Recent insights into the mucosal reactions associated with Giardia lamblia infections

Giardia lamblia is an intestinal protozoan parasite infecting humans and various other mammalian hosts. The most important clinical signs of giardiasis are diarrhoea and malabsorption. Giardia lamblia is able to undergo continuous antigenic variation of its major surface antigen, named VSP (variant surface protein). While intestinal antibodies, and more specifically anti-VSP IgA antibodies, were proven to be involved in modulating antigenic variation of the parasite the participation of the local antibody response in control of the parasite infection is still controversial. Conversely, previous studies based on experimental infections in mice showed that cellular immune mechanisms are essential for elimination of the parasite from its intestinal habitat. Furthermore, recent data indicated that inflammatory mast cells have a potential to directly, or indirectly, interfere in duodenal growth of G. lamblia trophozoites. However, this finding was challenged by other reports, which did not find a correlation between intestinal inflammation and resistance to infection. Since intestinal infiltration of inflammatory cells and/or CD8+T-cells were demonstrated to coincide with villus-shortening and crypt hyperplasia immunological reactions were considered to be a potential factor of pathogenesis in giardiasis. The contribution of physiological factors to pathogenesis was essentially assessed in vitro by co-cultivation of G. lamblia trophozoites with epithelial cell lines. By using this in vitro model, molecular (through surface lectins) and mechanical (through ventral disk) adhesion of trophozoites to the epithelium was shown to be crucial for increased epithelial permeability. This phenomenon as well as other Giardia-induced intestinal abnormalities such as loss of intestinal brush border surface area, villus flattening, inhibition of disaccharidase activities, and eventually also overgrowth of the enteric bacterial flora seem to be involved in the pathophysiology of giardiasis. However, it remains to be elucidated whether at least part of these pathological effects are causatively linked to the clinical manifestation of the disease.     

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.     

Virulence of Giardia lamblia: an in vitro study on host-parasite interaction

Trophozoites of G. lamblia, a human parasite, were lysed by polymorphonuclear leukocytes (PMNL) of healthy individuals during in vitro interaction. However, the parasite damaged PMNL of giardiasis patients. A prior treatment of giardia trophozoites with anti-giardia serum, caused agglutination of pathogen and, thereby, the cytotoxic capacity of the parasite was reduced. Interaction of giardia-trophozoites with peritoneal macrophage, derived from infected mouse, reduced the phagocytic activity of the latter to 43% (against 100% in control). Macrophage activity was, however, stimulated to 131% when the mice were immunized with giardia antigen prior to experimental infection. Giardia extract proved cytotoxic at a dose of 0.7 mg, to HeLa cells in tissue culture. These in vitro studies offer experimental evidence of the cytotoxic and immuno-toxic behaviour of G. lamblia towards the host cells.     

Giardia lamblia: evaluation of the in vitro effects of nocodazole and colchicine on trophozoites

Giardia lamblia is the most commonly detected parasite in the intestinal tract of humans and other mammals causing giardiasis. Giardia presents several cytoskeletal structures with microtubules as major components such as the ventral adhesive disk, eight flagella axonemes, the median body and funis. Many drugs have already been tested as antigiardial agents, such as albendazole and mebendazole, which act by specifically inhibiting tubulin polymerization and hence microtubule assembly. In the present work, we used the microtubule inhibitors nocodazole and colchicine in order to investigate their direct and indirect effects on Giardia ultrastructure and attachment to the glass surface, respectively. Axenically grown G. lamblia trophozoites were treated with nocodazole or colchicine for different time intervals and analyzed by light and electron microscopy. It was observed that trophozoites became completely misshapen, detached from the glass surface and failed to complete cell division. The main alterations observed included disc fragmentation, presence of large vacuoles, and appearance of electrondense deposits made of tubulin. The cytokinesis was blocked, but not the karyokinesis, and membrane blebs were observed. These findings show that Giardia behavior and cytoskeleton are clearly affected by the commonly used microtubule targetting agents colchicine and nozodazole.     

Intestinal mucus protects Giardia lamblia from killing by human milk

We have previously shown that nonimmune human milk kills Giardia lamblia trophozoites in vitro. Killing requires a bile salt and the activity of the milk bile salt-stimulated lipase. We now show that human small-intestinal mucus protects trophozoites from killing by milk. Parasite survival increased with mucus concentration, but protection was overcome during longer incubation times or with greater milk concentrations. Trophozoites preincubated with mucus and then washed were not protected. Protective activity was associated with non-mucin CsCl density gradient fractions. Moreover, it was heat-stable, non-dialyzable, and non-lipid. Whereas whole mucus inhibited milk lipolytic activity, protective mucus fractions did not inhibit the enzyme. Furthermore, mucus partially protected G. lamblia trophozoites against the toxicity of oleic acid, a fatty acid which is released from milk triglycerides by lipase. These studies show that mucus protects G. lamblia both by inhibiting lipase activity and by decreasing the toxicity of products of lipolysis. The ability of mucus to protect G. lamblia from toxic lipolytic products may help to promote intestinal colonization by this parasite.     

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.     

Interactions of Giardia lamblia with human intestinal mucus: enhancement of trophozoite attachment to glass

Giardia lamblia trophozoites frequently are associated with mucus in vivo. We investigated the effects of human intestinal mucus on parasite attachment and survival in vitro. All samples of mucus from the duodenum and ileum (from four humans and two rabbits) enhanced attachment at 100 micrograms/ml. Attachment increased with mucus concentrations from 1 to 1000 micrograms/ml but declined toward the unstimulated level at concentrations above 1000 micrograms/ml. Mucus from the small intestine also promoted the survival of the parasites during the 2-h incubation. In contrast, colonic mucus promoted survival, but inhibited attachment. Fractionation of mucus from the human small intestine by cesium chloride equilibrium density gradient ultracentrifugation revealed that both attachment- and survival-promoting activities were in the low density, protein-rich fraction. The high density fractions containing the mucins were devoid of activity. Thus, a non-mucin fraction of mucus from the human small intestine may promote colonization by G. lamblia.     

Respiration in the cysts and trophozoites of Giardia muris

Cysts and trophozoites of the parasitic protozoon Giardia muris both showed respiratory activity but respiration in cysts was only 10 to 20% that of trophozoites. The O2 dependence of respiration in cysts and trophozoites showed O2 maxima above which respiration decreased. The O2 concentration at which the respiration rate was greatest was higher for cysts than trophozoites. The effects of various inhibitors on cyst and trophozoite respiration suggested that flavoproteins and quinones play some role in respiration. The substrate specificities and the effects of inhibitors on G. muris trophozoites were similar to those observed for Giardia lamblia. Metronidazole, the drug most commonly used in the treatment of giardiasis completely inhibited respiration and motility in trophozoites; however, it had no effect on either respiration or viability in cysts. Menadione, a redox cycling naphthoquinone, stimulated then completely inhibited respiration in cysts and trophozoites; a complete loss of cyst viability or trophozoite motility was also observed. The effects of menadione on G. muris may indicate that redox cycling compounds have potential as chemotherapeutic agents for the treatment of giardiasis.     

Lactobacillus casei as a probiotic in malnourished Giardia lamblia-infected mice: a biochemical and histopathological study

The study describes the in vivo activity of Lactobacillus casei in malnourished Giardia lamblia-infected BALB/c mice. By experimentation, it was found that daily administration of the probiotic 7 days before inoculation with Giardia trophozoites in malnourished mice efficiently reduced both the severity and duration of giardiasis. More specifically, excretion of Giardia cysts and trophozoites counts were reduced, while faecal lactobacilli counts increased significantly in probiotic-fed malnourished mice, compared with control mice. Interestingly, it was also observed that oral feeding of the probiotic to malnourished mice abrogated all the anthropometric and biochemical anomalies. Histologically, morphological and cellular alteration of microvillus membrane integrity revealed that probiotic administration ameliorated the mucosal damage in malnourished, probiotic-inoculated, Giardia-infected mice compared with the severe microvillus atrophy, ?dematous and vacuolated epithelial cells, and ileitis in malnourished Giardia-infected mice. The results clearly show the antigiardial effect of the probiotic in vivo by modulating the gut cells to inhibit the colonization and multiplication of Giardia trophozoites, thus reducing the severity and duration of murine giardiasis.     

Giardia lamblia rearranges F-actin and alpha-actinin in human colonic and duodenal monolayers and reduces transepithelial electrical resistance

The mechanisms of epithelial injury in giardiasis remain unknown. The effects of live Giardia lamblia on cellular G-actin, F-actin, alpha-actinin, and electrical resistance of human intestinal epithelial monolayers were investigated using SCBN and Caco2 cell lines grown on chamber slides or Transwell filter membranes. In separate experiments, some monolayers were also exposed to sonicated trophozoites, some to supernatant from live G. lamblia cultures, and some with or without the Ca2+ channel blocker verapamil. After 2, 24, or 48 hr of coincubation with G. lamblia, monolayers were assessed for cytoskeletal arrangement under fluorescence and confocal laser microscopy, and transepithelial electrical resistance was measured. Exposure to live G. lamblia trophozoites induced localized condensation of F-actin and loss of perijunctional alpha-actinin while G-actin remained unchanged. Confocal laser microscopy indicated that F-actin rearrangement was not affected by verapamil and was localized within the terminal web area. Coincubation of monolayers with G. lamblia lysates or with spent medium alone similarly rearranged F-actin. Verapamil alone did not alter F-actin. Electrical resistance of SCBN and Caco2 monolayers exposed to G. lamblia was significantly decreased versus controls regardless of whether live or lysed trophozoite samples were used. The results indicate that G. lamblia-induced epithelial injury is associated with F-actin and alpha-actinin rearrangements in the terminal web area via mechanisms independent of extracellular Ca2+. These alterations are associated with reduced transepithelial electrical resistance and are due at least in part to trophozoite products.     

Giardia lamblia: electrophysiology and ultrastructure of cytopathology in cultured epithelial cells

The pathogenicity of Giardia lamblia is a subject of debate. Some studies of human biopsy material have mentioned the presence of trophozoites inside the intestinal mucosa, while in others, flagellates have only been found attached to the epithelium. To study the possible cytopathic effects of G. lamblia cultured under axenic conditions, trophozoites of the human 1/Portland and WB strains were placed in contact with monolayers of Madin Derby Canine Kidney cells, a well characterized cell strain with morphological and functional properties similar to those of a transporting epithelium. After 24 and 48 hr of interaction, the effect of the parasite on epithelial cells was assessed by transmission, scanning, and freeze fracture electron microscopy. In addition, the possible action of living trophozoites and sonicates of G. lamblia on the transepithelial resistance of MDCK monolayers mounted in Ussing chambers was analyzed for periods varying up to 48 hr. The results demonstrate that G. lamblia trophozoites do not invade epithelial monolayers. Furthermore, the parasites fail to produce cytoplasmic changes on target cells and have no effect on transepithelial resistance as judged both electrophysiologically and by the failure to open the occluding junctions that bind together epithelial cells. Damage induced by the parasites to cultured cells was limited to focal distortion or depletion of microvilli at the site of adhesion, which may progress to leave circular areas devoid of microvilli, different from the adhesion marks reported by others for G. muris. Therefore, under the in vitro conditions described here, giardias showed no toxic or invasive effect.     

Giardia lamblia: regulation of secretory vesicle formation and loss of ability to reattach during encystation in vitro

Encystation of Giardia lamblia is required for survival outside the host, as well as for initiation of new infections. Previously, we induced cultured G. lamblia trophozoites to encyst in vitro for the first time. During encystation, we observed the appearance of a new class of large secretory vesicle (encystation-specific vesicle; ESV) within which cyst antigens are concentrated and transported to the nascent wall. The present kinetic and physiologic studies now show that ESV are the earliest morphologic change observed in encystation. Expression of ESV, as well as subsequent encystation, are regulated by exposure to bile at the slightly alkaline pH which is typical of the human intestinal tract. ESV formation appears to be less stringently regulated than formation of water-resistant cysts because omission of either encystation stimuli or alkaline pH preferentially inhibits encystation. Since cysts do not attach, we asked when in encystation this physiologic transition occurs. We found that most encysting trophozoites remain attached until they begin to round up (greater than 24 hr). However, if they are made to detach, as early as 12 hr in encystation, well before they round up, they are defective in the ability to reattach. If trophozoites also become less able to reattach to the intestinal epithelium early in encystation in vivo, this would increase their exposure to lumenal encystation stimuli and promote encystation. These studies have provided new insights into the complex sequence of morphologic and physiologic alterations which occur during encystation of G. lamblia in vitro and their regulation by host intestinal factors.     

Giardia lamblia: stimulation of growth by human intestinal mucus and epithelial cells in serumfree medium

Giardia lamblia trophozoites specifically colonize the upper human small intestine which is normally serumfree but have been grown in vitro only in medium supplemented with serum or serum fractions. Recently, we demonstrated that biliary lipids will support the growth of G. lamblia without added serum. Now, we report that human duodenal jejunal mucus stimulates growth of Giardia in medium with biliary lipids. Stimulation by mucus was enhanced by inclusion of chymotrypsin or crude pancreatic proteases. Coculture of trophozoites with human intestinal epithelial cells also promoted growth, especially in the presence of mucus and/or biliary lipids. With biliary lipids alone, the mean increase in cell number was 3.2 fold and in the presence of mucus 8 fold (P less than 0.01) in 24 serial subcultures. Our demonstration that human intestinal mucus and epithelial cells promote serumfree growth of G. lamblia may help to explain specific colonization of the small intestine by G. lamblia.     

Uses and limitations of monoclonal antibodies to Giardia lamblia-specific 66-kDa copro-antigen in copro-immunodiagnosis of giardiasis

Abstract Antigen-capture enzyme-linked immunosorbent assay for the detection of Giardia lamblia -specific antigen in stool eluates from clinical subjects employing monoclonal antibody directed at 66-kDa G. lamblia copro-antigen has been evaluated. The G. lamblia copro-antigen was detected in 67% (31 of the 46 cases) of stool eluates from clinical cases, while none of the stool eluates from subjects with other intestinal parasites or from apparently healthy individuals, had detectable levels of G. lamblia copro-antigen. Monoclonal antibodies secreted by clones B4C5 and D3F4 recognised the periodate-sensitive and -insensitive epitopes of 66-kDa G. lamblia specific copro-antigen, respectively. Eight (73%) of the 11 symptomatic cases of giardiasis had trypsin-/periodate-sensitive epitopes of 66-kDa copro-antigen while 9 (92%) of 11 of the symptomatic cases and asymptomatic G. lamblia cyst carriers had trypsin-sensitive periodate-insensitive G. lamblia specific copro-antigen. The data tend to suggest that detection of periodate-insensitive epitopes of G. lamblia copro-antigen would indicate the presence of the parasite while the detection of periodate sensitive epitopes of G. lamblia copro-antigen would suggest symptomatic active giardial infection.     

Characterisation of alpha-1 giardin: an immunodominant Giardia lamblia annexin with glycosaminoglycan-binding activity

Alpha-1 giardin is an immunodominant protein in the intestinal protozoan parasite Giardia lamblia. The Triage((R)) parasite panel, used to detect copro-antigens in stool from giardiasis patients, reacts with an epitope between amino acids 160 and 200 in alpha-1 giardin. This region of the protein is also highly immunogenic during human infections. Alpha-1 giardin is related to annexins and like many other annexins it was shown to be plasma membrane associated. Immunoelectron and immunofluorescence microscopy revealed that some alpha-1 giardin are displayed on the surface of recently excysted cells. Recombinant alpha-1 giardin displayed a Ca(2+)-dependent binding to glycosaminoglycans (GAGs), in particular heparan sulphate, a common GAG in the intestinal tract. Recombinant alpha-1 giardin bound to thin sections of human small intestine, a binding which could be inhibited by adding increasing concentrations of sulphated sugars. A surface associated trypsin activated Giardia lectin (taglin) has been suggested to be important for G. lamblia attachment. In this study we show that a monoclonal antibody that inhibits taglin recognises alpha-1 and alpha-2 giardin. Thus, alpha-1 giardin is a highly immunoreactive GAG-binding protein, which may play a key role in the parasite-host interaction. Our results further show a conserved function of annexins from lower to higher eukaryotes.     

The biology of Giardia spp.

Gardia spp. are flagellated protozoans that parasitize the small intestines of mammals, birds, reptiles, and amphibians. The infectious cysts begin excysting in the acidic environment of the stomach and become trophozoites (the vegetative form). The trophozoites attach to the intestinal mucosa through the suction generated by a ventral disk and cause diarrhea and malabsorption by mechanisms that are not well understood. Giardia spp. have a number of unique features, including a predominantly anaerobic metabolism, complete dependence on salvage of exogenous nucleotides, a limited ability to synthesize and degrade carbohydrates and lipids, and two nuclei that are equal by all criteria that have been tested. The small size and unique sequence of G. lamblia rRNA molecules have led to the proposal that Giardia is the most primitive eukaryotic organism. Three Giardia spp. have been identified by light lamblia, G. muris, and G. agilis, but electron microscopy has allowed further species to be described within the G. lamblia group, some of which have been substantiated by differences in the rDNA. Animal models and human infections have led to the conclusion that intestinal infection is controlled primarily through the humoral immune system (T-cell dependent in the mouse model). A major immunogenic cysteine-rich surface antigen is able to vary in vitro and in vivo in the course of an infection and may provide a means of evading the host immune response or perhaps a means of adapting to different intestinal environments.     

The biology of Giardia spp.

Gardia spp. are flagellated protozoans that parasitize the small intestines of mammals, birds, reptiles, and amphibians. The infectious cysts begin excysting in the acidic environment of the stomach and become trophozoites (the vegetative form). The trophozoites attach to the intestinal mucosa through the suction generated by a ventral disk and cause diarrhea and malabsorption by mechanisms that are not well understood. Giardia spp. have a number of unique features, including a predominantly anaerobic metabolism, complete dependence on salvage of exogenous nucleotides, a limited ability to synthesize and degrade carbohydrates and lipids, and two nuclei that are equal by all criteria that have been tested. The small size and unique sequence of G. lamblia rRNA molecules have led to the proposal that Giardia is the most primitive eukaryotic organism. Three Giardia spp. have been identified by light lamblia, G. muris, and G. agilis, but electron microscopy has allowed further species to be described within the G. lamblia group, some of which have been substantiated by differences in the rDNA. Animal models and human infections have led to the conclusion that intestinal infection is controlled primarily through the humoral immune system (T-cell dependent in the mouse model). A major immunogenic cysteine-rich surface antigen is able to vary in vitro and in vivo in the course of an infection and may provide a means of evading the host immune response or perhaps a means of adapting to different intestinal environments.     

Survival of Giardia lamblia trophozoites after exposure to UV light

The ability of Giardia lamblia trophozoites to reproduce after exposure to different fluences of UV radiation was determined using an in vitro-cultured method. The rate of parasite reproduction following UV exposure was measured by direct enumeration of trophozoites cultured in Diamond's Trypticase Yeast extract-Iron (TYI)-S-33 medium. The results suggested that some G. lamblia trophozoites may survive or are reactivated following exposure to UV fluences up to 10 mJ cm(-2). In addition, trophozoites exposed to a UV fluence of 1 mJ cm(-2) were infectious to Mongolian gerbils. Evidence of survival or reactivation at UV fluences of 20 and 40 mJ cm(-2) was ambiguous and statistically inconclusive, while at 100 mJ cm(-2) there was no evidence of survival or reactivation. This finding may have implications for criteria used by the drinking water and wastewater treatment industry to ensure safe reduction of G. lamblia cysts by UV disinfection processes.     

Defective spontaneous but normal antibody-dependent cytotoxicity for an extracellular protozoan parasite, Giardia lamblia, by C3H/HeJ mouse macrophages

To understand murine host responses to extracellular protozoa, the capacity of peritoneal macrophages to exhibit cytotoxicity for [3H]thymidine-labeled Giardia lamblia trophozoites was investigated. Resident peritoneal macrophages from C3H/HeN mice expressed spontaneous cytotoxicity for G. lamblia in a manner that was dependent on both time and effector cell number; this cytotoxic activity was increased with cells elicited by an intraperitoneal injection of thio-glycollate. In contrast, spontaneous cytotoxicity for G. lamblia by resident and thioglycollate-elicited peritoneal macrophages from C3H/HeJ mice was markedly reduced. In the presence of anti-G. lamblia serum (ADCC), however, peritoneal macrophages from both C3H/HeN and C3H/HeJ mice exhibited striking augmentation of their cytotoxic activity for G. lamblia to equivalent levels. We conclude that macrophages from C3H/HeJ mice express defective spontaneous cytotoxicity but normal ADCC for the extracellular protozoan parasite, G. lamblia. The dissociation between the expression of these two effector cell functions suggests that macrophage spontaneous cytotoxicity and ADCC for extracellular protozoa are mediated by separate macrophage functions.     

Evaluation of ELISA for detection of Giardia lamblia-specific copro-antigen employing monospecific antibodies

An enzyme linked immunosorbent assay (ELISA) system, using monospecific antibodies for the detection of Giardia lamblia specific 66 kDa copro-antigen has been developed and evaluated. The assay detected the antigen in stool eluates of all the 24 microscopically confirmed cases of giardiasis and in 17 (68%) of the 25 microscopy-negative clinically suspected cases of giardiasis. None of stool eluates from 20 subjects infected with other protozoal/helminthic intestinal parasites or from 20 apparently healthy subjects had G. lamblia-specific copro-antigen. The ELISA employing monospecific antibodies is a sensitive and specific tool for the diagnosis of giardiasis and is especially useful for confirming microscopy-negative suspected cases of giardiasis.