Neutralizing antibody levels for protection against betanodavirus infection in sevenband grouper, Epinephelus septemfasciatus (Thunberg), immunized with an inactivated virus vaccine

An inactivated betanodavirus, red-spotted grouper nervous necrosis virus (RGNNV), is a vaccine candidate for viral nervous necrosis (VNN). The present study was conducted to examine inoculation doses of the vaccine and neutralizing antibody titre levels to protect fish against VNN. Young sevenband grouper, Epinephelus septemfasciatus, averaging 25.4 g, were immunized at 25 degrees C water temperature by a single intraperitoneal injection of formalin-inactivated RGNNV. Fish immunized at vaccine doses of 10(8.5), 10(8.0), 10(7.5), 10(7.0) and 10(6.5) TCID50 per fish produced antibodies at mean titres of 1:907, 1:511, 1:259, 1:197 and 1:96, respectively, at 20 days post-immunization (p.i.). Neutralizing antibodies were not detected in any control fish (titre < 1:80). When fish were challenged with RGNNV (10(5.0) and 10(4.0) TCID50/fish) at 20 days p.i., cumulative mortalities of the fish groups immunized with 10(8.5), 10(8.0), 10(7.5) and 10(7.0) TCID50 per fish were significantly lower than those of the control group, and the relative percent survival values were higher than 60% in fish groups immunized with 10(7.5) TCID50 per fish or higher doses. However, no significant differences were found in mortality between the group immunized with 10(6.5) TCID50 per fish and the control group. From these results, it was deduced that the minimum effective inoculation dose of the vaccine is 10(7.0) TCID50 per fish and the minimum mean neutralizing antibody titre giving significant protection is approximately 1:200. This antibody titre level is a possible measure of vaccine efficacy against VNN in sevenband grouper, instead of a virus challenge test.