Abstract
In recent years, Vibrio harveyi has become a significant cause of skin ulceration of Japanese pufferfish (Takifugu rubripes), and the outbreaks have been responsible for mass mortalities and high economic losses. Thus, the main purpose of this study was to evaluate the protective effect of chicken egg yolk immunoglobulins (IgY) against V. harveyi infection in the T. rubripes. In the present study, hyperimmune anti-V. harveyi IgY was extracted from eggs laid by Hy-Line Brown hens immunized with formaldehyde-inactivated whole V. harveyi cells. The purity of the IgY after separation from egg yolk was approximately 82.4% as assessed by SDS-PAGE. The maximum IgY titer (ELISA) in water-soluble fractions was up to 1:130000 at week 12th of immunization of hens. In-vitro experiment revealed that the IgY inhibited the growth of V. harveyi in liquid medium by a concentration-dependent manner in the range of 1 to 10 mg/mL and on agar plates (P < 0.05). The effect of 10 mg/mL of IgY was similar to the 0.1 mg/mL of streptomycin within 7 h (P > 0.05). Confocal laser scanning microscope (CLSM) assays revealed that anti-V. harveyi IgY caused bacterial agglutination. The T. rubripes intramuscularly injected with 100 μL of V. harveyi (5.4 × 107 CFU/mL) and the IgY (10 mg/mL) showed a survival rate of 80% (P < 0.001). Dietary supplementation of 10% egg yolk powder significantly enhanced the resistance to V. harveyi infection with a survival rate of 50.0% (P < 0.001), as against 100% death in the positive control group. Immersing in 0.5 g/L aqueous IgY resulted in a 60.0% survival in challenged fish (P < 0.001), along with increase in the levels of non-specific immune-related enzymes (SOD, POD, NOS, ACP and LYZ) (p < 0.05). While 1 mg/mL of IgY significantly enhanced the phagocytosis of V. harveyi by macrophages (p < 0.05). These results revealed that IgY has great prospects in protecting T. rubripes against V. harveyi infection.
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