FULL PAPER Bacteriology

Characterization of tarda Isolated from Farm-Cultured , Anguilla japonica, in the Republic of Korea

Seong-Joon JOH1)*, Min-Jeong KIM1), Hyuk-Man KWON1), Eun-Hye AHN1), Hwan JANG1) and Jun-Hun KWON1)

1)Laboratory of Aquatic Animal Diseases, Department of Veterinary Research, National Veterinary Research and Quarantine Service, Ministry of Food, Agriculture, Forestry and Fisheries, 480 Anyang Kyonggido, Republic of Korea

(Received 10 June 2010/Accepted 29 July 2010/Published online in J-STAGE 12 August 2010)

ABSTRACT. We surveyed the occurrence of edwardsiellosis on farms and investigated the characteristics of Edwardsiella tarda isolated from farm-cultured eels in the Republic of Korea. The occurrence rate of edwardsiellosis was 72% in the investigated samples. Among the edwardsiellosis cases, 46% were found to be mixed infections, with parasites and other kinds of . Some of the biochemical characteristics of the E. tarda isolates were different from those of the previously reported E. tarda isolated from several kinds of from different countries, especially in terms of hydrogen sulfide and indole production. The E. tarda isolated from the eels in the Repub- lic of Korea had the characteristics of two biogroups, the wild-type biogroup and biogroup 1. The enzymatic activity of the E. tarda showed similar patterns to previously reported E. tarda strains and ATCC strains. This is the first it has been reported that E. tarda isolated from farm-cultured eels had some different biochemical characteristics from those of previously reported E. tarda isolated from several kinds of fish. KEY WORDS: characteristics, Edwardsiella tarda, eel. J. Vet. Med. Sci. 73(1): 7–11, 2011

Edwardsiellosis, which is caused by the bacterium teristics of E. tarda isolated from farm-cultured eels in the Edwardsiella tarda (E. tarda), is a serious systemic bacte- Republic of Korea. rial disease that affects a variety of fish taxa and has a world- wide distribution in fresh and marine waters [3, 7]. MATERIALS AND METHODS Moreover, E. tarda may infect , , marine and other warm-blooded animals including birds Fish samples: Moribund or almost dead eels were pro- and humans [18]. Fish infections with economically impor- vided by eel culture farms in the Gochang and YoungK- tant losses have been reported in , Ictalurus wang areas of the Republic of Korea. In all cases, the fish punctatus (Rafinesque), catfish Clarias gariepinus, striped were stocked in concrete tanks with an open flow and heat- bass Morone saxatilis (Walbaum), European eel Anguilla ing systems and were fed farm-made feed regularly. The anguilla (L.) and Japanese eel Anguilla japonica (L.), olive outbreaks affected different batches of fish weighing from flounder Paralichthys olivaceus (Temminck & Schlegel) 50 to 200 g. and turbot Psetta maximus (L.) in different geographical Parasites examination and bacterial isolation: For para- areas [7, 17, 20]. E. tarda is one of the important bacterial sitical examination on the gills, only the left side of the gill pathogens for eels, Anguilla japonica, and olive flounder, apparatus was investigated for parasite distribution because Paralichthys olivaceus, in the Republic of Korea and Japan of technical reasons. The gill apparatus was divided into [5, 20]. E. tarda includes two biogroups, the wild and bio- four gill arches. Each arch was divided into two gill sur- group 1 types. The wild-type biogroup produces indole and faces (outer, inner), put on a glass slide and examined on a hydrogen sulfide. In contrast, biogroup 1 does not produce light microscope. For bacterial isolation, samples were hydrogen sulfide [16]. aseptically collected from the livers, ascites and kidneys and Eels are the most important freshwater fish in the aquac- then directly streaked onto blood agar plates (BAP; CoMed ulture industry in the Republic of Korea, and many losses of Co, Republic of Korea), MacConkey’s agar and tryptic soy farm-cultured eels have occurred in the Republic of Korea broth (TSB; BD – Diagnostic Systems, Sparks, MD, because of repeated outbreaks of edwardsiellosis [8, 11]. U.S.A.). Plates were incubated at 30°C overnight [6, 18]. However, there were very few reports about edwardsiellosis Gram-stained smears were prepared for microscopic exami- in farm-cultured eels. There have also been no reports about nation after incubation for 24 hr. Motile activity was deter- the characteristics of E. tarda in the last 10 years. We report mined after overnight incubation of an additional subculture here newly confirmed biochemical and enzymatic charac- in semisolid medium. We used the 40 E. tarda isolates and 2 ATCC strains (ATCC 23685 and 15947) in this study. *CORRESPONDENCE TO: JOH, S. J., Laboratory of Aquatic Animal Sequencing and identification of 16S rRNA: The overall Diseases, Department of Animal Health, National Veterinary procedures for molecular work were conducted as described Research and Quarantine Service, Ministry of Food, Agriculture, Forestry and Fisheries, 480 Anyang Kyonggido, Republic of by Sambrook and Russell [19] and were modified using kits. Korea. Pure culture bacterial isolates were grown at 26C for 24 hr e-mail: [email protected] in tryptic soy broth (TSB) and harvested, and the bacterial 8 S-J. JOH ET AL. cells were lysed. The DNA was extracted using a microbial RESULTS DNA isolation kit (iNtRON Biotechnology, Inc., Seoul, Republic of Korea). The DNA was checked for purity using Occurrence of edwardsiellosis and isolation of E. tarda: NanoDrop (NanoDrop Technologies, Inc., Wilmington, DE, We surveyed a total of 81 ponds at 18 farms in Gochang, U.S.A.). We submitted the DNA templates to Macrogen, Jeonbuk province, and YoungKwang, Jeonnam province, Inc. (Seoul, Republic of Korea) for sequencing, which was and investigated edwardsiellosis in eels that showed clini- performed as follows. The DNA templates were amplified cally abnormal signs and poor health conditions. The occur- by polymerase chain reaction (PCR) on an authorized ther- rence of edwardsiellosis was 72% (13/18 farms) at the farm mal cycler (Eppendorf, Hamburg, Germany) using universal level and 36% (29/81 ponds) at the pond level (Table 1). primers to amplify an expected 1,505-bp fragment of the Among the edwardsiellosis cases, 46% (6/13 farms) of the 16S rRNA. The primers, 27F (5’ AGR GTT TGA TCM cases were found to be coinfected with parasites and other TGG CTC AG 3) and 1492R (5’ GGY TAC CTT GTT ACG kinds of bacteria. The major agents of the mixed infections ACT T 3), were obtained from Bioneer (Bioneer Technolo- were the Pseudodactylogyrus sp. and Trichodina sp. of par- gies, Republic of Korea). The amplification mixture (100 asites and Aeromonas sp. of bacteria. µL) comprised 2 µL (50 pmol µ/L) each of the 27F and Identification of E. tarda: All the bacteria suspected to be 1492R primers, 0.5 µL (2 U µ/L) of Taq DNA polymerase, E. tarda on investigation of morphology and conventional 10 µL of 10 reaction buffer, 10 µL of dNTP mixture, 70 µL tests were identified as E. tarda by the 16S rRNA sequenc- of sterile filtered water and 5.5 µL of DNA template. The ing. All of the E. tarda isolates that were compared with the DNA templates were amplified by initial denaturation at GenBank database had identity rates from 0.987 to 1.000 on 94C for 10 min, followed by 20 cycles of denaturation at the RDP (Data not shown). 94C for 1 min, annealing at 55C for 1 min, extension at Biochemical and enzymatic characterization: Fifty-four 72C for 1 min and a final extension at 72C for 10 min. isolates of E. tarda showed the same morphologic Controls, without DNA, were simultaneously included in homogeneity on the blood agar and tryptic soy agar plates. the amplification process. The integrity of the PCR After incubation for 24 hr, the colonies were small, products was assayed by the development of single bands uniformly round, glistening, entire, convex, translucent and following electrophoresis for 1 hr at 100 V in 2% (w/v) moist. In the Gram staining, all isolates were Gram- agarose gels in Tris–borate–EDTA buffer. Each of the negative, with short-thick rods and non-spore forms on resulting products was then cloned into TA cloning vectors microscopic examination. We divided the E. tarda isolates (Promega Corporation, Madison, WI, U.S.A.) according to into 4 groups according to hydrogen sulfide and indole the manufacturer’s instructions. Nucleotide sequencing was production (Table 2). Most of the isolates were included in carried out by the dideoxynucleotide chain termination group 4, which had no production of hydrogen sulfide and method using T7 DNA and SP6 DNA polymerase with an indole. In the biochemical tests, eleven kinds of tests ABI PRISM 3730 (Applied Biosystems, Inc., Foster City, showed totally negative reactions, and the other biochemical CA, U.S.A.). The nucleotide sequences of 16S rRNA were tests showed variable results (Table 3). In the enzymatic analyzed and identified by Ribosomal Database Project activity tests, 11 of 19 tests showed negative reactions, and (RDP), Release 10 (Michigan State University, East Lan- two tests showed positive reactions (Table 4). sing, MI, U.S.A.). Biochemical and enzyme activity characterization: Fifty- DISCUSSION four isolates were subjected to phenotypic tests. We tested hydrogen sulfide and indole production in all 54 isolates Edwardsiellosis, which is caused by E. tarda, has been a using SIM medium (BD– Diagnostic Systems, Sparks, MD, major economic problem in the eel-farming industry in the U.S.A.) and an indole test kit (BD – Diagnostic Systems, Republic of Korea [8, 11]. We investigated the occurrence Sparks, MD, U.S.A.) according to the respective manuals. of edwardsiellosis at eel farms for 3 years from 2007 to Forty of the isolates were subjected to biochemical and 2009. In many cases, the eels infected with E. tarda were enzymatic characterization. The biochemical characteriza- co-infected with parasites in the gills (Table 1). The major tion was performed using some conventional tests and with coinfecting parasite was Pseudodactylogyrus sp. The coin- API 20E strips (bioMerieux, Madrid, Spain), following the fected cases showed more severe clinical signs. The E. manufacturer’s instructions (API manual ver. 4). API 20E tarda isolated from eels in the Republic of Korea showed strips containing E. tarda were incubated at 26°C overnight. some different biochemical characteristics and enzymatic The enzymatic characterization was carried out using API activities from those isolated from the flounder, Paralich- ZYM strips according to the manufacturer’s instructions. thys olivaceus, fall chinook salmon, Oncorhynchus tshaw- The ZYM strips were incubated at 37°C for 4 hr and then ytscha, turbot, Psetta maxima, tilapia, Sarotherodon illuminated with a strong light for more intense color devel- niloticus, and eels, Anguilla japonica. The isolates in the opment. When the color of a strip developed two or more present study showed just 35% positive results. Other levels in the reaction, we considered as positive. The API researchers reported that E. tarda isolated from the flounder, profiles were read and compared with the API database eel and snakehead, Channa argus, in the Republic of Korea (APIweb ver. 1.2.1, bioMerieux). [5, 11, 12, 15], from the flounder and eel in Japan [14, 20] CHARACTERIZATION OF EDWARDSIELLA TARDA FROM EELS 9

Table 1. Occurrence of edwardsiellosis in farm-cultured eels in the Republic of Korea Edwardsiellosis Mixed Areas Farms No. ponds Farms Ponds infections at farms A 2 + 1 Parasitesa), bacteriac) B 7 + 7 Parasitesb) C1 – – – D5 + 1 – E12+ 1 – Gochang, F 11 + 1 – Jeonbuk G 10 + 5 – H 1 + 1 Parasitesa), bacteria I1 – – – J 2 + 2 Parasitesa), bacteria K 7 + 1 Bacteria Iksan, Jeonbuk L 1 – – – M10+ 2 – Youngkwang, Jeonnam N1 + 1 – O2 – – – P3 + 3 – Naju, Jeonnam Q2 – – – Nonsan, R 3 + 3 Parasitesa) Chungnam Total 18 81 13/18 (72%) 29/81 (36%) 6/13 (46.2%) a) Pseudodactylogyrus spp. b) Trichodina spp. c) Aeromonas hydrophila.

Table 2. Grouping of E. tarda isolates according to hydrogen sulfide production and indole production Types Wild type Biogroup 1 Groups Group 1 Group 2 Group 3 Group 4 Total H2S + Indole + H2S + Indole - H2S - Indole + H2S - Indole - No. E. tarda 9 (16.7) 11 (20.4) 7 (13.0) 27 (50) 54 (100) isolates (%)

Table 3. Biochemical characteristics of E. tarda isolated from farm-cultured eels in the Republic of Korea No. positive (%) No. positive (%) Biochemical Biochemical Isolates ATCC Isolates ATCC characteristics characteristics (n=40) (n=2) (n=40) (n=2) Motility 38 (95) 0 (0) Tryptophan deaminase 0 (0) 0 (0) Oxidase 0 (0) 0 (0) Voges-Proskauer 4 (10) 0 (0) Catalase 33 (83) 2 (100) Gelatninase 0 (0) 0 (0) Hemolysis 38 (95) 2 (100) Glucose 40 (100) 2 (100) H2S production 14 (35) 2 (100) Mannitol 8 (20) 0 (0) Indole 12 (30) 2 (100) Inositol 0 (0) 0 (0) -galactosidase 0 (0) 0 (0) Sorbitol 0 (0) 0 (0) Arginine dihydrolase 4 (10) 0 (0) Rhamnose 0 (0) 0 (0) Lysine decarboxylase 36 (90) 2 (100) Saccharose 0 (0) 0 (0) Ornithine decarboxylase 34 (85) 2 (100) Melibiose 0 (0) 0 (0) Citrate utilization 0 (0) 0 (0) Amygdalin 4 (10) 0 (0) Urase 0 (0) 0 (0) Arabinose 2 (5) 0 (0) OF medium 36 (90) 2 (100) NO2 production 36 (90) 2 (100) and from the fall chinook salmon in the U.S.A. [2] were hydrogen sulfide production, but the A group did not [13]. 100% positive for hydrogen sulfide production. In another In regard to indole production, many researchers have previous study E. tarda isolates from tilapia were divided reported that E. tarda isolates are 100% positive, except E. into 2 groups, A and B. The B group of E. tarda showed tarda from tilapia [1, 2, 5–7, 10, 11, 13, 14, 20]. Based on 10 S-J. JOH ET AL.

Table 4. Enzymatic activity against E. tarda isolated from farm-cultured eels in the Republic of Korea No. positive (%) No. positive (%) Kinds of enzymatic Kinds of enzymatic Isolates ATCC Isolates ATCC activities activities (n=40) (n=2) (n=40) (n=2) Control 0 (0) 0 (0) Acid phosphatase 40 (100) 2 (100) Alkaline phosphatase 36 (90) 2 (100) Naphthol-AS-BI-phosphohydrolase 10 (25) 2 (100) Esterase 0 (0) 0 (0) -galactosidase 0 (0) 0 (0) Esterase Lipase 2 (5) 0 (0) -glucuronidase 0 (0) 0 (0) Lipase 0 (0) 0 (0) -glucosidase 0 (0) 0 (0) Leucine arylamidase 40 (100) 2 (100) -glucosidase 0 (0) 0 (0) Valine arylamidase 4 (10) 0 (0) -glucosidase 4 (10) 0 (0) Crystine arylamidase 0 (0) 0 (0) N-acetyl--glucosaminidase 4 (10) 2 (100) Trypsin 0 (0) 0 (0) -mannosidase 0 (0) 0 (0) -chymotrypsin 0 (0) 0 (0) -fucosidase 0 (0) 0 (0) hydrogen sulfide production, the positive samples corre- REFERENCES spond to the wild type, and the negative samples corre- sponded to biogroup 1. 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