50 SEPTEMBER 2016 • WORLD AQUACULTURE • WWW.WAS.ORG Infectivity experiments. To test the pathogenicity of P. putida obtained from hybrid catfish, one strain was selected for experimental infection. Six groups (20 fish in each group) of healthy, 200-300 g hybrid catfish were acclimated in separate 1-m3 tanks at 25-27 C for 7 d prior to bacterial challenges. Catfish were challenged with 0.1 mL bacterial suspensions of the strain in 0.8 percent NaCl solution at approximately 3.4×108, 3.4×107, 3.4×106, 3.4×105, 3.4×104 CFU/mL for each group via intraperitoneal injection. The control group received an intraperitoneal injection of the same volume of sterile 0.8 percent NaCl solution. Clinical signs and mortality were recorded daily for 14 days after the challenge. Re-isolation and identification of the bacteria from culture of kidneys and spleens from moribund experimental fish were performed on blood agar plates. All animal challenges were conducted following IACUC-approved protocols of Sichuan Agricultural University. Histopatholog y. Tissues for histopathology were collected and fixed immediately in 10 percent neutral buffered formalin (1:10 ratio of tissue to fixative). Tissues included gill, brain, eye, kidney, spleen, heart, liver, nares, stomach, intestine and body wall. Bone and cartilage (cranium, eye, body wall, and gill) were decalcified by immersion for 24 hours in a commercially available decalcification fluid. After decalcification, tissues were trimmed into cassettes, dehydrated in graded ethanol solutions, cleared in xylene, and embedded in paraffin wax. Tissues were cut to 5 μm and stained with hematoxylin and eosin. Antimicrobial susceptibility testing. Antibiotic susceptibilities of isolated strains were determined using the disc diffusion method and criteria specified by the National Committee for Clinical Laboratory Standards (CLSI 2006). Briefly, 5 mL of BHI broth were inoculated with one loop of culture. After incubation for 24 h at 28 C, the suspension obtained was uniformly spread onto the surface of dry Mueller-Hinton agar (Oxoid, Basingstoke, UK) plates containing 5 percent defibrinated sheep’s blood using broth-impregnated swabs. The inoculum concentration was approximately 1.0×108 cfu/mL. Disks (Hangzhou Taihe Microbiological Reagent, Hangzhou, China) of nine antimicrobial agents were used. Plates were incubated with antibioticimpregnated discs at 28 C for 24 h and inhibition of the bacteria by the antimicrobial drugs was scored. The sensitivity and resistance of each isolate were determined following the manufacturer’s instructions. bacteria (Weisburg et al. 1991), were used to amplify the 16S ribosomal gene (Jensen et al. 2002). The universal primers reported in a previous work (Yamamoto and Harayama 1995) for the amplification of partial gyrB genes from Escherichia coli, Pseudomonas putida, and Bacillus subtilis were used to amplify the gyrB gene. These primers were used to yield an expected approximately 1.5-kb segment of the 16S rRNA gene, and an expected approximately 1.2-kb segment of the gyrB gene. The PCR products were purified using the Gel DNA Purification Kit (TaKaRa, Dalian, China). Sequencing of PCR products was performed using a 3730 DNA sequencer (Shanghai Invitrogen Biotechnology Co., Ltd., Shanghai, China). The obtained 16S rDNA sequences and the gyrB gene sequences were aligned to related sequences of bacteria in GenBank using the BLAST program. The aligned 16S rDNA sequences and the gyrB gene sequences of the related species were retrieved from the National Center for Biotechnology Institution’s (NCBI) nucleotide database. Phylogenetic and distance analysis of the aligned sequences was performed using the program MEGA 4.1 (Tamura et al. 2007). The resulting unrooted tree topologies were evaluated using the bootstrap analysis of the neighbor-joining method based on 1000 re-samplings. FIGURE 3. The phylogenetic tree of P. putida, based on 16S rDNA sequences, was generated by using the neighbor joining method, maximum composite likelihood, and 1000 bootstrap replicates. Bootstrap values (percent) are shown besides the clades, accession numbers are written besides the name of strains, and scale bars represent distance values. FIGURE 4. The phylogenetic tree of P. putida, based on gyrB gene sequences, was generated by using the neighbor joining method, maximum composite likelihood, 1000 bootstrap replicates. Bootstrap values (percent) are shown besides the clades, accession numbers are written besides the name of strains, and scale bars represent distance values. FIGURE 5. Marked locally extensive integumental and skeletal necrosis, with exposed tissues heavily colonized by rod-shaped bacteria. Arrow shows invasive bacteria in muscle.
RkJQdWJsaXNoZXIy MjExNDY=