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研究生:謝宜峰
研究生(外文):Yi-Feng Hsieh
論文名稱:台灣原壁藻種別及基因型之鑑定與碘及氯己啶之最小殺藻濃度
論文名稱(外文):Identification of Prototheca species and genotypes of Prototheca zopfii and its minimal algaecide concentration to iodine and chlorhexidine in Taiwan
指導教授:莊士德莊士德引用關係
指導教授(外文):Shih-Te Chuang
口試委員:葉光勝陳鵬文
口試委員(外文):Kuang-Sheng YehJacky Peng-Wen Chan
口試日期:2015-07-21
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫學系暨研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:55
中文關鍵詞:原壁藻基因型最小殺藻濃度
外文關鍵詞:Protothecagenotypesminimal algaecide concentration
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原壁藻感染症是由單細胞、不具葉綠素且普遍存在於環境中的原壁藻 (Prototheca spp.)所造成,其於人類及其他動物皆能造成疾病。1952年,Lerche等人首次發現原壁藻會造成牛隻乳房炎以來,近二十年來,世界各國舉凡德國、美國、中國、日本等皆有關於爆發牛隻藻類乳房炎的報告,有鑑於其所帶來的牧場經濟損失,使得原壁藻更為科學家們重視。本篇報告為台灣第一篇針對牛隻原壁藻造成之乳房炎所做的分子流行病學調查。本研究的目的在於鑑定自台灣乳牛乳汁中所分離到的原壁藻的種別、調查P. zopfii不同基因型間的組成比例並評估以碘(iodine)及氯己啶(chlorhexidine)對於P. zopfii的最小殺藻濃度(minimal algaecide concentration, MAC)。牧場中所分離到的原壁藻株經過沙氏葡萄糖瓊脂培養基(Sabouraud dextrose agar)的繼代、濕片鏡檢以及利用VITEK® 2系統進行生化分析,實驗結果顯示,全部148株原壁藻株中有147株(99.3%)屬於P. zopfii,1株(0.7%)原壁藻株屬於P. wickerhamii。將上述147株P. zopfii透過特定基因型聚合酶鏈鎖反應(genotype-specific PCR)鑑定,全部P. zopfii皆鑑定為genotype 2。挑選16株P. zopfii分別針對碘以及氯己啶進行最小殺藻濃度試驗,碘的最小殺藻濃度範圍為0.0024%-0.0195%;氯己啶的最小殺藻濃度範圍為0.00015%-0.0003%。根據本實驗的結果顯示,台灣造成牛隻乳房炎的P. zopfii以genotype 2為主,而根據實驗的結果得知,於實驗環境下(in vitro)碘以及氯己啶對於P. zopfii皆具有良好的殺藻效果且可做為乳房內灌注治療的參考。

Protothecosis is an infection caused by unicellular, achlorophyllous, and ubiquitous algae of the genus Prototheca and has been reported in humans and in many other animal species. In 1952, Prototheca was first linked to bovine mastitis by Lerche, and since then there were several studies about outbreaks of bovine protothecal mastitis in many countries such as German, America, China and Japan in the past twenty years. No wonder it has gradually attracted attention of study because of its increasing incidence and economic loss. This study is the first report to provide molecular characterization of P. zopfii isolated from bovine mastitis in Taiwan. The aim of this study was to identify Prototheca species, to investigate the genotypic composition of the population of P. zopfii and to investigate minimal algaecide concentration (MAC) of iodine and chlorhexidine for P. zopfii isolated from bovine milk in Taiwan. A total of 148 Prototheca isolates collected from dairy farms were purely cultivated on Sabouraud dextrose agar, examined with microscopic methods, and then tested by VITEK® 2 system. The result revealed that 147 (99.3%) were P. zopfii and the other one (0.7%) was P. wickerhamii. All of the P. zopfii isolates were identified as P. zopfii genotype 2 and analyzed by genotype-specific PCR assays. The MACs for iodine and chlorhexidine of the 16 P. zopfii strains tested were 0.0024%-0.0195% and 0.00015%-0.0003%, respectively. According to the results, P. zopfii genotype 2 is predominant in bovine protothecal mastitis in Taiwan. The result of in vitro study indicated that iodine and chlorhexidine were effective against P. zopfii and were potential for its therapeutic effect on intramamary infusion.

目次
中文摘要........................................................................................................................... i
英文摘要.......................................................................................................................... ii
目次................................................................................................................................. iii
表圖目次......................................................................................................................... vi
第一章 前言.................................................................................................................. 1
第二章 文獻探討.......................................................................................................... 2
第一節 原壁藻簡介............................................................................................... 2
一、 原壁藻的分類.......................................................................................... 2
二、 原壁藻的形態與生活史.......................................................................... 3
三、 原壁藻分佈概況...................................................................................... 3
第二節 動物藻類感染症....................................................................................... 4
一、 犬藻類感染症.......................................................................................... 4
二、 貓藻類感染症.......................................................................................... 6
三、 牛藻類感染症.......................................................................................... 6
四、 羊藻類感染症.......................................................................................... 7
第三節 人類原壁藻感染症................................................................................... 9
一、 流行病學.................................................................................................. 9
二、 臨床症狀.................................................................................................. 9
1. 皮膚型.................................................................................................. 9
2. 鷹嘴滑液囊炎(Olecranonbursitis).................................................... 10
3. 瀰漫性、系統性感染.......................................................................... 10
三、 診斷方法................................................................................................ 11
四、 治療........................................................................................................ 11
第四節 藻類乳房炎的病原鑑定和分型方法..................................................... 12
一、 病原分離與形態學鑑定........................................................................ 12
二、 生化表現型鑑定.................................................................................... 13
三、 基因序列分析........................................................................................ 13
第五節 藻類感染症之治療................................................................................. 14
一、 原壁藻對藥物之敏感性........................................................................ 14
1. 紙錠擴散試驗(Disc diffusion method)............................................. 14
2. 最小抑制濃度試驗(Minimal inhibitory concentration; MIC).......... 14
3. 最小殺藻濃度試驗(Minimal algaecide concentration; MAC)......... 15
二、 臨床治療方法與疾病預防.................................................................... 16
1. 內科治療............................................................................................ 16
2. 外科治療............................................................................................ 17
3. 疾病預防............................................................................................ 17
第三章 材料與方法.................................................................................................... 20
第一節 原壁藻株來源......................................................................................... 20
一、 乳汁採樣................................................................................................ 20
二、 微生物培養............................................................................................ 20
第二節 藻株培養與鑑定..................................................................................... 21
一、 原壁藻之分離與判定............................................................................ 21
二、 VITEK® 2 Systems 生化鑑定............................................................... 21
三、 加做生化試驗........................................................................................ 22
四、 藻種保存................................................................................................ 22
第三節 以genotype-specific PCR區分Prototheca zopfii的兩種基因型以及Prototheca blaschkeae............................................................................................. 23
一、 原壁藻DNA萃取................................................................................. 23
二、 特定基因型聚合酶鏈鎖反應................................................................ 24
1. 使用特定基因型引子........................................................................ 24
2. 反應條件............................................................................................ 25
3. 電泳產物分析.................................................................................... 25
4. 18S rDNA區段基因序列定序........................................................... 25
三、 原壁藻親緣分析與樹狀圖之建構........................................................ 26
第四節 最小抑制濃度試驗(Minimal inhibitory concentration, MIC)與最小殺藻濃度試驗(Minimal algaecide concentration; MAC)............................................... 27
一、 選擇待測藻株........................................................................................ 27
二、 選擇消毒水種類與配製待測藥劑溶液................................................ 27
三、 接種菌液(Inoculum)準備...................................................................... 28
1. 待測藻株活化.................................................................................... 28
2. 調查藻類菌液於特定光密度(optical density; OD)下之生菌數............................................................................................................. 28
3. 接種菌液的配製................................................................................ 28
四、 微量肉湯連續稀釋法(Broth microdilution method)............................. 28
五、 最小殺藻濃度試驗(Minimal algaecide concentration; MAC).............. 29
第四章 結果................................................................................................................ 30
第一節 原壁藻分離結果..................................................................................... 30
一、 乳汁培養結果........................................................................................ 30
二、 菌種生化鑑定結果................................................................................ 30
第二節 特定基因型聚合酶鏈鎖反應................................................................. 31
第三節 18S rDNA區段基因序列定序結果....................................................... 31
第四節 最小抑制濃度試驗與最小殺藻濃度試驗之試驗結果......................... 32
第五章 討論................................................................................................................ 38
一、 原壁藻之分離及鑑定結果.................................................................... 38
二、 原壁藻株親緣關係................................................................................ 40
三、 原壁藻對消毒水之最小抑制濃度試驗及最小殺藻濃度試驗結果探討............................................................................................................................ 41
第六章 總結................................................................................................................ 44
參考文獻........................................................................................................................ 45


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