Identification of zoonotic genotypes of Giardia duodenalis

Abstract

Giardia duodenalis, originally regarded as a commensal organism, is the etiologic agent of giardiasis, a gastrointestinal disease of humans and animals. Giardiasis causes major public and veterinary health concerns worldwide. Transmission is either direct, through the faecal-oral route, or indirect, through ingestion of contaminated water or food. Genetic characterization of G. duodenalis isolates has revealed the existence of seven groups (assemblages A to G) which differ in their host distribution. Assemblages A and B are found in humans and in many other mammals, but the role of animals in the epidemiology of human infection is still unclear, despite the fact that the zoonotic potential of Giardia was recognised by the WHO some 30 years ago. Here, we performed an extensive genetic characterization of 978 human and 1440 animal isolates, which together comprise 3886 sequences from 4 genetic loci. The data were assembled into a molecular epidemiological database developed by a European network of public and veterinary health Institutions. Genotyping was performed at different levels of resolution (single and multiple loci on the same dataset). The zoonotic potential of both assemblages A and B is evident when studied at the level of assemblages, sub-assemblages, and even at each single locus. However, when genotypes are defined using a multi-locus sequence typing scheme, only 2 multi-locus genotypes (MLG) of assemblage A and none of assemblage B appear to have a zoonotic potential. Surprisingly, mixtures of genotypes in individual isolates were repeatedly observed. Possible explanations are the uptake of genetically different Giardia cysts by a host, or subsequent infection of an already infected host, likely without overt symptoms, with a different Giardia species, which may cause disease. Other explanations for mixed genotypes, particularly for assemblage B, are substantial allelic sequence heterogeneity and/or genetic recombination. Although the zoonotic potential of G. duodenalis is evident, evidence on the contribution and frequency is (still) lacking. This newly developed molecular database has the potential to tackle intricate epidemiological questions concerning protozoan diseases.

Figure 1. Phylogenetic analysis of assemblage A.

Phylogenetic trees of 84 isolates with 3 markers were inferred using Unweighted Pair Group Method with Arithmetic mean, corrected by complete linkage, which uses the lowest similarities found between two clusters. Individual and merged BG, GDH and TPI nucleotide sequences were used. Bootstrap values were calculated by the analysis of 1000 replicates. Only bootstrap values >60 are shown. The phylogenetic analysis of assemblage A shows that the three sub-assemblages clustered together with high bootstrap support (i.e., they are monophyletic). The genetic diversity of the multi-locus genotypes (isolates/subtypes: 9,3) is relatively low (see Table 13), and the maximum genetic distance is 4,0%.

Figure 2. Phylogenetic analysis of assemblage B.

Phylogenetic trees of 65 isolates of assemblage B with 3 markers were constructed using Unweighted Pair Group Method with Arithmetic mean, corrected by complete linkage, which uses the lowest similarities found between two clusters. Individual and merged BG, GDH and TPI nucleotide sequences were used. Bootstrap values were calculated by the analysis of 1000 replicates. Only bootstrap values >60 are shown. The phylogenetic analysis of the merged sequences shows significant mixtures of the BIII and BIV sub-assemblages. The genetic diversity of the multi-locus genotypes (isolates/subtypes: 2,1) is relatively high (see Table 13), and the maximum genetic distance is 1,7%.