Francisella noatunensis – Taxonomy and ecology Karl Fredrik Ottem The degree doctor philosophiae (dr.philos) University of Bergen, Norway 2011 2 Aknowlegdement The studies included in this thesis were conducted at the Fish Disease Group, Department of Biology, University of Bergen in the period 2006-2009. The project was financed by the Norwegian Research Council grant NFR174227/S40, Intervet Norbio AS and PatoGen Analyse AS. I am especially indebted to my supervisor Are Nylund for his guidance, support and enthusiasm over the years and in the completion of this thesis. I am also deeply indebted to Egil Karlsbakk for his guidance, enthusiasm, many contributions and efforts that have helped me in completing this thesis. Your help have been very valuable and much appreciated. I would also like to thank all the good friends and colleagues in the Fish Disease Group for all the fun between the work, and for all of the interesting discussions not always related to biology. Thanks go to Kuninori Watanabe, Linda Andersen, Marius Karlsen, Trond E. Isaksen, Stian Nylund, Øyvind Brevik, Henrik Duesund, Siri Vike, and Heidrun Nylund. It is fun working with you. I am also grateful to my parents, my brothers and sister and all of my friends for all support during these years. I am deeply grateful and indepted to my dearest Susanne for your love, support and encouragment during the completion of this thesis, a process which must have seem endless. Finally Folke, even though you do not realize it now, your laughter and good mood have been priceless after a long day at the office. I look forward to spend some much longed family time with the both of you. 3 List of Papers Paper-I. Nylund A, Ottem KF, Watanabe K, Karlsbakk E, Krossøy B (2006) Francisella sp. (Family Francisellaceae) causing mortality in Norwegian cod (Gadus morhua ) farming. Arch Microbiol 5:383-92. Epub 2006 Apr 14. Paper-II. Ottem KF, Nylund A, Karlsbakk E, Friis-Møller A, Krossøy B (2007) Characterization of Francisella sp., GM2212, the first Francisella isolate from marine fish, Atlantic cod ( Gadus morhua ). Arch Microbiol 5:343-50. Epub 2006 Dec 12. Paper-III. Ottem KF, Nylund A, Karlsbakk E, Friis-Møller A, Krossøy B, Knappskog D (2007) New species in the genus Francisella (Gammaproteobacteria; Francisellaceae); Francisella piscicida sp. nov. isolated from cod ( Gadus morhua ). Arch Microbiol 5:547-50. Epub 2007 Jul 10. Paper-IV. Ottem KF, Nylund A, Isaksen TE, Karlsbakk E, Bergh Ø (2008) Occurence of Francisella piscicida in farmed and wild Atlantic cod Gadus morhua L. in Norway. J Fish Dis 7:525-34. Epub 2008 May 13. Paper-V. Ottem KF, Nylund A, Karlsbakk E, Friis-Møller A, Kamaishi T (2009) Elevation of Francisella philomiragia ssp. noatunensis Mikalsen et al. 2007 to Francisella noatunensis comb. nov. (syn. Francisella piscicida Ottem et al. 2008 syn. nov.) and characterization of Francisella noatunensis ssp. orientalis ssp. nov., two important fish pathogens. J Appl Microbiol 4: 1231- 1243. 4 CONTENTS AKNOWLEGDEMENT..................................................................................................................................... 2 LIST OF PAPERS............................................................................................................................................... 3 INTRODUCTION............................................................................................................................................... 5 NORWEGIAN FISH -FARMING ........................................................................................................................... 5 DISEASES OF ATLANTIC COD .......................................................................................................................... 5 FRANCISELLA SPP . IN FISH ............................................................................................................................... 7 THE ECOLOGY OF FRANCISELLA SPP ............................................................................................................ 13 TRANSMISSION AND RESERVOIRS OF F. NOATUNENSIS ................................................................................ 16 FRANCISELLA SPP . AND HOST -CELL INTERACTIONS .................................................................................... 18 TAXONOMY OF GENUS FRANCISELLA ........................................................................................................... 19 PHYLOGENETIC RELATIONSHIP AMONG FRANCISELLA SPP . ....................................................................... 22 BACTERIAL TAXONOMY AND CURRENT PRACTICES .................................................................................... 25 BACTERIAL TAXONOMY AND CRITIQUES ..................................................................................................... 27 DNA-DNA hybridization........................................................................................................................... 27 Gene-sequence comparison and analysis................................................................................................. 28 EVOLUTION OF BACTERIA ............................................................................................................................ 30 AIMS OF THE PRESENT STUDY................................................................................................................. 33 SUMMARY OF PAPERS ................................................................................................................................ 34 SYNTHESIS ...................................................................................................................................................... 37 THE TAXONOMY OF GENUS FRANCISELLA .................................................................................................... 37 PHENOTYPIC CHARACTERISTICS OF FRANCISELLA SPP ............................................................................... 37 Phenotype and biochemical properties..................................................................................................... 37 Fatty acids content.................................................................................................................................... 39 Serology.................................................................................................................................................... 41 Conclusions phenotypic characteristics ................................................................................................... 42 GENETIC CHARACTERISTICS OF FRANCISELLA SPP . .................................................................................... 43 16S rRNA gene-sequences ........................................................................................................................ 43 DNA-DNA hybridization........................................................................................................................... 44 House-keeping genes ................................................................................................................................ 46 Conclusions genetic characteristics ......................................................................................................... 47 CRITERIA FOR SPECIES SEPARATION IN THE GENUS FRANCISELLA ............................................................ 49 SOME CLARIFICATIONS REGARDING THE TAXONOMY AND NOMENCLATURE OF FISH PARASITIC FRANCISELLA SPP .. ........................................................................................................................................ 51 CONCLUSION.................................................................................................................................................. 56 OUTLOOK........................................................................................................................................................ 57 SUPPLEMENTARY MATERIAL.................................................................................................................. 58 LITERATURE CITED..................................................................................................................................... 71 5 INTRODUCTION Norwegian fish-farming The Norwegian fish-farming industry has grown substantially during the last 30-40 years from the onset as a small industry until today where it represents a multi million dollar industry. During this time of development the industry has experienced numerous problems affecting the profitability, in particular those related to fish- health. From an industry mainly producing Atlantic salmon ( S. salar ), Norwegian aquaculture are now producing several marine species, such as Atlantic cod ( Gadus morhua ), Atlantic halibut ( Hippoglossus hippoglossus ), turbot ( Psetta maxima ), blue mussel ( Mytilus edulis ), oyster ( Ostrea edulis ) and the great scallop ( Pecten maximus ). In 2008 the sale of farmed Atlantic cod was the largest of these species. When including catch-based aquaculture, it amounted to 18052 metric tons or 2.14 % out of a total of 841776 metric tons of farmed fish sold (Statistics from Norwegian Directorate of Fisheries 2009). It was expected that the production of farmed Atlantic cod should increase further in the coming years, with a prognose for 2010 in the order 150-200 000 tons (Kjesbu et al. 2006). However, in 2009 and 2010 the industry experienced a serious set-back mainly due to poor economy