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

Molecular characterization and antimicrobial sensitivity of Ornithobacterium rhinotracheale strains isolated from poultry in Taiwan

林欣穎(Shin-Ying Lin)

指導教授 ： 蔡向榮

國立臺灣大學/生物資源暨農學院/獸醫學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.03027

摘要

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。

關鍵字

鳥鼻氣管桿菌

並列摘要

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.

並列關鍵字

ORT ； AFLP ； RAPD

參考文獻

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