臺灣博碩士論文加值系統

弧菌感染症（Vibriosis）為吳郭魚養殖過程中常見的細菌性疾病之一，抗生素處理為目前主要的疾病預防或治療的方式，但容易造成環境汙染甚至產生具有抗藥性之細菌，因此抗菌胜肽被認為具有發展潛力，能做為取代抗生素的抗菌製劑。利用兩種來自吳郭魚的抗菌胜肽：tilapia piscidin 3（TP3）與tilapia piscidin 4（TP4）。已經有研究指出TP3與TP4對革蘭氏陽性菌與陰性菌皆有良好的抑菌效果，本研究探討TP3與TP4是否對創傷弧菌具有抗菌效果，並且以吳郭魚作為模式生物，使用腹腔注射方式，分別在細菌感染前後給予TP3或TP4提供保護及預防，觀察是否能提升對創傷弧菌感染之抵抗力，並進一步改善存活率。實驗結果顯示，先將創傷弧菌與20（µg/尾）之TP3或TP4混合後，再注射至吳郭魚體內其存活率分別為95.3%及88.9%，在創傷弧菌感染前30分鐘先注射30（µg/尾）之TP3或20（µg/尾）之TP4，吳郭魚存活率能提升至28.9%與37.8%，而在細菌感染後30分鐘分別注射30（µg/尾）之TP3或20（µg/尾）之TP4，吳郭魚存活率分別提升至33.3%及48.9%。同時，在吳郭魚肝臟微生物檢測實驗中，觀察到吳郭魚在創傷弧菌感染下，利用不同方式給予TP3或TP4時，觀察到肝臟創傷弧菌含量均較未處理組別低。透過real-time PCR觀察免疫相關基因表現量，在創傷弧菌感染後免疫相關基因大量表現，如Il-1β、Il-6、Il-8等，在經過TP3與TP4處理後表現量均有降低。in vitro實驗結果顯示，TP3與TP4能透過破壞創傷弧菌細胞表面及改變細胞外膜（outer membrane）通透性，造成創傷弧菌死亡。此外TP3與TP4在不同溫度與酸鹼環境中仍維持一定的抗菌活性，更進一步探討TP3與TP4是否能與抗生素產生協同作用抑制創傷弧菌，在1/2× MIC濃度下的TP3、TP4、Kanamycin、Ampicillin單獨使用對創傷弧菌之生長抑制並無影響，但當結合TP3、TP4與抗生素時，能有效抑制創傷弧菌生長。經由上述研究，我們期許抗菌胜肽能夠廣泛抑制各式不同的水產病原菌，降低養殖過程中由細菌感染所造成的損失，並進一步取代抗生素使用，減少多重抗藥性細菌產生。

Vibriosis is one of the most ubiquitous and serious disease in tilapia culture worldwide. Antibiotics treatment has been the common way to control disease but leading to occurrence of resistant bacterium. Antimicrobial peptides are considered that develop as novel therapeutic agents substituted for conventional antibiotics. TP3 and TP4 are antimicrobial peptide derived from Nile tilapia and has been previously reported to possess bactericidal activity against broad spectrum bacterium. Here, we investigated the inhibitory effect of TP3 and TP4 on Vibrio vulnificus (V. vulnificus) and used tilapia as model to study the impacts of co-treatment, pretreatment, and post-treatment with TP3 and TP4 on its protective efficacy. The result showed that co-treatment with V. vulnificus and TP3 (20 µg/fish) or TP4 (20 µg/fish) achieved 95.3% and 88.9% survival rates, respectively. When we co-injected TP3 or TP4 and V. vulnificus into tilapia, then rechallenged the fish with V. vulnificus after 28 days, the tilapia had survival rates of 35.6% and 42.2%, respectively. Pre-treatment with TP3 (30 µg/fish) or TP4 (20 µg/fish) for 30 minutes before infection with V. vulnificus resulted in high survival rates of 28.9% and 37.8%, respectively. Post-treatment with TP3 (20 µg/fish or 30 µg/fish) or TP4 (20 µg/fish) for 30 minutes after infection with V. vulnificus yielded high survival rates of 33.3% and 48.9%. In addition, pre-treatment, co-treatment, and post-treatment with TP3 or TP4 were all effective for decreasing the number of V. vulnificus and for promoting a significant decrease in tilapia mortality. Real-time PCR results also indicated that TP3 and TP4 treatment suppressed V. vulnificus induced immune responsive genes expression like interleukin (IL)-1β, IL-6, IL-8. In vitro results showed that TP3 and TP4 decreased growth and viability of V. vulnificus. In addition, bacterial morphology and outer membrane permeability were affected by TP3 and TP4. TP3 and TP4 combined with antibiotics exert the synergistic activity against V. vulnificus. Furthermore, TP3 and TP4 remained its antimicrobial activity under the different temperature or acid-base environment. In this study, we demonstrate that TP3 and TP4 show bactericidal activity against V. vulnificus. The use of TP3 and TP4 may provide a strategy of decreasing fish disease problems in aquaculture.

目錄
第一章 前言 5
1.1 全球與台灣吳郭魚養殖現況 5
1.2 吳郭魚養殖疾病與宿主防衛機制 7
1.3 水產養殖抗生素使用及其隱憂 10
1.4 抗菌胜肽 11
1.5 tilapia piscidins 13
1.6 抗菌胜肽發展潛力與應用價值 15
1.7 研究目的 15
第二章 實驗材料與方法 17
2.1 實驗材料 17
2.1.1 實驗動物 17
2.1.2 實驗菌株 17
2.1.3 儀器設備 17
2.1.4 試劑與藥品 18
2.1.5 實驗用引子 20
2.1.6 使用軟體與統計軟體 21
2.2 實驗方法 21
2.2.1 培養基、培養液與吳郭魚抗菌胜肽(tilapia piscidin)製備 21
2.2.2 創傷弧菌培養與建立生長曲線 22
2.2.3 TP3與TP4對創傷弧菌最小抑菌濃度/最小殺菌濃度測試 22
2.2.4 TP3與TP4對創傷弧菌生長抑制測試 22
2.2.5 TP3、TP4與創傷弧菌DNA結合試驗 23
2.2.6 TP3、TP4與抗生素之協同作用 23
2.2.7 溫度、pH值與蛋白酶對TP3與TP4抗菌活性之影響 24
2.2.8 TP3、TP4對創傷弧菌外膜通透性之影響 24
2.2.9 掃描式電子顯微鏡操作 25
2.2.10 穿透式電子顯微鏡操作 25
2.2.11 TP3與TP4與創傷弧菌對吳郭魚存活率影響 26
2.2.12 吳郭魚肝臟器官微生物檢測 27
2.2.13 吳郭魚組織核醣核酸萃取 27
2.2.14 反轉錄與即時定量聚合酶連鎖反應 28
2.2.15 統計分析 29
第三章 實驗結果 30
3.1 TP4對創傷弧菌抑制效果較佳 30
3.2 TP3、TP4影響創傷弧菌外膜通透性與造成形態改變 30
3.3 TP3、TP4能與細菌DNA結合 31
3.4 TP3與TP4在不同溫度及酸鹼值環境中仍具抗菌活性 32
3.5 TP3、TP4能與抗生素產生協同作用 34
3.6 TP3、TP4提高被感染吳郭魚之存活率 35
3.7 處理TP3與TP4降低創傷弧菌感染後吳郭魚肝臟細菌量 37
3.8 TP3、TP4降低由創傷弧菌引起之免疫相關基因表現量 38
第四章 討論 41
4.1 探討TP3與TP4如何改善被細菌感染吳郭魚之存活率 41
4.2 探討TP3與TP4在宿主免疫反應過程扮演之角色 42
4.3 探討TP3與TP4對創傷弧菌之殺菌機制 43
4.4 探討TP3與TP4在不同環境中的穩定性與抗菌效果 45
第五章 結論與展望 48
第六章 參考文獻 49




圖表目錄
圖1-1. 全球捕撈與養殖吳郭魚產量 6
圖1-2. 吳郭魚在不同養殖時期易感染之病原微生物 7
圖1-3. 抗菌胜肽與細胞膜之交互作用 13
圖3-1. 2× MIC之TP3與TP4可以有效殺死LD90之創傷弧菌 55
圖3-2. TP3與TP4可以對創傷弧菌細胞型態造成影響 56
圖3-3. 利用1-N-phenylnaphthylamine（NPN）uptake方法來分析TP3與TP4對於
細菌外膜通透性之影響 57
圖3-4. TP3、TP4能與創傷弧菌genomic DNA及plasmid DNA結合 58
圖3-5. TP3及TP4可與Ampicillin和Kanamycin產生協同殺菌效果 59
圖3-6. 選擇感染吳郭魚最適創傷弧菌濃度以及TP3、TP4最佳注射劑量 60
圖3-7. 混合TP3、TP4與創傷弧菌再注射至吳郭魚體內並不造成明顯死亡，並且
提高第二次感染時之存活率 61
圖3-8. 感染創傷弧菌前注射TP3與TP4能提升細菌感染後之存活率 62
圖3-9. 感染創傷弧菌後注射TP3與TP4能提升細菌感染後之存活率 63
圖3-10. 處理TP3、TP4降低被創傷弧菌感染吳郭魚肝臟細菌量 64
圖3-11. 於創傷弧菌感染前後給予TP3能降低細菌引起之免疫基因表現 65
圖3-12. 於創傷弧菌感染前後給予TP4能降低細菌引起之免疫基因表現 68

表2-1. 吳郭魚抗菌胜肽合成序列 21
表3-1. TP3、TP4、Kanamycin與Ampicillin對於創傷弧菌之最小抑菌濃度（minimum
inhibition concentration, MIC）及最小殺菌濃度（minimum bactericidal
concentration, MBC） 71
表3-2. 在不同溫度處理下，TP3、TP4、Kanamycin與Ampicillin對於創傷弧菌之
最小抑菌濃度（minimum inhibition concentration, MIC）及最小殺菌濃度
（minimum bactericidal concentration, MBC） 71
表3-3. TP3、TP4、Kanamycin與Ampicillin在不同酸鹼環境中，對於創傷弧菌
之抗菌活性之最小抑菌濃度（minimum inhibition concentration, MIC）及
最小殺菌濃度（minimum bactericidal concentration, MBC） 72
表3-4. TP3、TP4在不同蛋白酶分別處理10、30分鐘後，對於創傷弧菌之抗菌活
性之最小抑菌濃度（minimum inhibition concentration, MIC） 72

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劉文御等﹙民92﹚，養殖水產生物病害防治，水產試驗所特刊。2：185-189
劉富光等﹙民99﹚，台灣淡水魚類養殖﹙上﹚，水產試驗所特刊。11：27-44