- 學位論文
養殖吳郭魚法朗西斯氏菌之表現型、基因型及其疫苗開發
Phenotyping, genotyping and vaccine development of Francisella noatunensis subsp. orientalis isolated from cultured tilapia (Oreochromis niloticus) in Taiwan.
摘要
法朗西斯氏菌（Francisella noatunensis subsp. orientalis）是一種會感染溫水性淡水與海水養殖魚類的細胞內寄生病原，且常見於吳郭魚（Oreochromis spp.）。魚類感染法朗西斯氏菌會引起全身性肉芽腫性疾病；並造成高死亡率及全球性巨大的經濟損失。本研究有四項主要目標：1. 分離並鑑定台灣罹病魚隻之法朗西斯氏菌種別； 2. 分析源自台灣不同魚種與地區之法朗西斯氏菌其表現型與基因型特徵；3. 台灣法朗西斯氏菌菌株致病性研究及建立吳郭魚實驗攻毒模式；4. 以台灣法朗西斯氏菌強毒株開發一有效且安全疫苗並應用在養殖吳郭魚。由台灣不同地區罹病魚隻共成功分離18株法朗西斯氏菌，所有標的菌株培養於含2%血紅蛋白之半胱氨酸-牛心瓊脂培養基 (cysteine heart agar) ，隨後以 API ZYM 試驗套組檢測其酵素活性，並利用16S rRNA 與4種housekeeping 基因分析其基因多樣性特徵。另，利用限制酶BamHI 與 XhoI 進行脈衝式電泳分析；完成標的菌株之基因組DNA指紋分析。結果顯示在酵素活性與脈衝式電泳圖譜結果不同於法朗西斯氏菌(Francisella philomiragia) 。在親緣樹狀分析中， 16S rRNA和housekeeping基因與 GenBank數據庫比對顯示99-100％核苷酸序列的相似性。 此外，在本研究中所建立之致病性和實驗攻毒模式可供後續半致死劑量(LD50)研究。 在疫苗研究方面，選擇強毒菌株(AOD104086)作為法朗西斯氏菌疫苗候選株並進行鑑定與發展為注射型福馬林滅活法朗西斯氏菌死菌疫苗，而商品化礦物油佐劑(MontanideTM ISA 763 AVG) 亦使用作為疫苗成分之一。概括而言，吳郭魚腹腔免疫注射0.1 mL含佐劑的疫苗與無菌磷酸鹽緩衝溶液，在免疫後第2週進行補強注射，而初次免疫後第2與6週採集魚血清並評估特異性IgM 抗體力價。免疫接種之吳郭魚隨後以腹腔注射與浸泡途徑進行攻毒並分別記錄攻毒後21天及60天期間的死亡率。攻毒組別魚隻的相對存活百分比與肉芽腫分數亦被研究與紀錄。收集攻毒後魚隻血液並利用平板計數法在魚隻攻毒後0、24、48、72、120和168小時測量每毫升血液中的菌落形成單位以進行宿主體內細菌侵襲與清除的研究。與對照組相比，法朗西斯氏菌疫苗在重複試驗其存活率分別為71％和76％的疫苗組中可誘發更高的抗體，而在疫苗組魚隻的血中細菌亦顯著降低且在腎、脾、肝臟與鰓可見較少肉芽腫。再者，接種疫苗吳郭魚之促炎症細胞激素與免疫相關基因包含 IL-1β、TNFα、CXCL8與 IL-17C 等轉錄調控顯著上升。本研究總結結果為發展具潛力特徵之法朗西斯氏菌作為疫苗候選株並進一步為未來台灣吳郭魚養殖產業之不活化法朗西斯氏菌疫苗研究提供了重要資源。
並列摘要
Francisella noatunensis subsp. orientalis (Fno) is an intracellular pathogen affecting warm water fish both in freshwater and seawater culture conditions. It is most prevalent in tilapia (Oreochromis spp.). Infection with Fno in fish causes systemic granulomatous disease resulting in high mortality and leading to huge economic losses worldwide. The present study was designed with four major objectives: i) isolate and identify species of Francisella spp. isolated from diseased fish in Taiwan ii) characterize the phenotypic and genetic characteristics of Francisella spp. obtained from different fish and geographic locations in Taiwan iii) investigate the pathogenicity and establish experimental challenge models of Taiwanese Fno strain in cultured tilapia iv) develop an efficacious and safe vaccine from the highly virulent strain of Taiwanese Fno in cultured tilapia. A total of 18 Fno isolates were successfully isolated from diseased fish from different geographical locations in Taiwan. All Fno isolates were cultured on cysteine heart agar supplemented with 2% hemoglobin (CHAH). Enzymatic activity was analyzed using the API ZYM test kit and genetic diversity was analyzed by sequencing the 16S rRNA and four housekeeping genes and analyzing the phylogenetics. Genomic DNA fingerprinting was performed by pulsed-field gel electrophoresis (PFGE) with BamHI and XhoI restriction enzymes digestion. The results revealed an identical profile of enzymatic and PFGE pattern which was distinct from F. philomiragia. In the phylogenetic tree analysis, we observed a nucleotide sequence similarity of 99-100 % for 16S rRNA and housekeeping genes when compared to the GenBank database. In this study, we also established the pathogenicity and experimental challenge model, determining the median lethal dose (LD50) for subsequent experimental challenge studies. In vaccine study, highly virulent bacterial strain (AOD104086) was chosen as the Fno vaccine candidate strain and was used to develop an injectable formalin-killed Fno vaccine. A commercial adjuvant (MontanideTM ISA 763 AVG) was used for vaccine formulation. Briefly, tilapia (Oreochromis niloticus) were intraperitoneally immunized with 0.1 mL of vaccine formulation in adjuvant and substituted with sterile phosphate-buffered saline (PBS) in the control group. A booster was administered at weeks 2 post-immunization. Fish sera were collected and evaluated for the specific IgM antibody titer at weeks 2 and 6 post-primary immunizations. At 6 weeks post-primary immunization, tilapia were subjected to intraperitoneal injection and immersion challenge with Fno, recording the mortality for 21 days and 60 days, respectively. The relative percentage of survival (RPS) and granuloma scores from both challenge groups of fish were also investigated and recorded. Bacterial invasion and clearance were analyzed from the collected fish blood after challenge, using plate count method at 0 h, 24 h, 48 h, 72 h, 120 h, and 168 h after challenge by measuring the number of colonies forming unit per mL of blood. Fno vaccine induced greater antibody production when compared to the control group with a survival rate of 71 % and 76 % in two vaccine group replicates. Bacterial load in blood was significantly lower in the vaccinated group and a lower granuloma score was observed in the kidney, spleen, liver, and gill in the vaccinated fish. Furthermore, the transcriptional of pro-inflammatory cytokine and immune-related genes including IL-1β, TNFα, CXCL8, and IL-17C were significantly up-regulated in tilapia after vaccination. The overall results of the present study provide the potential of characterized Fno isolates as vaccine candidates and as an essential resource for further studies on inactivated Fno vaccine in the Taiwanese tilapia farming industry.
參考文獻
延伸閱讀
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