Recently discovered Vibrio anguillarum phages can protect against experimentally induced vibriosis in Atlantic salmon, Salmo salar Gastón Higuera a, 1 , Roberto Bastías a, ⁎ , 1 , George Tsertsvadze b , Jaime Romero a , Romilio T. Espejo a a Laboratorio de Biotecnología, Instituto de Nutrición y Tecnología de los Alimentos (INTA), University of Chile, Santiago, Chile b George Eliava Institute of Bacteriophage, Microbiology and Virology, Tbilisi, Georgia abstract article info Article history: Received 23 January 2013 Received in revised form 8 February 2013 Accepted 11 February 2013 Available online 20 February 2013 Keywords: Vibrio anguillarum Bacteriophage Phage Salmo salar Vibriosis Vibrio anguillarum is a marine bacterium that can cause vibriosis in several fish species of economic impor- tance. The use of bacteriophage is an alternative strategy to control vibriosis in aquaculture systems. Here, we present the isolation and characterization of six phages that are able to infect the pathogenic strain of V. anguillarum, PF4. These phages all possess a similar double stranded DNA (dsDNA) genome but, according to their restriction pattern, can be differentiated into three types. The phages exhibited a similar host range, infecting both V. anguillarum and V. ordalii but not V. parahaemolyticus strains. The CHOED phage protected Salmo salar against experimentally induced vibriosis with the strain PF4. The presence of the phage increased the survival of fish to 100% when it was used with a MOI of 1 and 20, versus less than 10% of survival in the absence of the phage. To our knowledge, this is the first report of the ability of V. anguillarum phages to pro- tect fish against experimental infection with V. anguillarum, and our results support the development of phage therapy as a valid alternative for the control of vibriosis in salmonid aquaculture. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Vibrio anguillarum (also known as Listonella anguillarum) is a ma- rine Gram-negative bacterium that is the etiologic agent of vibriosis, a fatal hemorrhagic septicemia disease that affects more than 50 fresh- and salt-water fish species, including several species that are of eco- nomic importance to the aquaculture industry, such as the Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), turbot (Psetta maxima), sea bass, and sea bream (Sparus aurata)(Toranzo et al., 2005). Moreover, some bivalve mollusks and crustaceans are also affected by this bacteria (Paillard et al., 2004). To date, 23 O serotypes (O1-O23) have been found; however, the O1, O2 and, in a lesser way, the O3 serotype have been associated with vibriosis (Frans et al., 2011; Silva-Rubio et al., 2008). Recently, the genome of the V. anguillarum 775 strain, which is of the O1 sero- type, was sequenced, and the analysis of this genome demonstrated that the majority of the genes that encode essential cellular functions and pathogenicity are located on the first of the two chromosomes that the bacteria possess (Naka et al., 2011). Antibiotics are still the main therapeutic tool for controlling bacte- rial diseases in aquaculture. However, their use is becoming increas- ingly restricted, due to their negative environmental impact and the emergence of multi-resistant bacteria (Cabello, 2006). Although there are multiple commercial vaccines to protect fish against out- breaks of vibriosis that are caused by the O1 and O2 serotypes, the outbreaks that are caused by serotype O3 cannot be completely prevented (Mikkelsen et al., 2007). Because of this, there is increasing interest in the development of new strategies to control pathogenic bacteria in aquaculture. One option is the use of bacteriophages (or phages), which are viruses that infect bacteria and are present in all environments. Bacteriophages have the potential to kill their host bacteria and have therefore been used to control pathogenic bacteria in different fields with very promising results (Hagens and Loessner, 2007; Kutateladze and Adamia, 2010; Nakai and Park, 2002). Recently, several phages that infect various bacterial fish pathogens have been isolated (Vibrio harveyi, Flavobacterium psychrophilum, etc.), and they have shown potential in controlling these bacteria (Castillo et al., 2012; Crothers-Stomps et al., 2010; Karunasagar et al., 2007; Nakai and Park, 2002; Park and Nakai, 2003; Stenholm et al., 2008). One phage that infects Aeromonas salmonicida and exhibits a broad host range was isolated and was demonstrated to be effective against a V. anguillarum strain. However, its ability to protect fish from vibriosis has not yet been proven (Pereira et al., 2011). Although in Europe most of the pathogenic isolates of V. anguillarum correspond to the O1 and O2 serotypes, in Chile, where there is a grow- ing salmon farming industry, most of the pathogenic isolates exhibit an O3 serotype. Here, we describe seven strains of V. anguillarum and the closely related species Vibrio ordalii, which were isolated in Chile and have been phenotypically characterized previously (Silva-Rubio et al., 2008), as well as the isolation and characterization of six phages that Aquaculture 392-395 (2013) 128–133 ⁎ Corresponding author at: Institute of Marine Biology, Biotechnology, and Aquaculture, Hellenic Centre for Marine Research, Gournes, Heraklion 71003, Crete, P.O. BOX 2214, Greece. Tel.: +30 2810337761; fax: +30 2810 337778. E-mail addresses: robastias@gmail.com, roberto@hcmr.gr (R. Bastías). 1 Both authors contributed equally to this work. 0044-8486/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquaculture.2013.02.013 Contents lists available at SciVerse ScienceDirect Aquaculture journal homepage: www.elsevier.com/locate/aqua-online