52 SEPTEMBER 2016 • WORLD AQUACULTURE • WWW.WAS.ORG was constructed based on the gyrB gene sequences of the isolate (DYJ140914) and the homologous sequences of other strains of P. putida (Fig. 4). The isolate investigated in this study together with P. putida (KC189956.1) formed a tight cluster with 99.0 percent sequence similarities. On the basis of morphology, biochemical and physiological characteristics, and 16S rDNA and gyrB gene sequences analysis results, the isolate was identified as P. putida. Histopatholog y. All fish had multi-tissue and systematic marked lesions, especially in the liver and on the skin. Some moderate changes could also be seen in the spleen, kidney, heart and intestines. Skin tissue sections indicated marked necrotic dermal dermatitis with massive infiltration of bacteria (Fig. 5). All tissue sections of liver indicated necrotizing hepatitis with marked, multi-focal necrosis and numerous rod-shaped bacteria (Fig. 6). Diseased fish also had moderate splenitis, interstitial nephritis and endocarditis, and mild gastroenteritis. Rod-shaped bacteria were also easily found in the spleen, interstitial kidney, and sometimes in the heart and gastrointestinal tract. No significant morphological changes were found in the brain or gills. Pathogenicity. The challenge experiment indicated that strain DYJ140914 was lethal to hybrid catfish. Affected fish showed similar clinical signs of disease as naturally infected hybrid catfish. On the first day after challenge, affected fish displayed only less swimming and anorexia. The first fish died after the second day post-challenge and none died after day seven. Dead fish showed obvious multifocal round areas of skin discoloration and cutaneous hemorrhage at the edge of lesions. Some had obvious dermal ulcers. The bacterial strain could be re-isolated as pure colonies from the kidney and spleen of all challenged hybrid catfish, and the re-isolated strain was also strongly virulent to healthy channel catfish. No mortality or visible changes were observed in the control group. Antimicrobial susceptibility testing. Isolates were sensitive to doxycycline, norfloxacin, levofloxacin; had intermediate susceptibility to neomycin; and were resistant to florfenicol, sinomin, cefoxitin, azithromycin, and rifarnpin (Table 2). On the basis of the sensitivity results, doxycycline, norfloxacin, levofloxacin are recommended to producers to control outbreaks of this disease. Discussion Ulcerative dermatitis can be found frequently in catfish. However, disease outbreaks in cultured catfish have been reported without a definitive etiology. Vibrio mimicus can infect freshwater catfish and lead to skin ulcers (Geng et al. 2014, Zhang et al. 2014). Skin erosions or ulcers in southern catfish were caused by Edwardsialla ictaluri (Geng et al. 2013) or Aeromonas caviae (Ji et al. 2008). Aeromonas caviae-like bacterium were responsible for ulcerative disease in Indian catfish Clarias batrachus (Thomas et al. 2013). In this study, four bacterial isolates were obtained from diseased hybrid catfish and these were confirmed to be P. putida by morphological, biochemical, and molecular assays. To our knowledge, this is the first report of P. putida infection in hybrid catfish. Further studies are needed to evaluate the possibility that P. putida is a common pathogenic bacterium and can infect other freshwater catfish. Pseudomonas aeruginosa and Pseudomonas fluorescens are considered to be opportunistic pathogens in aquaculture (Doménech et al. 1999, Palleroni 2010, Thomas et al. 2014). However, other species of the genus may also induce serious infection. For instance, P. luteola could infect rainbow trout (Altinok et al. 2006), P. anguilliseptica infect farmed trout and salmon (Wiklund and Lonnstrom 1994), and P. alcaligenes infect Chinese sturgeon (Xu et al. 2015). Pseudomonas putida is not a common pathogen in aquaculture, but it can infect fish. It causes bloody ascites in ayu Plecoglossus altivelis (Wakabayashi et al. 1996), large abscesses in farmed yellowtail (Kusuda and Toyoshima 1976), and skin ulcers in rainbow trout (Altinok et al. 2006). Here we described clinical findings, histopathology and biochemical properties of P. putida, which can naturally infect hybrid catfish, causing severe mortality. Pseudomonas spp., especially P. putida, can result in mortality of fish in stressed and non-stressed environments (Smolowitz et al. 1998). The results support the idea that P. putida may be as an opportunistic pathogen in intensive aquaculture, but more proof is needed to demonstrate definitively. The results of the antibacterial drug sensitivity tests showed that all P. putida isolates from hybrid catfish were sensitive to only three antibiotics (doxycycline, norfloxacin and levofloxacin) and were resistant to most antibiotics tested. Compared to other bacteria, Pseudomonas spp. or closely related species had fast growth, versatile adaptation to different conditions, and can establish resistance to stresses, including antibiotics. Pseudomonas spp. showed the strongest potential to be an environmental reservoir of antibiotic resistance mechanisms (Meireles et al. 2013). Furthermore, the TABLE 2. The sensitivity of bacterial isolates to various antimicrobial agents. Antibiotics STANDARD (mm) Diameter of inhibition Sensitivity Resistant (R) Intermediate (I) Sensitive (S) zone (mm) Neomycin ≤12 13 16 ≥17 13 I Doxycycline ≤12 13 15 ≥16 20 S Florfenicol ≤12 13 17 ≥18 0 R Norfloxacin ≤12 13 16 ≥17 22 S Sinomin ≤12 13 16 ≥17 0 R Cefoxitin ≤14 15 17 ≥18 0 R Azithromycin ≤13 14 17 ≥18 0 R Levofloxacin ≤13 14 16 ≥17 24 S Rifarnpin ≤16 17 19 ≥20 13 R ~ ~ ~ ~ ~ ~ ~ ~ ~
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