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Allelic sequence heterozygosity in single Giardia parasites
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. (Prof Staffan Svärd laboratory)
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
2012 (English)In: BMC Microbiology, ISSN 1471-2180, Vol. 12, 65Article in journal (Refereed) Published
Abstract [en]

Background: Genetic heterogeneity has become a major inconvenience in the genotyping and molecular epidemiology of the intestinal protozoan parasite Giardia intestinalis, in particular for the major human infecting genotype, assemblage B. Sequence-based genotyping of assemblage B Giardia from patient fecal samples, where one or several of the commonly used genotyping loci (beta-giardin, triosephosphate isomerase and glutamate dehydrogenase) are implemented, is often hampered due to the presence of sequence heterogeneity in the sequencing chromatograms. This can be due to allelic sequence heterozygosity (ASH) and /or co-infections with parasites of different assemblage B sub-genotypes. Thus, two important questions have arisen; i) does ASH occur at the single cell level, and/or ii) do multiple sub-genotype infections commonly occur in patients infected with assemblage B, G. intestinalis isolates? Results: We used micromanipulation in order to isolate single Giardia intestinalis, assemblage B trophozoites (GS isolate) and cysts from human patients. Molecular analysis at the tpi loci of trophozoites from the GS lineage indicated that ASH is present at the single cell level. Analyses of assemblage B Giardia cysts from clinical samples at the bg and tpi loci also indicated ASH at the single cell level. Additionally, alignment of sequence data from several different cysts that originated from the same patient yielded different sequence patterns, thus suggesting the presence of multiple sub-assemblage infections in congruence with ASH within the same patient. Conclusions: Our results conclusively show that ASH does occur at the single cell level in assemblage B Giardia. Furthermore, sequence heterogeneity generated during sequence-based genotyping of assemblage B isolates may possess the complexity of single cell ASH in concurrence with co-infections of different assemblage B sub-genotypes. These findings explain the high abundance of sequence heterogeneity commonly found when performing sequence based genotyping of assemblage B Giardia, and illuminates the necessity of developing new G. intestinalis genotyping tools.

Place, publisher, year, edition, pages
2012. Vol. 12, 65
Keyword [en]
Giardia, ASH, assemblage B, genotyping, single cell, micromanipulation
National Category
Medical and Health Sciences Natural Sciences
Research subject
Epidemiology; Biology with specialization in Microbiology
URN: urn:nbn:se:uu:diva-167049DOI: 10.1186/1471-2180-12-65ISI: 000308649900001PubMedID: 22554281OAI: oai:DiVA.org:uu-167049DiVA: diva2:480614
Available from: 2012-01-19 Created: 2012-01-19 Last updated: 2015-05-26Bibliographically approved
In thesis
1. Inter and Intra-Assemblage Characterizations of Giardia intestinalis: from clinic to genome
Open this publication in new window or tab >>Inter and Intra-Assemblage Characterizations of Giardia intestinalis: from clinic to genome
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The protozoan parasite Giardia intestinalis (syn. G. lamblia, G. duodenalis) is one of the most common causes of diarrheal disease throughout the world, where an estimated 500 million people are infected annually. Despite efforts in trying to elucidate factors associated with virulence in G. intestinalis little is currently known. The disease outcome is highly variable in Giardia infected individuals, ranging from asymptomatic carriers to severe disease. The reasons behind the differences in disease outcome are vaguely understood and studies trying to link infectivity to different Giardia assemblages or sub-assemblages have rendered conflicting results. Prior to this study, little was known about the prevalence and genetic diversity of different G. intestinalis assemblages across the world.

In this thesis, molecular characterization of clinical G. intestinalis samples from Eastern Africa and Central America, has been performed, enabling a better understanding of the prevalence of different Giardia genotypes in endemic areas (Papers I and II). A correlation between Giardia colonization and the presence of Helicobacter pylori in the human host was established. We found that the currently available genotyping tools provide low resolution when used to characterize assemblage A Giardia. Also, genotyping of assemblage B isolates at these loci is troublesome due to the polymorphic substitutions frequently found in the sequencing chromatograms. This ambiguity was investigated by using micromanipulation to isolate single assemblage B Giardia cells (Paper III). Both cultured trophozoites and cysts from giardiasis patients were analyzed. The data showed that allelic sequence heterozygosity (ASH) does occur at the single cell level, but also that multiple sub-assemblage infections appear to be common in human giardiasis patients.

Furthermore, genome-wide sequencing followed by comparative genomics was performed in order to better characterize differences between and within different Giardia assemblages. The genome of a non-human infecting, assemblage E isolate (Paper IV) was sequenced.  The genomes of two freshly isolated human infecting assemblage AII isolates were also sequenced (Paper V). Subsequent, comparative analyses were performed and included the genomes of two human infecting isolates, WB (AI) and GS/M (B). Several important differences were found between assemblages A, B and E, but also within assemblage A; including unique gene repertoires for each isolate, observed differences in the variable gene families and an overall difference in ASH between the different isolates. Also, a new multi-locus genotyping (MLG) strategy for genotyping of assemblage A Giardia has been established and evaluated on clinical samples from human giardiasis patients.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. 85 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 893
Giardia, protozoa, parasite infection, diarrhea, genome sequencing, comparative genomics, genotyping, ASH, MLG
National Category
Natural Sciences Medical and Health Sciences
Research subject
Infectious Diseases; Molecular Medicine; Microbiology
urn:nbn:se:uu:diva-167063 (URN)978-91-554-8259-6 (ISBN)
Public defence
2012-02-23, B42, BMC, Husargatan 3, Uppsala, 10:15 (English)
Available from: 2012-02-02 Created: 2012-01-19 Last updated: 2012-02-15Bibliographically approved

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