Vibrio salmonicida pathogenesis analyzed by experimental challenge of Atlantic salmon (Salmo salar) Ane Mohn Bjelland a, * , Renate Johansen b , Espen Brudal a , Hilde Hansen c , Hanne C. Winther-Larsen d , Henning Sørum a a Section for Microbiology, Immunology and Parasitology, Department of Food Safety and Infection Biology, Norwegian School of Veterinary Science, PO Box 8146 Dep, 0033 Oslo, Norway b Section for Pathology, Department of Laboratory Services, Norwegian Veterinary Institute, PO Box 750 Sentrum, 0106 Oslo, Norway c The Molecular Biosystems Research Group, Institute of Chemistry, Faculty of Sciences, University of Tromsø, N-9037 Tromsø, Norway d Laboratory for Microbial Dynamics (LaMDa) and Department of Pharmaceutical Biosciences, University of Oslo, PO Box 1068, Blindern, 0316 Oslo, Norway article info Article history: Received 8 July 2011 Received in revised form 21 October 2011 Accepted 27 October 2011 Available online 4 November 2011 Keywords: Vibrio salmonicida Cold-water vibriosis Pathogenesis Virulence Motility abstract Cold-water vibriosis (CV) is a bacterial septicemia of farmed salmonid fish and cod caused by the Gram- negative bacterium Vibrio (Aliivibrio) salmonicida. To study the pathogenesis of this marine pathogen, Atlantic salmon was experimentally infected by immersion challenge with wild type V. salmonicida and the bacterial distribution in different organs was investigated at different time points. V. salmonicida was identified in the blood as early as 2 h after challenge demonstrating a rapid establishment of bacteremia without an initial period of colonization of the host. Two days after immersion challenge, only a few V. salmonicida were identified in the intestines, but the amount increased with time. In prolonged CV cases, V. salmonicida was the dominating bacterium of the gut microbiota causing a release of the pathogen to the water. We hypothesize that V. salmonicida uses the blood volume for proliferation during the infection of the fish and the salmonid intestine as a reservoir that favors survival and transmission. In addition, a motility-deficient V. salmonicida strain led us to investigate the impact of motility in the CV pathogenesis by comparing the virulence properties of the mutant with the wild type LFI1238 strain in both i.p. and immersion challenge experiments. V. salmonicida was shown to be highly dependent on motility to gain access to the fish host. After invasion, motility was no longer required for virulence, but the absence of normal flagellation delayed the disease development. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Cold-water vibriosis (CV) is a bacterial septicemia of farmed salmonid fish [1e4] characterized by anemia and extended pete- chial hemorrhages, especially in the integument surrounding the internal organs of the fish, in the vent region and at the base of the pectoral, pelvic and anal fins. As its name indicates, the disease occurs mainly in late autumn, winter and early spring when the seawater temperature is below 10  C [1]. Although affecting mainly farmed Atlantic salmon (Salmo salar), CV is identified in both farmed rainbow trout (Oncorhynchus mykiss) and wild caught captive and farmed Atlantic cod (Gadus morhua) [2,4]. The causal agent of CV is the cold- (psychrophilic) and salt- adapted (halophilic) bacterium Vibrio (Aliivibrio) salmonicida. This marine bacterium is a Gram-negative curved and motile rod which carries up to nine polar flagella [1]. V. salmonicida and its three closely related species Vibrio fischeri, Vibrio logei and Vibrio wodanis were recently proposed reclassified into a new genus, Aliivibrio gen. nov., resulting in the new name A. salmonicida [5]. The species designation A. salmonicida is, however, already occupied by the well-established abbreviation of A. salmonicida, the etiological agent of furunculosis in salmonids. To avoid possible nomenclature confusion, the name V. salmonicida will be used throughout this paper. V. salmonicida shows a high potential for starvation and survival in the ocean environment and the numbers of V. salmonicida in fish farm seawater ranges from 12 to 43 bacteria/ml with concentra- tions being highest during the winter period when the total bacterial count in seawater generally is at the lowest [6e8]. The pathogen is suggested to be transmitted through seawater between salmonids either as bacterioplankton or on the surface of particles Abbreviations: AFM, Atomic force microscopy; BA2.5, Blood agar with 5% ox blood and 2.5% NaCl; CFU, Colony forming units; CV, Cold-water vibriosis; LB1, Luria Bertani broth with 1% NaCl; LB3, Luria Bertani broth with 3% NaCl; OMVs, Outer membrane vesicles. * Corresponding author. Tel.: þ47 22597416; fax: þ47 22964818. E-mail address: ane.mohn@nvh.no (A.M. Bjelland). Contents lists available at SciVerse ScienceDirect Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath 0882-4010/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.micpath.2011.10.007 Microbial Pathogenesis 52 (2012) 77e84