Riemerella anatipestifer（RA）為革蘭氏陰性短桿菌，其可造成鴨傳染性漿膜炎或稱新鴨病，在鵝則可造成鵝傳染性感冒或滲出性敗血症。RA主要經由呼吸道感染後造成急性或慢性的傳染性敗血症，耐過之病禽常呈現生長遲鈍、發育不良，可造成嚴重的經濟損失。截至目前為止RA真正的毒力因子及抗藥性基因尚未被鑑定出來。本實驗由台灣地區六個縣市收集到66株RA菌株，首先分析收集菌株之質體相，發現有84.9％（56/66）菌株含有質體，質體相共分七型。除原有發表之質體外，另有分子量約為7 kb及9 kb的質體尚未被定序，遂著手進行定序並分別命名為pY1及pY2。結果pY1核酸序列全長6849 bp，pY2核酸序列全長8745 bp，其上除含有已發表之RA質體蛋白基因序列外，於此兩質體中各發現一新的蛋白基因序列，其所轉譯之蛋白分別命名為ORF651及ORF1233。隨後進行表現ORF651蛋白及製備抗血清以檢測其功能及位置，結果顯示其為可溶性蛋白且為質體相關蛋白，將此蛋白序列於NCBI網站上比對後得知其具有與FemABX family相似性之功能蛋白序列，含有可與雙糖六月生月太脂質結合的區域。由於此兩個質體上並無任何抗藥性基因存在，所以利用於GenBank已發表不同藥物之抗藥性基因，設計引子偵測RA基因體是否含有類似或相同片段，將增幅出之片段加以定序及比對，結果可成功增幅出兩種基因型之dihydropeteroate synthase片段，證實RA基因體上具有抗磺胺劑基因，此為第一個被發現的RA抗藥性基因。實驗中不論是分自鴨源或鵝源的菌株皆含有此兩型抗磺胺劑基因，且大部分的菌株具有第二型抗磺胺劑基因可抵抗磺胺劑。比較菌株PCR檢測基因型結果與抗生素感受性試驗結果，發現菌株抗磺胺劑之基因型與表型達90.3％之吻合度，故實驗中所建立之引子可對RA菌株以PCR方法進行初步之磺胺劑抗性檢測，有利於縮短檢測菌株抗藥性之時間。

Riemerella anatipestifer is a Gram-negative rod-shaped bacterium and may cause epizootic infections mostly in water fowls and others. R. anatipestifer infection characterized by infectious serositis, new duck syndrome in duck or exudative septicaemia, infectious influenza in geese. This disease causes acute or chronic contagious septicemic disease and accounts for major economic losses in industrialized duck forms due to weight loss. There has been little work on the molecular basis of the pathogenesis of this organism, and so far no virulence factors or drug resistance genes have been established. This study sixty-six strains of R. anatipestifer were isolated from ducks with infectious serositis and geese in Taiwan. All strains contain seven differently sized plasmids ,and eighty-five percent of the ioslates（56/66）contained plasmids. In this study find the new plasmids, 7-kb and 9-kb plasmids in R. anatipestifer strains. The 7-kb plasmid（designated as pY1）and the 9-kb plasmid（designated as pY2）were completely sequenced to determine if they encoded virulence factors or drug resistance genes. pY1 had 6849 bp and pY2 had 8745 bp. Six ORFs（open reading frames） were identified in the pY1 and eight ORFs were identified in the pY2. There were a new ORF（ORF651）in pY1 and a new ORF（ORF1233）in pY2. The results indicated that ORF651 was a plasmid-associated protein, and it was a water-soluble protein. After blasting ORF651 with other proteins, indicated that ORF651 has disaccharide hexapeptide lipid substrate binding site. There was no drug resistance gene in pY1 and pY2. Then designated PCR primers to amplify the drug resistance gene of R. anatipestifer genome. The results of PCR indicated that R. anatipestifer genome has encoded two types of dihydropeteroate synthase. This is the first report that R. anatipestifer genome encoded drug resistance gene, dihydropeteroate synthase. Compared the PCR results with antibiotic susceptibility test , there were 90.3％ similarity. Thus, using the PCR method could be short the testing time of antibiotic susceptibility.

第一章 序言…………………………………………………… 1
第二章 文獻探討……………………………………………… 2
第一節Riemerella anatipestifer分類學簡介與生物學特性……2
第二節Riemerella anatipestifer 感染症……………… 4
第三節 細菌質體探討…………………………… 5
第四節 Riemerella anatipestifer抗藥性…………………………………8
第三章 材料與方法…………………………………………………………10
第一節Riemerella anatipestifer之分離……………………………10
第二節Riemerella anatipestifer之鑑定…………………………………10
3-2.1聚合酶連鎖反應…………………………………………………..10
3-2.2 BIOLOG 96項生化測定鑑定試驗………………………………..12
第三節 質體相分析…………………………………………………………12
第四節 質體核酸序列定序分析……………………………………………13
3-4.1質體片段之分子選殖……………………………………………..13
3-4.2 質體核酸片段定序與整合分析…………………………………..14
第五節 重組質體蛋白之表現………………………………………………15
3-5.1重組表現載體之構築……………………………………………..15
3-5.2重組質體之表現與確認…………………………………………..16
第六節 表現蛋白抗血清製備及RA質體蛋白檢測……………………….18
3-6.1表現蛋白抗血清製備……………………………………………...18
3-6.2應用Western blot檢測RA質體蛋白表現……………………….19
第七節 藥物感受性試驗……………………………………………………20
3-7.1紙錠瓊脂擴散試驗………………………………………………..20
3-7.2微量培養液稀釋試驗……………………………………………..20
第八節 抗藥性基因偵測……………………………………………………21
3-8.1抗藥性基因引子之設計…………………………………………..21
3-8.2 PCR偵測RA抗藥性基因…………………………………………21
第四章 結果………………………..………………………………………24
第一節Riemerella anatipestifer之分離與鑑定…………………………24
4-1.1引子特異性分析…………………………………………………..24
4-1.2 分離鑑定結果…………………………………………………….24
第二節 質體相分析…………………………………………………………24
第三節 質體核酸序列定序分析……………………………………………25
4-3.1質體DNA內核酸限制酶圖譜分析………………………………25
4-3.2質體核酸序列分析………………………………………………..25
第四節 重組質體蛋白之表現………………………………………………26
4-4.1重組表現載體之構築……………………………………………..26
4-4.2重組質體之表現與純化…………………………………………..26
第五節 應用Western blot檢測RA質體蛋白表現………………………..27
4-5.1 RA菌株表現檢測………………………………………………….27
4-5.2 RA-ORF651蛋白可溶及不可溶性檢測…………………………27
第六節 藥物感受性試驗……………………………………………………27
4-6.1紙錠瓊脂擴散試驗………………………………………………..27
4-6.2微量培養液稀釋試驗……………………………………………...28
第七節 抗藥性基因偵測……………………………………………………28
第五章 討論………………………………………………………………53
參考文獻………………………………………………………………………61

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