Development of a PCR method for the specific identification of the marine fish pathogen Tenacibaculum soleae P. García-González ⁎, N. García-Lamas, C. Fuentes Edfuf, Y. Santos Departamento de Microbiología y Parasitología, Facultad de Biología, Edificio CIBUS, Campus Vida, Universidad de Santiago de Compostela, Spain abstract article info Article history: Received 14 March 2011 Received in revised form 8 June 2011 Accepted 9 June 2011 Available online 17 June 2011 Keywords: Identification Tenacibaculum soleae Tenacibaculosis Polymerase chain reaction Specific This work describes a new polymerase chain reaction (PCR) assay for the accurate and fast identification of the fish pathogen Tenacibaculum soleae. The selected primers amplified a 248 bp fragment of the T. soleae 16S rRNA gene. The PCR system was specific and very sensitive with a detection limit of approximately 1 to 10 bacterial cells per reaction when pure cultures of T. soleae are used. The PCR approach described in this paper allowed detection of the pathogen in mixed plate cultures obtained from diseased fish affected by tenacibaculosis, in which growth of this bacterium cannot always be visualized. Our results indicated that the specific primers and PCR method designed provide sensitive and fast diagnosis of the tenacibaculosis caused by the fish pathogen T. soleae. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Tenacibaculosis is one of the most important bacterial fish diseases in marine aquaculture, causing substantial economic losses in farms in Japan, Europe and North America (Bernardet et al., 1990; Ostland et al., 1999; Wakabayashi et al., 1986). The main affected fish species are turbot, Scophthalmus maximus L.; Dover sole, Solea solea L.; Sen- egalese sole, Solea senegalensis (Kaup); sea bass, Dicentrarchus labrax L.; Atlantic salmon, Salmo salar L.; and coho salmon, Oncorhynchus kisutch (Walbaum) (Bernardet, 1997; Bernardet et al., 1990; Cepeda et al., 2003; Cepeda and Santos, 2002; Devesa et al., 1989; McVicar and White, 1979, 1982; Ostland et al., 1999; Pazos et al., 1993; Santos et al., 1999; Toranzo et al., 2005; Wakabayashi et al., 1986). Although Tenacibaculum maritimum is the main causative agent of this dis- ease, other filamentous gliding bacteria belonging to the genus Tenacibaculum such as T. soleae, Tenacibaculum gallaicum and Tenacibaculum discolor have been isolated from diseased fish that had been vaccinated against T. maritimum and have been shown to be pathogenic for sole, S. senegalensis and turbot, S. maximus (López et al., 2010; Piñeiro-Vidal et al., 2007, 2008a, 2008b). Identification of the causative agent is usually carried out by agar cultivation and biochemical characterization of the isolated bacteria (Bernardet et al., 1990; Pazos et al., 1993; Santos et al., 1999; Toranzo et al., 2005), but differentiation of the different Tenacibaculum species from each other and from other marine flavobacteria on the basis of biochemical characteristics is problematic (Piñeiro-Vidal et al., 2007, 2008a, 2008b; Toranzo et al., 2005). Fatty acid analysis and molecular methods such as DNA–DNA hybridization, ribotyping, random amplified polymorphic DNA analysis (RAPD), could be of value for taxonomical and epizootiological differentiation of these bacterial fish pathogens (Bernardet et al., 2002; Buller, 2004; Piñeiro-Vidal et al., 2008a, 2008b, 2008c). However, these rather sophisticated methods are time consuming and the isolation of the microorganisms in pure culture is a prerequisite. Alternatively, Polymerase Chain Reaction (PCR) is successfully used for the diagnosis of the disease caused by T. maritimum using samples of mucus, infected tissue and mixed bacterial cultures (Avendaño-Herrera et al., 2004; Bader and Shotts, 1998; Cepeda et al., 2003; Toyama et al., 1996). In all these studies the targeted sequences are located in the variable regions of the 16S rRNA gene. Unfortunately, these PCR procedures do not allow the differ- entiation between T. maritimum and the other Tenacibaculum species also described as causative agents of the disease (Piñeiro-Vidal et al., 2007, 2008a, 2008b). The importance of a quick and differential method for diagnosis of the disease caused by Tenacibaculum species and the growing use of thermal cyclers in many research settings, motivated us to develop a pair of specie-specific primers, based on the 16S rRNA region, and a PCR assay for fast and reliable iden- tification of the fish pathogen Tenacibaculum soleae. 2. Materials and methods 2.1. Bacterial strains A total of 29 strains from 15 bacterial species were used in the study. The geographical origin and host species are given in Table 1. The bacterial strains of the Genera Tenacibaculum, Aeromonas, Vibrio, Aquaculture 319 (2011) 1–4 ⁎ Corresponding author. Tel.: + 34 981563100x16958/16912. E-mail address: ysabel.santos@usc.es (P. García-González). 0044-8486/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2011.06.013 Contents lists available at ScienceDirect Aquaculture journal homepage: www.elsevier.com/locate/aqua-online