Research paper
Strong genetic structure revealed by multilocus patterns of variation in Giardia duodenalis isolates of patients from Galicia (NW-Iberian Peninsula)

https://doi.org/10.1016/j.meegid.2016.12.014Get rights and content

Highlights

  • G. duodenalis assemblages A and B behave as two genetically isolated populations.

  • High between-isolate variation reflects a genetically structured population.

  • Infections are likely caused by a reduced number of distinct individuals.

  • Mechanisms for the homogenization of the genomes of the two nuclei must exist.

Abstract

We report a survey of genetic variation at three coding loci in Giardia duodenalis of assemblages A and B obtained from stool samples of patients from Santiago de Compostela (Galicia, NW-Iberian Peninsula). The mean pooled synonymous diversity for assemblage A was nearly five times lower than for assemblage B (0.77% ± 0.30% and 4.14% ± 1.65%, respectively). Synonymous variation in both assemblages was in mutation-drift equilibrium and an excess of low-frequency nonsynonymous variants suggested the action of purifying selection at the three loci. Differences between isolates contributed to 40% and 60% of total genetic variance in assemblages A and B, respectively, which revealed a significant genetic structure. These results, together with the lack of evidence for recombination, support that (i) Giardia assemblages A and B are in demographic equilibrium and behave as two genetically isolated populations, (ii) infections are initiated by a reduced number of individuals, which may be genetically diverse and even belong to different assemblages, and (iii) parasites reproduce clonally within the host. However, the observation of invariant loci in some isolates means that mechanisms for the homogenization of the genetic content of the two diploid nuclei in each individual must exist.

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.

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