Isolation of Vibrio alginolyticus and Vibrio splendidus from captive-bred seahorses with disease symptoms
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- Balcázar, J.L., Gallo-Bueno, A., Planas, M. et al. Antonie van Leeuwenhoek (2010) 97: 207. doi:10.1007/s10482-009-9398-4
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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.
KeywordsSeahorsesrep-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.
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.
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.