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請用此 Handle URI 來引用此文件: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/26763
完整後設資料紀錄
DC 欄位值語言
dc.contributor.advisor張繼堯
dc.contributor.authorChia-Huei Chenen
dc.contributor.author陳佳慧zh_TW
dc.date.accessioned2021-06-08T07:24:28Z-
dc.date.copyright2011-08-19
dc.date.issued2011
dc.date.submitted2011-08-10
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dc.identifier.urihttp://tdr.lib.ntu.edu.tw/jspui/handle/123456789/26763-
dc.description.abstractGrouper aquaculture has been threaten by grouper iridovirus (GIV) and nervous necrosis virus (NNV) for many years, and the insufficient apply of long chain unsaturated fatty acids for grouper metamorphosis development and neural growth, all result in a high mortality rate for grouper fry. Therefore, to culture a high quality grouper fry of specific pathogen free (SPF) becomes an important issue for nowadays grouper aquaculture. Rotifer and artemia nauplius, which are commonly used as live feed organisms are low contents of high unsaturated fatty acids (HUFA), so feeding
with these feed organisms effect the successful rate of fry metamorphosis. Although there are many ways of enrichment techniques and products develop to improve these feed organisms, it’s hard to provide proper nutrition composition for grouper fry. The abundant species of copepods, character of appropriate size for grouper fry and high quantity of HUFA all made copepods as the best natural foods for marine fishes fry. However, most of the feeding copepods in grouper cultivation are collected from fish and shrimp culture farm, therefore feeding grouper with these copepods is possible to transport viral pathogens. We had detected the existence of iridovirus from collected copepods in south Taiwan aquatic farm, and showing copepods can be a possible transmission vector for iridovirus. In this experiment, Tigriopus japonicus was used to investigate its possible role of iridovirus and nervous necrosis virus transmission. The results revealed that the best T. japonicus population growth rate was obtained from feeding Isochrysis galbana, then Tetraselmis chui. In addition, T. japonicus can also be cultivated with photosynthetic bacteria and the population growth rate is nearly to that of feeding with Tetraselmis chui. After virus experimental infection and different III periods of fresh seawater culture, PCR, cell titration and transmission electron microscopy were used to check out the existence of viruses in the infected T. japonica.The PCR result shows that GIV can be detected up to 96h in experimental infected T. japonica. The TEM result reveals that some viral like particle can be observed in the digestive tract of copepod. Furthermore, in the experiment of cell titration, both GIV and NNV have been demonstrated viral activity to infect grouper cell, but the viral activity cannot exist over time. It represent that both virus cannot be replicated in T.japonicus, and after periods new fresh seawater culture, the virus can be clean out in copepods. Furthermore, we can get virus-free copepods when T. japonicus is neutralized with anti-virus antibody. Those characteristic can help us to culture SPF copepods for further application.
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dc.description.tableofcontents目錄
摘要 ................................................................................................................ I
Abstract ........................................................................................................ II
目錄 ............................................................................................................. IV
圖目次 ......................................................................................................... VI
表目次 ......................................................................................................... VI
壹. 前言 ........................................................................................................................... 1
1.1 石斑魚水產養殖簡介 ...................................................................................... 1
1.2 虹彩病毒 ........................................................................................................... 1
1.3 神經壞死病毒 .................................................................................................. 3
1.4 不飽和脂肪酸和魚類營養需求 ...................................................................... 3
1.5 餌料生物 .......................................................................................................... 5
1.6 橈足類 .............................................................................................................. 6
1.7 台灣的橈足類培養 ........................................................................................... 8
1.8 日本虎斑猛水蚤 ............................................................................................... 8
貳、材料與方法 ............................................................................................................ 11
2.1 實驗材料 ......................................................................................................... 11
2.1.1 日本虎斑猛水蚤及海水藻種 ............................................................ 11
2.1.2 海水藻種培育 .................................................................................... 11
2.1.3 DNA 萃取及聚合酶連鎖反應 ........................................................... 13
2.1.4 RNA 萃取及反轉錄聚和酶連鎻反應 ............................................... 13
2.1.5 膠體電泳及染色 ................................................................................ 14
2.1.6 細胞培養和中和試驗 ........................................................................ 14
2.2 日本虎斑猛水蚤及海水藻之培育 ................................................................. 14
2.2.1 海水藻的培養 .................................................................................... 14
2.2.2 日本虎斑猛水蚤的培養及族群生長測試 ........................................ 15
2.3 日本虎斑猛水蚤對虹彩病毒(grouper iridovirus, GIV)之傳播性質 ........... 15
2.3.1 石斑魚虹彩病毒效價測定 ................................................................ 15
2.3.2 不同效價石斑魚虹彩病毒浸染水蚤不同時間之殘存實驗 ............ 16
2.3.3 抽取DNA ........................................................................................... 17
2.3.4 聚合酶連鎖反應(Polymerase Chain Reaction, PCR) ........................ 17
2.3.5 石斑魚虹彩病毒巢式聚合酶連鎖反應(GIV nested-PCR) ............... 18
2.3.6 瓊脂膠體電泳分析 ............................................................................ 18
2.3.7 石斑魚虹彩病毒活性測試 ................................................................ 18
2.3.8 石斑魚虹彩病毒中和試驗 ................................................................ 19
2.4 日本虎斑猛水蚤對神經壞死病毒(nervous necrosis virus, NNV)之傳播性質
V
............................................................................................................................... 19
2.4.1 神經壞死病毒效價測定 .................................................................... 19
2.4.2 不同效價神經壞死病毒浸染水蚤不同時間之殘存實驗 ................ 20
2.4.3 抽取RNA ........................................................................................... 21
2.4.4 反轉錄聚合酶連鎖反應(Reverse Transcription-Polymerase Chain
Reaction, RT-PCR) ...................................................................................... 21
2.4.5 神經壞死病毒之巢式聚合酶連鎖反應(NNV nested-PCR) ............. 22
2.4.6 神經壞死病毒活性測試 .................................................................... 22
2.4.7 神經壞死病毒中和試驗 .................................................................... 23
2.4.8 電子顯微鏡鏡檢日本虎斑猛水蚤超薄切片 .................................... 23
參、結果 ........................................................................................................................ 24
3.1 不同食物對日本虎斑猛水蚤之族群生長影響 ............................................ 24
3.2 日本虎斑猛水蚤及餵食日本虎斑猛水蚤藻類之病毒帶原測試 ................ 24
3.3 日本虎斑猛水蚤對石斑魚虹彩病毒之傳播性質 ........................................ 24
3.3.1 日本虎斑猛水蚤之石斑魚虹彩病毒殘存PCR 檢測 ...................... 25
3.3.2 日本虎斑猛水蚤之石斑魚虹彩病毒殘存活性檢測 ........................ 25
3.3.3 石斑魚虹彩病毒中和試驗之病毒殘存活性 .................................... 26
3.3.4 電子顯微鏡鏡檢日本虎斑猛水蚤殘存石斑魚虹彩病毒 ................ 26
3.4 日本虎斑猛水蚤對神經壞死病毒之傳播性質 ............................................ 26
3.4.1 日本虎斑猛水蚤之神經壞死病毒殘存RT-PCR 檢測 .................... 26
3.4.2 日本虎斑猛水蚤之神經壞死病毒殘存活性檢測 ............................ 27
3.4.3 神經壞死病毒中和試驗之病毒殘存活性 ........................................ 27
3.4.4 電子顯微鏡鏡檢日本虎斑猛水蚤殘存神經壞死病毒 .................... 27
肆、討論 ........................................................................................................................ 28
伍、參考文獻 ................................................................................................................ 32
VI
圖目次
Figure 1. Population growth of T. japonicus fed with different microalgae diets .......... 41
Figure 2. Population growth of developmental stages of T. japonicus fed with photo
synthetic bacteria ............................................................................................................ 43
Figure 3. Detection of GIV in T. japonicus .................................................................... 44
Figure 4. Detection of GIV in microalgaes by nested-PCR. .......................................... 45
Figure 5. Detection of GIV-DNA in experimentally GIV infected T. japonicus of
different virus titer and immersion time. ........................................................................ 46
Figure 6. Cell titration of experimentally GIV infected T. japonicus. ........................... 47
Figure 7. Cell titration of experimentally GIV infected T. japonicus following GIV
specific antibody neutralization. ..................................................................................... 48
Figure 8. TEM observation of experimentally GIV infected T. japonicus. ................... 49
Figure 9. Detection of NNV-RNA in experimentally NNV infected T. japonicus. ....... 50
Figure 10. Cell titration of experimentally NNV infected T. japonicus. ........................ 51
Figure 11. Cell titration of experimentally NNV infected T. japonicus following NNV
specific antibody neutralization. ..................................................................................... 52
Figure 12. TEM observation of experimentally NNV infected T. japonicus. ................ 53
Figure 13. TEM observation of NNV virus particle ....................................................... 54
表目次
Table1. Titration of copepod grinding tissue fluid following challenge copepods with
GIV immersion ............................................................................................................... 55
Table 2. Titration of copepod grinding tissue fluid following challenge copepods with
GIV immersion and antibody neutralization .................................................................. 56
Table 3. Titration of copepod grinding tissue fluid following challenge copepods with
NNV immersion ............................................................................................................. 57
Table 4. Titration of copepod grinding tissue fluid following challenge copepods with
NNV immersion and antibody neutralization ................................................................. 58
dc.language.isozh-TW
dc.title探討日本虎斑猛水蚤在石斑魚虹彩病毒及神經壞死病
毒傳播上的可能角色
zh_TW
dc.titleAssessment of the possible role of Tigriopus japonicus
in grouper iridovirus and nervous necrosis virus transmission
en
dc.typeThesis
dc.date.schoolyear99-2
dc.description.degree碩士
dc.contributor.oralexamcommittee林正輝,蘇建國
dc.subject.keyword橈足類,餌料生物,病毒傳播,石斑魚,虹彩病毒,神經壞死病毒,zh_TW
dc.subject.keywordcopepods,live food organism,virus transmission,grouper,irido virus,nervous necrosis virus,en
dc.relation.page58
dc.rights.note未授權
dc.date.accepted2011-08-10
dc.contributor.author-college生命科學院zh_TW
dc.contributor.author-dept漁業科學研究所zh_TW
顯示於系所單位:漁業科學研究所

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