Introductory studies on bacterial agents infecting cleaner fish Philosophiae Doctor (PhD) Thesis Snorre Gulla Department of Food Safety and Infection Biology Faculty of Veterinary Medicine and Biosciences Norwegian University of Life Sciences Adamstuen 2015 Thesis number 2015:98 ISSN 1894-6402 ISBN 978-82-575-1967-4 “I'd take the awe of understanding over the awe of ignorance any day.” - Douglas Adams (author, 1952-2001) Table of contents ACKNOWLEDGEMENTS ............................................................................................................................. II LIST OF ABBREVIATIONS ......................................................................................................................... IV LIST OF PAPERS ........................................................................................................................................... VI SUMMARY .................................................................................................................................................... VII SAMMENDRAG (SUMMARY IN NORWEGIAN) ................................................................................. VIII 1. INTRODUCTION ......................................................................................................................................... 1 1.1. NORWEGIAN SALMON FARMING – A BRIEF DISEASE HISTORY .................................................................... 1 1.2. LEPEOPHTHEIRUS SALMONIS – THE SALMON LOUSE ................................................................................... 2 1.2.1. The parasite and its impact .............................................................................................................. 2 1.2.2. Salmon lice control measures........................................................................................................... 3 1.3. CLEANER FISH USED FOR SALMON DELOUSING ......................................................................................... 4 1.3.1. History of use and relevant species .................................................................................................. 4 1.3.2. Supply channels and associated risks .............................................................................................. 6 1.3.3. Best practice care – health and welfare considerations ................................................................... 7 1.4. THE CURRENT HEALTH SITUATION OF CLEANER FISH ................................................................................ 8 1.4.1. Mortality levels and associated causes ............................................................................................ 8 1.4.2. Bacterial infections – common agents and current knowledge ........................................................ 9 1.5. AEROMONAS SALMONICIDA ....................................................................................................................... 10 1.5.1. Background .................................................................................................................................... 10 1.5.2. Susceptible fish species and the disease ......................................................................................... 11 1.5.3. The bacterium ................................................................................................................................. 12 1.5.4. Virulence factors ............................................................................................................................ 14 1.5.5. Vaccination .................................................................................................................................... 16 1.5.6. Epizootiology .................................................................................................................................. 17 1.6. VIBRIO SPLENDIDUS ................................................................................................................................. 17 1.6.1. Background .................................................................................................................................... 17 1.6.2. Relevance for fish disease .............................................................................................................. 18 1.6.3. The bacterium ................................................................................................................................. 18 1.6.4. Virulence factors ............................................................................................................................ 20 1.6.5. Vaccination .................................................................................................................................... 21 1.6.6. Ecology ........................................................................................................................................... 21 1.7. BACTERIAL DIAGNOSTICS ....................................................................................................................... 21 1.7.1. Isolation and identification methods .............................................................................................. 21 1.7.2. Non culture-based detection methods ............................................................................................ 23 1.8. BACTERIAL POPULATIONS AND POPULATION GENETICS .......................................................................... 24 1.9. BACTERIAL TYPING ................................................................................................................................. 26 1.9.1. Serotyping ...................................................................................................................................... 26 1.9.2. Molecular typing – available tools and methods............................................................................ 26 1.9.3. Molecular typing methods relevant to the present study ................................................................ 28 1.10. KNOWLEDGE GAPS ................................................................................................................................ 29 2. AIMS OF STUDY ........................................................................................................................................ 31 3. SUMMARY OF MATERIALS AND METHODS ................................................................................... 33 3.1. MATERIALS ............................................................................................................................................. 33 3.2. METHODS ............................................................................................................................................... 33 4. SUMMARY OF PAPERS ........................................................................................................................... 35 5. RESULTS AND GENERAL DISCUSSION.............................................................................................. 37 5.1. BACTERIAL INFECTION LEVELS IN CLEANER FISH .................................................................................... 37 5.2. MOLECULAR CHARACTERISATION OF BACTERIA FROM CLEANER FISH .................................................... 38 5.2.1. A. salmonicida A-layer typing ........................................................................................................ 38 5.2.2. MLSA and serotyping of V. splendidus .......................................................................................... 41 5.3. GENERALISED IMPACT OF WORK ............................................................................................................. 43 5.3.1. Killing the dogma of ‘typical’ and ‘atypical’ A. salmonicida ........................................................ 43 5.3.2. A-layer type as a putative epidemiological marker ........................................................................ 44 5.3.3. Heterogeneity within the Splendidus-clade .................................................................................... 46 6. METHODOLOGICAL CONSIDERATIONS .......................................................................................... 47 6.1. SAMPLING ............................................................................................................................................... 47 6.2. QPCR SCREENING ................................................................................................................................... 47 6.3. A-LAYER TYPING .................................................................................................................................... 47 6.4. MLSA LOCI ............................................................................................................................................ 48 6.5. ANTISERA USED FOR SEROTYPING ........................................................................................................... 48 7. MAIN CONCLUSIONS AND FUTURE PROSPECTIVES .................................................................... 49 8. REFERENCES ............................................................................................................................................ 51 9. ERRATA ...................................................................................................................................................... 69 10. SCIENTIFIC PAPERS I-III ....................................................................................................................