Although Yersinia ruckeri, the etiological agent of the enteric red mouth disease (ERM), has been isolated from freshwater fish in several countries, there are few reports of its presence in cultured sea and brackish water fish. The aim of this study was to isolate and identify the etio- logical agent of disease outbreaks that occurred in rainbow trout cage farms on the Black Sea coast of Turkey in 2002. Clinical observation, biochemical, API 20 NE, and agglutination tests allowed the diagnosis of ERM caused by Y. ruckeri, which was successfully treated with med- icated feed (oxytetracycline at 75 mg per kg body weight per day for 10 days)

Candan, Akin

Demircan, Didem

Karatas, Süheyla

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 56(3), 2004, 226-231.226SHORT COMMUNICATIONENTERIC RED MOUTH DISEASE IN CULTURED RAINBOW TROUT (ONCORHYNCHUS MYKISS) ON THE BLACK SEA COAST OF TURKEYSüheyla Karatas∗, Akin Candan and Didem DemircanDepartment of Fish Diseases, Faculty of Fisheries, Istanbul University, 200 Ordu Caddesi,34470 Laleli, Istanbul, Turkey(Received 25.12.03, Accepted 26.12.03)Key words: Black Sea, enteric red mouth, Oncorhynchus mykiss, Yersinia ruckeriAbstractAlthough Yersinia ruckeri, the etiological agent of the enteric red mouth disease (ERM), hasbeen isolated from freshwater fish in several countries, there are few reports of its presence incultured sea and brackish water fish. The aim of this study was to isolate and identify the etio-logical agent of disease outbreaks that occurred in rainbow trout cage farms on the Black Seacoast of Turkey in 2002. Clinical observation, biochemical, API 20 NE, and agglutination testsallowed the diagnosis of ERM caused by Y. ruckeri, which was successfully treated with med-icated feed (oxytetracycline at 75 mg per kg body weight per day for 10 days).IntroductionThe genus Yersinia, of the Enterobacteriaceaefamily consists of Gram negative short rods.Yersinia ruckeri is an important primarypathogen of fish and the causative agent ofenteric red mouth disease that primarily affectsrainbow trout and other salmonids. Yersiniosisor enteric red mouth (ERM) is a serious infec-tious disease in the rainbow trout farmingindustry and causes economic problems inmany countries. Y. ruckeri was reported inAtlantic salmon in Norway and Scotland, burbotin Canada, sturgeon in France, whitefish andAtlantic salmon in Finland, and rainbow trout inGreece, Turkey, Iran and Venezuela (Ross etal., 1966; Frerichs et al., 1985; Giorgetti et al.,1985; Bragg and Henton, 1986; Rintamaki etal., 1986; Sparboe et al., 1986; Dwilow et al.,1987; Vuillaume et al., 1987; Dear, 1988;*  Corresponding author. Fax: +90-212-5140379, e-mail: skaratas@istanbul.edu.tr227Bullock and Cipriano, 1990; Savvidis, 1990;Timur and Timur, 1991; Alvarez et al., 1992;McCormic and McLoughlin, 1993; Petrie et al.,1996; Soltani et al., 1999). Although known tobe a pathogen of freshwater fish, it was isolat-ed from marine fish as well (Sparboe et al.,1986). The disease was first reported in culturedrainbow trout in Turkey by Timur and Timur(1991) and later in cultured rainbow trout infreshwater (Cagırgan and Yüreklitürk, 1991;Karatas and Candan, 1997; Savaser andDiler, 1997). The mode of transmission of ERM hasbeen related to wild or farmed carrier fish andother putative vectors such as aquatic inverte-brate and birds (Willumsen, 1989). Thepathogen has been isolated from the feces ofcarrier fish even two months after an ERMoutbreak (Rodgers, 1992). The survival of Y.ruckeri was examined by Romalde et al.(1994) who concluded that salinity may effectsurvival time of Y. ruckeri and that the straincan survive in 15‰ salinity for three months.Diler and Ekici (2003) found that the optimalgrowth for Y. ruckeri strains occurs at 5‰ and15‰ salinity. Salinity of 9‰ did not preventERM, but mortality of rainbow trout was sig-nificantly reduced with each increase in salin-ity (Altınok and Grizzle, 2001). For many years, the inland Black Sea hascontributed about 70% of the total fish pro-duction of Turkey. Because of its low salinity(16‰) and appropriate water temperature,both fresh and marine fish are cultured inthese waters. The Black Sea coast has appro-priate salinity and temperature conditions forrainbow trout farming in net cages (Okumus etal., 1997; Sener, 2002). Rainbow trout pro-duction in net cages in the Black Sea isaround 1700 tons yearly (Memis, 2003). Theaim of this study was to isolate and identify theetiological agent of disease outbreaks amongrainbow trout cage farms on the Black Seacoast of Turkey. Materials and MethodsDuring May, 2002, unusual mortality out-breaks occurred among rainbow trout cagefarms in Ordu Peninsula on the Black Seacoast of Turkey to which cultured rainbowtrout were transferred during November 2001at approximately 70-80 g.During the outbreak, the seawater temper-ature was 16ºC and the fish averaged 250 g.Cumulative mortality reached approximately3%. Diseased fish were transported to the FishDiseases Laboratory and examined to diag-nose the responsible agent. Samples from thekidney, liver, and spleen were inoculated ontryptic soy agar (TSA, Merck). The agar plateswere incubated at 20ºC for 48 h. After purifyingthe cultures, biochemical and physiologicaltests were carried out on the isolates accordingto standard tube methods. Colonies were dilut-ed in sterile pure water and API 20NE stripswere inoculated according to the manufactur-er’s instructions at an incubation temperatureof 20ºC for 48 h. The isolated bacteria werecharacterized using the API 20NE system andthe identity was confirmed using a BionorMono Yr kit (BioNor Mono AS, BIONOR Aqua,Skien, Norway). Then the isolated bacteriawere inoculated into Shotts-Waltman medium. Susceptibility of the isolated strains to var-ious antibiotics and a test for sensitivityagainst O/129 Vibriostat 150 µg were deter-mined by the disc diffusion method onMüeller-Hinton agar (MHA) using commercial-ly available discs. Samples were taken from the skin, mus-cle, gills, heart, intestine, liver, kidney, andspleen, and fixed in 10% formaldehyde forhistopathological examination. Sections werestained with hematoxylin and eosin (Roberts,1978). Results and DiscussionsExophthalmus, hemorrhages at the base ofthe ventral fin and mouth, swelling in theabdominal region, prolapsus, and hemor-rhages in the anus were observed on post-mortem examination of fish. The internalpathology involved hemorrhages in the mus-cle and swim bladder, and enlarged bright redspleen, kidney, and liver. The stomach andintestine were full of fluid. These results agreewith earlier findings described by Bragg andHenton (1986), Sparboe et al. (1986),Vuillaume et al. (1987), Bruno (1990),Enteric red mouth disease in rainbow troutSavvidis (1990), Timur and Timur (1991),Alvarez et al. (1992), Inglis et al. (1993), andPetrie et al. (1996). Parasitological examina-tion was negative. Cream-colored, smooth, round colonieswere isolated from the liver and kidney of fourexamined fish. Gram negative, motile,cytochrome oxidase-negative, fermentative,rod-shaped bacterial strains were identified inthe isolated colonies. Green colonies with sur-rounding opaque haloes due to a zone ofhydrolysis appeared on Shotts-Waltmanmedium. The biochemical, morphological, andphysiological characteristics and API 20NEresults of the isolates are shown in Table 1.The isolates tested positive in the agglutina-tion tests for Y. ruckeri. Clinical observation,biochemical and API 20 NE test results, andthe agglutination test led to the diagnosis ofenteric red mouth disease (ERM) caused byY. ruckeri. The result of the biochemical testsagree with Stevenson and Daly (1982), De LaCruz et al. (1986), Vuillaume et al. (1987),Savvidis (1990), Timur and Timur (1991),Inglis et al. (1993), McCormick andMcLoughlin (1993), Savaser and Diler (1997),Soltani et al. (1999), and Oraic et al. (2002). Histologically, there were several necroticareas and a loss of hemapoietic tissue in theanterior and posterior parts of the kidney (Fig.1). The liver was also necrotic and diffusedvacuolation of hepatocytes were prominent(Fig. 2). Within the digestive tract, especiallythe intestine, necrosis of the mucosa andsloughing into the lumen occurred (Fig. 3).Our histological results were similar with thefindings of Wobeser (1973), Bruno (1990),Timur and Timur (1991), Petrie et al. (1996),and Horne and Barnes (1999). All strains were sensitive to trimethoprime(5 µg), flumequine (30 µg), and oxytetracy-cline (30 µg), and resistant to oxolinic acid (2µg). The disease outbreak was treated withmedicated feed (oxytetracycline, 75 mg/kgbody weight/day for 10 days). Bacterial sus-ceptibility to antimicrobial agents was similarto Giorgetti et al. (1985), Sparboe et al.(1986), Savvidis (1990), and Karatas andCandan (1997). In contrast, Dear’s isolateswere sensitive to oxolinic acid (Dear, 1988). 228 Karatas et al.Characteristic Liver, kidneyGram –Motility +TSI +Cytochrome oxidase –O/F test FNitrate +Triptophane –Urea +Esculine –Gelatin +Arabinose –Mannose +Gluconate –Caprate +Adipate –Citrate +Acid from glucose +Gas from glucose –Sucrose –Fructose +Mannitol +Hydrolysis of ornithine +Hydrolysis of lysine +Hydrolysis of arginine –H2S production –Indole –Metil red –Sorbitol –Starch –Hydrolysis of gelatin +Methyl red +Voges Proskauer –Oxytetracycline SFlumequine SOxolinic acid RSulphonamides RTrimethoprine SCatalase +Growth on Shotts-Waltman +Table 1. Biochemical, morphological char-acteristics, and API 20NE results of Yersiniaruckeri isolated from the liver and kidney ofrainbow trout cultured on the Black Sea coastof Turkey.+ Positive reaction; – Negative reaction; FFermentative, S Sensitive; R Resistant..229This first report of the outbreak of entericred mouth disease in rainbow trout raised inthe Black Sea indicates that the long term sur-vival of this bacterium in the salinity of theBlack Sea enables it to cause outbreaks, con-firming earlier reports that the salinity toler-ance of Y. ruckeri causes mortality in rainbowtrout (Thorsen at al., 1992; Altınok andGrizzle, 2001). It remains for future investiga-tions to determine the source of the infection.ReferencesAltınok I. and J.M. Grizzle, 2001. Effects ofsalinity on Yersinia ruckeri infection of rain-bow trout and brown trout. J. Aquatic AnimalHealth, 13:334-339. Alvarez J.D., Austin B. and D.A. Conroy,1992. First outbreak of enteric red mouth inrainbow trout (Oncorhynchus mykiss,Walbaum) cultured in Venezuela. Bull. Eur.Assoc. Fish Pathologists, 12(3):83.Enteric red mouth disease in rainbow troutFig. 1. Posterior kidney of diseased fish shows reduction of hemapoietic tissue and necrosis of the renaltubules: (a) hematoxylin and eosin stain x 10, (b) hematoxylin and eosin stain x 40.Fig. 2. Severe necrotic areas and extensive vacuolation of cytoplasm of hepatocytes: (a) hematoxylinand eosin stain x 10, (b) hematoxylin and eosin stain x 40.a ba bBragg R.R. and M.M. Henton, 1986. Isolationof Yersinia ruckeri from rainbow trout in SouthAfrica. Bull. Eur. Assoc. Fish Pathologists,6(1):5. Bruno D.W. 1990. Enteric red mouth disease.Aquacult. Information Ser., 10:1-5. Bullock G.L. and R.C. Cipriano, 1990.Enteric red mouth disease of salmonids. pp.1-5. In: Fish Disease Leaflet 82. United StatesDepartment of the Interior, Fish and WildlifeService. Çagırgan H. and O. Yüreklitürk, 1991. Firstisolation of Yersinia ruckeri from rainbow troutfarm in Turkey. p. 131. In: 5th Conf. EAFP,Disease of Fish and Shellfish - Book ofAbstracts. August 24-29, Hungary.Dear G. 1988. Yersinia ruckeri isolated fromAtlantic salmon in Scotland. Bull. Eur. Assoc.Fish Pathologists, 8(2):18. De La Cruz J.A., Rodriguez A., Tejedor C.,De Lucas E. and L.R. Orozco, 1986.Isolation and identification of Yersinia ruckeri,causal agent of enteric red mouth disease(ERM), for the first time in Spain. Bull. Eur.Assoc. Fish Pathologists, 6(2):43-44.Diler O. and S. Ekici, 2003. The effect of vari-ations in salinity and pH on the in vitro growthrate of Yersinia ruckeri strains. S.D.Ü. EgirdirSu Ürünleri Fakültesi Dergisi, 9:63-70.Dwilow A.G., Souter B.W. and K. Knight,1987. Isolation of Yersinia ruckeri from burbot,Lota lota (L.), from the Mackenzie River,Canada. J. Fish Dis., 10:315-317.Frerichs G.N., Stewart A. and R.O. Collins,1985. Atypical infection of rainbow trout,Salmo gairdneri Richardson, with Yersiniaruckeri. J. Fish Dis., 8:383-387. Giorgetti G., Ceschia G. and G. Bovo, 1985.First isolation of Yersinia ruckeri in farmedrainbow trout in Italy. pp. 161-166. In: A.E.Ellis (ed.). Fish and Shellfish Pathology.Academic Press, London. Horne M.T. and A.C. Barnes, 1999. Entericred mouth disease. pp. 455-477. In: P.T.K.Woo, D.W. Bruno (eds.). Fish Diseases andDisorders, vol. 3. CABI Publ., New York. Inglis V., Roberts R.J. and N.R. Bromage,1993. Bacterial Diseases of Fish, BlackwellSci. Publ., London. 312 pp.Karatas Dügenci S. and A. Candan, 1997.Marmara bolgesindeki bir gokkusagı alabalıgı(O. mykiss) isletmesinde yersiniozis vakası.IX. Ulusal Su Ürünleri Sempozyumu, Eylül17-19, Egirdir, Isparta.McCormic J.I. and M.F. McLoughlin, 1993.The characterization and pathogenicity of thefirst isolate of Yersinia ruckeri from rainbowtrout (Oncorhynchus mykiss Walbaum) inNorthern Ireland. Bull. Eur. Assoc. FishPathologists, 13(4):138. 230 Karatas et al.Fig. 3. Necrosis of the intestinal mucosa and sloughing into the lumen: (a) hematoxylin and eosin stainx 10, (b) hematoxylin and eosin stain x 40.a b231Memis D., 2003. Review of marine aquacul-ture development in Turkey. World Aquacult.,34:4. Okumus I., Küçük E., Basçınar N., Sahin T.and B. Akbulut, 1997. Deniz levregi(Dicentrarchus labrax) yavrularının dogukaradeniz de büyüme performansı. AkdenizBalıkçılık Kongresi, Nisan 9-11. E.Ü. Su Ürün-leri Fakültesi, Izmir. Oraic D., Zrncic S., Sostaric B., Bazulic D.and Z. Lipej, 2002. Occurrence of enteric redmouth disease in rainbow trout(Oncorhynchus mykiss) on farms in Croatia.Acta Veterinaria Hungarica, 50(3):283-291. Petrie J., Bruno D.W. and T.S. Hastings,1996. Isolation of Yersinia ruckeri from wild,Atlantic salmon, Salmo salar L., in Scotland.Bull. Eur. Assoc. Fish Pathologists, 16(3):83. Rintamaki P., Tellervo Valtonen E. andG.N. Frerichs, 1986. Occurrence of Yersiniaruckeri infection in farmed whitefish,Coregonus peled Gmelin and Coregonusmuksun Pallas, and Atlantic salmon, Salmosalar L., in northern Finland. J. Fish Dis.,9:137-140.Roberts R., 1978. Fish Pathology. BailliereTindall, London. 317 pp. Rodgers C.J., 1992. Development of a selec-tive-differential medium for the isolation ofYersinia ruckeri and its application in epidemi-ological studies. J. Fish Dis., 15:243-254. Romalde J.L., Barja J.L., Magarinos B. andA.E. Toranzo, 1994. Starvation survivalprocesses of the bacterial fish pathogen,Yersinia ruckeri. Syst. Appl. Microbiol.,17:161-168. Ross A.J., Rucker R.R. and W.H. Ewing,1966. Description of a bacterium associatedwith red mouth disease of rainbow trout(Salmo gairdneri). Canadian J. Microbiol.,12:763-770. Savaser S. and O Diler, 1997. An investiga-tion on the morphological and biochemicalproperties of the Yersinia ruckeri causing ofenteric red mouth. Mediterranean Fish.Congress - Abstracts. April 9-11, Izmir.Savvidis G.K., 1990. First isolation ofYersinia ruckeri from rainbow trout,Oncorhynchus mykiss, in Greece. Bull. Eur.Assoc. Fish Pathologists, 10(5):131.Sener E., 2002. Farming of the rainbow trout,Oncorhynchus mykiss, in the Black Searegion of Turkey. Turkish J. Fish. Aquatic Sci.,2:93-96. Soltani M., Fadaii Fard F. and M.R.Mehrabi, 1999. First report of a yersiniosis-like infection in Iranian farmed rainbow trout.Bull. Eur. Assoc. Fish Pathologists, 19(4):173.Sparboe O., Koren C., Hastein T., Poppe T.and H. Stenwig, 1986. The first isolation ofYersinia ruckeri from farmed Norwegiansalmon. Bull. Eur. Assoc. Fish Pathologists,6(2):41.Stevenson R.M.W. and J.G. Daly, 1982.Biochemical and serological characteristics ofOntario isolates of Yersinia ruckeri. CanadianJ. Fish. Aquatic Sci., 39:870-876.Thorsen B.K., Enger O., Norland S. andK.A. Hoff, 1992. Long-term starvation survivalof Yersinia ruckeri at different salinities stud-ied by microscopical and flowcytometric meth-ods. Appl. Environ. Microbiol., 58(5):1624-1628. Timur G. and M. Timur, 1991. An outbreak ofenteric red mouth disease in farmed rainbowtrout (O. mykiss) in Turkey. Bull. Eur. Assoc.Fish Pathologists, 11(5):182. Vuillaume A., Brun R., Chene P., Sochon E.and R. Lesel, 1987. First isolation of Yersiniaruckeri from sturgeon, Acipencer baeri Brandt,in south west. Bull. Eur. Assoc. FishPathologists 7(1):18. Willumsen B., 1989. Birds and wild fishpotential vectors of Yersinia ruckeri. J. FishDis., 12: 275-277.Wobeser G., 1973. An outbreak of red mouthdisease in rainbow trout (Salmo gairdneri) inSaskatchewan. J. Fish. Res. Board Canada,30(4):571-575.Enteric red mouth disease in rainbow trout

Enteric Red Mouth Disease In Cultured Rainbow Trout (Oncorhynchus Mykiss) On The Black Sea Coast Of Turkey

Although Yersinia ruckeri, the etiological agent of the enteric red mouth disease (ERM), has been isolated from freshwater fish in several countries, there are few reports of its presence in cultured sea and brackish water fish. The aim of this study was to isolate and identify the etiological agent of disease outbreaks that occurred in rainbow trout cage farms on the Black Sea coast of Turkey in 2002. Clinical observation, biochemical, API 20 NE, and agglutination tests allowed the diagnosis of ERM caused by Y. ruckeri, which was successfully treated with medicated feed (oxytetracycline at 75 mg per kg body weight per day for 10 days)

Candan, Akın

Demircan, D

İstanbul Üniversitesi Açık Erişim Sistemi

Enteric red mouth disease in cultured rainbow trout (Oncorhynchus mykiss) on the Black Sea coast of Turkey

http://evols.library.manoa.hawaii.edu/bitstream/10524/19113/1/56_3_Karatas.pdf

Enteric Red Mouth Disease In Cultured Rainbow Trout (Oncorhynchus Mykiss) On The Black Sea Coast Of Turkey

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