P2-08 Brucella melitensis immunoproteomics reveal strain-specific antigens suitable for the unbiased diagnosis in a diva strategy

Abstract

In the Mediterranean area, Brucella melitensis is among the most common species with sheeps and goats being the major reservoir of the pathogen. Diagnostic and prophylactic measures adopted for the control of brucellosis suffer from a lack of specificity and cross-reactivity, respectively. Also, live-attenuated B. melitensis Rev.1 administration in goat and sheep showed residual virulence and cross-reactivity withthe current diagnostics, raising the need for studies to amend both the vaccinal and diagnostic strategies. Here, we employed an immunoproteomic approach to underline the specific immunogenic proteins of both B. melitensis Rev.1 and B. melitensis 16M strains, meant respectively as the reference of the vaccinal and field strains, and discriminate their antigens from each other and from those of the most cross-reactive specimens (i.e. Escherichia coli O157:H7 and Yersinia enterocolitica O:9). Following the optimized resolution of the Brucellae proteomes, independent incubation of the bacterial protein profiles with properly depleted sera from either Rev.1 or 16M infected animals revealed a distinct map of immunogenic proteins for the assayed bacteria. Differential immunogenic proteins have been identified both computationally and analytically via MS/MS, yielding a list of suitable candidates for the design of novel diagnostics and/or prophylactic strategies. Universal stress protein and related nucleotide-binding proteins (Uspa), nucleoside diphosphate kinase and putrescine utilization regulator were uniquely identified as predictive for the presence of the vaccinal strain. On the other hand, ABC transporter-substrate-binding protein (cluster4_ leucine/isoleucine/valine/benzoate, broad-specificity amino acid ABC transporter-substrate-binding protein) and ABC transporter-substrate-binding pro (cluster1maltose/g3p/polyamine/iron) were identified as putative biomarkers of B. melitensis 16M. The independent identification of the immunogenic proteins confirms previous in-silico bioinformatic prediction performed on the same bacterial strains. Sorting the major immunogenic proteins of both the vaccinal and field strains is pivotal for the design of unbiased serodiagnostic strategies crowning the conventional diagnostics for Brucella. Also, these may serve as the starting point for drawing optimized prophylactic strategies targeting other molecules than those addressed by the diagnostic tests; thus, differentiating infected from vaccinated animals. This improves monitoring campaigns which represents the keystone for the fair and efficient control and eradication of brucellosis in animals and humans.