- 學位論文
神經壞死症病毒及虹彩病毒單價與雙價疫苗對龍膽石斑之保護效力
Protection efficacy of NNV, GIV and NNV-GIV vaccines in giant groupers
摘要
石斑魚為臺灣重要養殖魚種，具有高經濟價值。神經壞死症病毒(nervous necrosis virus, NNV)及石斑虹彩病毒(grouper iridovirus, GIV)是石斑魚最重要的兩種病毒性病原，特別是在幼魚的養成階段，常先後或一起感染而造成重大疫情。疫苗是防疫病毒性疾病的有效策略，但目前只有NNV及GIV單價疫苗之研究報告，尚無結合這兩種病毒之雙價疫苗的報告或商品。本研究目的為比較NNV 與GIV的單價不活化疫苗及這兩種病毒之雙價不活化疫苗對龍膽石斑的保護效力。實驗結果顯示，NNV單價及NNV-GIV雙價疫苗皆能誘發免疫魚產生高力價(ND50)之NNV中和抗體，且單價與雙價疫苗的中和抗體力價沒有顯著性差異。兩種疫苗免疫過的魚在NNV肌肉注射攻毒後，累積死亡率和對照組的落差大於40%，皆能達到有效保護之標準，免疫組魚血清的ND50都在473以上，且免疫魚中和力價高(ND50 ≥ 781)的組別，相對存活率(RPS)也較高(81.8~92.9)；以ELISA檢測這兩種疫苗誘發的NNV抗體總量，皆在免疫後第2~3週達到高峰，第4~24週的抗體力價雖下降，但仍顯著高於對照組血清中的NNV抗體總量。GIV單價與NNV-GIV雙價不活化疫苗誘發GIV中和抗體力價明顯低於不活化NNV疫苗所誘發的NNV中和抗體力價，但這兩種疫苗免疫魚經GIV攻毒後，RPS卻高達73.3~100。檢測免疫魚脾臟中細胞免疫相關基因的表現，發現雙價免疫組的MHC-I與MHC-II基因表現量在免疫後2週均顯著高於對照組，且CD8α在免疫後2週及CD4在免疫後4週的基因表現量相較於對照組都有上升的趨勢，顯示細胞免疫可能對雙價疫苗的保護效果有一部份貢獻。總結上述結果，NNV-GIV雙價疫苗對龍膽石斑苗具有抗NNV及GIV感染的保護效果，且效力相當於NNV與GIV單價疫苗，可減少分別接種NNV及GIV兩種單價疫苗所耗的人力成本及魚的緊迫次數。
並列摘要
Grouper is an economically important fish species for aquaculture industry in Taiwan. Nervous necrosis virus (NNV) and grouper iridovirus (GIV) are two major viral pathogens of grouper fries and fingerlings, and have caused mass mortality during epidemics. Vaccination is an effective prophylaxis strategy for viral disease. To date, many research papers focus on NNV and GIV monovalent vaccines, but no report concerns NNV-GIV bivalent vaccine. The aim of this study is to compare the protection efficacy of NNV-GIV bivalent vaccine with those of NNV and GIV monovalent vaccines in giant groupers. At 4 weeks post vaccination (wpv), both NNV monovalent and NNV-GIV bivalent vaccines could induce high neutralizing antibody titers (ND50 ≥ 473) against NNV, and protect fish against NNV infection with cumulative mortalities 40% lower than that of control fish. High relative percent survival (RPS = 81.8 and 92.9) after NNV challenge tests were consistently observed in the fish with high neutralizing antibody titers (ND50 ≥ 781). By indirect ELISA, NNV-specific antibody titers in the serum of immunized fish peaked at 2 and 3 wpv, and kept significantly higher than those of control fish until 24 wpv. On the contrary, the neutralizing antibody titers against GIV induced by GIV and NNV-GIV vaccines were much lower than those against NNV induced by NNV monovalent and bivalent vaccines; however, the RPS of GIV monovalent and bivalent vaccine groups were all higher than 70 in GIV challenge experiments. In bivalent vaccine-immunized fish, the expression levels of MHC-I and MHC-II gene at 2 wpv were significantly higher than those in the control fish; furthermore, the expression levels of CD8α gene at 2 wpv and CD4 gene at 4 wpv both elevated. Therefore, it is suggested that NNV-GIV bivalent vaccine may also provide protection through induction of cellular immunity. In conclusion, NNV-GIV bivalent vaccine could provide good protection against NNV and GIV similar to two monovalent vaccines, and it will reduce labor cost and injection stress of fish during vaccination.