| In recent years, aquatic animals’ diseases to the aquaculture development have caused great serious economic losses. To isolate the pathogen of diseased giant salamander(Andrias davidianus) came from Chongqing, identify the bacteria species, and determined its several extracellular enzymatic activities, two bacteria stains(K4L1 and CH6) were separated from the liver of diseased individuals. The morphological, physiological and biochemical characteristics, and 16 S r DNA sequence were analyzed for species identification. Its pathogenicity was tested by artificial infection. The extracellular enzymatic activities were detected by plate method. The results showed that one bacteria strain was Gram-negative short bacillus without spores, and the physiological and biochemical characteristics were consistent with Citrobacter freundii. Another bacteria strain was also Gram-negative short bacillus with flagellum were consistent with Aeromonas hydrophila.They 16 S r DNA sequence shared high homology of more than 99% with Citrobacter freundii and Aeromonas hydrophila, and were in the same cluster as Citrobacter freundii and Aeromonas hydrophila on the phylogenetic tree. Artificial infection with the bacteria to healthy frog, Cynops orientalis and giant salamander induced the same diseased symptoms as occurred naturally. The pathogen(K4L1) produced lecithinase and amylase, but no activity of lipases, proteases, gelatinase, and urease were detected. The another pathogen(CH6) produced lecithinase, lipases, proteases and gelatinase, but no activity of amylase and urease were detected. In conclusion, the bacteria stain isolated from diseased giant salamander were Citrobacter freundii and Aeromonas hydrophila, which showed strong pathogenicity to giant salamander. From the two aspects of the prevention and treatment of disease prevention and control of giant salamander were studied, including antibiotics, Chinese herbal medicine, disinfectants three aspects.The disc diffusion method was used to study the pathogenic bacteria resistance to 30 antibiotics. The experimental results showed that Citrobacter freundii had sensitive to ceftriaxone, ceftazidime and furazolidone, moderately sensitive to polymyxin B, neomycin and gentamycin, resistance to 24 kinds of antibiotics, drug resistance rate as high as eighty percent.We also have found that Aeromonas hydrophila had sensitive to 13 kinds of antibiotics as ceftriaxone, furazolidone and ciprofloxacin, moderately sensitive to piperacillin and kanamycin, resistance to sinomin compositea, carbenicillin and gentamycin of 15 kinds of antibiotics, drug resistance rate was fifty percent. These results suggested that the drug resistance of pathogenic bacteria has come to the point where serious giant salamander, may be caused by antibiotics abuse.The antibacterial effect of Chinese herbal medicines on Citrobacter freundii and Aeromonas hydrophila were detected through disc diffusion method(punch method). The results showed that most Chinese herbal medicines had the inhibitory activity on pathogenic bacteria strains, four species(F. schisandrae, F. Mume,R. chinensis Mill and T. chebula) had the best antibacterial role against two pathogenic bacterias,and the inhibitory circle of these four medicines exceeded 20 mm. The same effect of Chinese herbal medicine of two strains of bacterial pathogens were different, the inhibiting effect on Aeromonas hydrophila is significantly higher than Citrobacter freundii. The minimal inhibitory concentration(MIC) and the minimum bactericidal concentration(MBC) of Chinese herbal medicines which had significantly antibacterial effect against Citrobacter freundii and Aeromonas hydrophila were determined with the double dilution method. It was found that F. schisandrae had inhibitory and bactericidal actions against pathogenic bacteria strains, and MIC were both 7. 8125 mg /m L. Then 11 screened Chinese herbal medicines which had high efficiency bacteriostatic action on Citrobacter freundii were mixed by one based on chessboard experimental method to get 55 double combinations. To study synergism antibacterial activity of the compounds, the ratios of 0.5<FIC<1 and FIC≤0.5 to all FIC were 40% and 21.82% respectively. According to the result of MIC screen out 17 kinds of Chinese herbal medicine which highly sensitive to Aeromonas hydrophila, designing 136 kinds of double compounds and evaluation their efficacy.We found that 38.97% compounds had significant synergies and 36.03% compounds had synergistic effect.The study indicated that the rational use of Chinese herbal compounds could not only reduce drug antimicrobial efficacy, but also significantly increase the concentration of Chinese herbs in the compounds.The antibacterial effect of disinfectants on Citrobacter freundii and Aeromonas hydrophila were detected through disc diffusion method. The results showed that most disinfectants had the inhibitory activity on pathogenic bacteria strains, the same effect of disinfectants of two strains of bacterial pathogens were different, and bacteriostasis effect decreased with the disinfectant concentration reduce. MIC and MBC of disinfectants which had significantly antibacterial effect against Citrobacter freundii and Aeromonas hydrophila were also determined with the double dilution method. Evaluation disinfection effect, its found that such as formaldehyde and peracetic is efficient disinfectant. Na OH and symclosene were intermediate efficacy disinfectants, potassium permanganate was inefficient disinfectants. Then 8 screened disinfectants which had high efficiency bacteriostatic action were mixed by one based on chessboard experimental method to get 28 double combinations. To study synergism antibacterial activity of the compounds against Citrobacter freundii, the ratios of 0.5<FIC<1 and FIC≤0.5 to all FIC were 10.71% and 57.14% respectively. It’s found that 35.71% compounds had significant synergies and 25% compounds had synergistic effect against Aeromonas hydrophila. The study also indicated that the antibacterial effects were improved significantly after use of disinfectants by reasonable compatibility.In summary, the Citrobacter freundii and Aeromonas hydrophila from Andrias davidianus have developed resistance to many antibiotics. At present, more and more traditional Chinese herbal medicines treatment have been recognized to prevent bacteria diseases. Studying the drug sensitivity, resistance characteristics and selecting the disinfectants, drugs and Chinese herbal medicines with high antibacterial activity, can provide the theoretical basis for the prevention and controlling of disease research on Andrias davidianus. |
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