Antonie van Leeuwenhoek

, 97:207

Isolation of Vibrio alginolyticus and Vibrio splendidus from captive-bred seahorses with disease symptoms

Authors

    • Instituto de Investigaciones MarinasConsejo Superior de Investigaciones Científicas (CSIC)
  • Alfonso Gallo-Bueno
    • Instituto de Investigaciones MarinasConsejo Superior de Investigaciones Científicas (CSIC)
  • Miquel Planas
    • Instituto de Investigaciones MarinasConsejo Superior de Investigaciones Científicas (CSIC)
  • José Pintado
    • Instituto de Investigaciones MarinasConsejo Superior de Investigaciones Científicas (CSIC)
Short Communication

DOI: 10.1007/s10482-009-9398-4

Cite this article as:
Balcázar, J.L., Gallo-Bueno, A., Planas, M. et al. Antonie van Leeuwenhoek (2010) 97: 207. doi:10.1007/s10482-009-9398-4

Abstract

Vibrio species isolated from diseased seahorses were characterized by PCR amplification of repetitive bacterial DNA elements (rep-PCR) and identified by 16S ribosomal RNA gene sequence analysis. The results demonstrated that Vibrio alginolyticus and Vibrio splendidus were predominant in the lesions of these seahorses. To our knowledge, this is the first time that these bacterial species have been associated with disease symptoms in captive-bred seahorses.

Keywords

Seahorsesrep-PCRDisease symptomsVibrio alginolyticusVibrio splendidus

Members of the family Vibrionaceae are autochthonous to aquatic environments including estuarine, coastal waters and sediments, and some species are well-known pathogens of marine organisms including fish, crustaceans and molluscs (Thompson et al. 2004). The biochemical methods currently used to identify Vibrio species can be time-consuming, labour-intensive and hard to implement because of difficulties in distinguishing between closely related species given the wide variety of biochemical profiles obtained (Vandenberghe et al. 2003). For this reason, attention has focused on the application of molecular-based techniques. Protocols for bacterial typing using polymerase chain reaction (PCR) techniques are becoming increasingly valuable. Among these methods, PCR amplification of repetitive bacterial DNA elements (rep-PCR) has proven to be a powerful tool for studies of microbial ecology, environmental microbiology, molecular diagnostics, medical microbiology and epidemiological analyses (Ishii and Sadowsky 2009). The aim of this study was thus to analyze the Vibrio species associated with disease symptoms in Hippocampus guttulatus and Hippocampus hippocampus, two species of captive bred seahorses.

Eight seahorses with disease symptoms (lethargy, lack of appetite, white spots on the skin, and in some cases, necrotic tail lesions) were collected from aquaria at the Institute of Marine Research (IIM-CSIC, Spain) between March 2007 and May 2009. Seahorses were untreated as symptoms were sporadic in the sampling period. All samples for microbiological examination were taken from the white spots and necrotic lesions. Samples were then serially diluted in saline solution (0.85% NaCl) and plated on marine agar (Difco, Detroit, MI), tryptic soy agar supplemented with 1.5% NaCl (Cultimed) and Cytophaga agar prepared with 50% seawater [0.05% tryptone, 0.05% yeast extract, 0.02% sodium acetate, 1.5% agar, and adjusted to pH 7.2]. All plates were incubated for 3–7 days at 20°C. All the colonies were counted, isolated and stored at −80°C in a suitable medium supplemented with 15% glycerol (v/v).

The isolates were characterized by microscopic examination and by conventional biochemical and physiological tests. The cultures were examined for colony and cell morphology; motility; Gram reaction; catalase reaction; growth on thiosulfate-citrate-bile-sucrose (TCBS) agar; and glucose fermentation. Subsequently, we used rep-PCR analysis to group the isolates. Genomic DNA of each isolate was extracted and purified following the method described by Balcázar et al. (2007a). Amplification reactions were performed in a total volume of 25 μl containing 1.0 U of Taq polymerase, 2.0 mM MgCl2, a 0.2 mM concentration of each deoxynucleoside triphosphate, 0.8 μM (GTG)5 primer (5′-GTG GTG GTG GTG GTG-3′) (Versalovic et al. 1994), and 1.0 μl of DNA template. The thermal cycler temperature profile (GeneAmp PCR System 2700; Applied Biosystems) consisted of predenaturation at 95°C for 10 min followed by 30 cycles of 30 s at 95°C, 1 min at 47°C, 8 min at 65°C, and a final extension for 16 min at 65°C. The fragments were electrophoresed in a 2% agarose gel in Tris–acetate–EDTA buffer. Numerical cluster analysis was performed with GelCompar II software (v6.0; Applied Maths, Belgium). A dendrogram was obtained by means of the unweighted-pair group method using the arithmetic averages (UPGMA) algorithm, with correlation levels expressed as percentage values of the Pearson correlation coefficient.

Representative isolates from each rep-PCR genotype were selected and identified by 16S ribosomal RNA gene sequence analysis as described previously (Balcázar et al. 2007b). The sequences of these genes were compared against the sequences available in the GenBank, EMBL and DDBJ databases obtained from the National Center of Biotechnology Information database using the BLASTN programme (Altschul et al. 1990). Phylogenetic analysis was performed using the software MEGA version 4.0 (Tamura et al. 2007) after multiple alignments of data by CLUSTAL X (Thompson et al. 1997). Distances (distance options according to the Kimura two-parameter model) and clustering with the neighbour-joining method was determined by using bootstrap values based on 1,000 replications.

In total, 64 isolates were obtained from diseased seahorses. They were initially grouped on the basis of morphological and physiological characteristics. All isolates were curved Gram-negative rods, motile, and catalase- and oxidase- positive. Subsequently, 24 representative isolates were selected from the different media and were biochemically identified as members of the genus Vibrio, according to the method described by Gómez-León et al. (2005). The results of numerical analysis of the generated rep-PCR patterns grouped into two clusters at an 86.7% similarity level (Fig. 1). The analysis also demonstrated that 53.8% of the isolates belonging to Cluster 1 and 46.2% to Cluster 2 were isolated from necrotic tail lesions, whereas 45.5% of the isolates belonging to Cluster 1 and 54.5% to Cluster 2 were isolated from white spots on the skin.
https://static-content.springer.com/image/art%3A10.1007%2Fs10482-009-9398-4/MediaObjects/10482_2009_9398_Fig1_HTML.gif
Fig. 1

Dendrogram based on the numerical analysis of generated, digitized rep-PCR fingerprints. Banding patterns were clustered together with the reference strains by using UPGMA, with correlation levels expressed as percentage values of the Pearson correlation coefficient. Source: C, strains from culture collection; TS, isolates from tail lesions; WS, isolates from white spots

Sequencing of the 16S rRNA genes from representative isolates belonging to the two clusters confirmed that these isolates were affiliated to the genus Vibrio (Fig. 2). Strain N26-1, representing Cluster 1 (12 isolates), could be assigned to Vibrio alginolyticus with 99.0% rRNA gene sequence similarity to type strain ATCC 17749T. Strain N26-3, representing Cluster 2 (12 isolates), was closely related to Vibrio splendidus with 97.7% rRNA gene sequence similarity to type strain ATCC 33125T (=LMG 4042T).
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Fig. 2

Neighbour-joining tree based on 16S rRNA gene sequences of the isolates and the closest Vibrio species. The isolates are shown in boldface, and GenBank accession numbers are given in parentheses. Numbers at nodes indicate percentages of bootstrap support (1,000 replicates). Bar, 1% sequence divergence

These results were corroborated once the binary matrix was analyzed and a rep-PCR dendrogram was generated by UPGMA (Fig. 1). It shows, for example, that the isolates from Cluster 1 were related to V. alginolyticus ATCC 17749T. Interestingly, it also shows that all the isolates from Cluster 2 were more related to V. splendidus LMG 4042T, than to the other type strains.

We have recently observed that some members of the Alphaproteobacteria, notably representatives of the genera Antarctobacter, Roseobacter, Ruegeria, Sulfitobacter and Thalassobacter, are the predominant bacteria on the mucosal surfaces of healthy seahorses (Balcázar JL, Pintado J, Planas M, unpublished observations). In the present study, we found that V. alginolyticus and V. splendidus were the predominant bacteria on the mucosal surfaces of diseased seahorses; thus, these species may be associated with disease symptoms. Vibrio alginolyticus and V. splendidus are ubiquitous microorganisms in seawater and have been frequently isolated from marine fish and invertebrates (Thompson et al. 2004). However, some studies have suggested that these species are pathogenic to several marine organisms (Lee et al. 1996; Gómez-León et al. 2005; Thomson et al. 2005). To our knowledge, this is the first time that these bacterial species have been associated with disease symptoms in captive-bred seahorses.

These results presented (Fig. 1) also suggest that rep-PCR fingerprinting technique using (GTG)5-PCR allowed us to differentiate Vibrio strains, which is notable considering that this method was a very simple, rapid, specific, and low-cost tool. This method may therefore be helpful for differentiating V. alginolyticus and V. splendidus in epidemiological analyses, particularly in large studies and critical situations.

Nucleotide sequence accession numbers

The nucleotide sequences derived from this study have been deposited in the GenBank/EMBL/DDBJ databases under accession numbers FN436276 and FN436277.

Acknowledgments

The study was financed by the Spanish Ministry of Science and Technology (CGL2005-05927-C03-01), as part of a coordinated research project (Proyecto Hippocampus; 2005/PC091). Funding was also partially provided by the Regional Government of Galicia (Xunta de Galicia: PGIDIT06PXIC402106PN). We thank P. Quintas and A. Chamorro for their kind assistance in seahorse collection and maintenance.

Copyright information

© Springer Science+Business Media B.V. 2009