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Increased Survival of Juvenile Turbot Scophthalmus maximus by Using Bacteria Associated with Cultured Oysters a

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Rosa M. Farto Seguín , María Bobo Bermúdez , Leticia Rivera & Teresa P. Nieto a

Universidad de Vigo, Vigo 36310, Spain Published online: 31 Oct 2014.

Click for updates To cite this article: Rosa M. Farto Seguín, María Bobo Bermúdez, Leticia Rivera & Teresa P. Nieto (2014) Increased Survival of Juvenile Turbot Scophthalmus maximus by Using Bacteria Associated with Cultured Oysters, Journal of Aquatic Animal Health, 26:4, 251-262, DOI: 10.1080/08997659.2014.920734 To link to this article: http://dx.doi.org/10.1080/08997659.2014.920734

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Journal of Aquatic Animal Health 26:251–262, 2014  C American Fisheries Society 2014 ISSN: 0899-7659 print / 1548-8667 online DOI: 10.1080/08997659.2014.920734

ARTICLE

Increased Survival of Juvenile Turbot Scophthalmus maximus by Using Bacteria Associated with Cultured Oysters ´ Rosa M. Farto Segu´ın,* Mar´ıa Bobo Bermudez, Leticia Rivera, and Teresa P. Nieto

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Universidad de Vigo, Vigo 36310, Spain

Abstract Preventing vibriosis in juvenile cultured Turbot Scophthalmus maximus caused by Vibrio anguillarum frequently requires the use of feed supplemented with antibiotics in addition to vaccines. Whether the use of probiotics instead of antibiotics in juvenile Turbot is a safer strategy requires more study. The antibacterial potential of 148 Vibrio spp. strains (mostly isolated from cultures of healthy oysters, clams, and Turbot) was analyzed in vitro against V. anguillarum and other pathogens by means of an agar diffusion assay. A wide spectrum of inhibitory activity was shown by 9 strains. Based on their easy phenotypic differentiation from V. anguillarum, we selected two strains (S1 and S2, both isolated from the European flat oyster Ostrea edulis) for testing in juvenile Turbot (3 g). None of the strains were virulent by intraperitoneal or bath challenges, and all were susceptible to the antibiotics most frequently used in aquaculture. Three different stocks of Turbot, which were assayed separately, were significantly protected from infection with V. anguillarum. The final survival rates of fish treated in mixed challenges with S1 or S2 and V. anguillarum were 44% and 66%, respectively, whereas only 17% of the fish treated with only the pathogenic strain survived. The application of probiotic strains also increased the survival time of juvenile Turbot after infection with V. anguillarum. Both strains persisted in the epidermal mucus layer of the fish for 30 d, and they were not displaced by the pathogen. These data prove the efficacy of using bacteria well adapted to the dynamics of culture production as a way to provide juvenile Turbot immediate protection against infection by V. anguillarum. Moreover, the epidermal mucus sampling was useful for investigating the persistence of both probiotic strains when exposed to the pathogen.

The global production of Turbot Scophthalmus maximus in 2011 was 72,413 metric tons, and it was valued at US$600 million. The largest producer by far is Spain, whose annual production has nearly doubled since 1998 and represents more than 75.9% of the global total (Serrano 2014). Protecting juvenile Turbot during their development is necessary to avoid infection. The use of bacteria as probiotics in Turbot larval aquaculture has previously been examined for bacteria isolated from marine environments and other sources. Low survival rates were found in larvae treated with Streptococcus thermophilus, Lactobacillus helveticus, and Lactobacillus plantarum (Gatesoupe 1991). Larvae treated with Pediococcus acidilactici showed no significant differences in survival rate from the control (Villamil et al. 2010). On the contrary, a high survival rate was

obtained by using a combination of bacteria from this genus and Lactobacillus, although the beneficial effects have not been studied in challenge trials against pathogens (Dag´a et al. 2013). A few studies have also addressed the use of bacteria isolated from Turbot aquaculture. In particular, two strains of Vibrio have been selected, but the authors believed that they would provide a weak defense against highly virulent pathogens such as V. anguillarum (Gatesoupe 1997; Ringo and Vadstein 1998). Some more promising candidates were bacteria from the Roseobacter clade, which appear to be universal colonizers of marine larval rearing units and displayed antibacterial activity against V. anguillarum (Porsby et al. 2008). In fact, treatment with a Roseobacter strain (currently identified as Phaeobacter; Pintado et al. 2010) led to increased survival of larvae

*Corresponding author: [email protected] Received October 11, 2013; accepted March 10, 2014

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during experimental infection with that pathogen. However, the quantity and frequency of the additions were not optimized (Planas et al. 2006), nor was the effect on juvenile Turbot studied. Although ingestion of V. proteolyticus by juvenile Turbot tended to produce positive effects on its feed efficiency, its nutritional significance and potential as a prophylactic agent against vibriosis were not examined (Schrijver and Ollevier 2000). Pseudomonas spp. and Vibrio spp. were also recently administered to juvenile Turbot, which enhanced serum bactericidal activity against Edwardsiella tarda and Vibrio anguillarum. However, both strains were also able to disseminate into internal organs, and the immediate protection of the host was not evaluated (Wang et al. 2013).The probiotic effect in juvenile Turbot would have to be studied in detail to produce Turbot that were disease resistant until they reached market or broodstock size. Such study could provide important economic benefits for aquaculture. In this study, 148 Vibrio spp. strains previously isolated by us (mostly from healthy oysters, clams, and Turbot) were analyzed in vitro and in vivo in order to identify probiotic strains that might be useful in preventing vibriosis caused by Vibrio anguillarum in juvenile Turbot. The epidermal mucus of the Turbot was sampled to evaluate the persistence of the probiotics in this host, along with their capacity to resist being excluded by the pathogen. METHODS Bacterial Strains and Characterization We used 129 Vibrio spp. strains previously isolated by us (Guisande et al. 2004) from healthy oysters and clams at different stages of culture at several facilities in northwestern Spain between 2008 and 2010. A total of 110 strains were isolated from the European flat oyster Ostrea edulis (82 from broodstock, 24 from larvae, and 4 from spat), 6 from the Manilla littleneck clam Tapes japonica (larvae), 8 from the pullet carpet shell Venerupis pullastra (broodstock), and 5 from the grooved carpet shell Ruditapes decussatus (broodstock). Briefly, broodstock (50 g of flesh and intervalval liquid) and larvae or spat (50 units each) were homogenized in 100 mL of sterile marine phosphatebuffered saline (pH 7.1) and adequate serial dilutions were plated onto marine agar (MA; Cultimed) or thiosulfate–citrate– bile–sucrose agar (TCBS; Cultimed). Colonies selected from the TCBS plates were spread on MA to obtain pure cultures, which were inoculated on nutritive broth (Cultimed) supplemented with 2% (weight : volume) NaCl (Panreac) and 15% (volume : volume) of glycerol (Panreac) for preservation at −80◦ C. Thawing was avoided each time they were needed in order to preserve their original features. The strains were phenotypically characterized as Vibrio by using the classic tests: gram staining, oxidase, oxidative or fermentative glucose metabolism, motility, nitrate reduction, growth ability at 0% NaCl, and susceptibility to O129 vibriostatic agent (2,4-diamino-6,7-di-isopropylpteridine phosphate; 150 µg/disc). These assays indicated that all of the strains were facultative, anaerobic, gram-negative rods

and that they were positive for all tests except for being able to grow at 0% NaCl. We also analyzed Vibrio spp. strains that had previously been isolated and phenotypically characterized (Montes et al. 1999) from the skin mucus of Turbot (17) and the water (2) at the aquaculture experiment system at the Instituto Oceanogr´afico de Vigo in which they had been held. Reference strains pathogenic to fish or shellfish were used for the agar diffusion assays. These strains were previously identified and provided by national or international bacteria collections (see www.cect.org/english,http://bccm.belspo.be/db/ lmg search form.php, and http://www.ncimb.com/). Pathogenic V. lentus strains (P52, P54, and P58) previously isolated and identified by us were also used (Farto et al. 2003; Table 1). Characterization of strains.—The strains with strong inhibitory activity (described below) and the three V. anguillarum strains (CECT 7199, NCIMB 571, and NCIMB 6) were characterized by 90 physiological, morphological, and biochemical tests as previously described (Guisande et al. 2004). Cultures grown at 22◦ C for 24 h on tryptic soy agar (Cultimed) supplemented with up to 2% (weight : volume) NaCl (TSA-2) were used as inocula. Additionally, both of the strains used in the virulence assays (S1 and S2) were characterized by their susceptibility to the antibiotics most frequently used in aquaculture (ciprofloxacin [5 µg], florfenicol [30 µg], tetracycline [10 and 30 µg], and sulfamethoxazole/trimetropin [25 µg]) by using the disk diffusion susceptibility method on TSA-2 plates as described by Huys et al. (2005). After incubation at 22◦ C for 24 h, the diameter of the inhibited growth zone surrounding the antibiotic disk was measured and the presence of any inhibition area was taken as indicating susceptibility to the antibiotic. The strains S1 and S2 were also characterized by sequencing the 16S rRNA gene according to the method described by Guisande et al. (2008). Multiple alignment of sequences was created by ClustalW in Genious Editor version 7.0.6 (Biomatters; http://www.geneious.com/). This included 1.138 positions after the removal of ambiguous ones. A phylogenetic tree was constructed using Molecular Evolutionary Genetics Analysis (MEGA) version 6 (Tamura et al. 2013). This was performed using the neighbor-joining method and Tamura–Nei distance model, with the calculation of cluster stability by bootstrap analysis with 1,000 replicates. The partial 16S rRNA sequences of the S1 and S2 strains have been deposited in the GenBank (Mountain View, Maryland), EMBL (Heidelberg, Germany), and DDBJ (Mishima, Japan) nucleotide sequence databases under the accession numbers KJ364527 and KJ364528, respectively. Differentiation of probiotic strains from the pathogenic strain and other naturally occurring bacteria.—To distinguish the pathogenic strain (CECT 7199) from the potentially probiotic strains (S1 and S2) used in the virulence assays, appropriate tests were conducted after biochemical and physiological profiling. The S1 and S2 strains were positive for β-hemolysis, with susceptibility to ampicillin (TSA-2 supplemented with ampicillin [25 µg/mL]) and able to grow at 7% NaCl. By contrast,

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TABLE 1. Inhibitory activity in an agar diffusion assay of potentially probiotic strains (109 CFU/mL) against various pathogenic strains (109). Values are the mean ± SD diameters of the zones of inhibited growth from four different experiments; blank cells indicate the absence of inhibitory activity. Strains with inhibitory activity against pathogenic strains

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Pathogenic straina V. alginolyticus CECT 586 V. anguillarum CECT 7199 NCIMB 571 NCIMB 6 V. harveyi LMG 4044 T V. lentus CECT 5293 P52 P54 P58 V. neptunius LMG 20615 V. parahaemolyticus CECT 5306 V. splendidus LMG 16751 V. tapetis CECT 4600T V. tasmaniensis LMG 20012T

S1b

S2b

S3c

S4d

S5e

S6c

2.6 ± 2.0 5.5 ± 0.6 2.1 ± 2

6.5 ± 1.1 1.4 ± 2.1

3.3 ± 1.1 6.3 ± 1.0 2.3 ± 0.9

9.0 ± 0.0

5.0 ± 1.0 4.1 ± 2.3

S7c

S8c

S9c

S10b

S11c

S12c

2.9 ± 0.8 7.5 ± 0.6

4.1 ± 2.7 7.2 ± 1.4

3.8 ± 0.5

3.3 ± 2.8

2.1 ± 2.9

1.0 ± 0 3.0 ± 1.3 2.0 ± 0.0 3.5 ± 0.0

3.0 ± 0 3.8 ± 0.5

1.0 ± 0 1.8 ± 0.8

5.3 ± 3.7

2.8 ± 3.4

8.7 ± 5.1

5.1 ± 2.2

3.0 ± 1.1

3.2 ± 1.2

1.7 ± 1.1 2.5 ± 1.6 4.7 ± 2.4 5.7 ± 4.5

3.2 ± 1.7 1.0 ± 0

1.4 ± 1.0 1.0 ± 0

6.1 ± 1.0

8.1 ± 2.8 9.0 ± 2.5

5.0 ± 0

2.8 ± 1.3 2.5 ± 2.1

4.3 ± 0.9

4.2 ± 2.6

9.8 ± 2.6

2.3 ± 1.9

3.5 ± 2.7

1.9 ± 1.2

3.0 ± 2.0

1.5 ± 1.4

2.7 ± 1.2

1.5 ± 1.6

2.3 ± 1.6

2.6 ± 1.8

1.3 ± 1.4

1.2 ± 1.5

4.7 ± 1.8

4.7 ± 0.9

3.4 ± 1.0

5.0 ± 1.3

4.6 ± 0.8

4.0 ± 0.8

2.9 ± 1.2

3.7 ± 1.6

2.5 ± 0.7

4.0 ± 0.0

1.3 ± 0.6

3.0 ± 4.0 5.3 ± 2.0

4.5 ± 2.1

1.5 ± 1.2 1.5 ± 1.0

6.3 ± 4.1

2.0 ± 1.2

T = type strain; abbreviations are as follows: CECT = Spanish Type Culture Collection, LMG = Belgian Coordinated Collections of Microorganisms (University of Ghent), NCIMB = Scottish National Collections of Industrial and Marine Bacteria. b Ostrea edulis larvae. c Ostrea edulis broodstock. d Venerupis pullastra broodstock. e Tapes japonica larvae. a

the pathogenic strain was positive for α-hemolysis, resistant to ampicillin, and unable to growth at 7% NaCl. Naturally occurring bacteria that were susceptible to ampicillin and positive for α-hemolysis or resistant to ampicillin and positive for β-hemolysis could thus be distinguished from the probiotic and pathogenic strains. Additionally, to differentiate our probiotic strains from other closely related Vibrio species (V. artabrorum, V. celticus, V. chagasii, V gigantis, V. hemicentroti, V. pomeroyi, and V. splendidus), the following phenotypical tests from the literature (see Table S.1 in the supplement that appears in the online version of this article) were used: ability to grow at 6% NaCl; response to Thornley’s arginine dihydrolase and the Voges–Proskauer test; ability to produce acid from L-arabinose, D-lactose, D-melibiose, L-rhamnose, and sucrose; and degradation of esculin and gelatin. In Vitro Antibacterial Potential of Vibrio spp. Strains The 148 Vibrio spp. strains were tested against 14 strains pathogenic for fish and shellfish (Table 1) on MA plates. Briefly, the strains tested were cultured in M9 minimal media supplemented with up to 1.5% NaCl and incubated at 22◦ C for 24 h with shaking. The concentration of the bacterial cultures was adjusted (OD590 = 0.5), and the MA plates were covered with an agar layer containing 109 or 1010 CFU per plate that was prepared for each pathogenic strain. Following that, 109 CFU of each potentially probiotic strain were inoculated on these plates

using the Replicator Mast Multipointer Inoculator (MAST) and incubated at 22◦ C for 120 h. The diameters of the inhibited-growth zones surrounding the potentially probiotic strains were measured, and zones at least 1 mm in diameter were considered indicative of antibacterial activity. The inhibitory capacity of each tested strain was classified according to the total number of pathogenic strains that it inhibited (strong, medium, or weak depending on whether the strain inhibited >5, 3–5, or 1–2 pathogenic strains, respectively), independently of the diameters of the inhibited zones. In Vivo Antibacterial Potential of Vibrio spp. Strains Experimental fish.—Healthy, unvaccinated Turbot (3 ± 0.5 g [mean ± SE]) were used for all challenges. The fish were the offspring of Turbot reared at an experimental fish farm at the University of Vigo (Marine Sciences Station at Toralla) with no history of disease. Prior to challenge, 10 fish randomly selected from the stock were screened for bacterial pathogens using routine diagnostic procedures, as previously described (Lago et al. 2012). For each bacterial strain and experiment (intraperitoneal or bath challenge), fish were held for 2 weeks in separate experimental tanks containing 50 L of sterile, 18◦ C aerated (with an air stone) seawater with a salinity of 33‰ and were fed twice a day. These conditions were maintained during the challenges, except that tanks with noncirculating seawater were used. The environmental parameters

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were recorded daily in each tank; the values of NH4 + and NH3 were kept below 3.3 mg/L and 0.1 mg/L, respectively, at pH 7.7–8 and an oxygen concentration of 6 mg/L. Water was renewed after feeding when required. All animal experiments were performed according to practices approved by the Spanish Ethical Committee (http://www.boe.es/boe/dias/2013/02/08/). Preliminary challenges to confirm the absence of virulence in potentially probiotic strains.—For both the intraperitoneal and bath challenges, we used S1 and S2 isolated from Ostrea edulis larvae. These were selected since they inhibited the growth of pathogenic strain V. anguillarum (CECT 7199) and were easily differentiated from V. anguillarum by phenotypical tests. For the intraperitoneal challenge, all strains were grown in shaken M9 broth supplemented with up to 1.5% NaCl (M9B; 22◦ C, 24 h); cells were harvested and resuspended in sterilized seawater to an OD590 of 0.4 (109 CFU/mL). The bacterial suspension (0.1 mL) was inoculated in Turbot (n = 30) by injection into the abdominal cavity, with doses of 106 or 107 CFU per fish, which were monitored for 7 d. The surviving fish were euthanized by an overdose of tricaine methanesulfonate (MS-222; Sigma). For all moribund, dead, and euthanized fish, the liver and kidney tissues were sampled for bacteria by spreading them on blood agar (Oxoid) and TSA-2 supplemented with ampicillin (25 µg/mL; TSA-2A). After incubation at 22◦ C for 48 h, identification of suspicious re-isolated colonies was carried out as described above. Fish intraperitoneally injected (0.1 mL) with M9 broth were used as negative controls and sampled as described above. Virulent strains were defined as those showing any mortality after challenge at doses of 106 CFU per fish. For the bath challenge, the bacterial inoculum of the potentially probiotic or pathogenic strains was prepared by growth in shaken M9B supplemented with up to 1.5% NaCl (22◦ C, 24 h). The inoculum was added to each tank, which contained 30 fish, for 12 h (to a final concentration of 105 or 107 CFU/mL, adjusted as above). After that, the seawater was changed. The starting bacterial numbers were confirmed by determining the number of CFU per milliliter of each tank at 0 h. Each fish group was monitored for 30 d and sampled as described above. Fish exposed to M9 broth and maintained under the same conditions were used as negative controls. Virulent strains were defined as those showing any mortality after challenge at doses of 106 CFU/mL. Preliminary challenges to confirm the virulence of pathogenic strains.—Our model pathogen was V. anguillarum, which is a well-established pathogen of Turbot and considered one of the leading fish pathogens, with a broad host range among farmed and wild fish (reviewed by Austin and Austin 2007). The strains CECT 7199, NCIMB 571, and NCIMB 6 were tested in order to select one that would be adequate. Intraperitoneal and bath challenges and sampling were performed as above. Virulence assays mixing potentially probiotic and pathogenic strains.—Based on the results of the intraperitoneal and bath challenges, the pathogenic strain CECT 7199 was selected to perform these assays. Potentially probiotic (S1 and S2) and pathogenic strains were mixed in the same tank for a bath challenge (S1mP and S2mP tanks) as follows: the bacterial inocu-

lum of the potentially probiotic strains was prepared and added for 12 h as described above, with a final concentration of 105 CFU/mL. After this, the seawater was changed and the bacterial inoculum of the pathogenic strain was also added for 12 h to a final concentration of 106 CFU/mL (stocks 1 and 2) or 107 CFU/mL (stock 3). Then the seawater was changed. The starting bacterial numbers were confirmed by determining the number of CFU per milliliter of each tank at time 0. Each fish group was monitored for 30 d and sampled as described above. Fish exposed only to the pathogenic strain were used as a positive control (Pc tank). On the contrary, fish exposed only to potentially probiotic strains (S1c and S2c tanks) and those exposed only to M9 broth (M9B tank) were used as a negative control in each stock. All fish were maintained under the same conditions as in the mixed-challenge assay. All treatments, (M9B, S1c, S2c, Pc, S1mP, and S2mP) were administered to three different stocks at different times to investigate whether the protection of fish was reproducible or not. One tank per treatment was used for each stock, except for assays in which probiotic and pathogenic strains were mixed, where two tanks were used. The total numbers of fish used per treatment and stock depended on availability (Table 5). Additionally, the external surfaces of fish were examined during the experiments in order to determine the persistence of the bacteria tested and the degree to which they were excluded by the pathogen. At various times postchallenge (0, 12, 24, 36, 60, 84, and 156 h as well as 14, 16, and 30 d), the epidermal mucus layer close to the pectoral fin (1 cm2) was removed aseptically from three randomly selected fish. These samples were swabbed with a sterile inoculating cotton bud for direct plating of the bacteria on blood agar, TSA-2 supplemented with ampicillin (25 µg/mL), and TSA-2. The plates were incubated at 22◦ C for 72 h, and one swab per fish was analyzed. The presence of probiotic and pathogenic strains or naturally occurring bacteria was determined by their differential responses to blood agar (BA) and TSA-2 containing ampicillin (TSA-2-A), as described above. Predominance and morphological differences in colonies were confirmed on those plates and on TSA-2. Each colony from the BA, TSA-2-A, and TSA plates was compared with those previously cultured with probiotic or pathogenic strains. When the result was unclear, the colonies were isolated and cultured to purity on TSA-2. Then they were tested for their ability to grow at 7% NaCl, and their responses to the different tests were noted as described above for other Vibrio species closely related to our probiotic strains. The presence of probiotic and pathogenic strains or naturally occurring bacteria was considered confirmed when a colony of each one was identified in at least one of the three sampled fish at each time postchallenge. After 30 d the surviving fish from the three stocks previously exposed only to S1 (S1c tank) or S2 (S2c tank) were divided in two groups containing similar numbers of fish. One was bathchallenged again with V. anguillarum (5 × 106 CFU/mL)to determine whether fish retained their protection against vibriosis. Another, which was used as a negative control, was

INCREASED SURVIVAL OF JUVENILE TURBOT

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not exposed again (S1cp or S2cp tanks). Fish exposed only to M9 broth (M9B tank) or pathogenic strain (Pc tank) were also used as negative and positive controls, respectively, in each stock. Variable numbers of fish were used, depending on availability (see Results). Each fish group was monitored for 30 d and sampled as described above. Statistical Analysis Kaplan–Meier survival curves for the three stocks analyzed jointly were generated, and mean survival times were calculated. Comparisons of the survival curves and the final survival of fish were made by means of the log-rank (Mantel–Cox) test. Each curve was constructed from data for three stocks, which included data from two replicates in assays in which probiotic and pathogenic strains were mixed. Additionally, Kaplan–Meier survival curves were generated for each stock independently and mean survival was calculated. These analyses were calculated using SPSS version 15.0. Statistical differences in the persistence of probiotic or pathogenic strains in epidermal mucus among the three stocks were also evaluated using paired-sample t-tests and one-way analysis of variance (ANOVA; Tukey’s post hoc test). These tests were done in Sigma Plot version 12. In all cases, a significance level of 0.05 was used. RESULTS In Vitro Antibacterial Potential of Vibrio Strains Of the 148 strains tested, 12 (S1–S12) showed inhibitory activity against different pathogens, such as V. anguillarum. Interestingly, 9 of them (most of which were isolated from the broodstock of Ostrea edulis) had a strong inhibitory activity, with the ability to inhibit at least 6 of the 14 pathogenic strains tested. The other strains had weak inhibitory activity. The probiotic strains showed different inhibitory activity against each pathogenic strain (Table 1). All of the potentially probiotic strains retained their inhibitory ability even with a 10-fold increase in the number of cells (1010 CFU/mL) of V. anguillarum (data not shown). Characterization of Potentially Probiotic Strains and Confirmation of V. anguillarum Only strains S1 and S2 could be easily differentiated phenotypically from all of the pathogenic strains of V. anguillarum tested, since both showed β degradation of sheep erythrocytes, susceptibility to ampicillin (25 µg/mL), and the ability to grow at 7% NaCl. Conversely, the pathogenic strains showed α-hemolysis, were resistant to ampicillin, and were unable to tolerate 7% NaCl. The two potentially probiotic strains produced identical results in 83 tests. They consisted of motile short rods; were gram-negative, oxidase- and catalase-positive, and oxidative and fermentative in O/F medium with glucose; and grew in yellow on TCBS medium and in crystal violet (0.001%). They were able to tolerate from 3.5% to 7% NaCl or pH 7.5–10 but not 10% NaCl or pH 4.5. Other characteristics are shown in

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Table 2. Furthermore, both strains were susceptible to all of the antibiotics most frequently used in aquaculture (Table 2). The sequencing of 16S rRNA showed that the two probiotic strains have 100% identity in gene sequences. They were also closely related to the V. splendidus group (having 99.6–100% similarity with eight species) and showed the highest sequence similarity (100%) with V. crassostreae, V. celticus, V. gigantis, and V. pomeroyi (Figure 1). The phenotypic profile of all the V. anguillarum strains obtained in this study confirmed their previous identification. In Vivo Antibacterial Potential of Vibrio Strains Preliminary challenges to confirm the absence of virulence in potentially probiotic strains.—We injected the strains S1 and S2 into the abdominal cavities of the Turbot, and neither of them was virulent at 106 or 107 CFU per fish. Similar results were obtained after bath challenges with 105 or 107 CFU/mL. Bacteria were never recovered from the livers of euthanized fish injected with probiotic strains or from uninfected control fish. No clinical signs were seen. Preliminary challenges to confirm the virulence of pathogenic strains.—The NCIMB 571 and CECT 7199 strains of V. anguillarum caused 100% mortality after intraperitoneal challenge with 106 or 107 CFU per fish. Both were recovered in pure culture from all fish. These two strains were also tested by bath challenge; only CECT 7199 was virulent at 106 CFU/mL and was recovered from fish in pure culture. The lethal effect was detected within 3 and 8 d after the intraperitoneal and bath challenges, respectively. Clinical signs included anorexia, abnormal swimming behavior, hemorrhagic lesions on the liver and intestine, and pale liver. NCIMB 6 was avirulent according to both virulence assays. Bacteria were never recovered from the liver of euthanized fish infected with the avirulent strain or from uninfected control fish. Virulence assays mixing potentially probiotic and pathogenic strains.—The potentially probiotic and pathogenic (CECT 7199) strains were mixed in the same tank to analyze the antibacterial potential of the former against vibriosis in Turbot. Kaplan– Meier survival curves for the three stocks analyzed jointly are shown in Figure 2; mean survival times are given in Table 3. The comparisons of the survival curves by the log-rank test revealed that there were significant differences between all of the negative controls (S1c, S2c, and M9B) and fish treated with the pathogen alone (Pc) or with probiotic S1 (S1mP) or S2 (S2mP) in the mixed challenges. The analyses also showed significantly better survival among fish treated with S1 or S2 in mixed challenges than among those exposed only to the pathogen (Table 4). Whereas the final survival rates of fish treated with S1 and S2 in the mixed challenges were 44% and 66%, respectively, that of fish exposed to the pathogen alone was 17% (Figure 2; Table 3). Significant differences between fish treated with S1 and S2 were found as well (Table 4). Furthermore, the application of probiotic strains significantly increased survival time (Figure 2). Additionally, Kaplan–Meier survival curves were generated for each stock independently (data not shown) in order to

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TABLE 2. Phenotypic characteristics of the two potentially probiotic strains selected for analysis. A positive sign indicates that the strain tested positive for that characteristic, a negative sign that it tested negative.

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Test Growth at: 4, 10, 22, 28◦ C 37◦ C 44◦ C Thornley’s arginine dihydrolase Moeller’s lysine decarboxylase, ornithine decarboxylase, and histidine decarboxylase Methyl red test Voges–Proskauer test Nitrate reduction Indole production Utilization of D-lactose or production of H2 S on Kligler medium Hemolysis-α Acid from: arbutin, D-cellobiose, D-fructose, D-galactose, D-mannitol, D-mannose, ribose, sucrose, D-trehalose amygdaline, L-arabinose, ethanol, inositol, D-lactose, D-raffinose, L-rhamnose, and D-sorbitol glycerol D-melibiose salicin Hydrolysis of: casein, chitin, DNA, esculin, gelatin, lecithin, starch urea, Tween 20, chondroitin celullose Use as sole carbon source: aspartate, β-alanine, DL-alanine, alginate, L-arginine, glycine, L-lysine, malonate, L-phenylalanine, L-proline propanol, pyruvate, L-serine, succinate, L-tartrate, L-tryptophan, uracil acetate, citrate inulin Susceptibility to: O129 (150 µg/disc) ciprofloxacin (5 µg/disc) florfenicol (30 µg/disc) tetracycline (10 µg/disc) tetracycline (30 µg/disc) sulphamethoxazole/trimetropin (25 µg/disc) derive stock-specific final survival rates and mean survival times. The different treatments produced different values for each stock (Table 5). All dead fish exhibited the disease symptoms described above, and the pathogenic strain was recovered in pure culture from most fish. The behavior of the positive and negative controls was as expected. The persistence of bacteria in the joint analysis of the three stocks is shown in Figures 3 and 4. In challenges with only probiotics as well as in mixed challenges, both probiotic strains were able to persist in the epidermal mucus layer of fish for 30 d. In the mixed challenges, however, the cumulative presence of S1 was significantly higher than that of S2 after 0, 12, 336, and 384 h. In comparing the diversity of bacteria on plates derived from fish subjected to those challenges with that of fish subjected to M9 broth, clear differences were seen. Cultures

S1

S2

+ – – + – + – + + – –

+ + + + – + – + + – –

+ – – – –

+ – + + +

+ – +

+ – –

+

+

– –

– +

+ + + + + +

+ + + + + +

grown from M9 broth tanks showed higher diversity (>3 different colonies) in naturally occurring bacteria. By contrast, S1 or S2 was predominant on plates obtained from fish exposed only to probiotics or to mixed challenges. Interestingly, in mixed and control challenges with strain S1, naturally occurring bacteria were significantly absent from the skin of several sampled fish, at least for 36 h postchallenge (Figure 3). This was not the case with S2, however (Figure 4). The pathogenic strain was detected in all experiments from 0 h after infection to 14 d postchallenge and was able to significantly displace the naturally occurring bacteria for at least 60 h (Figures 3, 4). By contrast, S1 and S2 were not displaced by the pathogen, since they showed similar persistence when used alone and when mixed with the pathogenic strain in any of the stocks. These results were also confirmed by the mixed challenges involving S1 and S2. In fact,

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FIGURE 1. Phylogenetic tree based on partial 16S rRNA gene sequences obtained by the neighbor-joining method. Vibrio cholerae was used as an out group. Bootstrap percentages (≥50) after 1,000 simulations are shown at the nodes. The bar labelled 0.005 indicates the nucleotide substitution per position. GenBank sequence accession numbers are given in parentheses.

the cumulative presence of S1 was significantly higher than that of the pathogenic strain, though no significant differences between the persistence of S2 and that of the pathogenic strain were found. There were no significant differences in the rest of the comparisons. Thirty days postchallenge, the surviving fish from the three stocks that had been exposed to the probiotic strains in the TABLE 3.

control challenges still had the strains in their epidermal mucus layer (Figures 3, 4). However, when they were bath-challenged again with V. anguillarum, the resulting number of dead fish was similar to that among fish exposed only to the pathogen (Table S.2). The lethal effect was detected within 8 d. All of dead fish showed the disease symptoms described above, and the pathogenic strain was recovered in pure culture from most fish.

Case summaries, final survival rates, and mean survival times for all Turbot stocks by the Kaplan–Meier method.

Mean for survival timec 95% confidence limits Treatmenta M9B S1c S2c Pc S1mP S2mP

Total no. of fishb 49 49 49 58 95 73

No. of dead No. of Final survival fish surviving fish rate (%) 0 0 0 48 53 25

49 49 49 10 42 48

100 100 100 17.20 44.20 65.80

Estimate

SE

Lower

Upper

30 30 30 7.93 16.63 22.04

0 0 0 1.35 1.24 1.30

30 30 30 5.29 14.19 19.48

30 30 30 10.57 19.07 24.60

a M9B = control with M9 broth, S1c = control with S1, S2c = control with S2, Pc = control with pathogenic bacteria, S1mP = S1 mixed with pathogenic bacteria, and S2mP = S2 mixed with pathogenic bacteria. b One replicate per treatment was used per stock except for treatments S1mP and S2mP, for which there were two replicates. c Estimate limited to the longest survival time that was recorded (30 d).

FARTO SEGU´IN ET AL.

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FIGURE 2. Kaplan–Meier survival curves of juvenile Turbot treated with the potentially probiotic strains S1 and S2 (105 CFU/mL) to prevent infection with V. anguillarum (106 CFU/mL for stocks 1 and 2, 107 CFU/mL for stock 3). Each curve was constructed from data for three stocks, which included data from two replicates in assays for which probiotic and pathogenic strains were mixed. The number of fish per stock or treatment varied, depending on availability (Table 5). According to the log-rank analysis (Table 4), infected Turbot with no previous treatment with probiotic strains died sooner.

DISCUSSION This study demonstrates that bacteria present in marine rearing environments enhance the survival of juvenile Turbot exposed to a pathogenic strain of V. anguillarum, thus qualifying as probiotics (Verschuere et al. 2000). We chose bacteria isolated from healthy cultures of oysters and clams, the epidermal mucus of Turbot, and the ambient water at culture facilities. Thus, all strains are well adapted to the dynamics of Turbot culture. Interestingly, we found that 9 of 148 strains (most of them isolated from Ostrea edulis) exerted strong antibacterial activity

FIGURE 3. Cumulative presence of bacteria in the skin of Turbot after treatment with probiotic S1. The data are the means of three stocks, each with three fish randomly sampled at each sampling time postchallenge (error bars = SDs). Abbreviations are as follows: O (M9B) = naturally occurring bacteria (“other bacteria”) in the M9B tank; O (Pc) = other bacteria in the pathogen control tank; O (S1c) = other bacteria in the S1 control tank; O (mS1) = other bacteria in the mixed S1–pathogen tank; Pc = pathogenic bacteria in the pathogen control tank; P (mS1) = pathogenic bacteria in the mixed S1–pathogen tank; S1c = S1 in the S1 control tank; and S1 (mP) = S1 in the mixed S1–pathogen tank. On the y-axis, 1 means that a colony of bacteria was found in three of the three sampled fish at time 1 postchallenge and similarly for the other sampling times.

against V. anguillarum in vitro. Similar results have previously been found (Prado et al. 2009). Thus, this method has proved itself to be useful for reducing the number of potential probiotic strains to be tested in vivo and provides a means of quickly differentiating between them. It is generally accepted that laboratory cultures do not survive if they are reintroduced into the natural environment because naturally occurring bacteria outcompete or antagonize them (reviewed by Austin and Austin 2007). Thus, the assays in vivo were crucial. Based on an easy phenotypical differentiation from V. anguillarum, we selected the potentially probiotic strains S1 and S2. The 16S rRNA sequencing data revealed a close phylogenetic relationship (above the level proposed as the threshold

TABLE 4. Pairwise comparisons of survival curves by the log-rank test for Turbot stocks analyzed jointly by the Kaplan–Meier method. Blank cells indicate no significant differences (P > 0.05); df = 5. See Table 3 for treatments.

Treatmenta

M9B χ2

M9B S1c S2c Pc S1mP S2mP

75.64 39.65 20.41

S1c P

0.0 0.0 0.0

χ2

75.64 39.65 20.41

S2c P

0.0 0.0 0.0

χ2

75.64 39.65 20.41

Pc P

0.0 0.0 0.0

S1mP

S2mP

χ2

P

χ2

P

χ2

P

75.64 75.64 75.64

0.0 0.0 0.0

39.65 39.65 39.65 32.39

0.0 0.0 0.0 0.0

32.39 57.02

0.0 0.0

20.41 20.41 20.41 57.02 8.39

0.0 0.0 0.0 0.0 0.004

8.39

0.004

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FIGURE 4. Cumulative presence of bacteria in the skin of Turbot after treatment with the probiotic S2. See Figure 3 for a key to the abbreviations and other information.

for intraspecies variability [≥98%]) between our strains and most of the species in the Vibrio splendidus group, confirming the previously shown inability of such data to differentiate among these species (reviewed by Beaz-Hidalgo et al. 2010). However, our strains can be differentiated from V. celticus (using conventional phenotypical traits) by the Voges–Proskauer test and the production of acid from D-lactose, sucrose, and L-rhamnose—which are four of the five traits used to distinguish this species from V. crassostreae (Beaz-Hidalgo et al. 2010). They can be also differentiated from V. gigantis using their growth in 6% NaCl and the production of acid from sucrose (Le Roux et al. 2005). Similarly, the Voges–Proskauer test, acid production from L-arabinose, and the degradation of gelatin were useful for distinguishing our strains from V. artabrorum (Di´eguez et al. 2011). All of these, in addition to ADH, were unique discriminatory traits between this species and V. crassostreae. Our strains differ from V. pomeroyi and V. chagasii with respect to the degradation of esculin and, in the case of the latter, with respect to the production of acid from D-mannose and sucrose (Kim et al. 2013; Lasa et al. 2013). Likewise, the ability to produce acid from L-arabinose or sucrose was useful in differentiating our strains from V. hemicentroti (Kim et al. 2013). Finally, our strains were distinguished from V. splendidus by means of three of the four traits used to distinguish this species from V. crassostreae (ADH, growth in 6% NaCl, and production of acid from L-rhamnose; Table S.1). All these results and the source of isolation suggest that our probiotic strains could be identified as V. crassostreae. Further molecular studies are needed to confirm the identity of the species. Our potentially probiotic strains were included in the V. splendidus clade as expected. In fact, this group has been found to be the predominant one in the genus Vibrio in coastal marine environments (Lambert et al. 1998; Sobecky et al. 1998). Since several species in this clade have caused mortality in a wide

259

range of marine animals (reviewed by Lago et al. 2009; Kim et al. 2013), it is important to confirm the lack of virulence of S1 and S2 for Turbot. After being isolated our strains were kept under optimal conditions to preserve their original features, and they were used directly for in vivo virulence assays. They were avirulent by intraperitoneal challenge, which produced a faster development of disease and associated mortality than in the bath challenge (Lago et al. 2012). Similarly, they were avirulent by bath challenge (performed in a large number of treatments) and with different fish stocks (the immunological responses of the different stocks varied somewhat as a result of different rearing conditions). Furthermore, both strains were unable to invade fish tissues even when intraperitoneal assays were performed. Thus, it is unlikely that the strains will show virulence. All these data provide strong evidence that these strains, which were isolated from healthy Ostrea edulis larvae, are also safe for the stage of Turbot tested. Even so, the strains could be pathogenic to other stages and/or species different from those studied. Infection can be avoided if (1) they are used in closed hatcheries and (2) biosecurity standards are correctly applied. One effective tactic would be to segregate the fish groups by age in accordance with good practice on fish farms. Another would be the disinfection of water before it leaves the facility to diminish the total number of bacteria in it (Yanong 2012). Both strains were also susceptible to all of the antibiotics most frequently used in aquaculture and thus cannot transfer resistance genes to other bacteria. The in vivo assays revealed that fish can be protected by using one order of magnitude fewer probiotic cells than pathogenic ones (i.e., 105 probiotic cells versus 106 pathogenic cells). However, higher concentrations of probiotic were needed to obtain antibacterial activity against Vibrio species in different organisms (Gram et al. 1999; Vaseeharan and Ramasamy 2003; Kesarcodi-Watson et al. 2012). Although inhibitory activity was tested with 109 CFU of probiotic strains/mL in our in vitro assays, protection of Turbot was also found with a dose of 105 CFU/mL. As low doses would be preferable in aquaculture, the dosage that achieves the highest efficacy should be determined (Nikoskelainen et al. 2001). Promising probiotic bacteria isolated from marine environments have previously been selected for use in the larval aquaculture of this host (Gatesoupe 1994; Huys et al. 2001; Planas et al. 2006); despite the high commercial value of Turbot, however, the use of probiotics with juveniles has not yet been developed. The bacteria that we identified protected Turbot of three different stocks (which were assayed at different times) from infection with V. anguillarum. Although there were differences in the final survival percentages, this is not surprising, as variation among individual fish is to be expected (Svendsen and Bøgwald 1997; Defoirdt et al. 2005; Ogut and Reno 2005; Croxatto et al. 2007). Variations in the immune responses of the different stocks, along with the variable number of fish groups, could explain our results. Kaplan–Meier analysis is a powerful tool because it estimates the probability of individual survival

FARTO SEGU´IN ET AL.

260

TABLE 5. Case summaries, final survival rates, and mean survival times for individual Turbot stocks by the Kaplan–Meier method. See Table 3 for additional information.

Mean survival time Average for all stocks

Stock

Total no. of fish

No. of dead fish

No. of surviving fish

Final survival (%)

Estimate

SE

Lower

Upper

Mean

SE

M9B

1 2 3

21 11 17

0 0 0

21 11 17

100 100 100

30 30 30

0 0 0

30 30 30

30 30 30

30

0

S1c

1 2 3

21 11 17

0 0 0

21 11 17

100 100 100

30 30 30

0 0 0

30 30 30

30 30 30

30

0

S2c

1 2 3

21 11 17

0 0 0

21 11 17

100 100 100

30 30 30

0 0 0

30 30 30

30 30 30

30

0

Pc

1 2 3

32 10 16

31 5 12

1 5 4

3.1 50.0 25.0

3.4 17.1 11.3

0.9 4.3 2.8

1.7 8.7 5.8

5.1 25.5 16.8

10.6

2.7

S1mP

1 2 3

43 20 32

37 8 8

6 12 24

14.0 60.0 75.0

10.0 18.7 24.3

1.3 3.2 1.8

7.5 12.4 20.8

12.5 24.9 27.8

17.6

2.1

S2mP

1 2 3

21 20 32

13 7 5

8 13 27

38.1 65.0 84.4

15.1 22.4 26.4

2.6 2.4 1.5

9.9 17.7 23.4

20.2 27.1 29.3

21.3

2.2

Treatment

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95% confidence limits

independently of source (stock), and pooling data is recommended (Srinivasan and Zhou 1993). In fact, the accuracy of our results was improved by analyzing all three stocks together. All of the data revealed that there was significantly higher final survival when juvenile fish were treated with probiotics. Hence, they confirm the promise of using these strains as probiotics in aquaculture. Vaccines to prevent vibriosis in juvenile Turbot produce survival rates ≥60% (www.hipra.com/english; Santos et al. 1991). Achieving an acceptable level of protection requires that the vaccine be administered at least 1 month prior to exposure to a pathogen (Santos et al. 1991; Wang et al. 2013). It is not surprising that vaccination protocols are sometimes inadequate due to poor form management (e.g., insufficient bath exposure, incorrect fish sizes, stressful handling, etc.). As a result, it is a common practice to use both vaccines and feed supplemented with antibiotics. The use of our probiotic strains in juvenile Turbot could be a way to achieve protection both immediately and over the course of the fish’ development which would make them

preferable to antibiotics. Interestingly, our probiotics showed a wide spectrum of antibacterial activity, which could be useful against other pathogens that harm Turbot and other marine organisms. Further studies will confirm whether the probiotic effect can be achieved in other Turbot growth stages as well as their practical value against other pathogens. Both probiotic strains persisted in the epidermal mucus of Turbot for 30 d. Moreover, S1 caused the exclusion of natural occurring bacteria there, at least during the early stages of exposure. Competitive exclusion is widely thought to be an interference mechanism used by probiotic (Jin et al. 1996; Lee et al. 2003; Chabrillon et al. 2005; Capkin and Altinok 2009; reviewed by Verschuere et al. 2000) and pathogenic bacteria (Vine et al. 2004; reviewed by Austin and Austin 2007). Vibrio anguillarum was previously shown to use the epidermal mucus as an access point to fish (reviewed by Austin and Austin 2007). Interestingly, we found exclusion of V. anguillarum only during the late stages of infection (16–30 d). However, V. anguillarum was unable to displace any of the probiotic strains. It was also

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INCREASED SURVIVAL OF JUVENILE TURBOT

unable to overcome the protective effect of the probiotics S1 and S2 in mixed challenges. Although our data are qualitative and the potential mechanism of action was not investigated, they provide strong evidence that the presence of probiotic bacteria impedes the steps necessary for V. anguillarum to cause disease in fish. In fact, the probiotic strains were applied 12 h before the pathogenic strain and are well adapted to the marine medium. This allows them to establish immediate protection. Therefore, our strains are good candidates for probiotics to be used with juvenile Turbot. In the mixed challenges, significant differences were found between the two probiotic strains with respect to their persistence in the epidermal mucus layer at specific times during the experiment. Furthermore, although the cumulative presence of S1 was significantly higher than that of the pathogenic strain, it was similar for S2 and the pathogenic strain. Naturally occurring bacteria were also significantly absent from the skin of sampled fish after challenge with S1 but were never excluded by S2. Fish previously treated with this strain also showed a significantly higher final survival rate than those treated with S1. These results suggest that the mechanisms by which the pathogen is blocked are different for the two strains. Pilot plant–scale experiments could be useful in determining the best strain (or combination of them) (Dag´a et al. 2013). No protection against vibriosis was found in any stock more than 30 d postchallenge with either probiotic strain. Thus, although the strains have the ability to persist in the epidermal mucus layer, the data suggest that the presence of S1 or S2 at that time is insufficient to protect fish against a new exposure to V. anguillarum. Since we tested only a single application of probiotics, additional treatments before the end of the 30-d period could be assayed to determine whether they prevent infection by V. anguillarum. Although oral administration of live probiotic cells could be more effective than adding such cells to the rearing water (Taoka et al. 2006), immediate protection is not expected. Despite this, incorporation of our strains into fish feed would also be essential to ensure the colonization of fish guts and achieve the highest protection. Our data prove the efficacy of using bacteria present in oysterrearing environments to establish the immediate protection of juvenile Turbot against infection by V. anguillarum. Moreover, epidermal mucus sampling was useful in investigating the persistence of both probiotic strains and their resistance to being excluded by the pathogen. ACKNOWLEDGMENTS This study was supported by grants 10MMA312017PR from the Xunta de Galicia (Regional Government of Galicia) and 09VIA11 from the Universidad de Vigo. We thank D. Costas and A. G. Villanueva for valuable comments, and the staff of ECIMAT for their helpful technical support. The authors acknowledge M. Reigosa for valuable comments on the statistical analysis.

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