Whole genome sequencing of dog specific assemblages C and D of Giardia duodenalis from single and pooled cysts indicates host associated genes

Abstract

Giardia duodenalis (Syn. G. intestinalis or G. lamblia) infects over 280 million people each year and numerous animals. G. duodenalis can be subdivided into 8 assemblages with different host specificity. Unculturable assemblages have so far resisted genome sequencing efforts. In this study we isolated single and pooled cysts of assemblage C and D from dog faeces by FACS and sequenced them using multiple displacement amplification and Illumina paired end sequencing. The genomes of assemblages C and D were compared with genomes of assemblages A and B from humans and assemblage E from ruminants and pigs. The genomes obtained from the pooled cysts and from the single cysts were considered complete (>99% marker genes observed) and the allelic sequence heterozygosity (ASH) of assemblage C and D was 0.89% and 0.74%, respectively. Higher than for assemblage B (> 0.43%) and much higher than for assemblages A and E (<0.01%). The flavohemoglobin and 4Fe-4S binding domain family gene involved in O2 and NO detoxification were only present in assemblages A, B and E. Cathepsin-B orthologs were found in all genomes. Six clades of cathepsin-B orthologs contained one gene of each genome, while in three clades not all assemblages were represented. We conclude that whole genome sequencing from a single Giardia cyst results in complete draft genomes making the genomes of unculturable Giardia assemblages accessible. Observed differences between the genomes of assemblage C and D on one hand and the assemblages A, B and E on the other hand are possibly associated with host specificity.

A chromosome-scale reference genome for Giardia intestinalis WB

“…Since then, this genome has been used as the reference genome for Giardia research. Subsequently, a few other Giardia genomes have been published, including human isolates (GS, GS-B and BAH15c1) from assemblage B [3][4][5] , human isolates (DH, AS175 and AS98) from assemblage A2 4,6 , one pig isolate (P15) from assemblage E 7 and two dog isolates from assemblages C and D 8 . The availability of several genomes has advanced Giardia research and it is now possible to use different omics techniques during studies in vitro and in vivo.…”

Variation in haplotypes in single cysts of assemblages C and D, but not of assemblage E of Giardia duodenalis

“…All cysts were isolated from fecal samples from various animal species that were sent for diagnostic purposes to the Veterinary Microbiological Diagnostic Center (VMDC) (Table S 1 ). Cysts were isolated and stored in 70% ethanol at 4 °C as described [ 9 ].…”

“…This variation has been determined for various assemblages based on whole genome sequence data and is called the allelic sequence heterozygosity (ASH) [ 6 ]. The ASH in the assemblages varies over a wide range; with (sub)assemblages AI, AII and E showing low ASH rates: < 0.01%, 0.037% and 0.0023%, respectively, in contrast to assemblages B, C and D having a high ASH: 0.53%, 0.89% and 0.74%, respectively [ 5 – 9 ]. The ASH in assemblages C and D is based on single cysts, in contrast to assemblage A, B and E which have been determined based on pools of trophozoites grown in axenic culture.…”

“…Other studies found also signs of intra and inter-assemblage recombination in genome sequences [ 3 , 13 ]. When looking at the phylogenetic tree based on the whole genome, assemblages A and E with a low ASH, diverge late in the evolutionary timeline [ 9 ]. The genome of assemblage F is not known, but based on a single locus (glutamate dehydrogenase, gdh ), it groups together with assemblages A and E [ 14 ].…”

“…If true, we hypothesized that having a low ASH is a shared, derived character of the monophyletic clade containing the assemblages A, E and F. 2) Estimating the variation and number of haplotypes within single cysts of the assemblages C and D with a high ASH by cloning and Sanger sequencing of the single copy gene dis3 . For this, the same cysts from which the whole genome is published are used [ 9 ]. Because of the 4 N character of the binucleated Giardia , we hypothesized that there will be no more than four haplotypes of the dis3 gene within a single cyst.…”