J Vet Clin 27(5) : 618-621 (2010)
618
Isolation of Photobacterium Damselae Subsp.
Damselae from the Giant Grouper, Epinephelus Lanceolatus
Jin-Woo Jun, Ji-Hyung Kim, Jee-Eun Han, Sang-Phil Shin, Dennis K. Gomez, Casiano Choresca Jr., Kyu-Seon Oh* and Se-Chang Park1
Laboratory of Aquatic Animal Medicine, College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul 151-742, Korea
*Basic Research Laboratory, Center for Cancer Research, National Cancer Institute, Bethesda, USA (Accepted: September 16, 2010)
Abstract : A giant grouper (Epinephelus lanceolatus) that was reared for public exhibition in a private commercial aquarium in Seoul, Korea, was recently found dead. The fish had evidenced symptoms including anorexia, lethargy, and depression persisting for two weeks. A bacterial pathogen from fish organs (kidney, liver, spleen) was cultured, identified and confirmed as Photobacterium damselae subsp. damselae, using a Vitek System 2, API 20E test, multiplex PCR, and 16S rRNA gene sequencing. In this paper, we have described the isolation and identification of Photobacterium damselae subsp. damselae from a giant grouper reared in a private aquarium in Korea.
Key words : giant grouper, Photobacterium damselae subsp. damselae.
Introduction
Photobacterium damselae subsp. damselae, classified for- merly as Vibrio damsela, are gram-negative marine bacteria capable of producing a hemorrhagic septicemia in both warm- water and cold-water fish (5). P. damselae also encompasses the fish virulent isolates, P. damselae subsp. piscicida (for- merly Pasteurella piscicida). Despite its high degree of over- all DNA base sequence similarity, the two subspecies of P.
damselae are phenotypically dissimilar and develop different diseases; only one of them (the subsp. damselae) is patho- genic in humans (5,10). The giant grouper (Epinephelus lan- ceolatus) is one of the two largest species of groupers (Epinephelus) thus far known. Owing to its rapid growth and high price, the giant grouper is an economically important cultured finfish in East Asia (9). In this study, we describe the isolation and identification of P. damselae subsp. damse- lae from a giant grouper exhibited in a commercial aquar- ium in Korea.
Case
In September 2009, a giant grouper (total length 97 cm, body weight 19.8 kg) which was reared for public exhibition in a private commercial aquarium in Seoul, Korea was found dead. The fish had evidenced symptoms including anorexia, lethargy, and depression persisting for two weeks. Shortly after
the death of the fish, it was submitted to the College of Vet- erinary Medicine, Seoul National University for postmortem examination.
The water temperature during this month ranged from 22 to 24oC, and other water parameters such as DO, pH and ammonia were optimal. At necropsy, protrusion of the intes- tine, extensive hemorrhages on the skin of the left dorsal and ventral part, the presence of yellowish fluid in the abdominal cavity, pale and enlarged liver and severe hemorrhages inside the intestine were noted (Fig 1).
For bacterial isolation, sterile swabs from the kidney, liver, and spleen were streaked onto tryptic soy agar (TSA), and the inoculated plates were incubated for 24 h at 25oC. Suspected common colonies were re-streaked on TSA to acquire pure cultures, which were then simply identified on the basis of microscopic analysis results, with the aid of a Vitek System®2 (bioMérieux®, France) and an API 20E test for biochemical analysis. The bacterial DNA was extracted by boiling the cells followed by centrifugation. In order to identify accurately the isolated bacteria, P. damselae subsp. damselae from pure cul- ture, multiplex PCR was conducted as described previously (12). The purified PCR product was submitted to the Macro- gen Genomic Division (Korea) and nucleotide sequencing was conducted using an ABI PRISM Big Dye TM Termina- tor Cycle Sequencing Kit (Applied BioSystems, USA). Elec- trophoresis of the sequencing reactions was completed using an automated ABI PRISM 3730XL DNA Sequencing Sys- tem (Applied BioSystems, USA). The rRNA sequence gene of the bacterial strains acquired in this study was aligned with other bacteria of the same species (AY147861.1, EF635307.1
1Corresponding author.
E-mail: parksec@snu.ac.kr
Isolation of Photobacterium Damselae Subsp. Damselae from the Giant Grouper, Epinephelus Lanceolatus 619
and FJ971859.1) according to sequences contained in the Gen- Bank database; multiple alignment algorithms in the MegA- lign package (Windows Version 3.12e; DNASTAR Software Package, USA) were employed and the sequence similarity percentages were determined.
The antibiotic susceptibility of bacterial isolates was deter- mined via the disc diffusion method (2). The antibiotic discs (BBL, USA) employed in this study are shown in Table 2. The sensitivity and resistance of isolated bacteria and the zone diameter interpretive standards were determined in accor- dance with the CLSI (Clinical and Laboratory Standards Insti- tute) criteria for animal isolates (3).
After diagnostic examination, the observed clinical signs were similar to those described previously in conjunction with vibriosis and pasteurellosis in other fish (1). Streaking on TSA yielded an apparently pure transparent common bacterial growth from 3 organs of the fish sample. The results of our microscopic examination demonstrated that the isolates were gram-negative, motile, oxidase-positive, rod bacteria. Colo- nies measuring 2-3 mm in diameter formed on the TSA. These colonies induced β-hemolysis on blood agar. Additionally, according to the results generated with the Vitek System®2 (bioMérieux®, France), P. damselae was isolated with a prob- ability of 99%. According to the results of the API 20E test, the isolates differed slightly from the reference provided in
Bergey’s Manual of Determinative Bacteriology (8) (Table 1).
Collectively, these results indicate that the isolate was a mem- ber of P. damselae. The identification was further confirmed via a PCR assay using 16S rRNA gene and ureC gene prim- ers. The isolated strain evidenced both of the amplification products: 267bp of the 16S rRNA gene for Photobacterium damselae and 448bp of the ureC gene for Photobacterium damselae subsp. damselae (Fig 2). Moreover, as the result of 16S rRNA gene sequencing, P. damselae subsp. damselae consisting of approximately 950 nucleotides was isolated, and exhibited a sequence similarity of 100% with other P. damse- lae subsp. damselae strains contained in the GenBank data- base. As a result, the identification of P. damselae subsp.
damselae was confirmed.
The antibiotic resistance profile of the P. damselae subsp.
damselae strain is shown in Table 2. The bacterial isolate proved resistant to aminoglycosides. However, it was found to be sensitive to carbapenems, cephems, β-lactam, penicillins, quinolones, and tetracyclines.
Discussion
Confirmation via isolation in pure culture and the results of Fig 1. Postmortem examination of giant grouper. Hemorrhages
on the skin of left dorsal and ventral part (a). Protrusion of the in- testine (b). Yellowish fluid in the abdominal cavity (c). Discol- ored and enlarged liver (d). Hemorrhages inside the intestine (e).
Table 1. The characteristics of isolated strain as revealed by API 20E profile in comparison with the reference of Photobacterium damselae strain from Bergey’s Manual of Determinative Bac- teriology (8)
Characteristics API 20E Bergey’s Manuala
βD Galactosidase (ONPG) - -
A-Dihydrolase + +
L-Decarboxylase + +
O-Decarboxylase - -
Citrate - -
H2S - -
Urease + +
Tryptophane DeAminase + -
Indole production - -
VP + -
Gelatinase + -
Glucose + +/-
Mannitol - -
Inositol - -
Sorbitol - -
Rhamnose - -
Sucrose - -
Melibiose - -
Amygladin - -
Arabinose - -
aBergey’s Manual of Determinative Bacteriology.
−: negative, +: positive, +/−: positive or negative.
620 Jin-Woo Jun et al.
a PCR assay conducted on the giant grouper in this study indicated clearly that the isolated strain was P. damselae subsp. damselae. This bacterium has been recognized as an opportunistic pathogen for a broad variety of hosts, including fish and mammals (14). A number of marine animal diseases associated with this organism have been reported around the world (13). This bacterium was previously shown to cause septicemia in warm-water and cold-water fish such as dam- selfish (Chromis punctipinnis), eels (Anguilla anguilla), brown shark (Carcharhinus plumbeus), yellowtail (Seriola quinquer- adiata), seabream (Sparus aurata) and turbot (Scophthalmus maximus) (6). In all cases, the fish infected with this bacte- rium had skin ulcerative lesions in their mouths, eyes, and musculature, indicating that this bacterium may pose the risk of profound economic losses to aquaculture (6). Infection with this bacterium in private aquaria may be a more significant issue, as there are many types of valuable ornamental fishes.
This case occurred in the same private aquarium as a previ- ous case report regarding the isolation of P. damselae subsp.
damselae from the zebra shark (Stegostoma fasciatum) (7).
The giant grouper and the zebra shark had been reared in same water. So we assume that the giant grouper was infected by contact with the contaminated water. Although the LD50 of P. damselae subsp. damselae is relatively high and P. damse- lae subsp. damselae is commonly isolated in an aquatic environ- ment, this opportunistic bacterium can cause disease with acute stress-inducing factors, most notably overstocking and poor water quality (4,11). The P. damselae subsp. damselae which was isolated in this study was found to be sensitive to several commercial antibiotics. Thus, it is clearly necessary to develop a proper treatment for this condition using the most effective anti- biotics possible, along with the control of stress-causing factors.
Conclusion
In this paper, we have isolated and identified P. damselae subsp. damselae from a giant grouper reared in a private aquar- ium in Korea.
Acknowledgment
This study was financially supported by a Korean Research Foundation Grant (KRF-2008-331-E00385) and by Basic Sci- ence Research Program through the National Research Foun- dation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-0074437).
References
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multiplex PCR assay for detection of Photobacterium damselae subsp. damselae in giant grouper. Lane M, 100-bp DNA ladder;
lane P, positive control (Photobacterium damselae subsp. dam- selae ATCC 33539, 267bp of the 16S rRNA gene for Photobac- terium damselae, 448bp of the ureC gene for Photobacterium damselae subsp. damselae); lane K, amplification product (using bacteria from kidney); lane L, amplification product (using bac- teria from liver); lane S, amplification product (using bacteria from spleen); lane K, lane L and lane S, positive for Photobac- terium damselae subsp. damselae (267bp, 448bp); lane N, neg- ative control (no template DNA).
Table 2. Antimicrobial susceptibility test of Photobacterium damselae subsp. damselae isolated from giant grouper
Antibiotics (µg) Isolated strain
Amikacin (30) Ra
Amoxicillin/clavulanic acid (20/10) Sa
Ampicillin (10) Ia
Ampicillin/sulbactam (10/10) S
Cefazolin (30) R
Cefepime (30) S
Cefotaxime (30) S
Cefoxitin (30) S
Ceftazidime (30) S
Cefuroxime sodium (30) S
Cephalothin (30) S
Chloramphenicol (30) S
Ciprofloxacin (5) I
Gentamicin (10) R
Imipenem (10) S
Levofloxacin (5) S
Meropenem (10) S
Ofloxacin (5) S
Piperacillin (100) I
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Tetracycline (30) I
Trimethoprim/Sulfamethoxazole
(1.25/23.75) I
aThe category ‘S’ means sensitive to antibiotic; ‘R’ means resistant;
‘I’ means intermediate. Each category was decided by zone diam- eter interpretive standards (3).
Isolation of Photobacterium Damselae Subsp. Damselae from the Giant Grouper, Epinephelus Lanceolatus 621
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Giant Grouper (Epinephelus Lanceolatus) 에서 Photobacterium Damselae subsp. Damselae 분리 및 특성
전진우·김지형·한지은·신상필·데니스 고메즈·카시아노 허모피아·오규선*·박세창1
서울대학교 수의과대학 수생동물질병학연구실, *미국 국립보건원 암연구센터
요 약 : 서울 소재 수족관에서 관상용으로 사육되었던 giant grouper (Epinephelus lanceolatus) 한 마리가 폐사된 채 발견되었다. 폐사어는 2주간 식욕부진, 무기력 및 침울의 증상을 보였다. 폐사어의 장기 (신장, 간 및 비장)로부터 세 균을 순수 분리하여 Vitek System 2, API 20E test, multiplex PCR 및 16S rRNA gene sequencing을 수행하여 Photobacterium damselae subsp. damselae로 확진하였다. 본 증례에서, 우리는 국내 수족관에서 사육되었던 giant grouper에서의 Photobacterium damselae subsp. damselae의 분리 및 특성을 보고하고자 한다.
주요어 : giant grouper, Photobacterium damselae subsp. damselae
