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

harveyi, vaccines, immune responses, protective efficacy, orange-spotted grouper... harveyi formalin-killed cells + pCpG ODN 1668 vaccines .... harveyi formalin-killed cells + pCpG OD

國立屏東科技大學 兽医系 熱帶農業暨國際合作系 Department of Veterinary Medicine National Pingtung University of Science and Technology

National Pingtung University of Science and Technology

博士學位論文 Ph.D Dissertation

(書稿) )

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)

指導教授：陳石柱博士 Advisor: Shih-Chu Chen, Ph.D., Professor

Student: Hai Trong Nguyen

中華民國 107 年 02 月 26 日 February 26th, 2018

福馬林死菌混合富含 CpG ODN 1668 質體（p30CpG 與 p60CpG）及（3）

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 1 天與第 3 天可促使石斑魚腎臟與脾臟的細胞激素 IL-1β、IL-6、 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 6 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 2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

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％），

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 2 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

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

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 1β, IL-6, IL-8, and IL-10 in the groupers’ kidneys and spleens, as recorded after

(IL)-1 and 3 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

percentage survival values of 100% and 91.7% at 6 and 12 weeks immunization, respectively Vaccinated fish also demonstrated strong cross-

post-protection 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 2 weeks

post-immunization The expression levels of major histocompatibility complex

(MHC) class II, CD 8, and toll-like receptor 9 were significantly upregulated

in the spleen of fish immunized with the CpG ODN 1668-adjuvanted vaccines

at 6 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 2 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

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 do 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

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 1 1

INTRODUCTION 1

1.1 Research background 1

1.2 Research objectives 4

CHAPTER 2 5

REVIEW OF LITERATURE 5

2.1 Groupers aquaculture 5

2.2 Diseases in cultured groupers 6

2.2.1 Bacterial diseases 7

2.2.2 Parasitic diseases 7

2.2.3 Viral diseases 8

2.3 V harveyi 9

2.3.1 Characteristic of V harveyi 9

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 3 32

MATERIALS AND METHODS 32

3.1 Flow chart for development and evaluation of emulsified V harveyi formalin-killed cells vaccine 33

3.1.1 Fish husbandry for evaluation of emulsified V harveyi

formalin-killed 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

formalin-killed 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

formalin-killed 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

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 + pCpG ODN 1668 vaccines 42

3.2.6 RT-qPCR to analyze immune-related genes expression induced by

V harveyi formalin-killed cells + pCpG ODN 1668 vaccines 42

3.2.7 Challenge test to evaluate V harveyi formalin-killed cells + pCpG

ODN 1668 vaccines efficacy 43

3.2.8 Bacterial count from peripheral blood of grouper immunized with

V harveyi formalin-killed cells + pCpG ODN 1668 vaccines 43

3.2.9 Statistical analysis to compare immune responses and efficacy of

V harveyi formalin-killed cells + pCpG ODN 1668 vaccines 43

3.3 Flow chart for development and evaluation of emulsified V harveyi

rOMP vaccines 44

3.3.1 Bacteria and plasmids for development and evaluation of

emulsified V harveyi rOMP vaccines 44

3.3.2 Fish husbandry for evaluation of emulsified V harveyi rOMP

vaccines 45

3.3.3 Construction of recombinant plasmids to express V harveyi rOMPs

45

3.3.4 Expression and identification of V harveyi rOMPs 46

3.3.5 Preparation of emulsified V harveyi rOMP vaccines 47

3.3.6 Immunization of emulsified V harveyi rOMP vaccines and sample

collection 47

3.3.7 Measument of antibody production induced by emulsified V harveyi rOMP vaccines 48

3.3.8 RT-qPCR to analyze cytokine expression induced by emulsified V harveyi rOMP vaccines 48

3.3.9 Challenge tests for evaluation of emulsified V harveyi rOMP vaccines efficacy 49

3.3.10 Cross-reactivities of V harveyi rOmpK-OmpU antisera 49

3.3.10.1 Production of antisera 49

3.3.10.2 Preparation of heterologous rOmpK and rOmpU 50

3.3.10.3 Antibody cross-reactivity 51

3.3.10.4 Bactericidal effect 51

3.3.11 Statistical analysis to compare immune responses and protective efficacy of emulsified V harveyi rOMP vaccines 52

CHAPTER 4 54

RESULTS AND DISCUSSION 54

4.1 Immune responses and effectiveness of emulsified V harveyi formalin-killed cells vaccine 54

4.1.1 Identification and genomic characterization of V harveyi 54

4.1.2 Gene expression profiling after immunization of emulsified V harveyi formalin-killed cells vaccine 56

4.1.3 Agglutination titers induced by emulsified V harveyi

formalin-killed cells vaccine 60

4.1.4 Protective efficacy of emulsified V harveyi formalin-killed cells

vaccine 61

4.1.5 Bacterial invasion and clearance in grouper immunized with

emulsified V harveyi formalin-killed cells vaccine after challenge 63

4.1.6 Cross-protection of emulsified V harveyi formalin-killed cells

vaccine 64

4.2 Immune responses and effectiveness of V harveyi formalin-killed cells

+ pCpG ODN 1668 vaccines 66

4.2.1 Construction of CpG ODN 1668-enriched plasmids and preparation

of V harveyi formalin-killed cells + pCpG ODN 1668 vaccines 66

4.2.2 Gene expression following immunization of V harveyi

formalin-killed cells + pCpG ODN 1668 vaccines 68

4.2.3 Antibody titer induced by V harveyi formalin-killed cells + pCpG

4.3.1 Production of emulsified V harveyi rOMP vaccines 79

4.3.2 Immune responses after immunization with emulsified V harveyi

rOMP vaccines 81

4.3.2.1 Cytokine expression induced by emulsified V harveyi rOMP

vaccines 81

4.3.2.2 Antibody production induced by emulsified V harveyi rOMP vaccines 84

4.3.3 Safety and protective efficacy of emulsified V harveyi rOMP vaccines 85

4.3.4 Cross-reactivity of V harveyi rOmpK-OmpU antisera 87

CHAPTER V 91

CONCLUSION AND FUTURE STUDY 91

5.1 Conclusion 91

5.2 Future study 94

APPENDICES 92

REFERENCES 98

BIOSKETCH OF AUTHOR 118

Table 4 Pathogenicity mechanisms of V harveyi 16

Table 5 Application of immunostimulants to control V harveyi diseases in

aquatic animals 21

Table 6 Vaccines developed against V harveyi in fish 27

Table 7 Comparison of 4 classes of synthetic CpG oligodeoxynucleotide 31

Table 8 Primers used in PCR for identification and cloning 50

Table 9 Primers used in RT-qPCR for analyzing immune responses 52

Table 10 Investigation of V harveyi virulence in grouper for selection of

bacterial strains for vaccine development and evaluation 55

Table 11 Mortality and relative percentage survival of vaccinated grouper after challenge with vaccine strain (Vh) 62

Table 12 The mortality and relative percentage survival of groupers

challenged with heterologous V harveyi strain (Vh 20) after immunization of emulsified V harveyi FKC vaccine 65

Table 13 Cumulative mortality and relative percent survival of groupers

challenged with V harveyi after immunization of V harveyi FKC + pCpG

ODN 1668 vaccines 77

Table 14 The mortality and relative percent survival of challenged grouper

after immunization of emulsified V harveyi rOMP vaccines 87

List of Figures

Figure 1 Grouper production in countries surveyed by Global Aquaculture Alliance 6

Figure 2 Electron micrograph of V harveyi EmI82KL strain isolated from

grouper with gastroenteritis 10

Figure 3 Biochemical key for the identification of Vibrio sp 12

Figure 4 Schematic illustration of the steps for directional multiplication of

CpG ODN 1668 in the pcDNA3.1(+) plasmid 41

Figure 5 PCR analysis for the identification of representative V harveyi

strains isolated from orange-spotted grouper (A) and the DNA banding

dendrogram of V harveyi obtained by PFGE using SmaI enzyme digestion

(B) 55

Figure 6 The relative mRNA expressions of IL-1β (A, B); IL-6 (C, D); IL-8 (E, F) and IL-10 (G, H) in grouper kidneys and spleens after immunization of

emulsified V harveyi FKC vaccine 58

Figure 7 The antibody titer of grouper serum immunized with emulsified V harveyi FKC vaccine determined by agglutination assay 60

Figure 8 Cumulative mortality of orange-spotted grouper challenged with vaccine strain (Vh) at 6 weeks (A) and 12 weeks (B) post-immunization of

emulsified V harveyi FKC vaccine 62

Figure 9 Bacterial concentration in grouper peripheral blood samples after

challenge with V harveyi at 6 weeks post-immunization of emulsified V harveyi FKC vaccine 63

Figure 10 The cumulative mortality of grouper challenged with heterologous

V harveyi strain (Vh 20) at 6 (A) and 12 weeks (B) post-immunization of emulsified V harveyi FKC vaccine 65

Figure 11 Verification of CpG ODN 1668-enriched plasmid constructs 67

Figure 12 Comparative expression profiles of MHC I, MHC II, CD8, TLR-9,

TNF-α and IL-10 in the kidney of groupers following immunization of V harveyi FKC + pCpG ODN 1668 vaccines 70

Figure 13 Comparative expression profiles of MHC I, MHC II, CD8, TLR-9,

TNF-α and IL-10 in the spleen of groupers following immunization of V harveyi FKC + pCpG ODN 1668 vaccines 71

Figure 14 V harveyi-specific antibody titer in grouper sera after

immunization of V harveyi FKC + pCpG ODN 1668 vaccines 74

Figure 15 Protective efficacy of V harveyi FKC + pCpG ODN 1668 vaccines against V harveyi challenge (A) Cumulative mortality of vaccinated groupers after V harveyi challenge (B) V harveyi concentration in the peripheral

blood of challenged fish 76

Figure 16 Construction, expression, identification, and purification of

recombinant V harveyi outer membrane proteins (rOMPs) (A) Verification

of recombinant plasmids constructed by PCR (B) SDS-PAGE analysis of the

V harveyi rOMPs expressed in E coli (C) Identification of V harveyi rOMPs

by Western blotting using anti-His antibodies (D) Purified V harveyi rOMPs

prepared for immunization of groupers 80

Figure 17 Immune responses of groupers to vaccination with emulsified V harveyi rOMP vaccines The expression levels of IL-1β, Il-6, IL-8, and IL-10

mRNAs in the kidney (A) and spleen (B) of grouper following immunization 83

Figure 18 V harveyi-specific antibodies in grouper sera following

immunization of emulsified V harveyi rOMP vaccines 84

Figure 19 The cumulative mortality of challenged groupers after

immunization of emulsified V harveyi rOMP vaccines 86

Figure 20 Comparison of OmpK and OmpU of V harveyi (Vh),

V parahaemolyticus (Vp), and V alginolyticus (Va) strains 88

Figure 21 Cross-reactivity of the V harveyi rOmpK-OmpU antisera with

heterologous bacterial pathogens Identification of rOmpK (A) and rOmpU

(B) by SDS-PAGE and Western blotting using V harveyi rOmpK-OmpU

antisera 89

List of Abbreviation

• APC Antigen-presenting cell

• CFU Colony-forming unit

• ECP Extracellular product

• pDC Plasmacytoid dendritic cell

• PCR Polymerase chain reaction

• PFGE Pulsed-field gel electrophoresis

• PI Post-immunization

• RPS Relative percentage survival

• TCBS Thiosulfate citrate bile salts sucrose

• TNF Tumor necrosis factor

• TLR Toll-like receptor

• TTS Type III secretion system

• UPGMA Unweighted average pair-group method with arithmetic mean

Groupers are important fish species for aquaculture and widely cultured

in Southeast Asia (Huang et al., 2014) However, the rapid development of

intensive aquaculture often leads to the incidences of many infectious diseases caused by bacteria, viruses, and parasites, which have become increasingly

severe (Harikrishnan et al., 2011a) Vibriosis is one of the most common infections of bacterial diseases that threaten the grouper industry (Huang et al., 2014) This serious infection is caused by a major bacterial pathogen, Vibrio harveyi, that can result in high mortality in groupers (Yii et al., 1997; Lee et al., 2002), thus may lead to dramatic economic losses for the farmers In addition, vibriosis in this fish species was also reported to cause by Vibrio parahaemolyticus (Najiah et al., 2003; Li et al., 2010) and Vibrio alginolyticus infection (Lee, 1995; Li et al., 2010) in a number of studies

Some antibiotics effectively control vibrio infections (Zhang et al., 2007),

but the emergence of drug-resistant and multi-drug resistant strains poses

serious challenges (Weston, 1996; Harikrishnan et al., 2011b) Vaccination is

an alternative option that can significantly reduce specific disease-related losses, resulting in a reduction of antibiotics use, and become an increasingly important part of aquaculture (Muktar and Tesfaye, 2016) For effectively and sustainably controlling vibriosis, different types of vaccines were developed

and tested, including formalin-inactivated (Arijo et al., 2005), attenuated (Hu

et al., 2012), recombinant protein (Zhang et al., 2007; Sun et al., 2009; Wang

et al., 2011; Chuang et al., 2014) and DNA vaccines (Qin et al., 2009; Hu and Sun, 2011; Wang et al., 2017) However, several hurdles have to be overcome

with regards to the production of cheap but effective antigens and adjuvants, while bearing in mind numerous environmental and associated regulatory

concerns (Sommerset et al., 2005) Currently, there is still a need for an

effective vaccine with a broader range of protection to cover the genetically

diversified Vibrio sp pathogens Additionally, the safety issue is critical for the

vaccine to be utilized in the fish as animal welfare and human health issues are

of paramount importance

To achieve high level protection while maintaining safety and high feasibility for application in aquaculture, inactivated bacterin vaccines are good candidates This may explain why most bacterial vaccines used in aquaculture are inactivated ones (Muktar and Tesfaye, 2016) Alternatively, subunit vaccine using conserved antigens is a promising approach to achieve cross-protection

(Kawai et al., 2004; Qian et al., 2008; Ningqiu et al., 2010) and also can meet the safety criteria (Liang et al., 2017) Previous studies have demonstrated that

outer membrane proteins (OMPs) of Gram-negative bacteria are immunogenic

(Rahman and Kawai, 2000; Kawai et al., 2004) since they expose epitopes on

the cell surface and can be easily recognized by the immune system of the host

(Zhang et al., 2007) Studied have indicated that V harveyi outer membrane

protein K (OmpK) and outer membrane protein U (OmpU) are relatively

conserved and widely distributed in other Vibrio sp.; they also show an

enhanced ability to induce immune responses and elicit protection in fish (Qian

et al., 2008; Li et al., 2010) In addition, recent research has successfully

utilized fusion protein strategies for enhancing vaccine efficacy and

strengthening host immune responses (Zhang et al., 2007; Wang et al., 2011) Therefore, it is worthwhile to develop and investigate V harveyi inactivated

and fused outer membrane protein vaccines in our effort to develop more effective and feasible vaccines with a broader range of protection potential

Vaccines are not usually able to confer adequate protection on their own, especially those that are based on recombinant antigens or inactivated

pathogens (Huang et al., 2014) Therefore, there is a need for supplement

formulations with adjuvants to improve vaccine efficacy and prolong the

duration of protection (Evensen et al., 2005; Thim et al., 2014) MontanideTM

ISA 763 A VG (Seppic, France) is an injectable, metabolizable, oil adjuvant that does not contain mineral oil Research indicates that this adjuvant is excellent for stimulating a protective immune response with different kind of

antigens in fish models (Thim et al., 2014), including grouper (Huang et al., 2014) and rainbow trout (Jaafar et al., 2015)

CpG oligodeoxynucleotides (ODNs) have been demonstrated to function

as adjuvants are able to both accelerate and magnify immune responses when

co-administered with vaccines (Bode et al., 2011) in mammals Among

different classes of ODNs, class-B CpG is the most stable, has strong

stimulatory effects on B lymphocytes (Cuesta et al., 2008), and therefore, can

be a potential vaccine adjuvant CpG ODN 1668 belongs to CpG class B and has been shown to elicit strong immune responses against various infectious

pathogens in fish, including Photobacterium damselae subsp piscicida (Byadgi et al., 2014), Aeromonas hydrophila (Yogeshwari et al., 2015), and Miamiensis avidus (Kang et al., 2014a; Kang et al., 2014b) Against this

background, it is necessary to develop a possible strategy to improve the

immunity against V harveyi in order to effectively control its infection and

mitigate this bacterial disease in orange-spotted grouper with the following objectives

1.2 Research objectives

1) To 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 the immune responses, protective efficacy of

vaccines containing V harveyi formalin-killed cells formulated with ISA

MontanideTM 763 A VG adjuvant in orange-spotted grouper;

3) To develop plasmids carrying multiple copies of CpG ODN 1668 as an

adjuvant, examine the effectiveness of vaccines using V harveyi

formalin-killed cells formulated with the CpG-enriched plasmids constructed and immune responses induced by vaccination in orange-spotted grouper;

4) To 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

Our main goal is to develop good vaccines that can effectively control

V harveyi infection in orange-spotted grouper that can be applied in

aquaculture to reduce severe losses caused by vibriosis

CHAPTER 2

REVIEW OF LITERATURE

2.1 Groupers aquaculture

Groupers belong to the Serranid sub-family Epinephelinae, which is

composed of 15 genera and 159 species (Pierre et al., 2008) These are

characterized by large-mouthed, rather heavy bodied fishes, tend to remain in discrete areas, and are widely distributed in tropical or warm waters worldwide

(Harikrishnan et al., 2011a) They are principally distributed among the

Indo-Pacific region (110 species), the East Atlantic and Mediterranean regions (14

species) and the intertropical American zone (35 species) (Pierre et al., 2008)

There are approximately 15 grouper species being cultured in Southeast Asia

and the dominant species varies between are regions Epinephelus coioides and

E malabaricus are the most consistently abundant species captured wild for

culture and also reared in hatcheries, follow by other important species

including E bleekeri, E akaara, E awoara and E areolatus (Sadovy, 2001)

Owning to their desirable taste, high consumer demand, good price, efficient feed conversion, rapid growth, and hardiness in a crowded environment, groupers are good candidate species for aquaculture

(Harikrishnan et al., 2012) Additionally, the supply of wild caught fish is

usually insufficient to satisfy the strong demands of the market, thus there is a

need for expansion of aquaculture (Pierre et al., 2008) Grouper culture industry has been established in Asia in the 1980s (Pierre et al., 2008) and is widely

practiced in the tropical East Asia, such as China, Hong Kong, Taiwan, as well

as Southeast Asia, including Indonesia, Malaysia, Vietnam, Philippines,

Singapore, and Thailand (Sim et al., 2005) Groupers are also cultivated in other parts of the tropics in the Caribbean (Pierre et al., 2008) and south-eastern

USA (Tucker, 1999) The fishes are mostly cultured in floating net cages either

in the open sea or at the seaward end of estuaries (Pierre et al., 2008) Most

grouper species bring high prices in local or export markets, and so are an

attractive culture proposition for coastal aquaculture farmers (Sim et al., 2005)

The production of groupers in their main producer countries was predicted to keep increasing and reach approximately 155000 metric tons in the year 2018 (Figure 1)

Figure 1 Grouper production in countries surveyed by Global Aquaculture Alliance (Tveteras, 2016)

2.2 Diseases in cultured groupers

Groupers are cultured in industrial scale in many Asian countries

(Boonyaratpalin, 1997; Sim et al., 2005) In Taiwan, groupers are one of the

most expensive fish on the Taiwanese market, which has generated a great deal

of interest among breeders and research organizations (Pierre et al., 2008) The

average annual production of grouper was about 9,400 mt from years 2000 to

2004 and it is on the top-ten highest yielding aquaculture species in Taiwan (Liao, 2005) However, infectious disease outbreaks in cultured grouper have found significantly increased as the consequence of the rapid development of

intensive culture (Harikrishnan et al., 2012) The industry, therefore, has been severely hit by epidemics associated with bacteria (Lee, 1995; Lee et al., 2002), viruses (Fukuda et al., 1996), and parasites (Yambot et al., 2003), which have

become increasingly severe over the last several years leading to heavy

economic losses (Lee, 1995; Yeh et al., 2008)

2.2.1 Bacterial diseases

Various bacterial diseases have been reported in cultured groupers in the

Asian Pacific region (Harikrishnan et al., 2011a) Firstly, vibriosis caused by

V harveyi is one of the most serious diseases that commonly occur in various stages of grouper culture (Yii et al., 1997; Lee et al., 2002) V harveyi infection

is frequently associated with mass mortalities, which occur often in

brown-spotted grouper (Epinephelus tauvina) in Kuwait (Saeed, 1995) and spotted grouper (E coioides) in Taiwan (Yii et al., 1997) Other Vibrio species

orange-have also been demonstrated as causative agents of vibriosis in cultured

groupers (Lee, 1995; Najiah et al., 2003; Li et al., 2010) In addition, groupers are also susceptible to Pseudomonas sp (Nash et al., 1987) and Flexibacter sp

(Tendencia and Lavilla-Pitogo, 2004) Streptococcosis is also a big problem in groupers and other marine finfish in East and Southeast Asia, which caused by

Streptococcus sp (Bowater et al., 2012; Delamare-Deboutteville et al., 2015) The disease is often associated with vibriosis and it has been reported in E malabaricus and E bleekeri in Brunei Darussalam, Malaysia, Singapore and

Thailand (Tendencia and Lavilla-Pitogo, 2004)

2.2.2 Parasitic diseases

There is a wide variety of parasitic organisms causing significant problems reported in grouper aquaculture Parasitic diseases of groupers are predominantly caused by protozoans, particularly the ciliates in the hatchery and nursery stages When these fish are transferred to grow-out facilities after fry stage, which is subjected to handling and suffers from transport stress, a large variety of ciliated protozoans, skin and gill monogeneans, and caligid

copepods often infect the fish with high intensity (Harikrishnan et al., 2011a) Cryptocaryon irritans is a holotrichous ciliate that causes white spot disease in

marine fishes in temperate and tropical seas This parasite can overwhelm an

entire fish population in a few days under aquarium conditions C irritans

invades the skin, eyes, gills, and impairs the physiological function of these

organs of the hosts (Colorni and Burgess, 1997) Heavy infections of C irritans

trophonts in the epidermis of the skin and gills can lead to asphyxiation, osmotic imbalance, and secondary bacterial infections resulting in death of the

host (Diggles and Adlard, 1997) An initial outbreak of C irritans caused more

than 50% mortality of juvenile brown-spotted grouper in Kuwait (Rasheed,

1989) and infection of different strains of C irritans have been reported in groupers (Diggles and Adlard, 1997; Yambot et al., 2003; Harikrishnan et al.,

2011a)

2.2.3 Viral diseases

Diseases caused by viral pathogens are the other serious problems in grouper aquaculture Viral infections are often associated with high mortalities and lead to tremendous economic losses for farmers in Southeast Asia One of the most important pathogens of groupers in the last decade is Iridovirus, which

infected more than 30 grouper species worldwide (Harikrishnan et al., 2011a) The Iridoviridae family is currently subdivided into five genera, including Chloriridovirus, Iridovirus, Lymphocystivirus, Megalocytivirus, and Ranavirus containing great diversity in gene content between different genera (Eaton et al., 2010) Singapore grouper iridovirus is a novel virus that belongs to the

genus Ranavirus It is considered as an important pathogen in cultured groupers that causes serious systemic disease and capable of killing up to 96% of infected

grouper fry farm stocks (Chang et al., 2002; Qin et al., 2003) Megalocystivirus

infection was recorded in a variety of fish species, including brown-spotted grouper (E chlorostigma), malabar grouper (E malabaricus ), and Epinephelus

sp (Harikrishnan et al., 2011a)

Additionally, grouper aquaculture is also threatened by viral nervous necrosis (VNN) disease and caused by a new member of the Nodaviridae The disease is also found in many marine fish species world-wide leading to an

extremely high mortality rate in larvae and juveniles (Chi et al., 2001) Based

on the viral genome and its proteins properties, fish nodaviruses have been classified into four genotypes, including barfin flounder nervous necrosis virus, red-spotted grouper nervous necrosis virus, striped jack nervous necrosis virus,

and tiger puffer nervous necrosis virus (Nishizawa et al., 1997) In groupers,

this disease was first reported in 1994 and has continuously been identified in cultured groupers Infected with NNV resulted in 80-100% mortality in grouper

larvae within 1-2 months of age (Chi et al., 1997; Chi et al., 2001; Chi et al., 2003; Harikrishnan et al., 2011a).

2.3 V harveyi

2.3.1 Characteristic of V harveyi

V harveyi is a Gram-negative, motile bacterium, ubiquitous in marine and

estuarine aquatic ecosystems (Hashem and El-Barbary, 2013) It was originally

named as Achromobacter harveyi after E.N Harvey for the numerous

fundamental contribution in the systematic study of bioluminescence (Johnson

and Shunk, 1936) Afterward, Lucibacterium harveyi and Beneckea harveyi

names were given to the organism at various times The bacterium is also

known as the synonym of V carchariae (Pedersen et al., 1998) Currently, the taxonomic position of the bacterium is V harveyi after the identity of V carchariae with V harveyi was shown conclusively (Farmer et al., 2005;

Austin and Zhang, 2006)

The characteristics of V harveyi are as described for the genus Vibrio,

which is small, straight, slightly curved, curved, or comma-shaped rods, 0.5–

0.8 x 1.4–2.6 µm (Farmer et al., 2005) V harveyi EmI82KL strain isolated

from diseased grouper was swarming with polar and lateral flagella on tryptic soy agar supplemented with 2% NaCl as shown in Figure 2 The strain occasionally produces H2S and formed yellow and transparent colonies on Thiosulfate Citrate Bile Salts Sucrose (TCBS) and MacConkey agar plates Additionally, this strain produced no acetoin, was positive with methyl red and

gelatinase, fermented glucose, used starch, urea, and chitin (Yii et al., 1997)

Some biochemical characteristics of V harveyi and other related species in the family Vibrionacea are presented in Table 1 (Jayasinghe et al., 2010)

Figure 2 Electron micrograph of V harveyi EmI82KL strain isolated from

grouper with gastroenteritis Scanning electron microscopy (bar = 1 µm): P,

polar flagellum; L, lateral flagellum (Yii et al., 1997)

Table 1 Biochemical characteristics of several species of family Vibrionacea

(Jayasinghe et al., 2010)

2.3.2 Identification of V harveyi

A Selective and differential medium, termed V harveyi agar (VHA), was developed for the isolation and enumeration of V harveyi On VHA, colonies

of V harveyi displayed distinctive morphologies and could be visually differentiated from the colonies of 15 other Vibrio sp Additionally, the growth

of two strains of marine Pseudomonas sp and Flavobacterium sp was inhibited in this medium All of V harveyi colonies displayed typical

morphology on VHA, small (2 – 5 mm), light green colonies with dark green centers, most of them circular with an entire margin, were positively confirmed

by conventional tests This agar is a useful tool for primary isolation and

provided significant advantages over TCBS agar for differentiating V harveyi from other marine and estuarine Vibrio sp (Harris et al., 1996)

A set of biochemical keys providing fast and presumptive identification

of V harveyi and other species of Vibrio spp was developed by Alsina and

Blanch (1994) The testing screen specially designed for routine purposes of identifying environmental Gram-negative, oxidase-positive, TCBS positive, facultative anaerobe isolates, and can be used for studies with a high number

of isolates Similarly, a biochemical key using commonly available

biochemical tests in the laboratory and biochemical characteristics of Vibrio sp

was designed according to Bergey’s and FDA manuals In this study, seven biochemical tests were included in the screen and fifteen species in the family

Vibrionaceae can be identified as shown in Figure 3 (Jayasinghe et al., 2010)

Some enzyme-activity based kits could be used with biochemical keys

for identifying environmental Vibrio isolates; however, the results should be supplemented by further confirmatory standard methods (Alsina and Blanch, 1994) To reduce the number of biochemical tests and time consumed for

definitively identifying V harveyi, Oakey et al (2003) developed a PCR using 16S rDNA sequences from a number of V harveyi strains and other vibrios Positive results were obtained for all V harveyi strains tested; however, there was false-positive in a small number of V alginolyticus strains that need

additional biochemical tests for differentiation of these two species In 2006,

an effective method for identification of V harveyi was further developed based

on the toxR gene as a taxonomic marker The PCR enabled identification of V

harveyi within 5 h with high specificity, sensitivity, and had a detection limit

of 4.0 x 103 cells/mL (Pang et al., 2006) An improved one-step colony PCR was established allowing detection and differentiation of V harveyi from

related species Significant advantages of this PCR is the rapid and specific detection of the target pathogen without the need for bacterial isolation, DNA extraction, and assistance of biochemical tests (Fukui and Sawabe, 2007)

Figure 3 Biochemical key for the identification of Vibrio sp (Jayasinghe et al.,

2010)

Moreover, a hemolysin gene-based species-specific multiplex PCR was developed in order to obtain simple, rapid, cost-effective method for detection

of V harveyi V campbellii, and V parahaemolyticus The multiplex PCR

provided 100% specificity and sensitivity each and sufficient to be considered

as an effective tool in a prediction system for preventing potential disease

outbreak caused by Vibrio sp (Haldar et al., 2010) Recently, a TaqMan Time PCR assays for monitoring V harveyi infection and virulent strains

Real-harboring a plasmid was reported These were powerful and useful tools to

provide a better understanding of the epidemiology of diseases caused by V harveyi in European abalone (Haliotis tuberculata) and others cultured marine

species (Schikorski et al., 2013) Primers and probes designed for PCR and real-time PCR to identify V harveyi are listed in Table 2

Table 2 Primers and probes for PCR and real-time PCR to identify V harveyi

2.3.3 V harveyi diseases in fish

V harveyi is the major causative agent of luminous vibriosis affecting a wide range of marine vertebrates and invertebrates (Table 3) In addition to V harveyi, V parahaemolyticus and V alginolyticus infection were also reported

in grouper in a number of studies (Najiah et al., 2003; Li et al., 2010) (Lee, 1995; Li et al., 2010) V harveyi was initially known to cause of vasculitis in

sharks (Colwell and Grimes, 1984) and it was lethal for a physiologically

compromised lemon shark (Gkimes and Gruber, 1985) Additionally, V harveyi was also reported to be associated with skin ulcer in similar fish, Carcharhinus plumbeus, and it was predominantly isolated from the skin ulcer (Bertone et al., 1996) In cultured groupers (E coioides), V harveyi was

described to cause gastroenteritis syndrome with swollen intestine containing

yellow fluid (Yii et al., 1997); this disease was also recorded in moribund black sea bream, yellowfin sea bream, Japanese sea bass, red drum (Lee et al., 2002), and cobia (Liu et al., 2004)

V harveyi has also been attributed to the causative agent of infectious necrotizing enteritis in summer flounder (Paralichthys dentatus) This disease

is characterized by reddening around the anal area, distended abdomens filled with opaque serosanguineous fluid, enteritis, and necrosis of the posterior intestine Posterior intestines of the fish was detached from the anus and

prolapse in severe cases of the disease (Soffientino et al., 1999) In cultured Japanese abalone (Sulculus diversicolor supratexta), V harveyi was reported

to cause high mortality rates Abalone infected with V harveyi showed white

spots on the foot, detached from the surfaces, and died on the bottom of the

aquaria in the end (Lee et al., 2002) This bacteria was reported to cause a

systemic disease in Arabian surgeon (Hashem and El-Barbary, 2013),

European abalone (Cardinaud et al., 2014), and shrimp (Zhang et al., 2007)

The abundant presence of the microorganism was found in wild

specimens of Jack crevalle (Caranx hippos) in Florida, the USA with deep

dermal lesions and it was the most common and numerically predominant bacteria recovered from opaque corneas of all snook examined (Kraxberger-

Beatty et al., 1990) V harveyi was dominantly recovered from samples and

displayed a clear association with mortalities in diseased common dentex

(Dentex dentex) (Company et al., 1999) This pathogen was also identified in sole (Solea senegalensis) cultured in the south of Spain in a disease outbreak

with moderate mortalities; and the signs of the disease were also reproduced

and confirmed by infection with the isolated bacterium (Zorrilla et al., 2003)

V harveyi was also responsible for mortality in cultured marine fish, such as

silvery black porgy and brown-spotted grouper in Kuwait (Saeed, 1995),

and ulcer disease in grouper (E awoara) in China (Lee et al., 2002)

Table 3 Diseases associated with V harveyi in marine vertebrates and

invertebrates (Austin and Zhang, 2006)

1 Deep dermal lesions Jack crevalle (C hippos)

2 Gastroenteritis grouper (E coioides), black sea bream

(Acanthopagrus schlegeli), Japanese sea bass (Lateolabrax japonicus), yellowfin sea bream (Acanthopagrus latus),

summer flounder (P dentatus), red drum (Sciaenops ocellatus)

4 Infectious necrotizing

enteritis

Summer flounder (P dentatus)

shark (Negraprion brevirostris)

Sea cucumber (Holothuria scabra)

9 White spot on the foot Japanese abalone (Sulculus diversicolor

supratexta)

2.3.4 Pathogenicity of V harveyi

V harveyi is known as a serious pathogen of marine animals; however,

its pathogenicity mechanisms have not been fully elucidated yet There are many possibilities, including the ability to attach to host cells and form biofilms, quorum sensing, extracellular products (ECPs) notably proteases and hemolysins, lipopolysaccharide (LPS), and interaction with bacteriophage and BLIS It is still unclear which markers are the most important virulence determinants It is possible that there are differences in strains examined and their virulence that may be attributable to any of a number of factors Several

putative pathogenicity mechanisms or virulence factors of V harveyi was

summarized in Table 4 (Austin and Zhang, 2006)

Table 4 Pathogenicity mechanisms of V harveyi (Austin and Zhang, 2006)

1 Extracellular products (cysteine

protease, phospholipase, hemolysin)

(Liu et al., 1996; Rodriguez et al., 2003)

7 Ability to attach and form biofilms (Karunasagar et al., 1994)

V harveyi ECPs have been considered as important virulence factors of this pathogen The association of V harveyi with mortalities was demonstrated,

and the role of ECPs in inducing disease was proven in brown-spotted grouper

(Saeed, 1995) The pathogenicity of V harveyi strains isolated from tiger prawn, (Penaeus monodon) was studied and their ECPs exhibited stronger

proteolytic (caseinase), phospholipase and hemolytic activities than those of

the reference strains Results from this study indicated that proteases, phospholipases, hemolysins or exotoxins may exert significant roles in the

pathogenicity of V harveyi in tiger prawn (Liu et al., 1996) The mortality of

naupliar was found significantly associated with the production of proteases,

phospholipases or siderophores of V harveyi However, the correlation of

mortality with lipase production, gelatinase production, hydrophobicity or hemolytic activity was not observed suggesting that virulence of the pathogen

in this host was more related to the exoenzymes production than colonization

factors (Soto-Rodriguez et al., 2003)

The pathogenicity of V harveyi may also be attributed to the presence of hemolysins Duplication of these genes in a virulent isolate of V harveyi (VIB

645) was reported The bacteria strain contained two closely related hemolysin genes, had a high titer of hemolytic activity towards fish erythrocytes in its produced extracellular product, and was found very pathogenic in salmonids

(Zhang et al., 2001) V harveyi hemolysin was considered as an important

virulence determinant in fish pathogenesis and further characterized The enzyme was identified as a phospholipase B by gas chromatography; and a specific residue, Ser153, which is critical for its enzymatic activity (and for

virulence in fish) was revealed by site-directed mutagenesis (Sun et al., 2007)

Another extracellular virulence factor, serine protease (DegQVh), from a

pathogenic V harveyi strain was characterized The purified DegQVh protein

expressed in E coli showed serine protease activities and required the integrity

of the catalytic site and PDZ domains The purified recombinant DegQVh was subsequently demonstrated as a protective immunogen conferring high level

protection in fish challenged with V harveyi (Zhang et al., 2008)

Type III secretion system (TTS) and quorum-sensing system were

considered to contribute in pathogenicity of V harveyi through the regulation

of certain proteases production (Wang et al., 2011) Presentation of genes

encoding components of a putative type III secretion system was discovered in

V harveyi by autoinducer-regulated target genetic screens TTS system surely

exists in V harveyi and an intact quorum-sensing signal transduction cascade

is essential for expression of genes encoding secretion machinery However,

quorum sensing was found to suppress TTS in V harveyi and V parahaemolyticus at high cell density along with the presence of autoinducers

This finding was not in agreement with previous studies in enterohemorrhagic

and enteropathogenic E coli reporting that quorum sensing activates TTS at

high cell density (Henke and Bassler, 2004) A recent study suggested that repression of type III secretion, which is energy-consuming, by the quorum sensing master regulators could probably be a mechanism to save energy under

certain conditions where it provides no advantage to the cells (Ruwandeepik et al., 2015)

2.4 Control V harveyi infection

2.4.1 Antibiotic treatments

V harveyi disease outbreaks have occurred and caused serious mortality

in cultured grouper and cobia in Taiwan The pathogens were susceptible to some antibiotics, including chloramphenicol, doxycycline HCl, nalidixic acid, oxolinic acid, oxytetracycline, and sulfonamide, thus represent possibilities for

treatments (Yii et al., 1997; Liu et al., 2004) Short finish (M mola) infected with V harveyi showing ocular lesions recovered after an oral antibiotic treatment and topical application of a collyrium (Hispano et al., 1997)

However, not all of these antimicrobial agents are approved for aquaculture use

in Taiwan Of note, the outcome of treatment with antimicrobials in the field is

not consistent and the use of antimicrobial substances (Harikrishnan et al.,

2011a) Moreover, the extensive application of chemotherapy with antimicrobial agents to prevent bacterial diseases and promote growth has posed some problems in the emergence of drug-resistant microorganisms and accumulation of antibiotics in the farmed fish as well as the environment ;

(Weston, 1996; Harikrishnan et al., 2011b)

2.4.2 Probiotics and immunostimulants

Infectious diseases are major problems in aquaculture and cause heavy losses to fish farmers Administration of immunostimulants, for instance, probiotics and herbals, is a promising prophylactic option, which can be

adopted to control contagious diseases (Harikrishnan et al., 2011a) Under

immunosuppressive or stressful conditions, the supplementation of immunostimulants in diets is helpful to improve the immunity of animals Consequently, their resistance to infection increased, resulting in a reduction of mortality rates and providing economic benefits (Sahoo and Mukherjee, 2002) Therefore, immunostimulants are worthy of being taken into consideration as a great potential and alternative means for controlling infection in cultured

aquatic animals (Harikrishnan et al., 2011a) A list of immunostimulants used for controlling V harveyi diseases in aquatic animals is shown in Table 5

An equal proportion of all five herbal extracts, including Cynodon dactylon, Piper longum, Phyllanthus niruri, Tridax procumbens, and Zingiber officinalis was incorporated into diets for juvenile grouper Results from this study showed that the herbal diets effectively controlled V harveyi infection,

significantly enhanced the survival rate, growth performance, and immune responses in treated fish, and the optimum results were obtained the herbals at

concentrations of 400 mg per kg diet (Punitha et al., 2008) Higher cellular and humoral immune responses, and protection from infection of V harveyi, V anguillarum, V alginolyticus was recorded in kelp grouper (E bruneus) after feeding with Phellinus linteus-enriched diet for 30 days (Harikrishnan et al.,

2011b) Dietary administration of this fish species with 1.0% and 2.0%

Pueraria thunbergiana extract significantly influenced the growth, hematology, and enhanced the innate immune responses against V harveyi

(Harikrishnan et al., 2012) The use of dietary immunostimulants containing glucan, A3α-peptidoglycan, vitamin C and vitamin E not only improved the growth in cobia but also significantly enhanced immune function and resistance

β-to V harveyi infection (Dong et al., 2015)

V harveyi is associated with luminous vibriosis causing high mortality and morbidity in farmed shrimp (Kanjana et al., 2011), and known as a major

constraint on production of larval penaeid shrimp, in South America, and Asia (Austin and Zhang, 2006) Therefore, many studies have been conducted for selecting appropriate immunostimulants to control the disease in shrimp

Injection of solvent extracts of red seaweed (Gracilaria fisheri) significantly

increased the total number of hemocytes and proportion of semi-granulocytes

as well as granulocytes in juvenile black tiger shrimp (P monodon)

Furthermore, mortality of the shrimp was markedly reduced in the

extracts-treated shrimp upon being challenged with V harveyi (Kanjana et al., 2011) Dietary supplementation of V harveyi LPS, yeast β-glucan, and Fucus vesiculosus fucoidan was proven to boost immunological response and enhance the resistance of juvenile P monodon against V harveyi infection (Traifalgar

et al., 2013) In another study, application of bacterial LPS as an

immunostimulant induced elevated immune gene expression in intestines and

high resistance to V harveyi challenge providing clear evidence for potential

in black tiger shrimp (P monodon) farming (Rungrassamee et al., 2013)

Probiotics have also been widely employed in aquaculture industry for enhancement of growth performance and resistance to diseases Administration

of live microbial cell into aquatic animals results in colonization of useful microbes in the digestive tract of the host; improving their immunity

Specifically, supplementation of Lactobacillus plantarum in shrimp diet

positively modifies bacterial microbiota in its digestive tract and increase

resistance to V harveyi infection, while maintaining shrimp growth and survival rate (Vieira et al., 2010) Inhibitory effects of Bacillus probionts on growth and toxin production of V harveyi pathogens was demonstrated in shrimp Vibrio sp growth was inhibited by Bacillus subtilis, and the hemolytic activity suppression of this bacterial pathogen was observed in both B licheniformis and B megaterium Moreover, the cell-free supernatants produced by B probionts inhibited Vibrio sp diseases, and the probiotics might have an influence on communications of Vibrio (Nakayama et al., 2009)

Table 5 Application of immunostimulants to control V harveyi diseases in

aquatic animals

I Plant and biochemical extracts

1 Cynodon dactylon, Piper

longum, Phyllanthus niruri,

Tridax procumbens, and

7 Bacterial lipopolysaccharide Black tiger shrimp

Recently, phage cocktails including two bacteriophages of the family Myoviridae and one from the Siphoviridae family phages have been applied in

shrimp larvae The therapy offered protective efficacy against V harveyi and