Mahmoud Abd El Aziz Mabrok The host/pathogen interaction during experimental infection of Senegalese sole (Solea senegalensis) by Tenacibaculum maritimum Tese de Candidatura ao grau de Doutor em Ciência Animal, especialidade em: Morfologia e Fisiologia submetida ao Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto. Orientador - Doutor Benjamín Costas Refojos Categoria - Post-doctoral researcher Afiliação- Centro Interdisciplinar de Investigacão Marinha e Ambiental da Universidade do Porto. Co-orientador - Professor António Manuel dos Santos Afonso Categoria – Professor Associado Afiliação - Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto. Co-orientador - Professor Luísa Maria Pinheiro Valente Categoria - Professor Associado Afiliação - Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto. This thesis also includes one scientific paper published in an international journal originating from part of the results obtained in the experimental work referenced to as: Mabrok M., Machado M., Serra C.R., Afonso A., Valente L.M.P. & Costas B. (2016) Tenacibaculosis induction in the Senegalese sole (Solea senegalensis) and studies of Tenacibaculum maritimum survival against host mucus and plasma. Journal of Fish Diseases. (in press). doi:10.1111/jfd.12483. Contents Acknowledgements............................................................................................................VII Summary............................................................................................................................XI Resumo............................................................................................................................XV Chapter-1: General introduction 1. World Aquaculture ...................................................................................................... 3 2. Senegalese sole, Solea senegalensis (Kaup) nutritional value and economic importance ..................................................................................................................... 4 3. Senegalese sole: general aspects of its biology and feeding habitat .............................. 4 4. General feature about Gram-negative bacteria .............................................................. 5 5. Tenacibaculosis as an important disease affecting marine fishes .................................. 6 5.1. Taxonomy .............................................................................................................. 6 5.2. Species susceptibility ............................................................................................. 6 5.3. Clinical features ...................................................................................................... 7 5.4. Morphological and biochemical characterization of T. maritimum ........................... 8 5.5. Diagnostic tools ....................................................................................................... 8 5.6. T. martimum pathogenicity .................................................................................... 10 5.6.1. Natural reservoirs ............................................................................................ 10 5.6.2. Age susceptibility ............................................................................................ 11 5.6.3. Prevalence ...................................................................................................... 11 5.6.4. Virulence mechanisms .................................................................................... 12 5.7. T. maritimum vaccination ....................................................................................... 13 6. Fish immune system .................................................................................................... 14 7. Innate immune system recognition patterns (PAMPS) ................................................. 16 8. Innate immune system Pathogen recognition receptors (PRR) .................................... 17 9. PAMPS - PRR interaction and induction of Immune system ........................................ 19 10. Inflammatory responses and phagocytic activities against bacterial infection ............. 21 10.1. Initiation of the inflammatory processes ............................................................... 21 10.2. Control of inflammation ........................................................................................ 21 10.3. Phagocytic mechanism ........................................................................................ 22 Objectives ........................................................................................................................ 24 References ...................................................................................................................... 24 I Chapter-2: Tenacibaculosis induction in the Senegalese sole (Solea senegalensis) and studies of Tenacibaculum maritimum survival against host mucus and plasma Abstract ........................................................................................................................... 38 1. Introduction .................................................................................................................. 39 2. Material and Methods .................................................................................................. 40 2.1. Experimental fish ................................................................................................... 40 2.2. Bacterial strains ..................................................................................................... 40 2.3. Bacterial culture optimization ................................................................................. 41 2.3.1. Non-ionic surfactants treatment ......................................................................41 2.3.2. Cellulase hydrolysis treatment ........................................................................41 2.3.3. Detergent treatment ........................................................................................41 2.3.4. Another bacterial culture approach..................................................................41 2.4. Lipopolysaccharides extraction and purification from T. maritimum strains ............ 42 2.5. Pathogenicity assays ............................................................................................. 43 2.6. Bacteriological assays ........................................................................................... 43 2.7. Mucus and plasma collection ................................................................................. 43 2.8. Assessment of mucus and plasma bactericidal activities against T. maritimum ..... 44 2.9. Data analysis ......................................................................................................... 44 3. Results ......................................................................................................................... 45 3.1. Bacterial growth ..................................................................................................... 45 3.2. Pathogenicity assays ............................................................................................. 45 3.3. Mucus and plasma bactericidal activities ............................................................... 47 4. Discussion ................................................................................................................... 50 5. Conclusion ................................................................................................................... 51 References ...................................................................................................................... 52 II Chapter-3: In vitro assessment of Senegalese sole (Solea senegalensis) immune responses against different Tenacibaculum maritimum strains Abstract ........................................................................................................................... 56 1. Introduction .................................................................................................................. 57 2. Material and Methods .................................................................................................. 58 2.1. Bacterial strains and inoculum preparation ............................................................ 58 2.2. Experimental fish ................................................................................................... 58 2.3. Isolation of head-kidney leucocytes ....................................................................... 59 2.4. Nitric oxide assay ..................................................................................................59 2.5. ROS assay ............................................................................................................60 2.6. Killing assay ..........................................................................................................60 2.7. Lactate dehydrogenase assay ...............................................................................61 2.8. Gene expression analysis ..................................................................................... 61 2.9. Statistical analysis ................................................................................................. 64 3. Results ........................................................................................................................