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

Abstract

Background Giardia duodenalis, a single-celled intestinal parasite, is divided into eight assemblages (A-H), with differences in host specificity. Giardia duodenalis reproduces asexually and cycles between the binucleated trophozoite (4 N) and the infectious cyst with four nuclei (16 N). Interaction between the nuclei is limited. Therefore, genetic drift causes differences in genetic make-up between the non-daughter nuclei; the allelic sequence heterozygosity (ASH). The ASH is low (0.01%—0.0023%) for the related assemblages A and E, higher (0.43–0.53) for assemblage B and much higher (0.74% -0.89%) for the assemblage C and D at the root of the phylogenetic tree. The heterozygosity in assemblage F, in the same clade as assemblage A and E, was unknown. The heterozygosity in the sequences of the gdh and dis3 genes was used as proxy for the ASH and whole genome amplification of single cysts followed by cloning and Sanger sequencing of dis3 fragment could reveal the genetic variation within the cyst. The aim of the study was to determine the level of heterozygosity within pooled and single cysts of different assemblages. Results The heterozygosity in gdh and dis3 was determined in pooled cysts of the assemblages A to F. Heterozygosity in the isolates of the assemblages C (n = 2) and D (n = 1) ranged from 0.41% to 0.82% for gdh and dis3 and no heterozygosity was found in the isolates of the assemblages A (n = 4), E (n = 3) and F (n = 3). The heterozygosity in assemblage B (n = 7) was intermediate (0% to 0.62%). Next, the number of haplotypes of dis3 was determined for single cysts of assemblages C, D and E. In the assemblages C and D, two to four haplotypes were found per cyst, while in assemblage E only one haplotype was identified. Conclusions Having high heterozygosity is characteristic for the assemblages C and D, while having a low heterozygosity is characteristic for the clade with the assemblages A, E and F. Presence of more than 1 haplotype per cyst in assemblage C and D suggests differences between the non-daughter nuclei, in contrast to the one haplotype in assemblage E.

Cryptosporidium and Giardia in cats and dogs: What is the real zoonotic risk?

“…Many of the typing studies conducted to date on Giardia in cats and dogs are problematic due to (i) high levels of mixed infections with multiple Giardia assemblages, (ii) the use of only one locus or lack of agreement between multiple loci, and (iii) the high levels of allelic sequence hetrerozygosity (ASH) in assemblages C and D, which make assemblage assignment unreliable ( Morrison et al, 2007 ; Franzén et al, 2009 ; Kooyman et al, 2019 ; Veldhuis et al, 2022 ; Klotz et al, 2023 ). Therefore, it is difficult to draw firm conclusions on the zoonotic potential of Giardia infections in companion animals.…”