Abstract
Brucellosis is a neglected zoonotic disease caused by alpha proteobacterial genus Brucella comprising of facultative intracellular pathogenic species that can infect both animals and humans. In this study, we aimed to identify genome-wide unique insertion sequence (IS) elements among Brucella abortus, B. melitensis, B. ovis, B. suis and B. canis for use in species differentiation by conducting an intensive in silico-based comparative genomic analysis. As a result, 25, 27, 37, 86 and 3 unique ISs were identified respectively and they had a striking pattern of distribution among them. To explain, a particular IS would be present in four species with 100% identity whereas completely absent in the fifth species. However, flanking regions of that IS element would be highly identical and conserved in all five species. Species-specific primers designed on these flanking conserved regions resulted in two different amplicons grouping the species into two: one that possesses IS and the other that lacks it. Seeking for species-specific amplicon size for particular species was sufficient to identify it irrespective of biovar. A multiplex PCR developed using these primers resulted in successful differentiation of the five species irrespective of biovars with significant specificity and sensitivity when examined on clinical samples.
Highlights
Brucella, the etiological agent of a classical zoonotic disease named brucellosis, is an alpha-2 proteobacteria
We describe a novel in silico-based comparative genomic analysis approach to decipher species-associated unique and precise DNA block distributions among five species of Brucella, namely, B. abortus, B. melitensis, B. ovis, B. suis and B. canis irrespective of biovars
3405 fragments were obtained after dividing the genomes of B. abortus A13334, B. melitensis ATCC 23457, B. ovis ATCC 25840, B. suis 1330, B. suis ATCC 23445, B. suis VBI22 and B. canis ATCC 23365 into 1000 bp fragments
Summary
The etiological agent of a classical zoonotic disease named brucellosis, is an alpha-2 proteobacteria. In another study, novel insertion of IS711 within the Omp[31] gene of an atypical B. ovis isolate recovered from an infected ram in Hungary was encountered and was not correctly identified in the Bruce-ladder multiplex PCR assay[17] Based on these new case reports, the fact that the most acclaimed IS element, IS711, could be a transposon element in Brucella species was established, and its reliability in differentiating Brucella species was weakened as it demonstrated polymorphism within the strains of a species. We describe a novel in silico-based comparative genomic analysis approach to decipher species-associated unique and precise DNA block distributions among five species of Brucella, namely, B. abortus, B. melitensis, B. ovis, B. suis and B. canis irrespective of biovars. These highly specific nucleotide sequences were evaluated for their ability to detect targets, and a novel multiplex PCR was developed wherein the five species were differentiated irrespective of biovars
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