Identification of an Edwardsiella tarda surface antigen and analysis of its immunoprotective potential as a purified recombinant subunit vaccine and a surface-anchored subunit vaccine expressed by a fish commensal strain

Edwardsiella tarda is the etiological agent of edwardsiellosis, a systematic disease that affects a wide range of marine and freshwater fish cultured worldwide. In order to identify E. tarda antigens with vaccine potential, we in this study conducted a systematic search for E. tarda proteins with secretion capacity. One of the proteins thus identified was Esa1, which contains 795 amino acid residues and shares extensive overall sequence identities with the D15-like surface antigens of several bacterial species. In silico analyses indicated that Esa1 localizes to outer membrane and possesses domain structures that are conserved among bacterial surface antigens. The vaccine potential of purified recombinant Esa1 was examined in a Japanese flounder (Paralichthys olivaceus) model, which showed that fish vaccinated with Esa1 exhibited a high level of survival and produced specific serum antibodies. Passive immunization of naive fish with antisera raised against Esa1 resulted in significant protection against E. tarda challenge. Taking advantage of the secretion capacity of Esa1 and the natural gut-colonization ability of a fish commensal strain, we constructed an Esa1-expressing recombinant strain, FP3/pJsa1. Western immunoblot and agglutination analyses showed that FP3/pJsa1 produces outer membrane-localized Esa1 and forms aggregates in the presence of anti-Esa1 antibodies. Vaccination analyses showed that FP3/pJsa1 as an intraperitoneal injection vaccine and an oral vaccine embedded in alginate microspheres produced relative percent survival rates of 79% and 52%, respectively, under severe challenging conditions that resulted in 92-96% mortality in control fish. Further analyses showed that following oral vaccination, FP3/pJsa1 was able to colonize in the gut but unable to disseminate into other tissues. Together these results indicate that Esa1 is a protective immunogen and an effective oral vaccine when delivered by FP3/pJsa1 as a surface-anchored antigen. (c) 2010 Elsevier Ltd. All rights reserved.

On the evaluation of diffusivities in gels using the diffusion cell technique

Modeling of the gel-immobilized cell system requires accurate measurement of diffusion coefficients. Three methods of the quasi-steady-state (QSS) method, the time-lag (TL) method and a variant quasi-steady-state (VQSS) method were critically assessed and compared for the evaluation of diffusivities using the diffusion cell technique. Experimental data from our laboratory were used for the analysis of the influence of crucial theoretical assumptions not being fulfilled in each method. The results highlighted a risk in obtaining highly variable diffusion coefficients by not validating the QSS and the accuracy of the measurements. In the TL method, the estimation of diffusivities based on the plot intercept that was mostly used in the literature, results in a many fold lower value when compared to that based on the plot slope. The comparison with the QSS and VQSS methods confirmed similar diffusivity obtained by the TL method based on the plot slope. It thus suggested that the correct estimation of diffusivities by the TL method could be based on the plot slope only. Furthermore, the errors associated with the solute mass in the gel, the sample withdrawal and the non-negligible concentration changes in the chambers were also discussed. It is concluded that diffusion cell technique has to be employed cautiously for a correct evaluation of diffusivities. (C) 2001 Elsevier Science B.V. All rights reserved.