-dense precipitates on the cytoplasm and nucleus. The main changes observed in the treated group included the following: (1) trophozoites were rounder in shape and the endoplasmic reticulum was dilated, (2) enlarged trophozoites contained lamella structures and deformed nuclei, (3) trophozoites displayed dissolved cytoplasm with large vacuole spaces or decreased cytoplasmic volume, (4) adhesive disc bubbles or the lamella structures of cytoplasm were clearly observed, and (5) cell division was arrested. Using microscopy and cytometry techniques, we demonstrate that changes in G. lamblia morphology and cell cycle state were induced by DHA     

The anti-amoebic activity of some medicinal plants used by AIDS patients in southern Thailand

The anti-amoebic activities of chloroform, methanol and water extracts from 12 Thai medicinal plants (39 extracts) commonly used by AIDS patients in southern Thailand were screened, at a concentration of 1,000 μg/ml, against Entamoeba histolytica strain HTH-56:MUTM and strain HM1:IMSS growing in vitro. The extracts were incubated with 2×10⁵ E. histolytica trophozoites/ml of medium at 37°C under anaerobic conditions for 24 h. The cultures were examined with an inverted microscope and scored (1–4) according to the appearance and numbers of the trophozoites. The extracts that caused inhibition were selected and retested using the same conditions but with concentrations that ranged from 31.25 to 1,000 μg/ml using E. histolytica strain HM1:IMSS, and the IC₅₀ values for each extract were calculated. The chloroform extracts from Alpinia galanga (IC₅₀ 55.2 μg/ml), Barleria lupulina (IC₅₀ 78.5 μg/ml), Boesenbergia pandurata (IC₅₀ 45.8 μg/ml), Piper betle (IC₅₀ 91.1 μg/ml) and Piper chaba (IC₅₀ 71.4 μg/ml) and the methanol extract from B. pandurata (IC₅₀ 57.6 μg/ml) were all classified as “active”, i.e. with an IC₅₀ of less than 100 μg/ml, whereas those from Murraya paniculata (IC₅₀ 116.5 μg/ml) and Zingiber zerumbet (IC₅₀ 196.9 μg/ml) were classified as being “moderately active”. The IC₅₀ of a standard drug, metronidazole, was 1.1 μg/ml.     

In vitro and in vivo antileishmanial efficacy of nitazoxanide against Leishmania donovani

Control of Leishmania infection relies primarily on chemotherapy, and the current drug available for treating leishmaniasis is limited. Nitazoxanide (NTZ) is a broad spectrum antiparasitic agent with activity against protozoa, nematodes, cestodes, and trematodes. In the present study, the in vitro antileishmanial efficacy of NTZ was evaluated by incubation of Leishmania donovani promastigotes with NTZ, indicating that NTZ can affect the ultrastructure of parasite promastigote and efficiently inhibit the parasite growth. Moreover, 200 μg/ml NTZ inhibited >90% of promastigotes growth, showing similar activity of the reference drug amphotericin B (P > 0.05). Therapeutic efficacy of NTZ against L. donovani-infected BALB/c mice demonstrated that oral NTZ produced a significant reduction of parasite burden in spleen and liver from L. donovani-infected mice, compared with the untreated mice (P < 0.05). These results indicated NTZ may be a novel therapeutic drug for leishmaniasis.     

Reduced Release of DNA from Streptococcus pneumoniae After Treatment with Rifampin in Comparison to Spontaneous Growth and Ceftriaxone Treatment

 In order to study the release of DNA from Streptococcus pneumoniae in vitro during spontaneous growth and treatment with ceftriaxone or rifampin, a semiquantitative polymerase chain reaction was used. During spontaneous growth, high concentrations of bacterial DNA were released. Exposure to 10 μg/ml of ceftriaxone decreased the DNA release, in median, by 19 times (P=0.03 vs. spontaneous growth). Treatment with 10 μg/ml of rifampin led to a reduction of DNA release, in median, by a factor of 49 (P=0.03 vs. ceftriaxone; six experiments performed on different days).     

Influence of Antibodies in Mother’s Milk on Antigenic Variation of Giardia lamblia in the Murine Mother-Offspring Model of Infection

In the present study, neonatal ZU.ICR mice and their mothers were infected with trophozoites of Giardia lamblia clone GS/M-83-H7 expressing the variant surface protein (VSP) H7. The infection experiments included a detailed analysis of the specificities of anti-Giardia immunoglobulin A (IgA) antibodies in mother’s milk and a determination of the effects of the milk antibodies on both the growth of the parasite during in vitro cultivation and colonization of the parasite within the intestine of suckling offspring. These investigations revealed that transiently emerging milk IgA antibodies against a variant-specific 314-amino-acid N-terminal region of VSP H7 exhibit a strong parasiticidal effect on VSP H7-type trophozoites both in vitro and in vivo. These findings indicated that parasiticidal effects of local IgA antibodies against the N-terminal part of VSP H7 select for new variant types within the intestinal parasite population of suckling mice. The selective influence of such antibodies promotes in vivo antigenic variation of G. lamblia clone GS/M-83-H7 and modulates the early course of parasite infection in these animals.     

Effect of the antitubulin drug oryzalin on the encystation of Entamoeba invadens

We have recently demonstrated that the antimicrotubule drug oryzalin inhibits the growth of Entamoeba invadens as well as E. histolytica, the former being more resistant to the drug than the latter, and that effective doses of oryzalin are higher for Entamoeba than for the other parasitic protozoa examined thus far. The aim of the present study was to examine the effect of oryzalin on the encystation of E. invadens using an axenic encystation system in vitro. Oryzalin inhibited the encystation of E. invadens strain IP-1 in a dose-dependent manner. The addition of oryzalin after the induction of encystation was also inhibitory for encystation and cyst maturation. Trophozoites incubated for 1 day in encystation medium with oryzalin did not encyst after removal of the drug. Although trophozoites grown in the presence of 300 μM oryzalin for 2 days did not encyst after their transfer to encystation medium containing the same concentration of drug, a number of trophozoites survived for at least 3 days. In contrast, trophozoites grown in the absence of oryzalin neither survived nor encysted after their transfer to encystation medium supplemented with the drug, which suggests that pretreatment of trophozoites with oryzalin contributes to their continued survival as trophozoites, i.e., without their transforming into cysts, in encystation medium. Trophozoites grown with oryzalin did encyst after their transfer to encystation medium without the drug. Accumulation of trophozoites in the mitotic phase was observed after culture with oryzalin. When cysts prepared at day 1 of encystation, most of which were mononucleate, were reincubated in the presence of oryzalin for an additional 2 days, inhibition of their maturation was observed. Thus, oryzalin is a potent mitotic-phase inhibitor of E. invadens and may become a useful tool for studies on the relationship between the cell cycle and encystation of this parasite. Received: 29 November 1999 / Accepted: 28 January 2000     

Efficacy of ginkgolic acids against Cryptosporidium andersoni in cell culture

Cryptosporidium is a worldwide waterborne parasite and the treatment is a severe problem in immunocompromised patients. In this study, we used the in vitro culture system to evaluate the anti-Cryptosporidium activity of ginkgolic acids (GAs), nitazoxanide (NTZ), garlicin (GAR), and artemether (ART). The growth of Cryptosporidium andersoni in HCT-8 cells was determined by real-time PCR assay. When exposed to 5.00 μg/ml GAs or 10.00 μg/ml NTZ for 48 h, the number of C. andersoni in cultures was on a very low lever, but the number of parasites did not significantly decrease when exposed to GAR and ART. Our results indicate that GAs could be a potential drug for the treatment of cryptosporidiosis.     

Gedunin and photogedunin of Xylocarpus granatum possess antifilarial activity against human lymphatic filarial parasite Brugia malayi in experimental rodent host

The present study is aimed to evaluate antifilarial activity of Xylocarpus granatum (fruit from Andaman) against human lymphatic filarial parasite Brugia malayi in vivo. The in vitro antifilarial activity has already been reported earlier for this mangrove plant which has traditionally been used against several ailments. Aqueous ethanolic crude extract, four fractions (ethyl acetate fraction, n-butanol fraction, water-soluble fraction and water-insoluble fraction) and pure molecule/s of X. granatum (fruit) were tested in vitro on adult worms and microfilariae (mf) of B. malayi and the active samples were further evaluated in vivo in B. malayi (intraperitoneally) i.p. transplanted in the jird model (Meriones unguiculatus) and Mastomys coucha subcutaneously infected with infective larvae (L3). The crude aqueous ethanolic extract was active in vitro (IC50: adult = 15.46 μg/ml; mf = 13.17 μg/ml) and demonstrated 52.8% and 62.7% adulticidal and embryostatic effect on B. malayi, respectively, in Mastomys at a dose of 5 × 50 mg/kg by oral route. The antifilarial activity was primarily localized in the ethyl acetate-soluble fraction which revealed IC50 of 8.5 and 6.9 μg/ml in adult and mf, respectively. This fraction possessed moderate adulticidal and embryostatic action in vivo in Mastomys. Out of eight pure molecules isolated from the active fraction, two compounds gedunin (IC50 = 0.239 μg/ml, CC50 = 212.5 μg/ml, SI = 889.1) and photogedunin (IC50 = 0.213 μg/ml, CC50 = 262.3 μg/ml, SI = 1231.4) at 5 × 100 mg/kg by subcutaneous route revealed excellent adulticidal efficacy resulting in to the death of 80% and 70% transplanted adult B. malayi in the peritoneal cavity of jirds respectively in addition to noticeable microfilaricidalo action on the day of autopsy. The findings reveal that the extract from the fruit X. granatum contains promising in vitro and in vivo antifilarial activity against human lymphatic filarial parasite B. malayi which could be attributed to the presence of two pure compounds gedunin and photogedunin.     

Description of the gamonts of a small species of<Emphasis Type="Italic"> Hepatozoon</Emphasis> sp. (Apicomplexa,Hepatozoidae) found in<Emphasis Type="Italic"> Crotalus durissus terrificus</Emphasis> (Serpentes,Viperidae)

A small species of the genus Hepatozoon found in a specimen of Crotalus durissus terrificus from the Botucatu region, São Paulo State, Brazil is described. The morphologic alterations induced in the snake’s erythrocytes by the presence of this parasite are described. Morphology and morphometric analyses were performed using the Qwin Lite 2.5 computerized image analysis system (Leica). The Hepatozoon possessed a small and short body (8.1±0.5 μm long and 3.8±0.4 μm wide), with round extremities. The cytoplasm varied from pale blue to basophilic and had no granulations. Its nucleus was large, occupied a large area of the cytoplasm, and was irregular in shape and not condensed. Despite its small size, this parasite induced important changes in the host cell. Total parasitemia observed was 56.6%.     

Antigiardial activity of <Emphasis Type="Italic">Ocimum basilicum</Emphasis> essential oil

In this study, we investigated the effects of Ocimum basilicum essential oil on Giardia lamblia and on the modulation of the interaction of these parasites by peritoneal mouse macrophage. The essential oil (2 mg/ml) and its purified substances demonstrated antigiardial activity. Linalool (300 μg/ml), however, was able to kill 100% parasites after 1 h of incubation, which demonstrates its high antigiardial potential. Pretreatment of peritoneal mouse macrophages with 2 mg/ml essential oil dilution reduced in 79% the association index between these macrophages and G. lamblia, with a concomitant increase by 153% on nitric oxide production by the G. lamblia-ingested macrophages. The protein profiles and proteolitic activity of these parasite trophozoites, previously treated or not with 2 mg/ml essential oil or with the purified fractions, were also determined. After 1 and 2 h of incubation, proteins of lysates and culture supernatants revealed significant differences in bands patterns when compared to controls. Besides, the proteolitic activity, mainly of cysteine proteases, was clearly inhibited by the essential oil (2 mg/ml) and the purified linalool (300 μg/ml). These results suggest that, with G. lamblia, the essential oil from O. basilicum and its purified compounds, specially linalool, have a potent antimicrobial activity. Igor de Almeida and Daniela Sales Alviano contributed equally to this study.     

Preliminary experiments on use of zebrafish as a laboratory model for <Emphasis Type="Italic">Giardia duodenalis</Emphasis> infection

Although Giardia duodenalis is considered a parasite of mammals, different genotypes have been identified as infecting several species of freshwater and marine fish in Australia. Establishment of G. duodenalis infection in common laboratory zebrafish (Danio rerio), could provide an excellent tool for a range of studies on Giardia. We conducted preliminary experiments to investigate this possibility. Zebrafish were inoculated with viable G. duodenalis cysts from two different Assemblages (A and D) using a modified oro-gastric tube. Direct microscopy and immunofluorescent antibody test were used to check for Giardia cysts/trophozoites in the intestine, and histology was performed on intestinal mucosa to evaluate possible pathological changes. Giardia cysts were successfully deposited in the zebrafish alimentary tract using a modified oro-gastric tube, and were maintained in the fish gut for at least 8 days. Although a single trophozoite was observed in one fish three days post-exposure, we were unable to demonstrate established, propagative infection under the conditions tested.     

Development and validation of a cell culture based assay for in vitro assessment of anticryptosporidial compounds

In vitro culture of Cryptosporidium parvum oocysts in HCT-8 cells was combined with immunofluorescent labelling and digital image analysis to quantify the development of the parasite by detecting and measuring the labelled area in the respective cell cultures. The number of inoculated oocysts and the labelled area correlated reliably and significantly (R ², 0.98–0.99). The effects of various concentrations of halofuginone bromide (0.00039 to 50 μM) and monensin (0.00225 to 0.144 μM) on in vitro parasite development were determined in further trials in cultures inoculated each with 10⁵ oocysts. Monensin reduced the detected area in a dose-dependant manner. In comparison to the untreated controls, the area positive for C. parvum in the cultures treated with 0.144 to 0.009 μM monensin reached a maximum of 17%, and inhibition of 40% was observed at 0.0045 μM. Halofuginone bromide also efficiently inhibited parasite in vitro reproduction, albeit at higher concentrations. At 12.5 μM or more, inhibition was at least 90%; 0.05 μM still yielded 80% inhibition, whereas at concentrations below 0.00625 μM, labelled areas abruptly increased. Both drugs appeared efficient under in vitro conditions; the applied system is suited to screen drugs for their anti-cryptosporidial capacity.     

Increased Adherence of Fluconazole-Resistant Isolates of Candida Species to Explanted Esophageal Mucosa

 The adherence of fluconazole-resistant and fluconazole-susceptible isolates of Candida albicans to explanted rabbit esophageal mucosa was examined in vivo. Among six Candida albicans isolates collected from HIV-infected patients, three fluconazole-resistant (MIC>64 μg/ml) isolates attached more avidly than three fluconazole-susceptible strains (MIC≤0.5 μg/ml) to esophageal mucosa (P≤0.05). When three strains each of six different Candida spp. were compared, the more inherently fluconazole-resistant isolates adhered more avidly in the following order: Candida glabrata>Candida krusei>Candida albicans fluconazole-sensitive >Candida tropicalis>Candida parapsilosis. Nonetheless, fluconazole-resistant Candida albicans demonstrated the greatest degree of adherence in comparison to all fluconazole-susceptible Candida albicans (P<0.001) and to all Candida spp. tested (P<0.001). Thus, the refractoriness of esophageal candidiasis in patients infected with fluconazole-resistant isolates may be related to both in vitro drug resistance and increased mucosal adherence.     

Heterogeneity in the sensitivity of microtubules of Giardia lamblia to the herbicide oryzalin

Giardia lamblia is a protozoan that inhabits the small intestine of vertebrates, attaching to the epithelial cells by means of cytoskeletal elements. G. lamblia trophozoites possess several microtubular structures, namely the adhesive disc, the median body, the funis and the four pairs of flagella. Several drugs that target cytoskeletal proteins have been used in the study of cytoskeletal function and dynamics. In this work, we used oryzalin, which binds to α-tubulin, as a tool to study the Giardia cytoskeleton. The trophozoites were treated with oryzalin, and its effects were analysed by immunofluorescency, transmission and scanning electron microscopies. Oryzalin inhibited Giardia proliferation. Treated cells were not able to complete cell division and had flagella showing extensive shortening. Strikingly, the drug did not interfere with the adhesive disc, in contrast to what happens when other drugs are used.