Development and testing of new multi-locus sequence typing procedures for potentially zoonotic Giardia duodenalis genotypes

Abstract text

Introduction

Giardia duodenalis represents a species complex of 8 genetically distinguishable assemblages (A-H) with distinct host preferences. The two assemblages A and B are relevant to humans and are also found in a broad range of various mammals. Adequate procedures for molecular typing are only insufficiently developed to address epidemiological questions. Typing schemes require improvement of new marker genes allowing higher typing resolution, and need to account for allelic sequence heterozygocity (ASH) that can be a consequence of the tetraploid nature of the parasite or possible mixed infections.

Objectives

The aim was to test or develop new multi-locus sequence typing (MLST) procedures for assemblage A and B, respectively, as separate approaches are necessary for both assemblages.

Materials & methods

For assemblage B, based on newly generated genomes we developed a new MLST scheme comprising 7 genomic marker sequences. Nested PCR protocols were designed and the new procedure was evaluated on > 80 assemblage B isolates from various origins. For assemblage A, a recently published MLST procedure based on 6 marker genes was evaluated using > 70 human and animal derived isolates.

Results

The new assemblage B MLST scheme confirmed ASH as a hallmark of assemblage B, which occurred in approximately 70% of the isolates and further revealed a subpopulation of assemblage B parasites exhibiting very low to no ASH. Sequence analysis showed that these subpopulations clustered separately. Strikingly, sequences of isolates from an assemblage B outbreak clearly formed a sub-cluster within the high ASH cluster with significantly lower inter se SNP variation compared to non-outbreak high ASH cluster isolates.

The MLST scheme for assemblage A confirmed largely the lack of ASH in this assemblage and showed that sub-assemblage AII comprises only isolates from humans. All isolates from animals and a few from humans belonged to sub-assemblage AI. The discriminatory power within sub-assemblage AII was sufficient to group isolates from two separate outbreaks as distinct MLSTs.

Conclusion

Different typing protocols for assemblage A and B are necessary to retrieve informative MLST data for epidemiological purposes. The evaluation of relatedness of isolates based on SNPs depends on their respective subpopulation assignments. Both protocols will help to foster the generation of meaningful typing data much needed for future epidemiological studies.