| This research aimed to evaluate the immune response and efficacy of several vaccine preparations, including oral-oil-emulsified vaccines against infections of some important marine fish bacterial pathogens, and some outer membrane proteins against infection of E. tarda and A. hydrophila.An oil-emulsified bivalent vaccine was prepared comprised of inactivated bacterial cells of pathogenic Vibrio anguillarum strains M3 and SMP1. The oil-emulsified bivalent vaccine was embedded in the feed pellet and administered to cultured turbot (Scophthalmus maximus) by the route of oral. The protection against M3 and SMP1 was evaluated, and several parameters of the specific and non-specific immune response were characterized in the vaccinated and control fish. Vaccination administration for 7 days elicited higher protection against M3 and SMP1 (with relative percentage survival RPS of 100% and 50%, respectively) in the group of oil-emulsified bivalent vaccine than in the non-oil-emulsified bivalent vaccine (with RPS of 57.9% and 0%, respectively). The protection was correlated with the activation of mucosa and humoral immune response in the hindgut of the group of oil-emulsified bivalent vaccine, which showed more increasing lysozyme and antiprotease activities and higher levels of antibody against M3 and SMP1 (p<0.05), and moderately increasimg antibody titres against M3 in the serum. Hindgut-situ-hybridization also indicated that the IgM were produced and distributed with higher level in the lamina of hindgut of fish administered by oil-emulsified bivalent vaccine. The oil-emulsified bivalent vaccine in the present study is applicable in the cultured fish against the infection of V. anguillarum.Inactivated bacteria cells of M3 and SMP1, CF(Streptococcus sp.) and SMW7(Edwardsiella tarda) were used as antigens to prepare for the polyvalent oral-oil-emulsified vaccine. The cultured fingerling turbot were immunized with the polyvalent vaccine by oral route. Results showed that antibody level against M3 increased (P<0.05) in hindgut, while the antibody titer in bile, gill, mid-gut, surface mucus, foregut and serum had no change in the immunized turbot. No antibody titres against SMP1, CF and SMW7 were detected in the above tissues of the immunized fish. The RPS was 100% when challenged with M3 by immersion route but no protection was detected when challenged by the i.p. route. Although the mortalities were delayed in turbot challenged immersely with SMP1, CF and SMW7, respectively, all the fish died during the observation period.Meanwhile, live rotifer vaccine was prepared using the rotifers feed by cells of M3, SMP1, CF and SMW7. The newly hatched larvae turbot were immunized with the live rotifer vaccine by oral route, but no visible protection were detected against M3, SMP1, CF and SMW7.The genes of ahaI and gapA of Aeromonas hydrophila LSA34, and eseB of Edwardsiella tarda LSE40 were cloned and expressed in E. coli. The expressed proteins were used to immunize turbots by intraperitoneal (IP) injection route. Specific antibodies were detected in the immunized turbot, and challenged tests showed that proteins of AhaI and GapA could confer higher RPS (80% and 100% respectively) against infection of A. hydrophila, but lower cross prection against E. tarda challenge (RPS of 30% and 10% respectively). Although high antibody titres could be detected in the turbot immunized with eseB, but no visible protection against the infection of E.tarda was detected.Based on the results obtained above, proteins of AhaI and GapA were used for antigens to prepare oil-emulsified vaccine for further evaluation their efficacy. Japanese flounder fish were immunized with the oil- emulsified vaccine and non-oil- emulsified vaccine respectively, via the administration of oral route. Results showed that specific antibody titres were detected in hinds of both groups of fish, however, the antibody titre in group of oil- emulsified fish were higher than that of non-oil- emulsified fish. In contrast, antibody levels produced by GapA oil-emulsified vaccine and GapA normal vaccine had no difference in the serum. The serum antibody levels against AhaI in the group of oil-emulsified fish were higher than those of the non-immunized control fish in 21-day and 35-day, respectively; while the serum antibody levels against AhaI in the group of non-oil-emulsified fish showed no difference from those of non-immunized control fish. |
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