A SDS/Page/Western Blot/EIA Protocol for the Specific Detection of Shrimp Viral Pathogens

Abstract

Shrimp viral diseases have seriously impacted the sustainability and economic success of the shrimp aquaculture industry worldwide. Among several of the recent viral pathogens which caused massive mortalities in cultured shrimp were the yellow-head virus (YHV) and the white spot baculovirus (WSBV) (also called the Chinese baculovirus (CBV) or the systemic ectodermal and mesodermal baculovirus (SEMBV)) Both YHV and CBV have been reported by our laboratory to be highly pathogenic for Penaeus stylirostris (blue shrimp) and Penaeus vannamei (white shrimp), the two principal penaeid species commercially cultured in Hawaii and the Western Hemisphere. They are thus potentially a serious problem particularly to the brood stock industry. A major problem to the control and prevention of viral diseases is the lack of relatively simple and cost-effective technologies for the early detection and diagnosis of viral infections, particularly asymptomatic infections. In this paper, we report the successful development of a less invasive, combined Western blot and enzyme immunoassay protocol for the early detection of both YHV and CBV in experimentally infected animals before the appearance of clinical symptoms. Detection of both viruses were also examined in infected primary shrimp cell cultures. Employing the nitrocellulose-enzyme immunoassay/streptavidin (NC-EAI/SAB) protocol which has a sensitivity of 0.4 ng for YHV and 1 ng for CBV; and in conjunction with the PAGE-Western blot protocol, viral proteins for both viruses were detected in the hemolymph as early as 48 hr post-infection (pi.). In corroborating studies with the infected cell culture system, viral proteins were detected as early as 3 to 5 days pi. This highly specific combination protocol presents several advantages for the monitoring and surveillance of shrimp vial infections particularly of asymptomatic infections. The sampling of hemolymph is relatively simple and less invasive particularly for the monitoring of invaluable shrimp brood stock populations. Also, the reagents used are relatively more stable and the entire procedure from sampling to the reading of the Western blots requires a time frame of 6-8 hours. Lastly, the entire combination protocol does not require highly skilled personnel and can eventually be automated.