Detection of Nervous Necrosis Virus of Grouper Fish in Lombok Island, Indonesia Based on Serological and Molecular Studies

Abstract

Cantang grouper fish (Epinephelus fuscoguttatus x Epinephelus lanceolatus) is one of the marine fisheries commodities that have opportunities both in the domestic and international markets. The cultivation of marine fish in Indonesian water has been largely harmed by the outbreak of viral diseases, for example, those caused by Nervous Necrosis Virus (NNV). The aim of study is to: produce primary antibody of NNV; and to detect disease based on immunohistochemical (IHC) and molecular methods. Two New Zealand rabbits (Oryctolagus cuniculus) of 8 months old males, ± 2 kgs of weight were vaccinated against NNV disease each week in a dose of 0.50 cc, 1.00 cc, 2.00 cc, and 3.00 cc by intra-peritoneal injection. The rabbit serum was harvested in the fifth week activated, and filtered by membrane dialysis three times to obtain immunoglobulin as the primary antibody. The brain, eyes, and spleen of infected grouper, were processed for histological tissue and stained with Hematoxylin and Eosin and immunohistochemical methods. Positive results were supported by the molecular test of Polymerase Chain Reaction (PCR) with Nishizawa's primers: R3: 5 '-CGA GTC AAC ACG GGT GAA GA-3' and F2: 5 '-CGT GTC AGT CAT GTG TCG CT-3'. The NNV primary antibody was sensitive for the detection. The positive results of IHC stains were supported by the PCR test in grouper fish from Lombok.