Biochemical, pathogenical, antigenical, and molecular characteristics of Vibrio alginolyticus iso- lates from diseased cultured gilthead sea bream juveniles (4 and 20 g) were determined by lab- oratory challenge experiments. Intraperitonal challenge resulted in development of the disease but immersion did not. Electrophoresis of outer membrane protein (OMP) and lipopolysaccha- rides (LPS) was performed using sodium dodecyl sulphate poly-acrylamide gel electrophoresis (SDS-PAGE). Molecular typing of Vibrio isolates was conducted using random amplification of polymorphic DNA (RAPD). According to SDS-PAGE of surface antigens of the isolates, profiles of both OMP and LPS were heterogeneous. According to RAPD, isolated bacteria varied genet- ically. The findings reveal differences among V. alginolyticus isolates from diseased cultured gilt- head sea bream in Turkey

Akayli, T.

Albayrak, G.

Arican, E.

Aydemir, B.

Coskun, O.

Kiziler, A.R.

Timur, G.

Biochemical, pathogenical, antigenical, and molecular characteristics of Vibrio alginolyticus isolates from diseased cultured gilthead sea bream juveniles (4 and 20 g) were determined by laboratory challenge experiments. Intraperitonal challenge resulted in development of the disease but immersion did not. Electrophoresis of outer membrane protein (OMP) and lipopolysaccharides (LPS) was performed using sodium dodecyl sulphate poly-acrylamide gel electrophoresis (SIDS-PAGE). Molecular typing of Vibrio isolates was conducted using random amplification of polymorphic DNA (RAPD). According to SDS-PAGE of surface antigens of the isolates, profiles of both OMP and LIPS were heterogeneous. According to RAPID, isolated bacteria varied genetically. The findings reveal differences among V. alginolyticus isolates from diseased cultured gilthead sea bream in Turkey

Kiziler, A. R.

Coşkun, Oğuz Kağan

İstanbul Üniversitesi Açık Erişim Sistemi

Characterization of Vibrio alginolyticus isolates from diseased cultured gilthead sea bream, Sparus aurata

eVols at University of Hawaii at Manoa

The Open Access Israeli Journal of Aquaculture – BamidgehAs from January 2010 The Israeli Journal of Aquaculture - Bamidgeh (IJA) will be published  exclusively  as  an  on-line  Open  Access  (OA) quarterly  accessible  by  all AquacultureHub  (http://www.aquaculturehub.org)  members  and  registered  individuals and institutions.  Please visit our website (http://siamb.org.il) for free registration form, further information and instructions.  This transformation from a subscription printed version to an on-line OA journal, aims at supporting the concept that scientific peer-reviewed publications should be made available to all, including those with limited resources. The OA IJA does not enforce author or subscription fees and will endeavor to obtain alternative sources of income to support this policy for as long as possible.Editor-in-ChiefDan Mires Editorial BoardSheenan Harpaz Agricultural Research OrganizationBeit Dagan, IsraelZvi Yaron Dept. of ZoologyTel Aviv UniversityTel Aviv, IsraelAngelo Colorni National Center for Mariculture, IOLREilat, IsraelRina Chakrabarti Aqua Research LabDept. of ZoologyUniversity of DelhiIngrid Lupatsch Swansea UniversitySingleton Park, Swansea, UKJaap van Rijn The Hebrew University Faculty of AgricultureIsraelSpencer Malecha Dept. of Human Nutrition, Food and Animal SciencesUniversity of HawaiiDaniel Golani The Hebrew University of JerusalemJerusalem, IsraelEmilio Tibaldi Udine UniversityUdine, ItalyCopy Editor Ellen Rosenberg Published under auspices ofThe Society of Israeli Aquaculture and Marine Biotechnology (SIAMB), University of Hawaii at Manoa Library andUniversity of Hawaii Aquaculture Program in association withAquacultureHub http://www.aquaculturehub.org              ISSN 0792 - 156X Israeli Journal of Aquaculture - BAMIGDEH.PUBLISHER: Israeli Journal of Aquaculture - BAMIGDEH -Kibbutz Ein Hamifratz, Mobile Post 25210, ISRAELPhone: + 972 52 3965809http://siamb.org.il The Israeli Journal of Aquaculture – Bamidgeh  60(2), 2008, 89-94. 89Characterization of Vibrio alginolyticus Isolates fromDiseased Cultured Gilthead Sea Bream, Sparus aurataT. Akayli1*, G. Timur1, B. Aydemir2, A.R. Kiziler2, O. Coskun3, G. Albayrak4, and E. Arican41 Department of Fish Diseases, Fisheries Faculty, University of Istanbul, Istanbul, Turkey2 Department of Biophysics, Cerrahpasa Faculty, University of Istanbul, Istanbul, Turkey3 Department of Biophysics, Istanbul Faculty, University of Istanbul, Istanbul, Turkey4 Molecular Biology and Genetic Department, Science Faculty, University of Istanbul, Istanbul, Turkey(Received 26.8.07, Accepted 15.1.08)Key words: gilthead sea bream, Vibrio alginolyticus, electrophoresis, RAPDAbstractBiochemical, pathogenical, antigenical, and molecular characteristics of Vibrio alginolyticus iso-lates from diseased cultured gilthead sea bream juveniles (4 and 20 g) were determined by lab-oratory challenge experiments. Intraperitonal challenge resulted in development of the diseasebut immersion did not. Electrophoresis of outer membrane protein (OMP) and lipopolysaccha-rides (LPS) was performed using sodium dodecyl sulphate poly-acrylamide gel electrophoresis(SDS-PAGE). Molecular typing of Vibrio isolates was conducted using random amplification ofpolymorphic DNA (RAPD). According to SDS-PAGE of surface antigens of the isolates, profilesof both OMP and LPS were heterogeneous. According to RAPD, isolated bacteria varied genet-ically. The findings reveal differences among V. alginolyticus isolates from diseased cultured gilt-head sea bream in Turkey.IntroductionVibriosis is the most significant disease of cul-tured sea bream in the Mediterranean (Colorniet al., 1981; Paperna, 1984; Balebona et al.,1998a) and Aegean (Akayli and Timur, 2002,2004) areas. The widely distributed Vibrioalginolyticus is an important pathogen thatcauses severe vibriosis in cultured giltheadsea bream, Sparus aurata L. (Colorni et al.,1981; Balebona et al., 1998a; Zorrilla et al.,2003). However, many strains of V. alginolyti-cus are avirulent and could be used as probi-otic strains (Austin et al., 1995). As wide vari-ation exists among the strains, differentiatingbetween harmful and beneficial strains wouldhelp to find ways to control infection.Biochemical characteristics and outermembrane protein (OMP) profiling are valu-able methods for typing and differentiating*  Corresponding author. E-mail: takayli@yahoo.com90between bacterial fish pathogens.Lipopolysaccharides (LPS) and OMP arethought to relate to antigenic determinants ingram-negative bacteria (Zorrilla et al., 2003). Specific, sensitive, and rapid methods fordetecting and identifying pathogenic microor-ganisms are needed to control bacterial infec-tions in intensive fish culture. Random ampli-fication of polymorphic DNA (RAPD) can beused to compare microorganisms at the inter-species and intraspecies levels with high dis-crimination (Welsh and McClelland, 1990).RAPD has been used for intraspecies charac-terization of Vibrio harveyi (Pujalte et al.,2003), V. parahaemolyticus (Najiah et al.,2003), and V. vulnificus (Gutacker et al.,2003) isolated from diseased fish. The few reports on V. alginolyticus infec-tion in cultured gilthead sea bream in Turkeyfocus on the isolation and identification of theetiological agent (Akayli and Timur, 2002,2004). Some studies concentrate on theintraspecific characterization of V. alginolyti-cus strains isolated from cultured sea bream(Balebona et al., 1998b; Zorrilla et al., 2003).However, no attention has been given to themolecular characterization of the bacteria. Inthe present work, we applied several typingmethods to twelve strains of V. alginolyticusisolated from different epizootic outbreaksaffecting cultured gilthead sea bream inTurkey and compared those strains with fivereference strains.Materials and MethodsBacteria. Bacteria were isolated from giltheadsea bream showing clinical signs of vibriosis.The fish were collected from different farmsalong the southwestern coast of Turkey. Thebiochemical, pathogenical, antigenical, andmolecular properties of twelve bacteria iso-lates were examined and compared to fiverepresentative reference strains (Table 1).Biochemical analyses. The isolates wererecovered on TSA (T-TSA) and TSB (T-TSB)supplemented with 1% NaCI for 2 days at22°C. The physiological and biochemicalcharacterization of the isolates and the refer-ence strains was carried out using conven-tional bacteriological methods (Alsina andBlanch, 1994; Holt et al., 1994) and API 20Eand API 50CH test kits (MacDonell et al.,1982). Phenotypes of the isolates were com-pared using approximately 40 morphological,phenotypical, and biochemical tests describedby Alsina and Blanch (1994), Holt et al.(1994), and Austin and Austin (1999).Pathogenicity assay. Four isolates weretested for pathogenicity in sea bream juve-niles. For each isolate, two sizes of 30 fisheach were challenged. The first size (avg 4 g)was challenged by bacterial suspension in abath with approximately 107 cells/ml for 60min. The second (avg 20 g) was injectedintraperitonally with the same bacterial dose.There were two replicates of each challenge,a total of 480 fish.Electrophoresis. Outer membrane protein(OMP) and lipopolysaccharides (LPS) profilesof four of the twelve Turkish isolates wereanalyzed by electrophoresis using sodiumdodecyl sulphate poly-acrylamide gel elec-trophoresis (SDS-PAGE) in a Mini-Protein IIunit (BioRad). OMP and LPS from bacteriacultures of the isolates were obtained in tryp-tic soy broth (TSB) supplemented with 1.5%NaCI (TSAS) after 24 h incubation at 22°C.Akayli et al.Bacteria SourceV. alginolyticus CECT 521aV. damsela CECT 626a(=Photobacterium damselae)V. ordalii CECT 582aV. anguillarum (=Listonella anguillarum) serotype 01 IMRbV. anguillarumserotype 02 IMRba Prof. Dr. Federico Uruburu, Director,Spanish Type Culture Collection, Burjassot,Valencia, Spainb Prof. Dr. Sigmund Jensen; Institute ofMarine Research, Bergen, NorwayTable1. Sources of reference strains usedin this study.LPS samples were prepared by Proteinase Kdigestion of whole-cell lysates as described byHitchcock and Brown (1983). They were frac-tionated by 18% SDS-PAGE (Laemmli, 1970)and visualized with Silver Stain Plus (Merck-159437) following the manufacturer’s instruc-tions. OMP was determined using the methoddescribed by Arda and Ertan (2004). Thesamples were separated by 12.5% SDS-PAGE (Laemmli, 1970). The gels were fixedand stained with 0.1% Coomassie brilliantblue (Merck-112553).Random amplification of polymorphicDNA. RAPD was used to characterize the bac-teria to the genotype level. Genomic DNA wasextracted using the High Pure PCR TemplatePreparation Kit (Roche Cat. No.1796828). Thereaction procedure was modified fromSudheesh et al. (2002), Valle et al. (2002), andNajiah et al. (2003). Two randomly designed10-mer oligonucleotide primers (OperonTechnologies CA/USA), OPC-05 (5’ GAT-GACCGCC 3’) and OPC-19 (5’GTTGCCAGCC 3’), were used for amplifica-tion. These primers were preliminarily tested inPCR cycles at different temperatures. PCRreactions were run in a PHC-3 thermocycler(Techno, Princeton, N.J.), using the followingprogram: denaturation step at 94ºC for 5 min,45 cycles consisting of 94ºC for 1 min, 34ºCfor 1 min, and 72ºC for 2 min, and a final cycleconsisting of 72ºC for 15 min. The RAPDproducts were electrophoresized using aFisher Biotech Small Horizontal Gel System.ResultsBiochemical analyses. All the isolates sharedthe same general characteristics and wereclassified as typical V. alginolyticus (Table 2).All had gram-negative rods and were motile,sensitive to vibriostat (O/129), cytochromeoxidase positive, and fermentative by O/F glu-cose test. They all produced acid fromsucrose, grew at 37°C, and swarming growthdeveloped on the surface of the solid medium(TSA). Some isolates responded negatively toornithine decarboxylase, indol production, andcitrate reaction.Pathogenicity assay. The bath challengedid not lead to development of the diseasewhile intraperitonal injection did cause vibrio-sis. Infected fish were moribund seven daysafter injection. Their skin was dark with super-ficial lesions and their abdomens wereswollen with ascetic fluid.Electrophoresis. SDS-PAGE analysis of thefour isolates showed that the structures of LPSand OMP were heterogeneous (Fig. 1). Themajor OMP had molecular masses ranging 33-37 kDa while LPS bands ranged 20-34 kDa.Random amplification of polymorphicDNA. Using two primers, the PCR amplifica-tion technique successfully differentiatedbetween the twelve Vibrio strains, as validat-ed by RAPD. The RAPD-PCR profiles of thefour strains showed genetic variation (Fig. 2).DiscussionThe phenotypic identification of genera andspecies of Vibrionaceae is problematic, main-ly because of great variability of diagnosticphenotypic features such as arginine dihydro-lase, indole production, and carbon utilization(Austin and Lee, 1992; Austin et al., 1997).Our isolates had similar biochemical profilesand were all typed as V. alginolyticus. Therewas a close relationship between our V. algi-nolyticus isolates and those described byAlsina and Blanch (1994), Holt et al. (1994),and Balebona et al. (1998a). Some of ourstrains responded negatively to ornithinedecarboxylase, indol production, and citratereaction. In general, however, our resultsshowed that the biochemical profile allows alow degree of discrimination among isolates.Our pathogenicity results were similar tothose of Balebona et al. (1998ab) and  Zorrilaet al. (2003), i.e., V. alginolyticus is an impor-tant pathogen for gilthead sea bream. WhilePaperna (1984) isolated strains of V. algi-nolyticus from this fish, he did not performpathogenicity studies.There were several differences in OMPprofiles, as reported by Zorrilla et al. (2003).Likewise, the LPS profiles were heteroge-neous. Although we have no other reports onLPS profiles with which to compare, we con-clude that SDS-PAGE and silver staining ofpurified LPS is a useful tool for determiningantigenical properties of isolates.91Vibrio alginolyticus isolates from gilthead sea bream92 Akayli et al.Table 2. Selected biochemical characteristics of twelve Vibrio alginolyticus isolates and onereference strain.+ = possesses characteristic, - = lacks characteristic, v = variable resultsCharacteristic Isolate no. Reference1 2 3 4 5 6 7 8 9 10 11 12 CECT 521Swarming colonies + + + + + + + + + + + + +H2S + + + v + + + + + + + + +Arginine dihydrolase - - - - - - - - - - - - -Lysine decarboxylase + + + + + v + + + + + + +Ornithine decarboxylase + - + - + - - + + - + + +Indole + - + + - - + + + - + + +Hydrolysis of aesculine - - - - + - - - - - - - -Methyl red test + + + + + + + + + + + + +Voges-Proskuer + + + + + + + + + + + + +Citrate + + + - + + - + + + - + +Urea + + + + + + v + + + + + +Acid from salicin + v + + + + + + + + + + -Growth on 0% (w/v) sodium chloride - - - - - - - - - - - - -Growth on 7% (w/v) sodium chloride + + + + + + + + + + + + +Fig. 1. SDS-PAGE of (a) outer membrane protein (OMP) and (b) lipopolysaccharides (LPS) from fourVibrio alginolyticus strains isolated from diseased gilthead sea bream cultured in Turkey.452929a bVarying genetic profiles were obtainedfrom RAPD analysis in our study, similar tothe findings of Sudheesh et al. (2002) for V.alginolyticus isolates from cultured shrimp.Similarly, there were genetic variations in V.harveyi (Pujalte et al., 2003), V. para-haemolyticus (Najiah et al., 2003), and V. vul-nificus (Gutacker et al., 2003) isolated fromdiseased fish.In conclusion, the isolates of V. alginolyti-cus were phenotypically and antigenicallysimilar, but molecularly heterogeneous.RAPD was the most discriminating typingmethod, although biochemical and antigenicalprofiles were complementary for intraspecificcharacterization of V. alginolyticus isolates ingilthead sea bream.AcknowledgementsThis study was supported by the IstanbulUniversity Research Fund (project no.51/230120003). We are grateful to Dr.Sigmund Jensen and Dr. Federico Uruburu,who generously provided the bacterial strains.ReferencesAkayli T. and G. Timur, 2002. Vibriosis in thecultured sea bream (Sparus aurata L. 1758)farms at the Aegean sea coast of Turkey andaffecting factors at outbreak of the diseases.Turk. J. Fish. Aquat. Sci., 1(2):289-291.Akayli T. and G. Timur, 2004. A diagnosticstudy on vibriosis cultured gilt-head seabream (S. aurata) in the Aegean Sea coastfarms of Turkey. I.U. Su U. Der., 18:43-54. Alsina M. and A.R. Blanch, 1994.Improvement and update of a set of keys forbiochemical identification of environmentalVibrio species. J. Appl. Bacteriol., 77:719-721. Arda N. and H. Ertan, 2004. MolekulerBiyolojide Kullanilan Yontemler. I.U.Biyoteknoloji ve Genetik Muh. Arastirma veUygulama Merkezi (BIYOGEM). G. Temizkan,N. Arda (eds.). Nobel Tip Kitapevi. pp. 161-230.Austin B. and J.V. Lee, 1992. Aeromona-daceae and Vibrionaceae. pp. 163-182. In:R.G. Board, D. Jones, F.A. Skinner (eds.).Identification Methods in Applied and93Vibrio alginolyticus isolates from gilthead sea breamFig. 2. RAPD-PCR profiles of four strains of Vibrio alginolyticus (lanes 1-4) generated after amplificationwith primers (a) OPC-05 and (b) OPC-19, compared to a DNA molecular mass marker, λDNA/EcoRI+HindIII(lane M).a bM 1 2 3 4 M 1 2 3 4353020271375947bp94Environmental Microbiology. Blackwell Sci.Publ., Oxford, UK.Austin B. and D.A. Austin, 1999.Vibrionaceae representatives. pp. 102-118.In: Bacterial Fish Pathogens: Diseases ofFarmed and Wild Fish, 3rd ed. Praxis Publ.,Chichester. Austin B., Stuckey L.F., Robertson A.W.,Effendi I. and D.R.W. Griffith, 1995. A probi-otic strain of Vibrio alginolyticus effective inreducing diseases caused by Aeromonassalmonicida, Vibrio anguillarum and Vibrioordalii. J. Fish Dis., 18(1):93-96. Austin B., Austin D.A., Blanch A.R., CerdaM., Grimont P.A.D., Jofre J., Koblavi S.,Larsen J.L., Pedersen K, Tiainen T.,Verdonck L. and J. Swings, 1997. A com-parison of methods for the typing of fish-path-ogenic Vibrio spp. Syst. Appl. Microbiol.,20:89-101.Balebona M.C., Zorrilla I., Morinigo A. andJ.J. Borrego, 1998a. Survey of bacterialpathologies affecting farmed gilt-head seabream (Sparus aurata L.) in southwesternSpain from 1990 to 1996. Aquaculture,166:19-35.Balebona M.C., Andreu M.J., Bordas M.A.,Zorrilla I., Morinigo M.A. and J.J. Borrego,1998b. Pathogenicity of Vibrio alginolyticus forcultured gilt-head sea bream (Sparus aurataL.). Appl. Environ. Microbiol., 64(11): 4269-4275. Colorni A., Paperna I. and H. Gordin, 1981.Bacterial infections in gilt-head sea breamSparus aurata cultured at Eilat. Aquaculture,23:25-267.Gutacker M., Conza N., Benagli C., PedroliA., Bernasconi M.V., Permin L., Aznar R.and J.C. Piffaretti, 2003. Population geneticsof Vibrio vulnificus: identification of two divi-sions and a distinct eel-pathogenic clone.Appl. Environ. Microbiol., 69(6):3203-3212.Hitchcock P.J. and T.M. Brown, 1983.Morphological heterogeneity among Salmo-nella lipopolysaccaride chemotypes in silver-stained polyacrylamide gels. J. Bacteriol.,154:269-277.Holt J.G., Krieg N.R., Sneath A.P., StaleyJ.T. and S. Williams, 1994. Groups 5 faculta-tively anaerobic gram-negative rods, sub-group 2: Vibrionaceae. pp. 175-290. In: W.R.Hensly (ed.). Bergey’s Manual ofDeterminative Bacteriology, 9th ed. Williamsand Wilkins, Waverly Co., Baltimore.Laemmli U.K., 1970. Cleavage of structuralproteins during the assembly of the head ofbacteriophage T4. Nature, 227:680-688.MacDonell M.T., Singleton F.L. and M.A.Hood, 1982. Diluent composition for use ofAPI 20E in characterizing marine and estuar-ine bacteria. Appl. Environ. Microbiol., 44(2):423-427.Najiah M., Lee K.L., Hassan M.D., Shariff M.and M.L. Mohd-Azmi, 2003. Preliminarystudy on genetic distance of Vibrio para-haemolyticus isolates from diseased fish andshrimp brackish water ponds by randomamplified polymorphic DNA (RAPD) inMalaysia. Asian Fish. Sci., 16:299-305.Paperna I., 1984. Review of diseases affect-ing cultured Sparus aurata and Dicentrarchuslabrax. pp. 465-482. In: G. Barnabe, R. Billard(eds.). L’aquaculture du bar et des sparides.INRA Publ., Paris, France. Pujalte M.J., Sitja-Bobadilla A., MacianM.C., Belloch C., Alveroz-Pellietro P.,Perez-Sanchez J., Uruburu F. and E.Garay, 2003. Virulence and molecular typingof Vibrio harveyi strains isolated from cultureddentex, gilthead sea bream and Europeansea bass. System. Appl. Microbiol., 26:284-292.Sudheesh P.S., Jie K. and H.S. Xu, 2002.Random amplified polymorphic DNA-PCRtyping of Vibrio parahaemolyticus and V. algi-nolyticus isolated from cultured shrimps.Aquaculture, 207:11-17. Valle L.D., Zanella L., Belvedere P. and L.Colombo, 2002. Use of random amplificationto develop a PCR detection method for thecausative agent of fish pasteurellosis,Photobacterium damselae subsp. piscicida(vibrionaceae). Aquaculture, 207:187-202Welsh J. and M. McClelland, 1990.Fingerprinting genomes using PCR with arbi-trary primers. Nucleic Acids Res., 18:7213-7218.Zorrilla I., Morinigo M.A., Castro D.,Balebona M.C. and J.J. Borrego, 2003.Intraspecific characterization of Vibrio algi-nolyticus isolates recovered from cultured fishin Spain. J. Appl. Microbiol., 95:1106-1116.Akayli et al.

Characterization of Vibrio alginolyticus Isolates from Diseased Cultured Gilthead Sea Bream, Sparus aurata

http://evols.library.manoa.hawaii.edu/bitstream/10524/19245/1/60_2_Akayli.pdf

Characterization of Vibrio alginolyticus Isolates from Diseased Cultured Gilthead Sea Bream, Sparus aurata

Abstract

Similar works

Full text

Available Versions

İstanbul Üniversitesi Açık Erişim Sistemi

eVols at University of Hawaii at Manoa