Characterisation of the genetic diversity of Brucella by multilocus sequencing

Abstract

BackgroundBrucella species include economically important zoonotic pathogens that can infect a wide range of animals. There are currently six classically recognised species of Brucella although, as yet unnamed, isolates from various marine mammal species have been reported. In order to investigate genetic relationships within the group and identify potential diagnostic markers we have sequenced multiple genetic loci from a large sample of Brucella isolates representing the known diversity of the genus.ResultsNine discrete genomic loci corresponding to 4,396 bp of sequence were examined from 160 Brucella isolates. By assigning each distinct allele at a locus an arbitrary numerical designation the population was found to represent 27 distinct sequence types (STs). Diversity at each locus ranged from 1.03–2.45% while overall genetic diversity equated to 1.5%. Most loci examined represent housekeeping gene loci and, in all but one case, the ratio of non-synonymous to synonymous change was substantially <1. Analysis of linkage equilibrium between loci indicated a strongly clonal overall population structure. Concatenated sequence data were used to construct an unrooted neighbour-joining tree representing the relationships between STs. This shows that four previously characterized classical Brucella species, B. abortus, B. melitensis, B. ovis and B. neotomae correspond to well-separated clusters. With the exception of biovar 5, B. suis isolates cluster together, although they form a more diverse group than other classical species with a number of distinct STs corresponding to the remaining four biovars. B. canis isolates are located on the same branch very closely related to, but distinguishable from, B. suis biovar 3 and 4 isolates. Marine mammal isolates represent a distinct, though rather weakly supported, cluster within which individual STs display one of three clear host preferences.ConclusionThe sequence database provides a powerful dataset for addressing ongoing controversies in Brucella taxonomy and a tool for unambiguously placing atypical, phenotypically discordant or newly emerging Brucella isolates. Furthermore, by using the phylogenetic backbone described here, robust and rationally selected markers for use in diagnostic assay development can be identified.

Brucellosis at the animal/ecosystem/human interface at the beginning of the 21st century

“…While the above tools are undoubtedly valuable, none has significant resolution at the subspecies level. The use of multilocus sequence analysis (MLSA) opens the way to detailed characterization of the global population structure of Brucella (Whatmore et al, 2007). These analyses confirmed the status of the classical species as distinct genetic entities, began to index intra-species diversity and relate this to historical biovar designations, and provided a framework for the placement of atypical or emerging Brucella isolates Schlabritz-Loutsevitch et al, 2009;Tiller et al, 2010).…”

“…These repeats have been exploited in many bacteria to develop a new generation of VNTR (Variable Number of Tandem Repeat) based typing approaches but are likely to prove particularly valuable in Brucella which previously lacked any epidemiological tool with adequate resolution to facilitate reliable epidemiological trace-back (Bricker et al, 2003;Le Fleche et al, 2006;Whatmore et al, 2006). Both MLSA and VNTR based analyses question the validity of some of the biovars established by classical microbiological typing, particularly those of B. melitensis (Al Dahouk et al, 2007a;Whatmore et al, 2007). Such analysis applied to local epidemiological scenarios shall allow progress in a number of areas previously hampered by the lack of tools with adequate discriminatory capacity.…”

Isolation ofBrucella microtifrom Soil

“…from soil, 2 g each of 2 selected PCR-positive samples with the highest amplifi cation rate (both from the affected area) were thoroughly homogenized in 5 mL of phosphate-buffered saline (PBS), pH 7.2, in 50-mL tubes. To confi rm that strains BMS 17 and BMS 20 were B. microti, these strains were subjected to multilocus sequence analysis and multilocus variable number of tandem re-LETTERS peat analysis (MLVA) as described (1,(3)(4)(5) In summary, we successfully isolated B. microti from soil samples collected at the same site 7 years after primary isolation of this novel species from common voles. B. microti could still be isolated from the same soil samples 6 months after storage at 4°C.…”

Serological and Nucleic Acid Based Detection of Brucellosis in Livestock Species and Molecular Characterization of Brucella melitensis Strains Isolated from Pakistan

“…Multilocus Sequence Analysis (MLSA) was performed using nine unique genome sequences amplified through PCR according to the procedure illustrated by Whatmore et al (2007). The amplified PCR products were purified using the QIA quick 96 PCR purification kit (QIAGEN, cat.…”

“…In brief, genomic DNA was extracted from field isolates which grew on blood culture through procedures as explained earlier by Whatmore et al (2005). Based on single nucleotide polymorphism (SNP) identified by Whatmore et al (2007), species identification of field isolates and determination of possible vaccine markers were undertaken using previously described multiple outcome real-time PCR assays (Goupal et al, 2008(Goupal et al, , 2010. These competition assays were set up in final reaction volume of 12.5 µL containing 6.25 µL TaqMan genotyping mix (Applied Biosystems, Warrington, United Kingdom) using Agilent MX3005p platform (Agilent, La Jolla, CA) with working concentrations of primers, probes and cycling conditions as mentioned in above citation.…”