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Molecular targets for rapid identification of Brucella spp.

ABSTRACT: BACKGROUND: Brucella is an intracellular pathogen capable of infecting animals and humans. There are six recognized species of Brucella that differ in their host preference. The genomes of the three Brucella species have been recently sequenced. Comparison of the three revealed over 98% sequence similarity at the protein level and enabled computational identification of common and differentiating genes. We validated these computational predictions and examined the expression patterns of the putative unique and differentiating genes, using genomic and reverse transcription PCR. We then screened a set of differentiating genes against classical Brucella biovars and showed the applicability of these regions in the design of diagnostic tests. RESULTS: We have identified and tested set of molecular targets that are associated in unique patterns with each of the sequenced Brucella spp. A comprehensive comparison was made among the published genome sequences of B. abortus, B. melitensis and B. suis. The comparison confirmed published differences between the three Brucella genomes, and identified subsets of features that were predicted to be of interest in a functional comparison of B. melitensis and B. suis to B. abortus. Differentiating sequence regions from B. abortus, B. melitensis and B. suis were used to develop PCR primers to test for the existence and in vitro transcription of these genes in these species. Only B. suis is found to have a significant number of unique genes, but combinations of genes and regions that exist in only two out of three genomes and are therefore useful for diagnostics were identified and confirmed. CONCLUSION: Although not all of the differentiating genes identified were transcribed under steady state conditions, a group of genes sufficient to discriminate unambiguously between B. suis, B. melitensis, and B. abortus was identified. We present an overview of these genomic differences and the use of these features to discriminate among a number of Brucella biovars.

SUBMITTER: Ratushna VG

PROVIDER: S-EPMC1413539 | biostudies-literature | 2006

REPOSITORIES: biostudies-literature

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Molecular targets for rapid identification of Brucella spp.

Ratushna Vladyslava G VG Sturgill David M DM Ramamoorthy Sheela S Reichow Sherry A SA He Yongqun Y Lathigra Raju R Sriranganathan Nammalwar N Halling Shirley M SM Boyle Stephen M SM Gibas Cynthia J CJ

BMC microbiology 20060222

<h4>Background</h4>Brucella is an intracellular pathogen capable of infecting animals and humans. There are six recognized species of Brucella that differ in their host preference. The genomes of the three Brucella species have been recently sequenced. Comparison of the three revealed over 98% sequence similarity at the protein level and enabled computational identification of common and differentiating genes. We validated these computational predictions and examined the expression patterns of t ...[more]

PMID: 16504063

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Project description:BackgroundBrucellosis still remains an endemic disease for both livestock and human in Greece, influencing the primary sector and national economy in general. Although farm animals and particularly ruminants constitute the natural hosts of the disease, transmission to humans is not uncommon, thus representing a serious occupational disease as well. Under this prism, knowledge concerning Brucella species distribution in ruminants is considered a high priority. There are various molecular methodologies for Brucella detection with however differential discriminant capacity. Hence, the aim of this survey was to achieve nationally Brucella epidemiology baseline genotyping data at species and subtype level, as well as to evaluate the pros and cons of different molecular techniques utilized for detection of Brucella species. Thirty-nine tissue samples from 30 domestic ruminants, which were found positive applying a screening PCR, were tested by four different molecular techniques i.e. sequencing of the 16S rRNA, the BP26 and the OMP31 regions, and the MLVA typing panel 1 assay of minisatellite markers.ResultsOnly one haplotype was revealed from the 16S rRNA sequencing analysis, indicating that molecular identification of Brucella bacteria based on this marker might be feasible solely up to genus level. BP26 sequencing analysis and MLVA were in complete agreement detecting both B. melitensis and B. abortus. An interesting exception was observed in 11 samples, of lower quality extracted DNA, in which not all expected MLVA amplicons were produced and identification was based on the remaining ones as well as on BP26. On the contrary OMP31 failed to provide a clear band in any of the examined samples.ConclusionsThe present study reveals the constant circulation of Brucella bacteria in ruminants throughout Greece. Further, according to our results, BP26 gene represents a very good alternative to MLVA minisatellite assay, particularly in lower quality DNA samples.

Project description:Brucellosis is a highly contagious zoonosis worldwide with economic and public health impacts. The aim of the present study was to identify Brucella (B.) spp. isolated from animal populations located in different districts of Egypt and to determine their antimicrobial resistance. In total, 34-suspected Brucella isolates were recovered from lymph nodes, milk, and fetal abomasal contents of infected cattle, buffaloes, sheep, and goats from nine districts in Egypt. The isolates were identified by microbiological methods and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Differentiation and genotyping were confirmed using multiplex PCR for B. abortus, Brucella melitensis, Brucella ovis, and Brucella suis (AMOS) and Bruce-ladder PCR. Antimicrobial susceptibility testing against clinically used antimicrobial agents (chloramphenicol, ciprofloxacin, erythromycin, gentamicin, imipenem, rifampicin, streptomycin, and tetracycline) was performed using E-Test. The antimicrobial resistance-associated genes and mutations in Brucella isolates were confirmed using molecular tools. In total, 29 Brucella isolates (eight B. abortus biovar 1 and 21 B. melitensis biovar 3) were identified and typed. The resistance of B. melitensis to ciprofloxacin, erythromycin, imipenem, rifampicin, and streptomycin were 76.2%, 19.0%, 76.2%, 66.7%, and 4.8%, respectively. Whereas, 25.0%, 87.5%, 25.0%, and 37.5% of B. abortus were resistant to ciprofloxacin, erythromycin, imipenem, and rifampicin, respectively. Mutations in the rpoB gene associated with rifampicin resistance were identified in all phenotypically resistant isolates. Mutations in gyrA and gyrB genes associated with ciprofloxacin resistance were identified in four phenotypically resistant isolates of B. melitensis. This is the first study highlighting the antimicrobial resistance in Brucella isolated from different animal species in Egypt. Mutations detected in genes associated with antimicrobial resistance unravel the molecular mechanisms of resistance in Brucella isolates from Egypt. The mutations in the rpoB gene in phenotypically resistant B. abortus isolates in this study were reported for the first time in Egypt.

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Molecular targets for rapid identification of Brucella spp.

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Molecular targets for rapid identification of Brucella spp.

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