The use of sub-unit vaccines can solve some drawbacks associated with traditional attenuated or inactivated ones. However, in order to improve their immunogenicity, these vaccines needs to be associated to an appropriate adjuvant which, adequately selected, may also offer an alternative pathway for administration. The aim of this work was to evaluate the protection offered by the hot saline complex extracted from Brucella ovis (HS) encapsulated in mannosylated nanoparticles (MAN-NP-HS) when instilled conjunctivally in mice. Nanoparticles displayed a size of 300 nm and the antigen loading was close to 30 mu g per mg nanoparticle. Importantly, encapsulated HS maintained its protein profile, structural integrity and antigenicity during and after the preparative process of nanoparticles. The ocular immunization was performed on BALB/c mice. Eight weeks after vaccination animals were challenged with B. ovis, and 3 weeks later, were slaughtered for bacteriological examinations. Animals immunized with MAN-NP-HS displayed a 3-log reduction in spleen CFU compared with unvaccinated animals. This degree of protection was significantly higher than that observed for the commercial vaccine (Rev1) subcutaneously administered. Interestingly, the mucosal IgA response induced by MAN-NP-HS was found to be much more intense than that offered by Rev1 and prolonged in time. Furthermore, the elicited IL-2, IL-4 and.-IFN levels showed good correlation with the degree of protection. On the other hand, biodistribution studies in animals were performed with nanoparticles labelled with either (99m)technetium or rhodamine B isothiocyanate. The biodistribution revealed that, after instillation, MAN-NP-HS moved from the palpebral area to the nasal region and, the gastrointestinal tract. This profile of distribution was different to that observed for free (TcO4)-Tc-99m-colloids, which remained for at least 24 h in the site of administration. In summary, mannosylated nanoparticles appear to be a safe and suitable adjuvant for conjunctival vaccination. (C) 2012 Elsevier B. V. All rights reserved.