臺灣博碩士論文加值系統

雞球蟲病是造成養雞產業相當大損失的重要疾病之一。主要是造成雞隻腸道上皮細胞中度至嚴重損壞，導致增重與飼料效率降低，並經常引發雞群高的發病率和死亡率。本試驗應用Illumina MiSeq system 分析球蟲對於土雞盲腸與泄殖腔內微生物菌相的影響。DNA樣品取自雞隻盲腸有球蟲感染與沒有受到球蟲感染的盲腸與泄殖腔內容物。以16S rRNA共通的引子組與Illumina MiSeq system 定序分析獲得每個樣品組大量的細菌類群的DNA片段定量型材(quantitative profiles)。採用分層聚類和主成分分析已鑑定出的類群的相對百分比豐度。在盲腸沒有球蟲感染的雞隻盲腸內容物的微生物菌相中，以擬桿菌屬(Bacteroides)最豐富，其次分別為牛黄瘤胃球菌屬(Ruminococcus)、Alistipes及梭孢桿菌屬(Clostridium)。而在盲腸有球蟲感染的雞隻盲腸內容物的微生物菌相中，則以Clostridium最豐富，其次分別為Bacteroides及Alistipes。在盲腸沒有球蟲感染的雞隻泄殖腔內容物的微生物菌相中，以Clostridium最豐富，其次分別為 Bacteroides、Subdoligranulum及乳桿菌屬(Lactobacillus)。而在盲腸有球蟲感染的雞隻泄殖腔內容物的微生物菌相中，也是以Lactobacillus最豐富，其次為Clostridium、Turicibacter及Bacteroides。球蟲感染明顯地影響雞隻盲腸與泄殖腔內微生物菌相的分布與豐度。本研究所呈現球蟲感染對雞腸道菌群的影響，可作為雞隻球蟲病防治的重要參考資訊。

Coccidiosis, one of the most important chicken diseases, causes considerable economic loss in the chicken industry. Coccidiosis causes serious mucosal damage and predisposes chickens to enteropathogen infection. In this study, Illumina sequencing approach was used to evaluate effects of coccidiosis on the intestinal microflora of native chicken. Total DNA was isolated from the cecal and cloaca contents of infected and uninfected birds, respectively, PCR amplified with an universal 16S rRNA barcodes, and sequenced by Illumina MiSeq. Quantitative profiles of bacterial taxa were obtained using an analysis pipeline written in Perl. The relative abundance of the identified taxa was analyzed using hierarchical clustering and principal component analysis. Samples from the cecal contents of uninfected chickens were dominated by bacterial species belonging to the genera Bacteroides, Ruminococcus, Alistipes and Clostridium; cecal contents of infected samples were dominated by species from the genera Clostridium, Alistipes and Bacteroides. On the other hand, samples from the cloaca of uninfected chickens were dominated by bacterial species belonging to genera Clostridium, Bacteroides, Subdoligranulum and Lactobacillus; the infected cloaca samples were dominated by species from the genera Lactobacillus, Clostridium, Turicibacter and Bacteroides. Coccidia infection affects not only the richness of the microbial communities but also its composition in the cecum and cloaca. These results prove the coccidia influence on the microbial communities of chicken gut and provide important information for the prevention and cure of chicken coccidiosis.

目錄
中文摘要……………………………………………………………………………… Ⅰ
英文摘要……………………………………………………………………………… Ⅱ
致謝 ………………………………………………………………………………. … Ⅲ
目錄…………………………………………………………………………………… Ⅳ
表目錄………………………………………………………………………………… Ⅵ
圖目錄………………………………………………………………………………… Ⅷ

第壹章 緒言 …....…………..…………………………..………………. ………… 1
第一節 雞的重要性 …....…………..…………………………..………………. 1
第二節 球蟲對養雞產業的影響 …….………………………….………………. 3
第三節多源基因體應用於雞腸道之相關研究 …………………..……………. 4
第四節 研究目的….……………………………………………………………… 6
第貳章 材料與方法….……………………………………………………………… 6
第一節 研究材料………….……………………………………………………… 6
第二節 實驗方法………….……………………………………………………… 8
第三節 資料分析………….……………………………………………………… 13
第參章 結果….……………………………………………………………………… 14
第一節 球蟲感染率……………….……………..…………………………..…… 14
第二節 雞盲腸與泄殖腔內容物微生物序列分析 ………………………….… 15
第三節 雞盲腸內容物微生物多源基因體的比較 ……………………….…… 22
第四節 雞泄殖內容物微生物多源基因體的比較 ……………………….…… 26
第五節 以R程式OTU階層分群分析 ………………………………………… 30
第肆章 討論….……………………………………………………………………… 31
第一節 多源基因體資訊 ……………………………….……….…………….… 31
第二節 盲腸與泄殖腔微生物多源基因體 …………….……….……….……… 32
第三節 R程式OTU階層分群 …………………………….….….…………… 34
第伍章 結論 …………………………………………………………………….… 35
第陸章 參考文獻 …………………………………………………………………… 35
表 ………………………………………………………...………………………… 46
圖 …………..…………………………………………...…………………………… 82

表目錄
表1. 試驗雞隻各生長階段之飼糧組成 ……………………………..………… 46
表2. 試驗動物的防疫計畫 ……………………………………………….......... 46
表3. PCR反應添加物與添加量 ………………………………………………. 47
表4. 在分類門的階層CC、NS、NCC 及 NCS 四個樣點所得到的OUTs與 taxonomic assignments ….…….…………………………………………
47
表5. 在分類綱的階層CC, NCC, NS and NCS 四個樣點所得到的OUTs與 taxonomic assignments ………………………………………………….
48
表6. 在分類目的階層CC, NCC, NS and NCS 四個樣點所得到的OUTs與 taxonomic assignments .…….…………………………………………
49
表7. 在分類科的階層CC, NCC, NS and NCS 四個樣點所得到的OUTs與 taxonomic assignments .…….…………………………………………
50
表8. 在分類屬的階層CC, NCC, NS and NCS 四個樣點所得到的OUTs與 taxonomic assignments .…….…………………………………………
52
表9. 在CC組、CS組、NCC組及NCS組各組OUT數量豐度排名前10名的OUT編號與比對Ribosomal Database Project參照序列最接近的分類科屬 .…….…………………………………………………………………

56
表10. 比較盲腸有球蟲感染的雞隻盲腸內容物(CC)與盲腸無球蟲感染的雞隻盲腸內容物(NCC)之微生物菌相在分類門階層所獲得的taxonomic assignment ….……………………………….……………………………

58
表11. 比較盲腸有球蟲感染的雞隻盲腸內容物(CC)與盲腸無球蟲感染的雞隻盲腸內容物(NCC)之微生物菌相在分類綱階層所獲得的taxonomic assignment ….……………………………………………………………

59
表12. 比較盲腸有球蟲感染的雞隻盲腸內容物(CC)與盲腸無球蟲感染的雞隻盲腸內容物(NCC)之微生物菌相在分類目階層所獲得的taxonomic assignment ….……………………………………………………………

60
表13. 比較盲腸有球蟲感染的雞隻盲腸內容物(CC)與盲腸無球蟲感染的雞隻盲腸內容物(NCC)之微生物菌相在分類科階層所獲得的taxonomic assignment ….……………………………………………………………

62
表14. 比較盲腸有球蟲感染的雞隻盲腸內容物(CC)與盲腸無球蟲感染的雞隻盲腸內容物(NCC)之微生物菌相在分類屬階層所獲得的taxonomic assignment ….……………………………………………………………

65
表15. 比較盲腸有球蟲感染的雞隻泄殖腔內容物(CS)與盲腸無球蟲感染的雞隻泄殖腔內容物(NCS)之微生物菌相在分類門階層所獲得的taxonomic assignment ….……………………………………………………………

70
表16. 比較盲腸有球蟲感染的雞隻泄殖腔內容物(CS)與盲腸無球蟲感染的雞隻泄殖腔內容物(NCS)之微生物菌相在分類綱階層所獲得的taxonomic assignment ….……………………………………………………………

71
表17. 比較盲腸有球蟲感染的雞隻泄殖腔內容物(CS)與盲腸無球蟲感染的雞隻泄殖腔內容物(NCS)之微生物菌相在分類目階層所獲得的taxonomic assignment ….……………………………………………………………

72
表18. 比較盲腸有球蟲感染的雞隻泄殖腔內容物(CS)與盲腸無球蟲感染的雞隻泄殖腔內容物(NCS)之微生物菌相在分類科階層所獲得的taxonomic assignment ….……………………………………………………………

74
表19. 比較盲腸有球蟲感染的雞隻泄殖腔內容物(CS)與盲腸無球蟲感染的雞隻泄殖腔內容物(NCS)之微生物菌相在分類屬階層所獲得的taxonomic assignment ….……………………………………………………………

77



圖目錄
圖1. 雛雞飼養於以鐵皮圍成一圈的未消毒的水泥地，讓雛雞接受球蟲自然感染。 ........................................................................................................ 82
圖2. 試驗雞隻飼養於未消毒的放牧地，讓雛雞接受球蟲自然感染。 ............ 82
圖3. 死亡雞隻現場解剖。 .................................................................................. 83
圖4. 雞隻盲腸是位於小腸與大腸交接處的兩條長而肥大的盲袋。 …...… 83
圖5. 雞盲腸內容物鏡檢出球蟲(紅色箭頭所示)。 ………………………… 84
圖6. 在分類門的階層CC, NCC, NS and NCS 四個樣點所得到的OUTs與 taxonomic assignments 相對豐度。 ……………………………………
84
圖7. 在分類綱的階層CC, NCC, NS and NCS 四個樣點所得到的OUTs與 taxonomic assignments 相對豐度。 ……………………………………
85
圖8. 在分類目的階層CC, NCC, NS and NCS 四個樣點所得到的OUTs與 taxonomic assignments 相對豐度。 ……………………………………
86
圖9. 在分類科的階層CC, NCC, NS and NCS 四個樣點所得到的OUTs與 taxonomic assignments 相對豐度。 ……………………………………
87
圖10. 在分類屬的階層CC, NCC, NS and NCS 四個樣點所得到的OUTs與 taxonomic assignments 相對豐度。 ……………………………………
87
圖11. 以R 程式進行分群成4個cluster group。 …………………………… 88
圖12. 以R 程式進行分群的第1個cluster group。 …………………………… 89
圖13. 以R 程式進行分群的第2個cluster group。 …………………………… 89
圖14. 以R 程式進行分群的第3個cluster group。 …………………………… 90
圖15. 以R 程式進行分群的第4個cluster group。 …………………………… 90

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