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: ……………………………… ……………………………… Reviewer 2: ……………………………… ……………………………… Reviewer 3: ……………………………… ……………………………… 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 1 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 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). 3 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 4 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. 5 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. 6 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 10 2 , 10 3 , 10 4 and 10 5 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 7 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 1 st day and at 15 th 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 1 st and 15 th day after stopping the β-glucan diet supplement, 24 fish/group were injected with virulent V. parahaemolyticus V3 at 1.07 x 10 5 cfu/g and the mortality were monitored during the challenge period of 2 weeks. 2.2.3. Characteristic of the IgM immunoglobuline of orange- spotted grouper.  Grouper sera were collected according to Aakre et al. (1994); IgM was purified from serum on FPLC chromatography system 8 (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 -70 o C.  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 10 9 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 [...]... Post injection (day) Figure 3.12: Accumulative mortality of orange spotted grouper challenged with Vibrio parahaemolyticus at the 1st (a) and 15th (b) day stopping diet β-glucan supplement 17 3.3 Specific immune response of orange- spotted grouper to Vibrio parahaemolyticus 3.3.1 Characteristic of IgM of orange- spotted grouper IgM of orange- spotted grouper consists of a heavy chain and a light chain with... 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... function of this peptide in this molecular should be considered 4 Strain V parahaemolyticus V3 with high virulence, study on toxic proteins and immunogenetic of main proteins should be carried out to produce sub-unit vaccine on grouper LIST OF PUBLICATIONS RELATED TO THE DISSERTATION Local journals 1 Nguyen Thi Thanh Thuy, Nguyen Huu Dung, H.I Wergeland (2011) The immune response of orange spotted grouper. .. different from the control group when stopping diet 15 days 4 After immunized by 4 strains of bacterin V parahaemolyticus, levels of antibodies in orange- spotted grouper sera increased In particular, strains V3 stimulated grouper to produce the highest antibodies comparing with other strains 5 Antigen formalin inactivated V parahaemolyticus V3 with FIA adjuvant stimulated groupers E coioides to produce... chain; L: Light chain) 3.3.2 Antibody response of orange- spotted grouper to 4 strains Vibrio parahaemolyticus activated by formaline without FIA After 30 and 45 days post immunization by inactivated V parahaemolyticus, specific antibodies in grouper sera was higher than in the control group (P . 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. 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). 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