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Brucellosis is a zoonotic disease caused by Brucella spp. Brucella spp. can be sub-typed by multilocus sequence typing (MLST) method, which targets a set of housekeeping genes. We have developed a core genome MLST (cgMLST) typing scheme to distinguish and differentiate species of Brucella up to biovar level. A total of 407 whole (complete and draft) genome sequences of different Brucella strains were used in this study. Genome sequences were filtered using the BLAST score ratio (BSR)-based allele calling algorithm, and we found that 164 cgMLST target loci are shared in all the 407 genome sequences. These 164 loci were used to develop the cgMLST scheme and further evaluated to sub-type different species of Brucella. Based on our cgMLST scheme, Brucella spp. were classified into 287 sequence types (STs). A phylogenetic tree was constructed based on the STs derived from the cgMLST analysis. The phylogenetic tree differentiated all the 11 Brucella spp. and five biovars of B. suis. B. vulpis formed the outmost clade followed by B. inopinata and B. microti. Among the four subgroups of B. abortus, group A and B were differentiated based on their geographic origins. Similarly, three subgroups of B. melitensis were separated based on their geographical origins with few exceptions. B. neotomae that infect rodents were distinguished from other Brucella spp. B. canis showed the closest relationship with B. suis bv. 4, followed by B. suis bv. 3 and bv. 1. Brucella spp. associated with the marine mammals, such as B. ceti and B. pinnipedialis were closely related. Of these, B. ceti strains isolated from dolphins and porpoise were differentiated into two groups. We incorporated our cgMLST tool in BrucellaBase (http://www.dbtbrucellosis.in/brucella_cgmlst.html), which will be helpful to predict the cgMLST allelic profile and the ST of a newly sequenced genome., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Brucellosis is an important zoonotic disease caused by Brucella spp. We present a phylogeny of 552 strains based on genome-wide single nucleotide polymorphisms (SNPs) determined by an alignment-free k-mer approach. A total of 138,029 SNPs were identified from 552 Brucella genomes. Of these, 31,152 and 106,877 were core and non-core SNPs, respectively. Based on pan-genome analysis 11,937 and 972 genes were identified as pan and core genome, respectively. The pan-genome-wide analysis studies (Pan-GWAS) could not identify the group-specific variants in Brucella spp. Therefore, we focused on SNP based genome-wide association studies (SNP-GWAS) to identify the species-specific genetic determinants in Brucella spp. Phylogenetic tree representing eleven recognized Brucella spp. showed 16 major lineages. We identified 143 species-specific SNPs in Brucella abortus that are conserved in 311 B. abortus genomes. Of these, 141 species-specific SNPs were confined in the positively significant SNPs of B. abortus using SNP-GWAS. Since conserved in all the B. abortus genomes studied, these SNPs might have originated very early during the evolution of B. abortus and might be responsible for the evolution of B. abortus with cattle as the preferred host. Similarly, we identified 383 species-specific SNPs conserved in 132 Brucella melitensis genomes. Of these 379 species-specific SNPs were identified as positively associated using GWAS. Interestingly, >98% of the SNPs that are significantly, positively associated with the traits showed 100% sensitivity and 100% specificity. These identified species-specific core-SNPs identified in Brucella genomes could be responsible for the speciation and their respective host adaptation., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Brucella melitensis is an intracellular pathogen resides in the professional and non-professional phagocytes of the host, causing zoonotic disease brucellosis. The stealthy nature of the Brucella makes it's highly pathogenic, and it is hard to eliminate the bacteria completely from the infected host. Hitherto, no licensed vaccines are available for human brucellosis. In this study, we identified potential antigens for vaccine development from non-classically secreted proteins through reverse vaccinology approach. Based on the systemic screening of non-classically secreted proteins of B. melitensis 16M, we identified nine proteins as potential vaccine candidates. Among these, Omp31 and Omp22 are known immunogens, and its role in the virulence of Brucella is known. Roles of other proteins in the pathogenesis are yet to be studied. From the nine proteins, we identified six novel antigenic epitopes that can elicit both B-cell and T-cell immune responses. Among the nine proteins, the epitopes were predicted from Omp31 immunogenic protein precursor, Omp22 protein precursor, extracellular serine protease, hypothetical membrane-associated protein, iron-regulated outer membrane protein FrpB. Further, we designed a multitope vaccine using Omp31 immunogenic protein precursor, Omp22 protein precursor, extra cellular serine protease, iron-regulated outer membrane protein FrpB, hypothetical membrane-associated protein, and LPS-assembly protein LptD and polysaccharide export protein identified in the previous study. Epitopes were joined using amino acid linkers such as EAAAK and GPGPG. Cholera toxin subunit B, the nontoxic part of cholera toxin, was used as an adjuvant and it was linked to the N-terminal of the multitope vaccine candidate. The designed vaccine candidate was modeled, validated and the physicochemical properties were analyzed. Results revealed that the vaccine candidate is soluble, stable, non-allergenic, antigenic and 87% of residues of the designed vaccine candidate is located in the favored region. In conclusion, the computational analysis showed that the newly designed multitope protein could be used to develop a promising vaccine for human brucellosis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Brucella sp. causes a major zoonotic disease, brucellosis. Brucella belongs to the family Brucellaceae under the order Rhizobiales of Alphaproteobacteria. We present BrucellaBase, a web-based platform, providing features of a genome database together with unique analysis tools. We have developed a web version of the multilocus sequence typing (MLST) (Whatmore et al., 2007) and phylogenetic analysis of Brucella spp. BrucellaBase currently contains genome data of 510 Brucella strains along with the user interfaces for BLAST, VFDB, CARD, pairwise genome alignment and MLST typing. Availability of these tools will enable the researchers interested in Brucella to get meaningful information from Brucella genome sequences. BrucellaBase will regularly be updated with new genome sequences, new features along with improvements in genome annotations. BrucellaBase is available online at http://www.dbtbrucellosis.in/brucellabase.html or http://59.99.226.203/brucellabase/homepage.html., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Brucellosis is an important zoonotic disease caused by Brucella spp. Brucella suis is the etiological agent of porcine brucellosis. B. suis is the most genetically diverged species within the genus Brucella. We present the first large-scale B. suis phylogenetic analysis based on an alignment-free k-mer approach of gathering polymorphic sites from whole genome sequences. Genome-wide core-SNP based phylogenetic tree clearly differentiated and discriminated the B. suis biovars and the vaccine strain into different clades. A total of 16,756 SNPs were identified from the genome sequences of 54 B. suis strains. Also, biovar-specific SNPs were identified. The vaccine strain B. suis S2-30 is extensively used in China, which was discriminated from all biovars with the accumulation of the highest number of SNPs. We have also identified the SNPs between B. suis vaccine strain S2-30 and its closest homolog, B. suis biovar 513UK. The highest number of mutations (22) was observed in the phosphomannomutase (pmm) gene essential for the synthesis of O-antigen. Also, mutations were identified in several virulent genes including genes coding for type IV secretion system and the effector proteins, which could be responsible for the attenuated virulence of B. suis S2-30., (Copyright © 2016 Elsevier B.V. All rights reserved.)