WWW.WAS.ORG • WORLD AQUACULTURE • SEPTEMBER 2016 49 Pseudomonas putida is a rod-shaped, aerobic, Gramnegative bacterium commonly found in natural sources of water, soil and the surfaces of living organisms (Bouallègue et al. 2004). P. putida is commonly considered an important opportunistic human pathogen that can lead to sepsis and bacteraemia (Fujita et al. 1998, Korcova et al. 2005, Yoshino et al. 2011, Liu et al. 2014). However, the role of this bacterium in aquatic animal diseases is less clear. It has been reported as an opportunistic pathogen that infects some fish species. For instance, P. putida was considered to be the cause of hemorrhagic ascites of ayu (Wakabayashi et al. 1996), an epizootic in yellowtail (Kusuda and Toyoshima 1976), skin ulcer in rainbow trout (Altinok et al. 2006) and disease in oyster toadfish (Smolowitz et al. 1998) and large yellow croaker (Shen et al. 2008). Hybrid catfish Silurus asotus♀ × Silurus meridionalis♂ is one of the most numerous catfish species in China. Because of its good meat quality and wide consumer acceptance, this species has become an important foodfish in China. However, the rapid expansion and intensification of hybrid catfish aquaculture have led to a series of problems, including the occurrence of various diseases that have limited sustainable development of the industry. In the summer of 2014, a very serious infectious disease broke out on a freshwater hybrid catfish farm in Sichuan Province, China. Almost all ponds on the hybrid catfish farm were affected. The main clinical signs of disease were listlessness, loss of appetite, skin discoloration and obvious deep dermal ulcers with necrosis of the underlying musculature on the flank, head or caudal peduncle. During Outbreak of Severe Infection Caused by Pseudomonas putida in Cultured Hybrid Catfish Silurus asotus♀ × Silurus meridionalis♂ X. L. Huang , Y. J. Duan, Z. J. Du and D. F. Chen, Y. Geng and K. Y. Wang the disease outbreak, water temperature ranged from 25 to 28 C. Diseased fish often died within several days and cumulative mortality reached 70 percent. We conducted a study to definitively identify the pathogen of this disease and to evaluate the pathogenicity of the pathogen. This is the first report of P. putida as a pathogen of hybrid catfish. Materials and Methods Bacterial isolation. Fifty diseased and moribund hybrid catfish with typical clinical signs of disease were collected from affected ponds for bacterial isolation. Specimens were transferred alive to the laboratory in plastic bags provided with oxygen. Sterile swabs of the kidney and spleen were made during necropsy for routine bacteriology from each fish. Bacteria were grown on blood agar at 28 C for 24-48 h. Dominant isolates were purified by streaking and re-streaking on the same type of agar plates. Pure-stock isolates were stored at -80 C in sterile balanced salt solution (0.8 percent NaCl, 0.11 percent K2SO4, 0.135 percent NaH2PO4, 0.005 percent NaHCO3; pH 7.2) supplemented with 15 percent glycerol. Bacterial identification. After incubation for 24 h at 28 C on blood agar, colonies were characterized using Gram-stain and oxidase, catalase, motility tests, and standard biochemical tests according to standard methods. Identification to species level was performed using the 16S rRNA gene and the gryB gene sequence analysis. Both genes were amplified by polymerase chain reaction (PCR). The universal primers 27F (5-AGAGTTTGATCCTGGCTCAG-3) and 1492R (5-GGCTACCTTGTTACGACTT-3), specific for 16S rRNA gene of (CONTINUED ON PAGE 50) FIGURE 1. Mutifocal skin ulcers appeared on the body, with marked skin discoloration and hemorrhaging around the ulcers FIGURE 2. Rod-shaped bacteria were found in kidney imprints with Diff-quick staining.
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