| Aeromonas hydrophila is a common pathogenic bacterium in aquaculture bacterium which can lead to large-scale outbreak of disease and bring to huge economic losses in the breeding of the fishery. In addition, the bacterium can also cause the diarrhea and sepsis of birds and mammals in some reports, so Aeromonas hydrophila have attracted wide attention of domestic and overseas researchers.In the paper virulence factor test and artificial infection test, and tissue distribution of bacterium were studied. The result provided theoretical support for the transmission route and pathogenesis of Aeromonas hydrophila.The experimental bacterial stain was obtained from the professor Yang Xianle of Shanghai Ocean University. DNA of bacterium was extracted after NB liquid medium activatio n. Several specific primers were designed according to the reported conserved sequence of aerolysin(AH-78-2-Aer) gene, then the aero ly sin gene of Aeromonas hydrophila was amplified by PCR technique. The sequencing result proved that the amplified fragment was the specific gene of Aeromonas hydrophila. The homology of sequence was 99% with the reported sequence in the GenBank. The result indicated that the experimental strain of Aeromonas hydrophila carried virulence factor.Cyprinus carpio haematopterus was used as experimental material, and different concentrations of Aeromonas hydrophila has been injected into experimental groups carp, then the regular observation and record for fishy clinical symptoms and deaths were performed. The clinical symptoms of experiment group were showed as following:hyperemia in fish body surface, black body surface, rottenness of body, abdominal distention, anal swelling. The characteristics of anatomy in experimentl group were showed as following:peritoneal effusion, internal organs congestion, pale yellow mucus around intestinal wall, a bigger bladder volume, thick bladder wall, loss-blood and pale gills. The control group had no any adverse symptoms and death records. Tissue-isolation method was used in the infected fish body, at last, we obtained the pure culture bacteria. By colonial morphology observation, gram staining, physiological and biochemical reaction phenomena, scanning electron microscope observation and 16SrDNA sequencing analysis, and experimental strains can be isolated from sickness or death fishes. On the whole, the result shows that the strain, which causes disease for experimental groups was the inoculated strain of Aeromonas hydrophila, and the strain had a strong pathogenic effect to Cyprinus carpio haematopterus.Susceptibility-medicine paper method was used to detect 15 kinds of antibiotics in a drug sensitive experiment, and the result showed that only ammonia benzyl, cefalotin cephalosporin, rifampicin, amikacin and gentamicin had a sensitive effect to the strain. It interpreted that the strain had resistance to many antibiotics when a large-scale use of antibiotics was prevailing in the fishery breeding.The strain was directly used to infect the Cyprinus carpio haematopterus in aquarium in order to imitate natural infection condition and tissue frozen-sections of different tissues were prepared after experimental fish had symptoms of infection. Then in situ hybridization experiment of tissue was developed on the basis of a specific probe and analyzed the distribution of Aeromonas hydrophila in different organs. The result showed that Aeromonas hydrophila can been detected in the muscle, intestines, kidneys, liver, gill and brain tissue. More positive signals can been found in kidneys and liver, it indicated that the organs had severe infection of Aeromonas hydrophila. In kidneys and liver, some signals of in situ hybridization were violet black and developed a few large regions of precipitation. |
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