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PubMed2003

Tubulin diversity in trophozoites of Giardia lamblia.

Giardia lamblia is a diplomonad that parasitizes the small intestine of vertebrates. The trophozoite is multiflagellar and its cytoskeleton presents a complex organization of microtubular structures. One of these, the adhesive disk, consists of a microtubular spiral. The median body, whose function is not yet determined, is also composed by microtubules. The cell has eight flagella and two microtubule sheets, known as the funis. In this study we used several antibodies and immunofluorescence microscopy to help in the characterization of these structures. We observed that Giardia tubulin reacts with antibodies raised against very distinct immunogens. The antibodies used were against: (1) alpha-tubulin from chicken embryo brain, Trypanosoma brucei, sea urchin sperm, Paramecium, acetylated alpha-tubulin from Paramecium, and tyrosinated alpha-tubulin, (2) beta-tubulin from chicken embryo brain and Physarum polycephalum, and (3) an antibody with specificity to beta-tubulin having as immunogen the FtsZ bacterial protein. Each cytoskeletal structure of Giardia presented a distinct pattern of labeling by the several antibodies used. These data indicate that even being considered one of the most ancient of organisms, Giardia shares similarities (at least in relation to tubulin) with other organisms. They also open some questions about the organization and composition of its microtubular structures.

Animals↗
PubMed2005

Evaluation of silymarin in the treatment on asymptomatic Giardia infections in dogs.

We have reported previously the efficacy of antiprotozoal drugs against canine giardiasis (In press, Journal of Veterinary Clinic, the Korean Society of Veterinary Clinics). Fenbendazole was found to be the most efficacious for the treatment of canine giardiasis. There were no significant differences between the efficacy of albendazole and fenbendazole against canine giardiasis. On the other hand, the efficacy of metronidazole for the treatment of canine giardiasis, the efficacy was lower when compared to that of albendazole and fenbendazole. On the basis of these results, to evaluate clinical effect of silymarin, we evaluated the therapeutic efficacy of metronidazole alone, or combined with silymarin for 2 weeks for canine giardiasis. In addition, to observe effects on nutrition, we investigated the changes of body weight, the serum biochemical indicators for liver inflammation (GOT, GPT, NH3), the liver cell regeneration indicators (total protein, albumin) and the hematological changes during treatment (WBC, RBC, MCV, MCH and MCHC). The dogs were allocated to four groups; one group was treated with silymarin (3.5 mg/kg once a day, oral), another with metronidazole (50 mg/kg once a day, oral), and the other group with silymarin (3.5 mg/kg once a day, oral) plus metronidazole (50 mg/kg once a day, oral), while control group remained nontreated. The fecal samples from all the dogs were examined, using the ZSCT and giardia antigen test kit (SNAP(*) Giardia, IDEXX Laboratories), from each dog of each group for three times a week for 2 weeks. Dogs were considered to have giardiasis when one or more of the fecal samples had positive results for Giardia cysts. Seven days after treatment, the efficacy of silymarin plus metronidazole was found 79%, whereas that of metronidazole was 72%. Ten days post-treatment the efficacy of metronidazole plus silymarin (91%) was significantly different in comparison with that of metronidazole (75%). Two weeks post-treatment no cysts were detected in the fecal samples in the dogs of metronidazole or silymarin plus metronidazole-treated groups. Whereas, the fecal samples of all the dogs of the control and only silymarin-treated groups were giardia positive. Signs of side effects were not observed in silymarin plus metronidazole-treated dogs. But poor appetite and intermittent vomiting signs were observed in two dogs of the metronidazole-treated group that resolved when metronidazole administration was discontinued. The body weight of those treated with metronidazole was significantly decreased in comparison with those treated with silymarin and metronidazole plus silymarin. There were significant differences of body weight between the dogs treated with silymarin and metronidazole. Two weeks after metronidazole treatment, serum concentration of GOT, GPT and NH3 were significantly increased in comparison with those treated with silymarin. On the other hand, the serum concentration of GOT, GPT and NH3 were not significantly increased when treated with silymarin plus metronidazole compared to those treated with metronidazole. Serum total protein and albumin concentrations were decreased after metronidazole treatment as compared to those treated with silymarin and silymarin plus metronidazole. The concentrations of serum total protein and albumin decreased significantly in metronidazole-treated group as compared to that of treated with silymarin. The numbers of WBC and RBC did show significant differences in the dogs treated with metronidazole, while MCV, MCH were significant by different between silymarin and metronidazole-treated dogs. On the other hand, there were no significant differences in MCHC in any groups. These data suggest that silymarin, in supplement with antiprotozoal drugs, can influence the therapy of canine giardiasis.

Administration, Oral↗
PubMed2002

Post-translational glutamylation and tyrosination in tubulin of tritrichomonads and the diplomonad Giardia intestinalis.

Glutamylated and tyrosinated tubulin were localized in Giardia intestinalis and selected trichomonads of the Tritrichomonadinae subfamily, using specific monoclonal antibodies directed at each of the post-translational modifications. Analysis was carried out using indirect immunofluorescence microscopy. Although trichomonad tubulins remained unlabeled by anti-tyrosine tubulin (TUB-1A2), the presence of the glutamylation motif (GT 335) was confirmed and found to differ in distribution among tritrichomonads. Tritrichomonas muris was most heavily labeled with GT 335, while T. foetus was the least so. Like trichomonads, Giardia was unreactive to anti-tyrosine tubulin; however, the GT 335 antibody produced marked fluorescence in Giardia trophozoites. This study is the first to report immunofluorescent localization of tubulin glutamylation in Giardia and confirms previously reported mass spectrometry data.

Animals↗
PubMed1993

Molecular variation in Giardia.

Molecular characterisation of species within the genus Giardia has revealed that much of the phenotypic heterogeneity, particularly within the species G. duodenalis, has a genetic basis. The source of this genetic variation appears to arise from predominantly asexual, clonal reproduction, although occasional bouts of sexual reproduction cannot be ruled out. Genetic variation is extensive with some clones widely distributed and others seemingly unique and localised to a particular endemic focus. Little attention has been given to the molecular epidemiology of Giardia infections. Future studies should be directed at studying the ecology and dynamics of transmission of Giardia clones, particularly in localised areas, and to evaluating the factors that serve to maintain genetic diversity between clones, especially the role of inter-clonal competition. Future research using molecular techniques should aim to identify and follow Giardia clones in nature and correlate genetic typing with important clinical and epidemiological characteristics such as virulence, drug sensitivity and zoonotic potential.

Animals↗
PubMed1995

Biological and genetic analysis of a longitudinal collection of Giardia samples derived from humans.

Duodenal aspirates from children investigated for diarrhoea have been examined for the presence of Giardia over an eleven year period, and where possible, in vitro or in vivo Giardia cultures in mice were established. Based on biochemical characteristics of electrophoretic karyotype, RFLP analysis and rDNA hybridization studies of 40 stocks at least two major varieties, or demes, of Giardia have infected the population of South East Queensland and environs during this period. These demes carried different rDNA repeat units and differed markedly in both the electrophoretic karyotype pattern and the molar representation of chromosome bands. From 1983 to 1991 only one deme was documented. The first evidence of a new deme seen in local children occurred in 1991 and was followed by a predominance of this deme in 1993. These 40 stocks do not represent all positive samples. Other stocks established in vivo were not able to be cultured in vitro, and these probably represent other demes. Since all of the stocks established in vivo were not able to be cultured in vitro, and these probably represent other demes. Since all of the stocks were derived from children with similar chronic symptoms it appears that at least two demes of Giardia are pathogenic.

Animals↗
PubMed1982

The specificity of serum antibodies to Giardia lamblia and to enterobacteria in gastrointestinal disease.

A comparative evaluation was made of immunofluorescent serum antibodies against Giardia lamblia and enterobacteria isolated from the upper intestine of patients with severe giardiasis. The study was carried out on sera from 51 patients belonging to seven intestinal disease groups. Antibodies against Giardia and against some enterobacteria were present in all eight cases of giardiasis with malabsorption, and absent in all five cases of invasive amoebiasis and in six normal control sera. Bacterial antibodies were found in the other five groups of intestinal disease, in 29 out of 38 cases. Giardia antibodies were found in only two of the other groups, tropical sprue (two cases) and coeliac disease (two cases). Thus Giardia antibodies may not signify active giardiasis but they were of restricted distribution and were sometimes present at relatively high titre. Bacterial antibodies were widely distributed but always at low titre. Absorption experiments indicated that the two types of antibody did not cross-react.

Antibodies↗
PubMed1983

Mammalian bile promotes growth of Giardia lamblia in axenic culture.

Giardia lamblia was adapted to grow in modified Diamond's TYI-S culture medium with a mean generation time of 12.0 +/- 1.1 hours. Addition of bovine or porcine bile to this medium dramatically promoted growth with marked reduction in generation time. The shortest mean generation time (7.5 hours) was obtained with fresh bovine bile and to our knowledge is the lowest reported time for axenically cultivated G. lamblia. Trophozoite numbers were increased by a factor of 3.4 after 48 hours cultivation. Generation time lengthened as bile concentration was increased, the effect being most marked with commercial bile preparations. We consider that this bile-supplemented TYI-S medium will support growth more consistently than the usual Giardia culture medium TPS-1, and is also cheaper. This trophic effect of bile on Giardia growth may partly explain why Giardia trophozoites are found mainly in the duodenum and proximal jejunum where bile is plentiful.

Animals↗
PubMed1985

Detection of Giardia in human jejunum by the immunoperoxidase method. Specific and non-specific results.

Rabbit antiserum to cultured trophozoites of Giardia intestinalis (G. lamblia) was used to detect organisms in jejunal biopsies using the PAP immunoperoxidase technique. In 30 sections examined from seven cases of giardiasis associated with histological changes in malabsorption, trophozoites were seen in the lamina propria in one instance, although they were otherwise seen in the intestinal lumen and surface mucus. One anti-Giardia serum reacted with the neutrophil polymorphs in all infected jejunal biopsies, and in jejunal and rectal biopsies from patients not suffering from giardiasis. The reaction with Giardia and with polymorphs could be absorbed out using washed Giardia trophozoites but not with preparations of bacteria.

Giardia↗
PubMedDate not supplied

Heterogeneity in the sensitivity of stocks and clones of Giardia to metronidazole and ornidazole.

The sensitivity in vitro to metronidazole and ornidazole of 7 stocks and of the cloned lines of 5 stocks of Giardia isolated from humans, rodents and monkeys was studied by the growth inhibition test. All 7 stocks of Giardia, irrespective of the host, differed in their sensitivity to these drugs, commonly used in therapy of human giardiasis. The differences were greater with ornidazole than with metronidazole. The 5 Giardia stocks from which clones were prepared were found to consist of populations with significantly (P less than 0.05) differing sensitivities to ornidazole and metronidazole. There was a positive correlation between high resistance in vitro to both drugs of all clones of one parent stock and treatment failures of giardiasis in the patient from which the parasite stock had been isolated. The spectra of sensitivity of Giardia to anti-giardial drugs may have implications concerning the suspected zoonotic character of human giardiasis.

Animals↗
PubMedDate not supplied

Successful experimental infections of a human volunteer and Mongolian gerbils with Giardia of animal origin.

A human volunteer and Mongolian gerbils were shown to be susceptible to infection with Giardia from a Gambian giant pouched rat. The course of infection and the pattern of cyst excretion, as well as the number of cysts in gerbils which were given the same inocula of Giardia, differed from the course of infection in humans. The latent period in gerbils was longer and the gerbils continually excreted numerous cysts, whereas excretion of cysts, in smaller numbers, by the volunteer was intermittent. Moderate clinical symptoms were noted in the volunteer, and the infection was confirmed by the development of anti-Giardia antibodies. The study demonstrated the zoonotic character of giardiasis and has important implications for the epidemiology of Giardia.

Animals↗
PubMed1994

Evaluation of the Merifluor immunofluorescent assay for the detection of Cryptosporidium and Giardia in sodium acetate formalin-fixed stools.

The Merifluor Cryptosporidium/Giardia direct immunofluorescent assay (MFDFA) was used for the blinded detection of Cryptosporidium and Giardia in 100 previously tested sodium acetate formalin (SAF)-preserved fecal specimens. Examination for ova and parasites by formalin-ether concentration followed by iron-hematoxylin and Kinyon staining had demonstrated Cryptosporidium (n = 32), Giardia (n = 20) and both Giardia and Cryptosporidium (n = 5). The MFDFA detected one additional specimen with Cryptosporidium and another with Giardia and was rapid, accurate, and simple to perform.

Acetates↗
PubMedDate not supplied

In vitro activity of two phenyl-carbamate derivatives, singly and in combination with albendazole against albendazole-resistant Giardia intestinalis.

Giardia intestinalis is one of the most prevalent parasites in adults and children in Mexico. Benzimidazoles have been proposed as a therapeutic alternative in the treatment of giardiasis. However, high-dose related toxicity and the development of resistance have emerged in clinical trials using this therapy. In the search of alternative drugs, we found that benzimidazole-resistant strains of fungi have shown increased sensitivity to phenyl-carbamates, hence, we developed several substituted phenyl-carbamates, two of which were tested against the protozoan parasite G. intestinalis in susceptible and albendazole-resistant Giardia strains. 4-R-ethyl-phenyl-carbamates IRE-6A and IRE-7B demonstrated antigiardial, albeit modest, activity when compared with albendazole, against susceptible and albendazole-induced resistant Giardia. However, when albendazole 0.38 microg/mL (MIC(50)) was combined with each IRE compound, a significant antigiardial synergism (fractional inhibitory concentration index (FICI < 0.5)) was obtained not only with sensitive cultures but also with resistant Giardia parasites. The results described here suggest a potential role for a combined therapy with phenyl-carbamates and sub-doses of benzimidazoles in the treatment of giardiasis.

Albendazole↗
PubMed2005

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.

Animals↗
PubMed2006

The effects of metronidazole and furazolidone during Giardia differentiation into cysts.

The protozoon Giardia lamblia infects millions of people worldwide, most of them in underdeveloped countries, where it is frequently a hyperendemic disease. The search for an effective anti-Giardia treatment has been intense, but recurrent infections, virulence factors, and drug resistance imposed obstacles in the achievement of an efficient medication. Most papers about drug effects in Giardia are related to the trophozoite form, although viable cysts, the infective forms, are continuously eliminated in the stools during the treatment. Supported by this knowledge, we analyzed the inhibitory effects of metronidazole (MZ) and furazolidone (FZ) on the differentiation of Giardia into cysts and its viability. The presence of cavities, lamellar bodies and thread-like structures were the most frequent morphological alterations. The results showed also that FZ was more effective by 50% than MZ in inhibiting in vitro cyst differentiation.

Animals↗
PubMed2006

Clathrin-dependent pathways and the cytoskeleton network are involved in ceramide endocytosis by a parasitic protozoan, Giardia lamblia.

Although identified as an early-diverged protozoan, Giardia lamblia shares many similarities with higher eukaryotic cells, including an internal membrane system and cytoskeleton, as well as secretory pathways. However, unlike many other eukaryotes, Giardia does not synthesize lipids de novo, but rather depends on exogenous sources for both energy production and organelle or membrane biogenesis. It is not known how lipid molecules are taken up by this parasite and if endocytic pathways are involved in this process. In this investigation, we tested the hypothesis that highly regulated and selective lipid transport machinery is present in Giardia and necessary for the efficient internalization and intracellular targeting of ceramide molecules, the major sphingolipid precursor. Using metabolic and pathway inhibitors, we demonstrate that ceramide is internalized through endocytic pathways and is primarily targeted into perinuclear/endoplasmic reticulum membranes. Further investigations suggested that Giardia uses both clathrin-dependent pathways and the actin cytoskeleton for ceramide uptake, as well as microtubule filaments for intracellular localization and targeting. We speculate that this parasitic protozoan has evolved cytoskeletal and clathrin-dependent endocytic mechanisms for importing ceramide molecules from the cell exterior for the synthesis of membranes and vesicles during growth and differentiation.

Actins↗
PubMed2006

Prevalence and risk factors for Giardia and coccidia species of pet cats in 2003-2004.

Prevalence and risk factors for feline coccidia and Giardia species infections were estimated for cats visiting 434 Banfield hospitals in 40 states in 2003-2004. Evaluated were 631,021 cats making 1,456,712 office visits (encounters) and having 211,105 fecal examinations. The overall fecal prevalences of coccidia and Giardia species were 1.4% and 0.58%, respectively. Cats at increased risk of coccidia infection were under 4 years of age, intact, and seen during the summer, fall, and spring months compared to winter. Cats at increased risk of Giardia species infection were under 4 years of age. Those at decreased risk were mixed breed and seen during the summer, fall, and spring. The highest regional risk of coccidia and Giardia species infection was for cats in the East South Central region and Mountain region, respectively, compared to the South Pacific region.

Animals↗
PubMed2004

Cyst morphology and sequence analysis of the small subunit rDNA and ef1 alpha identifies a novel Giardia genotype in a quenda (Isoodon obesulus) from Western Australia.

Sequence analysis of the small subunit ribosomal DNA (SSU-rDNA) and elongation factor 1 alpha (ef1 alpha) was performed on Giardia cysts isolated from faeces collected from a quenda (Isoodon obesulus) in the southwest of Western Australia. The SSU-rDNA and ef1 alpha were sequenced in their entirety and correspondingly aligned with the published sequence information of other known species and genotypes of Giardia. Phylogenetic analysis of the SSU-rDNA and ef1 alpha sequences identified the quenda isolate as a novel genotype of Giardia not previously reported. We believe that this quenda Giardia isolate constitutes a distinct species, which may be endemic within the Australian native fauna.

Animals↗
PubMed2005

Influence of 5' sequences on expression of the Tet repressor in Giardia lamblia.

Gene expression is poorly understood in Giardia lamblia. Previously we utilized the Escherichia coli tetracycline regulatory elements to develop a giardial-inducible gene expression system. In this study, we tested the hypothesis that regions flanking the tet repressor (tet R) may influence its expression and affect inducibility of the regulatory system. We found that addition of a 6-His tag or nuclear localization signal (NLS) at the N- but not C-terminus of tet R, increased the induction ratios >100-fold. A non-specific sequence also increased the induction ratio. Fusing NLS at the N-terminus, also led to exclusively nuclear tet R localization. Changing the promoter from gdh or alpha-giardin to alpha2-tubulin increased the induction ratio slightly. Tet R expression at both RNA and protein levels correlated with repression efficiency, indicating that coding sequences of the 6-His tag or NLS may contribute to transcriptional activation of the exotic tet R gene in Giardia. In addition, we found that the tet R system mediated gene repression and induction during encystation. Previous studies used an artificial reporter gene. In this study, we were able to induce overexpression of epitope-tagged cyst wall protein 1 (CWP1) in vegetatively growing Giardia trophozoites. Moreover, we could repress or induce expression of exogenous CWP1 in encysting cells. Taken together, our data suggest that expression of tet R in Giardia is complex and can be strongly influenced by additional sequences, especially at its N-terminus. This system provides insights into expression of an alien gene and can be exploited to regulate gene expression and study important functions in G. lamblia.

5' Flanking Region↗