台灣不同養殖魚類發光菌之基因型、分子型、致病性特徵與疫苗開發

Genotypic, molecular and pathogenic characteristics of Photobacterium damselae subsp. piscicida isolated from different farmed fish in Taiwan and vaccine development against Photobacteriosis

方孝仲(Pham Trung Hieu)

指導教授 ： 陳石柱

國立屏東科技大學/國際學院/觀賞魚科技國際學位專班/博士(2021年)

https://doi.org/10.6346/NPUST202100114

摘要

魚類巴氏桿菌症(Pasteurellosis)又稱發光桿菌症(Photobacteriosis)，是由海水養殖魚類中危害最嚴重的細菌病原體發光桿菌 (Photobacterium damselae subsp. piscicida, Phdp)引起。自1963年美國首次從野生的白鱸（Morone americanus）和條紋鱸（M. saxatilis）發現以來，發光桿菌症已傳播到全球各地之不同魚種。在台灣，最早的文獻指出鱧魚（Channa maculata）感染病病症後，陸續在許多養殖魚類中，如海鱺（Rachycentron canadum）、蓋斑鬥魚（Macropodus opercularis）和疊波蓋刺魚（Pomacanthus semicirculatus；俗名：神仙魚）均有本病原感染病例被證實。然而台灣相關魚類細菌性病原研究中，截至目前為止，仍嫌少對於發光桿菌 (Phdp)基因性、分子性與致病性間相關流行病學探討其致病性，並進入研製對本病預防控制的有力工具〝疫苗〞的給予，期望對控制本病，減少水產養殖過程中化學藥品或抗生素的使用量與環境永續無汙染盡一份心力。
首先，從台灣的不同罹病魚種來源收集39株發光桿菌 (Phdp)分離株，利用單管多引子聚合酶連鎖反應技術(multiplex PCR)確認菌種為Phdp。繼之，進行來自不同宿主和年份間各分離株之表現型(phenotypic)和基因型(genotypic)特性及毒力基因(virulence genes)分布特性，並利用親源樹分析(phylogenetic analysis)和致病性測試。結果顯示，不同Phdp分離株間具有相同的表現型特性。但以限制酶SmaI和NotI進行脈衝式電泳（pulsed-field gel electrophoresis; PFGE）分析顯示染色體核酸片段(band patterns)均可分為2型。16S rDNA和Fur基因序列之親源分析顯示無法區辨至亞種，而ToxR基因則可區辨發光桿菌兩個亞種。偵測AIP56，P55，PDP_0080，Sod和Irp1等致病基因後發現，在所有分離株中均具有這些基因。但是，分別以海鱺和尖吻鱸進行九株候選Phdp分離株之致病力研究顯示，分離株間的致病力確實存在差異。使用不同分離株測試死亡率相近的海鱺與尖吻鱸顯示，尖吻鱸在實驗室條件下對此種細菌有較明顯感受性，可在往後的研究中用做實驗模型。
目前針對發光桿菌 (Phdp)疫苗開發中已記載有四種不同疫苗類型，如全細胞疫苗(Whole-cell vaccine)、減毒活疫苗(Live attenuated vaccine)、DNA疫苗(DNA vaccine)和次單位疫苗(Subunit vaccine)。其中又以重組次單位疫苗之製備成本低廉，安全性高，受污染機會少等優點，因此吾人就本菌 (Phdp) 熱休克蛋白(Heat-shock proteins)之重組次單位疫苗，包括HSP90、HSP33、HSP70與DnaJ等高免疫原性之重組蛋白研製，並以尖吻鱸(Lates calcarifer)作為實驗動物模組，進行疫苗保護效力評估。
Phdp四種候選重組疫苗(HSP90，HSP33，HSP70和DnaJ)以載體pET-151轉殖入大腸桿菌BL21（DE3）中進行蛋白質表現並純化，以30:70（v / v）的比例混合佐劑Montanide ISA 763 AVG，腹腔注射50尾尖吻鱸，對照組2組(各別注射PBS及佐劑ISA)。當魚體進行標的疫苗免疫後、確實可產生高抗體力價、血清溶菌酶活性(serum lysozyme activities) 以及殺菌作用；並可偵測出抗原蛋白（特別是rHSP33）。此外，接種疫苗後，免疫組魚隻淋巴器官的早期免疫反應中亦可偵測到相關免疫基因的高表現量。當以Phdp菌(編號AOD105021)進行各組疫苗(HSP90，HSP33，HSP70和DnaJ)之保護效力評比攻毒，各組之相對存活率分別為48.28％，62.07％，51.72％和31.03％。綜合上述「魚類發光桿菌疫苗」研發結果顯示，HSP33確實為優秀候選疫苗抗原。

關鍵字

發光桿菌 ； 基因型 ； 表現型 ； 熱休克蛋白 ； 疫苗開發

並列摘要

Photobacterium damselae subsp. piscicida (hereafter Phdp), the causative agent of fish Pasteurellosis or Photobacteriosis, is one of the most dangerous bacterial pathogens to marine fish farming worldwide. Since the first report in the USA from wild populations of white perch (Morone americanus) and striped bass (M. saxatilis) in 1963, the pathogen was spread to different countries throughout the world. In Taiwan, the disease was first described in snakehead fish (Channa maculata). Thereafter, this bacterium has been reported in several farmed fish in this country, including cobia (Rachycentron canadum), paradise fish (Macropodus opercularis) and semicircle angelfish (Pomacanthus semicirculatus). However, up to now, the epidemiological characterizations of this pathogen in Taiwan is still scarce. Such a study will be necessary to develop strategies for the prevention and control of the disease. On the other hand, vaccine development has been considered as a powerful tool, not only to control the disease outbreak but also to reduce the use of chemicals or antibiotics in aquaculture. Different types of vaccine have been developed against Phdp, including whole-cell vaccine, live attenuated vaccine, DNA vaccine and subunit vaccine. With the advantages of low cost, safety and less chance of contamination, recombinant subunit vaccine has been the choice of researchers in vaccine development against fish pathogens. In this thesis, firstly, the genotypic, molecular and pathogenic characteristics of Phdp isolated from different farmed fish in Taiwan were studied. Then, four Phdp Heat-shock proteins, including HSP90, HSP33, HSP70 and DnaJ, a group highly immunogenic protein, was chosen for recombinant vaccine development against Photobacteriosis using Asian sea bass (Lates calcarifer) as a fish model.
In the first study, a collection of 39 isolates from different fish in Taiwan, confirmed as Phdp by multiplex PCR was used to investigate the phenotypic and genotypic characteristics, the prevalence of virulence genes, molecular phylogenetic analysis and pathogenicity test of isolates from different host and year of isolation. The results showed that Phdp is a homogeneous group when shares the same phenotype among isolates, however, high genetic variability was revealed when 13 and 11 different band patterns were obtained using the pulsed-field gel electrophoresis (PFGE) analysis with SmaI and NotI as restriction enzymes, respectively. Phylogenetic analysis using 16S rDNA and the Fur gene could not differentiate between Taiwanese Phdp with its subspecies damselae. However, ToxR gene, a valuable molecular clock, could discriminate between the two subspecies. Virulence-associated genes detection for AIP56, P55, PDP_0080, Sod, and Irp1 showed that all these genes were found in all isolates. However, the pathogenicity test for nine representative isolates in Cobia (Rachycentron canadum) and Asian sea bass (Lates calcarifer) showed that there was a difference in terms of virulence among isolates. And the similar mortality rates in Cobia and Asian sea bass after challenge with different Phdp isolates indicated that Asian sea bass is susceptible to this bacterium under laboratory conditions, and can be used as an experimental model in future studies.
In the next study, four Heat-shock proteins of Phdp including HSP90, HSP33, HSP70 and DnaJ were selected for recombinant vaccine development. DNA sequences encoding for each gene were amplified, cloned into the expression vector pET-151 and then transformed into the E.coli BL21 (DE3) for protein expression. Purified proteins were immunized to a group of 50 Asian sea bass in the mixture with the adjuvant Montanide ISA 763 AVG at a ratio of 30:70 (v/v). Control groups are fish were intraperitoneally injected with PBS emulsified with ISA or PBS only. High antibody levels, serum lysozyme activities along with bactericidal effect in fish immunized with recombinant Heat-shock proteins, especially rHSP33 group, were observed. Early immune responses after vaccination in fish lymphoid organs with high expression levels of immune-related genes were also recorded in this group. The relative percent survival in each group after challenge with Phdp strain AOD105021 was 48.28%, 62,07%, 51,72%, and 31,03%, respectively. Taken together, these results suggested that HSP33 is a good candidate for vaccine development against Photobacteriosis in fish.

並列關鍵字

Photobacterium damselae subsp. piscicida, Phdp ； genotyping ； phenotyping ； heat-shock proteins ； vaccine development

參考文獻

Abdelsalam M, Chen S-C, Yoshida T (2010) Phenotypic and genetic characterizations of Streptococcus dysgalactiae strains isolated from fish collected in Japan and other Asian countries. FEMS Microbiol Lett 302:32–38.

Adams A (2019) Progress, challenges and opportunities in fish vaccine development. Fish Shellfish Immunol 90:210–214.

Adams A, Aoki T, Berthe F, Karunasagar I (2008) Recent technological advancements on aquatic animal health and their contributions toward reducing disease risks. In: Bondad-Reantaso MG, Mohan CV, Crumlish M and SRP (eds) Diseases in Asian aquaculture VI, Colombo, Sri Lanka. Fish Health Section, Asian Fisheries Society, Manila, Philippines, pp 71–88

Amagliani G, Omiccioli E, Andreoni F, et al (2009) Development of a multiplex PCR assay for Photobacterium damselae subsp. piscicida identification in fish samples. J Fish Dis 32:645–653.

Amemiya K, Meyers JL, DeShazer D, et al (2007) Detection of the host immune response to Burkholderia mallei heat-shock proteins GroEL and DnaK in a glanders patient and infected mice. Diagn Microbiol Infect Dis 59:137–147.