Computational protocol: Redescription of Cercopithifilaria bainae Almeida & Vicente, 1984 (Spirurida, Onchocercidae) from a dog in Sardinia, Italy

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Protocol publication

[…] Microfilariae (n = 5) and adult nematodes (n = 8; 4 females and 4 males) were isolated, genomic DNA was extracted using a commercial kit (DNeasy Blood & Tissue Kit, Qiagen, GmbH, Hilden, Germany). A cox1 (~689 bp) and 12S (~330 bp) gene fragment, were amplified as previously reported [] and the phylogenetic analysis carried out on a partial cox1 (pcox1; 304 bp) fragment of Cercopithifilaria spp. []. Following purification (Ultrafree-DA columns; Amicon, Millipore; Bedford, USA), amplicons were sequenced directly using the Taq DyeDeoxyTerminator Cycle Sequencing Kit (v.2, Applied Biosystems) in an automated sequencer (ABI-PRISM 377). Sequences were determined from both strands (using the same primers individually as for the PCR) and the electropherograms verified by eye. Following alignment using ClustalW program [], sequences were compared with those available in GenBank™ dataset by Basic Local Alignment Search Tool (BLAST - http://blast.ncbi.nlm.nih.gov/Blast.cgi).To investigate the relationships among filarioids of the Onchocercidae family, sequences of both genes were phylogenetically analysed with those available in GenBank™. Selected haplotypes of pcox1 and 12S previously sequenced for Cercopithifilaria sp. I (accession numbers: JF461457; JF461461), Cercopithifilaria sp. II (accession numbers: JQ837809, JQ837811) and C. grassii (accession numbers: JQ837810, JQ837812) were included in the analysis. The evolutionary history was carried out using the Neighbour-joining (NJ) method [] using the Tajima-Nei model [] and Maximum Likelihood method based on the Kimura 2-parameter model []. The evolutionary distances were computed by MEGA5 software []. The bootstrap consensus trees inferred from over 8,000 replicates were taken to represent the evolutionary history of the taxa analysed []. Dirofilaria immitis was chosen as an out-group (accession numbers: DQ358815). A bootstrap support of 50 was considered significant. The nucleotide sequences analysed in this study are available in the GenBank™ (accession numbers: KC880117, KC880118). […]

Pipeline specifications

Software tools Clustal W, BLASTN, MEGA
Application Phylogenetics
Organisms Canis lupus familiaris, Caenorhabditis elegans