Research paperStrong genetic structure revealed by multilocus patterns of variation in Giardia duodenalis isolates of patients from Galicia (NW-Iberian Peninsula)
Introduction
Giardia duodenalis (syn. G. intestinalis, G. lamblia; Eukaryota, Excavata, Diplomonadida) is a binucleated protozoan that infects mammals causing giardiasis (Adam, 2001), one of the most common intestinal parasitic diseases in humans in both developed and developing countries (Feng and Xiao, 2011). To date, eight assemblages (A–H) and numerous sub-assemblages (e.g. AI, AII, BIII, BIV) have been described based on the analysis of genetic variation (Andrews et al., 1989, Ey et al., 1997, Homan et al., 1992, Lasek-Nesselquist et al., 2010, Mayrhofer et al., 1995, Monis et al., 1999, Monis et al., 2003, Monis et al., 1998, Nash and Keister, 1985, Nash et al., 1985). Only assemblages A and B infect humans, but they also infect other mammals, having zoonotic potential (Ryan and Caccio, 2013). The taxonomical status of assemblages is still a matter of debate, as they have been proposed to correspond to different species (Monis et al., 2009, Ryan and Caccio, 2013, Xu et al., 2012), but also to sub-species entities such as “near-clades”, owing to their observed genetic integrity over space and time (Tibayrenc and Ayala, 2012, Tibayrenc and Ayala, 2014). The efficient colonization of the upper intestinal tract by trophozoites depends on both the strain of the infecting pathogen and the host (Nash et al., 1987), which means that our understanding of the epidemiology and pathogenic properties of Giardia strongly relies on a detailed knowledge of the patterns of genetic diversity and population structure of the parasite.
In recent years, an increasing effort has been devoted to describe the patterns of diversity in Giardia populations with a special focus on human-derived samples, usually by direct sequencing of PCR products. This wealth of data uncovered a very complex scenario: (i) nucleotide variation exists both within assemblages and within isolates –revealed by the presence of double peaks in the electropherograms– (Ankarklev et al., 2012, Caccio et al., 2008, Choy et al., 2015, de Lucio et al., 2015, Durigan et al., 2014, Huey et al., 2013, Lalle et al., 2005, Lebbad et al., 2008, Lebbad et al., 2011, Minetti et al., 2015, Robertson et al., 2007), (ii) mixed-assemblage infections have been described in humans and other hosts (Almeida et al., 2010, Sprong et al., 2009) and (iii) multilocus analyses have shown inconsistent assemblage assignment and incongruent phylogenies within assemblages (Sprong et al., 2009). All these results have put into question even the classical limits of the assemblage-host associations (Durigan et al., 2014, Feng and Xiao, 2011, Sprong et al., 2009). But a detailed quantitative description of the patterns of genetic diversity of Giardia populations has been hampered by the limited resolution of the direct sequencing methodology. In fact, only in a handful of studies PCR products were cloned prior to sequencing (Hussein et al., 2009, Kosuwin et al., 2010, Lasek-Nesselquist et al., 2009, Siripattanapipong et al., 2011, Teodorovic et al., 2007). The data thus obtained allowed an incipient description of the patterns of diversity in Giardia samples and raised new questions that are central to the understanding of giardiasis at the biological, clinical and epidemiological levels. For example, the low heterozygosity usually observed seemingly contradicts the high allelic divergence expected for long-term asexual polyploids (Birky, 2010, Mark Welch and Meselson, 2000, Mark Welch et al., 2004). This is particularly so in Giardia where differences should accumulate between alleles within the same nucleus but also between the two sister diploid nuclei, which segregate together during the entire life cycle of the parasite (Carpenter et al., 2012, Jirakova et al., 2012, Sagolla et al., 2006, Yu et al., 2002). These observations suggest a role in genome homogenization for mitotic recombination, gene conversion, other mechanisms of asexual genetic exchange between nuclei (Carpenter et al., 2012, Poxleitner et al., 2008), but also for meiotic recombination (Cooper et al., 2007, Ramesh et al., 2005).
A detailed description of the genetic structure of the parasite populations (i.e. how much of the extant variation corresponds to differences between isolates, between individuals and within individuals) would contribute to delimit the role of sex and to understand the demographic history of the parasite. Additional population genetics data are needed to boost our still limited understanding of these fundamental aspects of the biology and population dynamics of this organism. Here, we report a multilocus population genetics survey of Giardia isolated from human patients of Galicia (NW-Spain), by PCR, cloning and sequencing three single copy loci.
Section snippets
Giardia duodenalis isolates
The G. duodenalis isolates used in this study were selected from a collection of isolates from the Complexo Hospitalario Universitario de Santiago (CHUS, Santiago de Compostela, Spain) during years 2000–2010. Symptomatic patients were diagnosed with giardiasis by examination of fresh faecal samples by microscopy. Total genomic DNA was extracted from stool samples using the QIAamp DNA Stool Mini kit (QIAgen).
Gene selection and primers design
Primers were designed to amplify coding regions from four single copy loci: the widely
Assemblage genotyping, PCR amplification, cloning and sequencing
After assemblage genotyping of 119 isolates, eight assemblage A isolates were detected (122, 147, 152, 209, 251, 263, 321, 839) with assemblage-specific primers for the tpi locus, two of which were also positive for assemblage B (122, 321). Given the reduced number of assemblage A positive isolates, they were all included in the analysis along with four additional isolates (407, 704, 1221, 1343) randomly selected among the assemblage B positives.
PCR amplification of gdh, bg, and calt with
Discussion
Our analysis unveiled substantial levels of within-isolate diversity at the three loci analyzed, which justifies the cloning of the PCR products prior to sequencing instead of the more common practice of direct sequencing of the mixed PCR products. This latter procedure likely leads to the systematic underestimation of the true diversity values, as the signal of the most common allele in the chromatograms likely masks the variants in alleles present at low frequency in the PCR product mixture.
Conclusions
The results presented here draw an overall scenario where human giardiasis is caused by the ingestion of a reduced number of individuals, which represent a small fraction of the diversity of the parasite population. Differences in nucleotide diversity suggest that assemblages A and B have recently experienced different demographic histories –assemblage A has a smaller estimated effective population size–, and both are apparently in demographic equilibrium. There is no evidence for genetic
Financial support
This study was funded with grant 10CSA208038PR from Xunta de Galicia (Spain), co-financed by European Regional Development Fund, to XM. LG was supported by a Predoctoral Fellowship by Xunta de Galicia (Spain).
Aknowledgements
We would like to thank Dr. A. Campos (Servizo de Hematoloxía, CHUS, Santiago de Compostela, Spain) and Dr. A. Vidal and Dra. C. Carneiro (CIMUS, USC) for the use of their laboratory facilities for some procedures.
References (75)
- et al.
Giardia intestinalis: electrophoretic evidence for a species complex
Int. J. Parasitol.
(1989) Giardia Sex? Yes, but how and how much?
Trends Parasitol.
(2010)- et al.
Multilocus genotyping of Giardia duodenalis reveals striking differences between assemblages A and B
Int. J. Parasitol.
(2008) - et al.
Population genetics provides evidence for recombination in Giardia
Curr. Biol.
(2007) - et al.
Multilocus genotyping of Giardia duodenalis in Malaysia
Infect. Genet. Evol.
(2013) - et al.
Multiple-subgenotype infections of Giardia intestinalis detected in Palestinian clinical cases using a subcloning approach
Parasitol. Int.
(2009) - et al.
How nuclei of Giardia pass through cell differentiation: semi-open mitosis followed by nuclear interconnection
Protist
(2012) - et al.
Evolution of protein molecules
Bias and artifacts in multitemplate polymerase chain reactions (PCR)
J. Biosci. Bioeng.
(2003)- et al.
Clonal diversity in Giardia duodenalis isolates from Thailand: evidences for intragenic recombination and purifying selection at the beta giardin locus
Gene
(2010)
Genetic heterogeneity at the beta-giardin locus among human and animal isolates of Giardia duodenalis and identification of potentially zoonotic subgenotypes
Int. J. Parasitol.
The identification of a new Giardia duodenalis assemblage in marine vertebrates and a preliminary analysis of G. duodenalis population biology in marine systems
Int. J. Parasitol.
Dominance of Giardia assemblage B in Leon, Nicaragua
Acta Trop.
Genetic diversity within the morphological species Giardia intestinalis and its relationship to host origin
Infect. Genet. Evol.
Variation in Giardia: towards a taxonomic revision of the genus
Trends Parasitol.
A phylogenomic inventory of meiotic genes; evidence for sex in Giardia and an early eukaryotic origin of meiosis
Curr. Biol.
Molecular characterisation of Giardia isolates from clinical infections following a waterborne outbreak
J. Infect.
On the number of segregating sites in genetical models without recombination
Theor. Popul. Biol.
High prevalence Giardia duodenalis assemblage B and potentially zoonotic subtypes in sporadic human cases in Western Australia
Int. J. Parasitol.
Biology of Giardia lamblia
Clin. Microbiol. Rev.
Genome sequencing of Giardia lamblia genotypes A2 and B isolates (DH and GS) and comparative analysis with the genomes of genotypes A1 and E (WB and Pig)
Genome Biol. Evol.
Genotyping of Giardia duodenalis cysts by new real-time PCR assays for detection of mixed infections in human samples
Appl. Environ. Microbiol.
Allelic sequence heterozygosity in single Giardia parasites
BMC Microbiol.
GiardiaDB and TrichDB: integrated genomic resources for the eukaryotic protist pathogens Giardia lamblia and Trichomonas vaginalis
Nucleic Acids Res.
Nuclear inheritance and genetic exchange without meiosis in the binucleate parasite Giardia intestinalis
J. Cell Sci.
Elements Of Evolutionary Genetics
Population expansion and gene flow in Giardia duodenalis as revealed by triosephosphate isomerase gene
Parasit. Vectors
Molecular genotyping of Giardia duodenalis isolates from symptomatic individuals attending two major public hospitals in Madrid, Spain
PLoS One
Genetic diversity of Giardia duodenalis: multilocus genotyping reveals zoonotic potential between clinical and environmental sources in a metropolitan region of Brazil
PLoS One
Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows
Mol. Ecol. Resour.
Genetic analysis of Giardia from hoofed farm animals reveals artiodactyl-specific and potentially zoonotic genotypes
J. Eukaryot. Microbiol.
Confidence limits on phylogenies: an approach using the bootstrap
Evolution
Zoonotic potential and molecular epidemiology of Giardia species and giardiasis
Clin. Microbiol. Rev.
Draft genome sequencing of Giardia intestinalis assemblage B isolate GS: is human giardiasis caused by two different species?
PLoS Pathog.
BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT
Nucleic Acids Symp. Ser.
Comparison of Giardia isolates from different laboratories by isoenzyme analysis and recombinant DNA probes
Parasitol. Res.
Two-locus sampling distributions and their application
Genetics
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