CHARACTERISATION OF THE IMMUNE RESPONSE OF THE STRIPED CATFISH (Pangasianodon hypophthalmus, Sauvage) FOLLOWING IMMUNOMODULATION AND CHALLENGE WITH BACTERIAL PATHOGENS THESIS SUBMITTED TO THE UNIVERSITY OF STIRLING FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN AQUATIC VETERINARY STUDIES WANNA SIRIMANAPONG D.V.M. (Hons) VETERINARY MEDICINE, M.Sc. AQUATIC ANIMAL DISEASES 20 DECEMBER 2013 INSTITUTE OF AQUACULTURE SCHOOL OF NATURAL SCIENCES i | P a g e To my family - without your support, I never would have got this far. Many thanks x ขอบคุณสำหรับก ำลังใจจำกครอบครัวที่ท ำให้สำมำรถมำจนถึงวันนี้ ii | P a g e Declaration Declaration I declare that this thesis has been compiled by myself, and is the result of my own work and that it has not been submitted for any other degree and all sources of information have been duty acknowledged. Wanna Sirimanapong iii | P a g e Acknowledgements Acknowledgements I would like to thank my supervisors Dr. Kim Thompson and Professor Alexandra Adams (Institute of Aquaculture, School of Natural Sciences, University of Stirling) for their support and encouragement until the end, Dr. Ei Lin Ooi (Novus International, Novus Aqua Research centre) for her support during experiments in Vietnam and Dr. Bertrand Collet (Marine Scotland-Science, Scottish Government, Marine Laboratory) for his support throughout the course of this study. I would like also to express my gratitude to Dr. Olwyn Byron for her help and patience during the analytical ultracentrifugation, Miss Prawporn Thaijongrak and Miss Kan Kledmanee for their help with experiments in Thailand, Mr Nguyen Ngoc Phuoc for providing P. borcoti and P. hypophthalmus serum samples from Vitenam, Mr Nguyen Dang Khoa for his valuable work in taking care of the fish and sample collection in Vietnam, Professor. James E Bron for his help with experiment design and statistical analysis, Dr. Darren M Green for his assistance with statistical analysis, Dr. Michaël Bekaert for his support in identifying immune genes, Dr, Michael J Leaver for his help and patience during the immune gene expression work, Dr. John B Taggart for his help with experiment design and technical support, and Dr. Andrew Shinn for assistance with PCA statistical analysis. I would also like to acknowledge Mrs Hilary McEwan, Mrs Karen Snedden, Mrs. Rowena Hoare, Mrs Debbie Faichney and Mrs. Jacquie Ireland for their valuable help. I would like to extend my appreciation to the Thai Office of the Higher Education Commission for funding my PhD. scholarship, Mahidol University for giving me the opportunity to study a Ph.D. and place of work, and Novus International for funding a part of my research project. In addition, I would like to thank all the staff at the Novus International, Novus Aqua Research centre, Linh Trung Ward, Thu Duc District, Ho Chi Minh City, Vietnam, for making it such a good experience in Vietnam. My thanks also goes to all the staff at the Mahidol University, Salalya Campus, Thailand, for providing experiment facilities and support in Thailand and all staff in the Aquatic Vaccine Unit, Institute of Aquaculture, School of Natural Sciences, University of Stirling who provided experimental facilities and technical support in UK throughout the project. A big thank you to all my friends and colleagues who have also given me much emotional and laboratory technical support especially Christoforos Metochis, Nilantha Jayasuriya, Polyana Da Silva, Carina Duarte, Sean Monaghan, Ngozi Izuchkwu, Juliet Nattabi, Zinan Xu, Yu-Ching Chuang, Arlene Satapornvanit, Nattakan Saleetid, Phuoc Nguyen Ngoc and Thao Phuong Huynh Ngo, for which I am very appreciative. Finally, thank you goes to my family, in particular my mum Lumjohn Sirimanapong and my dad Piroj Sirimanapong who have always been an inspiration and very supportive throughout this period, and also to my sister Wannee Sirimanapong and my brother Hiran Sirimanapong who have also given love and support towards the end of my PhD. I would like to dedicate my PhD to my grandfather and grandmother, who have been an inspiration in my life. THANK YOU ขอบคุณ iv | P a g e Abbreviations Abbreviations AChE Acetylcholinesterase ADCC Antibody-dependent cellular cytotoxicity AGD Amoebic gill disease APCs Antigen-presenting cells B-cells B lymphocytes BCP 1-Bromo-3-chloropropane BCR B-cell receptor BCWD Bacterial cold water disease BNP Bacillary Necrosis of Pangasianodon BSA Bovine serum albumin C Constant region CHNV crucian carp haematopoietic necrosis virus COI Cytochrome oxidase subunit 1 CRP C-reactive protein CTLs Cytotoxic T-cells CYP1B Cytochrome P4501B D Diversity DEAE Diethylaminoethly cellulose d.p.i. Day post infection ECPs Extracellular products ESC Enteric septicaemia of channel catfish EST Expressed sequence tag FASL Fas ligand GCCHDV Grass carp haemorrhage disease G-CFB Gelatine-complement fixation buffer GMO Genetically Modified Organism (GMO) NGS Next-generation sequencing) HOCl Hypochlorous acid h.p.i. Hour post infection HPSEC High-performance size-exclusion chromatography HSP70 Heat shock protein 70 HSWB High salt wash buffer IFNγ Interferon-γ IgM Immunoglobulin M IHNV Infectious hematopoietic necrosis virus IL-1β Interleukin-1β IL-6 Interleukin-6 IL-10 Interleukin-10 i.p. Intraperitoneal injection IPN Infectious pancreatic necrosis ISAV Infectious salmon anemia virus J Joining LAMP Loop-mediated isothermal amplification method LPS Lipopolysaccharide v | P a g e Abbreviations LGBP LPS/b-glucan binding protein LSWB Low salt wash buffer LTα Lymphotoxin-α MAI Motile Aeromonas infection MAS Motile Aeromonas septicaemia MBL Mannose-binding lectins MASPs MBL-associated serine proteases MAITs Mucosal associated invariant T-cells MHC II Major histocompatibility complex class II MPO Myeloperoxidase NBT Nitroblue tetrazolium NCBI National centre for biotechnology information NK cells Natural killer cells NKT cells Natural killer T-cells NO Nitric oxide OD Optical density PBS Phaosphate buffer saline PCA Principal component analysis PDV Pancreas disease virus PMA Phorbol myristate acetate p.f. Post feeding poly I:C Polyinosinic:polycytidylic acid proPO Prophenoloxidase RI Refractive index ROS Reactive oxygen species RSIV Iridoviral disease virus RT Room temperature SAP Amyloid P component SOD Superoxide dismutase SOMO SOlution MOdeller SRBC Sheep red blood cells SV Sedimentation velocity SD Standard diviation T-cells T lymphocytes TCR T-cell receptor TCM Central memory T cells TEM Effector memory T cells Th cells Helper T-cells TMB 3’3’5’5’-tetramethylbenzidine dihydrochloride TNFα1 Tumor necrosis factor-α1 TNFα2 Tumor necrosis factor-α2 TREG Regulatory T-cells TRM Resident memory T cells TSCM Stem cell memory T cells USD US Dollar V Variable region VASEP Vietnam Association of Seafood Exporters and Producer vi | P a g e Abstract Abstract 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. vii | P a g e Abstract 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