養殖魚類乳酸乳球菌之基因特性、表面特性及疫苗效力評估

Genetic characteristics, cell-surface properties and vaccine efficacy of Lactococcus garvieae in cultured fish

羅迪瓦(Shreesha Sadashiva Rao)

指導教授 ： 陳石柱

國立屏東科技大學/獸醫學院/獸醫學系所/博士(2021年)

https://doi.org/10.6346/NPUST202100213

摘要

乳酸球菌(Lactococcus garvieae)，此病原體已有多種淡水、海水養殖魚類感染病例，因急性出血性敗血症及高死亡率而被重視。因此，本論文以四個主題進行分析研究：(1) 進行烏魚來源乳酸球菌分離株與其他不同魚種來源菌株間之基因特性、表面特性分析。(2) 以實驗室規格進行烏魚乳酸球菌福馬林不活化疫苗之保護效力評估。 (3) 烏魚候選疫苗進行田間免疫後1、3個月之保護效力評估。 (4) 研究分析台灣首例箱網養殖海鱺感染乳酸球菌症分離菌株之表現特性、基因特性及致病性。
來自台灣不同地理位置養殖烏魚感染乳酸球菌症病例所分離之41株乳酸球菌菌株之表現型分析包括革蘭氏染色、氧化酶(oxidase)、過氧化氫酶(Catalase)、運動性(Motility)、NaCl耐受性、bile-esculin水解能力等。以16S rRNA之pLG-1和pLG-2引子對以及針對編碼位於16S-23S rRNA基因內之內部轉錄間隔區Internal transcribed spacer (ITS) 區域設計之G1和L1特異性引子對分別鑑定分析不同分離菌株。透過GenBank數據庫相比及親緣關係樹分析結果，可獲得16S rRNA和內部轉錄間隔區(ITS)區域的核苷酸序列相似性為99.1-100%。同時以限制酶SmaI和ApaI分別進行菌株染色體核型之脈衝式電泳(PFGE)分析。結果大多數菌株之基因型呈現相同的脈衝型，有利於流行病學之研究。
依據菌株不同脈衝型分析結果挑選候選菌株，進行致病性研究及半致死劑量(median lethal dose (LD50)測試，並以不同毒力候選菌株進行細胞表面特性研究。以電子顯微鏡觀察候選菌株之細胞表面特殊結構特性，並評估具有該結構菌株免疫魚隻後，確實可提供使魚隻獲得對乳酸球菌較具高的特異型抗體力價。因此研製該候選菌株(OT103003-S3) 之福馬林不活化死菌疫苗以實驗室規格進行烏魚注射免疫及同源、異源菌株攻毒評比疫苗之保護效力。結果其疫苗保護之相對存活率 (Relative percent survival (RPS)分別為91.4%和100%，且於攻毒後24小時各疫苗組之相關促炎性和抗炎性免疫基因的表現均明顯上升，溶菌酶活性和特異性抗體力價亦隨免疫期程呈現正向提升。另，以異源菌株分別於疫苗免疫後1、3個月進行攻毒，結果確實可保護烏魚抵抗乳酸球菌感染，RPS均為100%。
首例台灣箱網養殖海鱺感染病例出現後進行病原菌之表現特性、基因特性、表面特性分析及致病性研究。結果確認海鱺分離株菌種為乳酸球菌(L. garvieae) 。本菌在表現型中非莢膜 (KG +) 已被證實為其潛在致病性的毒力因子之一，雖本研究中進行莢膜基因 (Capsular gene cluster, CGC) 檢測為陰性，但以莢膜染色和穿透式電子顯微鏡 (Transmission electron microscopy, TEM) 技術則證實海鱺分離株的類透明質酸莢膜 (Hyaluronic acid-like) 結構確實存在。另，致病性研究結果，以候選菌株(AOD109191) 以人工接種攻毒後可引發與自然界海鱺感染本菌病症之相同病理學病變，符合Koch 假說病原菌定義，證實乳酸球菌確實在箱網養殖海鱺為一新浮現病原菌，並值得未來投入本病防治疫苗研製以減少化學用藥、落實保護環境永續，同時可增進產業發展。

關鍵字

乳酸球菌 ； 致病性 ； 烏魚 ； 疫苗 ； 海鱺

並列摘要

Lactococcus garvieae is a persistent pathogen in aquaculture that causes lactococcosis in cultured fish, a disease characterized by hyperacute hemorrhagic septicemic infections and mass mortalities in fish cultivated both in fresh and marine waters. The present study was designed with four major objectives: 1) Epidemiological study of L. garvieae isolated from grey mullet and other fish species isolated within different geographic locations of Taiwan and to characterize the cell surface property of contagious isolate from grey mullet through electron microscopic visualization. 2) To determine the efficacy of a formalin-killed L. garvieae (FKC) vaccine in grey mullet in a laboratory trial, both with and without adjuvant, through multiple parameters. 3) To assess the efficacy of formalin-killed L. garvieae (FKC) in a field trial with double dose vaccination over a three-month period for cross-protection. 4) Phenotypic and genotypic comparisons of newly identified L. garvieae isolates from cobia to previously characterized isolates.
A total of 41 fish pathogenic L. garvieae isolates were successfully isolated from diseased fish from different geographical locations in Taiwan majorly isolated from grey mullet. For comparison and characterization of Taiwanese isolates, three Japanese strains of L. garvieae and one reference strain, ATCC 43921, were used. All isolates obtained from pure bacterial cultures were subjected to Gram staining and common bench tests, ability to develop on bile esculin agar was examined. pLG-1 and pLG-2 primers targeting 16S ribosomal RNA (rRNA) and G1 & L1 primers targeting the 16S–23S rRNA gene encoding the internal transcribed spacer (ITS) region were used for L. garvieae-specific PCR. For genotyping, genomic DNA fingerprinting was performed by pulsed-field gel electrophoresis (PFGE) by using the SmaI and ApaI restriction enzymes digestion. The results revealed an identical profile seemed to be more suitable for epidemiological studies of L. garvieae. The pathogenicity and cell surface property of selected pathogenic isolate (OT103003-S3) from grey mullet was established by performing lethal dose determination challenge. Further, the pathogenic isolate was observed for its cell surface property through electron microscopic visualization, of non-treated L. garvieae did not show the presence of capsule on the bacterial surface. The electron microscopic visualization of selected pathogenic isolate (OT103003-S3) was confirmed to be KG+ phenotype. upon incubation with fish serum, the ability of KG+ phenotypic L. garvieae to evade phagocytosis by cloaking itself against specific antibodies was observed.
To evaluate the ability of a formalin-killed L. garvieae vaccine; research was designed with an oil-based adjuvant in grey mullet in laboratory trail. Formalin-killed cells (FKC) of previously selected pathogenic isolate (OT103003-S3) was used for the study. Electron microscopy observation of the FKC revealed a peculiar membrane vesicle-like pimple structure on the surface of L. garvieae, possibly indicating a shift in cell surface property in formalin-killed cells. Grey mullet fish were immunized with formalin-killed L. garvieae vaccine an oil-based adjuvant for single-dose test in laboratory trials. When challenged with homologous and heterologous strains after one month, the adjuvant mixed formalin-killed L. garvieae resulted in 91.4 percent and 100 percent relative percent survival (RPS), respectively. The vaccinated community had substantially higher levels of specific antibody titer and lysozyme activity. The levels of pro-inflammatory and anti-inflammatory cytokines increased 24 hours after the challenge, according to immune gene expression. The long-term efficacy of the L. garvieae vaccine was investigated in a parallel field trial experiment. The results showed that 100 percent survival was observed in the vaccinated groups of one month and three months after heterologous challenge.
In conjunction with multiple host species susceptible to L. garvieae infection, an etiological detection in diseased cage-cultured cobia (Rachycentron canadum) indicated an infection with L. garvieae for the first time. All isolates recovered were confirmed to be L. garvieae through, API 32 strep system and pLG-1 and pLG-2 primers targeting 16S ribosomal RNA (rRNA) and G1 & L1 primers targeting the 16S–23S rRNA gene encoding the internal transcribed spacer (ITS) region were used for L. garvieae-specific PCR. Genotypic characterization of cobia isolates was performed in comparison with previously characterized pathogenic isolate from grey mullet (vaccine strain- OT103003-S3) along with three Japanese strains of L. garvieae and one reference strain, ATCC 43921. Similar to our previous observations with pathogenic isolate from grey mullet (OT103003-S3), transmission electron microscopy and multiplex PCR revealed no existence of the capsular gene cluster (CGC) in L. garvieae isolates(AOD109191) from cobia. Intraperitoneal injection challenges in cobia were used to assess the pathogenic potential of the representative isolate in order to fulfil the Koch postulate. The gross lesions and histopathological changes observed in experimentally infected cobia were comparable to those observed in naturally infected fish. As a result, it confirms that L. garvieae caused disease outbreaks in cage-cultured cobia.
The overall results of the present study revile the genotypic and phenotypic characterisation of Taiwanese isolates and potency of pathogenic L. garvieae isolates as vaccine candidates and as an essential resource for further studies on inactivated vaccine in the Taiwanese grey mullet farming industry exhibiting concern over emergence of new isolates within aquaculture fish species like Cobia.

並列關鍵字

Lactococcus garvieae ； pathogenicity, ； Grey mullet ； vaccine ； Cobia

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