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Multilocus sequence analysis of the central clade of the genus Vibrio by using the 16S rRNA, recA, pyrH, rpoD, gyrB, rctB and toxR genes The central clade of the genus Vibrio, also called the Vibrio core group, comprises six species that are tightly related (DNA-DNA reassociation values are very close to 70 % for most species pairs). Identification of novel strains to the species level within this group is troublesome and results are quite often dependent on the methodology employed. Therefore, this group represents an excellent framework to test the robustness of multilocus sequence analysis (MLSA) not only for inferring phylogeny but also as an identification tool without the need for DNA-DNA hybridization assays. The genes selected, 16S rRNA, recA, pyrH, rpoD, gyrB, rctB and toxR, were amplified by direct PCR from 44 Vibrio core-group strains. Subsequent analysis allowed us to recognize toxR and rpoD as the most resolving individual genes and showed that concatenated sequences of rpoD, rctB and toxR were more useful than concatenated sequences of all seven genes. To validate our conclusions, MLSA similarities have been correlated with DNA-DNA relatedness values obtained in this study and values taken from the literature. Although the seven concatenated genes gave the best correlation, the concatenated sequences of rpoD, rctB and toxR have the practical advantage of showing a considerable gap between the maximal interspecies similarity and the minimal intraspecies similarity recorded, meaning that they can be used quite conveniently for species identification of vibrios. INTRODUCTIONMLSA (multilocus sequence analysis) is a recently developed technique derived from multilocus sequence typing that has been used successfully in epidemiology studies. The use of MLSA as an alternative method for species delineation in bacteriology is currently being evaluated, following a recommendation of the ad hoc committee for the re-evaluation of the species definition in bacteriology (Stackebrandt et al., 2002). In MLSA studies, several housekeeping genes (more than five) are analysed and relationships between taxa are established. One important advantage of this kind of approach is the availability of genomic sequences from any laboratory, avoiding the problems of lack of comparability that are associated with DNA-DNA reassociation data. However, the usefulness of MLSA for describing bacterial species has to be demonstrated for each of the taxa or group of species studied and for the selected housekeeping genes. Authors should prove that there is a sufficient degree of congruence between the alternative technique (i.e. MLSA) and DNA-DNA reassociation data. To date, the technique has been applied to discrete bacterial taxa, mainly genera of lactic acid bacteria (Naser et al. et al., 2007, 2008) and the Vibrionaceae (Goarant et al., 2006;Urbanczyk et al., 2007;Thompson et al., 2008;Rameshkumar et al., 2008).In the family Vibrionaceae, sequence similarities for the 16S rRNA gene are ¢97.6 % among members of the so-called Vib...

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A bacteriological and parasitological study on cultured common dentex, Dentex dentex L., was conducted in a 2‐year survey. Two different groups of fish were studied: (1) sampled only when mortalities occurred; and (2) sampled periodi‐cally from 10days post‐hatch until the juvenile stage. In both groups, peaks of mortalities coincided with high water temperatures. The same parasites were detected in both groups, except Ichthyophonus sp., which was identified only in the trunk kidney of fish from group 1. Two myxosporeans, Ceratomyxa sp. and Lepthotheca sp., were found in the bile and the trunk kidney, respectively. Epitheliocystis infection was also very frequent in the gills. An unidentified microsporidium, which was found in the liver, occurred very infrequently. Vibrio harveyi was dominant in samples from diseased animals (79%) and clearly associated with mortalities. It was not detected in water or larval homogenates. A high percentage (60%) of the isolates of this species were sorbitol positive. Other less frequently occurring bacteria were V. alginolyticus and Photobacterium (V.) damselae. The disease conditions found in common dentex were considered to be multifactorial in origin.