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PubMed2000

Localization of gamma-tubulin in interphase and mitotic cells of a unicellular eukaryote, Giardia intestinalis.

Giardia intestinalis, a bi-nucleated amitochondrial flagellate, possesses a complex cytoskeleton based on several microtubular systems (flagella, adhesive disk, median body, funis, mitotic spindles). MTOCs of the individual systems have not been fully defined. By using monoclonal antibodies against a conserved synthetic peptide from the C-terminus of human gamma-tubulin we investigated occurrence and distribution of gamma-tubulin in interphase and mitotic Giardia cells. On the immunoblots of Giardia cytoskeletal extracts the antibodies bound to a single polypeptide of approximately 50 kDa. Immunostaining of the interphase cell demonstrated gamma-tubulin as four bright spots at the basis of four out of eight flagella. Gamma-tubulin label was associated with perikinetosomal areas of the ventral and posterolateral pairs of flagella which are formed de novo during cell division. Basal body regions of the anterolateral and caudal pairs of flagella which persist during the division and are integrated into the flagellar systems of the daughter cells did not show gamma-tubulin staining. At early mitosis, gamma-tubulin spots disappeared reappearing again at late mitosis in accord with reorientation of parent flagella and reorganization of flagellar apparatus during cell division. The antibody-detectable gamma-tubulin epitope was absent at the poles of both mitotic spindles. Albendazole-treated Giardia, in which spindle assembly was completely inhibited, showed the same gamma-tubulin staining pattern thus confirming that the fluorescent label is exclusively located in the basal body regions. Our results point to a role of gamma-tubulin in nucleation of microtubules of newly formed flagella and indicate unusual mitotic spindle assembly. Moreover, the demonstration of gamma-tubulin in Giardia shows ubiquity of this protein through the evolutionary history of eukaryotes.

Animals↗
PubMed2002

Improved method for concentration of Giardia, Cryptosporidium, and poliovirus from water.

Methods for the concentration of enteric viruses and the protozoan parasites, Giardia and Cryptosporidium, from drinking water currently require the use of two different types of filters. Electropositive or electronegative microporous filters (0.2-0.45 microm nominal porosity) are used for the collection of enteroviruses, while polypropylene spun-fiber filters (1 microm porosity) and small pleated cartridge filters are used for the collection of protozoan parasites from water. Since the filter mechanically traps the protozoa by size exclusion, a microporous filter with an appropriately small nominal porosity could possibly be used for co-collection of both protozoa and enteroviruses. This study compared the concentration efficiencies of a polypropylene fiber cartridge (DPPPY) filter and two different microporous filters (Filterite and IMDS) with poliovirus (type 1), with respect to their ability to concentrate Giardia and Cryptosporidium from water. Giardia cysts and Cryptosporidium oocysts were added to 4001 of either tap water or tertiary treated wastewater and passed through the test filter. The protozoa were eluted from the polypropylene filter by hand-washing in a detergent solution. Viruses and protozoa were eluted from the microporous filter by two consecutive back-washes with a 1.5% beef extract, 0.1% Tween 80 solution. The eluent was then centrifuged to remove the parasites and the supernatant assayed for viruses. The overall efficiency was greater for the Filterite filter (40.4% for Giardia; 36.6% for Cryptosporidium) when compared to the spun fiber filter (10.1% for Giardia; 16.0% for Cryptosporidium). The Filterite filters were easier and faster to process than the polypropylene spun fiber filters. There was no significant difference in the recovery of protozoa from 1MDS and DPPPY filters. Co-collection of viruses and protozoan parasites from water onto the same filter is possible and can reduce the time and cost of routine water monitoring.

Animals↗
PubMed1996

Albendazole resistance in Giardia is correlated with cytoskeletal changes but not with a mutation at amino acid 200 in beta-tubulin.

Albendazole resistance was induced in three different Giardia cultures following growth in successively increasing amounts of drug. One of the lines was previously resistant to high levels of metronidazole and was able to grow in 2 microM albendazole. The other two survived exposure to 0.8 microM, while normally lethal levels of albendazole against Giardia in vitro were around 0.1-0.2 microM. Albendazole-resistant Giardia were cross-resistant to parbendazole. Major chromosome rearrangements were evident in the line resistant to 2 microM albendazole and IFA with antitubulin antibody indicated differences in the cytoskeleton, particularly the median body, between sensitive and resistant lines. This implicates the cytoskeleton in the mechanism of resistance. Substitution of Tyr for Phe is a consistent beta-tubulin amino acid change in the benzimidazole-resistant helminths and fungi so far analyzed. PCR primers were designed from the published Giardia beta-tubulin gene sequence and spanned the region encoding Phe at position 200. Sequence data from albendazole-resistant Giardia demonstrated that the beta-tubulin gene did not carry a mutation in the codon for amino acid 200. These data suggest that Phe at position 200 in beta-tubulin is not necessary for benzimidazole resistance.

Albendazole↗
PubMed2004

Adaptor protein complex 1 mediates the transport of lysosomal proteins from a Golgi-like organelle to peripheral vacuoles in the primitive eukaryote Giardia lamblia.

Giardia lamblia is an early branching protist that possesses peripheral vacuoles (PVs) with characteristics of lysosome-like organelles, located underneath the plasma membrane. In more evolved cells, lysosomal protein trafficking is achieved by cargo recognition involving adaptor protein (AP) complexes that recognize specific amino acid sequences (tyrosine and/or dileucine motifs) within the cytoplasmic tail of membrane proteins. Previously, we reported that Giardia has a tyrosine-based sorting system, which mediates the targeting of a membrane-associated cysteine protease (encystation-specific cysteine protease, ESCP) to the PVs. Here, we show that Giardia AP1 mediates the transport of ESCP and the soluble acid phosphatase (AcPh) to the PVs. By using the yeast two-hybrid assay we found that the ESCP tyrosine-based motif interacts specifically with the medium subunit of AP1 (Gimicroa). Hemagglutinin-tagged Gimicroa colocalizes with ESCP and AcPh and coimmunoprecipitates with clathrin, suggesting that protein trafficking toward the PVs is clathrin-adaptin dependent. Targeted disruption of Gimicroa results in mislocalization of ESCP and AcPh but not of variant-specific surface proteins. Our results suggest that, unlike mammalian cells, only AP1 is involved in anterograde protein trafficking to the PVs in Giardia. Moreover, even though Giardia trophozoites lack a morphologically discernible Golgi apparatus, the presence of a clathrin-adaptor system suggests that this parasite possess a primitive secretory organelle capable of sorting proteins similar to that of more evolved cells.

Acid Phosphatase↗
PubMed1989

Rapid detection of giardia antigen in stool with the use of enzyme immunoassays.

Enzyme-linked immunosorbent assays (ELISAs) using sera from rabbits and goats immunized with Giardia trophozoites were compared for detection of Giardia antigen in 300 stool specimens, 80 of which had positive results for Giardia by microscopic examination. The diagnostic accuracy of the two assays was similar, with sensitivities of 92% and 87% and specificities of 87% and 91% with the use of rabbit and goat antisera, respectively. Both ELISAs detected Giardia antigen in stool samples from asymptomatic as well as symptomatic excretors and from treated patients after organisms were no longer visualized by microscopic examination. The specificity of the assays was confirmed by consistently negative results on stool specimens from 10 neonates and 27 patients with enteric parasitic infections other than Giardia. Negative results also occurred when stool specimens containing 21 bacterial enteropathogens were tested. Potential confounding variables including multiple freezing and thawing episodes, prolonged storage, and stool filtration did not affect test results from clinical specimens. Antidiarrheal compounds did not interfere with assay results. Preservation of specimens in formalin did interfere with the assay, even if formalinized stool specimens were dialyzed before testing. For diagnosis of giardiasis, the ELISA is a sensitive and specific test that is not influenced by many environmental factors or by other enteropathogens. This test provides a practical and reliable method for evaluating large numbers of specimens in a variety of clinical and epidemiologic settings.

Animals↗
PubMed1985

Restriction-endonuclease analysis of DNA from 15 Giardia isolates obtained from humans and animals.

The DNA banding pattern of 11 human and four animal isolates (two beaver, one cat, and one guinea pig) of Giardia were compared by using two related techniques. Patterns were compared after endonuclease restriction of DNA followed by agarose gel electrophoresis and ethidium bromide staining and after Southern blot analysis using recombinant plasmids containing Giardia DNA as probes. Two major groups could be distinguished with ethidium bromide staining of eight isolates. Southern blot analysis, however, could distinguish nine different patterns among the 15 isolates studied. One common banding pattern was seen in six isolates (two animal and four human); the remainder of the isolates were unique, with the exception of two identical isolates from sisters. Three isolates (one from a beaver and two from humans) were markedly different from Giardia with the common banding pattern, whereas the other six unique isolates varied moderately. Beavers and other mammals do not seem to possess their own species of Giardia. This methodology introduces a way of distinguishing one species of Giardia isolate from another and promises to be helpful in epidemiological investigations.

Animals↗
PubMed2004

Effect of Giardia infection on growth and psychomotor development of children aged 0-5 years.

Giardiasis, an intestinal protozoan infection caused by Giardia intestinalis, is common in southeastern Anatolia, Turkey. In this cross-sectional survey, to investigate the role of giardiasis on growth and psychomotor development, we studied 160 children aged 0-5 years. Data were collected using a structured questionnaire, anthropometry, Ankara Developmental Screening Inventory, and laboratory analysis of fecal samples. The results showed that 50 per cent of the subjects were infected with at least one pathogen of intestinal parasitic infections. Giardia intestinalis was the most frequent pathogenic parasite. Giardia-infected children had a risk for stunted (OR = 7.67, 95 per cent CI = 2.25-26.16; p = 0.001) and poor psychomotor development (OR = 2.68, 95 per cent CI = 1.09-6.58; p = 0.030). The data indicate that Giardia intestinalis infection has an adverse impact on child linear growth and psychomotor development. In the primary healthcare centers, during the programme of the monitoring growth and developmental status of children, following children in terms of Giardia, diagnosis and treatment will have a positive effect on child health.

Animals↗
PubMed1993

Unique phylogenetic position of Diplomonadida based on the complete small subunit ribosomal RNA sequence of Giardia ardeae, G. muris, G. duodenalis and Hexamita sp.

Complete small-subunit rRNA (SSU-rRNA) coding region sequences were determined for two species of the intestinal parasite Giardia: G. ardeae and G. muris, both belonging to the order Diplomonadida, and a free-living member of this order, Hexamita sp. These sequences were compared to published SSU-rDNA sequences from a third member of the genus Giardia, G. duodenalis (often called G. intestinalis or G. lamblia) and various representative organisms from other taxa. Of the three Giardia sequences analyzed, the SSU-rRNA from G. muris is the smallest (1432 bases as compared to 1435 and 1453 for G. ardeae and G. duodenalis, respectively) and has the lowest G+C content (58.9%). The Hexamita SSU-rRNA is the largest in this group, containing 1550 bases. Because the sizes of the SSU-rRNA are prokaryotic rather than typically eukaryotic, the secondary structures of the SSU-rRNAs were constructed. These structures show a number of typically eukaryotic signature sequences. Sequence alignments based on constraints imposed by secondary structure were used for construction of a phylogenetic tree for these four taxa. The results show that of the four diplomonads represented, the Giardia species form a distinct group. The other diplomonad Hexamita and the microsporidium Vairimorpha necatrix appear to be distinct from Giardia.

Animals↗
PubMed2005

Secretory antibodies against Giardia intestinalis in lactating Nicaraguan women.

Secretory IgA (sIgA) antibodies are important in the host defence against the intestinal protozoan parasite Giardia intestinalis. However, few antigens have been identified. In this study 100 milk and saliva samples from lactating women, living in an endemic region (León, Nicaragua), were screened for the presence of antibodies against G. intestinalis. Most milk and saliva samples contained anti-Giardia antibodies (59% and 52%, respectively), with a mean sIgA content 50 times higher in milk than in saliva. The positive samples reacted with trophozoite membrane, flagella and cytoplasmic antigens. Western blot analysis showed that milk and saliva anti-Giardia sIgA recognized up to 16 different Giardia proteins in the molecular weight region 20-165 kDa. Two-dimensional Western blotting showed that the major immunoreactive proteins were the same as the immunoreactive proteins identified by serum from acute giardiasis patients in a non-endemic country. The major difference was a stronger reactivity against the variant surface proteins (VSPs) in the milk samples. Milk sIgAs also recognized recombinant Giardia proteins such as alpha-1 giardin, ornithine carbamoyl transferase, VSP-4EX, arginine deaminase and alpha-enolase. These antigens will be important targets in the development of new immunodiagnostic tools and vaccines.

Animals↗
PubMedDate not supplied

Fibrillarin, a conserved pre-ribosomal RNA processing protein of Giardia.

The flagellated protozoan Giardia has been shown by 16S rRNA sequence analysis to be one of the most primitive of the eukaryotes. A gene encoding the protein fibrillarin, a pre-rRNA processing protein implicated in rRNA methylation and ribosome assembly, has been isolated. A genomic DNA fragment 1,240 base pairs long containing an open reading frame of 981 base pairs (327 amino acids) was sequenced. The deduced protein sequence of 35.3 kDa is similar to other known fibrillarin sequences. The Giardia sequence includes the amino terminal glycine/arginine rich domain characteristic of eukaryotic fibrillarins but is unique in having a large number of acidic residues in this domain. Phylogenetic analysis of the available fibrillarin sequences is consistent with the assignment of Giardia to a position close to the most primitive of the eukaryotes. A monoclonal antibody to yeast fibrillarin crossreacts with a 36 kDa polypeptide from Giardia on western blots and diffusely stains both nuclei of the organism by immunofluorescence microscopy. This result is consistent with the absence of well defined nucleoli in this organism. The evolutionary conservation of fibrillarin suggests an important function for this protein in ribosome biosynthesis, and this function appears to be maintained from the archaebacteria, which lack a nucleus, to Giardia, which contains a nucleus but lacks a prominent nucleolus, to higher mammals, which have both nucleus and nucleolus.

Amino Acid Sequence↗
PubMed2006

Detection of Cryptosporidium, Giardia and Enterocytozoon bieneusi in surface water, including recreational areas: a one-year prospective study.

Accidental ingestion of natural waters while bathing carries a risk of infection by waterborne protozoa such as Cryptosporidium, Giardia and, possibly, microsporidia. In order to evaluate this risk, we conducted a one-year prospective study of two recreational lakes and three river sites located near Paris, where bathing and boating are frequent. Twenty-litre water samples were collected monthly from each site. Concentrated samples were submitted to immunomagnetic separation followed by immunofluorescence (IMS-IF) for Cryptosporidium and Giardia detection. PCR and PCR restriction fragment length polymorphism (PCR-RFLP) were used for the genetic characterization of Cryptosporidium species on IMS-IF-positive samples. PCR were systematically performed to detect Enterocytozoon bieneusi. Bacteria counts were also determined. IMS-IF revealed low counts of Giardia cysts and Cryptosporidium oocysts in the recreational lakes, with occasional peaks (max. 165 cysts/10 L and 9 oocysts/10 L). By contrast, the river sites were consistently and sometimes heavily contaminated throughout the year. Enterocytozoon bieneusi was found in only two river samples. PCR-RFLP genotyping showed the presence of C. hominis and C. parvum. No correlation was found between the presence or counts of parasites and bacteria, except between the presence of Giardia and high counts of Escherichia coli and enterococci. Based on a previously developed model for quantitative risk assessment of waterborne parasitic infections, we estimated that the mean risk of infection by Cryptosporidium and Giardia associated with swimming was <10(-4) in the recreational lakes, and frequently higher at the river sites.

Animals↗
PubMed1999

Efficacy of new 5-nitroimidazoles against metronidazole-susceptible and -resistant Giardia, Trichomonas, and Entamoeba spp.

The efficacies of 12 5-nitroimidazole compounds and 1 previously described lactam-substituted nitroimidazole with antiparasitic activity, synthesized via SRN1 and subsequent reactions, were assayed against the protozoan parasites Giardia duodenalis, Trichomonas vaginalis, and Entamoeba histolytica. Two metronidazole-sensitive lines and two metronidazole-resistant lines of Giardia and one line each of metronidazole-sensitive and -resistant Trichomonas were tested. All except one of the compounds were as effective or more effective than metronidazole against Giardia and Trichomonas, but none was as effective overall as the previously described 2-lactam-substituted 5-nitroimidazole. None of the compounds was markedly more effective than metronidazole against Entamoeba. Significant cross-resistance between most of the drugs tested and metronidazole was evident among metronidazole-resistant lines of Giardia and Trichomonas. However, some drugs were lethal to metronidazole-resistant Giardia and had minimum lethal concentrations similar to that of metronidazole for drug-susceptible parasites. This study emphasizes the potential in developing new nitroimidazole drugs which are more effective than metronidazole and which may prove to be useful clinical alternatives to metronidazole.

Animals↗
PubMed1981

Effect of chlorine on Giardia lamblia cyst viability.

The effect of chlorine concentration on Giardia lamblia cyst viability was tested under a variety of conditions. The ability of Giardia cysts to undergo excystation was used as the criterion of viability. The experimental variables employed included temperature (25, 15, and 5 degrees C), pH (6, 7, and 8), chlorine-cyst contact time (10, 30, and 60 min), and chlorine concentration (1 to 8 mg/liter). In the pH range studied, cyst survival generally was observed to increase as buffer pH increased. Water temperature coupled with chlorination proved to be important in cyst survival. Results of these experiments at the three temperatures studied can be summarized as follows: at 25 degrees C, exposure to 1.5 mg/liter for 10 min killed all cysts at pH 6, 7, and 8. At 15 degrees C, 2.5 mg of chlorine per liter for 10 min killed all cysts at pH 6, but at pH 7 and 8 small numbers of cysts remained viable after 30 min but not after 60 min. At 5 degrees C, 1 mg of chlorine per liter for 60 min failed to kill all the cysts at any pH tested. At this temperature, 2 mg of chlorine per liter killed all cysts after 60 min at pH 6 and 7, but not at pH 8. A chlorine concentration of 4 mg/liter killed all the cysts at all three pH values after 60 min, but not after 30 min. A chlorine concentration of 8 mg/liter killed all Giardia cysts at pH 6 and 7 after contact for 10 min, and at pH 8 after 30 min. This study points up the role of temperature, pH, and chlorine demand in the halogen treatment of drinking water to destroy cysts. It also raises an epidemiological problem, namely: low water temperatures, where killing of Giardia requires relatively high chlorine concentrations and long contact times, are (i) to be expected in many areas where epidemic waterborne giardiasis has been reported and (ii) particularly conducive to the long-term survival of Giardia cysts.

Animals↗
PubMed1985

Use of immunofluorescence and phase-contrast microscopy for detection and identification of Giardia cysts in water samples.

A method was developed in which indirect immunofluorescence and phase-contrast microscopy are used for rapid detection and identification of Giardia cysts in raw and finished water supplies. When anti-Giardia cyst antiserum and fluorescein conjugate were applied to known Giardia cysts on membrane filters, the cysts fluoresced bright green when they were illuminated by UV light. This procedure permitted individual cysts to be quickly located even in samples heavily contaminated with other microorganisms and debris. The identity of presumptive Giardia cysts located in this way could then be confirmed by observing characteristic internal morphological features with phase-contrast microscopy. With this method, Giardia cysts were detected and their identities were confirmed in samples taken from raw and finished surface water supplies during several recent outbreaks.

Fluorescent Antibody Technique↗
PubMed1991

Occurrence of Giardia and Cryptosporidium spp. in surface water supplies.

Giardia and Cryptosporidium levels were determined by using a combined immunofluorescence test for source waters of 66 surface water treatment plants in 14 states and 1 Canadian province. The results showed that cysts and oocysts were widely dispersed in the aquatic environment. Giardia spp. were detected in 81% of the raw water samples. Cryptosporidium spp. were found in 87% of the raw water locations. Overall, Giardia or Cryptosporidium spp. were detected in 97% of the raw water samples. Higher cyst and oocyst densities were associated with source waters receiving industrial or sewage effluents. Significant correlations were found between Giardia and Cryptosporidium densities and raw water quality parameters such as turbidity and total and fecal coliform levels. Statistical modeling suggests that cyst and oocyst densities could be predicted on the basis of watershed and water quality characteristics. The occurrence of high levels of Giardia cysts in raw water samples may require water utilities to apply treatment beyond that outlined in the Surface Water Treatment Rule of the U.S. Environmental Protection Agency.

Animals↗
PubMed1991

Giardia and Cryptosporidium spp. in filtered drinking water supplies.

Giardia and Cryptosporidium levels were determined by using a combined immunofluorescence test for filtered drinking water samples collected from 66 surface water treatment plants in 14 states and 1 Canadian province. Giardia cysts were detected in 17% of the 83 filtered water effluents. Cryptosporidium oocysts, were observed in 27% of the drinking water samples. Overall, cysts or oocysts were found in 39% of the treated effluent samples. Despite the frequent detection of parasites in drinking water, microscopic observations of the cysts and oocysts suggested that most of the organisms were nonviable. Compliance with the filtration criteria outlined by the Surface Water Treatment Rule of the U.S. Environmental Protection Agency did not ensure that treated water was free of cysts and oocysts. The average plant effluent turbidity for sites which were parasite positive was 0.19 nephelometric turbidity units. Of sites that were positive for Giardia or Cryptosporidium spp., 78% would have been able to meet the turbidity regulations of the Surface Water Temperature Rule. Evaluation of the data by using a risk assessment model developed for Giardia spp. showed that 24% of the utilities examined would not meet a 1/10,000 annual risk of Giardia infection. For cold water conditions (0.5 degree C), 46% of the plants would not achieve the 1/10,000 risk level.

Animals↗
PubMed1997

Prevalence of and associated risk factors for shedding Cryptosporidium parvum oocysts and Giardia cysts within feral pig populations in California.

Populations of feral pigs (Sus scrofa) may serve as an environmental reservoir of Cryptosporidium parvum oocysts and Giardia sp. cysts for source water. We conducted a cross-sectional study to determine the prevalence of and associated demographic and environmental risk factors for the shedding of C. parvum oocysts and Giardia sp. cysts. Feral pigs were either live-trapped or dispatched from 10 populations located along the coastal mountains of western California, and fecal samples were obtained for immunofluorescence detection of C. parvum oocysts and Giardia sp. cysts. We found that 12 (5.4%) and 17 (7.6%) of 221 feral pigs were shedding C. parvum oocysts and Giardia sp. cysts, respectively. The pig's sex and body condition and the presence of cattle were not associated with the probability of the shedding of C. parvum oocysts. However, younger pigs (< or = 8 months) and pigs from high-density populations (> 2.0 feral pigs/km2) were significantly more likely to shed oocysts compared to older pigs (> 8 months) and pigs from low-density populations (< or = 1.9 feral pigs/km2). In contrast, none of these demographic and environmental variables were associated with the probability of the shedding of Giardia sp. cysts among feral pigs. These results suggest that given the propensity for feral pigs to focus their activity in riparian areas, feral pigs may serve as a source of protozoal contamination for surface water.

Animals↗
PubMed2002

Occurrence of Cryptosporidium and Giardia in wild ducks along the Rio Grande River valley in southern New Mexico.

Fecal samples were taken from wild ducks on the lower Rio Grande River around Las Cruces, N. Mex., from September 2000 to January 2001. Giardia cysts and Cryptosporidium oocysts were purified from 69 samples by sucrose enrichment followed by cesium chloride (CsCl) gradient centrifugation and were viewed via fluorescent-antibody (FA) staining. For some samples, recovered cysts and oocysts were further screened via PCR to determine the presence of Giardia lamblia and Crytosporidium parvum. The results of this study indicate that 49% of the ducks were carriers of Cryptosporidium, and the Cryptosporidium oocyst concentrations ranged from 0 to 2,182 oocysts per g of feces (mean +/- standard deviation, 47.53 +/- 270.3 oocysts per g); also, 28% of the ducks were positive for Giardia, and the Giardia cyst concentrations ranged from 0 to 29,293 cysts per g of feces (mean +/- standard deviation, 436 +/- 3,525.4 cysts per g). Of the 69 samples, only 14 had (oo)cyst concentrations that were above the PCR detection limit. Samples did test positive for Cryptosporidium sp. However, C. parvum and G. lamblia were not detected in any of the 14 samples tested by PCR. Ducks on their southern migration through southern New Mexico were positive for Cryptosporidium and Giardia as determined by FA staining, but C. parvum and G. lamblia were not detected.

Animals↗