| Skin ulceration syndrome causes great economic loss among sea cucumber{Apostichopus japonicus) aquaculture, owing to its high infection rate and the high mortality. It had been proved that Vibrio splendidus is one of the noxious pathogens of skin ulceration syndrome in the Apostichopus japonicus because of its high virulence and infectivity.This research was to investigate the immune protective effects of egg yolk immunoglobulin (IgY) against V. splendidus infection of the sea cucumber. Whole formalin-killed V. splendidus were used as an vaccine to immunize hens. IgY was produced from egg yolks obtained from these immunized hens using water dilution, two-step salt precipitation and ultrafiltration. ELISA experiment showed the IgY with a maximum titer of 1:320,000. The purity of the IgY produced was approximately 83%. The specific IgY significantly inhibited the growth and adhesion of V. splendidus in liquid and solid medium. The effects of IgY on biofilm formation and surface hydrophobicity of V. splendidus were determined. The results showed that IgY reduced significantly the biofilm formation and surface hydrophobicity of V. splendidus. Scanning electron microscope, immunofluorescence electron microscopy and transmission electron microscope indicated agglutination of V. splendidus treated with specific IgY and the surface structure of V. splendidus were influenced by specific IgY. The measurements of alkaline phosphatase activity showed the cell wall of V. splendidus were destroyed by specific IgY.The protective effects of specific IgY were evaluated in sea cucumber by intraperitoneally injecting anti-V. splendidus IgY antibodies (10 mg/mL) or immersing the sea cucumber in aqueous IgY (1 g/L) after an intraperitoneal injection of V. splendidus. The results showed that intraperitoneal injection and immersion brought about an 80% or a 75% survival, respectively. The survival rates were significantly higher than the control group with 20% or 15% (P<0.01). The protective effectiveness of specific IgY were investigated by immersing the sea cucumber in aqueous IgY before an intraperitoneal injection of V. splendidus, which brought about an 80% survival significantly higher than the control with 15% (P<0.01). The bacterial burden of the tissue treated with specific IgY was significantly lower than the group without IgY(P<0.05). The activity of phagocyte treated with specific IgY was significantly higher than the group without IgY (P<0.05). Specific IgY significantly enhanced activity of immune enzyme and immune factors by the coelomocytes. This research indicates that specific IgY has immune protective effectiveness against V. splendidus infection for sea cucumber. |
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