패류 내 축적 흰반점바이러스(WSSV)의 새우에 대한 위험 분석

Abstract

White spot syndrome virus (WSSV) is a major pathogen causing large economic losses in aquatic farms of shrimp of the world. Owing to the unavailability of immortal cell line for culturing of WSSV in vitro, transmission experiments with bivalve shellfish known as an important vector are hard to characterize. In this study, we developed a PCR method which is more sensitive for detection of WSSV compared to that of the conventional PCR of OIE utilizing as a stadard method, and applied to chase the potential risk of shellfish as a vector for transmission of WSSV in aquatic filed by accumulation of WSSV in tissues. Primers designed from the nucleotide sequence of WSSV VP28 showed higher sensitivity for detection of WSSV(85%, 17/20) in 2-step amplification compared to the results performed with OIE reference primers (75%, 15/20). We have also demonstrated the lowest PCR detection limit of WSSV in the infected shrimp when the subcuticular connective tissue among various tissues used as target sample for WSSV detection by 2nd PCR. For analysis of the risk in shellfish that might be induced by WSSV in shellfish, we compared the PCR detection rate of WSSV using gill, midgut and mantle tissues of 4 different shellfish species (oyster, mussel, clam, and bloody clam) during accumulation and depuration process. Except gill, two other tissues showed the same frequency of positive PCR result of WSSV in 5 samples of each different shellfish species exposed to 5000X diluted WSSV infected tissue homogenate for 24 hrs in 20ℓ tank (1/5000X, corresponding 1.00E + 10 copies / 20ℓ seawater). Thus, through the analysis of shellfish gill tissue for accumulation of WSSV, we found mussels showed 100% (5/5) detection rate since 12hrs accumulation process, and the lowest results in clam(20%, 1/5). But we were not able to see a significant different frequency of WSSV detection in all three different tissues from 4 shellfish species used to assess the depuration rate. Also we compared the level of accumulation between RSIV as an another aquatic fish pathogen and WSSV by comparison of the detection frequency of each pathogen in shellfish gill tissues. Shellfish were exposed to approximately 5.00E +04 copies per ㎖ of each WSSV and RSIV in seawater by immersion method. After 60hrs at 23℃, frequency of positive sample in shellfish (21/48, 43.8%) exposed to WSSV was almost twice as high as that of RSIV (12/48, 25.0%). To assess the risk of shellfish as a vector of WSSV, 20 individual of each oyster and mussel exposed to 5000X diluted WSSV-infected tissue homogenate for 24 hrs in 20ℓ tank (1/5000X, corresponding 1.00E + 10 copies / 20ℓ seawater). In each two 100ℓ tank, those shellfish were moved and cohabitated with 20 shrimp (20g of body weight) at 23℃ for 20 days and found the cumulative mortality of 44% (7/16) and 13 (2/16) with exposed oyster and mussel, respectively. Certainly, shellfish that can accumulate WSSV in tissue have the potential to act as a vector for transmission of WSSV.