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Title: Characterisation of the immune response of the striped catfish (Pangasianodon hypophthalmus, Sauvage) following immunomodulation and challenge with bacteria pathogens
Author: Sirimanapong, Wanna
Awarding Body: University of Stirling
Current Institution: University of Stirling
Date of Award: 2013
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In Southeast Asia, the family Pangasiidae is important for commercial fisheries and aquaculture. Pangasianodon hypophthalmus (striped catfish) is the most economically important species farmed in Vietnam, with a total export value of 1.7 billion USD in 2012. Intensive aquaculture can lead to problems with major outbreaks of disease and Edwardsiella ictaluri and Aeromonas hydrophila represent two important bacterial pathogens in P. hypophthalmus aquaculture. Immunostimulants have proven to be a very useful food additive for the aquaculture industry, since they can be easily fed to fish to enhance their immune response at times of stress and to improve resistance to disease. The immune system of pangasius catfish has not been fully described, despite the recent growth in aquaculture for this species, and little is known about the effects of immunostimulants on disease resistance. Understanding the immune response is very important in order to evaluate the health status of the fish and assist in control of disease (including prevention) so that production levels by the aquaculture industry can be sustained. The aims of this thesis were to develop and standardise methods to elucidate and measure immune responses in P. hypophthalmus and then to use these with relevant disease models (A. hydrophila and E. ictaluri) and immunomodulators (β-glucans from different sources and at different doses) to determine if bacterial diseases can be controlled, and which functional immune responses and immune genes could be correlated with disease resistance. As a variety of different species from family Pangasiidae are economically important for aquaculture, initial work focused on the characterisation of the immunoglobulin IgM molecule in these species, and anti-P. hypophthalmus IgM mAbs were tested to determine if they cross-reacted between different Pangasiidae species (Chapter 2). Although affinity purification of IgM from the different fish species resulted in a purer preparation ammonium sulphate precipitation (14% w/w), the latter proved faster and easier to perform. The heavy (H) and light (L) chains of IgM from P. hypophthalmus were estimated to be 70-72 kDa and 25-26 kDa, respectively, using SDS-PAGE (12.5%). The L chains of IgM in the other Asian fish species examined were similar in molecular weight to P. hypophthalmus, while the H chains varied (P. gigas and P. larnaudii 76kDa, P. sanitwongsei 69kDa, H. filamentus 73kDa, P. borcoti and H. wyckioides 75kDa, C. bactracus 74kDa, C. macrocephalus 73kDa and C. carpio 70kDa), as did the native IgM molecules. Sedimentation velocity ultracentrifugation was used to determine the molecular weight of the whole IgM molecule from P. hypophthalmus as an alternative to the more commonly used native gels that are run under non-denaturing conditions, although this technique proved more complex. Anti–P. hypophthalmus IgM monoclonal antibodies (mAbs) cross reacted with all of the Pangasiidae species and were successfully applied in an enzyme-linked immunosorbent assay (ELISA) using mAb 23 to measure serum antibody response of P. hypoophthalmus following experimental infection with A. hydrophila by interperitoneal (I.P.) injection in Chapter 3 and E. ictaluri by immersion in Chapter 4. As P. hypophthalmus is a relatively new aquaculture species, there are few reports evaluating its immune response to pathogens. Thus, functional assays were standardised to evaluate both innate and adaptive immune responses of this species and then these assays used to compare immune response following stimulation with live and killed A. hydrophila. (Chapter3). Four treatment groups of 40 fish per group (53.2 ± 14.8g.) consisting of an untreated control group, a group injected I.P. with adjuvant (Montanide ISA 760 VG) only, a group injected with heat-killed A. hydrophila (1 x109 cfu ml-1 mixed with adjuvant), and a group injected with a subclinical dose of live A. hydrophila 2.7 x105 cfu ml-1 were used in the study. Samples were collected 0, 1, 3, 7, 14 and 21 days post injection (d.p.i.) to assess the immune response of fish. The results indicated that challenge with live or/and dead bacteria stimulated the immune response in P. hypophthalmus significantly above control groups with respect to specific antibody titre, lysozyme activity, phagocytosis and plasma peroxidase at 7 or/and 14 d.p.i. Moreover, on 21 d.p.i. total IgM, specific antibody titre and lysozyme activity from both live and dead A. hydrophila challenge groups were significantly different to the control groups. Differential immune responses between live and dead bacterial challenges were also observed as only live A. hydrophila significantly stimulated WBC counts and plasma peroxidase at 3 d.p.i. with the greatest increase in WBC counts noted at 21 d.p.i. and in phagocytosis at 14 d.p.i. By 21 d.p.i. only the macrophages from fish challenged with dead A. hydrophila showed significantly stimulated respiratory burst activity. Immunostimulants are food additives used by the aquaculture industry to enhance the immune response, and β-glucan is now commonly used for this purpose in aquaculture. In Chapter 4 the effect of the prebiotic β-glucan on the immune response and disease resistance of P. hypophthalmus was evaluated. The fish (60.3 ± 11.7 g.) were fed with a basal diet (control) or diets supplemented with fungal derived β-glucan at concentrations of 0.05 %, 0.1 %, or 0.2 % g/kg for four weeks. Fish fed 0.1 % commercial yeast derived β-glucan were also included as a positive control group. Samples were collected from fish on Days 0, 1, 3, 7, 14, 21 and 28. The results showed that fish fed with the highest two levels of fungal derived β-glucan had enhanced immune responses compared to the control group, with respiratory burst activity on all days examined and lysozyme activity on 7 days post feeding (d.p.f.) being significantly elevated (P<0.05) in the group fed with 0.2 % fungal derived β-glucan, while plasma anti-protease activity on 21 d.p.f., natural antibody titre on 3 d.p.f. and complement activity 7 d.p.f. and 14 d.p.i. were significantly enhanced (P<0.05) in the group fed 0.1 % fungal derived β-glucan. The lowest dose of fungal derived β-glucan (0.05 %) appeared insufficient to effectively stimulate the fish’s immune response. WBC count, respiratory burst, lysozyme activity and complement were useful as an early indication of immunostimulation (1 to 7 days). Four weeks after feeding with the different diets, the fish were experimentally infected with E. ictaluri by immersion using 8 x104 cfu ml-1 for 1 h and mortalities were monitored for 14 days. There was a great deal of variation in the level of mortalities within the four replicate tanks for each dietary group. Although the in vivo challenge results showed no statistical differences between the groups fed on the different diets, the highest mortalities were observed in group fed with the control diet and the lowest mortalities were observed in the groups fed with commercial yeast derived β-glucan and 0.2 % fungal derived β glucan. Immune gene expression following stimulation with β-glucan and challenge with E. ictaluri was investigated in Chapter 5.
Supervisor: Adams, Alexandra; Thompson, Kimberly; Ooi, Ei Lin; Collet, Bertrand Sponsor: Thai Official of Educational Affairs
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Pangasianodon hypophthalmus ; Aeromonas hydrophila ; Edwardsiella ictaluri ; Beta glucan ; Immune genes ; Vaccine ; Catfishes ; Bacterial diseases in fishes