Genotypic diversity of culturable Vibrio species associated with the culture of oysters and clams in Galicia and screening of their pathogenic potential.

ABSTRACT The genotypic diversity of seventy-one Vibrio strains isolated from the culture of oysters and clams was analyzed by using ribotyping and sequencing of 16S rRNA gene. The combination of both techniques led to clustering, the separation of V. splendidus-related species and the identification of 91.5% of studied strains. New genotypic, phenotypic and biotypic information on V. tasmaniensis, V. kanaloae, V. pomeroyi, and V. neptunius was provided. Vibrio splendidus biotype 1 and Vibrio mediterranei were also identified. Different riboclusters of V. splendidus, V. tasmaniensis, and V. neptunius were obtained showing genotypic diversity of these species. A unique ribocluster in V. kanaloae, V. pomeroyi, and V. mediterranei was obtained. Venerupis rhomboides (P.) was susceptible to experimental infections with strains of V. kanaloae, V. neptunius, V. pomeroyi, and V. splendidus biotype 1. Mortalities of challenged clams and the isolation of strains from internal organs confirmed the presence of virulent strains among the isolates and pointed to the risk of molluscan culture outbreaks.

KEY WORDS: ribotyping, 16S rRNA sequence, diversity, Vibrio, oysters, clams, mortalities, pathogenic potential

INTRODUCTION

Molluscan aquaculture represents an economically relevant sector for Galicia (NW Spain) and also Europe, because an elevated culture volume is concentrated in this region. The study of the microbial diversity is the first step to be able to manipulate a microbial population with a certain aim: identification of possible pathogens, probiotics, and similar. Then, the knowledge of microbiota associated with aquaculture will be essential for improving the industrial culture production.

In a previous paper, we analyzed by numerical taxonomy a total of 488 strains associated with the culture of oysters and clams reared in Galicia (Guisande et al. 2004). Eighty-eight per cent of 397 anaerobic facultative isolates were included in 41 phena (using a [S.sub.J]/UPGMA similarity level of 69% for phena establishment), Vibrio being the genus identified in 39 phena. The identification of species was only possible in 8 phena and the application of a further analysis such as with molecular methods would be necessary to complete the identification of phena assigned as Vibrio spp.

Reliable molecular methods, such as ribotyping, have been widely used to group environmental isolates by using the restriction enzyme Hind III because it has been found to be highly discriminatory for typing Vibrio species (Austin et al. 1997, Pedersen et al. 1998, Farto et al. 2003, Montes et al. 2003, Montes et al. 2006). 16S rRNA gene sequence comparison was used to provide the genetic diversity of organisms and to identify indigenous bacteria population isolated from the natural environment (Kita-Tsukamoto et al. 1993, Schauer et al. 2003, Montes et al. 2006, Farto et al. 2006).

The aim of this study is to analyze the genotypic diversity of representative culturable facultative anaerobic environmental strains associated with the culture of oysters and clams reared in northwest Spain by ribotyping and 16S rRNA gene sequence analysis. Both techniques were evaluated as tools for identifying the isolates of Vibrio species associated with these culture productions. Phenotypic and biotypic information of isolates was also monitored. In addition, experimental infections using representative identified strains of V. kanaloae, V. neptunius and V. splendidus biotype 1 were carried out in Venerupis rhomboides clams to evaluate their virulence and the risk of outbreaks. All these results would improve the culture quality controls.

MATERIALS AND METHODS

Bacterial Strains and Conservation Conditions

This study analyzed 71 culturable Vibrio strains representative of main phena reported in our previous numerical taxonomy paper (Guisande et al. 2004). The number of selected strains was proportional to the number of isolates of each phenon obtained, and one strain of each five was chosen. This study comprises facultative anaerobic isolates from Atlantic oysters (larval, seed, and reproductive stages of Ostrea edulis L.), clams (larval, seed, and reproductive stages of Ruditapes decussatus L., Venerupis pullastra M., and Tapes japonica) and their sea water culture in different mollusc culture systems located on the Galician coast (NW Spain): Bueu, O Grove, Ribadeo, Couso, Malpica, and Vilagarcia de Arousa (Table 1). Sampling was carried out over 12 consecutive months (1 sample per month). The seawater had a temperature from 12[degrees]C to 18[degrees]C (corresponding to the minimum temperature in the winter season and the maximum in the summer season) and a salinity between 30 [per thousand] to 33 [per thousand]. Processing and isolation of strains were previously reported by ourselves (Guisande et al. 2004).

Pure cultures of selected strains were obtained on Marine Agar (Cultimed, Barcelona, Spain) and inoculated on Nutritive Broth (Cultimed, Barcelona, Spain) with 2% (w/v) NaCl (Panreac, Barcelona, Spain) and 15% (v/v) of glycerol (Panreac) for conservation at -80[degrees]C.

Growth Conditions and Phenotypic Characterization

Bacterial strains and the reference strains were previously characterized by 92 physiological, morphological, and biochemical tests (Guisande et al. 2004). Cultures grown during 24 h at 22[degrees]C on Tryptic Soy Agar (TSA, Cultimed) and supplemented up to 2% (w/v) NaCl (Panreac) (TSA 2%) were used as innocula.

Ribotyping

Isolation of genomic DNA of 71 strains was performed according to the method by Montes et al. (2003). The restriction enzyme Hind III (Roche Diagnostics, Mannheim, Germany) was used for ribotyping of isolates. DNA fragments were separated by horizontal electrophoresis through a 0.8% agarose gel in Tris-acetate buffer, transferred onto a positively charged nylon membrane (Roche Diagnostics), fixed to the membrane by baking for 30 min at 120[degrees]C, hybridized with a digoxigenin-labeled probe complementary to 16S and 23S rRNA genes of Escherichia coli and detected with alkaline phosphatase-labeled antidigoxigenin and nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate (Roche Diagnostics). Digoxigenin-labeled phage lambda Hind III DNA fragments (Roche Diagnostics) were used as size markers.

Hybridization patterns were recorded, as previously described (Shieh et al. 2003) and analyzed by using the software package ImageMaster 1D and Database (Amersham Pharmacia Biotech Europe, Freiburg, Germany). Similarity matrix was performed by using the Dice coefficient ([S.sub.D]). A dendrogram was constructed by using the UPGMA method.

Sequencing of 16S rRNA Gene

Sequencing of 16 strains was performed according to the method described by Montes et al. (2003) using a 310 Genetic Analyzer (Applied Biosystems, Darmstadt, Germany) automated sequencer. Representatives of all riboclusters and some unclustered isolates, including one strain of each five, were chosen.

The sequence of the 16S rRNA gene of representative isolates of each ribocluster was determined by using four primers (37F, 344F, 344R, and 1096R; Dewhirst et al. 1989, Paster & Dewhirst 1988) and compared with sequences in public databases of GenBank, EMBL, DDBJ and PDB with BLAST, version 2.2.6. (Altschul et al. 1997). Multiple alignment of sequences was created by ClustalX, version 1.81 (Higgins & Sharp 1988). This included 1,010 positions after removal of ambiguous positions (Hall 2001) utilizing BioEdit Sequence Alignment Editor, version 5.0.9 (Hall 1999). A phylogenetic tree was constructed by using MEGA (Molecular Evolutionary Genetics Analysis), version 2.1 (Kumar et al. 2001). This was performed using the neighbor-joining method (Saitou & Nei 1987) and Tamura-Nei distance model (Tamura & Nei 1993), with the calculation of cluster stability by bootstrap analysis with 1,000 replicates (Nei & Kumar 2000). The identification of sequenced isolates was performed according to the highest sequence identity with the type strain of Vibrio species when the limit value of intraspecies variability ([greater than or equal to] 98%; Stackebrandt & Embley 2000) was achieved.

Nucleotide sequence accession numbers: the partial 16S rRNA sequences of environmental isolates reported in this paper (strains 562, 673, 373.11, 48, 364.11, 236.10, 497, 337.98, 636, 685, 630, 257.11, 671, 258.11, 268.10, and 322.10) have been deposited in the GenBank (Mountain View, USA), EMBL (Heidelberg, Germany) and DDBJ (Mishima, Japan) nucleotide sequence data bases under accession numbers AY620964 to AY620979, respectively.

Experimental Infections

Bacterial cultures from strains 497 (V. kanaloae), 636 and 630 (V. splendidus biotype 1), 322.10 and 258.11 (V. neptunius), and 337.98 (V. pomeroyi) were selected for experimental infections in healthy clams (Venerupis rhomboides), which were kept for 5 days in tanks with aerated filtered (0.2 [micro]m) seawater, before experiments were performed. The seawater, with a salinity of 33[per thousand], was maintained at 18[degrees]C, as in the summer season, because of the faster growth of clams.

A group of 20 clams per assay was used for each strain and control, and kept in different tanks. The bacterial innoculum of the strains was prepared by growth in 60 mL of tryptic soy broth (TSB; Cultimed. Barcelona) supplemented up to 2% of NaCl at 22[degrees]C for 24h. The bacterial suspension was added to each tank containing 540 mL of sterile sea water, the final dose being 2-6 x [10.sup.7] CFU [mL.sup.-1] per bath. Each group of 20 clams was bath challenged for 3 h in noncirculating seawater conditions. Each group was then transferred to empty tanks for 1h and then to the tanks containing aerated filtered seawater at 18[degrees]C. All the clams were monitored for mortality over a 14 day period. Clams bath challenged with the type strain of Vibrio tapetis (CECT 4600) were used as a positive control.

Clams bath challenged with 600 mL of sterile seawater were used as a negative control.

Samples from internal organs were taken from all dead clams and after the 14-day period from all live clams. Identification of reisolates was conducted with specific phenotypical tests of each inoculated strain.

RESULTS

Genotypic Analysis

The assignment of strains by sequencing and ribotyping are shown in Table 1. The restriction enzyme Hind III showed reproducible results and yielded a sufficient number of appropriately sized fragments for strain differentiation, allowing an accurate analysis of the strains. A similarity level of [greater than or equal to] 71.3% was used for ribocluster establishment. A dendrogram constructed using the SD/UPGMA analysis grouped the strains in nine riboclusters (Fig. 1). Each ribocluster was given an arbitrary number from 1-9. Ribocluster 5 included V. splendidus-related species. A higher similarity level SD/UPGMA ([greater than or equal to] 71.5%) than 71.3% was needed for separating these species. The ribocluster was subgrouped in five subriboclusters and each subribocluster was arbitrarily named from 5A to 5E. Subribocluster 5A was formed with a 84% SD/UPGMA similarity level, subribocluster 5B with a 78%, subribocluster 5C and 5E with a 72% and subribocluster 5D with a 71.5%. Ribotyping was useful to make groups of strains closely related by 16S and 23S restriction pattern bands. A high heterogeneity of ribotyping profiles was found in each ribocluster and only two strains of ribocluster 6 showed an identical ribotype profile.

The phylogenetic position of sequenced strains is shown in the Figure 2. The strains: 562, 673, 373.11, 48, 364.11, 236.10, 497, 337.98, 636, 685, and 630 formed a clade with the high bootstrap support of 74%. The sequence of each strain analyzed was compared with that of the type strains also included in this group: V. tasmaniensis (LMG 20012), V. kanaloae (LMG 20539), V. pomeroyi (LMG 20537), V. splendidus biotype 1. The identification of sequenced isolates was performed according to the highest sequence identity with the type strain of these Vibrio species when the limit value of intraspecies variability ([greater than or equal to] 98%; Stackebrandt & Embley 2000) was achieved. The strain 257.11 and the type strain of V. mediterranei (CIP 103203) showed a strong bootstrap (99%) and a high value (99.69%) of sequence identity. The strains 258.11, 268.10, 322.10, and the type strain of V. neptunius (LMG 20536) also showed a strong bootstrap (99%) and a high value ([greater than or equal to] 99.3%) of sequence identity, being a monophyletic clade.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

The strain 671 of ribocluster 3 was not identified by sequencing, because sequencing analysis revealed their far off phylogenetic accommodation previously described in Vibrio species (<98% level). Therefore, ribocluster 3 remained as Vibrio sp.

Genotypic diversity of V. splendidus, V. tasmaniensis and V. neptunius species was shown. Several strains from each species belonging to identical phenon were clustered in different riboclusters and subriboclusters and identified as the same species. For example, several strains of phenon 5 were clustered in riboclusters 1 and 7 and subribocluster 5B and identified as V. tasmaniensis. One ribocluster of V. kanaloae, V. pomeroyi, and V. mediterranei was reported. (Fig. 1).

All V. splendidus-related, V. mediterranei and V. neptunius strains have exhibited a considerable variety of isolation origins in the NW of Spain. No specificity of bacteria according to the geographical area was found.

Phenotypic Differentiation

The 71 environmental isolates examined in this study (Table 1) had the main phenotypic features of the genus Vibrio: they were Gram-negative, oxidase and catalase positive, facultative anaerobic and motile.

Strains from different phena were clustered in the same subribocluster and identified by sequencing as identical species. So, several strains of phena 1, 5, 14, and 26 were clustered in the subribocluster 5B and identified as V. tasmaniensis. Similar results were found for the rest of identified species, showing a high phenotypic diversity (Fig. 1).

For each individual species shown in Tables 2 and 3, the results obtained for every phenotypic character were compared with those described by other authors for the species description, and only those found to be common in all studies were considered. Isolates identified as V. tasmaniensis (a sole unclustered strain and isolates belonging to riboclusters 2, 5B, 5C, 7, and 8) shared nine of the 12 phenotypic characteristics among them and only 7 with the reference strain (Table 2).

Isolates identified as V. kanaloae (ribocluster 5A) shared three of differential phenotypic characteristics with the reference strain of V. kanaloae. Five differential features were in common between these isolates and the reference strains of V. tasmaniensis or V. lentus (Table 2).

Strains identified as V. pomeroyi (ribocluster 5D) shared seven differential phenotypic characteristics with strains of V. pomeroyi, but nine tests were shared with the reference strain of V. splendidus biotype 1 (Table 2).

Isolates identified as V. splendidus biotype 1 (riboclusters 1, 5E, and 6) shared five differential features with the reference strain of V. splendidus biotype 1, characterized in this study. Six differential tests were shared between these isolates and V. kanaloae or V. lentus (Table 2).

Isolates included in ribocluster 9 were phylogenetically positioned closer to V. mediterranei (99.69% identity) than to V. tapetis (98.56% identity), but shared a phenotypic behavior closer to V. tapetis than to the type strain of V. mediterranei (Table 3). Isolates included in this ribocluster only presented one differential test in common with the reference strain of V. mediterranei but shared eight differential tests with the reference strain of V. tapetis analyzed in this study.

Isolates identified as V. neptunius (two unclustered strains and isolates belonging to ribocluster 4) were coincident in five phenotypic characteristics with those previously described for the differentiation of V. neptunius from closely related Vibrio species: they were unable to grow at 4[degrees]C or in 10% NaCl, they produced no acid from amygdaline or D-mannitol and showed a variable result for ADH test.

Experimental Infections

Mortalities and reisolation of inoculated strains were shown in Table 4. Reisolation of inoculated strains of V. kanaloae (497) and V. pomeroyi (337.98) from the internal organs as major component achieved 53% and 91% of dead clams. Values of 100% for strains 636 and 630 of V. splendidus biotype 1 were recorded and 74% and 15% for strains 322.10 and 258.11 of V. neptunius, respectively. A low or absent reisolation was obtained from the survivor clams. Clinical signs were not recorded. Mortality of clams bath challenged with V. tapetis strain was 82% and reisolation from internal organs as major component achieved 5%.

Neither mortality nor reisolation from the internal organs of clams was recorded in the negative control group.

DISCUSSION

A high number of unidentified isolates, belonging to genus Vibrio, were reported in our previous paper on bacterial community associated with Galician oyster and clam production when analyzed by numerical taxonomy (Guisande et al. 2004). To complete the diversity study and the identification of a higher number of isolates, this study analyzed 71 and 16 representative strains by ribotyping and by sequencing of 16S rRNA gene respectively, allowing for the identification of 91.5% of isolates. This and the previous analysis noted the main culturable representative species of Vibrio associated with the culture of oyster and clams in Galicia that would not be possible if any step in both studies were omitted.

The species V. tasmaniensis, V. kanaloae, V. pomeroyi, V. neptunius, V. splendidus biotype 1 and V. mediterranei were identified, some of them not having been reported previously in association with these cultures. Vibrio vulnificus, V. parahaemolyticus, or V. cholerae were not identified although these species were previously reported associated with seafood-producing estuarine and coastal areas of the world (Sindermann 1990). This is the first study to report the ribotype diversity of the V. tasmaniensis, V. kanaloae, V. pomeroyi, and V. neptunius species. The ribocluster 3 and two unclustered strains by ribotyping remained as Vibrio sp. Further molecular studies such as DNA-DNA hybridization or a multilocus analysis will be necessary to establish their exact identification.

The selection of a correct SD makes it possible to group strains closely related by ribotyping and makes it easier to apply another analysis such as sequencing, to complete the identification of a high number of isolates. Other authors have defined the ribotype cluster in isolates belonging to the same species at the [greater than or equal to] 70% similarity level using this coefficient (Austin et al. 1997, Farto et al. 2003). However, a So value of 71.3% did not allow for separating some V. splendidus-related species (ribocluster 5), such as Montes et al. (2003, 2006) had already described. Nor did we note a clear discrimination among V. splendidus-related strains found by using biochemical methods, as was previously found (Le Roux et al. 2004). They were differentiated by authors (Macian et al. 2001, Thompson et al. 2003a, 2003b) from the most closely phylogenetically related Vibrio species (Fig. 2), using the features shown in Table 2, but using the higher coincidental phenotypic result would give a misidentification, as was previously found (Montes et al. 2003). We have proved the high variation of these tests when applied to a broad spectrum of strains belonging to these species (Farto et al. 1999, Montes et al. 2003). This study confirmed that the phenotypic tests are not discriminatory for the separation of closely related Vibrio species.

In this study, isolates belonging to this group were separated and identified by using the combination of ribotype analysis, and their closest sequence identity with type strain of Vibrio species. The 16S sequencing data revealed the close phylogenetic relationship above the level proposed as the intraspecies variability (i.e., [greater than or equal to] 98%) between V. tasmaniensis, V. kanaloae, V. pomeroyi, and V. splendidus biotype 1, as authors have previously noted (Macian et al. 2001, Montes et al. 2003, Montes et al. 2006, Thompson et al. 2003a, Thompson et al. 2003b).

The selection of representative strains of each ribocluster, subribocluster, and unclustered strains allowed for the identification of closely related genetic strains sequencing 22% of isolates. The high number of sequenced strains and the inclusion of a unique identified species in each ribocluster and subriboclusters confirmed the validity of the identification because both techniques (ribotyping and sequencing analysis) analyze the same 16S rRNA gene.

This is the first study to report the isolation of V. tasmaniensis from the larvae and reproductive stages in oysters and clams, V. kanaloae from the larva stage of clams and reproductive stage in oysters, V. pomeroyi from larva in oyster and reproductive stages in oysters and reproductive stage in clams and V. neptunius from oyster cultures in reproductive stage, seed, and larval stage of clams and their culture water. These Vibrio species had been isolated from Atlantic salmon (Salmo salar L.) and gut of turbot larvae (Scophthalmus maximus L.) (Thompson et al. 2003a), diseased oyster (Ostrea edulis) larvae in France (Thompson et al. 2003b), healthy bivalve (Nodipecten nodosus) larvae and turbot (Scophthalmus maximus) in Brazil and Spain (Thompson et al. 2003b), diseased bivalve (Nodipecten nodosus L.) larvae in Brazil (Thompson et al. 2003c) and diseased oyster (Ostrea edulis) larvae in Galician (Prado et al. 2005), respectively. Also, the association of V. splendidus biotype 1 with disease in culture of Crassostrea gigas L. and Ruditapes deeussatus L. has already been well documented (Lacoste et al. 2001, Le Roux et al. 2002, Waechter et al. 2002, Gomez-Leon et al. 2005). Because our strains were isolated from healthy larvae and reproductive clams and oysters and their culture water, experimental infections were carried out in Venerupis rhomboides to confirm their pathogenic potential. The six assayed strains, 497, 636, 630, 322.10 258.11, and 337.98 caused mortality and were able to colonize the organs of clams. This confirms their virulence for Venerupis rhomboides and points to the risk of molluscan culture outbreaks. Interestingly, differential virulence levels were found among riboclusters of the same species (V. neptunius and V. splendidus biotype 1).

Although a high mortality was recorded from clams inoculated with V. tapetis, it was reisolated as major type of colony from one dead clam. Therefore mortalities of clams caused by this species were not confirmed and low susceptibility of Venerupis rhomboides to V. tapetis was found. Different levels of susceptibility among clam species to these bacteria were reported previously (Allam et al. 2002, Gay et al. 2004).

The study provides new genotypic, phenotypic, and biotypic information of recently described species of Vibrio. This study underlines the importance of using both techniques, ribotyping and 16S rRNA gene sequencing, for clustering, separation of V. splendidus-related species, and the identification of closely related Vibrio species. The ability of some strains, belonging to V. kanaloae, V. neptunius, V. pomeroyi, and V. splendidus biotype 1, to colonize reproductive stage of clams was shown. All this information would be useful to improve industrial culture production and to control culture quality.

ACKNOWLEDGMENTS

The authors thank J. Montes and CECT for kindly providing bacterial isolates and reference strains respectively, and also to P. Moran for providing the automated sequencer. This work was supported by grants PGIDIT00MAR2002PR and PGIDIT02RMA30102PR from the Xunta de Galicia (Regional Government of Galicia).

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JOSE ANTONIO GUISANDE, (1) ESTELA PEREZ LAGO, (1) SUSANA PRADO, (2) TERESA PEREZ NIETO (1) AND ROSA FARTO SEGUIN (1) *

(1) Area de Microbiologia, Departamento de Biologia Funcional y Ciencias de la Salud, Facultad de Ciencias, Universidad de Vigo, Lagoas Marcosende s/n 36310 Vigo, Spain; (2) Departamento de Microbiologia y Parasitologia. Universidad de Santiago de Compostela, Spain

* Corresponding author. E- mail: [e-mail]

TABLE 1.
Vibrio strains used in this study clustered by sequencing and
ribotyping analyses listed in alphabetical order.

                                   Ribocluster   Phenon
     Sequencing Assignment         (a)           (a)      Strain

V. kanaloae (99.52%) (d)           5A            22       497#
                                   5A            33        44
                                   5A            --       300.98
V. mediterranei (99.69%)           9             29       257.11#
                                   9             5        161
                                   9             37       539
V. neptunius (99.56%)              4             15       258.11#
                                   4             15       326.11
                                   4             21       240.11
                                   4             21       229.11
                                   4             24       187
                                   4             25       322.98
V. neptunius (99.31%)              --            14       322.10#
V. neptunius (99.36%)              --            7        268.10#
V. pomeroyi (99.63%)               5D            6        337.98#
                                   5D            5        106
                                   5D            9        660
                                   5D            28       303.10
                                   5D            30       321.11
                                   5D            37       579
V. splendidus biotype 1 (99.68%)   1             5        630#
                                   1             34       204
                                   1             39       646
V. splendidus biotype 1 (99.64%)   5E            5        685#
                                   5E            1        555
                                   5E            1        597
                                   5E            5         41
                                   5E            5        184
                                   5E            5        339.98
                                   5E            5        166
                                   5E            7        321.10
                                   5E            10       658
                                   5E            12       327.98
                                   5E            15       274.11
                                   5E            26       584
                                   5E            31       186
V. splendidus biotype 1 (99.64%)   6             5        636#
                                   6             1        559
                                   6             5         79.98
                                   6             5        156
                                   6             6        319.10
V. tasmaniensis (99.53%)           2             18       364.11#
                                   2             18       243.11
V. tasmaniensis (99.79%)           5B            1        562#
                                   5B            5        162
                                   5B            5         72.98
                                   5B            14       307.10
                                   5B            26       669
V. tasmaniensis (99.57%)           5C            21       236.10#
                                   5C            4        681
                                   5C            17       679
V. tasmaniensis (99.71%)           7             6        373.11#
                                   7             1        527
                                   7             2        568
                                   7             4        604
                                   7             5          5.98
                                   7             8        343.98
                                   7             11       313.98
                                   7             14       274.10
                                   7             15       267.11
                                   7             25       319.98
                                   7             36       347.98
V. tasmaniensis (99.69%)           8             4        673#
                                   8             32       308.10
V. tasmaniensis (99.36%)           --            23        48#
Vibrio sp. (< 98%)                 3             16       671#
                                   3             17       678
                                   3             17       696
                                   3             17       697
                                   --            21       233.10
                                   --            41       326.98

     Sequencing Assignment         Source (b)        Sampling (c)

V. kanaloae (99.52%) (d)           Oyster (R.)       Grove
                                   Oyster (R.)       Couso
                                   Clams (L.)        Grove
V. mediterranei (99.69%)           Clams (L.)        Grove
                                   Oyster (R.)       Couso
                                   Clams (R.)        Ribadeo
V. neptunius (99.56%)              Clams (L.)        Grove
                                   Oyster (R.)       Couso
                                   Oyster (R.)       Couso
                                   Oyster (R.)       Couso
                                   Sea water         Grove
                                   Oyster (R.)       Couso
V. neptunius (99.31%)              Oyster (R.)       Couso
V. neptunius (99.36%)              Clams (S.)        Grove
V. pomeroyi (99.63%)               Oyster (R.)       Couso
                                   Oysters (L.)      Couso
                                   Oysters (L.)      Ribadeo
                                   Oyster (R.)       Couso
                                   Oyster (R.)       Couso
                                   Clams (R.)        Ribadeo
V. splendidus biotype 1 (99.68%)   Sea water         Ribadeo
                                   Sea water         Grove
                                   Oysters (L.)      Ribadeo
V. splendidus biotype 1 (99.64%)   Oyster (R.)       Ribadeo
                                   Oyster (R.)       Bueu
                                   Clams (R.)        Ribadeo
                                   Oyster (R.)       Couso
                                   Sea water         Grove
                                   Oyster (R.)       Couso
                                   Oyster (R.)       Couso
                                   Oyster (R.)       Couso
                                   Oyster (R.)       Ribadeo
                                   Oyster (R.)       Couso
                                   Clams (L.)        Grove
                                   Oyster (R.)       Ribadeo
                                   Sea water         Grove
V. splendidus biotype 1 (99.64%)   Sea water         Ribadeo
                                   Oyster (R.)       Ribadeo
                                   Oysters (L.)      Grove
                                   Oysters (S.)      Grove
                                   Oyster (R.)       Couso
V. tasmaniensis (99.53%)           Oyster (R.)       Ribadeo
                                   Oyster (R.)       Couso
V. tasmaniensis (99.79%)           Oyster (R.)       Grove
                                   Oyster (R.)       Couso
                                   Oysters (L.)      V. de Arousa
                                   Oyster (R.)       Couso
                                   Oyster (R.)       Ribadeo
V. tasmaniensis (99.57%)           Oysters (L.)      Couso
                                   Oyster (R.)       Ribadeo
                                   Oyster (R.)       Ribadeo
V. tasmaniensis (99.71%)           Oyster (R.)       Ribadeo
                                   Clams (R.)        Ribadeo
                                   Clams (R.)        Ribadeo
                                   Oyster (R.)       Bueu
                                   Oysters (L.)      Grove
                                   Oyster (R.)       Couso
                                   Oyster (R.)       Couso
                                   Clams (L.)        Grove
                                   Clams (L.)        Grove
                                   Oyster (R.)       Couso
                                   Oyster (R.)       Couso
V. tasmaniensis (99.69%)           Oyster (R.)       Grove
                                   Oyster (R.)       Couso
V. tasmaniensis (99.36%)           Oyster (R.)       Couso
Vibrio sp. (< 98%)                 Oyster (R.)       Ribadeo
                                   Oyster (R.)       Ribadeo
                                   Oyster (R.)       Ribadeo
                                   Oyster (R.)       Ribadeo
                                   Oyster (R.)       Couso
                                   Oyster (R.)       Cousoa

(a) The symbol "--" means that one isolate was unclustered.

(b) "S." means seed stage, "L" means larval stage and "R."
means reproductive stage in the culture of oysters and clams.

(c) Sampling locations along the Galician coast.

(d) In brackets is shown the sequence identity (%) of the
isolate analyzed with the closest neighbor.

In bold print, isolate analyzed by sequencing of 16S rRNA gene.

In bold print, isolate analyzed by sequencing of 16S rRNA gene
indicated with #.

TABLE 2.
Presence of previously proposed differentiating features among
the isolates of Vibrio tasmaniensis, V. kanaloae, V. pomeroyi,
V. lentus and V. splendidus biotype 1 of this study.

                           1     2     3     4     5

Growth at:
  4[degrees]C              v     v    (+)    v     +
  28[degrees]C             +     +    (+)    +     +
  37[degrees]C            (-)    -     -     -     -
ADH                       (+)    v     +     +     -
Indole production         (+)    v    (+)   (+)    +
Gelatinase                (-)    v    (-)    v     -
Acid from:
  L-arabinose              -     -     -     -     -
  D-melibiose              -     -     v     -     -
  Sucrose                  v     -     v     v     -
Use of:
  L-alanine                v     v    (-)    v     +
  L-proline               (+)    v     v    (+)    -
Susceptibility to 0/129   (+)    v     +     v     +

                           6     7     8     9

Growth at:
  4[degrees]C              +     +     +     +
  28[degrees]C             +     +     +     +
  37[degrees]C             -     -     -     -
ADH                        +     +     +     +
Indole production          +     +     +     +
Gelatinase                 +     +     +     -
Acid from:
  L-arabinose              +     -     -     -
  D-melibiose              -     v     -     +
  Sucrose                  +     v     -     -
Use of:
  L-alanine                +     +     -     -
  L-proline                +     -     -     -
Susceptibility to 0/129    v     +     -     +

1. Isolates identified as V. tasmaniensis (Number of strains: n =
24); 2. Isolates identified as V. kanaloae (n = 3); 3. Isolates
identified as V. pomeroyi (n = 6); 4. Isolates identified as V.
splendidus biotype 1 (n = 21); 5. V. tasmaniensis LMG 20012T
(Thompson et al. 2003a); 6. V. kanaloae LMG [20539.sup.T] (Thompson
et al. 2003b); 7. V. pomeroyi strains (Thompson et al. 2003b); 8. V.
lentus CECT 5110T (Macian et al. 2001); 9. V. splendidus biotype 1
ATCC [33125.sup.T] (this study). Criteria (data are expressed in
percentage of positive results): ND: No data; +: Positive result
([greater than or equal to] 90% of positive results); -: Negative
result ([less than or equal to] 10% of positive results); (+):
Mainly positive results ([greater than or equal to] 70%<90% of
positive results); (-): Mainly negative results (>10% [less than or
equal to] 30% of positive results); v: Variable results (>30%<70%
of positive results).

TABLE 3.
Presence of previously proposed differentiating features between
the isolates of Vibrio mediterranei and V. tapetis of this study.

                  1   2   3

Growth at:
  44[degrees]C    -   -   +
Degradation of:
  Casein          v   -   +
  Esculin         v   +   -
  Chitin          -   +   -
Acid from:        -       -
  D-galactose     -   +   -
  Lactose         -   +   -
  D-melibiose     -   +   -
  Salicin         -   +   -
  Sorbitol        -   +   -
Use of:           -       -
  L-alanine       -   +   -
  L-serine        -   +   -

1. Isolates identified as V. mediterranei (Number of strains: n = 3);
2. V. mediterranei strains (Pujalte et al. 1992); V. tapetis CECT
[4600.sup.T] (this study).
The same criteria as indicated in Table 2 were applied.

TABLE 4.
Susceptibility of Venerupis rhomboides to live cells
of potentially pathogenic strains by bath challenge.

 Species and                      Dose by Bath         Reisolation
   Strain       Ribocluster   Challenge CFU (a)/mL     Rate % (b)

V. kanaloae
  497               5A          3.5 x [10.sup.7]      53 (10/19) (b)
V. neptunius
  322.10            --          3.2 x [10.sup.7]      74 (14/19)
  258.11             4          3.4 x [10.sup.7]      15 (3/20)
V. pomeroyi
  337.98            5D          7.0 x [10.sup.6]      91 (10/11)
V. splendidus
  636                6          5.1 x [10.sup.7]     100
  630                1          2.9 x [10.sup.7]     100 (18/18)
V. tapetis
  CECT 4600         --          3.8 x [10.sup.7]       5 (1/20)

(a) CFU: colony-forming units.

(b) % of dead clams from which the major type of colony reisolated
from internal organs, was the inoculated strain; in brackets No.
of positive strains/total number of dead clams.