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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳媺玫 | |
| dc.contributor.author | Ting-Fu Wang | en |
| dc.contributor.author | 王廷輔 | zh_TW |
| dc.date.accessioned | 2021-06-16T10:22:38Z | - |
| dc.date.available | 2016-08-23 | |
| dc.date.copyright | 2013-08-23 | |
| dc.date.issued | 2013 | |
| dc.date.submitted | 2013-08-16 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/60590 | - |
| dc.description.abstract | 格氏乳酸球菌 (Lactococcus garvieae) 為重要的水產動物病原，其感染對象包含多種淡、海水魚宿主，在夏季高水溫期經常因為疾病爆發而造成嚴重經濟損失，臨床上對於發病魚隻雖然可以選擇有效的抗生素治療，但往往投藥成效有限，並且在疾病控制後又可能反覆發生。早期文獻指出許多細菌性病原可能藉由在體內形成生物膜，而增加對抗菌劑之抵抗力，導致投藥治療效果不佳。因此本實驗針對近年來臨床上分離之L. garvieae進行最佳生物膜的培養條件測定，評估其生物膜形成能力，分析比較不同生物膜形成能力菌株在生長曲線、藥物感受性以及對巨噬細胞之抗吞噬與清除能力，再分別挑選形成能力較強與較弱之菌株，以兔抗偵測生物膜與細菌懸浮型態之抗體，比較細菌與生物膜之表面抗原之差異，並偵測體內生物膜之分布與形成。結果顯示不同生物膜形成能力之菌株，在生長速度、抗藥性、抗吞噬能力與清除能力皆可見顯著性差異，並於臨床發病與不顯性感染之魚隻皆可偵測到體內生物膜之訊號，且不顯性感染之魚隻受到相當之環境壓力時又會重新發病，顯示體內生物膜之形成確實可能成為臨床上L. garvieae難以治療，又反覆發病造成持續性感染的原因。 | zh_TW |
| dc.description.abstract | Lactococcus garvieae is one of the important aquaculture pathogen in Taiwan. It causes illness including freshwater and seawater teleosts. It causes high mortality while the water temperature rising, and latent infection on effective antibiotic treatment. The recurrence of diseases usually could be seen in practice. Literature review shows many bacterial pathogens form biofilm to enhance their resistant against antibiotics, and resulted into a worse prognosis. In view of this, we isolated several L. garvieae strains from clinical cases, and we tried to find the best biofilm formation condition, then compared the characteristics between the strong and weak biofilm formation ability strains such as growth curve, drug susceptibility, anti-phagocytosis and cell surviving experiment. To detect the distribution of biofilm formation in vivo, we produced an antibody to detect biofilm and planktonic bacteria. The results showed a significant difference in growth curve, drug susceptibility, anti-phagocytosis and cell surviving experiment between strong and weak biofilm formation ability strains. Besides, we located the biofilm signal not only in clinical cases, but also in latent infection which caused illness recurrence while stress increasing. In our conclusion, the biofilm formation of L. garvieae in vivo is the reason of control difficultly, breaking out recurrently, and persistent infection. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T10:22:38Z (GMT). No. of bitstreams: 1 ntu-102-R00629013-1.pdf: 4315276 bytes, checksum: a595b91510ef1c352432285f6fa47d1e (MD5) Previous issue date: 2013 | en |
| dc.description.tableofcontents | 目錄
摘要…………………………………………………………………………………….I Abstract………………………………………………………………………………..II 目錄…………………………………………………………………………………..III 圖次…………………………………………………………………………………VII 表次………………………………………………………………………………...VIII 第一章 緒論………………………………………………………………………1 第二章 文獻回顧…………………………………………………………………3 2.1 格式乳酸球菌………………………………………….…..…….…...……..3 2.1.1 背景與生化性狀………………..…………………………………..3 2.1.2 宿主與流行病學……………………………………………………3 2.1.3 乳酸球菌感染症與剖檢病變………………………………………4 2.2 生物膜介紹………………………………………………………………….5 2.2.1 生物膜簡介…………………………………………………………5 2.2.2 生物膜基質…………………………………………………………6 2.2.3 生物膜與水產養殖環境之相關性…………………………………8 2.2.4 生物膜在臨床上的問題……………………………………………9 2.2.5 生物膜產生抵抗性之機制………………………………….……..11 2.2.6 生物膜的控制……………………………………………….……..12 2.2.7 生物膜的偵測……………………………………………….……..16 第三章 材料與方法 3.1 實驗設計及流程……………………………………………………….…...18 3.2 魚類來源乳酸球菌株收集與菌種鑑定…………………………….……...19 3.2.1 溶血型判定……………..…………………………………….……19 3.2.2 革蘭氏染色…………………………………………………….…..19 3.2.3 觸酶試驗…………………………………………………...………..20 3.2.4 聚合酶鏈鎖反應……………..……………...………….…...………20 3.3 實驗室培養條件……………………………………...………………...……23 3.3.1 最佳生物膜形成培養條件分析……………...…………………...….23 3.3.2 細菌數定量-稀釋平板培養法………………..…….………………...24 3.3.3 生長曲線之測定………………………………...….………………...24 3.4 藥物感受性試驗………………………………………...…………………...25 3.4.1 抗生素最小抑菌濃度試驗………………………...…………………25 3.4.2 最小生物膜清除濃度試驗…………………………...………………26 3.5 抗體製備及免疫細胞化學染色偵測比較…………………...…………...…27 3.5.1 偵測生物膜之多株抗體製備………………………….……………..27 3.5.2 偵測細菌顆粒之多株抗體製備……………………………………...27 3.5.3 免疫細胞化學染色………………………………………………..….28 3.6 細菌表面抗原偵測及比較……………………………..……………………30 3.6.1 聚丙烯醯胺膠片電泳………………………………..……………….30 3.6.2 蛋白質轉印法…………………………………………..…………….33 3.6.3 西方墨點法………………………………………………..………….35 3.7 生物膜體內偵測……………………………………………………..………36 3.7.1 動物感染試驗……………………………………………………..….36 3.7.2 潛伏感染後以緊迫因子誘導發病試驗……………………………...36 3.7.3 細菌凝集試驗………………………………………………………...37 3.7.4 免疫組織化學染色…………………………………………………...38 3.8 巨噬細胞吞噬與清除能力之分析………………………………………..…40 3.8.1 分離吳郭魚頭腎巨噬細胞…………………………………………...40 3.8.2 抗吞噬能力分析-免疫細胞化學染色…………………..……………41 3.8.3 抗吞噬能力分析…………………………………...……………….43 3.8.4 細胞內存活試驗…………………………………………...……….44 3.8.5 細胞存活量…………………………………………………...…….45 第四章 實驗結果 4.1 菌株收集與種別鑑定……………………………………………………...46 4.2 實驗室生物膜最佳培養條件結果………………………………………...46 4.3 生長曲線之測定…………………………………………………………...46 4.4 抗生素感受性試驗結果…………………………………………………...47 4.5 偵測生物膜抗體製備與免疫細胞化學染色法分結果…………………...47 4.6 細菌顆粒與生物膜表面抗原比較結果………………...…………………48 4.7 生物膜體內偵測…………………………………………………………...49 4.7.1實驗魚感染試驗…………………………………………………….49 4.7.1.1 實驗魚感染後臨床症狀……………………...……………..49 4.7.1.2 實驗魚感染後病原分離…………………………………….49 4.7.1.3 實驗魚感染之血清凝集試驗…....………………………….50 4.7.2 臨床L.garvieae發病魚隻之檢體收集……………..…...................50 4.7.3 潛伏感染後以緊迫因子誘導發病試驗……………………………50 4.7.4 組織病理學變化……………………………………………………51 4.7.5 免疫組織化學染色…………………………………………………51 4.8 巨噬細胞之吞噬與清除能力分析………………………………………...53 第五章 討論 5.1 菌種鑑定與生物膜形成能力分析………………………………………..56 5.2 藥物敏感性試驗…………………………………………………………..57 5.3 細菌顆粒與生物膜表面抗原比較………………………………………..58 5.4 動物感染試驗……………………………………………………………..59 5.5 巨噬細胞之吞噬與清除能力分析………………………………………..62 第六章 參考文獻…………………………………………...…….…………….86 圖次 圖一、生物膜形成示意圖……………………………………………………..………6 圖二、生物膜與懸浮形態被吞噬後引起之免疫反應途徑…………………………11 圖三、數量感測系統抑制之示意圖…………………………………………………13 Fig 1 生長曲線之比較…..………………………………………………………..…..65 Fig 2 不同抗體對L. garvieae之免疫細胞化學染色結果……………………..……67 Fig 3 細菌與生物膜表面抗原之西方墨點法結果………………………………..…69 Fig 4 組織病理學病變…………………………………………………………..……70 Fig 5 組織病理學病變 (B&B染色)……………………………………………..…..71 Fig 6 免疫組織化學染色 (偵測生物膜與懸浮細菌之染色結果比較)………….....72 Fig 7 免疫組織化學染色 (偵測生物膜與懸浮細菌之染色結果比較，)...……......73 Fig 8 免疫組織化學染色 (其他臟器)…………………………………………….....74 Fig 9 免疫細胞化學染色 (抗吞噬能力分析)…………………………………….....75 Fig 10 巨噬細胞之吞噬與清除能力分析 (抗吞噬能力分析結果)………...............76 Fig 11 巨噬細胞之吞噬與清除能力分析 (細胞內存活結果)……………………...78 表次 Table 1 本實驗研究使用菌株一覽表………………………………………………78 Table 2 生物膜形成能力分級……………………………………………………....81 Table 3 藥物感受性結果 (最小抑細濃度與最小生物膜清除濃度)……………...81 Table 4 動物感染試驗 (Day1-10之細菌分離與免疫組織化學染色結果)………82 Table 5 動物感染試驗 (Day15-60之細菌分離與免疫組織化學染色結果)……..83 Table 6 臨床發病魚隻 (細菌分離與免疫組織化學染色結果)…………………...84 Table 7 實驗感染吳郭魚潛伏後誘導發病試驗結果……………………………...84 | |
| dc.language.iso | zh-TW | |
| dc.subject | 生物膜 | zh_TW |
| dc.subject | 格氏乳酸球菌 | zh_TW |
| dc.subject | 持續感染 | zh_TW |
| dc.subject | 免疫組織化學染色 | zh_TW |
| dc.subject | 抗吞噬能 | zh_TW |
| dc.subject | Lactococcus garvieae | en |
| dc.subject | biofilm | en |
| dc.subject | persistent infection | en |
| dc.subject | anti-phagocytosis | en |
| dc.subject | immunohistochemistry | en |
| dc.title | 比較不同生物膜形成能力之魚類來源乳酸球菌在體內持續感染期間與體外抗吞噬能力之差異 | zh_TW |
| dc.title | Compare the Difference of Biofilm Formation Ability from Fish Lactococcus spp. during the Period of Persistent Infection in vivo and Anti-phagocytosis in vitro | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 101-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 蔡信雄,潘銘正,李國誥 | |
| dc.subject.keyword | 格氏乳酸球菌,生物膜,持續感染,抗吞噬能,免疫組織化學染色, | zh_TW |
| dc.subject.keyword | Lactococcus garvieae,biofilm,persistent infection,anti-phagocytosis,immunohistochemistry, | en |
| dc.relation.page | 101 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2013-08-16 | |
| dc.contributor.author-college | 獸醫專業學院 | zh_TW |
| dc.contributor.author-dept | 獸醫學研究所 | zh_TW |
| 顯示於系所單位： | 獸醫學系 | |
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