Giardia peramelis molecular

Hillman, Alison, Ash, Amanda, Elliot, Aileen, Lymbery, Alan, Perez, Catherine & Thompson, R. C. Andrew, 2016, Confirmation of a unique species of Giardia, parasitic in the quenda (Isoodon obesulus), International Journal for Parasitology: Parasites and Wildlife 5 (1), pp. 110-115 : 112

3.2. Giardia peramelis molecular characterisation

Of the 111 individual quenda faecal samples found positive for Giardia spp. by immunofluorescence microscopy or PCR, 75 (67.6%) were successfully amplified and sequenced at one or more loci. Sixty-four samples were sequenced at the 18s rRNA locus, 50 samples sequenced at ITS1-5.8s-ITS2 and two samples sequenced at the gdh locus ( Table 1). Two of the 75 samples sequenced at all three loci, 37 samples sequenced at both 18S and ITS1-5.8s-ITS2, and the remaining 36 samples sequenced at only one locus ( Table 2).

Of the 116 sequences obtained, 114 were the novel G. peramelis ; two sequences obtained at the 18S rRNA locus were suspected to be mixed infections of Giardia duodenalis / G. peramelis and Giardia canis / G. peramelis . These ‘mixed infection’ samples were also amplified at the ITS1-5.8s-ITS2 region locus-the resultant sequences were clearly identified as G. peramelis , with no ambiguous nucleotides evident.

Utilizing the BLAST tool within NCBI, the 18S rRNA sequences were highly similar to the published sequence AY309064, previously reported as the ‘quenda genotype’. Alignment in Sequencher™ revealed one single nucleotide polymorphism (SNP) between sequences obtained in this study (represented by QBY95 and QM22) and AY309064. In addition, one SNP was identified within a small subset of sequences obtained in this study, represented by sequence QBN13. A large region of extreme variability was identified between all sequences and another reported ‘quenda genotype’. Of the 292 bp published for this sequence (accession number HQ398319), a region of approximately 150 bp showed extreme mismatch.

Sequences obtained at ITS1-5.8S-ITS2 required the BLAST program selection of ‘somewhat similar sequences’ in order to achieve a result. The ‘ G. duodenalis species complex’ was the primary match, but achieved low coverage and identities with all comparisons. Alignment of these sequences in Sequencher™ revealed one SNP, represented by samples QBY95/QM22 and QBN13. Similar BLAST results were obtained with the two gdh sequences.

3.2.1. Phylogenetic analysis Very similar trees were obtained by neighbour-joining, maximum likelihood and maximum parsimony methods; only the neighbour-joining trees are presented here. Phylogenetic analysis of sequence data obtained at 18s rRNA confirmed that the Giardia genotype obtained from quenda in this study ( G. peramelis ) formed a separate clade with the ‘quenda genotype’ reference AY309064, and was distinct from all assemblages within the ‘ G. duodenalis species complex’ and G. microti ( Fig. 3 ). A similar topology was observed based on genetic data obtained at ITS1-5.8S-ITS2, with the exception that G. peramelis was also placed external to G. ardeae ( Fig. 4 ). Phylogenetic analysis of gdh also placed the G. peramelis external to all assemblages within the ‘ G. duodenalis species complex’ (results not shown).

Figures

Fig. 3. Phylogenetic relationships of Giardia peramelis isolates obtained in this study (quenda QBN13, QM22, QBY95) with published reference material available at the 18s rRNA locus. Evolutionary history inferred using the neighbour-joining method supported with bootstrap test of 1000 replicates (values> 50% shown). G. muris is used as the out group.

Fig. 4. Phylogenetic relationships of Giardia peramelis isolates obtained in this study (quenda QBN13, QM22, QBY95) with published reference material available at the ITS1-5.8S- ITS2 locus. Evolutionary history inferred using the neighbour-joining method supported with bootstrap test of 1000 replicates (values> 50% shown). G. muris is used as the out group.