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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: 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.

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.)

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.)

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.

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.

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.