| Megalobrama amhlycephala is an important pond-reared freshwater fish in China. However, large-scale, high density culturing of Megalobrama amhlycephala has resulted in the occurrence of bacterial diseases, especially bacterial septicemia, causing huge losses to farmers. During August 2014, serious mortality of cultured Megalobrama amhlycephala occurred in a farm in Changzhou City, Jiangsu Province and infected fish presented mild symptoms of bacterial septicemia： white gills, blood spots on the abdomen and fin base, no food visible in the gut and slight hepatopancreatic congestion. To identify the pathogen infecting the Megalobrama amhlycephala and find effective inspection and prevention strategies for the disease, the dominant pathogenic strain was isolated from the liver and spleen of diseased Megalobrama amhlycephala and labeled the CZBY-1 strain. The phenotypic characteristics of the pathogenic strain were examined, including the morphological, physiological and biochemical characteristics, virulence factor detection and susceptibility test. The 16S rRNA gene was amplified and compared with sequences deposited in GenBank databases. Simultaneously, an artificial infection study was carried out on healthy Megalobrama amhlycephala by injecting different concentrations of a bacterial suspension of incubated CZBY-1 strain. Megalobrama amhlycephala, with body mass （100?5）g, were randomly assigned to five test groups (1×109, 1×108, 1×107, 1×106, 1×105 CFU/mL) and a control group, with 10 Megalobrama amhlycephala in each group and each test run in replicate. The Megalobrama amhlycephala in each test group were injected with 100μL of the appropriate bacterial suspension. Symptoms of the diseased Megalobrama amhlycephala and the mortality rate of each group were recorded daily, and the pathogenic strain from the infected Megalobrama amhlycephala was isolated to confirm the cause was strain CZBY-1. After one week, the mortality rate of Megalobrama amhlycephala in the test groups receiving 1×109 CFU/mL, 1×108 CFU/mL and 1×107 CFU/mL were 100%, 70% and 40%, respectively, and no deaths were observed in the 1×106 CFU/mL, 1×105 CFU/mL or control groups. Clinical symptoms in artificially infected Megalobrama amhlycephala were similar to those observed in naturally infected fish and the morphological, physiological and biochemical characteristics of the pathogenic strain isolated from the artificial test study were identical with strain CZBY-1. Bacterial strain CZBY-1 is smooth, opaque, round, gray-white color and the16S rDNA sequence is 1 440bp (GenBank accession No.: KF413424). The phylogenetic tree analysis, based on 16S rRNA gene sequences, shows that CZBY-1 strain clusters with a strain of Klebsiella Pneumoniae with homology more than 99%. The phenotypic characteristics and molecular biological analyses also show that CZBY-1 strain belongs to Klebsiella Pneumoniae. By using specific primers for the PCR amplification of ompC (Outer membrane protein C) gene, the 252bp gene fragments were amplified, indicating that CZBY-1 strain carried virulence factors of ompC. The drug susceptibility of the CZBY-1 strain was tested by paper extension methods using 44 test antibiotics and the results show that Klebsiella Pneumoniae was highly sensitive to 27 antibiotics including Fosfomycin and Cotrimoxazole, but has strong resistance to Streptomycin, Vancomycin and Tobramycin. |
