Paramoebiasis of sea-farmed salmonids in Tasmania - a study of its aetiology, pathogenicity, and control. By Teresa S. Howard B.Sc. (Latrobe University, Victoria) Submitted in fulfillment of the requirements for the Degree of Doctor of Philosophy. University of Tasmania, Hobart. June 2001 This thesis contains no material which has been accepted for a degree or diploma by the university or any other institution, except by way of background information and duly acknowledged in the thesis, and to the best of my knowledge and belief no material previously published or written by another person except where due acknowledgement is made in the text of the thesis. Teresa Howard University of Tasmania HOBART June 2001 This thesis may be made available for loan and limited copying in accordance with the Copyright Act, 1968. Table of Contents I Acknowledgements 1 Abstract 3 Abbreviations 5 Chapter 1. Background and review of literature 1.1 Sea-farming of salmonids 1.1.1 The Salmonidae ......................................................................................... 6 1.1.2 Introduction of salmonids to Aqstralia ...................................................... ? 1.1.3 History of sea-water salmon farming ......................................................... 9 1.1.4 Salmonid farming in Australia ................................................................... 10 1.1.5 Economics of the Tasmanian salmonid industry ....................................... 13 1.1.6 Sea-farming methods ................................................................................. 16 1.2 Major diseases of sea-farmed salmonids .................................... 19 1.2.1 Bacterial diseases ................. ..... .. ......................................... ...................... 20 1.2.2 Viral diseases ............................................................................................. 26 1.2.3 Diseases caused by protozoans .................................................................. 30 1.2.4 Other diseases ............... ........... .. .... ............................................................ 36 1.2.5 Diseases common in Tasmania ..................... ... ... ........... .... ... ....... .. ............ 38 1.3 Paramoebiasis of aquatic animals 1.3.1 The amoebae ................ .. ............................................................ ... ... .......... 39 1.3.2 Paramoeba species .... .. ..................................................................... ........ .41 1.3.3 Diseases of aquatic animals caused by amoebae .... ................................... 51 1.3.4 Paramoebiasis of crabs .............. ......... .. ................ ... ............. ... .. ................ 58 1.3.5 Paramoebiasis of sea urchins ............. .. .. .................................................... 63 1.3.6 Paramoebiasis of salmonids and turbot .. ....................................... ... ... .... .. 67 1.4 Objectives for this study .................................................................. 80 Chapter 2. Methods 2.1 Culture of marine amoebae ............................................................ 84 2.1.1 Seeding agar plates .................................................................................... 85 2.1.2 Subculture .................................................................................................. 85 2.1.3 Large-scale production ............................................................................... 86 2.1.4 Contamination control ............................................................................... 87 2.1.5 Revival of failing cultures ......................................................................... 88 2.2 Isolation of amoebae from fish gills 2.2.1 Ammonium chloride wash ......................................................................... 88 2.2.2 Cloning of amoebae ................................................................................... 89 2.3 Identification of Paramoeba species 2.3.1 Phase contrast microscopy ......................................................................... 90 2.3.2 DAPI-staining ............................................................................................ 91 2.3.2 Gomori's trichrome stain ........................................................................... 91 2.4. Cryopreservation of amoebae 2.4.1 Cryopreservation ........................................................................................ 92 2.4.2 Culture retrieval ......................................................................................... 93 2.5 Antigen preparation and antiserum production 2.5.1 Antigen Preparation ................................................................................... 93 2.5.2 Antiserum production in rabbits ................................................................ 94 2.5.3 Antiserum production in fish ..................................................................... 95 2.5.4 Serum preparation ...................................................................................... 96 2.6 Gill health assessment methods 2.6.1 Gross examination ..................................................................................... 97 2.6.2 Preparation of gill tissue for histological examination .............................. 97 2.6.3 Immunofluorescence antibody test (IF AT) ................................................ 98 2. 7 Immunostaining techniques 2.7.1 Immunofluorescence .................................................................................. 100 2.7.2 Immunoperoxidase .................................................................................... 100 2.7.3 Alkaline phosphatase ................................................................................. 101 2.8 Preparation of Paramoeba for electron microscopy .............. 102 2.9 Chemical screening assays 2.9.1 Preparation of chemicals ............................................................................ 103 2.9.2 Growth inhibition assay ............................................................................. 103 2.9.3 Contact inhibition assay ............................................................................. 106 2.10 ·Mucus antibody extraction 2.10.1 Gill perfusion ............................................................................................. 110 2.10.2 Antibody extraction from mucus ............................................................... 111 2.11 ELISA to detect Paramoeba antibodies in fish ........................ 111 Chapter 3. Isolation and verification of the pathogen 3.1 Introduction .......................................................................................... 113 3.2 Methods .................................................................................................. 114 3.2.1 Isolation and characterisation .................................................................... 114 3.2.2 Verification of the pathogen ...................................................................... 121 3.2.3 Additional investigations ........................................................................... 127 3.3 Results ..................................................................................................... 131 3.3.1 Isolation and characterisation .................................................................... 131 3.3.2 Verification of the pathogen ...................................................................... 143 3.3.3 Additional investigations ........................................................................... 156 3.4 Discussion .............................................................................................. 163 3.4.1 Isolation and characterisation .................................................................... 163 3.4.2 Verification of the pathogen ...................................................................... 168 3.4.3 Gill health .................................................................................................. 177 3.4.4 Additional investigations ........................................................................... 181 3.5 Conclusions ........................................................................................... 184 Chapter 4. Pathogenicity of Paramoeba sp. 'AGD' 4.1 Introduction .......................................................................................... 186 4.2 Methods .................................................................................................. 187 4.2.1 Tank systems .............................................................................................. 187 4.2.2 The fish ...................................................................................................... 191 4.2.3 Disease assessment .................................................................................... 192 4.2.4 Culture challenges ..................................................................................... 192 4.2.5 Cohabitation challenges ............................................................................. 198 4.2.6 Electron microscopy .................................................................................. 199 4.3 Results ..................................................................................................... 200 4.3.1 Culture challenges ..................................................................................... 200 4.3.2 Cohabitation challenges ............................................................................