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Giardia duodenalis, a major cause of waterborne infection, infects a wide range of mammalian hosts and is subdivided into eight genetically well-defined assemblages named A through H. However, fragmented genomes and a lack of comparative analysis within and between the assemblages render unclear the molecular mechanisms controlling host specificity and differential disease outcomes. To address this, we generated a near-complete de novo genome of AI assemblage using the Oxford Nanopore platform by sequencing the Be-2 genome. We generated 148,144 long-reads with quality scores of > 7. The final genome assembly consists of only nine contigs with an N50 of 3,045,186 bp. This assembly agrees closely with the assembly of another strain in the AI assemblage (WB-C6). However, a critical difference is that a region previously placed in the five-prime region of Chr5 belongs to Chr4 of Be-2. We find a high degree of conservation in the ploidy, homozygosity, and the presence of cysteine-rich variant-specific surface proteins (VSPs) within the AI assemblage. Our assembly provides a nearly complete genome of a member of the AI assemblage of G. duodenalis, aiding population genomic studies capable of elucidating Giardia transmission, host range, and pathogenicity., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Giardia is a prevalent single-celled microaerophilic intestinal parasite causing diarrheal disease and significantly impacting global health. Double diploid (essentially tetraploid) Giardia trophozoites have presented a formidable challenge to the development of molecular genetic tools to interrogate gene function. High sequence divergence and the high percentage of hypothetical proteins lacking homology to proteins in other eukaryotes have limited our understanding of Giardia protein function, slowing drug target validation and development. For more than 25 years, Giardia A and B assemblages have been readily amenable to transfection with plasmids or linear DNA templates. Here, we highlight the utility and power of genetic approaches developed to assess protein function in Giardia, with particular emphasis on the more recent clustered regularly interspaced palindromic repeats/Cas9-based methods for knockdowns and knockouts. Robust and reliable molecular genetic approaches are fundamental toward the interrogation of Giardia protein function and evaluation of druggable targets. New genetic approaches tailored for the double diploid Giardia are imperative for understanding Giardia's unique biology and pathogenesis., Competing Interests: Conflicts of interest. The authors declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Background & Aims: The protozoa Giardia duodenalis is a major cause of gastrointestinal illness worldwide, but underlying pathophysiological mechanisms remain obscure, partly due to the absence of adequate cellular models. We aimed at overcoming these limitations and recapitulating the authentic series of pathogenic events in the primary human duodenal tissue by using the human organoid system., Methods: We established a compartmentalized cellular transwell system with electrophysiological and barrier properties akin to duodenal mucosa and dissected the events leading to G. duodenalis-induced barrier breakdown by functional analysis of transcriptional, electrophysiological, and tight junction components., Results: Organoid-derived cell layers of different donors showed a time- and parasite load-dependent leak flux indicated by collapse of the epithelial barrier upon G. duodenalis infection. Gene set enrichment analysis suggested major expression changes, including gene sets contributing to ion transport and tight junction structure. Solute carrier family 12 member 2 and cystic fibrosis transmembrane conductance regulator-dependent chloride secretion was reduced early after infection, while changes in the tight junction composition, localization, and structural organization occurred later as revealed by immunofluorescence analysis and freeze fracture electron microscopy. Functionally, barrier loss was linked to the adenosine 3',5'-cyclic monophosphate (cAMP)/protein kinase A-cAMP response element-binding protein signaling pathway., Conclusions: Data suggest a previously unknown sequence of events culminating in intestinal barrier dysfunction upon G. duodenalis infection during which alterations of cellular ion transport were followed by breakdown of the tight junctional complex and loss of epithelial integrity, events involving a cAMP/protein kinase A-cAMP response element-binding protein mechanism. These findings and the newly established organoid-derived model to study G. duodenalis infection may help to explore new options for intervening with disease and infection, in particular relevant for chronic cases of giardiasis., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The deep-branching protozoan parasite Giardia lamblia is the causative agent of the intestinal disease giardiasis. Consistent with its proposed evolutionary position, many pathways are minimalistic or divergent, including its actin cytoskeleton. Giardia is the only eukaryote known to lack all canonical actin-binding proteins. Previously, our lab identified a number of noncanonical Giardia lamblia actin ( Gl Actin) interactors; however, these proteins appeared to interact only with monomeric or globular actin (G-actin) rather than with filamentous actin (F-actin). To identify F-actin interactors, we used a chemical cross-linker to preserve native interactions followed by an anti- Gl Actin antibody, protein A affinity chromatography, and liquid chromatography coupled to mass spectrometry. We found 46 putative actin interactors enriched under the conditions favoring F-actin. Data are available via ProteomeXchange with identifier PXD026067. None of the proteins identified contain known actin-interacting motifs, and many lacked conserved domains. Each potential interactor was then tagged with the fluorescent protein mNeonGreen and visualized in live cells. We categorized the proteins based on their primary localization; localizations included ventral disc, marginal plate, nuclei, flagella, plasma membrane, and internal membranes. One protein from each of the six categories was colocalized with Gl Actin using immunofluorescence microscopy. We also co-immunoprecipitated one protein from each category and confirmed three of the six potential interactions. Most of the localization patterns are consistent with previously demonstrated Gl Actin functions, but the ventral disc represents a new category of actin interactor localization. These results suggest a role for Gl Actin in ventral disc function, which has previously been controversial. IMPORTANCE Giardia lamblia is an intestinal parasite that colonizes the small intestine and causes diarrhea, which can lead to dehydration and malnutrition. Giardia actin ( Gl Actin) has a conserved role in Giardia cells, despite being a highly divergent protein with none of the conserved regulators found in model organisms. Here, we identify and localize 46 interactors of polymerized actin. These putative interactors localize to a number of places in the cell, underlining Gl Actin's importance in multiple cellular processes. Surprisingly, eight of these proteins localize to the ventral disc, Giardia 's host attachment organelle. Since host attachment is required for infection, proteins involved in this process are an appealing target for new drugs. While treatments for Giardia exist, drug resistance is becoming more common, resulting in a need for new treatments. Giardia and human systems are highly dissimilar, thus drugs specifically tailored to Giardia proteins would be less likely to have side effects.

Background: Giardia duodenalis is a widespread protozoan parasite affecting humans and many species of animals, including dogs and cats. Due to its zoonotic potential, it is important to know the frequency of this parasite in companion animals. The aim of this study was to determine current epidemiological status of G. duodenalis in household dogs and cats., Methods: In this study, 293 fecal samples from pet dogs and cats were collected from January 2017 to July 2019 and tested for G. duodenalis by PCR (using β-giardin gene). The animals were divided into groups depending on their age, breed and fecal consistency., Results: The examination allowed for detection of G. duodenalis in 6.0% of canine and 3.9% of feline fecal samples. The highest frequency was revealed in young (under one-year old) dogs. Sequencing confirmed the presence of assemblages C and D in dogs and A and F in cats., Conclusion: The study showed current frequency of G. duodenalis in dogs and cats and also revealed the occurrence of host-specific assemblages as well as zoonotic assemblage A.

Giardia intestinalis has been observed in human stools since the invention of the microscope. However, it was not recognized as a pathogen until experimental infections in humans in the 1950s resulted in diarrheal illness [1]. We now know that this protozoan is capable of inducing a malabsorptive diarrhea and that the parasite is a major contributor to stunting in young children [2]. However, the majority of infections with this parasite are not accompanied by overt diarrhea and several studies indicate that it actually has a protective effect against moderate-severe diarrhea [3]. There is therefore significant interest in the mechanisms responsible for the wide variation observed in the clinical outcomes of infection with Giardia. This review will highlight recent work on the interactions among the parasite, the host microbiome and the immune response as contributing to this variation., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

New information generated since 2016 from the application of molecular tools to infections with Giardia and Cryptosporidium is critically summarised. In the context of molecular epidemiology, nomenclature, taxonomy, in vitro culture, detection, zoonoses, population genetics and pathogenicity, are covered. Whole genome sequencing has had the greatest impact in the last three years. Future advances will provide a much better understanding of the zoonotic potential of both parasites, their diversity and how this is linked to pathogenesis in different hosts., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.)

Giardia lamblia is the causal agent of giardiasis, one of the most prevalent parasitosis in the world. Even though effective pharmacotherapies against this parasite are available, the disadvantages associated with its use call for the development of new antigiardial compounds. Based on the Giardia dependence on glycolysis as a main energy source, glycolytic enzymes appear to be attractive targets with antiparasitic potential. Among these, fructose 1,6-biphosphate aldolase (GlFBPA) has been highlighted as a promising target for drug design. Current efforts are based on the design of competitive inhibitors of GlFBPA; however, in the kinetic context of metabolic pathways, competitive inhibitors seem to have low potential as therapeutic agents. In this work, we performed an experimental and in silico structure-based approach to propose a non-catalytic binding site which could be used as a hot spot for antigardial drug design. The druggability of the selected binding site was experimentally tested; the alteration of the selected region by site directed mutagenesis disturbs the catalytic properties and the stability of the enzyme. A computational automated search of binding sites supported the potential of this region as functionally relevant. A preliminary docking study was performed, in order to explore the feasibility and type of molecules to be able to accommodate in the proposed binding region. Altogether, the results validate the proposed region as a specific molecular binding site with pharmacological potential.

Down syndrome is the most common human chromosomal disorder. Among clinical findings, one constant concern is the high prevalence of gastrointestinal system alterations. The aim of this study was to determine the prevalence of gastrointestinal disorders at a Down syndrome outpatient clinic during a 10-year follow-up period. Data from medical files were retrospectively reviewed from 1,207 patients. Gastrointestinal changes occurred in 612 (50.7%). The most prevalent disorder was chronic intestinal constipation. Intestinal parasite occurred in 22% (mainly giardiasis), gastroesophageal reflux disease in 14%, digestive tract malformations occurred in 5%: 13 cases of duodenal atresia, 8 of imperforate anus, 4 annular pancreases, 2 congenital megacolon, 2 esophageal atresias, 2 esophageal compression by anomalous subclavian and 1 case of duodenal membrane. We had 38/1,207 (3.1%) patients with difficulty in sucking and only three with dysphagia that resolved before the second year of life. Peptic ulcer disease, celiac disease, and biliary lithiasis were less prevalent with 3% each. Awareness of the high prevalence of gastrointestinal disorders promotes outstanding clinical follow-up as well as adequate development and greater quality of life for patients with Down syndrome and their families., (© 2019 Wiley Periodicals, Inc.)

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 Mbl2 and the secretion of parasite-specific IgA.

Innate lymphoid cells (ILCs) are key players during an immune response at the mucosal surfaces, such as lung, skin, and gastrointestinal tract. Giardia lamblia is an extracellular protozoan pathogen that inhabits the human small intestine. In this study, ILCs prepared from the lamina propria of mouse small intestine were incubated with G. lamblia trophozoites. Transcriptional changes in G. lamblia-exposed ILCs resulted in identification of activation of several immune pathways. Secretion of interleukin (IL)-17A, IL-17F, IL-1β, and interferon-γ was increased, whereas levels of IL-13, IL-5, and IL22, was maintained or reduced upon exposure to G. lamblia. Goup 3 ILC (ILC3) was found to be dominant amongst the ILCs, and increased significantly upon co-cultivation with G. lamblia trophozoites. Oral inoculation of G. lamblia trophozoites into mice resulted in their presence in the small intestine, of which, the highest number of parasites was detected at the 5 days-post infection. Increased ILC3 was observed amongst the ILC population at the 5 days-post infection. These findings indicate that ILC3 from the lamina propria secretes IL-17 in response to G. lamblia, leading to the intestinal pathology observed in giardiasis.

An infection of digestive system, Giardiasis, caused by tiny parasites called Giardia lamblia (also known Giardia intestinalis or Giardia duodenalis). Giardia sp. is the most common intestinal parasite of humans and other animals throughout the world. Isolates of G. lamblia are classified into eight assemblages based on isoenzyme and DNA analyses. Assemblages A and B infect humans and a broad range of other hosts. The purpose of this study was to genotype human isolates of G. lamblia by PCR-RFLP in Karaj city. 60 positive fecal samples of G. lamblia were collected. DNA extraction and amplification of TPI gene were successfully conducted by nested-PCR. Subsequently, all samples were positive. Sequencing on 5 samples was conducted to determine genetic differences. The presence of 2 genotypes of G. lamblia (A and B) was revealed by the alignment of the TPI sequences obtained with reference sequences. The results of RFLP technique show that 35 of 60 (58.3%) isolates belonged to assemblage A, and 17 of 60 (28.3%) belonged to assemblage B but 1(1.7%) sample was not determined. Whereas, 7 (11.6%) specimens were detected as mixed infections. The latter RFLP was carried out to identify subtypes.The final results were 100% (35/35) AII, 82.3% (14/17) BIII, and 17.7% (3/17) BIV. This study suggests that the modified RFLP method is favorably time saving and easily achievable and highly economical. Hence, the sub-assemblage AII might be dominant in Karaj city., (Copyright © 2018. Published by Elsevier B.V.)

Background: Giardia is a protozoan parasite of public health relevance that causes gastroenteritis in a wide range of hosts. Two genetically distinct lineages (assemblages A and B) are responsible for the human disease. Although it is clear that differences in virulence occur, the pathogenesis and virulence of Giardia remain poorly understood., Results: The genome of Giardia is believed to contain open reading frames that could encode as many as 6000 proteins. By successfully applying quantitative proteomic analyses to the whole parasite and to the supernatants derived from parasite culture of assemblages A and B, we confirm expression of ∼1600 proteins from each assemblage, the vast majority of which are common to both lineages. To look for signature enrichment of secreted proteins, we considered the ratio of proteins in the supernatant compared with the pellet, which defined a small group of enriched proteins, putatively secreted at a steady state by cultured growing trophozoites of both assemblages. This secretome is enriched with proteins annotated to have N-terminal signal peptide. The most abundant secreted proteins include known virulence factors such as cathepsin B cysteine proteases and members of a Giardia superfamily of cysteine-rich proteins that comprise variant surface proteins, high-cysteine membrane proteins, and a new class of virulence factors, the Giardia tenascins. We demonstrate that physiological function of human enteric epithelial cells is disrupted by such soluble factors even in the absence of the trophozoites., Conclusions: We are able to propose a straightforward model of Giardia pathogenesis incorporating key roles for the major Giardia-derived soluble mediators.

Microevolutionary data of Giardia duodenalis sub-assemblages is a prerequisite for determining the invasion zoonotic patterns of the parasite. To infer transmission patterns that could not be differentiated by the phenotypic features, a population genetic investigation is crucial for the elucidation of the genetic structure of G. duodenalis among the continents. Forty G. duodenalis positive fecal samples were collected from different foci of Northwest Iran. The specimens were subjected to Trichrome staining and sucrose gradient flotation. DNA samples were extracted, amplified, and sequenced by targeting glutamate dehydrogenase (gdh) gene. The global gdh sequences of sub-assemblages AII and BIV retrieved from NCBI GenBank were analyzed to estimate diversity indices, neutrality indices, and gene migration tests. Sequencing analyses indicated various levels of genetic variability of sub-assemblages AII and BIV among the five continents. Sub-assemblage BIV had greater genetic variability (haplotype diversity: 0.975; nucleotide diversity: 0.04246) than sub-assemblage AII. The statistical Fst value demonstrated that the genetic structure of sub-assemblages AII and BIV are moderately differentiated between European-American populations (Fst: 0.05352-0.15182), whereas a significant differentiation was not seen among other geographical population pairs. We conclude that a high gene flow of G. duodenalis sub-assemblages AII and BIV is unequivocally sharing among the continents. The current findings strengthen our knowledge to assess the evolutionary patterns of G. duodenalis in endemic foci of the world and it will become the basis of public health policy to control human giardiasis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The intestinal mucous layer provides a critical host defense against pathogen exposure and epithelial injury, yet little is known about how enteropathogens may circumvent this physiologic barrier. Giardia duodenalis is a small intestinal parasite responsible for diarrheal disease and chronic postinfectious illness. This study reveals a complex interaction at the surface of epithelial cells, between G. duodenalis and the intestinal mucous layer. Here, we reveal mechanisms whereby G. duodenalis evades and disrupts the first line of host defense by degrading human mucin-2 (MUC2), depleting mucin stores and inducing differential gene expression in the mouse small and large intestines. Human colonic biopsy specimens exposed to G. duodenalis were depleted of mucus, and in vivo mice infected with G. duodenalis had a thinner mucous layer and demonstrated differential Muc2 and Muc5ac mucin gene expression. Infection in Muc2 -/- mice elevated trophozoite colonization in the small intestine and impaired weight gain. In vitro, human LS174T goblet-like cells were depleted of mucus and had elevated levels of MUC2 mRNA expression after G. duodenalis exposure. Importantly, the cysteine protease inhibitor E64 prevented mucous degradation, mucin depletion, and the increase in MUC2 expression. This article describes a novel role for Giardia's cysteine proteases in pathogenesis and how Giardia's disruptions of the mucous barrier facilitate bacterial translocation that may contribute to the onset and propagation of disease., (Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Current control policies for intestinal parasitosis focuses on soil-transmitted helminths, being ineffective against Giardia intestinalis, a highly prevalent protozoon that impacts children's nutritional status in developing countries. The objective of this study was to explore spatial and molecular epidemiology of Giardia intestinalis in children of Amerindian descent in the Brazilian Amazon., Methodology/principal Findings: A cross sectional survey was performed in the Brazilian Amazon with 433 children aged 1 to 14 years. Fecal samples were processed through parasitological techniques and molecular characterization. Prevalence of G. intestinalis infection was 16.9% (73/433), reaching 22.2% (35/158) among children aged 2-5 years, and a wide distribution throughout the city with some hot spots. Positivity-rate was similar among children living in distinct socioeconomic strata (48/280 [17.1%] and 19/116 [16.4%] below and above the poverty line, respectively). Sequencing of the β-giardin gene revealed 52.2% (n = 12) of assemblage A and 47.8% (n = 11) of assemblage B with high haplotype diversity for the latter. The isolates clustered into two well-supported G. intestinalis clades. A total of 38 haplotypes were obtained, with the following subassemblages distribution: 5.3% (n = 2) AII, 26.3% (n = 10) AIII, 7.9% (n = 3) BIII, and 60.5% (n = 23) new B genotypes not previously described., Conclusions/significance: Giardia intestinalis infection presents a high prevalence rate among Amerindian descended children living in Santa Isabel do Rio Negro/Amazon. The wide distribution observed in a small city suggests the presence of multiple sources of infection, which could be related to environmental contamination with feces, possibly of human and animal origin, highlighting the need of improving sanitation, safe water supply and access to diagnosis and adequate treatment of infections.

Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi are common gastrointestinal protists in humans and animals. Two hundred and three fecal specimens from 80 wildlife species were collected in Zhengzhou Zoo and their genomic DNA extracted. Three intestinal pathogens were characterized with a DNA sequence analysis of different loci. Cryptosporidium felis, C. baileyi, and avian genotype III were identified in three specimens (1.5%), the manul, red-crowned crane, and cockatiel, respectively. Giardia duodenalis was also found in five specimens (2.5%) firstly: assemblage B in a white-cheeked gibbon and beaver, and assemblage F in a Chinese leopard and two Siberian tigers, respectively. Thirteen genotypes of E. bieneusi (seven previously reported genotypes and six new genotypes) were detected in 32 specimens (15.8%), of which most were reported for the first time. A phylogenetic analysis of E. bieneusi showed that five genotypes (three known and two new) clustered in group 1; three known genotypes clustered in group 2; one known genotype clustered in group 4; and the remaining four genotypes clustered in a new group. In conclusion, zoonotic Cryptosporidium spp., G. duodenalis, and E. bieneusi are maintained in wildlife and transmitted between them. Zoonotic disease outbreaks of these infectious agents possibly originate in wildlife reservoirs., (© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists.)

Background: Food handlers (FHs) may facilitate transmission and dissemination of pathogens. The importance of FHs as a link in the epidemiological chain of transmission of Giardia duodenalis and other intestinal protozoa was assessed., Methods: Fecal and subungual material from 27 FHs were analyzed using parasitological methods. G. duodenalis was identified by direct immunofluorescence and genotyped by PCR-RFLP for the bg and gdh genes, and gdh was sequenced., Results: At least one protozoan was detected in 30% (8/27) of the FHs and G. duodenalis (19%; 5/27) was the most common species. The AII and BIV genotypes were found in 20% (1/5) and 60% (3/5) of FHs infected with G. duodenalis, respectively., Conclusions: FHs can be involved in the chain of transmission of G. duodenalis and other protozoa., Genbank Accession Numbers: KJ741310 - KJ741313., (© The Author 2015. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Our first experience in genotyping Giardia from Moscow residents, has shown that 4 and 2 of seven samples belong to G. duodenalis genotype A and genotype B, respectively; one sample was negative during amplification with two types of primers. Genotyping was Carried out using the specific primers TPIA and TPIB to the gene encoding for the enzyme triosephosphate isomerase from the parasite. Thus, further such investigations using a larger number of samples will be able to complement the epidemiology of Lamblia infection in Moscow residents.

Serial Analysis of Gene Expression (SAGE) was used to quantify transcriptional changes in Giardia intestinalis during its interaction with human intestinal epithelial cells (IECs, HT-29) in serum free M199 medium. Transcriptional changes were compared to those in trophozoites alone in M199 and in TYI-S-33 Giardia growth medium. In total, 90 genes were differentially expressed, mainly those involved in cellular redox homeostasis, metabolism and small molecule transport but also cysteine proteases and structural proteins of the giardin family. Only 29 genes changed their expression due to IEC interaction and the rest were due to M199 medium. Although our findings generated a small dataset, it was consistent with our earlier microarray studies performed under different interaction conditions. This study has confined the number of genes in Giardia to a small subset that specifically change their expression due to interaction with IECs., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The ubiquitous protozoan parasites Giardia and Cryptosporidium have been detected from many species of captive and free-living wildlife, representing most mammalian orders. Twenty species of marine mammals have been reported to inhabit Galician waters and the region has one of the highest rates of stranding in Europe. Evidence from stranding, reported by-catches and sightings, suggests that the common dolphin (Delphinus delphis) is the most abundant cetacean on the Galician coast (Northwest Spain). The objective of this study was to detect and molecularly characterize isolates of Giardia and Cryptosporidium obtained from common dolphins stranded in this area. Between 2005 and 2012, sections of large intestine from 133 common dolphins stranded along the Galician coast were collected by the personnel of the Galician Stranding Network (Coordinadora para o Estudo dos Mamíferos Mariños, CEMMA). Using direct immunofluorescence antibody test (IFAT) and PCR amplification and sequencing of the SSU-rDNA, β-giardin genes and the ITS1-5.8S-ITS2 region, Giardia and Cryptosporidium were detected in 8 (6.0%) and 12 samples (9.0%), respectively. In two samples, co-infection by both parasites was observed. The molecular characterization revealed the presence of Giardia duodenalis assemblages A (genotypes A1 and A2) and B and Cryptosporidium parvum in these samples. This constitutes the first study in which the presence of Giardia and Cryptosporidium has been investigated in common dolphins on the European Atlantic coast, and it is also the first report of C. parvum in this host. Our findings indicate that these animals could act as reservoir of these waterborne parasites or could be victims of the contamination originated by anthropogenic activities., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A 40-year-old NIH male scientist camped and fished in a remote lake in Alaska. On his return, he developed diarrhea, cramps, and loose stools without blood or mucus in the absence of fever and was diagnosed with giardiasis. A 3-year-old female living in the Florida Keys complained of intermittent stomachaches over a 2-month period. Her stools were variably loose. The patient was diagnosed with giardiasis, which led to examination of her mother, father, and brother, who were mildly symptomatic; all 3 were subsequently diagnosed with giardiasis. The child's only exposure was from swimming in a local community pool. A 40-year-old male from Mexico, who resided in Virginia and worked as a cook in a fast food restaurant, was diagnosed with giardiasis. He denied any symptoms and was not allowed to prepare food. Treatment with metronidazole, nitazoxanide, and albendazole failed to eradicate the infection. He was successfully treated with the combination of paromomycin and metronidazole.

Giardia lamblia (syn G. intestinalis, G. duodenalis) is the most common pathogenic intestinal parasite of humans worldwide and is a frequent cause of endemic and epidemic diarrhea. G. lamblia is divided into eight genotypes (A-H) which infect a wide range of mammals and humans, but human infections are caused by Genotypes A and B. To unambiguously determine the relationship among genotypes, we sequenced GS and DH (Genotypes B and A2) to high depth coverage and compared the assemblies with the nearly completed WB genome and draft sequencing surveys of Genotypes E (P15; pig isolate) and B (GS; human isolate). Our results identified DH as the smallest Giardia genome sequenced to date, while GS is the largest. Our open reading frame analyses and phylogenetic analyses showed that GS was more distant from the other three genomes than any of the other three were from each other. Whole-genome comparisons of DH_A2 and GS_B with the optically mapped WB_A1 demonstrated substantial synteny across all five chromosomes but also included a number of rearrangements, inversions, and chromosomal translocations that were more common toward the chromosome ends. However, the WB_A1/GS_B alignment demonstrated only about 70% sequence identity across the syntenic regions. Our findings add to information presented in previous reports suggesting that GS is a different species of Giardia as supported by the degree of genomic diversity, coding capacity, heterozygosity, phylogenetic distance, and known biological differences from WB_A1 and other G. lamblia genotypes.

The purpose of this study was to determine the prevalence, associated risk factors and genotype of Giardia duodenalis infection in children attending public daycare centers in the city of Araguari, state of Minas Gerais, Brazil. Fecal samples were collected from 245 children aged 0-5 years, and questionnaires were asked about sociodemographic and hygiene-related characteristics. At the daycare centers where children tested positive, fecal samples were collected from the staff handling food, and from family members and domestic animals. Positive samples were analyzed at the dehydrogenase glutamate (gdh) locus to determine the genotype. The prevalence of G. duodenalis was 51.8%, and drinking unfiltered and unboiled water (OR 2.12, CI 1.26-3.69, p<0.001) and washing hands only with water (OR 2.14, CI 1.19-4.04, p<0.001) were related risk factors. No association was found between test-positive children and their family members, domestic animals and food handlers. An analysis of the sequences of 30 samples revealed that they all belonged to genotype B., (Copyright © 2012 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.)

Giardia duodenalis genotypes and Cryptosporidium species were studied in humans and free-ranging animals living in closed enclaves in Côte d'Ivoire. Three hundred and seven stool samples were tested from humans, and 47 from freely roaming domestic animals (dogs, goats, ducks, chickens). Molecular characterization of the isolates was performed by sequence analysis of a portion of the SSU-rDNA for Giardia and the COWP gene for Cryptosporidium, and a β-giardin SYBR-green real-time PCR was also used to confirm the assignment of Giardia isolates to Assemblages. In humans, genotyping of Giardia assigned many of the sequences (43/56 by the SSU-rDNA gene, and 36/61 by the β-giardin gene) to Assemblage B. The animal species harboured only zoonotic Assemblages A and B, except for dogs, in which host specific Assemblages C and D were also detected. Cryptosporidium meleagridis, C. parvum and C. hominis were detected in humans, while among the animals only chickens were found positive for oocysts, identified as C. meleagridis and C. parvum. The results provide further evidence about the role of free-ranging domestic animals living closely with humans in the environmental dissemination and potential transmission of these anthropozoonotic pathogens to humans., (Copyright © 2011 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.)

The protozoan Giardia lamblia differentiates from a pathogenic trophozoite into an infectious cyst to survive outside of the host. During encystation, genes encoding cyst wall proteins (CWPs) are coordinately induced. Pax family transcription factors are involved in a variety of developmental processes in animals. Nine Pax proteins have been found to play an important role in tissue and organ development in humans. To understand the progression from primitive to more complex eukaryotic cells, we tried to identify putative pax genes in the G. lamblia genome and found two genes, pax1 and pax2, with limited similarity. We found that Pax1 may transactivate the encystation-induced cwp genes and interact with AT-rich initiatior elements that are essential for promoter activity and transcription start site selection. In this study, we further characterized Pax2 and found that, like Pax1, Pax2 was present in Giardia nuclei and it may specifically bind to the AT-rich initiator elements of the encystation-induced cwp1-3 and myb2 genes. Interestingly, overexpression of Pax2 increased the cwp1-3 and myb2 gene expression and cyst formation. Deletion of the C-terminal paired domain or mutation of the basic amino acids of the paired domain resulted in a decrease of nuclear localization, DNA-binding activity, and transactivation activity of Pax2. These results are similar to those found in the previous Pax1 study. In addition, the profiles of gene expression in the Pax2 and Pax1 overexpressing cells significantly overlap in the same direction and ERK1 associated complexes may phosphorylate Pax2 and Pax1, suggesting that Pax2 and Pax1 may be downstream components of a MAPK/ERK1 signaling pathway. Our results reveal functional redundancy between Pax2 and Pax1 in up-regulation of the key encystation-induced genes. These results illustrate functional redundancy of a gene family can occur in order to increase maintenance of important gene function in the protozoan organism G. lamblia.

Background and Aims: Understanding the interplay between genetic susceptibility, the microbiome, the environment and the immune system in Crohn's Disease (CD) is essential for developing optimal therapeutic strategies. We sought to examine the dynamics of the relationship between inflammation, the ileal microbiome, and host genetics in murine models of ileitis., Methods: We induced ileal inflammation of graded severity in C57BL6 mice by gavage with Toxoplasma gondii, Giardia muris, low dose indomethacin (LDI; 0.1 mg/mouse), or high dose indomethacin (HDI; 1 mg/mouse). The composition and spatial distribution of the mucosal microbiome was evaluated by 16S rDNA pyrosequencing and fluorescence in situ hybridization. Mucosal E. coli were enumerated by quantitative PCR, and characterized by phylogroup, genotype and pathotype., Results: Moderate to severe ileitis induced by T. gondii (day 8) and HDI caused a consistent shift from >95% gram + Firmicutes to >95% gram - Proteobacteria. This was accompanied by reduced microbial diversity and mucosal invasion by adherent and invasive E. coli, mirroring the dysbiosis of ileal CD. In contrast, dysbiosis and bacterial invasion did not develop in mice with mild ileitis induced by Giardia muris. Superimposition of genetic susceptibility and T. Gondii infection revealed greatest dysbiosis and bacterial invasion in the CD-susceptible genotype, NOD2(-/-), and reduced dysbiosis in ileitis-resistant CCR2(-/-) mice. Abrogating inflammation with the CD therapeutic anti-TNF-α-mAb tempered dysbiosis and bacterial invasion., Conclusions: Acute ileitis induces dysbiosis and proliferation of mucosally invasive E. coli, irrespective of trigger and genotype. The identification of CCR2 as a target for therapeutic intervention, and discovery that host genotype and therapeutic blockade of inflammation impact the threshold and extent of ileal dysbiosis are of high relevance to developing effective therapies for CD.

Unlike superoxide dismutases (SODs), superoxide reductases (SORs) eliminate superoxide anion (O(2)(•-)) not through its dismutation, but via reduction to hydrogen peroxide (H(2)O(2)) in the presence of an electron donor. The microaerobic protist Giardia intestinalis, responsible for a common intestinal disease in humans, though lacking SOD and other canonical reactive oxygen species-detoxifying systems, is among the very few eukaryotes encoding a SOR yet identified. In this study, the recombinant SOR from Giardia (SOR(Gi)) was purified and characterized by pulse radiolysis and stopped-flow spectrophotometry. The protein, isolated in the reduced state, after oxidation by superoxide or hexachloroiridate(IV), yields a resting species (T(final)) with Fe(3+) ligated to glutamate or hydroxide depending on pH (apparent pK(a)=8.7). Although showing negligible SOD activity, reduced SOR(Gi) reacts with O(2)(•-) with a pH-independent second-order rate constant k(1)=1.0×10(9) M(-1) s(-1) and yields the ferric-(hydro)peroxo intermediate T(1); this in turn rapidly decays to the T(final) state with pH-dependent rates, without populating other detectable intermediates. Immunoblotting assays show that SOR(Gi) is expressed in the disease-causing trophozoite of Giardia. We propose that the superoxide-scavenging activity of SOR in Giardia may promote the survival of this air-sensitive parasite in the fairly aerobic proximal human small intestine during infection., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Giardia intestinalis is one of the most common diarrhea-related parasites in humans, where infection ranges from asymptomatic to acute or chronic disease. G. intestinalis consists of eight genetically distinct genotypes or assemblages, designated A-H, and assemblages A and B can infect humans. Giardiasis has been classified as a possible zoonotic disease but the role of animals in human disease transmission still needs to be proven. We tried to link different assemblages and sub-assemblages of G. intestinalis isolates from Swedish human patients to clinical symptoms and zoonotic transmission., Methodology/principal Findings: Multilocus sequence-based genotyping of 207 human Giardia isolates using three gene loci: ß-giardin, glutamate dehydrogenase (gdh), and triose phosphate isomerase (tpi) was combined with assemblage-specific tpi PCRs. This analysis identified 73 patients infected with assemblage A, 128 with assemblage B, and six with mixed assemblages A+B. Multilocus genotypes (MLGs) were easily determined for the assemblage A isolates, and most patients with this genotype had apparently been infected through anthroponotic transmission. However, we also found evidence of limited zoonotic transmission of Giardia in Sweden, since a few domestic human infections involved the same assemblage A MLGs previously reported in Swedish cats and ruminants. Assemblage B was detected more frequently than assemblage A and it was also more common in patients with suspected treatment failure. However, a large genetic variability made determination of assemblage B MLGs problematic. Correlation between symptoms and assemblages was found only for flatulence, which was significantly more common in children less than six years of age infected with assemblage B., Conclusions/significance: This study shows that certain assemblage A subtypes are potentially zoonotic and that flatulence is connected to assemblage B infections in young children. Determination of MLGs from assemblages A and B can be a valuable tool in outbreak situations and to help identify possible zoonotic transmission.

The effectiveness of molecular methods for the detection of species of Giardia and Cryptosporidium in fecal samples is often reduced by low or intermittent cyst and oocyst shedding, and/or the presence of polymerase chain reaction (PCR) inhibitors. The present study investigates the use of immunomagnetic separation (IMS) as an additional concentration step before PCR in the detection of these common protozoan parasites in dairy cattle. The IMS-PCR assays were optimized for amplifying fragments of the 16S ribosomal RNA (rRNA), β-giardin, and glutamate dehydrogenase (GDH) genes of Giardia duodenalis, as well as fragments of the 18S rRNA, heat shock protein (HSP)-70, and Cryptosporidium oocyst wall protein (COWP) genes of Cryptosporidium spp. In all cases, IMS-PCR was more sensitive than PCR alone. A significantly greater number of Giardia-positive samples were identified using IMS-PCR of the 16S rRNA gene (P < 0.01) and of the GDH gene (P < 0.01), as compared with PCR without any additional concentration step. In the case of Cryptosporidium, IMS-PCR of the COWP gene (P  =  0.02) resulted in a significantly greater number of positives than did PCR without the IMS concentration step. The greatest number of positives, however, was obtained using IMS-PCR to amplify a portion of the 16S rRNA gene of Giardia and a portion of the HSP-70 gene of Cryptosporidium. A further comparison of the optimized IMS-PCR assays to immunofluorescence microscopy suggested that the IMS-PCR assays were considerably more sensitive than microscopy was in the detection of Giardia cysts and Cryptosporidium oocysts in fecal samples.

Cryptosporidium and Giardia are two of the most common enteric pathogens of domestic and wild animals and humans. However, little is known on the prevalence, clinical manifestations and economic and zoonotic significance of these infections in horses. This study was undertaken to investigate the prevalence, excretion patterns and risk factors related to the faecal shedding of Cryptosporidium oocysts and Giardia cysts in horses and the zoonotic potential of species/genotypes isolated. The survey was performed on 120 foals and 30 broodmares reared in five Italian farms. Foals were divided in four homogeneous groups of 30 animals each (age classes: 0-2, 2-4, 4-8, >8 weeks). Three sequential faecal samples were collected from each animal and analysed by three techniques: direct fluorescent antibody test (DFA), faecal flotation (FF) and stained faecal smears (SFS). The DFA results showed a prevalence of 8% for Cryptosporidium and of 13.33% for Giardia; the prevalence values obtained by FF and SFS were lower and in poor agreement with DFA results. Giardia and Cryptosporidium infections were more common in foals (23.33% and 26.66% respectively) and higher excretions were observed in the youngest foals. Distribution of Cryptosporidium prevalence was statistically related to farms (P < 0.01), age of animals (P < 0.01), but was unrelated to the presence of diarrhoea. In the case of Giardia, the prevalence was only related to age (P < 0.01). Pattern sheddings were related to intestinal diseases and horse age (P < 0.01). Risk factors for shedding included residence farms and age older than 8 weeks for both parasites. All DFA-positive faecal samples were submitted to DNA extraction and PCR to determine Giardia and Cryptosporidium species/genotypes. Sequence analysis of the COWP gene of Cryptosporidium and of the SSU-rRNA gene of Giardia revealed that they were identical to each other and identified Cryptosporidium parvum and Giardia duodenalis assemblage E. The potential role of infected horses in zoonotic transmission of Cryptosporidium was supported by the findings of this study., (© 2009 Blackwell Verlag GmbH.)

Background: Giardia lamblia is ubiquitous in multiple communities of nonindustrialized nations. Genotypes A1, A2, and B (Nash groups 1, 2, and 3, respectively) are found in humans, whereas genotypes C and D are typically found in dogs. However, genotypes A and B have occasionally been identified in dogs., Methods: Fecal Giardia isolates from 22 families and their dogs, living in Pampas de San Juan, were collected over 7 weeks in 2002 and 6 weeks in 2003. Samples were genotyped, followed by sequencing and haplotyping of many of these isolates by using loci on chromosomes 3 and 5., Results: Human infections were all caused by isolates of genotypes A2 and B. Human coinfections with genotypes A2 and B were common, and the reassortment pattern of different subtypes of A2 isolates supports prior observations that suggested recombination among genotype A2 isolates. All dogs had genotypes C and/or D, with one exception of a dog with a mixed B/D genotype infection., Conclusions: In a region of high endemicity where infected dogs and humans constantly commingle, different genotypes of Giardia are almost always found in dogs and humans, suggesting that zoonotic transmission is very uncommon.

This study was undertaken to determine the genetic diversities of Giardia intestinalis isolated in Thailand. G. intestinalis cysts were collected from stool samples of 61 subjects residing in Bangkok or in rural communities of Thailand with and without gastrointestinal symptoms. All the cyst samples gave positive tpi amplicons (100% sensitivity), either of the 148- or the 81-bp tpi segments. Cyst assemblage identification of the 148- and 81-bp tpi gene segments by polymerase chain reaction showed that 8% of the cysts were assemblage A, 41% assemblage A and B combined, and 51% assemblage B. The prevalence of assemblage A was significantly lower than that of assemblage B and the mixed types. Restriction fragment length polymorphism (RFLP) of the 384-bp beta-giardin gene segment revealed that 12% and 88% of the assemblage A cysts were AI and AII respectively. RFLP, based on the 432-bp gdh gene segment, showed 45.5% of the assemblage B cysts to be BIII and 54.5% to be BIV. The AI sub-assemblage was less prevalent than the others. All subjects with AI and 50% of the subjects with BIII sub-assemblage cysts were symptomatic; 80% of symptomatic Bangkok residents were adults/elderly while 85% of the rural cases were children.

Cholesterol and bile salts are relevant modulators of Giardia encystation. Although several molecules within signaling cascades have been identified, and changes in their expression observed during giardial encystation, their underlying interactions leading to expression of cyst wall markers (CWPs and precursors of the GalNAc homopolymer) are not well defined. Recent experimental data and the completion of the Giardia Genome Project Database (GiardiaDB) allow us now to consider the role of bile salts as "natural stimuli" and the potential involvement of a Raf/MEK/ERK pathway mediating cholesterol-regulated expression of cyst-specific genes. These new findings may provide promising targets for diagnostics, drug design and prophylactic intervention against giardiasis.

Secretory immunoglobulin A (S-IgA) antibodies have a central role in anti-Giardial defence. It has been demonstrated that transforming growth factor-beta1 (TGF-beta1) stimulates B lymphocytes to produce and secrete S-IgA. We sought to determine the association between TGF-beta1 polymorphism (T+869C) with susceptibility to Giardiasis. The TGF-beta1 genotypes and levels of salivary (S-IgA) were analysed in individuals with Giardiasis (97 symptomatic and 57 asymptomatic) and controls (n = 92). Individuals with symptomatic Giardiasis had the lowest levels of S-IgA compared to individuals in asymptomatic Giardiasis and control groups (97%, 73% and 43%, <1 g L(-1), respectively, P = 0.002). The frequency of allele C and CC genotypes of TGF-beta1 polymorphism was significantly higher among symptomatic patients than asymptomatic and control groups. Logistic regression analysis demonstrated that the individuals homozygous for allele C of TGF-beta1 had a significantly higher risk for symptomatic Giardiasis with odds ratio of 2.76 (95% CI: 3.88, 1.71, P = 0.007). Among the participants with TT genotype per cent of individuals with S-IgA level of more than 1 g L(-1) was almost twice the percentage in CC genotype individuals (14% versus 7% respectively P = 0.01). Our data suggest that CC genotype of TGF-beta1 polymorphism at codon 10 is associated with occurrence of Giardiasis.

Giardia is frequently detected in stools of non-human primates (NHP). However, a molecular identification has been rarely applied to Giardia isolates from NHP, and the distribution of the zoonotic assemblages A and B remains unclear. Moreover, little is known about the genetic variability among the isolates, although this may contribute to the elucidation of the different transmission pathways, including the role of NHP as a reservoir for human giardiasis. Therefore, 258 Giardia samples from 31 NHP species housed in nine zoological gardens and one sanctuary in Belgium and The Netherlands were characterised based on an assemblage-specific PCR targeting the triose phosphate isomerase (tpi) gene to identify both assemblage A and B infections. In addition, a multi-locus sequencing approach based on the glutamate dehydrogenase, the tpi and the beta-giardin genes was used to examine both the genetic variability and the ability to allocate these isolates to different NHP groups. Overall, assemblage B was the most prevalent (78.6%), but mixed assemblage A and B infections occurred in 32.7% of the samples. Sequencing of the isolates revealed the presence of new polymorphisms for both assemblages and at the three loci examined. The majority of the assemblage B isolates could not be grouped into recently described sub-assemblages, particularly at the tpi gene. Isolates could only be allocated to a specific group when polymorphisms of the three loci were combined. The results confirm that NHP are a potential reservoir for zoonotic transmission and advocate the use of assemblage-specific primers in molecular epidemiological surveys, as mixed infections are likely to be underestimated. The high level of heterogeneity within assemblages indicates that a revised nomenclature of these sub-assemblages is needed, but points out the potency of a multi-locus sequencing approach to unravel the complex epidemiology of Giardia duodenalis.

Human giardiasis, the gastrointestinal infection caused by two genetically different groups (or assemblages) of Giardia duodenalis, is very common worldwide, and its prevalence is higher in developing countries. However, few surveys in these regions have been performed to include a genetic characterization of the parasite, which is necessary to unravel the complex epidemiology of the infection. In this work, we screened 120 faecal samples collected from Sahrawi children in 2003-2005, and found 41 (34.2%) of them to be positive, using immunofluorescent microscopy, for the presence of G. duodenalis cysts. Molecular characterization of the isolates was performed by RFLP and/or sequence analysis of the triose phosphate isomerase (tpi) and the glutamate dehydrogenase (gdh) genes. The results disclosed an unexpectedly high genetic polymorphism among isolates of both assemblages A and B, and a large percentage of the sequences (50% for the tpi gene, and 90% for the gdh gene) from assemblage B isolates characterized by the presence of overlapping nucleotide peaks at specific positions in the chromatograms, which can be attributed to mixed infections or to allelic sequence heterozygosity of single cysts. Notably, this phenomenon was not observed in sequences from assemblage A isolates. These results suggest that the genetic structure is different in isolates of assemblages A and B.

Giardia duodenalis causes diarrhoea and malabsorption. The objectives of the study were to detect local isolates of G. doudenalis by polymerase chain reaction (PCR) and to determine their restriction fragment length polymorphisms (RFLP). G. doudenalis isolated from stools of patients from Hospital Orang Asli Gombak were cultured axenically using TYI-S-33 medium with 10% foetal calf serum. The commercially designed primer-pair 432/433 was used to amplify a 0.52 kb segment known to encode the homologous cysteine-rich trophozoite surface antigen (tsp11 and tsa417). Results showed that the primer-pair 432/433 could amplify the target region of the local isolates. RFLP study on the identical isolates showed that all the restriction enzymes tested ( HindIII, ClaI, PstI and Kpn) gave a banding pattern similar to that of the WB strain a reference pathogenic strain from human. The reference pathogenic strain were commercially obtained from the American Type Culture Collection (ATCC).

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.

To determine the genotypes of Giardia intestinalis from domestic and wild animals in Japan, Giardia isolates obtained from feces of 24 dogs kept in households and breeding kennels, three companion cats, five dairy calves and three wild monkeys, Macaca fuscata, were genotyped using the 177 bp sequence of the glutamete dehydrogenase gene (gdh). The genotypes were assemblages A, C, D or A/D for dog isolates, Assemblage F for cat isolates, assemblages A or E for calf isolates and assemblage B for monkey isolates. This is the first report on the genotypes of Giardia isolates from cats, calves and wild monkeys in Japan.

The flagellated protozoan Giardia intestinalis is one of the most prevalent human-infective parasites with a worldwide distribution. This parasite must encyst to complete the life cycle and N-acetylgalactosamine is produced from endogenous glucose for cyst wall synthesis during the transformation. UDP-N-acetylglucosamine pyrophosphorylase in G. intestinalis (GiUAP, EC 2.7.7.23) is the fourth enzyme in the inducible pathway of N-acetylgalactosamine biosynthesis, catalysing the conversion of N-acetylglucosamine-1-P to UDP-N-acetylglucosamine. In this study the gene GiUAP was cloned and sequenced from the Portland 1 strain using PCR techniques. It has an ORF of approximately 1.3 kb and contains no introns. BLAST and ClustalW analysis of the deduced amino acid sequence revealed significant similarities to other eukaryotic UAPs with putative active sites identified. Southern hybridization showed that GiUAP exists as a single-copy gene and it was shown to have two transcripts by RT-PCR and Northern hybridization. RLM-RACE identified both 5' and 3' untranslated regions and suggested the transcripts exist as a 5'-capped mRNA, with the use of two tandem polyadenylation sites to generate two unusually long giardial 3' untranslated regions of approximately 522 bp and approximately 3 kb. Moreover, a recombinant protein (rGiUAP) was expressed in E. coli and subjected to physical characterizations. Surprisingly the results obtained in this study were significantly different from those reported for the GiUAP in MR4 strain, suggesting this gene is under different transcription control in different strains of G. intestinalis. This report describes the molecular characterization of GiUAP and provides an opportunity to explore the control of gene expression during encystation of the parasite.

The process of encystation is a key step in the Giardia duodenalis life cycle that allows this intestinal protozoan to survive between hosts during person-to-person, animal-to-person, waterborne, or food-borne transmission. The release of cysts from infected persons and animals is the main contributing factor to contamination of the environment. Genes coding for cyst wall proteins (CWPs), which could be used for developing a transmission-blocking vaccine, have been cloned. Since the immunogenicity of recombinant Giardia CWP is unknown, we have investigated the immunogenicity of recombinant CWP2 (rCWP2) and its efficacy in interfering with the phenomenon of encystation taking place in the small bowels of BALB/c mice vaccinated with the recombinant protein. Here we report that the immunization of BALB/c mice with rCWP2 stimulated the immune system in a manner comparable to that for a live infection with Giardia muris cysts. Fecal and serum anti-rCWP2 immunoglobulin A (IgA) antibodies were detected in the immunized mice. In addition, anti-rCWP2 IgG1 and IgG2a antibodies were detected in the serum. mRNAs coding for Th1 and Th2 types of cytokines were detected in spleen and Peyer's patch cells from immunized mice. When the vaccinated mice were challenged with live cysts, the animals shed fewer cysts. We conclude that rCWP2 is a possible candidate antigen for the development of a transmission-blocking vaccine.

A comparison of random amplified polymorphic DNA (RAPD) was used to investigate genetic polymorphisms among 25 isolates of Giardia intestinalis and to assess the utility of RAPD for subtype detection and genealogical analysis. Using data obtained for six human and 19 animal-derived isolates in polymerase chain reactions using 13 different primers, phylogenetic trees were constructed and bootstrap values computed by the program FreeTree. Three major clades were distinguished, corresponding to previously defined genetic assemblages A, B, and E. The purported specificity of assemblage E genotypes for artiodactyl hosts was supported. Assemblages A and B showed wide host spectra, including human and animal hosts. No correlation was found between the genotype of analyzed isolates and the presence or absence of the double-stranded RNA Giardiavirus. The results indicate that RAPD data provide reliable genetic information that can be used for both "fingerprinting" and genealogical purposes.

Giardia duodenalis infection in humans can cause a variety of clinical symptoms. The relation between clinical symptomatology and the Giardia isolate genotype was studied in 18 Dutch patients infected with G. duodenalis who visited their general practitioner. Contrary to earlier studies, a 100% correlation between severity of diarrhoeal complaints and genotype was found: assemblage A isolates were solely detected in patients with intermittent diarrhoeal complaints, while assemblage B isolates were present in patients with persistent diarrhoeal complaints. These results are significant because they show for the first time that genetically linked features of G. duodenalis are major determinants in the severity of infection in human giardiasis.

Fifty patients suffering from persistent giardiasis and 257 healthy persons from Uzbekistan were examined. HLA-typing was carried out by the Terasaki microlymphocytotoxicity technique. The patients with persistent giardiasis were characterized as a significant prevalence of HLA-B5, HLA-B14, HLA-DR3, DR4, DR7 and haplotypes HLA-A9-B5 and A1-B5. These haplotypes and HLA antigens can be considered as markers of predisposition to persistent giardiasis.

Wide variation has been observed in the natural history of human and murine giardiasis, which could be due to factors associated with the parasite or with the host. In humans, prolonged infections with Giardia duodenalis have been associated with hypogammaglobulinemia, protein-calorie malnutrition, and prior gastrectomy. The duration of infection or severity of symptoms may also be influenced by the humoral immune response and by other factors such as ABO blood group and HLA antigen type. Elimination of Giardia muris is impaired in hypothymic mice, B-cell-deficient mice expressing the xid gene, and in mice deficient in mast cells. Prolonged infections also occur following depletion of helper/inducer T cells and suppression of antibody production with antisera to IgM. However, quantitative or qualitative defects in immune function do not appear to account for prolonged cyst excretion in C3H/He, C3H/HeJ, and A/J mice. The defect in these strains is influenced by several genes and may involve intermediate or late stages of macrophage activation leading to impaired elimination of the parasite.