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標題: | 口服生物膜疫苗的研發-以烏魚格氏乳酸球菌為模板 Development of Oral Biofilm Vaccines - Using the Lactococcus garvieae as a Model in Mullet (Mugil cephalus) |
其他標題: | Development of Oral Biofilm Vaccines - Using the Lactococcus garvieae as a Model in Mullet (Mugil cephalus) |
作者: | 蘇豐傑 Feng-Jie Su |
指導教授: | 陳媺玫 Meei-Mei Chen |
關鍵字: | 格氏乳酸球菌,生物膜,浮游態,幾丁聚醣,生物膜疫苗, Lactococcus garvieae,Biofilm,Planktonic,Chitosan particle,Biofilm vaccine, |
出版年 : | 2022 |
學位: | 博士 |
摘要: | 格氏乳酸球菌為常見的水產動物病原菌,常造成感染魚隻大量死亡,存活的魚隻容易帶有病原以慢性感染的形式在夏季時反覆發病。經由哺乳動物研究得知當細菌在宿主體內形成生物膜後,容易使宿主免疫反應發生改變形成慢性感染,但關於生物膜在魚類所造成的免疫反應的改變與阻止生物膜生成的方法目前還缺乏探討,因此本研究主要分成兩部份(一)生物膜在魚隻體內所造成的免疫反應之變化,(二)開發生物膜疫苗對抗細菌生物膜的生成。
生物膜在魚隻體內所造成的免疫反應之變化方面,本實驗利用RNA定序比較格氏乳酸球菌浮游型態與生物膜型態感染烏魚後,其脾臟轉錄因子之間的差異,免疫相關基因的表現於感染生物膜後:類鐸受體2、介白素-1β、腫瘤壞死因子-α、補體蛋白7和主要組織相容性複體-II相關基因表現量下降,浮游態組基因表現量上升,並且在後續的不同時間點(6、12、24和48小時)分析上,更進一步發現浮游態組在48小時後的免疫反應與生物膜組的相同,此實驗也確認了當浮游態格氏乳酸球菌進入烏魚體內48小時可能會形成生物膜,並且導致宿主的免疫反應無法有效的清除細菌,因此後續我們需要開發疫苗進而防止生物膜的生成。 在生物膜疫苗建立的研究上,本實驗開發出高效率的生物膜增培技術,主要利用幾丁聚醣顆粒為基質並藉由懸浮培養方式,使格氏乳酸球菌在幾丁聚醣顆粒上形成生物膜,之後我們利用掃描式電子顯微鏡觀察與DMMB染色法定量確認生物膜的生成,並以福馬林不活化製成疫苗,經投餵不同劑量生物膜疫苗,測試抗體的生成、溶菌酶及吞噬能力分析,結果顯示連續口服1010 CFU/g的劑量14天為最佳的口服劑量與投餵時間。 在口服生物膜疫苗的免疫效果分析中,烏魚分別口服生物膜疫苗、全菌疫苗、幾丁聚醣顆粒與PBS連續投餵14天,並分析抗體的生成、吞噬能力、白蛋白/球蛋白、免疫相關基因及魚隻相對保護效果,結果顯示,生物膜疫苗組的吞噬能力為84%,顯著高於對照組,且該組的抗體產量顯著高於控制組且能持續32天以上,投予生物膜疫苗組別脾臟免疫相關基因(類鐸受體2、介白素-1β、腫瘤壞死因子-α)的mRNA相對表現量與控制組相比具有顯著上升,在攻毒實驗中,連續口服生物膜疫苗14天後的相對存活率為 74%,全細胞疫苗組為 42%,幾丁聚醣顆粒組為26%。此外免疫後 32 天的攻毒試驗結果,生物膜疫苗組的相對存活率為 77%,全細胞疫苗組為 18%,幾丁聚醣顆粒組為 0%。 因此,本實驗所開發的幾丁聚醣顆粒懸浮培養生物膜的方法與口服生物膜疫苗飼料皆可以做為後續大量生產口服疫苗的方式與預防生物膜生成,使水產養殖產業對於疾病預防能獲得控制並提升疫苗給予的便利性。 Lactococcus garvieae, an important pathogen affecting fish, is associated with high mortality rates and infection recurrence in summer. Biofilms are also known to cause chronic infection and disease recurrence. However, the effects of biofilms on fish immune response and factors that control biofilm formation remain unclear. Therefore, in this study, we aimed to (1) evaluate the changes induced by biofilms in fish immune response and (2) develop novel biofilm vaccines against bacterial biofilm formation. RNA sequencing (RNA-Seq) was used to compare the spleen transcriptome of planktonic- and biofilm-infected mullets. Additionally, genes encoding toll-like receptor 2(TLR2), interleukin-1β(IL-1β), tumour necrosis factor -α(TNF-α), complement 7 (C7), and major histocompatibility complex class I (MHC I) were downregulated in response to a biofilm infection. Subsequent analyses at different time points (6、12、24 and 48 hr) revealed that the immune response in the planktonic group was the same as that in the biofilm group after 48 hours. Furthermore, biofilm formation was observed in mullets infected by planktonic L. garvieae that could not be effectively eliminated by the host's immune response. Therefore, vaccines that prevent biofilm formation are required. In this study, we developed a high efficiency biofilm culture technology in which L. garvieae could form biofilms on chitosan particles in suspension culture. Biofilm formation was confirmed by scanning electron microscopy and quantify dimethylmethylene blue (DMMB) staining. An analysis of antibody generation and lysozyme and phagocytic activities after administration of formalin-inactivation different biofilm vaccine doses demonstrated that the most effective dose and administration time were a continuous oral dose of 1010 CFU/mL for 14 days. To confirm the effectiveness of the oral biofilm vaccine, experiments were conducted using biofilm vaccine, Whole-cell vaccine, chitosan particles, and phosphate- buffered saline (PBS). The production of antibodies, phagocytic ability, albumin/globulin ratio, immune-related genes, and relative survival rate were analysed. The phagocytic capacity of the biofilm vaccine group was 84%, which was significantly higher than that of the control group. Moreover, antibody production in this group was significantly higher than that in the control group and lasted for more than 32 days. The relative expression of spleen immune-related genes (TLR2, IL-1β, TNF-α) in the biofilm vaccine group was significantly higher than that in the control group. In the challenge experiment, the relative percent survival (RPS) rates 1 days post-vaccination were 74%, 42%, and 26% in the biofilm, Whole-cell, and chitosan particle vaccine groups, respectively. Additionally, the RPS rates 32 days post-vaccination were 77%, 18%, and 0% in the biofilm, Whole-cell, and chitosan particle vaccine groups, respectively. Therefore, our methods of producing biofilm on chitosan particles in suspension culture and administering oral biofilm vaccine may be utilized for mass production of oral vaccines and prevention of biofilm formation. This would aid the aquaculture industry in disease control and prevention as well as operational improvement and enhancement. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/79227 |
DOI: | 10.6342/NTU202210010 |
全文授權: | 同意授權(全球公開) |
顯示於系所單位: | 獸醫學系 |
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