We report the first isolation of Vibrio harveyi from wedge sole Dicologoglossa cuneata. The pathogen was recovered from ulcers and internal organs of ailing cultured fish, from 7 different outbreaks between 2004 and 2006. The 15 isolates found were phenotypically characterized using biochemical tests and BIOLOG GN plates, which revealed high phenotypic diversity. Diagnosis was confirmed with PCR using V. harveyi specific primers and partial 16S and 23S rRNA gene sequencing. A virulence evaluation of the isolates was also performed using fry and juvenile wedge sole. Significant mortalities were recorded by intraperitoneal injection; however, no mortalities were recorded by bath immersion.
KEY WORDS: Vibrio harveyi · Characterization · Wedge sole · Dicologoglossa cuneata · VirulenceResale or republication not permitted without written consent of the publisher Dis Aquat Org 84: 209-217, 2009 veyi is often problematic because of both phenotypic and genotypic diversity (Austin & Austin 1989, Oakey et al. 2003. In particular, V. harveyi displays a high phenotypic heterogeneity and has been extremely difficult to identify using only conventional bacteriological tests (Vandenberghe et al. 2003). Several speciesspecific PCR methods have been developed to identify V. harveyi isolates, although in some cases false-positive (e.g. Oakey et al. 2003) or false-negative results (Conejero & Hedreyda 2003) have been recorded. Other molecular tools, such as amplified fragment length polymorphism (AFLP), multilocus sequence analysis (MLSA), DNA-DNA hybridization and 16S rDNA gene sequencing have also been used for identification of this species (Gomez-Gil et al. 2004, 2007.The present paper reports the first description of Vibrio harveyi as responsible for vibriosis in cultured wedge sole Dicologoglossa cuneata, an economically valuable flatfish species belonging to the Family Soleidae, which is being cultured in some farms in southwestern Spain in an effort to diversify marine aquaculture. The production of this species is affected by pathological problems that cause severe losses, necessitating the identification of the pathogens and the development of prevention and treatment techniques to optimise farming conditions (Herrera et al. 2008). Studies carried out to confirm the identification of the isolates as V. harveyi as well as its involvement in the disease are reported here.
MATERIALS AND METHODSBacterial isolation. Sampled fish came from 7 epizootic outbreaks that occurred between August 2004 and April 2006 in wedge sole cultured in 2 marine farms located in southwestern Spain (Table 1). Mortalities occurred in fry, juvenile and adult stages of wedge sole (0.6 to 150 g) stocked at 0.8 to 3 kg m -2. Water temperature varied from 10 to 22°C and water salinity was 32 ± 4 ‰. Samples for bacterial isolation were taken from ulcers, liver and kidney of moribund wedge sole and cultured on Flexibacter maritimus medium (FMM) (Pazos et al. 1996) at 20°C for 24 to 96 h. Isolates were subcultured onto tryptic soy agar (TS...