Immunogenic antigens of the eel pathogen Vibrio vulnificus serovar E

The immunogenic antigens of Vibrio vulnificus serovar E were investigated in the eel. Fish were vaccinated by immersion with Vulnivaccine (V), revaccinated 2 years later by intraperitoneal injection (RV) and bath infected 15 days post-revaccination (RVI). The specific immune response in serum was followed in all groups, and selected sera were used for immunostaining of surface (SA) and extracellular antigens (ECA). Bacteria were grown in iron-rich (TSB and MSWYE) and iron-poor media (TSB and MSWYE plus human transferrin (TSB-T and MSWYE-T)) as well as eel serum (ES), and their SA and ECA were extracted and electrophoretically analysed. Cells grown in MSWYE-T and ES presented the same antigenic profiles, which suggests that iron-restriction is the main growth-limiting factor in vivo. The electrophoretic pattern of SA, but not that of ECA, varied with iron-availability in the growth medium. Further, SA extracted from bacteria grown in iron restriction were strongly immunogenic for eels, especially after vaccination and infection. Among the immunogenic antigens over expressed in iron-restriction, three outer membrane proteins of around 70-80 kDa, including the putative receptor for vulnibactin, together with the rapid and slow migrating forms of the lipopolysaccharide (LPS), were identified. The response was not so evident in the case of capsule, which was not clearly stained with any of the eel sera. With respect to ECA, two proteins, identified as the V. vulnificus protease (Vvp) and the major outer membrane protein (OMP), probably liberated to the medium after cell death, were recognised by RV and, more strongly, by RVI sera. The specific antibodies against the mentioned OMPs, LPS bands and the Vvp may contribute to the protection of vaccinated eels against infection, giving a reasonable explanation for the high effectiveness of Vulnivaccine.