The secondary PCR products were purified by using Microcon PCR Centrifugal Filter Devices (Millipore Corp., Bedford, MA) and sequenced on an ABI 3100 automated sequencer by using the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer). Sequence accuracy was confirmed by two-directional sequencing of two separate PCR products. Multiple alignment of the nucleotide sequences was performed by using Wisconsin Package Version 9.0 program (18 ). A phylogenetic analysis was performed on the aligned sequences to assess the extent of genetic diversity within G. duodenalis parasites as well as their evolutionary relationships with other Giardia species. In this analysis, published TPI nucleotide sequences representing G. duodenalis (from humans, cattle, cat, dog, muskrat, pig, and rat), G. muris, and G. ardeae were aligned with TPI sequences of Giardia parasites obtained in this study.
A neighbor-joining tree (19 (link)) was constructed on the basis of the evolutionary distances calculated by the Kimura-2-parameter model using the TreeconW program (20 (link)). A sequence of G. ardeae (GenBank accession no. AF069564) was used as the outgroup since the construction of an unrooted tree showed it to be the most divergent member under analysis. The reliability of these trees was assessed by using the bootstrap method (21 (link)) with 1,000 pseudoreplicates; values >70% were reported (22 (link)). Nucleotide sequences of the TPI gene of G. duodenalis from humans, cattle, dogs, muskrat, rat, and rabbit, representing different genotypes, were deposited in GenBank under accession numbers AY228628 to AY228649.
A similar phylogenetic analysis was carried out on the nucleotide sequences of the SSU rRNA gene from G. microti in muskrats. SSU rRNA nucleotide sequences were deposited in GenBank under accession numbers AY228332 and AY228333.
A neighbor-joining tree (19 (link)) was constructed on the basis of the evolutionary distances calculated by the Kimura-2-parameter model using the TreeconW program (20 (link)). A sequence of G. ardeae (GenBank accession no. AF069564) was used as the outgroup since the construction of an unrooted tree showed it to be the most divergent member under analysis. The reliability of these trees was assessed by using the bootstrap method (21 (link)) with 1,000 pseudoreplicates; values >70% were reported (22 (link)). Nucleotide sequences of the TPI gene of G. duodenalis from humans, cattle, dogs, muskrat, rat, and rabbit, representing different genotypes, were deposited in GenBank under accession numbers AY228628 to AY228649.
A similar phylogenetic analysis was carried out on the nucleotide sequences of the SSU rRNA gene from G. microti in muskrats. SSU rRNA nucleotide sequences were deposited in GenBank under accession numbers AY228332 and AY228333.
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