30,960 research outputs found
The Design of Giardia and the Genesis of Giardiasis
Giardia is a genus of protozoa discovered in 1681. Six morphologically distinct species are recognized. It mainly attaches in the upper GI tract of a wide variety of vertebrates (including zebrafish), often with beaver and muskrat as reservoirs/carriers but exhibiting minimal—if any—disease in some animals. Giardia is usually non-pathogenic in the human population, even in children if exposed early in life. Although Giardia can be pathogenic, some strains colonize the gut with no malady. This parasite is not invasive and only serious infections depress the small intestine. Giardia are pear-shaped, have an adhesive disc for attaching to enterocyte cells in the small intestine villus, and move with eight designed flagella. In the post-Fallen world, Giardia infection occasionally has resulted in digestive dysfunction. However, Giardia may function in non-parasitic, possibly mutualistic, ways. For example, it may have been designed to aid digestion having a role as a “primer.” The presence of Giardia muris causes a fundamental change in the microbiome in mice and Giardia may have other influences on the microbiome such as enhancing digestion in certain animals and possibly shifting ratios of bacteria from anaerobic to aerobic. Giardia may play a role in host metabolism and provide nutritional enhancement via its association with enteric bacteria, like E. coli. The function of Giardia may parallel with non-parasitic tasks found in Trypanosoma lewisi, and also termite systems that contain protozoa and bacteria for plant digestion. Giardiahas two “faces” even in today\u27s world: a harmless commensal in wildlife and a pathogenic parasite in humans
Prevalence of Enteropathogens in Dogs Attending 3 Regional Dog Parks in Northern California.
BackgroundThe prevalence and risk factors for infection with enteropathogens in dogs frequenting dog parks have been poorly documented, and infected dogs can pose a potential zoonotic risk for owners.Hypothesis/objectivesTo determine the prevalence and risk factors of infection with enteropathogens and zoonotic Giardia strains in dogs attending dog parks in Northern California and to compare results of fecal flotation procedures performed at a commercial and university parasitology laboratory.AnimalsThree-hundred dogs attending 3 regional dog parks in Northern California.MethodsProspective study. Fresh fecal specimens were collected from all dogs, scored for consistency, and owners completed a questionnaire. Specimens were analyzed by fecal centrifugation flotation, DFA, and PCR for detection of 11 enteropathogens. Giardia genotyping was performed for assemblage determination.ResultsEnteropathogens were detected in 114/300 dogs (38%), of which 62 (54%) did not have diarrhea. Frequency of dog park attendance correlated significantly with fecal consistency (P = .0039), but did not correlate with enteropathogen detection. Twenty-seven dogs (9%) were infected with Giardia, and genotyping revealed nonzoonotic assemblages C and D. The frequency of Giardia detection on fecal flotation was significantly lower at the commercial laboratory versus the university laboratory (P = .013), and PCR for Giardia was negative in 11/27 dogs (41%) that were positive on fecal flotation or DFA.Conclusions and clinical importanceEnteropathogens were commonly detected in dogs frequenting dog parks, and infection with Giardia correlated with fecal consistency. PCR detection of Giardia had limited diagnostic utility, and detection of Giardia cysts by microscopic technique can vary among laboratories
Genotypic characterisation of Giardia from domestic dogs in the USA
The first large-scale urban survey of Giardia infections in dogs was undertaken in the USA. It involved several locations in the Western United States with Giardia isolates from microscopy-positive samples characterised by multi-locus PCR and sequencing. A high prevalence of Giardia was confirmed in asymptomatic domestic dogs, and for the first time, provides evidence that zoonotic assemblages/subgroups of Giardia occur frequently in domestic dogs living in urban environments, and more frequently than the dog specific assemblages
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Giardia Colonizes and Encysts in High-Density Foci in the Murine Small Intestine.
Giardia lamblia is a highly prevalent yet understudied protistan parasite causing significant diarrheal disease worldwide. Hosts ingest Giardia cysts from contaminated sources. In the gastrointestinal tract, cysts excyst to become motile trophozoites, colonizing and attaching to the gut epithelium. Trophozoites later differentiate into infectious cysts that are excreted and contaminate the environment. Due to the limited accessibility of the gut, the temporospatial dynamics of giardiasis in the host are largely inferred from laboratory culture and thus may not mirror Giardia physiology in the host. Here, we have developed bioluminescent imaging (BLI) to directly interrogate and quantify the in vivo temporospatial dynamics of Giardia infection, thereby providing an improved murine model to evaluate anti-Giardia drugs. Using BLI, we determined that parasites primarily colonize the proximal small intestine nonuniformly in high-density foci. By imaging encystation-specific bioreporters, we show that encystation initiates shortly after inoculation and continues throughout the duration of infection. Encystation also initiates in high-density foci in the proximal small intestine, and high density contributes to the initiation of encystation in laboratory culture. We suggest that these high-density in vivo foci of colonizing and encysting Giardia likely result in localized disruption to the epithelium. This more accurate visualization of giardiasis redefines the dynamics of the in vivo Giardia life cycle, paving the way for future mechanistic studies of density-dependent parasitic processes in the host. IMPORTANCEGiardia is a single-celled parasite causing significant diarrheal disease in several hundred million people worldwide. Due to limited access to the site of infection in the gastrointestinal tract, our understanding of the dynamics of Giardia infections in the host has remained limited and largely inferred from laboratory culture. To better understand Giardia physiology and colonization in the host, we developed imaging methods to quantify Giardia expressing bioluminescent physiological reporters in two relevant animal models. We discovered that parasites primarily colonize and encyst in the proximal small intestine in discrete, high-density foci. We also show that high parasite density contributes to encystation initiation
Eight unique basal bodies in the multi-flagellated diplomonad Giardia lamblia.
Giardia lamblia is an intestinal parasitic protist that causes significant acute and chronic diarrheal disease worldwide. Giardia belongs to the diplomonads, a group of protists in the supergroup Excavata. Diplomonads are characterized by eight motile flagella organized into four bilaterally symmetric pairs. Each of the eight Giardia axonemes has a long cytoplasmic region that extends from the centrally located basal body before exiting the cell body as a membrane-bound flagellum. Each basal body is thus unique in its cytological position and its association with different cytoskeletal features, including the ventral disc, axonemes, and extra-axonemal structures. Inheritance of these unique and complex cytoskeletal elements is maintained through basal body migration, duplication, maturation, and their subsequent association with specific spindle poles during cell division. Due to the complex composition and inheritance of specific basal bodies and their associated structures, Giardia may require novel basal body-associated proteins. Thus, protists such as Giardia may represent an undiscovered source of novel basal body-associated proteins. The development of new tools that make Giardia genetically tractable will enable the composition, structure, and function of the eight basal bodies to be more thoroughly explored
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
Computational Identification of Four Spliceosomal snRNAs from the Deep-Branching Eukaryote Giardia intestinalis
Funding: Marsden Fund New Zealand Allan Wilson Centre The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.RNAs processing other RNAs is very general in eukaryotes, but is not clear to what extent it is ancestral to eukaryotes. Here
we focus on pre-mRNA splicing, one of the most important RNA-processing mechanisms in eukaryotes. In most eukaryotes
splicing is predominantly catalysed by the major spliceosome complex, which consists of five uridine-rich small nuclear
RNAs (U-snRNAs) and over 200 proteins in humans. Three major spliceosomal introns have been found experimentally in
Giardia; one Giardia U-snRNA (U5) and a number of spliceosomal proteins have also been identified. However, because of
the low sequence similarity between the Giardia ncRNAs and those of other eukaryotes, the other U-snRNAs of Giardia had
not been found. Using two computational methods, candidates for Giardia U1, U2, U4 and U6 snRNAs were identified in this
study and shown by RT-PCR to be expressed. We found that identifying a U2 candidate helped identify U6 and U4 based on
interactions between them. Secondary structural modelling of the Giardia U-snRNA candidates revealed typical features of
eukaryotic U-snRNAs. We demonstrate a successful approach to combine computational and experimental methods to
identify expected ncRNAs in a highly divergent protist genome. Our findings reinforce the conclusion that spliceosomal
small-nuclear RNAs existed in the last common ancestor of eukaryotes
Giardia Cyst Wall Protein 1 Is a Lectin That Binds to Curled Fibrils of the GalNAc Homopolymer
The infectious and diagnostic stage of Giardia lamblia (also known as G. intestinalis or G. duodenalis) is the cyst. The Giardia cyst wall contains fibrils of a unique β-1,3-linked N-acetylgalactosamine (GalNAc) homopolymer and at least three cyst wall proteins (CWPs) composed of Leu-rich repeats (CWPLRR) and a C-terminal conserved Cys-rich region (CWPCRR). Our goals were to dissect the structure of the cyst wall and determine how it is disrupted during excystation. The intact Giardia cyst wall is thin (~400 nm), easily fractured by sonication, and impermeable to small molecules. Curled fibrils of the GalNAc homopolymer are restricted to a narrow plane and are coated with linear arrays of oval-shaped protein complex. In contrast, cyst walls of Giardia treated with hot alkali to deproteinate fibrils of the GalNAc homopolymer are thick (~1.2 µm), resistant to sonication, and permeable. The deproteinated GalNAc homopolymer, which forms a loose lattice of curled fibrils, is bound by native CWP1 and CWP2, as well as by maltose-binding protein (MBP)-fusions containing the full-length CWP1 or CWP1LRR. In contrast, neither MBP alone nor MBP fused to CWP1CRR bind to the GalNAc homopolymer. Recombinant CWP1 binds to the GalNAc homopolymer within secretory vesicles of Giardia encysting in vitro. Fibrils of the GalNAc homopolymer are exposed during excystation or by treatment of heat-killed cysts with chymotrypsin, while deproteinated fibrils of the GalNAc homopolymer are degraded by extracts of Giardia cysts but not trophozoites. These results show the Leu-rich repeat domain of CWP1 is a lectin that binds to curled fibrils of the GalNAc homopolymer. During excystation, host and Giardia proteases appear to degrade bound CWPs, exposing fibrils of the GalNAc homopolymer that are digested by a stage-specific glycohydrolase. Author SummaryWhile the walls of plants and fungi contain numerous sugar homopolymers (cellulose, chitin, and β-1,3-glucans) and dozens of proteins, the cyst wall of Giardia is relatively simple. The Giardia wall contains a unique homopolymer of β-1,3-linked N-acetylgalactosamine (GalNAc) and at least three cyst wall proteins (CWPs), each of which is composed of Leu-rich repeats and a C-terminal Cys-rich region. The three major discoveries here are: 1) Fibrils of the GalNAc homopolymer are curled and form a lattice that is compressed into a narrow plane by bound protein in intact cyst walls. 2) Leu-rich repeats of CWP1 form a novel lectin domain that is specific for fibrils of the GalNAc homopolymer, which can be isolated by methods used to deproteinate fungal walls. 3) A cyst-specific glycohydrolase is able to degrade deproteinated fibrils of the GalNAc homopolymer. We incorporate these findings into a new curled fiber and lectin model of the intact Giardia cyst wall and a protease and glycohydrolase model of excystation.National Institutes of Health (AI048082, AI44070, GM31318, RR1088
Viability of Giardia intestinalis cysts : assessing viability under environmental conditions : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, Palmerston North, New Zealand
Much work has been put into the detection and monitoring of Giardia, but once found, it is not easy to tell whether the cysts are viable and thus infective. There are fluorescently labelled monoclonal antibody kits which can be used to identify Giardia, but are the Giardia cysts viable? Excystation has been the main method used to determine the viability of cysts. This is quite unreliable as varying excystation conditions seem to be required for different strains of cysts. Using samples of fresh cysts, certain batches consistently measured 80-95% viable, while others resulted in viability measurements of 0-10%. The cysts themselves displayed the normal morphology of viable cysts. The assumption that partially excysted trophozoites as well as completely excysted trophozoites are viable may also lead to over-estimation of viable cyst numbers. Another commonly used method for estimating the viability of Giardia is staining with vital dyes, in particular the combination of fluorescein diacetate (FDA) and propidium iodide (PI). These also gave unexpected results where none of the cysts in a fresh sample stained with FDA, which usually stains viable cysts. An alternative dye, 4',6-diamidino-2-phenylindole (DAPI) was used in the place of FDA. The combination of DAPI and PI showed viabilities of 85.7% for cyst samples. This correlated well with 88% viability using excystation. Using the DAPI/PI combination, the viability of G. intestinalis cysts over time was monitored under different temperature conditions, and in sea water. Temperature was quite significant in the viability of the cysts – cysts stored at 4°C remained viable for 62 days, while those stored at 25°C were non-viable after 5 days. Sea-water had an immediately lethal effect on the G. intestinalis cysts, with all cysts non-viable after 45 minutes. Giardia intestinalis trophozoites can be cultured in the laboratory. By the addition of bile to the growth media, it is possible to transform these into cysts. Over the course of four days in encystation media, a large proportion of the trophozoites in the culture were converted into cysts, 3.5 X 10⁵ cysts/ml from an initial trophozoite concentration of 7.2 X 10⁵ organisms/ml. However, the cysts generated from the strains of G. intestinalis used were completely non-viable, compared with viability rates for fresh in vivo cysts of 80-95%. A population of hamsters was found to be carrying a Giardia which seemed different to recognised species. An analysis was carried out by PCR and sequencing of sections of the ribosomal DNA of this Giardia. Through this it was found to be closely related to Giardia muris, but perhaps not as closely related as to be a species of G. muris, possibly a sub-species. The rDNA analysis used may be very useful in typing other strains and species of Giardia
Delayed development of the protective IL-17A response following a Giardia muris infection in neonatal mice
Giardia is an intestinal protozoan parasite that has the ability to infect a wide range of hosts, which can result in the clinical condition 'giardiasis'. Over the years, experimental research has shown the crucial involvement of IL-17A to steer the protective immune response against Giardia. The development of the protective response, as reflected by a significant drop in cyst secretion, typically takes around 3 to 4 weeks. However, early-life infections often have a more chronic character lasting for several weeks or months. Therefore, the aim of the current study was to investigate the dynamics of a Giardia muris infection and the subsequent host immune response in neonatal mice infected 4 days after birth. The outcome of the study showed that a G. muris infection in pre-weaned mice failed to trigger a protective IL-17A response, which could explain the prolonged course of infection in comparison to older mice. Only after weaning, a protective intestinal immune response started to develop, characterized by an upregulation of IL-17A and Mb12 and the secretion of parasite-specific IgA
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