In silico design and immunogenic assessment of a strain-specific peptide in Brucella melitensis 16 M for DIVA diagnosis

Brucella melitensis is a facultative intracellular pathogen that causes brucellosis in small ruminants and humans. Strain-specific molecular targets are needed to improve vaccination monitoring and epidemiological surveillance, particularly for DIVA strategies aimed at distinguishing naturally infected animals from Rev.1-vaccinated animals. Here, we performed a computational, hypothesis-generating characterization of the RD3-associated genomic interval 824,171 − 824,131 on chromosome II of the virulent reference strain Brucella melitensis 16 M. Comparative sequence analyses were conducted across a selected DIVA-oriented panel of 56 publicly available bacterial chromosomal/genomic sequence records, including Brucella melitensis biovar 3 strain Ether chromosome II (NZ_CP007761.1), and the 16 M/Rev.1 locus was further inspected using the NC_003318.1 and CP024716.1 chromosome II FASTA sequences. The 16 M interval contains a 39-nt ATG-initiated coding segment on the negative strand that translates into the 13-amino-acid peptide MDHRSSGRGYDEK, whereas the homologous Rev.1 locus lacks the corresponding 40-bp ATG-containing segment. IEDB Next-Generation tools prioritized three MHC class I candidates, HRSSGRGY, MDHRSSGRGY, and DHRSSGRGY, while cross-species mapping using bovine MHC alleles as exploratory proxies identified Ovar-N*14:01 as the closest ovine sequence comparator. VaxiJen and BepiPred analyses provided complementary antigenicity and linear-epitope support, and host-proteome similarity screening against Ovis aries and Capra hircus did not identify full-length host matches or exact 8-13-mer host-protein matches. These findings support MDHRSSGRGYDEK as a panel-level, 16 M/RD3-positive-context computational candidate for DIVA-oriented assay development, but not as a validated diagnostic marker. No experimental evidence is currently available for peptide expression, antigen processing, MHC presentation, antibody reactivity, ELISA performance, PCR/qPCR/ddPCR diagnostic accuracy, or biological absence of cross-reactivity; therefore, no diagnostic performance or broad field applicability is claimed here.