DEVELOPMENT OF ORAL VACCINATION METHOD AGAINST VIRAL NERVOUS NECROSIS OF FISH (Betanodavirus INFECTION) OF CULTURED MARINE FISH WITH THE AID OF EXTRACTS OF NATURAL PRODUCT.

For these two decades, betanodavirus infections (viral nervous necrosis: VNN) have emerged as major constraints on the culture of marine fish all over the world, causing severe economic losses. This worldwide finfish disease has primarily been associated with larval and juvenile stages in most of fish species. Recently an injection vaccine was commercialized, but the vaccine cannot be available for fish at un-injectable size. In order to develop efficacious oral vaccine for VNN, we investigated the effect of some inflammatory substances, which cause reversible pathology to the intestinal epithelium, on the epithelial passage of vaccine particles.

Juvenile sevenband grouper (Epinephelus septemfasciatus) and inactivated sevenband grouper nervous necrosis virus (RGNNV; strain SGEhi00) were used in this study. Capsaicin (methyl vanillyl nonenamide) (1mg/fish), piperine (1-[5-(1,3-Benzodioxol-5-yl)-1-oxo-2,4-pentadienyl]piperidine) (2mg/fish), and c- okadaic acid (9,10-Deepithio-9,10-didehydroacanthifolicin) (1µg/fish) were used as natural inflammatory substances. They were introduced individually or mixed with the vaccine (10¹⁰ TCID₅₀/fish) to fish via anal intubation. After 3 hours, fish were dissected for intestine which was subjected to histological (H&E) and immuno-histological (IFT, IHC) examinations. Each substance caused moderate congestion, increased mucous cell secretions and frequent intestinal villar destruction. In substance-vaccine mixture groups, the virus antigen was detected sub-epithelially in capsaicin mixture and intra-epithelally in piperine mixture, but no antigen was detected in okadaic acid mixture.

Capsaicin; as a promising candidate; impregnated commercial fish feed was sprayed with the vaccine, 200 juvenile fish were divided into 4 groups, (50 fish per group), placed in 8 seawater aquaria, fish were fed 1% of their total body weight once daily for consecutive 7 days.

two weeks later, all the fish groups were continuously exposed to the drainage of the same tank in which artificially NNV-infected fish were placed (cohabitation infection), then the fish were regularly checked for mortality for 3 weeks, the results were as the following.

Conclusion: capsaicin as oral vaccine adjuvant resulted in 100% protection against viral infection.