Infectivity of Giardia duodenalis Assemblages A and E for the gerbil and axenisation of duodenal trophozoites.

Establishing in vitro cultures of Giardia duodenalis trophozoites from faecal cysts remains very difficult due to poor excystation and bacterial contamination. This study investigated an alternative approach starting from duodenal trophozoites of gerbils that were artificially infected with field isolates from humans (Assemblages A and B) and cattle (Assemblage E and mixed E/A). Gerbil infection was successful for Assemblages A (1/1) and B (1/3) from humans, and for E (1/2) and mixed E/A (6/6) from cattle. Despite the fact that some isolates subsequently failed or were difficult to establish in vitro, several Assemblage A and Assemblage E in vitro trophozoite cultures were successful, however, subsequent cloning required adaptation of the standard TYI-S-33 medium whereby different medium supplements were required for promoting growth. Excess of l-cysteine (2mg/ml) and ascorbic acid (0.2mg/ml) supported cloning of Assemblage A, while l-glutathione (7.8 mg/ml) was required for Assemblage E. This is a first description of in vitro axenisation of Assemblage E trophozoites from cattle.

In vitro effects of propolis on Giardia duodenalis trophozoites.

In order to improve the current chemotherapy of Giardia infection, potential antigiardial agents have been screened, including natural products. Propolis, a resinous hive product collected by bees, has attracted attention as a useful and popular substance with several therapeutic activities. The present study was carried out aiming to evaluate the in vitro effects of an ethanolic extract of propolis on the growth and adherence of Giardia duodenalis trophozoites. Propolis inhibited the growth of trophozoites and the level of inhibition varied according to the extract concentration and incubation times. The highest reduction of parasite growth was observed in cultures exposed to 125, 250 and 500 microg/ml of propolis, in all incubation periods (24, 48, 72 and 96 h). Growth reduction by 50% was observed in 125 microg/ml propolis-treated cultures, while the concentrations of 250 and 500 microg/ml were able to inhibit growth by more than 60%. Propolis also inhibited parasite adherence and all assayed propolis concentrations promoted the detachment of trophozoites. Light microscope observations revealed changes of the pear-shaped aspect of the cell and reduction of flagellar beating frequency in the great part of the trophozoites. Our results hold the perspective for the utilization of propolis as an antigiardial agent.

The effects of saturated fatty acids on Giardia duodenalis trophozoites in vitro.

Giardia duodenalis is a protozoal, intestinal parasite that is a common aetiological agent of infectious diarrhoea in humans worldwide. Chemotherapeutic intervention presently offers a limited range of drugs and these are usually only employed after clinical diagnosis. Moreover, these drugs are ineffective against the infectious cysts, can produce unpleasant side effects, and are expensive with limited availability in developing countries. Frequent reports of drug toxicity, treatment failure and parasite drug resistance have, in some instances, also resulted in the increasing reluctance to over-prescribe synthetic anti-microbials. Alternatively, there is now mounting evidence to suggest that some of the naturally derived, medium-chain, saturated fatty acids (MCSFAs) possess anti-microbial and anti-parasitic properties. We have therefore examined the effects of four different fatty acids on G. duodenalis trophozoites in vitro. Cytotoxicity was determined using fluorescence, scanning and transmission electron microscopic techniques and standard cytotoxicity assays. Our studies have confirmed that the MCSFA, dodecanoic acid (C: 12) (common name: lauric acid), is anti-giardial, with an LD50 concentration comparable to that of metronidazole, the drug of choice in the treatment of giardiasis. Dodecanoic acid appeared to induce trophozoite death by accumulating within the parasite cytoplasm resulting in rupture of the cell membrane. This study has opened fresh avenues for development of natural drug therapy in which food supplementation may augment, or even replace, some of the standard chemotherapeutic agents presently employed in the treatment of giardiasis and possibly other infectious intestinal diseases.

Modification of RPMI 1640 for use in vitro immunological studies of host-parasite interactions in giardiasis.

Incubation of trophozoites for 6 h in RPMI 1640 affected the viability of the parasite; however, RPMI 1640 supplemented with L-cysteine did not affect trophozoite viability, ability to grow when transferred to fresh TYI-S-33, or ability to infect gerbils. Similarly, incubation of murine spleen cells in modified medium did not affect the viability of the cells or proliferative responses to mitogens. RPMI 1640 supplemented with 11.4 mM L-cysteine is a suitable maintenance medium for in vitro studies in immunoparasitology because it maintains viability as well as some of the physiological functions of both trophozoites and lymphocytes.

N-acetyl-D-glucosamine is present in cysts and trophozoites of Giardia lamblia and serves as receptor for wheatgerm agglutinin.

Previously, on the basis of lectin binding and glycosidase digestion assays, we have suggested that N-acetyl-D-glucosamine residues (GlcNAc) are major structural components of both trophozoites and in vivo cysts of the intestinal parasite Giardia lamblia. In this report we confirm that GlcNAc is present both in trophozoites and in vitro cysts as assessed by lectin binding and glycosidase digestion assays, galactosyltransferase labeling, immunochemical analysis using antibodies specific for GlcNAc and its beta 1-4 oligomers, and by gas chromatography/mass spectrometry (GC/MS). The results show that wheatgerm agglutinin (WGA) binds specifically to intact trophozoites and in vitro cysts as well as to SDS-PAGE separated proteins. WGA binding to the separated proteins was markedly reduced after their digestion with N-acetyl-beta-D-glucosaminidase, supporting the conclusion that WGA is reacting with terminal beta-linked GlcNAc residues. Labeling of trophozoites and cysts by 3H-exogalactosylation with galactosyltransferase further confirmed the presence of terminal GlcNAc in both surface and intracellular glycoproteins. The presence of GlcNAc is also supported by microfluorometric analysis using antibodies to (GlcNAc)1, (GlcNAc)2, and (GlcNAc)3, which revealed a sugar-inhibitable binding of the antibody to live trophozoites. Finally, the presence of GlcNAc in both cysts and trophozoites was unequivocally confirmed by GC/MS analysis of detergent-extracted membranes and of glycoproteins isolated by affinity chromatography on WGA-agarose. GC/MS analysis also revealed mannose (Man), N-acetyl-D-galactosamine (GalNAc), fucose (Fuc), galactose (Gal), glucose (Glc) and N-acetylneuraminic acid (NANA) to be present in cysts. All these sugars were also present in trophozoites, except for GalNAc. The glycoproteins isolated by WGA affinity chromatography were 5- to 40-fold enriched in GlcNAc, further supporting the conclusion that WGA reacts with GlcNAc in Giardia. In summary, the data presented here provide biological and chemical evidence for GlcNAc in both cysts and trophozoites of G. lamblia and are consistent with previously published results from this and other laboratories.

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.

Effects of bile and bile salts on growth and membrane lipid uptake by Giardia lamblia. Possible implications for pathogenesis of intestinal disease.

We have shown previously that ox and pig bile accelerate in vitro growth of Giardia lamblia. We have now investigated the possible mechanisms by which mammalian biles promote parasite growth. Growth effects of (a) ox, pig, guinea pig, and human biles, (b) pure bile salts, and (c) egg and soybean lecithins were studied in the presence of a lecithin-containing growth medium. Individually, dilute native bile and pure sodium taurocholate (TC), glycocholate (GC), and taurodeoxycholate (TDC) promoted parasite growth; growth was most marked with biles of high phospholipid content, with biles enriched in more hydrophobic bile salts (ox approximately equal to human greater than pig greater than guinea pig) and with micellar concentrations of GC and submicellar concentrations of TC and TDC. By measuring uptake of radiolabeled biliary lipids from bile and bile salt-supplemented growth medium, we showed that the parasite consumed bile lipids, with the rank order lecithin greater than bile salts. Apparent net uptake of cholesterol was considered to be due to exchange, since net loss of cholesterol from the growth medium was not detected. Although bile and bile salt-stimulated parasite growth was associated with enhanced lecithin uptake, reduction in generation time was observed at low bile and bile salt concentrations when lecithin uptake was similar to bile free controls. Thus, bile salts may stimulate Giardia growth initially by a mechanism independent of enhanced membrane phospholipid uptake. However, since Giardia has no capacity to synthesize membrane lipid, biliary lecithin may be a major source of phospholipid for growth of this parasite.