Computational protocol: Intestinal spirochaetes (genus Brachyspira) colonise wild birds in the southern Atlantic region and Antarctica

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Protocol publication

[…] Boiled bacterial lysates were prepared from washed bacterial cells of the 14 studied isolates () and were used as templates at 1:1 and 1:10 dilution for polymerase chain reaction (PCR) detection of the tlyA gene of B. hyodysenteriae (), the 16S rRNA gene of B. pilosicoli (, ), the nox gene of B. intermedia (, ), and the nox gene of B. innocens and/or murdochii () with some modifications as described previously (). Appropriate negative and positive controls of porcine and avian origin and water were included. All PCR reactions were duplicated. To obtain the near-complete 16S rRNA (1,433–1,434 nts) and partial nox (893 nts) and tlyA (487 nts) sequences, PCR, DNA purification, and sequencing were performed as previously described (, –). The data were edited with the CLC Main Workbench version 6.8.3 (CLC bio, www.clcbio.com/index.php?id=28). The selection of strains used for comparative phylogeny and sequence analyses was based on NCBI GenBank nBLAST results (April 2015) and downloaded. For ‘B. hampsonii’, sequences were retrieved from the available contigs by blasting the B. hyodysenteriae WA1 16S rRNA and nox gene sequences against them. Accession numbers to sequence data downloaded from GenBank are given in . All sequences were aligned using MUSCLE () and conserved blocks were selected using Gblocks () with default parameters. Phylogenetic inference was performed using the maximum-likelihood method and the general time reversible model for the 16S rRNA gene, and the Jukes–Cantor model for the nox gene. Analyses were performed in MEGA6 (). Bootstrap analyses were performed using 1,000 replicates ( and ). […]

Pipeline specifications

Software tools CLC Main Workbench, CLC Assembly Cell, MUSCLE, Gblocks, MEGA
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Brachyspira intermedia, Homo sapiens, Gallus gallus, Brachyspira murdochii
Diseases Colonic Diseases
Chemicals Doxycycline, Lincomycin, NAD, Tylosin