Giardia peramelis subsp. 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 : 111-112

2.4. G. peramelis molecular characterisation

2.4.1. DNA extraction and PCR ampli fi cation

Amplification by PCR was attempted on all immunofluorescence microscopy positive faecal samples and the ten randomly selected immunofluorescence negative samples. Ethanol-preserved faecal samples were centrifuged to separate ethanol from faeces, with the ethanol supernatant discarded. Samples were then twice rehomogenised in distilled water, centrifuged and supernatant discarded. DNA extraction was then conducted using the Maxwell ® 16 Instrument (Promega, Madison, USA) as per manufacturer's instruction.

Amplification by PCR was attempted at three loci: 18S rRNA, ITS1-5.8s-ITS2 and glutamate dehydrogenase (gdh). Initially, a semi-nested PCR protocol was employed to amplify a 130 bp product of the 18s rRNA, with primers RH11/RH4 and RH11/GiarR ( Hopkins et al., 1997; Read et al., 2002). The PCR reaction was performed in 25 M l volumes, consisting of 1 ‾ 2 M l of extracted DNA, 2.0 mM MgCl 2, 1 × reaction buffer (20 mM Tris-HCL, pH 8.5 at 25 ǫC, 50 mM KCl), 400 M M of each dNTP, 0.4 M M of each primer, 0.5 units of Taq DNA polymerase (Fisher Biotec, Perth, Australia), and DMSO 5%. Amplification conditions were modified from Hopkins et al. (1997), and involved a denaturing step of 95 ǫC for 6 min, then 40 cycles of 95 ǫC for 30 s, 53 ǫC for 30 s (56 ǫC in the secondary round) and 72 ǫC for 30 s, followed by a final extension of 72 ǫC for 7 min.

A nested PCR protocol was conducted to amplify a 330 bp product of the ITS1-5.8S-ITS2 region of the ribosomal gene, with primers developed by Caccio et al. (2010). The PCR reactions were the same as those used for 18s rRNA, but performed in 50 M l volumes. Conditions for amplifications were modified from Caccio et al. (2010), and involved an initial denaturing step of 95 ǫC for 5 min, then 40 cycles of 95 ǫC for 45 s, 59 ǫC for 30 s and 72 ǫC for 30 s, followed by a final extension of 72 ǫC for 7 min.

Finally, for gdh, a nested PCR protocol was used to amplify a 530 bp product, using the primer pairs Gdh1/Gdh2 and Gdh3/Gdh4 for the primary and secondary rounds respectively, as per Caccio and Ryan (2008). The PCR reaction was performed in 25 M l volumes, consisting of 2 M l of extracted DNA, 1.5 mM MgCl 2, 1 × reaction buffer, 200 M M of each dNTP, 0.4 M M of each primer, 1 unit of Taq DNA polymerase (Fisher Biotec, Perth, Australia) and DMSO 5%. Conditions for amplifications were the same for both rounds, and involved an initial denaturing step of 94 ǫC for 2 min, then 35 cycles of 94 ǫC for 30 s, 50 ǫC for 30 s and 72 ǫC for 60 s, followed by a final extension of 72 ǫC for 7 min.

2.4.2. Sequencing of ampli fi ed product

PCR products were purified using an Agencourt AMPure XP system (Beckman coulter, Beverly, USA). Sequence reactions were performed using the Big Dye Terminator Version 3.1 cycle sequencing kit (Applied Biosystems), according to the manufacturer's instructions. Reactions were electrophoresed on an ABI 3730 48 capillary machine. Amplicons were sequenced in both directions, with resultant nucleotide sequences compared with published sequences on NCBI GenBank ® using the basic alignment search tool (BLAST). Further sequence analysis was conducted using the sequence alignment program Sequencher™ 4.8 (Gene Codes, Ann Arbour, MI, USA).

Nucleotide sequence data reported in this paper are available in the NCBI GenBank ® database under accession numbers KU306911, KU306912, KU306913, KU306914 and KU306915.

2.4.3. Phylogenetic analyses

Phylogenetic analyses of sequences obtained in this study were conducted using the programme MEGA6 ( Tamura et al., 2013). Phylogenetic trees were inferred with the neighbour-joining method, with a bootstrapping of 1000 replicates. In addition, analyses were conducted using the maximum likelihood and maximum parsimony methods. Evolutionary distances were calculated using the Kimura 2-parameter method ( Kimura, 1980). Published sequences representing Giardia muris , Giardia ardeae , Giardia microti and all assemblages within the ‘ Giardia duodenalis species complex’ ( G. duodenalis , Giardia enterica , G. canis , Giardia bovis , G. cati , Giardia simondi and the ‘pinniped genotype’) were retrieved from GenBank ® (accession numbers for 18S rRNA: X65063, Z17120, AF006676, X52949, AF199446, DQ100287, AF199447, U09491, AF199443, AF199449, AF199448, AF199444, AF199450 and AY309064; accession numbers for ITS1-5.8s-ITS2: GU126450, X65063, X58290, M73684, GU124448, X52949, GU126432/33/34/35, AF239840, U09491, GU126437/38/40/43/44/ 45).