Computational protocol: Genotyping of Burkholderia mallei from an Outbreak of Glanders in Bahrain Suggests Multiple Introduction Events

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Protocol publication

[…] Glanders is a notifiable disease to the World Organization for Animal Health (OIE). As the official OIE reference laboratory for glanders in the Arabic region, the Central Veterinary Laboratory (CVRL) in Dubai is the officially authorized institution for glanders research, surveillance and eradication. All procedures involving animals were performed in strict accordance with the OIE guidelines for animal welfare using prescribed protocols.An ethic commission comprising 4 veterinarians of the Central Veterinary Research Laboratory (CVRL) and a government veterinarian from the Ministry of Environment and Water of the UAE followed the Ministerial Decree No 384 of the year 2008 on the executive by-law of the Federal Law No 16 of the year 2007 concerning Animal Welfare. All experimental animals and treatment in this study were reviewed and approved by the Animal Ethic Committee of CVRL, and Ministry of Environment and Water of the UAE (Permit Number: 550353).Isolation of the strains, DNA extraction, and MLVA were performed as described previously (8). A total of nine B. mallei isolates, each from a different horse and one from the camel from both Bahrainian outbreak events (2010 and 2011) were analyzed along with 15 isolates from the 2004 outbreak in the UAE by applying the same high-resolution 23-marker VNTR assay used to type the strain from the camel . To put the Bahrainian strains in a larger phylogeographic context, MLVA23 data from nine B. mallei isolates from the strain collection of the Bundeswehr Institute of Microbiology in Munich and previously published data from an additional 42 samples , were included in the phylogenetic reconstruction (). Analysis of VNTR data was performed as previously described in Hornstra et al. .To assess phylogenetic relationships we sequenced and compared eight genomes from the outbreak in the United Arab Emirates from 2004 to seven genomes from the Bahrain outbreak. Using ATCC 23344 genome as a reference, we identified homologous regions using MUMmer , and found single nucleotide polymorphisms (SNPs) using SolSNP (( After eliminating potential paralogs and positions with missing or ambiguous data among isolates, raw reads containing SNPs were aligned to the reference and inspected to verify allele calls and eliminate mixed alleles (when the minor allele frequency was greater than 10%). The final dataset was composed of 242 SNPs of which only 44 were variable among the Bahrain and Dubai genomes. Phylogenetic reconstruction was achieved using the maximum parsimony method implemented in MEGA 5.2.2 (, panel B). The consistency index was 1.0 for all sites, indicating that the final dataset is devoid of homoplasy and that all sites are in agreement with the topology. For datasets with little homoplasy, the consistency index is a more appropriate and direct measure of accuracy than bootstrapping, however 500 bootstrap replicates revealed that the clades of interest clustered together in greater than 98% of replicate trees. […]

Pipeline specifications

Software tools MUMmer, SolSNP, MEGA
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Burkholderia mallei, Equus caballus, Homo sapiens, Camelus dromedarius
Diseases Infection