WWW.WAS.ORG • WORLD AQUACULTURE • SEPTEMBER 2016 51 Results Necropsy findings. Diseased fish had remarkable multifocal whitish skin discoloration and deep dermal skin ulcers on the dorsal fin, tail fin, caudal peduncle, peri-eye, and flank (Fig. 1). The skin of the ulcers was obviously necrotic, revealing the underlying musculature. The edge of ulcer appeared congested. Internal organs including liver, spleen, and kidney were moderately enlarged. The stomach and intestine were empty. Rod-shaped bacteria were easily found in the kidney imprint (Fig. 2). Bacterial isolation and identification. More than twenty colonies grew on each plate in all samples after incubation at 28 C for 24 h on blood agar. However, only one type of colony was dominant on each plate. These were faint yellow in color, translucent, round, convex and 1.0~2.2 mm in diameter. Four isolates were obtained from different samples and numbered DYJ140914, DYJ140915, DYJ140916 and DYJ140917. All isolates had the same basic physiological and biochemical characteristics, indicating characteristics of P. putida. The bacteria were cytochrome oxidase, arginine dihydrolase, and urease positive; fermentation of glucose, utilization of citrates and arabinose, reduction of nitrate; ornithine decarboxylase, and lysine decarboxylase negative; a lack of indole and H2S production; and non-utilization of malonate, sucrose, sorbitol and melibiose (Table 1). For similar physiological and biochemical characteristics, one isolate (DYJ140914) was chosen to be identified with 16S rRNA gene sequences and gyrB gene sequences analysis. The nearly fulllength 16S rRNA gene sequences of DYJ140914 were amplified and compared with the related 16S rRNA sequences of bacteria in GenBank. It was submitted to NCBI and subsequently assigned NCBI accession number KP693689.1. A phylogenetic tree was constructed based on the 16S rDNA sequences of the isolate and the homologous sequences of other strains of P. putida (Fig. 3). The isolate investigated in this study, together with the other three strains, formed a tight cluster with 99.9 percent sequence similarities. The partial gyrB gene sequences (1.2 kbp) of the isolate were amplified and sequenced. It was submitted to NCBI and subsequently assigned NCBI accession numbers KP693690.1. A phylogenetic tree (CONTINUED ON PAGE 52) TABLE 1. Characteristics of P. putida isolates from hybrid catfish compared with published P. putida isolates. Items DYJ140914- P. putidaa P. putidab DYJ140917 Arginine dihydrolase + + + Ornithine decarboxylase + n.a. + Lysine decarboxylase + n.a. + Cytochrome oxidase + + n.a. Urease + + + Indole test + + n.a. H2S + + + Glucose fermentation + + + Citrates + n.a. + Malonate + n.a. + Sucrose + + + Sorbitol + + + Melibiose + n.a. + Mannose + + n.a. Mannitol + + + Maltose + + + Arabinose + + + Nitrate + n.a. + MR test + n.a. n.a. VP test + n.a. n.a. Xylose + n.a. + Rhamnose + + + Inositol + n.a. + Galactose + n.a. n.a. Gelatin liquefaction + n.a. + a, b Phenotypic characteristics of P. putida obtained from Altinok et al. (2006) and Shen et al (2008). n.a.: not applicable. This test was not done for reference strain. FIGURE 6. Necrotizing hepatitis with rod-shaped bacteria, with multifocal are marked. Multifocal necrosis in the liver (A) and the magnification of the foci (B). Hepatic cells were markedly necrotic, with chromatin condensed, karyoclasis and massive rod-shaped bacteria. Arrows in figure A showed the necrotic foci and Arrows in B show the invasive bacteria.
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