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Preventing disease outbreaks in cultured turbot Psetta maxima L. caused by Aeromonas salmonicida subsp. salmonicida (ASS) requires a better understanding of how this pathogen colonizes its host. Distribution of 1 virulent and 2 avirulent ASS strains in turbot tissues was investigated during early and late stages of infection following an immersion challenge. To track bacteria within the turbot, the ASS strains were tagged with green fluorescent protein (GFP). Both virulent and avirulent strains colonized the epidermal mucus, gills, and intestine within the first 12 h post challenge, suggesting that these sites may serve as points of entry into turbot. Although the avirulent strains colonized these initial sites in the turbot tissues, they were rarely found in the internal organs and were cleared from the host 4 d post challenge. In contrast, the virulent ASS strain was found in the liver and kidney as early as 12 h post challenge and was found in the muscle tissue at very late stages of infection. The virulent strain persisted in all tested host tissues until death occurred 7 d post challenge, suggesting that ASS must colonize and survive within the turbot tissues for an infection to result in death of the fish. Comparisons of the distribution profiles of both virulent and avirulent strains during early and late stages of an infection in turbot has provided important information on the route and persistence of an ASS infection in this host.KEY WORDS: Aeromonas salmonicida · Persistence · Turbot · Green fluorescent protein · GFP · Immersion challenge Resale or republication not permitted without written consent of the publisherDis Aquat Org 95: [167][168][169][170][171][172][173] 2011 for ASS in other internal organs and did not analyze the transmission of the bacteria through the internal tissues. Interestingly, Hodgkinson et al. (1987) compared infectivity of virulent and avirulent strains in salmonids but only during the first 24 h post challenge, showing inconclusive results since the virulent strain showed an avirulent behavior. Thus, additional studies are needed to investigate the fate of both virulent and avirulent ASS strains during the progression of early and late stages of disease. In particular, studies using non-salmonid fish such as turbot will give a broader understanding on how ASS colonizes its host and will provide information for the design of new therapeutic strategies to protect against this pathogen in aquaculture.The green fluorescent protein (GFP) from Aequorea victoria has been used successfully as a genetic marker for the detection of bacterial pathogens during the infection of fish (Ling et al. 2000, 2001, O'Toole et al. 2004, Welch & Wiens 2005, Chu & Lu 2008. In most cases, expression of GFP in the bacterial cell does not interfere with growth or virulence (Chalfie et al. 1994, Valdivia et al. 1996, Ling et al. 2000, Chu & Lu 2008. In addition, GFP fluorescence, which is induced by UV light, facilitates visualization of bacteria in fish tissues and the quantification o...

Aim:The aim of the present study was to clarify the taxonomic status of Vibrio strains isolated from an aquaculture system and to compare the results of the identifications made by phenotypic and molecular methods. Methods and Results: Fifty-one Vibrio strains isolated from a turbot (Scophthalmus maximus) aquaculture system were characterized by ribotyping and 16S rRNA gene sequencing. The strains had been identified phenotypically in a previous numerical taxonomy analysis as Vibrio anguillarum, V. mediterranei, V. splendidus, V. aestuarianus, V. ordalii, V. fischeri and V. scophthalmi. Cluster analysis of ribotype patterns showed that the strains were separated into two main groups: V. splendidus-V. lentus and V. scophthalmi groups. The use of 16S rRNA gene sequence allowed differentiation among V. splendidus biovar I and V. lentus strains. Conclusions: The molecular methods identified strains of V. splendidus biovar I, V. lentus and V. scophthalmi, showing discrepancies with phenotypic characterization. Significance and Impact of the Study: The molecular methods, as 16S rRNA gene sequence analysis, are necessary for the identification of phenotypically close species to avoid mis-identifications. Interestingly, this is the first report of V. lentus strains associated to turbot culture.

R. FARTO, M. MONTES, M.J. PÉ RE Z , T .P . NI ET O , J .L . LA RS E N A ND K . P ED E RS EN . 1999. TwelveVibrio strains were examined phenotypically in 91 biochemical characters and genotypically by ribotyping. Ten were isolated from sea water and two from diseased turbot (Scophthalmus maximus). All isolates originated from one experimental system located in Ría de Vigo (Galicia, north-west Spain). Different type strains were used for comparative purposes. The taxonomic position was analysed with the NTSYST-pc and similarities among strains were calculated by the Simple Matching coefficient (S SM ). rRNA gene restriction patterns were performed with the HindIII enzyme. The S SM coefficient separated the 12 Vibrio strains into two groups which included strains that showed a S SM coefficient quite similar to V. splendidus biovar I (ATCC 33125) and V. scophthalmi (CECT 4638). None of 91 phenotypical characters were specific in distinguishing both species. The ribotyping confirmed the taxonomic classification of strains. The pathogenicity of each strain was evaluated; 10 environmental strains were avirulent and two, isolated from diseased turbot, were virulent. Different biotypes and ribotypes were found among the avirulent isolates. This work showed ribotyping to be a valuable tool for identification and confirmed the necessity of extending the ribotype database within closely related Vibrio species in order to clarify the taxonomic position.

Aims: This study aimed to select and validate different methodological strategies to quantify the expression of the virulence genes ascC and ascV by qPCR in Aeromonas salmonicida subsp. salmonicida (Aer. salmonicida). Methods and Results: Using the geNorm, Normfinder and BestKeeper algorithms, reference genes for the qPCR were selected based on their in vitro expression stabilities in three Aer. salmonicida strains. Gene amplification efficiency was calculated by Real-time PCR Miner and LinReg PCR programmes, which have not been used previously in the analysis of bacterial gene expression. The expression of the ascC and ascV virulence genes in a virulent Aer. salmonicida strain was evaluated by three quantification models, including single (least or most stable) or three most stable reference genes, combined with constant or specific gene amplification efficiency. The most stable reference genes were gyrB, proC and rpoC, while rpoD and fabD were the least stable. Quantification models showed different expression patterns. Conclusions: The optimal strategy to quantify mRNA expression was to use a combination of the three algorithms and the quantification model including the three most stable reference genes. Real-time PCR Miner or LinReg PCR were valuable tools to estimate amplification efficiency. Significance and Impact of the Study: The methods used in this study gave more reliable expression data using qPCR than previously published methods. The quantification and expression dynamics of virulence genes will contribute to a better understanding of how Aer. salmonicida interacts with its host and the environment, and therefore to the prevention of epizootics due to this pathogen.