魚病研究 Fish Pathology, 49 (1), 16–22, 2014. 3 © 2014 The Japanese Society of Fish Pathology

Research article First Isolation and Characterization of from Sea Bass Dicentrarchus labrax

Nuria Castro*, Sabela Balboa, Soledad Núñez, Alicia E. Toranzo and Beatriz Magariños

Department of Microbiology and Parasitology, Faculty of Biology-CIBUS & Institut of Aquaculture University of Santiago de Compostela, Santiago de Compostela, 15782, Spain

(Received November 22, 2013)

ABSTRACT—Tenacibaculum soleae is a recently described pathogen that has been reported as the causative agent of considerable losses in sole cultures in Spain. This report documents the first case of T. soleae as an etiological agent of tenacibaculosis in farmed sea bass Dicentrarchus labrax. Its identi- fication was performed employing phenotypical, serological and molecular methods. Although the sea bass isolates were homogeneous from a biochemical, chemotaxonomic and molecular point of view, they belonged to a serological group different from the type strain CECT7292, which can be of great impor- tance in the development of future vaccines and other methods of disease prevention in sea bass industry. Virulence assays with a representative isolate confirmed the pathogenic potential for sea bass. The disease was experimentally reproduced by prolonged bath, while no mortalities could be recorded by intraperitoneal injection. The results presented here show that T. soleae must be taken into account as an important pathogen in the marine aquaculture industry.

Key words: Tenacibaculum soleae, Dicentrarchus labrax, fish pathogen, aquaculture, virulence

Tenacibaculosis, a bacterial disease mainly caused reports are available at present for studying T. soleae: by is one of the most devas- the first description of the (Piñeiro-Vidal et al., tating infectious diseases of farmed marine finfish world- 2008) and a study in which serological and molecular wide (Avendaño-Herrera et al., 2006a). T. maritimum characterization of isolates from flatfish was performed has been described as the causative agent of gliding (López et al., 2010). Therefore, the number of cur- bacterial disease (or tenacibaculosis) in a great variety rently available isolates of T. soleae for these studies is of valuable marine fish species such as turbot very small, and limited only to strains isolated from Scophthalmus maximus, salmon Salmo salar and flatfish. For the better understanding of this species, Oncorhynchus kisutch, Senegalese sole Solea the isolation and characterization of new isolates from senegalensis, Dover sole Solea solea, gilthead other sources would be a great contribution to deter- seabream Sparus aurata, red seabream Pagrus major, mine their possible intraspecific diversity, from a pheno- black seabream Acanthopagrus schlegelii, sea bass typic, serological and molecular point of view. Dicentrarchus labrax and flounder Paralichthys Sea bass culture, along with the sea bream, repre- olivaceus (McVicar and White, 1979; Wakabayashi et sents the largest marine fish farming in Europe. Pas- al., 1984; Devesa et al., 1989; Bernardet et al., 1990; teurellosis (Photobacterium damselae subsp. piscicida Chen et al., 1995; Ostland et al., 1999; Santos et al., infection), vibriosis (Vibrio anguillarum infection) and 1999; Bader and Starliper, 2002; Avendaño-Herrera et tenacibaculosis (i.e. T. maritimum infection) are consid- al., 2004). In the last decade, the genus Tenacibaculum ered until date the most important bacterial diseases of has grown rapidly with the description of several new this industry (Toranzo et al., 2005). However, as hap- memb ers, being currently formed by 15 species. One pens with other fish cultures, once controlled these of them is Tenacibaculum soleae which has been pathologies by developing of prophylactic measures reported to be pathogenic for Senegalese sole, and/or suitable vaccination programs, other new dis- turbot, wedge sole Dicologoglossa cuneata and brill eases may emerge. In fact, recently a novel bacterial Scophthalmus rhombus (Piñeiro-Vidal et al., 2008; pathogen in sea bass cultures belonging to the genus López et al., 2010). To our knowledge, only two Tenacibaculum has been described, which has been termed as T. dicentrarchi (Piñeiro-Vidal et al., 2012). * Corresponding author The present study is the first description of T. E-mail: [email protected] soleae as the etiological agent of tenacibaculosis in cul- Tenacibaculum soleae isolated from sea bass 17 tured sea bass. Biochemical, chemotaxonomic and as previously described (Romalde et al., 2004). DNA molecular identification/characterization of the isolates from the T. soleae type strain CECT7292 was used as are presented. positive control and sterile water instead of DNA as negative control. PCR amplification and sequencing of the 16S rRNA Material and Methods gene were performed as previously described (Pascual Examination of diseased fish and isolation of et al., 2010). All PCR amplifications were carried out in During 2010, several episodes of mortalities a T Gradient thermocycler (Biometra), and the PCR (approximately 15%) occurred in sea bass reared in two products were electrophoresed in a 1.0% agarose gel different marine nurseries in Spain (average weight and stained with ethidium bromide (Bio-Rad). A 7–12 g). Water temperature was around 18–20°C FastRuler™ Low Range DNA Ladder (Fermentas) was and salinity 33‰. Examination of affected fish used as a molecular mass marker. Gels were revealed the presence of superficial lesions in the fish scanned and the images were captured by a Gel-Doc- body, tail and fins i.e. corroded and discoloured areas 2000 Gel Documentation System (Bio-Rad). on the skin. Internally, fish presented abundant ascitic Sequence data analysis was carried out with fluid, anemic liver and kidney with petechial haemorrhages. DNASTAR LasergeneA SEQM N program. Sequence With the aim to determine the causative agent of similarities of 16S rRNA gene were determined with the these mortalities, internal organs (liver and kidney) as EzTaxon-e server. Sequences were aligned with well as external lesions of diseased sea bass were those from related organisms and a phylogenetic tree streaked onto Tryptic Soy Agar suplemented with 1% was obtained according to the neighbour-joining method NaCl (TSA-1) (Difco Laboratoires), Thiosulphate Citrate (Saitou and Nei, 1987) and using the program MEGA Bile Sucrose (TCBS) Agar (Oxoid), Marine Agar (MA, version 5.05 (Tamura et al., 2011). The evolutionary Difco) and FMM (Flexibacter Maritimum Medium; Pazos distance matrix was generated according to Kimura’s et al., 1996). The agar plates were incubated at 25°C two parameter model (Kimura, 1980). Bootstrap value for 24 to 72 h. The isolates were maintained frozen at was 1,000. –70°C in Criobille tubes (AES Laboratory). Determination of the fatty acid methyl ester composition Phenotypic characterization For analysis of fatty acid methyl esters (FAMEs), The isolates were characterized employing pheno- the isolates were grown on MA plates at 25°C for 48 h. typic tests (Suzuki et al., 2001). The presence of FAMEs extraction and GC analysis were performed fol- flexirubin-type pigments was determined by using the lowing the procedures of the Microbial Identification KOH test as described by Reichenbach (1989). Other System (MIDI, Microbial ID Inc.) (Sasser, 1990) and enzymatic activities were evaluated with the API ZYM using an Agilent 6890 gas chromatographic station system (BioMérieux) following the manufacturer’s (Agilent Technologies) equipped with split injector, instructions, except that sterile seawater was used as flame ionization detector and a fused silica column (30 the suspension medium and 25°C as incubation m × 0.2 mm × 0.33 mm). The operating systems temperature. employed were ChemStation (Agilent) and Sherlock The drug sensitivities of the isolates were deter- MIS (MIDI). FAME profiles obtained from the sea bass mined by the disc diffusion method on MA following the isolates were compared with that obtained from the type procedures of the Clinical Laboratory Standards Insti- strain of T. soleae CECT7292 under the same conditions. tute (CLSI, 2006). The following chemotherapeutic agents (micrograms per disc) were used: flumequine Serological analysis (30), florfenicol (30), enrofloxacin (5), oxytetracycline Serological identification was conducted by slide (30), ampicillin (10), doxycycline (30), erythromycin (5) agglutination test as previously described (Toranzo et and trimethoprim-sulfamethoxazole (23.75/1.25). The al., 1987) employing the thermostable O-antigen from MA plates were incubated at 25°C for 48 h prior to each isolate which was obtained by heating the bacte- determining the resistance/sensitivity of the isolates. rial suspension (10% v/v in PBS) at 100°C for 1 h. Specific polyclonal antiserum raised against the T. 16S rRNA gene sequencing and phylogenetic analysis soleae type strain CECT7292 was prepared by intrave- Chromosomal DNA of bacterial cultures was nous injection of New Zealand rabbits with formalin- obtained employing Insta-Gene Matrix (Bio-Rad) and killed cells according to the procedure described by subjected to specific PCR with the two pairs of primers Sørensen and Larsen (1986). Only a strong and rapid targeting the 16S rRNA gene of T. soleae: Sol-FW/Sol- agglutination was registered as a positive result. Rv and G47F/G47R (García-González et al., 2011; Moreover, dot blot assay was performed as López et al., 2011). PCR reactions were performed described by Cipriano et al. (1985). Only a reaction using Ready-To-Go PCR Beads (Pharmacia Biotech) similar to that exhibited by the homologous strain was 18N. Castro, S. Balboa, S. Núñez, A. E. Toranzo and B. Magariños recorded as positive. MA and FMM plates from internal organs of all exam- ined fish from both nurseries. No growth was Virulence assays observed on TSA-1 or TCBS plates. Six representa- Evaluation of the virulence of the isolates was per- tive isolates were subjected to biochemical, serological, formed using healthy tenacibaculosis-free juvenile sea chemotaxonomic and molecular characterization in bass (weighing 10–12 g) obtained from an experimental order to identify the causative agent of the mortalities. hatchery with no history of tenacibaculosis problems, Biochemical characterization of the isolates located in North-West Spain. In addition, to ensure showed that all were Gram-negative and oxidase and that the fish were uninfected with Tenacibaculum spp., catalase positive. Results obtained from plate and fish were subjected to standard bacteriological tube tests as well as API ZYM system are shown in examination. Groups of 10 fish were maintained in Table 1. These results were homogeneous among six plastic tanks with aerated sea water, and acclimatized isolates examined and matched with those of T. soleae for 48 h prior to bacterial challenge. The representa- type strain CECT7292 except for some differences in tive T. soleae isolate, designated as strain TS21–10, the enzymatic activity. Therefore, the isolates were from sea bass was selected for the experimental presumptively identified as T. soleae. Morphological, infections. Two types of challenge were developed: physiological and biochemical characteristics of the iso- Injection challenge: Duplicate groups of 10 sea lates are in agreement with previous descriptions of the bass were challenged by intraperitoneal injection (i.p.) pathogen (Piñeiro-Vidal et al., 2008; López et al., 2010). with 0.1 mL of 10-fold dilutions of T. soleae ranging from All the isolates showed a similar drug susceptibility 6.9 × 105 to 6.9 × 108 cells/mL. Two tanks with fish pattern, being sensitive to florfenicol and trimethoprim- injected with sterile saline solution (SS) were included sulfamethoxazole, intermediate to enrofloxacin, erythro- as controls. mycin and flumequine and resistant to oxytetracycline, Bath challenge: Duplicate groups of 10 sea bass ampicillin and doxycycline. To our knowledge, only were bath challenged in sea water following the proce- López et al. (2010) published the results of drug suscep- dures of Avendaño-Herrera et al., (2006b) with different tibility patterns of T. soleae isolates from wedge sole dilutions of T. soleae to obtain final concentrations rang- and brill, where the majority of their isolates were sensi- ing from 1.4 × 106 to 1.4 × 108 cells/mL. After an 18-h tive to novobiocin (30 m g) and ampicillin (10 m g). exposure, the water was changed and fish were main- In the specific PCR amplification with the two tained in the same tank. Control consisted of two primer sets, all the sea bass isolates and the reference tanks in which the same amount of SS was added to strain gave the characteristic 1,555 bp and 248 bp the sea water. bands respectively, confirming their identification as T. All trials were maintained in a closed system with soleae (Fig. 1). No PCR products were obtained in the sea water for up to 21 days at 18–20°C with pH from negative controls. 7.8 to 8.2, and salinity 35‰, with a 16L:8D light regime. The results of the phylogenetic analysis based on The fish were maintained in tanks of 50 L and fed daily the 16S rRNA gene clearly showed that the six strains at 1.5% body weight, and the water in each tank was isolated from sea bass and characterized in the present changed once every 2 days to remove faecal matter. study belonged to the species T. soleae. A phyloge- Sea bass were examined daily and dead or mori- netic tree derived from these sequences illustrates that bund fish were removed and analysed to confirm if the the sea bass isolates were grouped in a unique robust inoculated T. soleae strain was the cause of mortality. monophyletic branch with a bootstrap value of 100% The kidney and external lesions were streaked onto MA together with the T. soleae type strain (Fig. 2). Moreo- plates and incubated at 25°C for 72 h. Bacterial colo- ver, these sequences were clearly differentiated from nies were identified as described above. The lethal other Tenacibaculum species, grouping in a separate dose that killed 50% of challenged fish (LD50) was calcu- cluster. The 16S rRNA gene sequences similarities lated according to the method of Reed and Müench were in all cases between 99.16–99.44% values with (1938). the T. soleae type strain CECT7292. The similarities with other closely related species were consistently lower than 96.5%. Results and Discussion The fatty acid profiles obtained for the six sea bass In the present work, we report the first description isolates showed only very little variations among them of sea bass as a new host for T. soleae. The phenotypic and were quite similar to the pattern obtained for the T. and genetic characteristics of six isolates recovered soleae type strain (Table 2). The cellular fatty acid pro- from this fish species are described. files were characterized by the presence of large After 24 h incubation, large amounts of bacterial amounts of five main fatty acids: iso-C15:0, C15:1w6c, growth consisting of single type yellow-pigmented colo- iso-C15:03-OH, summed feature three (a component nies which did not adhere to the agar were obtained in that contains C16:1w7c and/or C16:1w6c) and iso- Tenacibaculum soleae isolated from sea bass 19

Table 1. Phenotypic characteristics of T. soleae isolates from sea bass with reference of the T. soleae type strain CECT7292T Sea bass Sea bass Test isolates CECT7292T API ZYM Test isolates CECT7292T (n = 6) (n = 6) Colony colour by by Gram – – Alkaline phosphatase + + Gliding motility + + Esterase (C4) + + Oxidase + + Esterase lipase (C8) – + Catalase + + Lipase (C14) + + O/F O O Leucine arylamidase + + Congo Red + + Valine arylamidase + + Flexirubin – – Cystine arylamidase + + Nitrate reduction + + Trypsin – – Hydrolysis of a -chymotrypsin – – Gelatin + + Acid phosphatase + – Esculin – – Phosphohydrolase + – Starch – – a -galactosidase – – Tween 80 – – b -galactosidase – – Growth at b -glucuronidase – – 4°C – – a -glucosidase – – 15°C + + b -glucosidase – – 25°C + + N-acetyl-b -glucosaminidase – – 37°C – – a -mannosidase – – Growth with a -fucosidase – – Only NaCl – – API ZYM reaction scores 0 and 1 were considered negative; scores 2 to 5 were considered positive. by, bright yellow; +, positive; –, negative; O, oxidative.

248 bp

1,555 bp

Fig. 1. 16S rRNA PCR amplification with specific T. soleae primers Sol-FW/Sol-Rv (a) and G47F/G47R (b). Lanes M: 50 to 1,500 bp molecular weight marker FastRulerTM Low Range DNA Ladder (Fermentas); Lane 1: T. soleae CECT7292T; Lane 2 to 7: T. soleae isolates from sea bass; Lane 8: negative control. 20N. Castro, S. Balboa, S. Núñez, A. E. Toranzo and B. Magariños

Fig. 2. Neighbour-joining phylogenetic tree based on 16S rDNA sequences showing the relationships between T. soleae isolates from sea bass, T. soleae type strain and other members of the genus Tenacibaculum. The Flavobacterium psychrophilum sequence was used as an outgroup. The numbers at the nodes indicate the levels of bootstrap support based on 1,000 replicates. Sequences from relative species were obtained fro m GenBank-EMBL data base. Accession numbers are indi- cated in brackets.

C16:03-OH (Table 2). This composition is in general gested by Lopez et al. (2010). agreement with the data previously reported for this bac- The strain TS21–10 selected for experimental terial species by Piñeiro-Vidal et al. (2008) and López et infections caused significant mortalities in sea bass only al., (2010), except for the absence of C15:0 and the when fish were bath-challenged. By this route, the presence of one (summed feature SF1) of fatty acid lethal dose 50 was 1.4 × 107 cells/mL, recording mortali- groups that cannot be separated by GLC using the MIDI ties between 8–15 days postinfection. Mortalities system (Table 2). As pointed out by Jantzen and were 40%, 50% and 60% when fish were inoculated Lassen (1980), this could be explained by the fact that with 1.4 × 106, 1.4 × 107 and 1.4 × 108 cells/mL, the biosynthesis of cyclopropane fatty acids is much respectively. The inoculated T. soleae was recovered dependent on the growth stage and the culture medium from the internal organs of all dead fish which showed of the bacterial populations. haemorrhages in the ventral surface and fins. How- Serological characterization using slide agglutina- ever, by the intraperitoneal route, no mortalities were tion and dot blot assays revealed that none of the sea recorded during 15 days postinfection, regardless of the bass isolates reacted with the antiserum raised to the T. doses used. None of the control fish died in the differ- soleae type strain. Only the O-antigen obtained from ent assays. These findings could be in agreement with the homologous strain showed positive reactions. previous studies in other Tenacibaculum species (T. These findings reveal the existence of antigenic variabil- maritimum), which reported that the disease was not ity among T. soleae and confirm the existence of more induced by intraperitoneal or intramuscular injection than one serotype in this species as previously sug- (Avendaño-Herrera et al., 2006b). According to our Tenacibaculum soleae isolated from sea bass 21

Table 2. Cellular fatty acid compositions (%) of sea bass iso- (2006a): Tenacibaculosis infection in marine fish caused T lates and the type strain of T. soleae CECT7292 by Tenacibaculum maritimum: a review. Dis. Aquat. T Org., 71, 255–266. Fatty acid Sea bass isolates CECT7292 Avendaño-Herrera, R., A. E. Toranzo and B. Magariños Straight chain (2006b): A challenge model for Tenacibaculum maritimum C15 : 0 – 4.8 infection in turbot, Scophthalmus maximus (L.). J. Fish

C16 : 0 tr tr Dis., 29, 371–374. Branched chain Bader, J. A. and C. E. Starliper (2002): The genera Flavobacterium and Flexibacter. In “Molecular diagnosis of salmonid dis- iso-C 1.06–1.26 tr 13 : 0 eases” (ed. by C. O. Cunninghan). Kluwer Academic iso-C14 : 0 2.82–3.41 2.4 Publishers, Dordrecht, pp. 99–139. iso-C15 : 0 25.73–26.18 23.1 Baxa, D. V., K. Kawai and R. Kusuda (1987): Experimental

anteiso-C15 : 0 1.06–1.08 tr infection of Flexibacter maritimus in black sea bream (Acanthopagrus schlegeli) fry. Fish Pathol., 22, 105– iso-C15 : 1 4.05–6.92 5.7 109. iso-C 1.45–1.59 1.7 16 : 0 Bernardet, J. F., A. C. Campbell and J. A. Buswell (1990): iso-C16 : 1 2.34–2.93 2.4 Flexibacter maritimus is the agent of “black patch necro- iso-C17 : 1w9c – − sis” in Dover sole in Scotland. Dis. Aquat. Org., 8, 233–237. Unsaturated Bernardet, J. F., B. Kerouault and C. Michel (1994): Compara- tive study on Flexibacter maritimus strains isolated from C15 : 1w6c 14.51–15.91 12.2 farmed sea bass (Dicentrarchus labrax) in France. Fish C w6c 1.53–1.73 1.7 17 : 1 Pathol., 29, 105–111. C18 : 3w6c – − Clinical and Laboratory Standards Institute (CLSI) (2006): Meth- Hydroxylated ods for antimicrobial disk susceptibility testing of bacteria

iso–C15 : 0 3-OH 11.87–12.25 10.6 isolated fro m aquatic animals; Approved Guideline. CLSI document M42-A. Clinical and Laboratory Standards C15 : 0 2-OH tr tr Institute, 940, West Valley Road, Suite 1400, Wayne, C 3-OH 3.55–4.65 3.2 15 : 0 Pennsylvania, 19087-1898, USA1-56238-612-3. iso-C16 : 0 3-OH 8.56–9.00 8.4 Chen, M. E., D. Henry-Ford and J. M. Groff (1995): Isolation C16 : 0 3-OH 1.05–1.11 2.2 and characterization of Flexibacter maritimus from marine

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