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Concurrent infections of Giardia and Cryptosporidium on two Ohio farms with calf diarrhea.

Giardia and Cryptosporidium infections were diagnosed by immunofluorescence assay on two Ohio dairy farms with calf diarrhea problems. On the first farm, all nine diarrheic calves sampled once in June had Giardia cysts in their feces. On the second farm, all five diarrheic calves examined at the beginning of the diarrhea outbreak in March had Giardia infection. When resampled, the overall infection rate of normal and diarrheic calves was 82.4% in April, and 40.0% in August after the diarrhea subsided. Positive calves ranged from 11 to 164 days of age, and 22.2% of them were as young as 1 to 3 weeks of age. Eight of nine diarrheic calves (88.8%) on the first farm had Cryptosporidium infection. Lower infection rates (< 30%) were found on the second farm. Six of 10 positive calves were 11-22 days old, three were 164-177 days old, and one was 71 days old. Five of these 10 positive calves were also positive for Giardia infection. Five diarrheic calves on the northern Ohio farm and one diarrheic calf on the central Ohio farm were treated with metronidazole after failing to respond to antibiotic therapy. Clinical improvement was observed in all calves within 48 h after the start of treatment. The high Giardia infection rates and intensities in calves of a wide age range and the clinical response to metronidazole suggest that Giardia infection contributed to the outbreaks of diarrhea.

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Occurrence of Giardia and Cryptosporidium in water supplies of Russia and Bulgaria.

The aim of the present study was to investigate water supplies in southern Russia and Bulgaria, in order to estimate the occurrence of Giardia and Cryptosporidium in drinking water resources from these countries. A total of 166 water samples of different origin (surface, tap, bottled, well, spring and waste water) were collected from Rostov (southern Russia), Sofia and Varna (Bulgaria) Greater Areas and screened for the detection of Giardia cysts and Cryptosporidium oocysts. The method incorporated concentration of water samples by filtration and flocculation, sucrose purification, (oo)cyst detection/identification by immunofluorescence test and differential interference contrast. Sixteen out of 166 samples (9.6%) were positive for Giardia and 30 (18.1%) positive for Cryptosporidium. Both Giardia and Cryptosporidium were detected in tap, river, well and waste waters. Giardia cysts were additionally detected in bottled water. Particularly some river, waste and well water samples were highly contaminated with (oo)cysts. This study has shown that drinking water supplies in Russia and Bulgaria are subject to contamination with Giardia and Cryptosporidium, with potential hazards for the public health.

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Genotyping of Giardia in Dutch patients and animals: a phylogenetic analysis of human and animal isolates.

Giardia duodenalis (syn. Giardia lamblia, Giardia intestinalis) is a protozoan organism that can infect the intestinal tract of many animal species including mammals. Genetic heterogeneity of G. duodenalis is well described but the zoonotic potential is still not clear. In this study, we analysed 100 Giardia DNA samples directly isolated from human stool specimens, to get more insight in the different G. duodenalis assemblages present in the Dutch human population. Results showed that these human isolates could be divided into two main Assemblages A and B within the G. duodenalis group on the basis of PCR assays specific for the Assemblages A and B and the DNA sequences of 18S ribosomal RNA and the glutamate dehydrogenase (gdh) genes. Genotyping results showed that G. duodenalis isolates originating from Dutch human patients belonged in 35% of the cases to Assemblage A (34/98) and in 65% of the cases to Assemblage B (64/98) whereas two human cases remained negative in all assays tested. In addition, we compared these human samples with animal samples from the Netherlands and human and animal samples from other countries. A phylogenetic analysis was carried out on the DNA sequences obtained from these Giardia and those available in GenBank. Using gdh DNA sequence analysis, human and animal Assemblage A and B Giardia isolates could be identified. However, phylogenetic analysis revealed different sub-clustering for human and animal isolates where host-species-specific assemblages (C, D, E, F and G) could be identified. The geographic origin of the human and animal samples was not a discriminating factor.

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Prevalence of Giardia and Cryptosporidium in dairy calves in three areas of Norway.

A study was undertaken to determine the prevalences of Cryptosporidium and Giardia in dairy calves less than 6 months of age in Norway. Faecal samples were collected from a total of 1386 calves, between 3 and 183 days of age, in 136 dairy farms from three different areas of Norway. Faecal samples were examined for Cryptosporidium oocysts and Giardia cysts after concentration and immunofluorescent staining. Giardia was found in 93% (127 out of 136) of the farms and in 49% (679 out of 1386) of the calves. Cryptosporidium was found in 53% (72 out of 136) of the farms and in 12% (167 out of 1386) of the calves. The level of Giardia and/or Cryptosporidium was low in the majority of the infected calves. Infection peaked in the age group 2-3 months for both Cryptosporidium and Giardia. The prevalences of both parasites were higher in samples taken during winter than in samples taken during summer, and statistically significant differences were found when prevalences in different age groups of calves were compared between the three areas. A significantly lower prevalence of Cryptosporidium was found in calves housed in shared pens that were thoroughly washed more than three times a year than in calves from pens washed less often. For Giardia there was a trend for decreasing intensity of infection with increasing age in the sampled calves. For Cryptosporidium there was a trend for increasing herd prevalence with increasing number of calves in the herd, but this trend was not statistically significant. Other parameters which were investigated such as housing, feeding or management routines were not associated with prevalence or intensity of infection with either parasite.

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Prevalence of Cryptosporidium and Giardia in free-ranging wild cervids in Norway.

Faecal samples were collected from 1,190 wild cervids in Norway and analyzed for cysts/oocysts of the protozoan parasites Giardia and Cryptosporidium. Samples were from calves, yearlings and adults of moose (Alces alces), red deer (Cervus elaphus), roe deer (Capreolus capreolus) and reindeer (Rangifer tarandus) shot during the hunting season. Cryptosporidium was found in 15 (3.3%) of 455 moose, 1 (0.3%) of 289 red deer, 18 (6.2%) of 291 roe deer, but was not found in any of 155 reindeer. Giardia was found in 56 (12.3%) moose, 5 (1.7%) red deer, 45 (15.5%) roe deer and 11 (7.1%) reindeer. The calves had the highest prevalence of infection, but this was only statistically significant for Giardia in moose and for Cryptosporidium and Giardia in roe deer. Calves generally had the highest intensity of infection, but this difference was only statistically significant for calves with Giarda and the highest intensity of infection. Both Giardia and Cryptosporidium were found in samples from several geographical areas, indicating that these parasites are distributed among the cervid population in all parts of Norway, especially in moose and roe deer. This is the first published report of Cryptosporidium in moose and of Giardia in reindeer.

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The cytoskeleton of Giardia lamblia.

Giardia lamblia is a ubiquitous intestinal pathogen of mammals. Evolutionary studies have also defined it as a member of one of the earliest diverging eukaryotic lineages that we are able to cultivate and study in the laboratory. Despite early recognition of its striking structure resembling a half pear endowed with eight flagella and a unique ventral disk, a molecular understanding of the cytoskeleton of Giardia has been slow to emerge. Perhaps most importantly, although the association of Giardia with diarrhoeal disease has been known for several hundred years, little is known of the mechanism by which Giardia exacts such a toll on its host. What is clear, however, is that the flagella and disk are essential for parasite motility and attachment to host intestinal epithelial cells. Because peristaltic flow expels intestinal contents, attachment is necessary for parasites to remain in the small intestine and cause diarrhoea, underscoring the essential role of the cytoskeleton in virulence. This review presents current day knowledge of the cytoskeleton, focusing on its role in motility and attachment. As the advent of new molecular technologies in Giardia sets the stage for a renewed focus on the cytoskeleton and its role in Giardia virulence, we discuss future research directions in cytoskeletal function and regulation.

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Modelling the sewage discharge and dispersion of Cryptosporidium and Giardia in surface water.

Modelling the discharge of parasitic protozoa into surface water and the dispersion in rivers and streams gives insight into the contribution of the different sources of environmental contamination and in the transmission of these organisms from the point of discharge to drinking water abstraction points and bathing sites. We tested the applicability of emission (PROMISE) and dispersion (WATNAT) models developed for chemical pollutants to describe the environmental behaviour of Cryptosporidium and Giardia in the Netherlands. The annual load of Cryptosporidium and Giardia in domestic wastewater was 3.2 x 10(13) and 3.8 x 10(14) respectively. The majority (85%) of the Cryptosporidium oocysts was discharged with effluent of wastewater treatment plants. while the majority (82%) of the Giardia cysts was discharged with untreated wastewater discharges and sewer overflows. The estimated annual import through the river Rhine and Meuse was 3.2 x 1014 Cryptosporidium oocysts and 2.1 x 10(15) Giardia cysts, of which the river Rhine contributed 87 and 66%, respectively. This outweighed the total load of the discharges of treated and untreated wastewater in the Netherlands. The combination of PROMISE and WATNAT predicted concentrations of Cryptosporidium and Giardia in surface water that were in the same order of magnitude as the concentrations that were observed at 5 of the 6 sites compared. At a site with primarily agricultural contamination, the models predicted concentrations that were 1 10log-unit lower than the observed concentrations. This is a first step in the direction of a quantitative description of the transmission cycle of Cryptosporidum and Giardia through water. The use of these models combines observational occurrence data and experimental data from laboratory survival studies into a single integrated description. The description needs further improvement by incorporation of agricultural run-off and increasing the number and time frame of input monitoring data.

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Recovery of Cryptosporidium oocysts and Giardia cysts from source water concentrates using immunomagnetic separation.

Immunomagnetic separation (IMS) procedures for the simultaneous isolation of Cryptosporidium oocysts and Giardia cysts have recently become available. We validated Dynal's GC-Combo IMS kit using source water at three turbidity levels (5000, 500 and 50 nephelometric turbidity units [ntu]) obtained from different geographical locations and spiked with approximately 9--11 (oo)cysts per ml. Mean recoveries of Cryptosporidium oocysts and Giardia cysts in deionized water were 62% and 69%, respectively. In turbid water matrices, mean recoveries of Cryptosporidium oocysts were between 55.9% and 83.1% while mean recoveries of cysts were between 61.1% and 89.6%. Marginally higher recoveries of the heat inactivated (oo)cysts were observed (119.4% Cryptosporidium oocysts and 90.9% Giardia cysts) in deionized water when compared with recoveries of viable (oo)cysts (69.7% Cryptosporidium oocysts and 79% Giardia cysts). Age of (oo)cysts on recoveries using the GC-Combo IMS kit demonstrated no effects up to 20 months old. Recovery of Giardia cysts was consistent for isolates aged up to 8 months (81.4%), however, a significant reduction in recoveries was noted at 20 months age. Recoveries of low levels (5 and 10 (oo)cysts) of Cryptosporidium oocysts and Giardia cysts in deionized water using IMS ranged from 51.3% to 78% and from 47.6% to 90.0%, respectively. Results of this study indicate that Dynal's GC-Combo IMS kit is an efficient technique to separate Cryptosporidium/Giardia from turbid matrices and yields consistent, reproducible recoveries. The use of fresh (recently voided and purified) (oo)cysts, aged (oo)cysts, viable and heat-inactivated (oo)cysts indicated that these parameters do not influence IMS performance.

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Giardia and Cryptosporidium in backwash water from rapid sand filters used for drinking water production.

Backwash water from rapid sand filters of a treatment plant using surface water from small rivers for drinking water production was examined with the aim of determining the degree of their potential contamination with Giardia cysts and Cryptosporidium oocysts. Simultaneous investigations were carried out for both protozoans from November 1993 to February 1994. Water samples were concentrated by continuous flow centrifugation (11 backwash water samples) or by using polypropylene cartridge filters (12 raw water samples and 39 backwash water samples). Parasites were identified by the direct immunofluorescence assay. Ten out of 12 raw water samples tested were positive for Giardia (range: 2-103/ 100 L) and 8 out of 12 were positive for Cryptosporidium (range: 0.8-109/100L). Eight of 11 backwash water samples collected by continuous flow centrifugation were positive for Giardia (range: 3-86/100 L) or Cryptosporidium (range: 1-69/100 L). Out of 39 samples collected using cartridge filters, 34 were positive for Giardia (range: 1.4-374/100 L) and 33 for Cryptosporidium (range: 0.8-252/100 L). Overall, Giardia, Cryptosporidium, or both were detected in 92% of the backwash water samples. The results have clearly shown that backwash waters were contaminated with Giardia and Cryptosporidium and the supernatant returned to the raw water after the sedimentation process was not free from cysts and oocysts.

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Giardia and Cryptosporidium removal from waste-water by a duckweed (Lemna gibba L.) covered pond.

AIMS: To determine the ability of duckweed ponds used to treat domestic waste-water to remove Giardia and Cryptosporidium. METHODS AND RESULTS: The influent and effluent of a pond covered with duckweed with a 6 day retention time was tested for Giardia cysts, Cryptosporidium oocysts, faecal coliforms and coliphage. Giardia cysts and Cryptosporidium oocysts were reduced by 98 and 89%, respectively, total coliforms by 61%, faecal coliforms by 62% and coliphage by 40%. There was a significant correlation between the removal of Giardia cysts and Cryptospordium oocysts by the pond (P < 0.001). Influent turbidity and parasite removal were also significantly correlated (Cryptosporidium and turbidity, P=0.05; Giardia and turbidity, P=0.01). CONCLUSIONS: The larger organisms (parasites) probably settled to the bottom of the pond, while removal of smaller bacteria and coliphages in the pond was not as effective. SIGNIFICANCE AND IMPACT OF THE STUDY: Duckweed ponds may play an important role in wetland systems for reduction of Giardia and Cryptosporidium.

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An ancestral secretory apparatus in the protozoan parasite Giardia intestinalis.

The protozoan parasite Giardia intestinalis belongs to one of the earliest diverged eukaryotic lineages. This is also reflected in a simple intracellular organization, as Giardia lacks common subcellular compartments such as mitochondria, peroxisomes, and apparently also a Golgi apparatus. During encystation, developmentally regulated formation of large secretory compartments containing cyst wall material occurs. Despite the lack of any morphological similarities, these encystation-specific vesicles (ESVs) show several biochemical characteristics of maturing Golgi cisternae. Previous studies suggested that Golgi structure and function are induced only during encystation in Giardia, giving rise to the hypothesis that ESVs, as a Giardia Golgi equivalent, are generated de novo. Alternatively, ESV compartments could be built on the template structure of a cryptic Golgi in trophozoites in response to ER export of cyst wall material during encystation. We addressed this question by defining the molecular framework of the Giardia secretory apparatus using a comparative genomic approach. Analysis of the corresponding transcriptome during growth and encystation revealed surprisingly little stage-specific regulation. A panel of antibodies was generated against selected marker proteins to investigate the developmental dynamics of the endomembrane system. We show evidence that Giardia accommodates the export of large amounts of cyst wall material through re-organization of membrane compartment(s) in trophozoites with biochemical similarities to ESVs. This suggests that ESVs are selectively stabilized Golgi-like compartments in a unique and archetypical secretory system, which arise from a structural template in trophozoites rather than being generated de novo.

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Mitosis in Giardia lamblia: multiple modes of cytokinesis.

Mitosis in Giardia is poorly understood. Until today, it is still controversial whether Giardia divides with a mirror-image symmetry (ventral-ventral or dorsal-dorsal) or in a dorsal-ventral mode. Here, we report the different modes by which cytokinesis takes place in Giardia lamblia. To determine how Giardia divides, video microscopy, scanning electron microscopy, semi-thick sections and freeze-fracture replicas were analyzed by transmission electron microscopy. Between 12 and 15% of the cells cultivated for 24-48 h were found in the process of division. Three types of cytokinesis were found: (1) ventral-ventral, where the discs face each other; (2) dorsal-dorsal, where the discs are in opposite directions; and (3) ventral-dorsal. Giardia divides with mirror-image symmetry either in ventral-ventral or dorsal-dorsal modes. During ventral-ventral type of division, Giardia becomes detached and swims freely in the culture medium, whereas, in the other modes of division, the cells can be found either adhered or swimming.

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Occurrence of Cryptosporidium and Giardia in irrigation water and its impact on the fresh produce industry.

In Mexico, relatively few studies have examined the presence of protozoan parasites from surface waters with irrigation and wash-water applications in the fresh produce industry. In this study, the presence of Cryptosporidium oocysts and Giardia cysts in surface water for irrigation, washing and disinfecting applications was evaluated. In addition, the occurrence of Cryptosporidium oocysts and Giardia cysts was determined in wash-water tanks at selected packinghouses. The occurrence of both Cryptosporidium and Giardia was determined by an immunofluorescent technique. Forty-eight percent of the surface water samples tested positive for Cryptosporidium oocysts and 50% tested positive for Giardia cysts. Concentration ranges for Cryptosporidium oocysts and Giardia cysts were 17-200 and 17-1633 per 100 litres, respectively. Sixteen percent of the wash-water tanks tested positive for Cryptosporidium oocysts and 83% were positive for Giardia cysts with concentrations ranging from 1-133 oocysts and 100-533 cysts per 100 litres, respectively. The presence of protozoan parasites in surface water for irrigation and packinghouse operations suggests that there may be a risk of contamination of fresh produce as protozoan oocysts/cysts might come in contact with and attach to crop surfaces posing a risk of infection to consumers who eat these products.

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Evolution of eukaryotic transcription: insights from the genome of Giardia lamblia.

The Giardia lamblia genome sequencing project affords us a unique opportunity to conduct comparative analyses of core cellular systems between early and late-diverging eukaryotes on a genome-wide scale. We report a survey to identify canonical transcription components in Giardia, focusing on RNA polymerase (RNAP) subunits and transcription-initiation factors. Our survey revealed that Giardia contains homologs to 21 of the 28 polypeptides comprising eukaryal RNAPI, RNAPII, and RNAPIII; six of the seven RNAP subunits without giardial homologs are polymerase specific. Components of only four of the 12 general transcription initiation factors have giardial homologs. Surprisingly, giardial TATA-binding protein (TBP) is highly divergent with respect to archaeal and higher eukaryotic TBPs, and a giardial homolog of transcription factor IIB was not identified. We conclude that Giardia represents a transition during the evolution of eukaryal transcription systems, exhibiting a relatively complete set of RNAP subunits and a rudimentary basal initiation apparatus for each transcription system. Most class-specific RNAP subunits and basal initiation factors appear to have evolved after the divergence of Giardia from the main eukaryotic line of descent. Consequently, Giardia is predicted to be unique in many aspects of transcription initiation with respect to paradigms derived from studies in crown eukaryotes.

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Activity of metronidazole, azithromycin and three benzimidazoles on Giardia lamblia growth and attachment to a human intestinal cell line.

BACKGROUND: Attachment of Giardia lamblia trophozoites to enterocytes is essential for colonization of the small intestine and is considered a prerequisite for Giardia-induced enterocyte damage. Inhibition of attachment may therefore have therapeutic potential. METHODS: Enterocyte-like differentiated Caco-2 cells were used as a biologically appropriate attachment surface to determine the effect of three benzimidazole compounds (albendazole, mebendazole and thiabendazole), azithromycin and metronidazole on Giardia attachment. The results were compared with the ability for each drug to inhibit Giardia growth, measured using [3H]-thymidine uptake. RESULTS: The benzimidazoles inhibited Giardia attachment at much lower concentrations than did metronidazole. However, metronidazole was a much more potent inhibitor of growth than any of the benzimidazoles. Azithromycin did not significantly impair Giardia attachment or growth. The benzimidazoles decrease attachment but are less giardiacidal than metronidazole. CONCLUSION: This model appears useful for testing potential antigiardial compounds and investigating mechanisms of drug action.

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Evaluation of seven commercial antigen detection tests for Giardia and Cryptosporidium in stool samples.

Stool samples from patients with abdominal symptoms were used to evaluate different copro-diagnostic assays for the detection of Giardia and Cryptosporidium. Results from microscopical examination following conventional stool concentration and direct fluorescent-antibody methods were compared with various commercially available immunochromatographic and enzyme immunoassays. Of 220 samples, 45 were positive for Giardia and 17 for Cryptosporidium. For Giardia, the sensitivities obtained by Ridascreen Giardia, Rida Quick Giardia, Rida Quick Combi and Giardia-Strip were 82%, 80%, 80% and 44%, respectively. For Cryptosporidium, the sensitivities obtained by Rida Quick Cryptosporidium, Ridascreen Cryptosporidium, Rida Quick Combi and Cryptosporidium-Strip were 88%, 82%, 82% and 75%, respectively. The specificity of all tests was > or = 98%. Other intestinal parasites were present in 68 samples, but cross-reactions with other protozoan or helminthic parasites were not observed. Overall, the copro-antigen assays were less time-consuming and easier to perform, but were less sensitive than conventional microscopical methods. Thus, these tests might be a useful addition to, but not a substitute for microscopical methods in the diagnosis of travel-associated giardiasis and cryptosporidiosis.

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The Giardia genome project database.

The Giardia genome project database provides an online resource for Giardia lamblia (WB strain, clone C6) genome sequence information. The database includes edited single-pass reads, the results of BLASTX searches, and details of progress towards sequencing the entire 12 million-bp Giardia genome. Pre-sorted BLASTX results can be retrieved based on keyword searches and BLAST searches of the high throughput Giardia data can be initiated from the web site or through NCBI. Descriptions of the genomic DNA libraries, project protocols and summary statistics are also available. Although the Giardia genome project is ongoing, new sequences are made available on a bi-monthly basis to ensure that researchers have access to information that may assist them in the search for genes and their biological function. The current URL of the Giardia genome project database is www.mbl.edu/Giardia.

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Detection of Giardia cysts with a cDNA probe and applications to water samples.

Giardiasis is the most common human parasite infection in the United States, causing a lengthy course of diarrhea. Transmission of Giardia species is by the fecal-oral route, and numerous waterborne outbreaks have been documented. The Environmental Protection Agency has regulated Giardia species in drinking water through the Surface Water Treatment Rule. Current methods for detection of Giardia species in water rely primarily on microscopic observation of water concentrates with immunofluorescence techniques. We evaluated the efficacy of using a gene-specific probe for the detection of Giardia species in water. A cDNA probe, 265 bp long, from the small subunit of rRNA of Giardia lamblia was used for detection of cysts. The replicative form of the M13 vector with an insert was isolated from lysed host Escherichia coli XL1-Blue and used for production of the cDNA probe by nick translation with 32P-labeled nucleotides. Six different protocols were tested for extracting nucleic acids from the cysts. With the most efficient procedure, disrupting Giardia cysts with glass beads in the presence of proteinase K, as few as 1 to 5 cysts per ml can be detected in water sample concentrates with dot blot hybridization assays.

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