Giardia duodenalis (syn. G. lamblia G. intestinalis) is the etiological agent of giardiasis, a gastrointestinal infection of humans, companion animals, livestock and wildlife. Symptoms of a G. duodenalis infection range from asymptomatic to severe diarrhea as well as chronic disease . G. duodenalis has a simple life cycle comprising rapidly multiplying, non-invasive trophozoites on the mucosal surface of the intestine, and the production of environmentally resistant cysts that are shed with the host faces. Infectious cysts are transmitted by the faecal-oral route either by direct contact or by ingestion of contaminated food or water . G. duodenalis is considered as a species complex, whose members show little variation in their morphology, yet can be assigned to eight distinct assemblages (A to H) based on enzyme electrophoretic and genetic studies [3, 4]. Assemblages A and B can infect and multiply in humans and are also found in a wide range of mammals. The remaining assemblages show more restricted host ranges: C and D are predominantly found in canids, E in livestock, F in cats, G in rodents and H in marine vertebrates (seal and gull) [5, 6]. In endemic areas where humans and animals live closely together, transmission from human to animals or vice versa may occur [7–9]. Direct evidence for transmission from animals to human is lacking, because Giardia cysts are shed into the environment, making it very difficult to determine the primary source of the infection. Genetic characterization has been extensively used to assess the role of animals in the epidemiology of human infection and to develop tools for tracing sources of infection. However, the zoonotic potential of G. duodenalis remains a major and unresolved issue [1, 10, 11].
Many molecular epidemiological studies have been based on the analysis of a single marker, often from a limited number of isolates. Using single locus approaches, the zoonotic potential of G. duodenalis assemblage A and B appears to be high: Irrespective of the genetic marker used, sequences from human and animal field isolates frequently appeared similar, if not identical . In order to increase the accuracy of genotyping of G. duodenalis isolates, multi-locus sequence typing strategies were introduced [13–18]. When genotypes from field isolates were defined using a multi-locus sequence typing scheme, only 2 from the 84 multi-locus genotypes (MLG) of assemblage A and none MLGs (n = 99) of assemblage B appear to have a zoonotic potential . Surprisingly, the genotypes of Giardia field isolates repeatedly constituted a combination of loci derived from different assemblages . The latter finding can be explained by two biological phenomena.
One explanation is that a Giardia field isolate is not a singular clone, but consists of a mixture of different Giardia genotypes. A Giardia field isolate is often not more than a DNA extract, obtained either directly from a stool sample or indirectly after (immune-) isolation of faecal cysts. For this situation, the uptake of genetically different Giardia cysts from the environment by a host, or subsequent infection of an already infected host, likely without overt symptoms, with a different Giardia genotype, must occur. As starting in vitro cultures from field samples prove to be very difficult due to variations in excitation and adaptation, and the ever-present bacterial and fungal contamination, it is hard to affirm the clonality of Giardia field isolates [19, 20]. Alternatively, the Giardia isolates are clonal, but the mixing of loci from different assemblages have arisen by (para) sexual recombination, i.e. genetic exchange, between G. duodenalis assemblages. Although G. duodenalis shows no cytological evidence of meiotic and sexual recombination, several studies challenged the idea that G. duodenalis is a strictly clonal organism [21, 22]. These studies have demonstrated: (i) the presence in the G. duodenalis genome of true homologs of genes involved in meiosis in other eukaryotes [23, 24]; (ii) the exchange of genetic material in different chromosomal regions among human isolates of the parasite [25, 26]; (iii) the fusion between cyst nuclei (karyogamy) and the transfer of genetic material (episomal plasmids) between them . These results are pivotal for the existence of sexual recombination. Recombination may take place at three levels within G. duodenalis: (i) between the two nuclei at an individual level, (ii) between individuals of the same assemblage and (iii) between individuals of different assemblages.
Here, we only address the latter situation, i.e. recombination between assemblages, as this mostly complicates the interpretation of the molecular epidemiological data. Genetic exchange between different assemblages may occur in laboratory cultures or in nature, but it remains to be determined to what extent this occurs in natural populations . Genetic exchange between isolates of different assemblages was addressed using two approaches: The detection of mosaic sequences in three loci and the performance of several tests for clonal reproduction at the population level.