데이터마이닝을 통한 넙치(Paralichthys olivaceus) 양식장의 바이러스성출혈성패혈증 관련 위험인자와 그들의 연관성에 관한 연구

Abstract

The diagnosis of viral hemorrhagic septicemia virus (VHSV) infection requires the detection of viral RNA by reverse transcription- quantitative polymerase chain reaction (RT-qPCR) performed mainly using lymphoid organs including the spleen and/or kidney of fish. However, this procedure requires separate analysis per each individual, and dissection of individual fish is always needed even for pooled samples. In this study, an alternative approach termed “non-invasive risk indicator-based VHSV surveillance” was explored to investigate risk and environmental factors in association with VHSV infection in olive flounder farms. Briefly, a total of 180 and 60 fish were challenged with VHSV at a final concentration of 105 or 107 copies/fish. Then, 30 fish were moved to tanks maintained at 8, 15, and 22°C (duplicated), and another 30 fish were experienced increases in water temperature from 15°C to 22°C and decrease from 22°C to 15°C by change 1 degree/day for 7 days. The spleen of five random fish and 1 L of tank water from each group were sampled at 1, 3, 7, and 14 days post-infection (dpi) and 1, 4, 8, and 13 days dpi, respectively, to quantify VHSV using Taq-Man based real-time PCR assay. Field olive flounder and potential risk factors samples including outlet water, seawater in the vicinity, biofilm in water tank and feed (extruded pellet and moist pellet made of low value fish) were obtained from each farm 14 times from March to October to quantify VHSV. Correlation analysis between VHSV infection of fish and each potential risk factors were calculated using SPSS Statistics. As a result, regardless of viral concentration for challenge test, VHSV genome copy number in the spleen of olive flounder increased at 8°C as time goes on, but it was maintained at 15°C and decreased at 22°C, indicating clear temperature dependence of this pathogenic virus. Likewise, fish maintained at 8°C tend to release more virus into water than fish at 15°C. Infection rate of fish in the increasing temperature group (from 15°C to 22°C) was higher than that of fish in the decreasing temperature group (from 22°C to 15°C), indicating that water temperature in the early stage of VHSV infection is very essential. However, the virus was not detected from rearing water in the both groups, suggesting that VHSV-infected fish did not shed enough or at all. These results suggested that although olive flounder are infected with VHSV at 15°C, If water temperature rises above 15°C soon after VHSV infection, infection rate and degree will be significantly reduced. As a result of field sampling, VHSV infection rate and intensity of olive flounder was higher in the spring season (March - May), when sea water temperature was 15-19°C. Interestingly, only some of fish less than 250 g of body weight were heavily infected, indicating its dependence on the susceptibility of olive flounder to VHSV. Also, VHSV was detected in some samples of potential risk factors, which were mostly collected in the spring season. In particular, correlation coefficient of outlet water, biofilm, and low value fish used as feed to VHSV infection rate was higher than other potential factors. Taken together from results obtained in this study, sampling of fish of less than 250 g during March-May can be used for targeted surveillance of VHSV as it will increase possibility of the viral detection. Sampling of outlet water and tank biofilm of fish farm as a VHSV risk indicator might be useful for future surveillance plan. This study provide insight into dynamics of VHSV infection in olive flounder under different water temperature-related conditions, and fish body weights. Results obtained in this study will contribute for a related competent authority to develop “non-invasive risk indicator-based VHSV surveillance” policy in the future.