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PubMed2005

Use of artificial neural networks to accurately identify Cryptosporidium oocyst and Giardia cyst images.

Cryptosporidium parvum and Giardia lamblia are protozoa capable of causing gastrointestinal diseases. Currently, these organisms are identified using immunofluorescent antibody (IFA)-based microscopy, and identification requires trained individuals for final confirmation. Since artificial neural networks (ANN) can provide an automated means of identification, thereby reducing human errors related to misidentification, ANN were developed to identify Cryptosporidium oocyst and Giardia cyst images. Digitized images of C. parvum oocysts and G. lamblia cysts stained with various commercial IFA reagents were used as positive controls. The images were captured using a color digital camera at 400 x (total magnification), processed, and converted into a binary numerical array. A variety of "negative" images were also captured and processed. The ANN were developed using these images and a rigorous training and testing protocol. The Cryptosporidium oocyst ANN were trained with 1,586 images, while Giardia cyst ANN were trained with 2,431 images. After training, the best-performing ANN were selected based on an initial testing performance against 100 images (50 positive and 50 negative images). The networks were validated against previously "unseen" images of 500 Cryptosporidium oocysts (250 positive, 250 negative) and 282 Giardia cysts (232 positive, 50 negative). The selected ANNs correctly identified 91.8 and 99.6% of the Cryptosporidium oocyst and Giardia cyst images, respectively. These results indicate that ANN technology can be an alternate to having trained personnel for detecting these pathogens and can be a boon to underdeveloped regions of the world where there is a chronic shortage of adequately skilled individuals to detect these pathogens.

Animals↗
PubMed2004

Systemic and mucosal responses to oral administration of excretory and secretory antigens from Giardia intestinalis.

Giardia, a flagellated protozoan that infects the upper small intestine of its vertebrate host, is the most common parasitic protist responsible for diarrhea worldwide. Molecules released by the parasite, particularly excretory and secretory antigens, seemed to be associated with pathogenesis as well as with the expression of Giardia virulence. In the present work, we examined the effect of oral administration of Giardia intestinalis excretory and secretory antigens on systemic and local antibody response as well as on mucosal injuries in BALB/c mice. Significant titers of serum-specific immunoglobulin G1 (IgG1) and specific IgG2a were observed. Systemic and mucosal specific IgA antibodies were also recorded. A transient production of serum-specific IgE antibody and high total IgE levels were also detected, suggesting the presence in excretory and secretory proteins of factors promoting a specific IgE response. The sera of excretory and secretory antigen-treated mice recognized proteins of 50 and 58 kDa as well as electrophoretic bands of 15, 63, and 72 kDa that could support a proteinase activity. The in vitro exposure of G. intestinalis trophozoites to heat-inactivated sera from mice orally inoculated with excretory and secretory antigens induced a decrease of growth, revealing a complement-independent inhibitory activity of specific serum antibodies. Furthermore, histological evaluation performed on the small and large intestines revealed moderate to acute histological changes comparable to those observed in natural or experimental Giardia infection characterized by eosinophilic infiltration, hypercellularity, and enterocytic desquamation. The present results suggested that Giardia excretory and secretory antigens stimulate a preferential Th2 response, which is probably involved in the intestinal alterations associated with giardiasis.

Administration, Oral↗
PubMed2002

Evidence for lateral transfer of genes encoding ferredoxins, nitroreductases, NADH oxidase, and alcohol dehydrogenase 3 from anaerobic prokaryotes to Giardia lamblia and Entamoeba histolytica.

Giardia lamblia and Entamoeba histolytica are amitochondriate, microaerophilic protists which use fermentation enzymes like those of bacteria to survive anaerobic conditions within the intestinal lumen. Genes encoding fermentation enzymes and related electron transport peptides (e.g., ferredoxins) in giardia organisms and amebae are hypothesized to be derived from either an ancient anaerobic eukaryote (amitochondriate fossil hypothesis), a mitochondrial endosymbiont (hydrogen hypothesis), or anaerobic bacteria (lateral transfer hypothesis). The goals here were to complete the molecular characterization of giardial and amebic fermentation enzymes and to determine the origins of the genes encoding them, when possible. A putative giardia [2Fe-2S]ferredoxin which had a hypothetical organelle-targeting sequence at its N terminus showed similarity to mitochondrial ferredoxins and the hydrogenosomal ferredoxin of Trichomonas vaginalis (another luminal protist). However, phylogenetic trees were star shaped, with weak bootstrap support, so we were unable to confirm or rule out the endosymbiotic origin of the giardia [2Fe-2S]ferredoxin gene. Putative giardial and amebic 6-kDa ferredoxins, ferredoxin-nitroreductase fusion proteins, and oxygen-insensitive nitroreductases each tentatively supported the lateral transfer hypothesis. Although there were not enough sequences to perform meaningful phylogenetic analyses, the unique common occurrence of these peptides and enzymes in giardia organisms, amebae, and the few anaerobic prokaryotes suggests the possibility of lateral transfer. In contrast, there was more robust phylogenetic evidence for the lateral transfer of G. lamblia genes encoding an NADH oxidase from a gram-positive coccus and a microbial group 3 alcohol dehydrogenase from thermoanaerobic prokaryotes. In further support of lateral transfer, the G. lamblia NADH oxidase and adh3 genes appeared to have an evolutionary history distinct from those of E. histolytica.

Alcohol Dehydrogenase↗
PubMed1991

Variant surface antigens of Giardia lamblia are associated with the presence of a thick cell coat: thin section and label fracture immunocytochemistry survey.

Giardia lamblia undergoes surface antigenic variation. The ultrastructural location of antigens on four different variants was studied by label fracture and immunocytochemistry with four monoclonal antibodies (MAbs), each of which recognized the predominant variant in a particular clone. Each Giardia clone and its reacting MAb showed similar findings. The entire surface of the organism was covered by a surface coat which contained the variant surface protein. The surface coat was densely and uniformly labeled. Unreacting MAbs failed to label the surface. Label-fractured Giardia trophozoites exposed to reactive MAb revealed a planar distribution of reactivity with no discernible relationship to visualized intramembranous particles. Unexpectedly, some Giardia trophozoites lacked a surface coat and consequently failed to react with the appropriate MAb. The biological relevance of coatless Giardia trophozoites is unknown. These findings localize the variant antigens to the surface coat of the parasite and identify a minority of the population which lacks a surface coat.

Animals↗
PubMed2006

Detection of Giardia lamblia antigens in human fecal specimens by a solid-phase qualitative immunochromatographic assay.

The SIMPLE-READ Giardia rapid assay (Medical Chemical Corporation) is a solid-phase qualitative immunochromatographic assay that detects Giardia lamblia in aqueous extracts of human fecal specimens. Testing 106 Giardia-positive and 104 Giardia-negative stool specimens yielded a sensitivity of 97.2% and a specificity of 100% for the SIMPLE-READ Giardia rapid assay.

Animals↗
PubMed1990

High-resolution immunogold localization of Giardia cyst wall antigens using field emission SEM with secondary and backscatter electron imaging.

We describe here the ultrastructural localization of Giardia cyst antigens in the filaments associated with the outer portion of intact cysts and on developing cyst wall filaments in encysting trophozoites. Post-embedding immunogold labeling of thin sections of intact Giardia cysts with polyclonal and monoclonal antibodies specific for cyst wall antigens (major protein bands of approximately 29, 75, 88, and 102 KD on Western blots) showed strong labeling of the filamentous cyst wall, whereas no labeling was seen on the membranous portion. High-resolution field emission scanning electron microscopy (FESEM) of Giardia cysts revealed that the cyst wall-specific polyclonal rabbit antisera and monoclonal mouse antibody produced gold labeling of 20-nm filaments in the cyst wall as detected with secondary electron imaging (SEI) and backscatter electron imaging (BEI) at 10 kV, despite coating of the cells with platinum by ion sputtering. FESEM studies of encysting Giardia trophozoites demonstrated that immunostaining with antibodies to cyst wall antigens produced colloidal gold labeling of developing cyst wall filaments on the cell surface; however, the intervening membrane domains were unlabeled. Substitution of normal serum for cyst wall-specific antibodies, or preabsorption of specific antibodies with Giardia cysts, eliminated immunolabeling of the filaments.

Animals↗
PubMed1994

The DNA content of trophozoites and cysts of Giardia lamblia by microdensitometric quantitation of Feulgen staining and examination by laser scanning confocal microscopy.

We investigated direct measurement of the DNA content of the parasitic intestinal flagellate Giardia lamblia through quantitation by Feulgen microspectrophotometry and also by visualization of Feulgen-stained DNA chromosomes within dividing cells by laser scanning confocal microscopy. Individual trophozoites of Giardia (binucleate) contained 0.144 +/- 0.018 pg of DNA/cell or 0.072 pg DNA/nucleus. Giardia lamblia cysts (quadranucleate) contained 0.313 +/- 0.003 pg DNA or 0.078 pg DNA/nucleus. The genome size (C) value per nucleus ranged between 6.5-7.1 x 10(7) BP for trophozoites and cysts, respectively. Confocal microscopic examination of Giardia trophozoites undergoing binary fission revealed five chromosome-like bodies within each nucleus. Further information about genome size and DNA content within different Giardia species may help to clarify the pivotal role of these primitive eukaryotic cells in evolutionary development.

Animals↗
PubMed2006

Waterborne microbial risk assessment: a population-based dose-response function for Giardia spp. (E.MI.R.A study).

BACKGROUND: Dose-response parameters based on clinical challenges are frequently used to assess the health impact of protozoa in drinking water. We compare the risk estimates associated with Giardia in drinking water derived from the dose-response parameter published in the literature and the incidence of acute digestive conditions (ADC) measured in the framework of an epidemiological study in a general population. METHODS: The study combined a daily follow-up of digestive morbidity among a panel of 544 volunteers and a microbiological surveillance of tap water. The relationship between incidence of ADC and concentrations of Giardia cysts was modeled with Generalized Estimating Equations, adjusting on community, age, tap water intake, presence of bacterial indicators, and genetic markers of viruses. The quantitative estimate of Giardia dose was the product of the declared amount of drinking water intake (in L) by the logarithm of cysts concentrations. RESULTS: The Odds Ratio for one unit of dose [OR = 1.76 (95% CI: 1.21, 2.55)] showed a very good consistency with the risk assessment estimate computed after the literature dose-response, provided application of a 20 % abatement factor to the cysts counts that were measured in the epidemiological study. Doing so, a daily water intake of 2 L and a Giardia concentration of 10 cysts/100 L, would yield an estimated relative excess risk of 12 % according to the Rendtorff model, against 11 % when multiplying the baseline rate of ADC by the corresponding OR. This abatement parameter encompasses uncertainties associated with germ viability, infectivity and virulence in natural settings. CONCLUSION: The dose-response function for waterborne Giardia risk derived from clinical experiments is consistent with epidemiological data. However, much remains to be learned about key characteristics that may heavily influence quantitative risk assessment results.

Adolescent↗
PubMed1997

Role of nitric oxide and superoxide in Giardia lamblia killing.

Giardia lamblia trophozoites were incubated for 2 h with activated murine macrophages, nitric oxide (NO) donors or a superoxide anion generator (20 mU/ml xanthine oxidase plus 1 mM xanthine). Activated macrophages were cytotoxic to Giardia trophozoites (approximately 60% dead trophozoites). The effect was inhibited (> 90%) by an NO synthase inhibitor (200 microM) and unaffected by superoxide dismutase (SOD, 300 U/ml). Giardia trophozoites were killed by the NO donors, S-nitroso-acetyl-penicillamine (SNAP) and sodium nitroprusside (SNP) in a dose-dependent manner (LD50 300 and 50 microM, respectively). A dual NO-superoxide anion donor, 3-morpholino-sydnonimine hydrochloride (SIN-1), did not have a killing effect in concentrations up to 1 mM. However, when SOD (300 U/ml) was added simultaneously with SIN-1 to Giardia, a significant trophozoite-killing effect was observed (approximately 35% dead trophozoites at 1 mM). The mixtures of SNAP or SNP with superoxide anion, which yields peroxynitrite, abolished the trophozoite killing induced by NO donors. Authentic peroxynitrite only killed trophozoites at very high concentrations (3 mM). These results indicate that NO accounts for Giardia trophozoites killing and this effect is not mediated by peroxynitrite.

Animals↗
PubMed1998

Occurrence of Cryptosporidium oocysts and Giardia cysts in water supplies of San Pedro Sula, Honduras.

During June 1996, water supplies of the city of San Pedro Sula, Honduras, were sampled to obtain an assessment of Cryptosporidium oocyst and Giardia cyst concentrations. Each sample was concentrated and stained with an indirect immunofluorescent antibody, and parasites were counted through microscopic analysis. In three surface water supplies, Cryptosporidium oocyst concentrations ranged from 58 to 260 oocysts per 100 L, and Giardia cysts were present in concentrations ranging from 380 to 2100 cysts per 100 L. Unlike the surface water samples, groundwater had a higher concentration of Cryptosporidium oocysts (26/100 L) than Giardia cysts (6/100 L), suggesting that the groundwater aquifer protects the water supply more effectively from larger Giardia cysts. Cryptosporidium oocyst concentrations are within the typical range for surface water supplies in North America whereas Giardia cyst concentrations are elevated. Efforts should be made to protect raw water from sources of contamination.

Animals↗
PubMed2006

Polymeric immunoglobulin receptor in intestinal immune defense against the lumen-dwelling protozoan parasite Giardia.

The polymeric Ig receptor (pIgR) is conserved in mammals and has an avian homologue, suggesting evolutionarily important functions in vertebrates. It transports multimeric IgA and IgM across polarized epithelia and is highly expressed in the intestine, yet little direct evidence exists for its importance in defense against common enteric pathogens. In this study, we demonstrate that pIgR can play a critical role in intestinal defense against the lumen-dwelling protozoan parasite Giardia, a leading cause of diarrheal disease. The receptor was essential for the eradication of Giardia when high luminal IgA levels were required. Clearance of Giardia muris, in which IgA plays a dominant role, was severely compromised in pIgR-deficient mice despite significant fecal IgA output at 10% of normal levels. In contrast, eradication of the human strain Giardia lamblia GS/M, for which adaptive immunity is less IgA dependent in mice, was unaffected by pIgR deficiency, indicating that pIgR had no physiologic role when lower luminal IgA levels were sufficient for parasite elimination. Immune IgA was greatly increased in the serum of pIgR-deficient mice, conferred passive protection against Giardia, and recognized several conserved giardial Ags, including ornithine carbamoyltransferase, arginine deiminase, alpha-enolase, and alpha- and beta-giardins, that are also detected in human giardiasis. Corroborative observations were made in mice lacking the J chain, which is required for pIgR-dependent transepithelial IgA transport. These results, together with prior data on pIgR-mediated immune neutralization of luminal cholera toxin, suggest that pIgR is essential in intestinal defense against pathogenic microbes with high-level and persistent luminal presence.

Animals↗
PubMed2005

Prevalence of Giardia and Cryptosporidium in beef cows in southern Ontario and in beef calves in southern British Columbia.

In 1998 and 1999, fecal samples were collected from 669 beef cows on 39 farms located within 10 counties of Ontario. Overall prevalences of Giardia, Cryptosporidium muris, and Cryptosporidium parvum in cows were 8.7%, 10.6%, and 18.4%, respectively. Of the 39 farms sampled, Giardia was detected on 64%, Cr. muris on 72%, and Cr. parvum on 90%. Cryptosporidium parvum was detected in 28% of the cows in 1998 and in 5.2% in 1999. Differences between the 2 y were attributed to sampling during calving in 1998 and during gestation in 1999. In 1998, Giardia, Cr. muris, and Cr. parvum were detected in herds provided with municipal water. In 1998, 193 calves were sampled from 10 farms, representing 4 watersheds, in British Columbia. Thirty-six percent of the calves exhibited signs of diarrhea. Overall prevalences of Giardia and Cryptosporidium spp. in calves were 36% and 13%, respectively. There was evidence that calves with Giardia were more likely to develop scours. Restricting cattle from surface water during periods of high shedding may reduce watershed contamination.

Animals↗
PubMed1987

Prevalence of Giardia sp. in a beaver colony and the resulting environmental contamination.

The prevalence of Giardia sp. in a beaver (Castor canadensis) colony in Colorado was determined by the collection and analysis of fecal samples over a period of 14 mo. Environmental contamination was monitored through the use and analysis of water filter samples. Beaver shed cysts of Giardia sp. in their feces throughout the year with temporal variations in the prevalence, and became infected as kits and remained infected as juveniles and adults. Beaver served as amplification hosts for Giardia sp. and contaminated surface waters downstream from their dams in late spring and early fall. In slow moving waters the cysts of Giardia sp. settled rapidly. Muskrats (Ondatra zibethicus) were the only other species of wildlife shedding cysts of Giardia sp. on the study area.

Animals↗
PubMed2001

Occurrence of Cryptosporidium oocysts and Giardia cysts in a conventional water purification plant.

A one-year monitoring of Cryptosporidium oocysts and Giardia cysts was conducted at a water purification plant. A total of thirteen 50 L samples of river source water and twenty-six 2,000 L samples of filtered water (treated by coagulation-flocculation, sedimentation and rapid filtration) were concentrated using a hollow fibre ultrafiltration membrane module at a purification plant. Cryptosporidium oocysts were detected in all raw water samples with a geometric mean concentration of 400 oocysts/m3 (range 160-1,500 oocysts/m3). Giardia cysts were detected in 12/13 raw waters (92%) with a geometric mean concentration of 170 cysts/m3 (range 40-580 oocysts/m3). Probability distributions of both Cryptosporidium oocysts and Giardia cyst concentration in raw water were nearly log-normal. In filtered water samples, Cryptosporidium oocysts were detected in 9/26 samples (35%) with a geometric mean concentration of 1.2 oocysts/m3 (range 0.5-8 oocysts/m3) and Giardia cysts in three samples (12%) with 0.8 cysts/m3 (range 0.5-2 oocysts/m3). The estimated removal of Cryptosporidium oocysts and Giardia cysts was, respectively, 2.54 log 10 and 2.53 log10 on the basis of geometric means, 3.20 and 3.57 log10 on the basis of 50% observation level and 2.70 and 2.90 log10 on the basis of 90% observation level.

Animals↗
PubMed2001

Occurrence of Cryptosporidium oocysts and Giardia cysts in raw waters in Norway.

AIMS: This paper reports the first investigation into the occurrence of Cryptosporidium and Giardia in Norwegian raw water sources. METHODS: Between June 1998 and October 1999, 408 raw water samples, collected from 147 different sites across Norway, were analysed for these parasites. Analysis was based upon US EPA Method 1623. RESULTS: In 305 samples (75%), parasites were not detected. In 55 samples (13.5%), Cryptosporidium only was detected. In 38 samples (9%), Giardia only was detected. In 10 samples (2.5%) both Cryptosporidium and Giardia were detected. Of the sites sampled, parasites were not detected at 100 (68%) of them, Cryptosporidium only was detected at 20 (13.5%). Giardia only was detected at 11 (7.5%), and both Cryptosporidium and Giardia were detected at 16 (11%). Concentrations of parasites were low; usually one cyst/oocyst detected per 10 litres of water. CONCLUSIONS: Significant associations were demonstrated for these samples between the detection of these parasites and (a) turbidity > or =2.0 NTU, and (b) high numbers of domestic animals within the catchment area. No association between seasonality and the occurrence of these parasites could be detected. The results are discussed in relation to other studies and the potential public health implications for Norway.

Animals↗
PubMedDate not supplied

[Phylogenetic distance between Giardia intestinalis isolates from symptomatic and asymptomatic children].

The purpose of the present investigation, was to analyze the sequences of 16S ribosomal genes partially amplified from 17 isolated Giardia intestinalis obtained from faces of 13 children with intestinal pathology and four symptoms-free children with intestinal pathology. Analysis was made also with Giardia Portland-I and four Giardias isolated from dogs. Children ages in both groups ranged from six to twelve years. Genomic DNA was isolated using the phenol-chloroform-isoamyl alcohol technique, and partial amplification of 16S rRNA ribosomal gene was carried out by the Polymerase Chain Reaction. Sequences of rDNA were compared with Portland-I by using CLUSTAL-W (1.81) and PHYLIP (3.6) software in order to determine phylogenic associations. Our results showed that only one isolate from symptoms-free children and three from the symptomatic children were associated with Portland I. The other isolated Giardias were associated between them and with two samples obtained from dogs that are phylogenetically distant from Portland-I. Two isolates from dog constituted a different group. These results suggest a zoonotic infection and presence of symptoms in children from the present investigation; probably it was associated with host or special characteristics of Giardia strains. The last two situations have a special epidemiological and public health interest.

Abdominal Pain↗
PubMedDate not supplied

Comparison of conventional coproscopical methods and commercial coproantigen ELISA kits for the detection of Giardia and Cryptosporidium infections in dogs and cats.

Faecal samples of 270 dogs and 100 cats from 3 animal shelters in Germany were comparatively examined using conventional coproscopical methods and commercial coproantigen ELISA kits for the detection of Giardia and Cryptosporidium infections. Giardia cysts were found in 9.5% and 0% of the faecal samples in dogs and cats, respectively, examined once using the ZnCl2-NaCl flotation. However, the Giardia coproantigen ELISA (ProSpecT Giardia Microplate Assay) was positive in 29.5% and 22.4% of the samples from dogs and cats, respectively. Direct faecal smears stained with carbol fuchsin showed Cryptosporidium oocysts in one dog (0.4%) and one cat (1%). In contrast, the Cryptosporidium coproantigen ELISA (ProSpecT Cryptosporidium Microplate Assay) reacted positively in 23% of the samples from dogs and 30% of the samples from cats. Both coproantigen ELISAs were more often positive in coproscopically Giardia-negative canine faecal samples that contained Isospora burrowsi/ohioensis oocysts than in faecal samples without any parasite stage. Possible reasons for these observations are discussed.

Animals↗
PubMed2005

[Obtention, purification and characterization of IgY polyclonal antibodies, developed in hens, directed against Colombian isolates of Giardia duodenalis].

INTRODUCTION: The development of polyclonal antibodies requires laboratory animals, as the usually rabbits, which are bled for obtaining the immunoglobulins. Hens are an alternative to developing IgY polyclonal antibodies. OBJECTIVE: In the current study, hens were used to develop IgY anti-Giardia duodenalis antibodies and to evaluate five protocols for its purification. MATERIALS AND METHODS: Three hens were immunized intramuscularly immunized with Colombian Giardia duodenalis isolates trophozoites on day 0, 15, 30, 45, 60, 90 and 120. The hen eggs were collected before each immunization, and IgY purified from yolk by delipidation (D) and precipitation (P) using five different protocols: M1: (P: ammonium sulfate/D:dextran sulfate-calcium chloride), M2: (D: dextran sulfate-calcium chloride/P: ammonium sulfate), M3: (D: chloroform/P: ammonium sulfate 50%), M4: (D: solution A/P: solution B) and M5: (D: chloroform/P: ammonium sulfate 30%). The immunochemistry evaluation of IgY anti-Giardia duodenalis was assayed by immunodiffusion, counterimmunoelectrophoresis (CIE) and Western blot. The purity of IgY was assayed by SDS-PAGE under reducing and nonreducing conditions. Immunoglobulin concentration (mg/mL) was estimated by spectrophotometry and densitometry. RESULTS: IgY anti-Giardia duodenalis by CIE had titres up to 1:32. SDS-PAGE, without 2-mercaptoethanol showed a 180 kd band characteristic of the whole to IgY and, with 2-mercaptoethanol, two bands of 68 and 30 kd, characteristic of its light and heavy chains, respectively. The greatest concentrations of immunoglobulin were recovered by method two (M2), producing 4.6 mg of IgY per mL of initial egg yolk. CONCLUSIONS: The easy and inexpensive production of IgY anti-Giardia duodenalis in hens is an advantage for using it to develop immunoassays that detect the parasite in fecal eluates.

Animals↗