Molecular variation in Giardia

doi:10.1016/0001-706X(93)90028-A

Acta Tropica

Volume 53, Issues 3–4, May 1993, Pages 167-184

https://doi.org/10.1016/0001-706X(93)90028-A Get rights and content

Abstract

Molecular characterisation of species within the genus Giardia has revealed that much of the phenotypic heterogeneity, particularly within the species G. duodenalis, has a genetic basis. The source of this genetic variation appears to arise from predominantly asexual, clonal reproduction, although occasional bouts of sexual reproduction cannot be ruled out. Genetic variation is extensive with some clones widely distributed and others seemingly unique and localised to a particular endemic focus. Little attention has been given to the molecular epidemiology of Giardia infections. Future studies should be directed at studying the ecology and dynamics of transmission of Giardia clones, particularly in localised areas, and to evaluating the factors that serve to maintain genetic diversity between clones, especially the role of inter-clonal competition. Future research using molecular techniques should aim to identify and follow Giardia clones in nature and correlate genetic typing with important clinical and epidemiological characteristics such as virulence, drug sensitivity and zoonotic potential.

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Although both major G. duodenalis assemblages A and B have been found in humans throughout the world, their propensity to cause disease might vary. Assemblage A appears to be more widespread, whereas assemblage B is more restricted to localised endemic loci (Thompson and Meloni, 1993; Meloni et al., 1995). It remains to be established whether there is geographical variation in the prevalences of different G. duodenalis genotypes, as assemblage A has been found to be more prevalent in Canada (van Keulen et al., 2002), Ethiopia (Gelanew et al., 2007), Italy (Lalle et al., 2005), Mexico (Eligio-Garcia et al., 2005) and the USA (van Keulen et al., 2002).
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Multi-locus analysis of Giardia duodenalis intra-Assemblage B substitution patterns in cloned culture isolates suggests sub-Assemblage B analyses will require multi-locus genotyping with conserved and variable genes
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A total of 11 cloned cultured isolates sourced from humans in Western Australia were analysed (Table 1 and Fig. 1). Two Assemblage A isolates (AI and AII) and nine Assemblage B isolates selected from as broad a genetic range as possible based on previous isozyme data were chosen (Thompson and Meloni, 1993). No clinical information was available for the isolates, only their collection locations.
Recent research concerning Giardia duodenalis has focused on resolving possible sub-assemblages within Assemblages A and B to better understand host-specific and zoonotic relationships. In the present study nine cloned, cultured, Assemblage B isolates were used to investigate the intra-Assemblage B substitution patterns of conserved (ssrDNA, ef, h2b, h4) and variable (tpi, gdh, bg) genes to assess their suitability for further application to sub-assemblage analyses. The resolution of each gene was found to be proportional to its substitution rate and for the genetically narrow sample set examined, the variable genes best represented the consensus phylogeny while the conserved genes only established fractions. However it was demonstrated that the spectra of conserved and variable genes were required to ensure accuracy of inferred phylogeny and it was therefore concluded that further research into sub-Assemblage B groups would require a mixture of conserved and variable genes for the multi-locus analyses of this genetically broad assemblage.
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The susceptibility of Giardia duodenalis trophozoites exposed in vitro to sublethal concentrations of metronidazole (MTZ) and albendazole (ABZ) may exhibit inter-culture (variability) and intra-culture (variation) differences in drug susceptibility. It was previously reported that MTZ-resistant trophozoites may display changes in pyruvate:ferredoxin oxidoreductase (PFOR) expression while changes at the β-tubulin molecule are apparently absent in ABZ-resistant cultures. To assess the levels of gene expression of these molecules, we obtained cloned cultures growing at concentrations up to 23 μM MTZ (WBRM23) and up to 8 μM ABZ (WBRA8) and gene sequence and expression of pfor and β-tubulin loci were compared with these of drug-susceptible clone WB1. Neither the pfor nor the β-tubulin genes showed changes at sequence level but the MTZ-resistant clones WBRM21 and WBRM23 showed up-regulation of the pfor RNA using the gdh gene as reference. By using WB1 and WBRA8 clones in representational difference analyses of gene expression (RDA) an insert referred to as ARR-VSP was selected and sequenced. It showed the highest homology to one VSP molecule in the Giardia Genome Database (orf GL50803_101765). This isogene was up-regulated in five ABZ-resistant clones and the clone WBRA8 exhibited the highest RNA expression level. When successive progenies of clones WB1, WBRM23 and WBRA8 were analyzed in Northern blot assays to detect pfor and ARR-VSP RNAs respectively, the expression patterns showed variation for both genes but it was much lower in the clone WBRA8. These results suggest that G. duodenalis cultures either susceptible or resistant to MTZ and ABZ may display variability and variation at RNA expression levels albeit these were more marked in the MTZ-resistant parasites. These data might have further implications defining major mechanisms involved in drug resistance of Giardia.
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The role of Giardia duodenalis surface molecules in the attachment of trophozoites to epithelial cells has been established through the dual strategies of characterizing G. duodenalis clones with deficient adhesion and blocking experiments with surface-specific monoclonal antibodies. Also, the infectivity of the analyzed clones was tested using Mongolian gerbils as experimental model. Two adhesion-deficient G. duodenalis clones, C6 and C7, were isolated from the wild type C5 clone which in turn was obtained from the WB strain. The adhesion efficiencies of C6 and C7 clones (48.2 ± 4.9 and 32.6 ± 2.4, respectively) were significantly lower as compared with WB strain or C5 clone (82.8 ± 6.4 and 79.9 ± 7.9). Analysis of radiolabel surface proteins by 1D and 2D SDS–PAGE revealed prominently labelled 28 and 88 kDa components in C6 and C7 clones and a major 200 kDa protein in the C5 clone and the WB strain. The 88 and 200 kDa components are acidic proteins by two-dimensional electrophoretic analyses. The most striking difference between wild-type and adhesion-deficient Giardia trophozoites was the reduced expression of a 200 kDa surface protein in the latter. Significantly, a mAb (IG3) specific for the 200 kDa protein that reacted with more than 99% of WB and C5 trophozoites and less than 1% of C6 and C7 trophozoites as determined by indirect immunofluorescence inhibited the adhesion of trophozoites from WB and C5 clone to Madin Darby Canine Kidney cells by 52% and 40.9%, respectively, suggesting a participation of this antigen in adherence. Finally, the functional relevance of trophozoite adhesion to epithelial cells was indicated by the reduced capacity of the adhesion-deficient clones to establish the infection in Mongolian gerbils.
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ReviewMolecular variation in Giardia

Abstract

Int. J. Parasitol.

Int. J. Parasitol.

Int. J. Parasitol.

Trans. R. Soc. Trop. Med. Hyg.

Trans. R. Soc. Trop. Med. Hyg.

TREE

Exp. Parasitol.

Trans. R. Soc. Trop. Med. Hyg.

Eur. J. Protist.

Parasitol. Today

TREE

Trans. R. Soc. Trop. Med. Hyg.

Int. J. Parasitol.

J. Pediatr.

Trans. R. Soc. Trop. Med. Hyg.

Trans. R. Soc. Trop. Med. Hyg.

Exp. Parasitol.

Mol. Biochem. Parasitol.

Trans. R. Soc. Trop. Med. Hyg.

Int. J. Parasitol.

Mol. Biochem. Parasitol.

Int. J. Parasitol.

Parasitol. Today

Parasitol. Today

Parasitol. Today

Int. J. Parasitol.

Int. J. Parasitol.

Mol. Biochem. Parasitol.

Exp. Parasitol.

Zoonoses and communicable diseases common to man and animals

The biology of Giardia spp

Microbiol. Rev.

The Giardia lamblia trophozoite contains sets of closely related chromosomes

Nucleic Acids Res.

Variable virulence of isolates of Giardia lamblia in mice

Ann. Trop. Med. Parasitol.

Comparison of two antigenically distinct Giardia lamblia isolates in gerbils

Am. J. Trop. Med. Hyg.

The genetic structure and evolutionary fate of parthenogenetic amphibian populations as determined by Markovian analysis

Am. Zool.

Isoenzyme studies of Giardia lamblia isolated from symptomatic cases

Aus. J. Exp. Biol. Med. Sc.

Giardia lamblia infections in Mongolian gerbils: an animal model

J. Inf. Dis.

Some perspectives on the transmission of giardiasis

A comparison of isozymes of five axenic Giardia isolates

J. Parasitol.

Giardia isozymes

A simple method for cloning Giardia duodenalis from cultures and fecal samples

J. Parasitol.

DNA fingerprinting in clonal organisms

Mol. Ecol.

First evidence for diploidy and genetic recombination in free-living amoebae of the genus Naegleria on the basis of electrophoretic variation

Genetics

Differentiation of aphid clones using DNA fingerprints from individual aphids

Origins and organization of genetic diversity in natural populations of Trypanosoma brucei

Parasitology

Giardia isolates from primates and rodents display the same molecular polymorphisms as human isolates

Mol Biochem. Parasitol.

When are parasites clonal?

Nature

Cross-species transmission of Giardia spp.: Inoculation of beavers and muskrats with cysts of human, beaver, mouse and muskrat origin

App. Env. Microbiol.

Comparative studies on the pattern of infection with Giardia spp. in Mongolian gerbils

J. Parasitol.

Studies on the cytology and life history of a Giardia from the laboratory rat

Univ. California Publn. Zool.

Review
Molecular variation in Giardia