Disease of rainbow trout caused by Pseudomonas luteolaIlhan Altinok a,⁎,Fikri Balta b ,Erol Capkin a ,Sevki Kayis baKaradeniz Technical University,Faculty of Marine Sciences,61530Surmene,Trabzon,TurkeybRize University,Department of Fisheries and Aquatic Sciences 53100,Rize,TurkeyReceived 13August 2007;received in revised form 21September 2007;accepted 2October 2007AbstractBacteria isolated from rainbow trout,Oncorhynchus mykiss ,kept in a farm,in Turkey.During the outbreak,40%of the rainbow trout (10–40g)died.Typical clinical signs were exophthalmia,dark pigmentation,hemorrhage at the base of the pectoral,pelvic,anal fins and around the vent.Internal signs were enlarged spleen,pale liver and intestine filled with yellowish fluid.Liver,kidney and spleen of diseased fish,were aseptically streaked on Tryptic Soy Agar.After incubation,pure cultured colonies were observed and biochemically characterized with API 20NE and other biochemical tests.Cultured bacterial 16S rDNA gene was sequenced.Based on biochemical characteristics and sequence of 16S rRNA,the causative bacteria were identified as Pseudomonas luteola .This study reports the first P .luteola infection in fish.©2007Elsevier B.V .All rights reserved.Keywords:Pseudomonas infection;Chryseomonas luteola ;Histopathology1.IntroductionPseudomonas luteola ,known also as known as Chryseomonas luteola ,a Gram-negative rod,is increas-ingly recognized as an important cause of hospital-acquired infections,especially among immunocompro-mised patients (Anzai et al.,1997;Kiska and Gilligan,1999).P .luteola is aerobic,non-spore-forming,motile,oxidase-negative and catalase-positive (Kiska and Gilligan,1999).Although the normal habitat of P .luteola is unclear,it is frequently found in soil,on plants,in aqueous environments and other damp environments (Freney et al.,1988;Silver et al.,1985;Hawkins et al.,1991).P .luteola has been known to be associated with human clinical infections,including bacteraemias (Con-nor et al.,1987;Hawkins et al.,1991;Rohav et al.,1995)that occur in association with pancreatic abscess,prosthetic valve endocarditis,peritonitis,osteomyelitis,indwelling vascular catheters and facial cellulites (Rastogi and Sperber,1998).However it is not a common pathogen in aquaculture and P.luteola infection in fish has not been reported.In the present study,P .luteola infection in rainbow trout,Oncor-hynchus mykiss and biochemical and 16S rDNA gene properties of P .luteola was described.2.Methods2.1.Isolation of bacteriaA disease outbreak occurred during the spring of 2004in a rainbow trout farm,Rize,Turkey.Affected fish size ranged between 10g and 40g.Thirty six moribund fish were necropsied.All sampled fish were examined externally and internally.The gills and body surface were examinedAvailable online at Aquaculture 273(2007)393–397/locate/aqua-onlineCorresponding author.Tel.:+904627522805;fax:+904627522158.E-mail address:ialtinok@.tr (I.Altinok).0044-8486/$-see front matter ©2007Elsevier B.V .All rights reserved.doi:10.1016/j.aquaculture.2007.10.025microscopically for the presence of parasites and bacteria. Then body surface of the fish was swabbed using70%ethyl alcohol to prevent contamination of the culture by normal external bacterial flora.Liver,trunk kidney,and spleen,were aseptically streaked on Tryptic Soy Agar(TSA).After incu-bation at25°C for2days,bacteria isolated from fish were subcultured on the same medium to check purity of the isolate although virtually pure culture growth was obtained from the fish on TSA.Pure cultured colonies were biochemically cha-racterized with API20NE(Biomerieux,Marcy l'Etoile, France)and other biochemical tests(Holt et al.,1994;Kiska& Gilligan1999).The identification was confirmed by sequenc-ing the16S rDNA as described in Section2.3.Antimicrobial test were applied on Muller Hinton agar.2.2.DNA isolation from cultured bacteriaIsolated bacteria from fish were grown in Tryptic Soy Broth(TSB)at22°C for24h,and then transferred to1.5ml centrifuge tubes,and centrifuged at9000×g for5min.Super-natant was discarded and the pellet was dissolved in567μl TE buffer,30μl of10%sodium dodecyl sulfate,and3μl Protei-nase K(60μg).After1h of incubation at37°C,100μl of5M NaCl was added and mixed thoroughly.Then,80μl cetyl-trimethylammonium bromide was mixed with the sample,which was incubated at65°C for10min(Ausubel et al.,1993).Th DNA was then purified as previously described(Altinok et al., 2001).2.3.DNA sequencingTo identify bacteria that caused mortality of fish,universal primers specific for16S rRNA gene of eubacteria were used. PCR primers were designed by Drancourt et al.(2000)to amplify the highly conserved5′and3′regions of the eubacterial16S rRNA gene.These primers were then used to yield a1496-bp16S rRNA gene product by PCR.These reactions were performed in a thermal cycler(Model Px2 ThermoHybaid;Thermo Electron Inc.,Waltham,MA,USA). The1496-bp PCR product was purified with a NucleoSpin PCR purification kit(Macherey–Nagel)and directly se-quenced with an ABI PRISM310genetic analyzer(Applied Biosystems).Both strands of templates were sequenced.The derived nucleotide sequences were analyzed and aligned with BioEdit Sequence Alignment Editor(North Carolina State University,Raleigh,North Carolina).The results of the sequencing were used for homology searches by the methods of Altschul et al.(1990).2.4.Infectivity experimentsThree-month-old rainbow trout weighing 3.88±0.16g (mean±SD)were housed at the research station of the Karadeniz Technical University,Faculty of Marine Sciences, Turkey.Fish were kept in two200l tanks supplied with aerated flow-through spring water at an average temperature of18°C.Fish were fed twice a day to satiation with commercial trout feed.These fish had no history of untoward mortalities or abnormalities.Fish were maintained and handled according to Institutional Animal Care and Use Committee approved guidelines.Fifty fish were exposed to P.luteola to test Koch's postulate of P.luteola isolated from rainbow trout as described above.Water flow was stopped and a24h culture of P.luteola in TSB was added to the water to give a final concentration of 6.4×106colony forming units(CFU)ml−1.Bacterial CFU were counted by plating serial dilutions as described by APHA (1998).One hour after bacteria were added to the water,water flow to the tank was re-established.Fifty control fish from the same source were exposed to same amount of TSB,which did not contain P.luteola.Once fish were exposed to P.luteola, Table1Comparison of phenotypic characteristics of P.luteola isolated from rainbow trout in the present study with the published description of P.luteola aPhenotypic characteristics P.luteola P.luteola a Arginine dihydrolase++Lysine decarboxylase−−Ornithine decarboxylase−−Citrate utilization++H2S production−−Urease−n.a Tryptophane deaminase−n.a Indole−−V oges–Proskauer−Gelatinase++Gas from glucose−−β-galactosidase++Acid from:Glucose++ Mannitol++ lnositol++ Rhamnose−n.a Sucrose+n.a Arabinose++D-mannose++ Assimilation of:Potassium gluconate+n.a Capric acid+n.a Adipic acid−n.a Malate++ Phenylacetic acid−n.a Glucose++ Denitrification+n.a Esculin hydrolyze++ Motility++TSA+MacConkey's++OF-oxidation++OF-fermentation−−Cytochrome oxidase−−Gram stain−−n.a.:not applicable.This test was not done for reference strain.a Phenotypic characteristics of P.luteola was obtained from Holt et al.(1994).394I.Altinok et al./Aquaculture273(2007)393–397they were observed twice a day for 42days.Twenty out of 38dead fish were necropsied and the liver and kidney were aseptically streaked on TSA agar.After incubation at 25°C for 48h,bacteria were identified as described above.During the 42days of experiment,water characteristics were as following:dissolved oxygen 8.30mg L −1,temperature 18.2°C,pH 7.61,total hardness 93.8±2.9mg L −1as CaCO 3and alkalinity 58.3±2.6mg L −1as CaCO 3.Un-ionized ammonia and nitrite in the water were below detectible levels.2.5.HistopathologyNaturally infected 10moribund (lethargic and loosing balance)fish were removed from the tank,anaesthetized withMS-222and then the second gill arch,liver and kidney were removed and fixed in 10%neutral buffered formalin (NBF)and processed for paraffin embedding.Sections were cut and stained with hematoxylin and eosin.Five healthy rainbow trout were also fixed in 10%NBF as controls.3.ResultsMass mortalities occurred in juvenile size rainbow trout and cumulative mortality reached 40%between March and April 2004.Externally,affected fish had exophthalmia,dark pigmentation hemorrhage at the base of the pectoral,pelvic,and anal fins and surrounding the vent.Internally,clinical signs included enlarged spleen,pale liver,empty stomachandparison of partial 16S rRNA gene sequences of Pseudomonas luteola isolated in the present study (KTU-PL)and P .luteola in the GenBank database (accession number D84002).Total of 1496base pair were compared and similarity between 2isolates was99.8%.Fig.2.Cumulative survival (Kaplan –Meier)of rainbow trout after infection with Pseudomonas luteola.Fish were observed for 42days and no fish died after 14days.395I.Altinok et al./Aquaculture 273(2007)393–397yellowish fluid filled intestine.Pure colonies of gram negative bacteria were isolated on TSA from internal organs of fish. Characteristics of the bacteria were Cytochrome oxidase posi-tive,non-reduction of nitrates to nitrites,lack of indole production,non-fermentation of glucose,utilization of citrate, liquefaction of gelatin,acid production from glucose,manni-tol,inositol,and arabinose,xylose;no acid from lactose. Bacterial isolates are characterized as P.luteola by API20NE (code1577651)and by other biochemical tests(Table1).It grew well on MacConkey agar as it formed yellow-pigmented colonies,which indicates lactose non-fermenter status.A1496base pair DNA fragment was amplified form the bacterial DNA extracted from a pure culture of P.luteola. Compared to the sequences available in GenBank,the ampli-fied nucleotide sequence expressed a99.8%homology with the16S rDNA sequence of P.luteola(GenBank accession number D84002).After comparison of2sequences,only4 bases differences were found(Fig.1).Thus,bacteria isolated from the diseased rainbow trout were confirmed as P.luteola.Based on antibiotic susceptibility studies,P.luteola were susceptible to entamicin,kanamycin,and ampicillin while it had an intrinsically high resistance to oxytetracycline,sulfa-methoxazole,tetracycline,streptomycin,cephalothin,and neomycin sulphate.Prior to bacterial challenge in the current study,no fish died during the acclimatization period.None of the control fish died during the experiment.The first incidence of dead fish was observed4days after exposure to P.luteola,and no fish died after14days postexposure(Fig.2).The cumulative percent mortality was76%.P.luteola was isolated from liver and kidney of all artificially infected moribund fish sampled microbiologically.There were no noteworthy histopathological changes found in gills compared to control fish.Histological lesions were observed in the liver and trunk kidney of the infected fish.The liver had a high number of necrotic hepatocytes and enlarged hepatic perisinusoidal areas containing eosinophilic material. Vacuolar dystrophies of hepatocytes were also observed.The trunk kidney of fish had enlarged sinusoids.Necrosis was present in glomerular cells and tubular cells.Glomeruli had eosinophilic exudates.None of these lesions were observed in control fish.4.DiscussionAmong the Pseudomonas species,Pseudomonas aeruginosa and P.fluorescens have been considered as opportunistic pathogenic species in aquaculture(Shiose et al.,1974;Alderman and Polglase,1988;Angelini and Seigneur,1988).However,other species of the genus may also induce serious infection e.g.P.putida infection in rainbow trout(Altinok et al.,2006),P.anguilliseptica in eel and Anguilla japonica(Wakabayashi and Egusa, 1972),P.chlororaphis in amago trout and Oncor-hynchus rhodurus(Hatai et al.,1975),P.plecoglossicida in ayu(Wakabayashi et al.,1996)and Plecoglossus altivelis(Kobayashi et al.,2000).The present study reports first that P.luteola may also cause bacterial septicemia in rainbow trout.The decisive diagnosis was accomplished by PCR analysis,using universal primers specific to16S rRNA gene of eubacteria.PCR amplified only16S rRNA gene of P.luteola which was subsequently confirmed by sequencing of PCR product.Pure culture of P.luteola was isolated and confirmed by biochemical tests and sequencing of16rRNA gene of bacteria.Koch's postu-late was also confirmed P.luteola infection on rainbow trout.Further more,ability to hydrolyse o-nitrophenyl-b-D-galactopyranoside(ONPG)and esculin can differ-entiate P.luteola from P.oryzihabitans,which is phenotypically related to P.luteola(Kiska and Gilligan, 1999).P.luteola isolated from fish in the present study was resistant to most commonly used antibiotics:thus, treatment of P.luteola infections can be painstakingly tough and expensive.Instead,farmers should establish a good hatchery management in order to prevent P.luteola infection.5.ConclusionDuring the outbreak,bacteria isolated from rainbow trout,kept in a farm.Based on biochemical character-istics and sequence of16S rRNA,the causative bacteria were identified as P.luteola.This study reports the first P.luteola infection in fish.AcknowledgementThis project was funded by the State Planning Organization(Devlet Planlama Teskilati;Project no:2003K120750).ReferencesAlderman,D.J.,Polglase,J.L.,1988.Pathogens,parasites and commen-sals.In:Holdich DM,Lowry RS(Eds)Freshwater crayfish—biology,management and exploitation.Timber Pres,Portland, Oregon,p.168.187.Altinok,I.,Grizzle,J.M.,Liu,Z.,2001.Detection of Yersinia ruckeri in rainbow trout blood by use of polymerase chain .44,29–34.Altinok,I.,Kayis,S.,Capkin,E.,2006.Pseudomonas putida infec-tion in rainbow trout.Aquaculture261,850–855.Altschul,S.F.,Gish,W.,Miller,W.,Myers,E.W.,Lipman,D.J.,1990.Basic logical alignment search tool.J.Mol.Biol.215,403–410. Angelini,N.M.,Seigneur,G.N.,1988.Disease of the fins of Rhamdia sapo.Isolation of the etiological agents and experimental infection.Rev.Argent.Microbiol.20,37–48.396I.Altinok et al./Aquaculture273(2007)393–397Anzai,Y.,Kudo,Y.,Oyaizu,H.,1997.The phylogeny of the genera Chryseomonas,Flavimonas,and Pseudomonas supports synonymy of these three genera.Int.J.Syst.Bacteriol.47,249–251. APHA(American Public Health Association),American Water Works Association,and Water Environment Federation,1998.Standard Methods for the Examination of Water and Wastewater,20th Edn.APHA,Washington,DC.Ausubel,F.M.,Brent,R.,Kingston,R.E.,Moore,D.D.,Seidman,J.G., Smith,J.A.,Struhl,K.,1993.Current Protocols in Molecular Biology.Wiley and Sons,New York.Connor,B.J.,Kopecky,R.T.,Frymoyer,P.A.,Forbes,B.A.,1987.Recurrent Pseudomonas(CDC Group Ve-1)peritonitis in a patient undergoing continuous ambulatory peritoneal dialysis.J.Clin.Microbiol.25,1113–1114.Drancourt,M.,Bollet,C.,Carlioz,A.,Martelin,R.,Gayral,J.P., Raoult,D.,2000.16S ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates.J.Clin.Microbiol.38,3623–3630.Freney,J.,Hansen,W.,Etienne,J.,Vandenesch,F.,Fleurette,J.,1988.Postoperative infant septicemia caused by Pseudomonas luteola (CDC group Ve-1)and Pseudomonas oryzihabitans(CDC group Ve-2).J.Clin.Microbiol.26,1241–1243.Hawkins,R.E.,Moriarty,R.A.,Lewis,D.E.,Oldfield,E.C.,1991.Serious infections involving the CDC group V e bacteria Chryseomonas luteola and Flavimonas oryzihabitans.Rev.Infect.Dis.13,257–260. Holt,J.G.,Krieg,N.R.,Sneath,P.H.A.,Staley,J.T.,Williams,S.T., 1994.Bergey's Manual of Determinative Bacteriology.Lippincott Williams and Wilkins,Baltimore,MD,p.84.Hatai,K.,Egusa,S.,Nakajima,M.,Chikahata,H.,1975.Pseudomo-nas chlororaphis as a fish pathogen.Bull.Jpn.Soc.Sci.Fish.41, 12–15.Kiska,D.L.,Gilligan,P.H.,1999.Pseudomonas.In:Murray,P.R., Baron,E.J.,Pfaller,M.A.,Tenover,F.C.,Yolken,R.H.(Eds.), Manual of Clinical Microbiology,7th Edn.American Society for Microbiology Press,Washington DC,pp.517–525. Kobayashi,T.,Imai,M.,Ishitaka,Y.,Kawaguchi,Y.,2000.Histopathological studies of bacterial haemorrhagic ascites of ayu,Plecoglossus altivelis(Temminck and Schlegel).J.Fish Dis.27,451–457.Rohav,G.,Simhon,A.,Mattan,Y.,Moses,A.E.,Sacks,T.,1995.Infections with Chryseomonas luteola(CDC group Ve-1) and Flavimonas oryzihabitans(CDC group Ve-2).Medicine74, 83–88.Rastogi,S.,Sperber,S.J.,1998.Facial cellulitis and Pseudomonas luteola bacteremia in an otherwise healthy patient.Diagn.Micro-biol.Infect.Dis.32,303–305.Silver,M.R.,Felegie,T.P.,Sorkin,M.I.,1985.Unusual bacterium, group Ve-2,causing peritonitis in a patient on continuous ambulatory peritoneal dialysis.J.Clin.Microbiol.21,838–839. Shiose,J.,Wakabayashi,H.,Tominaga,M.,Egusa,S.,1974.A report on a disease of cultured carp due to a capsulated Pseudomonas.Fish.Pathol.9,79–83.Wakabayashi,H.,Egusa,S.,1972.Characteristics of a Pseudomo-nas sp.from an epizootic of pond-cultured eels(Anguilla japonica).Bull.Jpn.Soc.Sci.Fish.38,577–587. Wakabayashi,H.,Sawada,K.,Ninomiya,K.,Nishimori,E.,1996.Bacterial hemorrhagic ascites of ayu caused by Pseudomonas sp.Fish Pathol.31,239–240.397I.Altinok et al./Aquaculture273(2007)393–397。
