Immunoserology of European seabass (Dicentrarchus labrax) and white grouper (Epinephelus aeneus) as a non-lethal diagnostic tool for viral nervous necrosis

Viral nervous necrosis (VNN) is a lethal fish disease that has spread worldwide over the last two decades, causing severe losses in aquaculture. Diagnosis of the infection is generally made by sampling brain tissue, which involves sacrificing often valuable fish. Aiming at developing a non-lethal diagnostic method, the immune responses to an experimental nervous necrosis virus (NNV) infection in sea bass Dicentrarchus labrax and white grouper Epinephelus aeneus, two species most susceptible to the disease, were studied. RT-qPCR revealed presence of NNV in the fish brain within 24 h post-infection, the virus titer remaining high up to 30-35 days post-infection. In D. labrax blood, the virus was detectable within the first 5 days, after which its presence declined rapidly. Mx gene expression correlated to the virus presence in the blood and brain. An indirect ELISA was developed that quantified anti-NNV IgM in the fish blood. In D. labrax, anti-NNV IgM titer increased significantly within 5 days post-infection, and presence of specific IgM was detectable for 180 days. A sandwich ELISA was developed for E. aeneus. In this latter species, anti-NNV IgM titer increased significantly within the first 12 days and was detectable for 208 further days. The sandwich ELISA can be used as a diagnostic tool for detecting NNV exposure in all fish species for which specific antibodies against their IgMs are not yet commercially available. Our immunoserological method can reliably be used for diagnosis of VNN infection and does not require sacrificing the fish.