| Summary: | 博士 === 國立臺灣大學 === 動物學研究所 === 101 === Nervous necrosis virus (NNV) has caused mass mortality in many species of cultured marine fish, especially at the larval and juvenile stages. Fish larvae can be infected with NNV through horizontal and vertical transmission. To prevent the NNV infection of early larvae, bath immunization of inactivated vaccine and oral immunization of recombinant vaccine have been reported to be effective in protection. However, the information of immune response of grouper early larvae after bath or oral immunization is still limited. In this study, grouper larvae were bath or orally immunized with inactivated NNV and the expression levels of immune genes were analyzed by real-time PCR. The early expressions of innate immunity genes (IL-1β, MX) and adaptive immunity genes (MHC-I, MHC-II, CD8, IgT and IgM) of the bath and orally immunized groupers were found to manifest on the 3rd and 5th day post immunization. In the viscera of both immunized fish, the expression levels of cellular immunity marker (MHC-I, CD8), humoral immunity marker (IgM), and innate immunity marker (IL-1β and MX) were all significantly higher than that of the non-immunized fish from one to four weeks post immunization. In addition, only bath immunization induced the elevation of IgT gene expression in the gill; while, only oral immunization raised the IgT gene expression level in the gut. The above data suggested that the previously reported protection against NNV infection through bath
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and oral immunization in grouper larvae, are related to the contribution of vaccine-induced elevation of cellular immunity genes (MHC-I, MHC-II and CD8) and immunoglobulin genes (IgM and IgT).
To prevent the vertical transmission of NNV, an effective immunization program was developed and pre-monitored in adult groupers (Epinephelus coioides) with average body weight of 1.35 kg. The highest neutralizing antibody titers were found in the fish intramuscularly injected with adjuvanted NNV vaccine at the dose of 109 TCID50 kg−1, and the enhanced 4-fold neutralization antibody titer could sustain up to 17 months post-vaccination (mpv). The same immunization program was then applied to grouper broodstocks of Epinephelus tukula with body weights among 35-60 kg. The levels of NNV-specific antibodies in the homogenates of the eggs were chased for 5 months. The neutralizing antibody titers found in the eggs from the vaccinated broodfish were higher than that from the non-vaccinated fish. By nested RT-PCR, NNV became detectable in the eggs from the non-vaccinated fish in Month 5, but not in the eggs of the vaccinated fish. It is therefore suggested that vaccination is potentially a practical measure to reduce the risk of vertical transmission of NNV from the grouper broodfish under repeated spawning stress.
We discovered that the pathogenicity of NNV to the 80 days post-hatch (dph) barramundi is lower than that to the 14 dph barramundi. Following NNV challenge,
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no mortality occurred in the 80 dph barramundi, but NNV RNA2 and barramundi Mx (BMx) gene expression was detectable in the brain and liver. The 80 dph barramundi pre-challenged with NNV became more resistant to the following RSIV challenge (mortality: 62%) compared to the NNV-free barramundi challenged with RSIV (mortality: 100%). A similar phenomenon was revealed in NNV-persistently infected barramundi brain (BB) cell line that RSIV proliferated less progeny virions in BB cells than in NNV-free cured BB (cBB) cells. The potential factors involved in the resistance of the persistently NNV-infected barramundi and BB cells to the secondary RSIV infection were examined in this study. The results indicated that barramundi anti-NNV polyclonal antibodies did not cross-neutralize RSIV, and NNV infection did not interfere with RSIV replication. However, the interferon (IFN) response and BMx gene expression in NNV-infected cBB cells could suppresses the RSIV proliferation. It is therefore suggested that the NNV-induced IFN response and BMx expression are responsible for the resistance against RSIV infection.
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