study on the immune response of orange spotted grouper (epinephelus coioides hamilton, 1822) cultured in khanh hoa province to bacteria vibrio parahaemolyticus

MINISTRY OF EDUACATION AND TRAINING NHA TRANG UNIVERSITY NGUYEN THI THANH THUY STUDY ON THE IMMUNE RESPONSE OF ORANGE -SPOTTED GROUPER Epinephelus coioides Hamilton, 1822 CULTURED IN

MINISTRY OF EDUACATION AND TRAINING

NHA TRANG UNIVERSITY

NGUYEN THI THANH THUY

STUDY ON THE IMMUNE RESPONSE OF ORANGE

-SPOTTED GROUPER (Epinephelus coioides Hamilton,

1822) CULTURED IN KHANH HOA PROVINCE TO

BACTERIA Vibrio parahaemolyticus

Speciality : Aquaculture Code : 62 62 03 01

ABSTRACT OF DOCTORAL DISSERTATION

KHANH HOA - 2014

The dissertation was finished at Nha Trang University

Supervisors:

1 Dr Nguyen Huu Dung - Nha Trang University

2 Prof.Dr Heidrun I Wergeland- Bergen University, Norway

Reviewer 1: ………

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Reviewer 2: ………

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Reviewer 3: ………

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The dissertation was defended at Dissertation Assessment Committee of Nha Trang Uiniversity At ………, date …… ……… …

The dissertation can be found at:

National Library

Nha Trang University’s Library

INTRODUCTION

1 Necessity of research

Orange-spotted grouper Epinephelus coioides has high

nutritional value for human consumption, grow fast and is cultured in many countries In recent years, various infectious diseases has caused major problems for the farming of grouper Among these, a

continuously occurring disease is, ulcerative disease caused by Vibrio

bacteria The economic loses for grouper farmers, due to this disease, have been extensive

Up to now, antibiotics and chemicals for prevention and treatment of Vibriosis in grouper have not showed suitable or had an acceptable effect In addition, it is important to keep in mind that preventive use of antibiotics is not acceptable and continuous use often result in occurrence of resistant bacteria Immunoprophylactic measures like vaccines are therefore highly recommended Thus, basic research on grouper immunology and responses to pathogens will provide more information about the mechanism involved in disease protection The characterization of the pathogen(s) with regard to pathogenicity and immunostimulatory abilities to obtain the required protection is therefore necessary The knowledge and results obtained by such studies can be applied for fish vaccine development and vaccine production Such new vaccine approach is highly demanded for development of marine fish farming The use of vaccines to avoid diseases and spread of pathogens also lead to reduced or no use of antibiotics and chemicals and will thus promote sustainable and ecologically acceptable environment in farming areas

2 Aims and contents of research

2.1 Aims of research

Study the immune response of orange-spotted grouper to Vibrio

parahaemolyticus

2.2 Contents of research

 Study on characteristics of V parahaemolyticus causing the

ulcerative disease in grouper

 Study on the effect of oral administration β-glucan on the native immune response of orange-spotted grouper

 Study on the specific immune response of orange-spotted

grouper to formalin killed cells of V parahaemolyticus

3 Significance and new conclusions from this dissertation

 The results of the dissertation contribute with scientific information about the immune response of orange-spotted grouper to

Vibrio This provide a scientific basis for vaccine production and for

the use immunostimulants to prevent vibiosis in marine fish in Vietnam

 This is the first publication about immune response as well as

effect of oral administration β-glucan in cultured grouper in Vietnam

4 Structure of dissertation

The dissertation includes 127 pages, 27 figures, 6 tables, and 219 references The structure of dissertation consists of introduction (3 pages); Chapter 1: Litterateur review (36 pages); Chapter 2: Materials and Methods (17 pages); Chapter 3: Results and Discussion (46 pages); Chapter 4: Conclusions and Suggestions (2 pages); References (23 pages) and Indexes (19 pages)

CHAPTER 1: LITTERATEUR REVIEW

1.1 Orrange spotted grouper Epinephelus coioides

1.1.1 Biological characteristics

Biological characteristics such as morphology, distribution, growth, nutrition, reproduction and larval characteristics of grouper generation and orange-spotted grouper specificity has been reviewed

from previous reports (Randall et al, 1991; Grandcourt et al, 2003; Lauro et al, 2012; Nguyen Nhat Thi, 1991 )

1.1.2 Grouper farming industry in the world and Vietnam

Grouper has been cultured since 1970s in some Asian countries and in Vietnam, since 1988 Main cultured areas for grouper farming are Hai Phong, Phu Yen, Khanh Hoa, Vung Tau

1.1.3 Common diseases in grouper

Infections in grouper by virus, bacteria, parasites have been reported from many countries including Vietnam Among the

pathogens, Vibrio was the most common agents with fast spread and

caused lot of fish health problems and losses for the farmers (Sarjito

et al., 2009; Sun et al., 2011; Yambot and Song, 2006; Do Thi Hoa et al., 2008 )

1.2 Vibrio and vibriosis in marine fish

1.2.1 Vibrio

Characteristics of structure, morphology, physiology,

bioecology, biochemical and virulent factors of Vibrio are described

in many publications including from Vietnam

1.2.2 Vibriosis in marine fish

Many marine fishes in different areas have shown to be

infected with Vibrio bacteria Extensive haemorrhagic septicemia and

ulceration of the skin, fins and tail are common symptoms of this

disease The disease causes severe infection and often result in high mortality The common agents of this disease are V parahaemolyticus, V vulnificus, V alginolyticus, V cachariae The

extensive use of chemicals and antibiotics for prevention and treatment ulcerative disease have resulted in reduced or no effect

1.3 Fish immune systems

1.3.1 Non-specific immune system

The difference aspects of fish non-specific immune system

have been reviewed by Ellis (2001), Rombout et al (2005), Magnadottir (2006), Whyte (2007), Chistiakov et al (2007), Van

Muiswinkel (2008)

1.3.2 Specific immune system

Up to now, most research related to fish specific immune responses are conducted on salmon, common carp and catfish

(Rombout et al., 2005; Swain et al., 2007; Uribe et al., 2011 )

A few studies on the immune system and responses of grouper have been published such as ontogenesis of lympho organs, IgM

characteristic, trials vaccine and immunostimulants (Lin et al., 2005; Kato et al., 2004; Harikrishnam et al., 2011)

1.3.3 Influence factors for fish immune responses

Factors affecting fish immune responses include factors related

to fish ontogenesis Among environmental factors, temperature has been shown to greatly influence the fish immune responses

1.4 Vaccine and immunostimulant trials for fish

1.4.1 Immunostimulation of fish

There are 22 immunostimulants, which have been applied to grouper by the farming industry

1.4.2 Vaccine for fish

The use of commercial fish vaccines and experimental vaccine testing are conducted many places in the world Most vaccines are developed and tested for cold water fish such as salmon, cod, catfish, carp

1.5 Research on fish immune system and fish vaccination in Vietnam

In Vietnam, the research in fish immunology and vaccination has still relatively new A few studies on freshwater fish were carried out while research on the immunological characteristics of marine fish has still uninterested, including grouper The first commercial

fish vaccine for Pangasius against Edwardsiella ictaluri has now

been approved in Vietnam

CHAPTER 2: MATERIALS AND METHODS

2.1 Object, materials, location and period of research

2.1.1 Object: Orange-spotted grouper Epinephelus coioides

2.1.2.Materials:

i) Orange-spotted grouper: Infected fish were collected from cage culture fish in Khanh Hoa province and used for isolation of bacteria Healthy orange-spotted grouper were purchased from hatcheries in Khanh Hoa province and were acclimatized before start

of the experiments

ii) Bacteria, V parahaemolyticus, strain V3 was isolated from

ulcerative infected grouper which were collected in Khanh Hoa; Strain V1 was kindly supplied by The center of monitoring environment and disease in the North Vietnam; Strain V2 was kindly supplied by The center of monitoring environment and disease in the South Vietnam;

Strain A was reference strain V parahaemolyticus 17802 ATCC

iii) Immunostimulant: β-glucan was kindly supplied by Macrogard-Biorigin, Norway

2.1.3 Research location: Research Institute for Aquaculture No 3 and

Fish immune laboratory, Biology section, Math and Natural science department, Bergen University, Norway

2.1.4 Research period: From January, 2008 to December, 2012 2.2 Research methods

2.2.1 Characteristics of V parahaemolyticus

 Morphological, physiological and biochemical characteristics

Isolate and study the morphological, physiological and biochemical characteristics of the bacteria according to Whitman (2004) Identification of bacteria is based on Bergey’s taxonomy keys and sequence 16S rDNA gene according to Harris and Hartley (2003)

 Analysis protein profiles of bacteria

Follow the SDS-PAGE method of Tsang et al., 1983

 Challenging testing of grouper by using V parahaemolyticus

Orange-spotted grouper were challenged by V parahaemolyticus V1, V2, V3 and A at 102, 103, 104 and 105 cfu/g by intramuscular injection The control group was injected with sterile Phosphate Buffered Saline (PBS) There were 6 fish and two parallels per group

The virulence of the bacteria was determined by lethal dose 50% following the procedure according to Reed and Muench (1938)

2.2.2 The effect of oral administration β-glucan on non-specific immune responses of orange-spotted grouper

 The experiment was carried out including 3 differential concentrations of oral administration β-glucan (500 ppm, 1000 ppm

and 2000 ppm) and control group (without using β-glucan) There were 30 fish/treatment in double replicates The pellet feed (M503, Uni-president Vietnam) mixed β-glucan with squid oil outside was fresh prepared Fish were given this diet during 2 weeks with 2 % BW/day After the diet period, the fish was feed with pellet feed

 Sampling: At 1st day and at 15th day after stopping diet supplemented with β-glucan, 6 fish/treatment were used for blood samples and sampled of head kidney were used for isolation of leucocytes

 Determination of WBC in blood: Blood smears were used

for counting blood cells according to Selvaraj et al (2006)

Identification of leucocytes was done according to Ainsworth (1992)

 Analysis non-specific immune parameters of head kidney leucocytes (HKL): HKL were isolated by centrifugation on percoll

gradient following the procedure of Yeh et al (2008); Observation of

phagocytic activity was based on the ability to ingest the fluorescent

beads of HKL according to Yeh et al (2008); The respiratory burst activity of HKL were measured according to Cheng et al (2007)

 Survey of disease resistance of orange-spotted grouper after eating β-glucan: At the 1st

and 15th day after stopping the β-glucan

diet supplement, 24 fish/group were injected with virulent V

parahaemolyticus V3 at 1.07 x 105 cfu/g and the mortality were monitored during the challenge period of 2 weeks

2.2.3 Characteristic of the IgM immunoglobuline of spotted grouper

orange- Grouper sera were collected according to Aakre et al (1994); IgM was purified from serum on FPLC chromatography system

(Pharmacia, Uppsala, Sweden) according to Havarstein et al (1988) Analyze IgM by SDS-PAGE according to Tsang et al (1983)

 Production of rabbit antiserum to grouper IgM: The rabbit was immunized 3 times (at 4 week intervals) by subcutaneous injection with 0.2 mg IgM and adjuvant (FCA or FIA) with a ratio 1:1 (v /v) Rabbit sera were collected 2 weeks after the last injection and stored

at -70oC

 Immunoreaction between grouper IgM and rabbit antiserum

was detected by Western Blot according to Towbin et al (1979)

2.2.4 Study on the specific immune response of orange-spotted

grouper to V parahaemolyticus

 Antigen: Bacteria was inactivated by 0.5% formalin (v/v) Bacterin was resuspended in PBS and mixed 1:1 (v/v) in Freundʼ s incomplete adjuvant (FIA, Difco) to 109 cfu/mL

 Antibody response of orange-spotted grouper to bacterin without FIA: Bacterin of strains V1, V2, V3 and A were used to immunized for 20 fish/strain with 0.1 mL/fish Control group was injected 0.1 mL sterilized PBS The experiment was carried out with two replicates At 30, 45 and 60 days post vaccination, blood from the caudal vein of 10 fish from each treatment was sampled for sera collection Specific antibodies in fish sera were analyzed by ELISA

following Jakobsen et al (1999)

 The antibody response of orange-spotted grouper to the bacterin containing FIA and the protective efficacy testing:

The experiment was conducted with 4 treatments including

V3F (fish were immunized with bacterin V parahaemolyticus V3 mixed FIA), V3 (fish were immunized with bacterin V

parahaemolyticus V3 without FIA), PBS-C (control group were

injected by PBS) and C (control group without PBS injection) Each treatment contained 50 fish ( 49.8 ± 12.8 g) using two paralells

At 30, 45 and 60 days post vaccination, blood from the caudal vein of 10 fish from each treatment was sampled for collection of serum Immunoreaction between antibodies in fish sera and the

antigen’s protein was detected by Western Blot following Towbin et

al (1979) and specific antibodies were measurement in fish sera by

ELISA according to Jakobsen et al (1999)

Twenty fish/treatment were randomly selected for challenge by

intramuscular (i.m.) injection with V parahaemolyticus 2.5 x 105

cfu/g at 30 days post immunization and 2.0 x 105 cfu/g at 60 days post immunization The relative percent survival (RPS) that considered as the protective efficacy was calculated according to Ellis (1988)

2.3 Data analysis:

Data were analysed by Chi- test and ANOVA-one factor with Tukey HSD Post hoc test on SPSS version 15.0 (P< 0.05)

CHAPTER 3: RESULTS AND DISCUSSION

3.1 Characteristics of Vibrio parahaemolyticus causing ulcerative disease on orange-spotted grouper

3.1.1 The ulcerative disease on cultured grouper in Khanh Hoa province

Among 24 infected grouper in Khanh Hoa collected from Match to July, 2008, the frequency of signs of disease was different depended on infectious situation Particularly, 100 % samples were hemorrhage on the mouth and fin; 75 % samples with ulcer on skin; 62.5 % samples with pale liver; 58.3 % samples with dark kidney and 33.3 % samples with accumulate fluid in the peritoneal cavity

From infected grouper, 16 strains of V parahaemolyticus

were isolated with frequency at 66.7% In particular, strains C07K isolated from kidney exhibited the highest virulent with LD50 at 2.14

x 103 cfu/g This strain is named as V3 and is used for comparison

with strain V parahaemolyticus V1, V2 and A

3.1.2 Characteristics of Vibrio parahaemolyticus

3.1.2.1 Morphological, physiological, biochemical characteristics

Morphological, physiological, ecological characteristics of 4 studied strains (V1, V2, V3 and A) showed similar such as negative Gram, short rod-shaped, mobility, green colonies on TCBS with high salt tolerance to 8 % in TSB and rabbit erythrocyte lysis on agar However, strain V3 showed β erythrocyte lysis type while 3 other strains of α type

Strain V1, V2, V3 are similar to the reference strain ATCC 17802

and were identified V parahaemolyticus according to Bergey’s

taxonomic key and sequence of 16S rDNA gene

3.1.2.2 Protein profile of V parahaemolyticus

Four strains of V parahaemolyticus showed similar protein

profiles Among these, proteins with molecular weight as 66, 47, 31,

28 and 21 kDa were homologous to toxic and high antigenic proteins

as previous reports (Figure 3.5) This finding is significant discovery for selection of potential strains for fish vaccine production

Figure 3.5: Protein profiles of Vibrio parahaemolyticus

3.1.3 Virulence of V parahaemolyticus on orange-spotted grouper

The challenging test showed that strain V3 exhibited the highest virulence comparing with other strains V1, V2 and A with

LD50 as 1.78 x 103 cfu/g (Figure 3.6)

Figure 3.6: Accumulative mortality of orange-spotted grouper

challenged with Vibrio parahaemolyticus (a: Strain V1; b: Strain

V2; c: Strain V3; d: Strain A)

3.2 Effect of oral administration β-glucan to non-specific immune parameters and disease resistance of orange spotted grouper to V parahaemolyticus

3.2.1 Composition, morphological characteristics and size of

blood cells of orange-spotted grouper

Similar to many other fish bones species, orange-spotted grouper has adequate types of blood cells Composition, size and image of grouper’s blood cells were showed in Table 3.2 and Figure 3.8

Post injection (h)