White Sport Syndrome Virus Disease of Shirmp and Diagnostic Methods

Since 1993, the White Spot Syndrome Virus (WSSV) disease occurred in China among cultured shrimps resulting in mass mortality. Epizootiological surveys undertaken during the outbreak period of 1993-1994 indicated that all stages of Penaeus chinensis, P. japonicus and P. monodon were infected. Consequent to the transport of contaminated shrimp seedlings and seawater, the disease spread all over the farms of China. The disease was more rapidly transmitted at temperatures above $25^{\circ}C$. Challenge experiments showed the causative agent was highly virulent. White spots appeared on the carapace of both span-taneous and experimentally infected shrimps. Moribund shrimps contained turbid hemolymph, hypertrophied Iymphoid organ and a necrotic mid-gut gland. Electron microscopy showed the presence of viral particles in the gills, stomach, lymphoid organ, and epidermal tissue of the infected shrimp. The visions were slightly ovoid with an envelope and averaged 350 $\times$ 150 nm; nucleocapsids measured 375 $\times$ 157 nm. With discontinuous sucrose gradient of 35, 50 and 60% (w/v), the virus was separated from hemolymph of the infected shrimp. The estimated molecular weight of genomic DNA was 237 Kb with EcoR I, 247 Kb with Hind III and 241kb with Pst I. A total of 9 hybridoma colones secreting monoclonal antibodies (MAbs) were produced from mouse myeloma and spleen cells immunized with WSSV. The immunofluorescence assay of gill tissue showed that the MAbs reacted with diseased but not with healthy shrimp. The MAbs belonged to IgGl, IgG2b subclass and IgM class, all with kappa light Immune-electron-microscopy with colloidal gold marker showed the presence of 5 MAbs epitopes on the envelope and one on the capsid of the virus. Baculoviral mid-gut gland necrosis showed the specificity of the MAbs produced. For diagnosis 5 different methods were selected. Using Kimura primers for PCR, or MAbs for immunoblot, ELISA or FAT method, in situ hybridization was carried out to show the gene. All these methods detected WSSV in the organ samples of the diseased shrimp but not in healthy one.