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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 蔡向榮 | |
| dc.contributor.author | Shin-Ying Lin | en |
| dc.contributor.author | 林欣穎 | zh_TW |
| dc.date.accessioned | 2021-06-13T02:07:13Z | - |
| dc.date.available | 2007-07-16 | |
| dc.date.copyright | 2007-07-16 | |
| dc.date.issued | 2007 | |
| dc.date.submitted | 2007-06-29 | |
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Rapid identification of pathogenic bacteria by single-enzyme amplified fragment length polymorphism analysis. Diagn Microbiol Infect Dis 39 (2): 77-83, 2001. Zorman-Rojs O, Zdovc I, Bencina D, Mrzel I. Infection of turkeys with Ornithobacterium rhinotracheale and Mycoplasma synoviae. Avian Dis 44 (4): 1017-22, 2000. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/30528 | - |
| dc.description.abstract | 鼻氣管鳥桿菌 (Ornithobacterium rhinotracheale, ORT) 為一家禽呼吸道疾病之病原菌，近年來已為全球性分佈。ORT於國內有自雞、鴿子及火雞之分離報告，本研究於九十四年七月至九十五年八月間，分別至台灣地區屠宰場及禽鳥養殖場進行採樣，涵蓋物種包括雞隻、鴕鳥、鵪鶉及鴨等，此外亦進行人工飼養寵物鳥或野外鳥隻之病原分離。從各物種所採集之906個喉頭拭子樣本中，其分離率分別為雞20.7 % (29/140)、鴕鳥12.1 % (4/33)、鵪鶉2.8 % (6/211)、觀賞鳥1.2 % (1/87) 及鴨0 % (0/435)。上述分離株利用生化試驗套組API 20NE進行生化試驗，52.6 % (20/38) 生物碼為0-2-2-0-0-0-4、39.5 % (15/38) 生物碼為0-0-2-0-0-0-4及7.9 % (3/38) 生物碼為0-2-2-0-0-0-0；血清學分型方面，利用A血清型ORT兔源高免血清進行快速平板凝集試驗，結果顯示84.2 % (32/38)分離株屬於A血清型。38株分離株經瓊脂藥片擴散法進行12種抗菌劑之感受性試驗後，發現大多數分離株(＞80 %)對於colistin、gentamicin、neomycin、lincomycin及sulfamethoxazol/trimethoprim有高比例之抵抗性，對amoxycillin及ceftiofur則有較佳之感受性 (＞89 %)。將各分離株以OR16S-F1/R1引子對所增幅出之16S rRNA核酸片段經定序並與GenBank所得序列比對後，得知在不同分離株間其核苷酸相似度極高 (97.0-100 %)，但由親緣樹狀圖可發現台灣火雞分離株、鴿分離株、雲林縣為主的雞分離株則各自分屬於不同的cluster；為探討ORT基因多樣性，利用SE-AFLP及RAPD兩種分子生物學分型方式檢測ATCC 51464標準株及九十三至九十五年間之台灣ORT分離株。結果顯示雞及鵪鶉分離株與鴕鳥、鴿及鳳頭蒼鷹分離株為不同基因分型，而部分火雞分離株則與雞及鵪鶉分離株為相同分型，以AFLP及RAPD方式均能將不同物種所分離之ORT依基因輪廓分為不同分型，因此推論本菌具有亞種或其他種別的可能。為了解本菌對quinolone類藥物抗藥性的產生，以enrofloxacin E-test試條檢測九十三至九十五年間台灣ORT分離株最小抑制濃度，15.2 % (14/92) 對enrofloxacin具感受性 (≦ 0.25 µg/mL)，26.1 % (24/92) 具抵抗性 (≧ 2 µg/mL)；挑選部分分離株利用ORTgyrAF/R增幅出gyrA基因片段，經定序及與國外研究結果比對後，推論ORT之gyrA胺基酸位83 S → F的改變可能與本菌對quinolone類藥物的抗藥性有相當大的關係。 | zh_TW |
| dc.description.abstract | Ornithobacterium rhinotracheale (ORT), a poultry respiratory disease, is worldwidely distributed in commercial poultry industires as well as in wild birds. In Taiwan, ORT has been found in chickens, pigeons and turkeys. In this study, tracheal swabs were collected from different avain species from 2005 to 2006. The isolation rate were 20.7% (29/140), 12.1% (4/33), 2.8% (6/211), 1.2% (1/87) and 0% (0/435), from chickens, ostrichs, quails, pet and wild birds, and ducks respectively. Identification were done by API 20NE and the result showed that 52.6% (20/38) of the isolates belonged to biocode 0-2-2-0-0-0-4, and 39% (15/38) of them for biocode 0-0-2-0-0-0-4 and 7.9% (3/38) of them beloged to biocode 0-2-2-0-0-0-0. Rapid slide agglutination test were done by using standar serotype A antiserum and 84.2% (32/38) belonged to serotype A. More than 80% of the isolates were resistant to colistin, gentamicin, neomycin, lincomycin and sulfamethoxazol/trimethoprim, however, they were susceptible to amoxycillin, and ceftiofur based on the agar disc diffusion method. The isolated ORT strains of each species (chicken, pigeon, ostrich, quail, turkey and Asian Crested Goshawk) were further differentiated by molecular typing methods including 16S rRNA sequence, random amplified polymorphic DNA (RAPD), and single enzyme amplified fragment length polymorphism (SE-AFLP). In the results of 16S rRNA sequence, high genetic similarity (97-100%) were revealed except those of chicken-origin isolates from Yunlin county, pigeon-origin and turkey-origin isolates which were also divided into different clusters within the phylogenetic tree. In order to investigate the genetic polymorphysm among the ORT isolates, SE-AFLP and RAPD methods were conducted and analyzed with the ATCC 51464 strain. The results showed that the DNA patterns of chicken-origin quail-origin and partial turkey-origin isolates were different from those of ostrich-origin, pigeon-origin and Asian Crested Goshawk-origin isolates. Base on the feature that different-origin ORT isolates were separated by the SE-AFLP and RAPD methods, it is suggested that intraspecies differerce within species rhinotracheale or even other species than rhinotracheale may exist. Furthermore, to investigate the resistance to quinolones antibiotics of ORT, E-test were performed to exam the MIC values for the ORT isolates, 15.2% (14/92) of isolates were determined susceptible and 26.1% (24/92) were determined being resistant to enrofloxacin. Moreover, 30 ORT isolates including enrofloxacin-susceptible and enrofloxacin-resistant strains were selected randomly for further examination of the existence of gyrA gene fragment after the amplification by primers ORTgyrAF/R. By the results of sequencing and comparing with other previous studies, it is our speculation that the change of nucleic acid position 635 (C635T) play an important role in the quinolone resistance of ORT. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T02:07:13Z (GMT). No. of bitstreams: 1 ntu-96-R94629035-1.pdf: 1747275 bytes, checksum: 8c10f684628bf27d1c8fdb41a81f99ad (MD5) Previous issue date: 2007 | en |
| dc.description.tableofcontents | 頁次
誌謝------------------------------------------------------------------------------------------ I 中文摘要------------------------------------------------------------------------------------ II 英文摘要------------------------------------------------------------------------------------ III 目錄------------------------------------------------------------------------------------------ V 圖次------------------------------------------------------------------------------------------ IX 表次------------------------------------------------------------------------------------------ X 第一章 序言------------------------------------------------------------------------------- 1 第二章 文獻探討------------------------------------------------------------------------- 2 第一節 歷史回顧------------------------------------------------------------ 2 第二節 病原分類與特性--------------------------------------------------- 2 第三節 感染途徑------------------------------------------------------------ 3 第四節 臨床症狀------------------------------------------------------------ 4 第五節 病理學變化--------------------------------------------------------- 4 5.1 肉眼病變------------------------------------------------------------ 4 5.2 組織病理學病變--------------------------------------------------- 4 第六節 診斷------------------------------------------------------------------ 5 6.1 病原直接偵測------------------------------------------------------ 5 6.1.1 聚合酶鏈鎖反應---------------------------------------- 5 6.1.2 免疫組織化學染色------------------------------------- 5 6.1.3 免疫螢光染色------------------------------------------- 6 6.2 病原間接偵測------------------------------------------------------ 6 6.2.1 病原分離------------------------------------------------- 6 6.2.2 生化鑑定------------------------------------------------- 7 6.2.2.1 API 20NE系統----------------------------- 7 6.2.2.2 API-ZYM系統----------------------------- 7 6.2.2.3 其他方式------------------------------------ 7 6.2.3 抗體監測------------------------------------------------- 8 6.2.3.1 血清學檢測--------------------------------- 8 第七節 血清學分型--------------------------------------------------------- 9 7.1 利用非血清學方式來區別血清型------------------------------ 10 7.1.1 蛋白質輪廓分析---------------------------------------- 10 7.1.2 基因比對------------------------------------------------- 10 7.2 各國血清學陽性率檢測結果------------------------------------ 11 第八節 分子生物學分型--------------------------------------------------- 12 8.1 逢機增幅多型性核酸--------------------------------------------- 13 8.2 增幅片段長度多型性--------------------------------------------- 13 8.3 O. rhinotracheale於分子生物學分型研究------------------- 14 第九節 預防與控制--------------------------------------------------------- 15 9.1 抗菌劑感受性------------------------------------------------------ 15 9.1.1 各國抗菌劑感受性報告------------------------------- 16 9.1.2 Quinolone類藥物抗藥性基因------------------------ 18 9.2 臨床治療------------------------------------------------------------ 20 9.3 疫苗防治------------------------------------------------------------ 20 第十節 未來方向------------------------------------------------------------ 22 第三章 材料方法------------------------------------------------------------------------- 24 第一節 細菌分離與鑑定--------------------------------------------------- 24 1.1 檢體採集------------------------------------------------------------ 24 1.2 病原菌分離--------------------------------------------------------- 24 1.3 聚合酶鏈鎖反應鑑定--------------------------------------------- 24 1.3.1 DNA之萃取---------------------------------------------- 24 1.3.2 引子之選用---------------------------------------------- 25 1.3.3 PCR之反應條件---------------------------------------- 25 1.4 瓊脂醣凝膠電泳分析--------------------------------------------- 26 1.5 陽性結果之判讀與保存------------------------------------------ 26 1.6 生化鑑定------------------------------------------------------------ 26 1.6.1 API 20NE------------------------------------------------ 26 1.6.2 其他生化試驗------------------------------------------- 27 1.6.2.1 觸酶試驗------------------------------------ 27 1.6.2.2 氧化酶試驗--------------------------------- 27 1.7 快速平板凝集鑑定------------------------------------------------ 27 1.8 紅血球凝集試驗--------------------------------------------------- 28 1.8.1 紅血球懸浮液之製備---------------------------------- 28 1.8.2 血球凝集試驗------------------------------------------- 28 1.9 統計分析------------------------------------------------------------ 28 第二節 核酸序列分析比較及演化分析--------------------------------- 28 2.1 16S rRNA基因片段之選殖------------------------------------- 28 2.1.1 TA-cloning------------------------------------------------ 28 2.1.2 Colony PCR--------------------------------------------- 29 2.2 選殖基因核苷酸定序--------------------------------------------- 30 2.3 基因序列分析------------------------------------------------------ 30 第三節 分子生物學分型--------------------------------------------------- 31 3.1 高濃度DNA之萃取----------------------------------------------- 31 3.2 逢機增幅多型性核酸--------------------------------------------- 31 3.3 增幅片段長度多型性--------------------------------------------- 32 3.3.1 限制酵素切割片段------------------------------------- 32 3.3.2 接合反應------------------------------------------------- 32 3.3.3 聚合酶鏈鎖反應---------------------------------------- 33 3.4 多型性條帶結果分析--------------------------------------------- 34 第四節 抗菌劑感受性------------------------------------------------------ 34 4.1 瓊脂藥片擴散法--------------------------------------------------- 34 4.2 Enrofloxacin E-test------------------------------------------------ 34 4.3 Quinolone類藥物抗藥性基因偵測----------------------------- 35 第四章 結果------------------------------------------------------------------------------- 36 第一節 細菌分離與鑑定--------------------------------------------------- 36 1.1 細菌分離------------------------------------------------------------ 36 1.1.1 陽性分離株---------------------------------------------- 36 1.2 鑑定------------------------------------------------------------------ 36 1.2.1 聚合酶鏈鎖反應檢測結果---------------------------- 36 1.2.2 生化鑑定------------------------------------------------- 37 1.2.2.1 API 20NE----------------------------------- 37 1.2.2.2 其他生化試驗------------------------------ 37 1.2.3 快速平板凝集鑑定------------------------------------- 37 1.2.4 紅血球凝集試驗---------------------------------------- 37 第二節 16S rRNA片段核酸序列分析比較---------------------------- 38 第三節 分子生物學分型--------------------------------------------------- 38 3.1 逢機增幅多型性核酸條帶分析--------------------------------- 39 3.2 增幅片段長度多型性條帶分析--------------------------------- 40 第四節 抗菌劑感受性------------------------------------------------------ 40 4.1 瓊脂藥片擴散法--------------------------------------------------- 40 4.2 Enrofloxacin最小抑制濃度-------------------------------------- 41 4.3 抗藥性基因gyrA核酸及胺基酸序列分析比較-------------- 42 第五章 討論------------------------------------------------------------------------------- 44 參考文獻------------------------------------------------------------------------------------ 67 附錄一 台灣地區O. rhinotracheale分離株資料一覽表--------------------------- 81 附錄二 抗菌劑感受性試驗瓊脂錠擴散法抑制圈直徑參考表-------------------- 82 附錄三 台灣地區與國外O. rhinotracheale分離株16S rRNA核酸序列表-- 83 附錄四 O. rhinotracheale分離株之gyrA核酸序列及其胺基酸序列表-------- 87 附錄五 2005年7月-2006年8月間採集檢體原始資料--------------------------- 89 | |
| dc.language.iso | zh-TW | |
| dc.subject | 鳥鼻氣管桿菌 | zh_TW |
| dc.subject | RAPD | en |
| dc.subject | ORT | en |
| dc.subject | AFLP | en |
| dc.title | 台灣地區禽類鳥鼻氣管桿菌之分子生物學分析
與抗菌劑感受性調查 | zh_TW |
| dc.title | Molecular characterization and antimicrobial sensitivity of Ornithobacterium rhinotracheale strains isolated
from poultry in Taiwan | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 95-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張紹光,陳秋麟,蘇鴻麟,徐媛曼 | |
| dc.subject.keyword | 鳥鼻氣管桿菌, | zh_TW |
| dc.subject.keyword | ORT,AFLP,RAPD, | en |
| dc.relation.page | 80 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2007-07-03 | |
| dc.contributor.author-college | 生物資源暨農學院 | 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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