由於養殖漁業的快速發展，為了有效防治疾病，提高漁獲量，目
前全球有相當多研究團隊致力於研究魚類免疫學與疫苗技術開發，使
用疫苗來刺激宿主適應性免疫系統，不但可以主動去預防養殖過程中
疾病的發生，且不會留下不好的副作用，像是抗藥性基因和藥物殘留
問題等。然而，不同地區可以引起疾病的病原並不相同，在不同魚種
之間的免疫反應亦存在著差距。所以，欲建立以免疫方法防治疾病，
區域性的病原及目標魚種的免疫反應相關研究是不可或缺的重要主
題。
此研究導因在2007 年10 月，台南七股地區石斑魚養殖場發生了
嚴重的疾病，在藥物的控制下，死亡率仍高達六成以上，且疾病的蔓
延十分迅速，經訪查後發現，此疾病可能是區域性流行的魚類疾病。
病原經分離純化以柯霍法則確認後，發現該疾病的半致死量LD50 在
浸泡時約為10e6 CFU / ml，在注射時約為10e4 CFU，且疾病的發生極
為迅速，屬於高致病力的病原菌，且病徵和臨場所觀察到的現象相
符，經初步鑑定可以確定該病原屬於弧菌的一支，並進一步利用生化
反應，分子生物學鑑定方法分析確定引起此疾病的病原為坎氏弧菌
(Vibrio campbellii) 。在與坎氏弧菌標準株進行致病力分析比對，發
現此菌的致病力為標準株的 1000 倍以上，顯示此菌為高致病性的坎
氏弧菌。我們進一步將坎氏弧菌製成去活化疫苗，對健康的魚隻進行
不同途徑的免疫實驗，以不同的攻毒途徑來去評估各免疫途徑疫苗使
用上的效果，結果顯示應用腹腔注射型疫苗進行免疫，在不同的攻擊
試驗中 (浸泡及注射攻毒) 皆表現出高度保護力，而在浸泡疫苗則沒
有明顯保護效果。再進一步利用免疫蛋白質體學方法分析坎氏弧菌，
找出可能的致病因子或是可引起強烈免疫反應的免疫抗原，藉此進一
步了解坎氏弧菌可能的致病機轉，以及與魚隻免疫反應的關連性，以
利於疫苗開發上的應用，結果找出十個與血清免疫反應高度相關的抗
原。在本研究中，首先發現一種高致病力的坎氏弧菌，其次建立坎氏
弧菌的攻毒系統，以及疫苗生產應用的基本相關技術，最後應用免疫
蛋白質體學的方法，快速篩選與魚隻免疫反應相關的抗原分子作為未
來應用目標。

Since aquaculture developed quickly in the past years, nowadays
there are lots of teams making efforts in the immunity of fish and vaccine
development in the world. In the resent study, using vaccine to prevent
disease outbreak actively does not remain side effects, like antibiotic
resistance pathogens emerged and chemical substance remained in the
process of aquaculture. Nevertheless, not only pathogens are various in
different areas but also the immune responses are changeful in different
species of fish. If desire set up immune methods to prevent diseases, the
research in relationship of different pathogens and various immune
responses is an important issue in the future.
In October 2007, there was a critical disease of groupers that broke
out in Tainan. There is a regional epidemic disease and possessing high
mortality. The tentative results point out the disease was caused by
unknown bacterial pathogens. In this study, we isolated the major
pathogen by Koch’s postulates and found out the LD50 were 10e6 CFU / ml
in immersion and 10e4 CFU in I.P. challenge. Characterization the
pathogen is Vibrio campbellii according to the Bergey’s Manual of
Systematic Bacteriology and the identification result of chemical methods
and molecule methods. Then the vaccine by utilizing the pathogenic
Vibrio campbellii was development and evaluating the protection in
different administration and challenge methods in grouper and the
optimal administrated and dosage was be established. In the final of this
research, immunogenic antigen of pathogens were be evaluated by using
immunoproteomic assay that may provide useful information about the

pathogen-host interaction and virulence factors, it could help to make use
of develop efficacy sub-unit vaccines. Herewith this research to know the
practicability about the prevention of the same disease happening again
by vaccination.

FAO全球魚訊水產養殖新聞月刊 (www.fao.org/fishery/aquaculture)。
行政院農委會漁業署 (www.fa.gov.tw/chnn)。
台灣魚類資料庫 (www.fishdb.sinica.edu.tw)。
James G. Cappuccino, Natalie Sherman. Microbiology- a laboratory manual second
edition. Canada: The Benjamin / Cummings Publishing Company, Inc. (1987) .
Gudding R, Lillehaug A, Midtlyng PJ, Brown F (eds) : Fish Vaccinology. Dev Biol
Stand. Basel Karger. (1997) .
王渭賢，陳明輝，鄭清福，劉正義。魚類弧菌對Fluoroquinolone 類藥物之感受
性試驗。台灣省畜牧獸醫學會會報。 (1995)，65，27-34。
陳明輝。弧菌對Fluoroquinolone 類藥物之感受性試驗。國立中興大學獸醫學研
究所碩士論文。 (1994)，1-89。
黃貴民。水產動物疾病學。水產出版社出版。 (1995)。
黃銀河。虱目魚越冬期間細菌性疾病之研究–初步報告。魚病研究專集。
(1997)，1，50-54。
蘇惠美。石斑魚繁養殖及病害防治講習班。台灣水產試驗所會報。 (2003)，1-5。
Anderson E. D., Mourich D. V., Fahrenkrug S.C., LaPatra S., Shepherd J. and Leong
J.C. Genetic immunization of rainbow trout (Oncorhynchus mykiss) against
infectious hematopoietic necrosis virus. Mol Mar Biol Biotechnol. (1996) 5,
114-122.
Austin Brian, Austin Dawn, Sutherland Rowan, Thompson Fabiano and Swings Jean.
Pathogenicity of vibrios to rainbow trout (Oncorhynchus mykiss, Walbaum) and
Artemia nauplii. Environmental Microbiology. (2005) 7(9), 1488-1495.
Awong-Taylor J., Craven K.S., Griffiths L., Bass C. and Muscarella M..
Comparison of biochemical and molecular methods for the identification of
bacterial isolates associated with failed loggerhead sea turtle eggs. Journal of
Applied Microbiology ISSN. (2007) 104(5), 1364-5072.
Bergmann Simone, Rohde Manfred, Chhatwal Gursharan S. and
Hammerschmidt Sven. α-Enolase of Streptococcus pneumoniae is a
plasmin(ogen)-binding protein displayed on the bacterial cell surface. Molecular
Microbiology. (2001) 40(6), 1273-1287.
Caipang Christopher Marlowe A., Hirono Ikuo, Aoki Takashi. Immunogenicity,
retention and protective effects of the protein derivatives of formalin-inactivated
red seabream iridovirus (RSIV) vaccine in red seabream, Pagrus major. Fish and
Shellfish Immunology. (2006) 20(4), 597-609.
Castroverde Christian Danve M., San Luis Boris B., Monsalud Rosario G., and
Hedreyda Cynthia T.. Differential detection of vibrios pathogenic to shrimp by
multiplex PCR. J. Gen. Appl. Microbiol. (2006) 52, 273–280.
Chakrabarti Sabyasachi, Sengupta Nilanjan, and Chowdhury Rukhsana. Role of DnaK
in In Vitro and In Vivo Expression of Virulence Factors of Vibrio cholerae.
Infection and Immunity. (1999) 67(3), 1025-1033.
Corripio-Miyar Y., Mazorra de Quero C., Treasurer J.W., Ford L., Smith P.D.,
Secombes C.J.. Vaccination experiments in the gadoid haddock,
Melanogrammus aeglefinus L., against the bacterial pathogen Vibrio
anguillarum. Veterinary Immunology and Immunopathology. (2007)
118(1-2), 147-153.
Culp Sandra J., Blankenship Lonnie R., Kusewitt Donna F., Doerge Daniel R.,
Mulligan Louis T., Beland Frederick A.. Toxicity and metabolism of malachite
green and leucomalachite green during short-term feeding to Fischer 344 rats and
B6C3F1 mice. Chemico-Biological Interactions. (1999) 122(3), 153-170.
De Lillo A , Ashley F. P., Palmer R. M., Munson M. A., Kyriacou L.,
Weightman A. J. , Wade W. G.. Novel subgingival bacterial phylotypes detected
using multiple universal polymerase chain reaction primer sets. Oral
Microbiology Immunology. (2006) 21, 61-68.
Dumetz Fabien, Duchaud Eric, Claverol Ste´phane, Orieux Nicolas, Papillon Sandrine,
Lapaillerie Delphine and Le He´naff Michel. Analysis of the Flavobacterium
psychrophilum outer-membrane subproteome and identification of new antigenic
targets for vaccine by immunomics. Microbiology. (2008) 154, 1793-1801.
Ehinger Stefanie, Schubert Wolf-Dieter, Bergmann Simone, Hammerschmidt Sven
and Heinz Dirk W.. Plasmin(ogen)-binding α-Enolase from Streptococcus
pneumoniae: Crystal Structure and Evaluation of Plasmin(ogen)-binding Sites. J.
Mol. Biol. (2004) 343, 997-1005.
Falkow Stanley. Molecular Koch’s postulates applied to bacterial pathogenicity — a
personal recollection 15 years later. Nature Reviews Microbiology. (2004) 2(1),
67-72.
Feldman-Salit Anna, Wirtz Markus, Hell Ruediger and Wade Rebecca C.. A
Mechanistic Model of the Cysteine Synthase Complex. J. Mol. Biol. (2009) 386,
37-59.
Foley Janet, Nieto Nathan C., Madigan John, and Sykes Jane. Possible
Differential Host Tropism in Anaplasma phagocytophilum Strains in the
Western United States. Animal Biodiversity and Emerging Diseases: Ann. N.Y.
Acad. Sci. (2008) 1149, 94-97.
Frank Jeremy A., Reich Claudia I., Sharma Shobha, Weisbaum Jon S., Wilson Brenda
A., and Olsen Gary J.. Critical Evaluation of Two Primers Commonly Used
for Amplification of Bacterial 16S rRNA Genes. Applied and Environmental
Microbiology. (2008) 74(8), 2461-2470.
Frost P. and Ness A. Vaccination of Atlantic salmon with recombinant VP2 of
infectious pancreatic necrosis virus (IPNV), added to a multivalent vaccine,
suppresses viral replication following IPNV challenge. Fish and Shellfish
Immunology. (1997) 7, 609-618.
Gabbianelli R., Signoretti C., Marta I., Battistoni A., Nicolini L.. Vibrio cholerae
periplasmic superoxide dismutase: isolation of the gene and overexpression of
the protein. Journal of Biotechnology. (2004) 109(1-2), 123-130.
Goulhen F., Hafezi A., Uitto V. J., Hinode D., Nakamura R., Grenier D., and
Mayrand D.. Subcellular Localization and Cytotoxic Activity of the GroEL-Like
Protein Isolated from Actinobacillus actinomycetemcomitans. Infection and
Immunity. (1998) 66(11), 5307-5313.
Gravningen K., Sakai M., Mishiba T., Fujimoto T.. The efficacy and safety of an
oil-based vaccine against Photobacterium damsela subsp. Piscicida in yellowtail
(Seriola quinqueradiata): A field study. Fish and Shellfish Immunology. (2008)
24, 523-529.
Hseu Jinn-Rong, Yeh Shinn-Lih, Chu Yeong-Torng And Ting Yun-Yuan.
Comparison of Efficacy of Five Anesthetics in Goldlined Sea Bream,
Sparus sarba. Acta Zoologica Taiwanica. (1998) 9(1), 35-41.
Ichise Nobutoshi, Morita Naoki, Hoshino Tamotsu, Kawasaki Kosei, Yumoto Isao,
and Okuyama Hidetoshi. A Mechanism of Resistance to Hydrogen Peroxide in
Vibrio rumoiensis S-1. Applied and Environmental Microbiology. (1999) 73-79.
JÄGER Heike, BIRKENHÄGER Ralf, STALZ Wolf-Dieter, ALTENDORF
Karlheinz and DECKERS-HEBESTREIT Gabriele. Topology of subunit a of the
Escherichia coli ATP synthase. Eur. J. Biochem. (1998) 251, 122-132.
Kai Yu-Hsuan, Chi Shau-Chi. Efficacies of inactivated vaccines against
betanodavirus in grouper larvae (Epinephelus coioides) by bath immunization.
Vaccine. (2008) 26, 1450-1457.
Kim Ju-Sim, Sung Moon-Hee, Kho Dhong-Hyo, and Lee Jeong K.. Induction of
Manganese-Containing Superoxide Dismutase Is Required for Acid Tolerance in
Vibrio vulnificus. Journal of Bacteriology. (2005) 187(17), 5984-5995.
Kumaran Sangaralingam, Yi Hankuil, Krishnan Hari B., and Jez Joseph M..
Assembly of the Cysteine Synthase Complex and the Regulatory Role of
Protein-Protein Interactions. The Journal of Biological Chemistry. (2009)
284(15), 10268-10275.
Lin C.C., Lin J.H., Chen M.S and Yang H.L. An oral nervous necrosis virus vaccine
that induces protective immunity in larvae of grouper (Epinephelus coioides).
Aquaculture. (2007) 268, 265-273.
Madetoja J., Lcnnstrcm L-G, Bjcrkblom C, Ulukcy G, Bylund G, Syvertsen C,
Gravningen K, Norderhus E-A and Wiklund T. Efficacy of injection vaccines
against Flavobacterium psychrophilum in rainbow trout, Oncorhynchus mykiss
(Walbaum). Journal of Fish Diseases. (2006) 29, 9-20.
Movahedi Abdolreza (Reza), Hampson David J.. New ways to identify novel
bacterial antigens for vaccine development. Veterinary Microbiology. (2008)
131(1-2), 1-13.
Navot Noy, Kimmel Eitan, Avtalion Ramy R.. Enhancement of antigen uptake and
antibody production in goldfish (Carassius auratus) following bath
immunization and ultrasound treatment. Vaccine. (2004) 22(20), 2660-2666.
Salonius K, Siderakis C, MacKinnon AM, Griffiths SG. Use of Arthrobacter
davidanieli as a live vaccine against Renibacterium salmoninarum and
Piscirickettsia salmonis in salmonids. Dev. Biol. (Basel). (2005) 121, 189-197.
Sandaa RA, Torsvik VL, Goksoyr J. Transferable drug resistance in bacteria from
fish-farm sediments. Canadian Journal of Microbiology. (1992) 38, 1061-1065.
Shao Z.J. Aquaculture pharmaceuticals and biologicals: current perspectives and
future possibilities. Adv Drug Deliv. Rev. (2001) 50(3), 229-243.
Shin Gee-Wook, Palaksha K.J., Kim Young-Rim, Nho Sung-Won, Kim Suk, Heo
Gang-Joon, Park Se-Chang, Jung Tae-Sung. Application of immunoproteomics
in developing a Streptococcus iniae vaccine for olive lounder (Paralichthys
olivaceus). Journal of Chromatography B. (2007) 849(1-2), 315-322.
Silva-Rubio A, Avendano-Herrera R, Jaureguiberry B, Toranzo AE and Magarinos B.
First description of serotype O3 in Vibrio anguillarum strains isolated from
salmonids in Chile. Journal of Fish Diseases. (2008) 31(3), 235-239.
Sorensen Uffe B. Skov and Larsen Jens Laurits. Serotyping of Vibrio
anguillarum. Applied and Enviromental Microbiology. (1986) 51(3), 593-597.
Van Der Ark A.A.J., I van straaten-van de kappel.e, van de Donk H.J.M. and
Hendriksen C.F.M.. Evaluation of whole cell vaccine-induce humoral antibody
response in the pertussis serological potency test in relation to mouse protection
test. National Institute of Public Health and The Environment Bilthoven. (1997)
1-29.
Weber JT, Mintz ED, Canizares R, Semiglia A, Gomez I, Sempertegui R, Davila A,
Greene KD, Puhr ND, Cameron DN. Epidemic cholera in Ecuador:
multidrug-resistance and transmission by water and sea food. Epedemiology and
Infection. (1994) 112(1), 1-11.
Wong Hin-Chung, Lu Kai-His. Cloning, sequencing and expression of GroEL-like
protein gene of Vibrio parahaemolyticus. J Microbiol Immunol Infect. (2004)
37(4), 203-207.
Zhang Anding, Chen Bo, Mu Xiaofeng, Li Ran, Zheng Pei, Zhao Yaxin, Chen
Huanchun, Jin Meilin. Identification and characterization of a novel protective
antigen, Enolase of Streptococcus suis serotype 2. Vaccine. (2009) 27(9),
1348-1353.