Proceedings of the Third World Fisheries Congress: Feeding the World with Fish in the Next Millenium—The Balance between Production and Environment
Using Monoclonal Antibodies to Diagnose White Spot Syndrome Virus Disease of Shrimp
Wenbin Zhan, Jing Chen, Jing Xing, Li Zhou
Shrimp diseases occur more frequently and seriously as the shrimp culture industry in China expands. Comprehensive research in aquatic pathology began in 1982, focused on identifying various shrimp diseases and investigating their etiology, diagnosis, treatment, prevention, and pathogenic ecology. In the 1980s, the diseases were mainly caused by bacteria, fungi, and ciliate parasites; although the negative impact was noticed, diseases were somewhat controlled. But since early 1993, outbreak of epidemic disease caused by white spot syndrome virus (WSSV) has brought mass mortality and led to severe losses in shrimp culture. The disease has been reported in cultured penaeid shrimp such as fleshy shrimp Penaeus chinensis, kuruma shrimp P. japonicus, giant tiger shrimp P. monodon, and other crustaceans (Zhan et al. 1995). There is nearly no effective drug for this virus disease, so the prevention and control of its spread are imperative, as is the demand for sensitive and rapid diagnostic methods.
Many methods have been developed to diagnose WSSV disease. Histology is the most commonly used method, and molecular methods (gene probes and DNA amplification using polymerase chain reaction) are comparatively new methods, but these methods need a long time to give results. Methods based on monoclonal antibodies (Mab’s) are thought to be promising for their speed, versatility, relatively low cost, simplicity, and reasonably good sensitivity (Lightner 1999). We used anti-WSSV Mab’s to develop a fluorescent antibody technique (FAT), an immunodot blot assay (dot blot), a staphylococcus protein A-coagglutination test (SPA-CoA), and an immuno-electroosmophoresis test (IEOP). Comparing these methods, we tried to develop a convenient, rapid, and accurate diagnostic method.
Morbid fleshy shrimp (mean weight 5.6 g) with obvious cuticular white spots were collected from shrimp farms in Shandong Province. Healthy fleshy shrimp (mean weight 8.2 g) used in the trial were collected from a shrimp farm in Hebei Province where no viral disease had been observed, and these shrimps showed no signs of virally induced lesions. Gills of morbid and healthy shrimps were homogenized in a glass homogenizer with phosphate-buffered saline (PBS, pH 7.4) for 20 min on ice, at a concentration of 20% (weight/volume), then centrifuged at 3,000 × gravity for 15 min at 4°C, and the supernatants were collected.
A mixture of six anti-WSSV Mab’s (hybridoma culture fluid) was used (produced by Zhan et al. 1999a).
The cryosections made from shrimp gills (morbid and healthy) were fixed with acetone for 20 min, and Mab’s were applied as primary antibody. Samples were incubated for 60 min at 37°C, then washed three times with PBS for 5 min. Secondary antibody, sheep anti-mouse immunoglobulin (Ig) serum conjugated with fluorescein isothiocyanate (diluted 1:200) was applied and incubated for 60 min at 37°C. After washing three more times, the slides were mounted in glycerol and examined under a fluorescence microscope (Zhan et al. 1999a).