Giardia duodenalis (syn. G. intestinalis, G. lamblia) is a widespread gastrointestinal protozoan parasite with debated taxonomic status. Currently, eight distinct genetic sub-groups, termed assemblages A-H, are defined based on a few genetic markers. Assemblages A and B may represent distinct species and are both of human public health relevance. Genomic studies are scarce and the few reference genomes available, in particular for assemblage B, are insufficient for adequate comparative genomics. Here, by combining long- and short-read sequences generated by PacBio and Illumina sequencing technologies, we provide nine annotated genome sequences for reference from new clinical isolates (four assemblage A and five assemblage B parasite isolates). Isolates chosen represent the currently accepted classification of sub-assemblages AI, AII, BIII and BIV. Synteny over the whole genome was generally high, but we report chromosome-level translocations as a feature that distinguishes assemblage A from B parasites. Orthologue gene group analysis was used to define gene content differences between assemblage A and B and to contribute a gene-set-based operational definition of respective taxonomic units. Giardia is tetraploid, and high allelic sequence heterogeneity (ASH) for assemblage B vs. assemblage A has been observed so far. Noteworthy, here we report an extremely low ASH (0.002%) for one of the assemblage B isolates (a value even lower than the reference assemblage A isolate WB-C6). This challenges the view of low ASH being a notable feature that distinguishes assemblage A from B parasites, and low ASH allowed assembly of the most contiguous assemblage B genome currently available for reference. In conclusion, the description of nine highly contiguous genome assemblies of new isolates of G. duodenalis assemblage A and B adds to our understanding of the genomics and species population structure of this widespread zoonotic parasite.
- MeSH
- genomika MeSH
- Giardia lamblia * genetika MeSH
- Giardia genetika MeSH
- giardiáza * parazitologie MeSH
- lidé MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The availability of high quality genomic DNA in sufficient amounts to perform Next Generation Sequencing (NGS) experiments is challenging for pathogens that cannot be cultivated in vitro, as is the case for many parasites. Therefore, Whole Genome Amplification (WGA) of genomic DNA is used to overcome this limitation. In this study, we evaluated the effect of WGA using the intestinal flagellated protozoan Giardia duodenalis as a model, due to its genome compactness (12 Mb), the presence of two diploid nuclei with variable levels of allelic sequence heterogeneity (ASH), and the availability of reference genomes. We selected one isolate (ZX15) belonging to the same genetic group of the reference isolate WB, namely Assemblage A, sub-Assemblage AI. Genomic DNA from the ZX15 isolate (GEN dataset) and that obtained by WGA of 1 ng of the same genomic DNA (WGA dataset) were sequenced on a HiSeq Illumina platform. Trimmed reads from the GEN and WGA experiments were mapped against the WB reference genome, showing the presence of a very small number of mutations (846 and 752, respectively). The difference in the number of mutations is largely accounted by local variation in coverage and not by bias introduced by WGA. No significant difference were observed in the distribution of mutations in coding and non-coding regions, in the proportion of heterozygous mutations (ASH), or in the transition/transversion ratio of Single Nucleotide Variants within coding sequences. We conclude that the quantitative and qualitative impact of WGA on the identification of mutations is limited, and that this technique can be used to conduct comparative genomics studies.
- MeSH
- celogenomová asociační studie MeSH
- Giardia lamblia genetika MeSH
- giardiáza parazitologie MeSH
- lidé MeSH
- mutace MeSH
- otevřené čtecí rámce genetika MeSH
- předškolní dítě MeSH
- protozoální DNA chemie genetika izolace a purifikace MeSH
- strukturální variace genomu MeSH
- techniky amplifikace nukleových kyselin MeSH
- výpočetní biologie MeSH
- Check Tag
- lidé MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
Giardiasis is a common gastrointestinal infection of humans and animals with a worldwide distribution. Eight genetic groups (known as assemblages A to H) are currently recognised within the species complex of Giardia duodenalis (Lambl, 1859), of which assemblages A and B are responsible for infection of humans and other mammalian hosts. Genotyping data on giardiasis are not available from Slovenia. In this work, we have characterised isolates of G. duodenalis from 85 human symptomatic cases collected during 2002-2013. Genomic DNAs were first tested by a real-time (rt) PCR assay and then by conventional PCR at three loci (beta-giardin, bg; triose phosphate isomerase, tpi; and glutamate dehydrogenase, gdh). We found that the threshold cycle (Ct) values in rt-PCR testing were higher for samples collected during 2002-2005 and that this was paralleled by a low amplification rate in conventional PCR (6 of 32, i.e. 19%). In contrast, lower Ct values and higher amplification rate (45 of 53; 85%) were observed for samples collected during 2006-2013, suggesting an adverse effect of prolonged freezing of stools. Assemblages A and B were found with an almost identical frequency in the 51 genotyped samples. In agreement with previous studies, sequences from assemblage B isolates were characterised by larger genetic variability and by the presence of heterogeneous positions, which made assignment to specific genotypes difficult. Less variability was observed in sequences from assemblage A isolates, which belonged to the human-specific subassemblage AII. These data showed that the genotypes of G. duodenalis that circulate in humans in Slovenia are similar to those previously identified in Europe.
- MeSH
- diagnostické techniky molekulární MeSH
- dospělí MeSH
- fylogeneze MeSH
- genotyp MeSH
- genotypizační techniky * MeSH
- Giardia lamblia * genetika izolace a purifikace patogenita MeSH
- giardiáza epidemiologie etiologie genetika parazitologie MeSH
- lidé MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- práce podpořená grantem MeSH
- Geografické názvy
- Slovinsko MeSH
Faecal samples from 162 wild animals were collected from 32 distinct sites of Łęczyńsko-Włodawskie Lakeland (eastern Poland). The presence of Giardia duodenalis (Stiles, 1902) was assessed by a Direct Fluorescence Assay (DFA) and by Polymerase Chain Reaction (PCR) and sequencing of a fragment of the beta-giardin gene. DFA showed the presence of cysts of G. duodenalis in 12 of 162 faecal samples (7%), namely in four wild boars (15%), four foxes (19%), two roe deer (4%), and two wolves (29%). PCR identified 34 of the 162 (21%) samples as positive, including 11 wild boars (41%), five red deer (18%), 11 roe deer (23%), four moose (17%), two wolves (29%) and a single sample from the European badger. Thus, PCR detected a significantly higher number of infection than DFA (P = 0.0005). However, 14 of 34 PCR products could not be sequenced because of their insufficient amount; the low number of cysts, poor conservation of the faeces or presence of PCR inhibitors may have contributed to weak DNA amplification. Sequence analysis of the remaining 20 products showed the presence of assemblage B in wild boars, red deer and roe deer, whereas samples from wolves were identified as assemblage D. This is the first detection of assemblage B in wild boars and deer. As assemblage B has zoonotic potential, wild animals from eastern Poland may act as reservoirs of cysts of G. duodenalis infectious for humans.
- MeSH
- divoká zvířata klasifikace parazitologie MeSH
- feces parazitologie MeSH
- fluorescenční protilátková technika přímá statistika a číselné údaje MeSH
- Giardia lamblia * cytologie patogenita MeSH
- giardiáza epidemiologie etiologie MeSH
- polymerázová řetězová reakce statistika a číselné údaje MeSH
- sekvenční analýza statistika a číselné údaje MeSH
- statistika jako téma MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Polsko MeSH
