| Summary: | 碩士 === 國立成功大學 === 生物科技研究所碩博士班 === 92 === Oral administration is a convenient, labor-saving vaccination method for aquaculture as compared to injection or immersion, and is less stress to the animals. In this study, we elaborated a previously developed oral vaccine
construction method based on the food chain concept. Recombinant antigen protein was expressed in the bacteria, and fed to the Artemia. Antigen encapsulated in Artemia can evade the gastro-intestinal digestion of target
animals. In addition, various subunit oral vaccines for different origins of pathogens can be prepared by cloning the putative antigen proteins. To evaluate the efficacy of oral vaccine, grouper (Epinephelus sp.) and viral
nervous necrosis disease (VNN) was chose as the challenge model. VNN caused mass mortalities of hatchery-reared grouper larva and juveniles in
Taiwan, and neither injection nor immersion vaccine was practical because the infection usually took place in very early stage. To test the efficacy of vaccine, a challenge model has to be established, infection by intraperitoneal injection of nervous necrosis virus (NNV) was done. RT-PCR was used to follow the infection, positive signals were detected at brain, eyes, intestine and muscle 7 days after challenge; signals were detected at brain, eyes and intestine but not in muscle 14 days after challenge. Immunohisochemistry analysis of diseased grouper’s tissue section proved that the pathological changes of nervous tissues
(including brain and eyes) were caused by nervous necrosis virus. These observations, indicated that the laboratory infection was similar to the natural infection. Age of fish was also a critical factor of NNV infection. 8 weeks after challenge （dosage:1×107 TCID50/fish）, younger grouper (average body length shorter than 2.5 cm, 40 days post hatch) suffered a higher mortality than older
grouper (average body length longer than 2.5 cm, 60 days post hatch) usually was resistant to infection. Oral vaccine encapsulated in Artemia can protect antigen proteins from the digestive tract and be absorbed at the hindgut of
grouper as demonstrated in two experiment where green fluorescent protein marker was used as marker and an immunohistochemistry examination using NNV antigen as marker. Enzyme-linked immunosorbent assay showed that the grouper’s anti-green fluorescent protein specific antibody was increased 7 days after vaccination. In the challenge test, 20 grouper juveniles were orally vaccinated by feeding on E.coli encapsulated with recombinant NNV coat protein, and then challenged. 60 days after vaccination, the survival rate of control and vaccinated group was 45% and 80%, respectively, and RPS was
63.6%, that demonstrated the NNV oral vaccine was an effective way to immunize groupers.
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