흰반점 바이러스의 전파 매개체로서의 새우 및 이매패류의 위험 평가
- Alternative Title
- Risk evaluation of shrimp and bivalve shellfish as white spot syndrome virus intermediates for viral transmission
- Abstract
- White spot syndrome virus (WSSV) has spread through the shrimp trade, causing large economic losses in the aquatic industry all over the world. The potential of WSSV transmission via frozen shrimp has been revealed in a recent study. And bivalve mollusc, due to the possibility of accumulation and release of disease-causing agents in their tissue, could be a route for virus transmission. Under these backgrounds, we examined the possibility of WSSV transmission by frozen shrimps and bivalves for human consumption.
To investigate the WSSV in frozen shrimp and shellfish, we collected domestic and imported samples (shrimp, n=86; shellfish, n=186) in Korean market between 2010 and 2018. First-step PCR detection rates identified from domestic shrimps was 36.8% (7/19), a different finding from imported shrimps which showed 0.01% (1/67). Interestingly, WSSV particles were identified from a variety of shellfish samples (15.6%, 23/147) in Korea. From the genetic relatedness of InDel-II regions between detected WSSV isolates from shrimps, four genotypes were determined by comparing with a reference strain from Taiwan (WSSV-TW, an ancestor WSSV strain). Furthermore, in addition to including all existing types of WSSV isolates from shrimp, three additional types were found in the isolates of the shellfish. Notably, it suggests that shrimp as well as shellfish could be applied for important factors for tracking the WSSV origin.
To estimate the potential transmission of WSSV in the environment, stability tests in various conditions were conducted. Firstly, the stability of WSSV genome was confirmed by degradation copy value in filtered seawater and freshwater at 18 °C and 23 °C. WSSV genome reduction in freshwater and 18℃ was less than that of seawater and 23℃. Second, in the digestion rate analysis using various digestive enzymes of shellfish, there was a significant decrease in all groups from 3 days post-incubation. Interestingly, WSSV has a lower digestion rate (Average 13.8% of three shellfish species) in digestive enzymes compared to the reduction rate (52.5%) in seawater at a day of incubation, suggesting the stability of WSSV was prolonged in the shellfish digestive tract. Furthermore, WSSV was identified from the exudate produced during the frozen shrimp thawing process as 1.29×105 genome copies/mL and PBS-containing-infected shrimp tissue (107 genome copies/g of tissue). Therefore, the possibility of transmission via wastewater and by-product produced during the processing and repacking of frozen shrimp was confirmed.
To investigate WSSV transmission between WSSV-accumulated shellfish and shrimp, shellfish (oyster, mussel, and clam) were cohabitated with WSSV-infected shrimp for bioaccumulation. Notably, in the higher WSSV concentrations in bioaccumulation process (107 viral genome copies/mL), accumulated WSSV in the shellfish gill and digestive gland tissues increased to 2.52 and 3.90×105 viral genome copies/mg, respectively, and can be detected from shellfish tissues at 120 hrs after depuration. These results suggest that accumulated WSSV in shellfish is dependent on the amount of WSSV in seawater, and even remains in shellfish tissues for a longer time. Notably, WSSV-accumulated shellfish tissues could induce 100% of cumulative mortality in healthy shrimp, shellfish that accumulate WSSV in tissue have the potential to act as a vector for transmission of WSSV.
Using the results of previous research, we investigated the likelihood that WSSV would be introduced with imported shrimp and bivalves to Korea. Through applying standardized risk analysis, we found that both frozen shrimp and bivalves have the potential risk of import in Korea and proposed risk management measures to reduce the risks.
Overall, this study evaluated the possibility of both the frozen shrimp and shellfish for human consumption as a transmitter for WSSV, and proposed risk management measures to reduce the impact on the domestic aquaculture industry.
- Issued Date
- 2023
- Awarded Date
- 2023-02
- Type
- Dissertation
- Publisher
- 부경대학교
- Alternative Author(s)
- Joon Gyu Min
- Affiliation
- 부경대학교 대학원
- Department
- 대학원 수산생명의학과
- Advisor
- 김광일
- Table Of Contents
- 제 1장 서 문 １
1. 서 론 １
2. 국내 새우 및 패류 산업 현황 ６
2.1. 국내 양식 생산량 현황 ６
2.1.1 국내 새우류 생산 현황 ６
2.1.2 이매패류의 생산 현황 10
2.2. 국내 수산생물 검역 현황 13
2.2.1 수산물 검역 관련 법률 조항 및 과정 13
2.2.2. WSSV 지정 검역물(갑각류 및 이매패류) 검역 현황 17
3. 수산동물전염병 수입위험평가 방법 22
3.1 수입 위험 평가 22
3.2 수입위험분석의 절차 및 방법 24
3.2.1 위해요소 확인 24
3.2.2 위험 평가 25
3.2.3 위험 추정 31
4. 위해요소 확인 35
제 2장 흰반점 바이러스 유입 평가 39
1. 서 론 39
2. 재료 및 방법 42
2.1 냉동새우 42
2.2 패류 44
2.3 바이러스 핵산 분리 46
2.4 새우 및 패류에서의 WSSV 검출 47
2.4.1 WSSV 검출을 위한 PCR 47
2.4.2 바이러스의 정량 분석 48
2.5 WSSV InDel-II region 분석 50
2.5.1 Primer design 50
2.5.2 PCR for Indel-II variation region 52
2.5.3 Cloning 53
2.5.4 Sequencing 54
2.6 InDel-II region에 대한 병원성 및 유전적 변동성 확인 55
2.6.1. 실험 새우 및 접종액 55
2.6.2 InDel-II region의 결손 크기에 따른 흰다리새우에 대한 병원성 비교 56
2.7 흰다리새우 인위감염을 통한 InDel-II region의 변동성 확인 57
2.8 통계 분석 58
2.9 유입 평가 59
3. 결과 62
3.1 모니터링 62
3.1.1 냉동새우에서의 WSSV 검출 62
3.1.2 국내 유통 패류에서의 바이러스 검출 66
3.1.3 양식장 근처 패류에서의 바이러스 검출 71
3.1.4 냉동 새우에서 검출된 WSSV의 정량적 분석 73
3.2 WSSV의 InDel-II 유전자 분석 75
3.2.1 냉동 새우에서의 유전자 분석 75
3.2.2 패류에서의 유전자 분석 78
3.3 InDel-II region의 deletion size에 따른 병원성 확인 80
3.4 InDel-II region의 변동성 확인 82
3.5 유입 평가 84
4. 고찰 91
제 3장 흰반점바이러스 노출 평가 99
1. 서 론 99
2. 재료 및 방법 103
2.1 간접적 전이 위험성 평가 103
2.1.1 바이러스 103
2.1.2 패류 소화선에서의 PCR inhibition rate 104
2.1.3 해수 및 담수에서의 WSSV 안정성 확인 105
2.1.4 패류 소화 효소에 대한 안정성 분석 106
2.1.4.1 패류 소화 효소에서의 WSSV 소화율 분석 106
2.1.4.2 다른 수생 바이러스와의 digestive rate 비교 107
2.1.5. WSSV 감염 냉동 새우의 전파 위험성 확인 108
2.1.5.1 냉동 새우의 삼출액에서의 WSSV 확인 108
2.1.5.2 조직에 따른 삼출된 WSSV 량 확인 109
2.2. 직접적 전이 위험성 평가 110
2.2.1 다양한 Indel-II variant의 접종에 따른 해수 중 바이러스 농도 확인 110
2.2.2 실험 패류 111
2.2.3 WSD 인위 감염 새우와 패류의 cohabitation 112
2.2.3.1. 인위감염 새우와 패류의 cohabitation을 통한 축적 및 방출 112
2.2.3.2. 높은 농도에서의 cohabitation에 의한 축적 및 방출 114
2.2.4 패류 조직 내 축적된 WSSV의 감염력 확인 116
2.3 통계 분석 117
2.4 노출 평가 118
3. 결과 122
3.1 간접적 전이 위험성 평가 122
3.1.1 패류 조직에서의 inhibition rate 확인 122
3.1.2. 해수 및 담수에서의 WSSV 안정성 분석 124
3.1.3 다른 수생 바이러스와의 digestive rate 비교 129
3.1.4 냉동 새우 삼출액에서의 WSSV 검출 131
3.1.5 WSSV 감염 냉동 새우의 조직별 WSSV shedding 133
3.2 직접적 전이 위험성 평가 135
3.2.1 다양한 InDel-II variant WSSV strain 접종에 따른 해수에서의 바이러스 농도 확인 135
3.2.2 WSSV 인위감염 새우와 패류의 cohabitation을 통한 축적 및 방출 137
3.2.3 높은 농도에서의 cohabitation을 통한 축적 및 방출 141
3.2.4 패류 조직 내 축적된 WSSV의 감염 및 전파력 분석 146
3.3 노출 평가 148
4. 고찰 155
제 4장 위험 추정 및 위험관리 방안 165
1. 수입 위험 분석 165
1.1. 본 연구에서의 전염성 관련 연구 요약 165
1.2. 새우 및 이매패류의 흰반점바이러스에 대한 정착 및 확산 가능성 170
1.3 결과 평가 172
1.4 위험 추정 173
2. 국내 흰반점병에 관한 관리 방안 및 고찰 175
요 약 189
참고문헌 193
- Degree
- Doctor
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