Development and evaluation of vaccines against vibrio harveyi in orange spotted grouper (epinephelus coioides)

國立屏東科技大學 兽医系 Department of Veterinary Medicine 熱帶農業暨國際合作系 National of Science Science and and Technology National Pingtung Pingtung University University of Technology 博士學位論文 Ph.D Dissertation 點帶石斑魚哈維氏弧菌之疫苗開發與評估 Development and evaluation of vaccines against Vibrio harveyi in orange-spotted grouper (Epinephelus coioides) (書稿) ) 指導教授：陳石柱博士 Advisor: Shih-Chu Chen, Ph.D., Professor 研究生：阮崇海 Student: Hai Trong Nguyen 中華民國 107 年 02 月 26 日 February 26th, 2018 中文摘要 學號： P10316008 論文題目:點帶石斑魚哈維氏弧菌之疫苗開發與評估 總頁數: 122 系所名稱：國立屏東科技大學 獸醫學系 時間及摘要別：106 學年度第二學期博士學位論文摘要 研究生：阮崇海 指導教授： 陳 石 柱 論文摘要內容： 哈維氏弧菌（Vibrio harveyi）是造成養殖石斑嚴重弧菌感染的主要 細菌性病原，所以實際上亦投入相當資源來控制本病原。在本研究中， 我們開發哈維氏弧菌的不活化疫苗和重組外膜蛋白（rOMPs）疫苗並評 估對點帶石斑魚免疫反應和保護效力。候選疫苗有（1）哈維氏弧菌福 馬林死菌 （FKC） 混合佐劑 MontanideTM ISA 763 AVG（2）哈維氏弧菌 福馬林死菌混合富含 CpG ODN 1668 質體（p30CpG 與 p60CpG）及（3） 哈維氏弧菌重組外膜蛋白混合佐劑 MontanideTM ISA 763 AVG。研究結果 顯示哈維氏弧菌福馬林死菌混合佐劑 Montanide ISA 763 AVG 的疫苗在 免疫後第 天與第 天可促使石斑魚腎臟與脾臟的細胞激素 IL-1β、IL6、 IL-8 與 IL-10 顯著高度表現。疫苗接種魚隻其抗體力價顯著增高，同 時亦可顯見此疫苗對同源哈維氏弧菌菌株攻毒之石斑魚具高保護力，在 免疫後第 週與第 12 週之相對存活百分比分別為 100％和 91.7％。免疫 魚隻亦被證實對異源性哈維氏弧菌菌株攻毒具強交叉保護力。接種哈維 氏弧菌福馬林死菌混合富含 CpG ODN 1668 質體疫苗的魚隻在免疫後 週可刺激抗體顯著增升。而魚隻接種具佐劑 CpG ODN 1668 疫苗 6週後， 脾臟中第二型主要組織相容性複合體（MHC）、CD8 和第九型類鐸受體 的表現程度顯著上升。此外免疫 FKC 混合 p60CpG 疫苗魚隻其第二型主 要組織相容性複合體與腫瘤壞死因子 α 顯示出更高量的 mRNA 表現。以 i 哈維氏弧菌攻毒後之相對存活百分比，FKC + p60CpG 免疫組（96.2％） 顯著高於 FKC + p30CpG 免疫組（79.8％）和 FKC 免疫組（59.9％）， 疫苗效果相當好。混合佐劑 MontanideTM ISA 763A VG 之重組哈維氏弧 菌外膜蛋白 rOmpK，rOmpU 和 rOmpK-OmpU 之融合蛋白疫苗不僅在免 疫後 24 小時即可顯著提升 IL-1β 與 IL-8 表現量在免疫後 週亦可誘導大 量抗體產生。特別的是以融合蛋白疫苗免疫的魚隻可觀察到強化的免疫 反應與對哈維氏弧菌感染具顯著保護效力，其相對存活百分比值達 81.8 ％。此外，rOmpK-OmpU 抗血清不僅對哈維氏弧菌具有很高的殺菌效力， 對副溶血弧菌和溶藻弧菌亦然。這些結果證明不活化哈維氏弧菌疫苗十 分具有潛力，其可以誘發良好的免疫反應並對養殖石斑魚提供顯著的保 護效力，而 CpG ODN 1668 可作為哈維氏弧菌疫苗之具潛力佐劑。此外 融合蛋白 rOmpK-OmpU 為另一具多重保護性之有效候選疫苗，可進一步 開發來控制養殖海水魚類的弧菌感染。 鍵詞: 哈維氏弧菌、疫苗、免疫反應、保護效力、點帶石斑 ii Abstract Student ID: P10316008 Title of dissertation: Development and evaluation of vaccines against Vibrio harveyi in orange-spotted grouper (Epinephelus coioides) Total page: 122 Name of Institute: National Pingtung University of Science and Technology Department of Veterinary Medicine Graduation date: February 26th, 2018 Dissertation abstract Name of student: Hai Trong Nguyen Name of advisor: Dr Shih-Chu Chen The content of abstract in this dissertation: Vibrio harveyi is a major bacterial pathogen that causes serious vibriosis in cultured groupers, so considerable efforts are in practice to control this pathogen In the present study, we developed and evaluated the immune responses and protective efficacy of inactivated and recombinant outer membrane protein (rOMP) vaccines against V harveyi in orange-spotted groupers The vaccine candidates were (1) V harveyi formalin-killed cells (FKC) plus MontanideTM ISA 763 AVG, (2) V harveyi FKC combined with CpG ODN 1668-enriched plasmids (p30CpG and p60CpG), and (3) V harveyi rOMPs in addition to MontanideTM ISA 763 AVG Our results indicated that the vaccine containing V harveyi FKC formulated with Montanide ISA 763 AVG adjuvant triggered remarkably high expression levels of interleukin (IL)1β, IL-6, IL-8, and IL-10 in the groupers’ kidneys and spleens, as recorded after and days of immunization Antibody titers were significantly elevated in the vaccinated fish A pivotal observation was that the vaccine highly protected the grouper from a homologous V harveyi strain challenge with relative iii percentage survival values of 100% and 91.7% at and 12 weeks postimmunization, respectively Vaccinated fish also demonstrated strong crossprotection against a heterologous V harveyi isolate challenge The vaccines containing V harveyi FKC and CpG ODN 1668-enriched plasmids stimulated remarkably greater antibody titers in vaccinated fish weeks postimmunization The expression levels of major histocompatibility complex (MHC) class II, CD 8, and toll-like receptor were significantly upregulated in the spleen of fish immunized with the CpG ODN 1668-adjuvanted vaccines at weeks after immunization Additionally, the FKC + p60CpG-vaccinated fish displayed greater mRNA levels of MHC I and tumor necrosis factor-alpha Of note, the relative percent survival after V harveyi challenge was significantly higher in FKC + p60CpG-vaccinated fish (96.2%) than in FKC + p30CpG-vaccinated (79.8%) and FKC-vaccinated fish (59.9%) The emulsified rOMP vaccines containing recombinant V harveyi outer membrane protein K (rOmpK), rOmpU, and rOmpK-OmpU fusion protein in addition to the MontanideTM ISA 763 A VG adjuvant resulted in a remarkably higher expression of IL-1β and IL-8 at 24 h, and greater antibody production, as early as weeks post-immunization Notably, an enhanced immune response and significant protective efficacy against V harveyi infections were observed in the fusion protein vaccine-injected fishes with relative percent survival value of 81.8% Additionally, the rOmpK-OmpU antisera presented a high bactericidal effect on not only V harveyi, but also V parahaermolyticus and V alginolyticus These results demonstrate that the inactivated V harveyi vaccines are promising candidates that could stimulate strong immune responses and confer high level protection in farmed groupers, and CpG ODN 1668 is a potential adjuvant for vaccines against V harveyi Additionally, the fusion protein rOmpK-OmpU is an alternative and effective vaccine candidate that exhibited potentially great versatility and can be developed further for controlling Vibrio sp infection in cultured marine fish Keywords: V harveyi, vaccines, immune responses, protective efficacy, orange-spotted grouper iv Acknowledgements This work was carried out in the Laboratory of aquatic animal diseases, at the Department of Veterinary Medicine, National Pingtung University of Science and Technology from March 2015 to January 2018 I would like to show my immense gratitude, first and foremost, to my advisors, Professor Dr Shih-Chu Chen for his guidance, support, constructive comments, and constant understanding You enlighten and lead me to go forward in my specialty and on the avenue of life filled with gleam victories but also contains many difficulties as well as challenges This study would not have been possible without your help I appreciate all your advice and it will prepare me for whatever obstacles I will face in the future In addition, I would like to extend our appreciation to all of the professors at Department of Veterinary Medicine, National Pingtung University of Science and Technology for all the lectures and knowledge that you taught I am especially indebted to Dr Pei-Chi Wang for all of your kind help during the time that I study in the school and research in the laboratory My Ph.D would have remained a dream if I did not have a great opportunity to study at National Pingtung University of Science and Technology I also greatly appreciate the financial support from Vietnam government for my Ph.D studying program at the NPUST and good condition provided by the Institute of Veterinary Research and Development of Central Vietnam I cannot find words to express my gratitude to my parents and my family for always standing by me in every moment of my life and keeping me going in the right tracts Last but not least is my special thanks to my dear colleagues and friends for all their help and cooperation For those who have assisted me during my study and are not listed here, please received by sincere acknowledgment and wish you all the best v Table of Contents 中文摘要 i Abstract iii Acknowledgements v Table of Contents vi List of Tables xiii List of Figures xv List of abbreviation xviii CHAPTER INTRODUCTION 1.1 Research background 1.2 Research objectives CHAPTER REVIEW OF LITERATURE 2.1 Groupers aquaculture 2.2 Diseases in cultured groupers 2.2.1 Bacterial diseases 2.2.2 Parasitic diseases 2.2.3 Viral diseases vi 2.3 V harveyi 2.3.1 Characteristic of V harveyi 2.3.2 Identification of V harveyi 10 2.3.3 V harveyi diseases in fish 13 2.3.4 Pathogenicity of V harveyi 16 2.4 Control V harveyi infection 18 2.4.1 Antibiotic treatments 18 2.4.2 Probiotics and immunostimulants 19 2.4.3 Development of vaccines against V harveyi 22 2.4.3.1 Inactivated vaccines 22 2.4.3.2 Recombinant subunit vaccines 23 2.4.3.3 DNA vaccines 24 2.4.3.4 Attenuated vaccines 25 2.5 Adjuvants for fish vaccines 28 2.5.1 Montanide ISA763 adjuvant 28 2.5.2 CpG ODNs adjuvant 30 CHAPTER 32 MATERIALS AND METHODS 32 3.1 Flow chart for development and evaluation of emulsified V harveyi formalin-killed cells vaccine 33 vii 3.1.1 Fish husbandry for evaluation of emulsified V harveyi formalinkilled cells vaccine 34 3.1.2 Bacteria isolation 34 3.1.3 Analysis of V harveyi genotype by pulsed-field gel electrophoresis 34 3.1.4 Preparation and vaccination of emulsified V harveyi formalinkilled cells vaccine 35 3.1.5 Agglutination assay to analyze antibody production induced by emulsified V harveyi formalin-killed cells vaccine 36 3.1.6 RT-qPCR to analyze immune-related genes expression induce by emulsified V harveyi formalin-killed cells vaccine 36 3.1.7 Challenge trials to evaluate emulsified V harveyi formalin-killed cells vaccine efficacy 37 3.1.8 Bacterial count in grouper immunized with emulsified V harveyi formalin-killed cells vaccine after challenge 37 3.1.9 Statistical analysis to compare immune responses and protective efficaccy of emulsified V harveyi formalin-killed cells vaccine 38 3.2 Flow chart for development and evaluation of V harveyi formalinkilled cells + pCpG ODN 1668 vaccines 38 3.2.1 Bacterial strain and plasmid 39 3.2.2 Fish husbandry for evaluation of V harveyi formalin-killed cells + pCpG ODN 1668 vaccines 39 3.2.3 Construction of plasmids carrying multicopies CpG ODN 1668 39 viii 3.2.4 Preparation of V harveyi formalin-killed cells + pCpG ODN 1668 vaccines 40 3.2.5 Immunization of V harveyi formalin-killed cells + pCpG ODN 1668 vaccines and sample collection 41 3.2.6 Agglutination assay to analyze antibody production induced by V harveyi formalin-killed cells + 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extracellular products Journal of Fish Diseases, 26, 103-108 117 BIOSKETCH OF AUTHOR Full name: Hai Trong Nguyen Date of birth: October 8th, 1983 Natoinality: Vietnamese Place of work: Department of Bacteriology Institute of Veterinary Research and Development of Central Vietnam Address: Km 4, 2/4 street, Vinh Hoa, Nha Trang city, Vietnam Contact no.: +886 965 455465 / +84 977 440048 Email: nguyentronghaipvtymt@gmail.com Education and training Ph.D Aquatic animal Vaccine Development, 2018 National Pingtung University of Science and Technology, Taiwan Thesis: “Development and evaluation of recombinant protein, DNA, and inactivated vaccines against V harveyi in orange-spotted grouper” M.S Animal Vaccine Technology, 2013 National Pingtung University of Science and Technology, Taiwan Thesis: “Expression of porcine circovirus type capsid protein in insect and mammalian cells using baculovirus expression vector system” B.S Doctor of Veterinary Medicine, 2007 Hue University of Agriculture and Forestry, Vietnam Thesis: “Isolation and identification of major virulent factors of Escherichia coli from poultry in highland provinces, Vietnam” Training Ethical care and use of animals in research, teaching, and testing, 2016 National Pingtung University of Science and Technology, Taiwan 118 Teaching assistant 2016 National Pingtung University of Science and Technology, Taiwan Antibiotic susceptibility testing for Salmonella sp., 2014 Italian health authority and research organization for animal health and food safety, Italy Diagnosis of aquatic diseases 2009 Nha Trang University, Vietnam Skills Construction of recombinant plasmids and expression of recombinant proteins using E coli, baculovirus, and mammalian expression systems Development of recombinant protein, DNA, and inactivated vaccines Evaluation of vaccine efficacy and immune responses in fish, mice, and livestock Isolation of bacteria, investigation of bacterial antibiotic resistance and virulence Analysis of bacterial genomic DNA profiles using pulsedfield gel electrophoresis Experiences Expression, identification, and purification of recombinant V harveyi, V alginolyticus, and V parahaemolyticus outer membrane proteins (OMPs) Evaluation of immune responses, protective efficacy of the V harveyi OMPs in orange-spotted grouper, and investigation of the serological cross-activity of antibody from the vaccinated fish with the OMPs of different Vibrio species Development of CpG ODN 1668-enriched plasmids adjuvant and evaluation of their adjuvanticity when formulated with V harveyi formalin-inactivated cells, recombinant proteins and DNA plasmids in orange-spotted grouper 119 Development and evaluation of V harveyi OmpK codon-optimized DNA vaccine efficacy and immune responses in orange-spotted grouper Analysis of DNA genomic profiles of S dysgalactiae, V parahaemolyticus, N seriolae, V harveyi and P damselae subsp piscicida using pulsed-field gel electrophoresis Investigation of antibiotic resistant and putative virulence genes in S dysgalactiae isolated from different aquatic animals and their virulence in vivo Development and evaluation of outer surface proteins and inactivated vaccines against S dysgalactiae in cobia Evaluation of immune responses and protective efficacy of a formalinkilled bivalent vaccine against V harveyi and S iniae in giant groupers Development of a DNA vaccine against Taiwan grouper iridovirus using major capsid protein, identification of protein expression eukaryotic cells and evaluation of the immune response postimmunization in orange-spotted grouper Expression and identification of porcine circovirus type (PCV2) major capsid protein in insect and mammalian cells (Sf9, CHO-k1, and Vero cells) Isolation of E coli O157:H7 from healthy cattle and buffaloes in some provinces of Central Vietnam using immunomagnetic cell separation (IMS) method Investigation of virulence genes and antibiotic resistance of E coli O157:H7 Isolation of E coli, Salmonella sp., and C perfringens from diarrhea calves Investigation of their virulence, prevention, and treatment approaches 120 Investigation of bacterial prevalence in the respiratory system of cattle with pneumonia syndrome in south central and highland provinces of Vietnam Isolation and examination of bacterial virulence in mice and cattle Development and evaluation of inactivated vaccine against P multocida in cattle Isolation of E coli from swine and poultry in central Vietnam Identification of main virulent genes of E coli and investigation of their virulence Investigate the prevalence of antibodies against E coli F4 and F18 antigens by ELISA from swine sera Awards M.S student with highest academic performance 2012, 2013 Department of Tropical Agriculture and International Cooperation, National Pingtung University of Science and Technology, Taiwan Excellent student 2009, 2010 Hue University of Agriculture and Forestry, Vietnam Publications Nguyen TH, Nguyen TTT, Wang YT, Wang PC, Chen SC 2017 Effectiveness of formalin-killed vaccines containing CpG oligodeoxynucleotide 1668 adjuvants against Vibrio harveyi in orange-spotted grouper Fish & shellfish immunology, 68: 124-131 DOI: 10.1016/j.fsi.2017.07.018 Nguyen TH, Nguyen TTT, Tsai MA, Ya-Zhen E, Wang PC, Chen SC 2017 A formalin-inactivated vaccine provides good protection against Vibrio harveyi infection in orange-spotted grouper (Epinephelus coioides) Fish & shellfish immunology, 65: 118-126 DOI: 10.1016/j.fsi.2017.04.008 Nguyen TTT, Nguyen TH, Wang PC, Chen SC 2017 Identification and expression analysis of two pro-inflammatory cytokines, TNF-alpha and IL-8, in cobia (Rachycentron canadum L.) in response to Streptococcus dysgalactiae 121 infection Fish & shellfish immunology, 67: 159-171 DOI: 10.1016/j.fsi.2017.06.014 Nguyen TTT, Nguyen TH, Tsai MA, Byadgi O, Wang PC, Yoshida T, Chen SC 2017 Genetic diversity, virulence genes, and antimicrobial resistance of Streptococcus dysgalactiae isolates from different aquatic animal sources Aquaculture, 479: 256-264 DOI: 10.1016/j.aquaculture.2017.06.002 Nguyen TH, Dao HT, Phan TH, Ho VH, Nguyen KP, Do VT, Le DH, Vu KH 2011 The prevalence and antibiotic resistance of E coli O157:H7 strains isolated from healthy cattle and buffaloes in some provinces of Central Vietnam Veterinary Science and Techniques (Vietnam), 3: 31-37 Vu KH, Nguyen TH, Dao HT, Phan TH, Ho VH, Nguyen KP, Do VT, Le DH 2011 Investigation of virulence genes of E coli O157:H7 strains isolated from healthy cattle and buffaloes in some provinces in central Vietnam Journal of Agriculture and Rural Development (Vietnam), 14: 63-67 Vo TT, Le DH, Dang TH, Dang VT, Truong CT, Nguyen TH, Vu KH 2009 Pathogen factors of Salmonella isolated from diarrheic calves in the central region and highland of Vietnam Veterinary Sciences and Techniques (Vietnam), 16 (2), 32-38 Vo TT, Le DH, Dang TH, Dang VT, Truong CT, Nguyen TH, Vu KH 2008 Prevalence and some virulence genes of E coli isolated from diarrheic calves in the central region and highland of Vietnam Journal of Agricultural and Rural Development (Vietnam), 12: 52-55 References Shih-Chu Chen, Ph.D Professor Department of Veterinary Medicine NPUST, Pingtung, Taiwan scchen@mail.npust.edu.tw +886 87703202 ext 5076, 5095 122 ... P10316008 Title of dissertation: Development and evaluation of vaccines against Vibrio harveyi in orange- spotted grouper (Epinephelus coioides) Total page: 122 Name of Institute: National Pingtung University... investigate virulence of V harveyi in groupers and screen a good candidate strain for development of effective vaccines against V harveyi infection in orange- spotted grouper; 2) To develop and evaluate... develop recombinant V harveyi outer membrane protein vaccines and study the immune responses and protective efficacy of the recombinant vaccines against V harveyi infection in orange- spotted grouper