Viabilidad de los quistes de lamblia intestinalis bajo diferentes condiciones / Survival of lambia intestinalis cysts under different condictions

Se estudió la resistencia de los quistes de Lamblia intestinalis a distintas condiciones. La viabilidad de los quistes fue mayor a temperaturas bajas; a 10 C los quistes permanecieron viables por 40 días. A pH7 cincuenta por ciento de los quistes permanecieron viables hasta por 22 días. Concentraciones de kilol de 5% al 0,05% fueron 100% efectivas en los quistes de L. intestinalis. Se discute la importancia epidemiológica de estos hallazgos

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.

Electrophoretic characterization of Giardia isolated from humans, cattle, sheep, and a dog in Switzerland.

Nine isolates of Giardia lamblia from humans, cattle, sheep, and 1 dog were compared by employing agarose gel isoenzyme electrophoresis and isoelectric focusing of total soluble cell protein on polyacrylamide gels. The banding patterns of the 14 enzymes examined showed remarkable similarities among the Swiss Giardia isolates. This was true also of the total soluble trophozoite proteins. The electrophoretic mobilities of most enzymes and other proteins obtained for the Swiss isolates were the same as those of 2 isolates from humans in other geographical areas, the WB and the Portland-1 strains. Only the human isolate CH-H2 could clearly be distinguished from all other isolates analyzed. The great biochemical similarities observed among the Swiss isolates contrast with the extensive heterogeneity previously demonstrated for G. lamblia by other investigators who used similar analytical techniques. These data are consistent with recent transmission studies of Giardia and suggest that in Switzerland domestic animals may serve as a reservoir of human Giardia infections and that cross-transmission between humans and animals is likely to occur.

Characterization of a 29.4-kilodalton structural protein of Giardia lamblia and localization to the ventral disk [corrected].

The amino acid sequence of a 29.4-kilodalton [corrected] structural protein located in the ventral disk and axostyle of Giardia lamblia was determined. Clone lambda M16 from a mung bean expression library in lambda gt11 expressed a fusion protein recognized by three different isolate-specific antisera and sera from G. lamblia-infected gerbils. One of the three EcoRI fragments (M16; 1.26 kilobases) encoded the recognized protein. Sequence analysis revealed a single open reading frame of 813 base pairs. Two areas showed conservation of the positions of some amino acids. The abundance of arginine, glutamic acid, and threonine was increased. Two potential alpha-helical regions were deduced in the regions of repeats. Antisera to the M16 fusion protein reacted specifically with internal components of the ventral disk and axostyle, as well as Giardia fractions enriched for ventral disk structural proteins. An identical protein was recognized in different isolates by anti-M16, and a single identical band was recognized in Southern blots using the M16 1.26-kilobase fragment as a probe. Therefore, the 29.4-kilodaltion [corrected] protein appears to be highly conserved compared with variant surface proteins.

Detection of giardia antigen in stool samples by a semi-quantitative enzyme immunoassay (EIA) test.

A semi-quantitative enzyme immunoassay (EIA) test for the detection of Giardia intestinalis antigen in faeces was developed. In order to avoid unspecific reactions due to anticalf serum activity, IgG fractions of anti-giardia rabbit and sheep sera were purified from antibovine antibodies by immunoadsorption. Faecal specimens tested in the assay were mixed with normal horse serum to avoid unspecific and proteolytic effects of stool components. Out of a range of bacterial and parasitic antigens tested, only high concentrations of Ascaris suum egg antigen and Shigella sonnei gave slight unspecific reactions. During an outbreak of waterborne giardia infection faecal samples from 49 individuals were tested. Giardia cysts were found by microscopy in 22 individuals and specimens from 24 persons were positive by EIA. The estimated amount of antigen found in the positive samples ranged from 50 to 5,000 ng giardia protein/ml. After filtration through a 0.45 microns filter antigenic activity was found in the filtrate of 10 samples. In 3/6 samples with no cysts detected by microscopy, mainly soluble giardia antigen was found.

Giardia cyst wall-specific carbohydrate: evidence for the presence of galactosamine.

Gas chromatographic (GC), mass spectrometric (MS), lectin binding and enzymatic analyses of the carbohydrates from Giardia cyst walls, intact cysts and trophozoites were performed to investigate the carbohydrate composition of Giardia cyst walls and to test the hypothesis that the Giardia cyst wall is composed largely of chitin. Galactosamine, verified by MS, was present in Giardia cyst walls and intact cysts (ca. 47 nmol 10(-6) cysts). Since not even trace amounts of it were detected in trophozoites by either GC or lectin binding, galactosamine is hypothesized to be a cyst wall-specific amino hexose. Based on the putative binding affinity of Phaseolus limensis lectin, galactosamine may be present in cyst walls as N-acetylgalactosamine. Neither glucosamine nor sialic acid were detected in as much as 11 mg dry weight of cysts, cyst walls, or trophozoites. Glucose, the most abundant carbohydrate, and ribose were detected in Giardia cysts and trophozoites. Galactose (ca. 10 nmol 10(-6) cysts) was detected in cysts but not in trophozoites. The lack of detectable levels of (1) glucosamine in cyst wall hydrolysates, (2) cyst staining by Calcofluor M2R, (3) endogenous chitinase activity and (4) N-acetylglucosamine when cysts served as a substrate for exogenous chitinase suggests that the Giardia cyst wall is not composed largely of chitin as previously reported. beta-N-Acetylgalactosaminidase, EC 3.2.1.32, activity was detected in cysts and trophozoites and represents the first carbohydrate splitting hydrolase detected in Giardia.

Isoelectric focusing of ten strains of Giardia duodenalis.

Ten strains of human- and animal-source Giardia duodenalis were evaluated using an isoelectric focusing technique. Banding patterns obtained from total cell proteins of trophozoites demonstrated both similarities and differences between strains. This confirms the heterogeneity of this morphological group of Giardia sp. demonstrated by others. Heterogeneity was demonstrated among the strains retrieved from human and animal hosts and from hosts within the same geographical region.

Giardiasis / Giardiasis

Una vez que los quistes de Giardia son expulsados del huesped mueren rapidamente si son deshidratados, pero pueden sobrevivir hasta dos meses en aguas frias con temperaturas de 8 C. Por tanto, las giardiasis se transmiten por la ingestion de heces o aguas contaminadas. Generalmente la infeccion produce diarrea, en las formas subaguda o cronica se pueden desarrollar signos y sintomas adicionales de malestar intestinal

A comparison of Giardia microti and Spironucleus muris cysts in the vole: an immunocytochemical, light, and electron microscopic study.

We have shown that cysts of the genus Spironucleus share many common morphological features with Giardia cysts including: 2-4 nuclei, flagellar axonemes, a distinct cyst wall, and they even display the same immunostaining as Giardia cysts when labeled with antibodies specific for Giardia cyst wall. A direct comparison of Spironucleus muris and Giardia microti cysts have revealed that cysts of S. muris are significantly smaller than cysts of G. miroti. At the ultrastructural level, the cyst walls are similar in fibrillar appearance, but the width of the S. muris cyst wall is significantly less than that of G. microti. The cysts of S. muris also differ from G. microti in that they contain a striated rootlet fiber, flagellar sheath, and numerous glycogen rosettes. Characteristic features of Giardia include the adhesive disc and median body. Although the cysts of Spironucleus and Giardia are similar in appearance, these unique morphological features can be used to distinguish between the 2 protozoa and should be employed in the detection of Giardia cysts in water samples.

Prevalence of giardiasis in patients with cystic fibrosis.

A group of 107 patients with cystic fibrosis and a control group of 64 normal members of households of patients with cystic fibrosis were surveyed for Giardia lamblia cysts and trophozoites by counterimmunoelectrophoresis of fecal samples. The patient group had a significantly higher rate of infestation than the control group (28.0% vs 6.3%, P = 0.0006), and the disparity between the two groups increased with age (P = 0.005). Aside from cystic fibrosis, all risk factors examined were without influence, except for the presence of household members less than or equal to 5 years of age. We conclude that our patients with cystic fibrosis have a previously unrecognized increased prevalence of giardiasis compared with that in a control population.

Lipid analysis of Giardia lamblia and its culture medium.

The neutral lipid and phospholipid composition of Giardia lamblia and its culture medium were analysed by a thin-layer chromatography flame-ionized detection system. Both lipid compositions of the parasite differed from that of the culture medium. Sterol was found to be the major neutral lipid, and phosphatidylcholine, phosphatidylethanolamine and sphingomyerin were also present. Fatty acid composition of G. lamblia and its culture medium were also analysed by gas liquid chromatography. Oleic and palmitic acid were the major fatty acids in the total lipid of the organism. The influence of porcine and bovine bile extracts on the lipid composition of G. lamblia was studied. The addition of bile to the medium caused no change in lipid composition of the parasite. Lipid composition of the culture medium was studied both before and following growth of the parasite, and it became evident that consumption of phosphatidylcholine occurred in the growth medium supplemented with bile extract. These results indicate that the presence of bile extract may possibly accelerate consumption by G. lamblia of lipid in the environment.

Biology of Giardia lamblia. Detection of N-acetyl-D-glucosamine as the only surface saccharide moiety and identification of two distinct subsets of trophozoites by lectin binding.

Lectins and glycosidases of known sugar specificity were used as probes to analyze the surface carbohydrate moieties of G. lamblia trophozoites and in particular to determine whether chitin or oligomeric D-GlcNAc is present in the trophozoite form of the parasite as well as on the cyst. Of 13 lectins with varying sugar specificity, only D-GlcNAc-specific lectins bound specifically to the trophozoite surface as determined by light microscopy and EM. A striking finding was the identification of two distinct subsets of trophozoites, distinguished by reactivity with WGA and detected by light microscopy and EM as well as by flow cytometry. Unlike the cyst wall, the trophozoite D-GlcNAc residues were resistant to chitinase treatment. In contrast N-acetyl-beta-D-glucosaminidase abolished WGA binding suggesting that the lectin binds to terminal beta-linked D-GlcNAc residues. These residues were identified as being present on surface glycoproteins by Western blotting of parasite membrane proteins using WGA as a probe. This study identifies D-GlcNAc as the only saccharide moiety detectable by lectin binding on the surface of G. lamblia trophozoites and demonstrates that in contrast to the cyst, chitin is not present in the trophozoite. In addition two distinct subsets of trophozoites were identified based on reactivity with WGA and may represent varying stages of differentiation from trophozoite to cyst.

Unusual ribosomal RNA of the intestinal parasite Giardia lamblia.

The anaerobic protozoan Giardia lamblia is a common intestinal parasite in humans, but is poorly defined at molecular and phylogenetic levels. We report here a structural characterization of the ribosomal RNA (rRNA) and rRNA genes of G. lamblia. Gel electrophoresis under native or non-denaturing conditions identified two high molecular weight rRNA species corresponding to the 16-18S and 23-28S rRNAs. Surprisingly, both species (1300 and 2300 nucleotides long, respectively) were considerably shorter than their counterparts from other protozoa (typically 1800 and 3400 nucleotides), and from bacteria as well (typically 1540 and 2900 nucleotides long). Denaturing polyacrylamide gel electrophoresis identified a major low molecular RNA of 127 nucleotides and several minor species, but no molecules with the typical lengths of 5.8S (160 nucleotides) and 5S (120 nucleotides) rRNA. The G. lamblia 1300, 2300, and 127 nucleotide RNAs are encoded within a 5.6 kilobase pair tandemly repeated DNA, as shown by Southern blot analysis and DNA cloning. Thus, the rRNA operon of this eukaryotic organism can be no longer than a typical bacterial operon. Sequence analysis identified the 127 nucleotide RNA as homologous to 5.8S RNA, but comparisons to archaebacterial rRNA suggest that Giardia derived from an early branch in eukaryotic evolution.

Localization of acid phosphatase activity in Giardia lamblia and Giardia muris trophozoites.

Numerous membrane-bounded vacuoles are found adjacent to the plasma membrane of the pathogenic protozoan Giardia lamblia. The function of these vacuoles has been discussed by several authors. Approximately 100-400 nm in diameter with a core of low electron density, they have been suggested to be mitochondria, mucocysts, lysosomes, and endocytotic vacuoles. Enzyme cytochemical localization for acid phosphatase activity using cerium as a capturing agent demonstrates reaction product in these vacuoles as well as in the endoplasmic reticulum and nuclear envelope cisternae. The distribution of reaction product suggests the vacuoles are lysosome-like; however, their function and development remain in question.

Giardia lamblia: detection and characterization of calmodulin.

Calmodulin was detected in Giardia lamblia by radioimmunoassay and cyclic AMP phosphodiesterase activation. This protein was purified to apparent homogeneity by fast protein liquid chromatography with a yield of 260 ng of calmodulin/mg of protozoan protein. Purity was established by gel electrophoresis, gel filtration, and ion exchange chromatography. The parasite calmodulin has properties characteristic of calmodulin isolated from other eukaryotes, e.g., an apparent molecular weight of 16.7 kD; activation in calcium dependent manner of bovine heart cyclic nucleotide phosphodiesterase; and sensitivity to known calmodulin antagonists.

Plasma membrane isolated from Giardia lamblia: identification of membrane proteins.

Two methods are introduced for preparing plasma membranes from Giardia lamblia trophozoites. Isolated membranes were purified by centrifugation on either a sucrose step-gradient or a self-generated Percoll gradient, where they band at a density of approximately 1.04 g ml-1. In pure fractions, membranes formed vesicles or extensive sheets. Electron microscope profiles show that they are asymmetric with a thin filamentous coat on one side. Membrane proteins were resolved by SDS/PAGE. They included a major component of apparent Mr 75,000 (75 kDa), and additional bands detectable by gel staining at 58 kDa, 54 kDa, 32 to 38 kDa (5 bands), 22 kDa, and 15 to 20 kDa. To probe the surface location of proteins, gels were also prepared from Giardia cells that were surface radio-iodinated using the immobilised catalyst IODOGEN. The 75 kDa membrane protein was strongly labelled in the corresponding autoradiograph, also the bands at 58 kDa and 54 kDa, the 22 kDa polypeptide, and some faint bands not resolved in the isolated membrane preparations. The set of close-running bands at 32 to 38 kDa were not iodinated. The labelled 58 kDa and 54 kDa proteins comigrated with alpha and beta-tubulins. Controls showed that cytoskeleton and flagellar tubulins were not iodinated in this experiment, indicating that the labelled tubulin is surface-derived. The principal approximately 75 kDa surface protein identified in isolated membranes probably corresponds to an iodinatable and antibody-precipitated "82 kDa" antigen reported previously.

Description and characterization of a surface lectin from Giardia lamblia.

The mechanisms by which the human enteric pathogen Giardia lamblia colonizes the proximal small intestine are poorly understood. Although the parasite possesses an attachment organelle on its ventral surface, the "sucking" disk, we considered that like many bacteria and some protozoa, G. lamblia might also have a surface membrane-associated modality for adherence to its host. Using an erythrocyte mixed-agglutination model, we demonstrated a parasite surface lectin with specificities for D-glucosyl and D-mannosyl residues. This lectin is soluble in Triton X-100, is calcium dependent, and is maximally active at pH 5.5 to 6.0. Partial purification was achieved by serial extraction of parasites in Triton X-100 followed by Sephadex G-150 affinity chromatography. The lectin could not be surface radiolabeled with 125I-Bolton-Hunter reagent, but radiolabeling of the hapten eluate from an affinity column produced four bands of 57,000 to 78,000 Mr on sodium dodecyl sulfate-polyacrylamide gels under reducing conditions. The biological function of this lectin is unknown. The presence of mannosyl residues on the luminal surface of human small intestinal epithelial cells suggests that there are receptors for Giardia lectin at the site of colonization.

Identification of chitin as a structural component of Giardia cysts.

The intestinal parasite Giardia lamblia is a significant cause of diarrheal disease, which is perpetuated by the infective cyst form of the parasite. Although a rational approach to the control of giardiasis would be to inhibit cyst formation, nothing is known of the chemical composition of the cyst wall or of its biosynthesis. In these studies, we have shown that chitin is a major structural component of G. lamblia and G. muris cyst walls. This conclusion is based on the finding that chitinase specifically destroys the cyst wall, as revealed by electron microscopy. The presence of chitin was also shown directly by lectin binding studies. Of 12 lectins with diverse carbohydrate recognition specificity, only the N-acetylglucosamine-specific lectins wheat germ agglutinin, succinylated wheat germ agglutinin, and tomato lectin bound to cyst walls, as shown by fluorescence microscopy and cytochemistry. Wheat germ agglutinin binding was completely abolished by treatment of the cysts with purified chitinase. This effect was specific since it could be prevented by incubating the enzyme with chitin before treatment of the cysts. Treatment of cysts with N-acetyl-beta-glucosaminidase partially inhibited wheat germ agglutinin binding, whereas other glycosidases and proteases had no effect. These findings indicate that chitin is a major structural component of Giardia cyst walls and raise the possibility that inhibitors of chitin synthesis may be of use in preventing encystation and thus controlling spread of the disease.

Thiol groups on the surface of anaerobic parasitic protozoa.

Evidence is presented that Giardia lamblia and Entamoeba histolytica, phylogenetically unrelated aerotolerant anaerobes, have crucial thiol groups on or easily accessible to their external surface. Both parasites were killed by three structurally unrelated thiol-blocking reagents which penetrate intact cells poorly or not at all. The parasites were protected from p-chloromercuribenzenesulfonic acid (10-100 microM) by cysteine or by reduced glutathione. Killing was arrested with identical kinetics by addition of either cysteine (which quickly penetrates the cells) or bovine serum albumin (which does not penetrate intact cells) at various times after p-chloromercuribenzenesulfonic acid, indicating that the reactive site may be on the outer surface of the cell. Proteins lacking cysteine did not protect. Sensitivity of three other protozoa to p-chloromercuribenzenesulfonic acid was also tested. Trichomonas vaginalis (anaerobic) was at least as sensitive as E. histolytica and G. lamblia, while Crithidia fasciculata and Paramecium tetraurelia (both aerobic) were less sensitive. Thiol groups on the G. lamblia surface were demonstrated directly by fluorescence-activated cell sorter analysis of trophozoites which had been modified with a thiol-specific hapten, N-iodoacetyl-N'-(5-sulfonic-1-naphthyl)ethylenediamine and reacted with fluorescent antibody to this hapten.

Selective extraction with Sarkosyl and repolymerization in vitro of cytoskeleton proteins from Giardia.

Sarkosyl has been used to dissociate structures in cytoskeletons isolated from Giardia lamblia. Results from sodium dodecyl sulphate/polyacrylamide gel electrophoresis and from electron microscopy of insoluble residues show that the solubilization of components is partly selective. At low concentrations of detergent (less than 0.3%), microribbons and microtubules of the disc cytoskeleton disappear, but doublet microtubules from axonemes resist extraction. Consequently, the microribbon protein giardin is extracted into solution more completely than tubulin. Soluble proteins in 0.1% Sarkosyl have been fractionated by gel filtration chromatography in Bio-Gel P300. Giardin elutes in two positions: as a low molecular weight subunit, and in early fractions corresponding to a larger particle size in which subunits might be forming oligomers. Supernatants prepared in 0.5% Sarkosyl were diluted with 0.1 M-KCl or 0.1 mM-MgCl2 to bring about reaggregation of the cytoskeleton proteins. Reassembled structures seen in negatively stained preparations were polymorphic. Some tubulin ribbons of 5 nM protofilaments were identifiable' also there were large fibres and some flat sheets of very thin filaments. Electron micrographs of sheets have been analysed by optical diffraction. The transforms show that the lateral separation of the fine filaments is about 2.5 nm. Axial periodicities from features spaced along filaments were weak. A 3.75 nm layer-line has been detected, corresponding to a similar periodicity found earlier in microribbons.