Flavobacterium johnsoniae (formerly Cytophaga john-sonae) belongs to a large, diverse group of aerobic Gram-negative bacteria known as the Cytophaga-Flavobacterium-Bacteroides group (Bernardet et al., 1996). F. johnsoniae commonly exists in soil and is also frequently isolated from the external lesions of several fish species (Flemming et al., 2007). Generally, F. johnsoniae is not considered a serious fish pathogen, but the species has been associated with sev-eral disease outbreaks. For example, F. johnsoniae infection in farmed barramundi Lates carcalifer with 2-5% mortality was reported in Australia (Carson et al., 1993). Thereafter, these bacteria have been found in Atlantic salmon Salmo salar, sea trout S. trutta m. trutta and brown trout S. trutta m. lacus-tris with more than 5% mortality during outbreaks in Finland (Rintamaki-Kinnunen et al., 1997). Recently, F. johnsoniae was also isolated from the external lesions of fish from aqua-culture systems in South Africa (Flemming et al., 2007).
The rainbow trout Oncorhynchus mykiss is the most im-portant freshwater fish species in aquaculture in Korea, with annual production increasing annually (Yoon, 2008). The spe-cies is susceptible to the fish-pathogenic bacteria of the ge-nus Flavobacterium, i.e., F. columnare (Suomalainen et al., 2005), F. psychrophilum (Misaka and Suzuki, 2007), and F. branchiophilum (Ko and Heo, 1997), but F. johnsoniae has not been reported in rainbow trout to date.
The purpose of this study is to report the isolation and char-acterization of F. johnsoniae from farmed rainbow trout in Korea.
Sixty-four juvenile rainbow trout (0.4-0.7 kg body weight) were collected from four hatcheries in Gangwon Province, South Korea, in 2010 for routine disease monitoring. Fish were packed individually in plastic bags, transported to the laboratory, and processed immediately. Gill and kidney tissues were aseptically removed from individual fish and placed in 1.5-mL microcentrifuge tubes.
Gill and kidney tissue samples (100 mg each) were homog-enized with 0.9% (w/v) NaCl and then spread on trypticase soy agar (TSA) plates at 15℃ for 2-7 days. Among the colo-nies that appeared, yellow-pigmented colonies, characteristic of the genus Flavobacterium, were selected. These colonies were then subcultured on TSA plates at 15℃ for an additional 2-7 days and examined for biochemical characterization. A presumptive identification was carried out by Gram staining and oxidase and catalase tests, as described by Austin and Austin (2007). Colonies were tested for the presence of flexi-rubin pigments by being exposed to 10 N KOH, followed by neutralization with 12 N HCl. The biochemical characteristics of the bacteria were then determined using API 20E and 20NE kits (BioMerieux, Marcy l’Etoile, France) with incubation at 15℃ for 48-72 h. The chitinolytic activity (Richter and Pate, 1988), Congo red absorption (Crump et al., 2001), and gelatin and casein hydrolysis (Cepeda et al., 2004) of the colonies were also tested. The published biochemical characteristics of F. columnare, F. psychrophilum, F. johnsoniae, and F. branchiophilum were used as references (Ko and Heo, 1997; Tamaki et al., 2003; Austin and Austin, 2007; Kubilay et al., 2008; Karatas et al., 2010).
Genomic DNA was extracted from 5 mL of cultured bac-terial cells by the AccuPrep Genomic DNA Extraction Kit (Bioneer, Daejeon, Korea). Extracted nucleic acids were con-centrated to a final concentration of 100 ng/μL. PCR targeting the 16S rRNA was conducted using the universal bacterium-specific primer set, 20F (5′-AGA GTT TGA TCA TGG CTC AG-3′) and 1500R (5′-GGT TAC CTT GTT ACG ACT T-3′) (Weisburg et al., 1991). PCR was performed using the lyophi-lized AccuPower PCR PreMix tube (Bioneer) containing 20 μL of reaction mixture, which comprised 25 μM of each prim-er, 5 μL of extracted DNA and DEPC-treated water, with a DNA Engine Peltier Thermal Cycler (BioRad, Hercules, CA, USA). Amplification was carried out with an initial cycle at 95℃ for 5 min, followed by 30 cycles of 95℃ for 30 s, 51℃ for 1 min, and 72℃ for 2 min, with a final extension step at 72℃ for 5 min.
PCR products were purified by an AccuPrep Gel Purification Kit (Bioneer). Then, 10 ng/μL of purified PCR products were directly sequenced (ABI 3730xl; PE Applied Biosystems, Foster City, CA, USA; available at the National Instrumentation Cen-ter for Environmental Management, Seoul National University, Korea). The nucleotide sequences of the 16S rRNA genes were aligned with those available in the GenBank database (NCBI) according to Clustal W by MEGA version 4 (Tamura et al., 2007). This alignment was used to infer phylogenetic relation-ships among the sequences by the neighbor-joining algorithm with bootstrap values determined by 1,000 replicates. The 16S rRNA gene sequences of several F. psychrophilum, F. colum-nare, and F. branchiophilum strains were included as references, because these species are genetically very close to F. johnsoniae. The nucleotide sequence was deposited in GenBank under ac-cession no. GU461280.
From 64 fish samples, a total of 231 colonies were observed on TSA plates after 72 h at 15℃, and 18 fish samples (28.1%) from two hatcheries had yellow-pigmented colonies, which were selected for further identification (Table 1). All 18 iso-lates showed the same biochemical types with characteristics including rapid gliding motility, Gram-negative rods, oxidase-positive, catalase-positive, flexirubin pigment, nitrate reduc-tion, β-galactosidase-positive, acid production from glucose, Congo red absorption, and hydrolysis of gelatin and casein. Growth occurred with 1% NaCl in tryptic soy broth (TSB) but not with 1.5% NaCl in TSB and no growth occurred at 4℃. The biochemical characteristics of the bacteria are sum-marized in Table 2.
The amplification of 16S rRNA from all 18 yellow-pig-mented colonies showed a band of approximately 1,300 bp (data not shown). Highly conserved sequences (97-99%) were found among the 18 isolates (Fig. 1), and one isolate was selected and named FjRt09 (GenBank accession no. GU461280). Phylogenetic tree analysis showed that FjRt09 had 98% homology and grouped more closely with F. johnso-niae than with F. columnare, F. psychrophilum, and F. bran-chiophilum (Fig. 2).
Gram-negative yellow-pigmented bacteria belonging to the genus Flavobacterium have been reported from external
lesions on fish, e.g., gill erosion, fin or tail rot, and saddle-like lesions (Rintamaki-Kinnunen et al., 1997). However, the symptoms are usually not specific and sometimes individu-als present with no external symptoms. Thus, it is difficult to identify the bacteria, and they are frequently misidentified (Austin and Austin, 2007).
Flavobacterium johnsoniae is known as an opportunistic skin pathogen in certain fish species (Carson et al., 1993). Rintamaki-Kinnunen et al. (1997) showed that brown trout with lesions caused by this bacteria have a high risk of mor-tality, which can be accelerated by high water temperature
and rearing density. F. johnsoniae has been detected in bar-ramundi, Atlantic salmon, sea trout, brown trout, brook trout, and even in the freshwater environment (Carson et al., 1993; Rintamaki-Kinnunen et al., 1997; Hahn et al., 2004; Clark et al., 2009; Seo et al., 2009). However, there has been no report of the isolation of F. johnsoniae from rainbow trout to date. Hence, this is the first report documenting F. johnsoniae in rainbow trout. Of the four hatcheries tested, two were found to harbor F. johnsoniae, based on rainbow trout gill and kidney samples. It is still unclear if these bacteria strains are patho-genic to rainbow trout in those hatcheries, because the suscep-tibility of rainbow trout to infection by these bacterial isolates was not tested in this study. F. johnsoniae can be opportunistic under certain conditions, such as a sudden change in water temperature, stress caused by handling, or negative environ-mental factors (Carson et al., 1993; Karatas et al., 2010).
In this study, 18 out of 64 rainbow trout sample plates de-veloped yellow-pigmented colonies. The affected samples originated from two hatcheries, although the fish showed no external clinical signs. Biochemical characterization was conducted on the 18 yellow-pigmented colonies, and the bio-chemical characteristics of the bacterial isolates corresponded to those of F. johnsoniae (Carson et al., 1993; Karatas et al., 2010). PCR and subsequent sequencing of 16s rRNA genes also identified all 18 yellow-pigmented colonies as F. johnso-niae with similarities of 97-99%; thus, one strain was selected and named FjRt09. A phylogenetic tree based on 16s rRNA gene sequences was constructed for FjRt09 and other F. john-soniae, F. columnare, F. psychrophilum, and F. branchiophi-lum. The phylogenetic tree demonstrated that the FjRt09 strain clustered in the F. johnsoniae group and shared a branch with F. johnsoniae P550 isolated from brook trout in the United States (Clark et al., 2009). It remains unclear how FjRt09 shares a close relationship with P550, and it would be interest-ing to investigate the relationship between these isolates from geographically distant areas, because there have been no fur-ther reports of F. johnsoniae in the Asia-Pacific region to date. Moreover, the pathogenic potential of the F. johnsoniae isolate in Korea also needs to be studied.