| Yersinia ruckeri is one genus of Yersinia, as a common pathogenic bacteria of coldwater fish, infection of the bacteria cause fish death and serious economic losses in aquaculture industry, It is necessary to prevent and control the disease caused by the pathogenic bacteria in coldwater aquaculture, epidemiological investigation of bacterial coldwater disease was carried out and found that a bacterial strain named BH1206, which was isolated from the liver and spleen of infected rainbow trout in July 2013. The strain BH1206,exhibited pathogenicity to rainbow trout. Clinical signs in BH1206 infected fish including punctuature on the body surface, especially on the abdominal and rostral side, different degree of hemorrhages in all visceral organs, these symptoms same as Enteric redmouth.Because of large economic losses for fisherman, at present, the disease has be focused as an important disease of cold-water fish farming. Therefore, it is necessary to study of the etiology of the disease and prophylaxis, to prevention provide a theoretical support.Under sterile conditions, a bacterial strain named BH1206 was isolated by TSA from the liver and spleen of the diseased rainbow trout. No parasite was found under microscope with the sick rainbow trout parasite, after infecting the normal rainbow trout and observing the symptom of the disease, a high pathogenicity was isolated, and test the toxicity of the bacteria, and then different concentration undetermined bacteria infected normal tilapias and did the qualitative probability date analysis, determining the lethal concentration of 50%(LD50), Observing the pathological structure of the liver, spleen and other different tissue and organs from infected rainbow trout to ensure the position of pathology; From the test of biochemistry, physiology and 16 S r RNAto identify the property of the strain; by the test of controlling different temperature, salinity, p H three factors to determine the bacteria cultures most suitable growth conditions; Using the diffusion method(KB method) susceptibility testing, specifically the sensitivity of bacteria to antibiotics; Immunization by injection of immune and soak two methods rainbow trout awakened vaccine. The experimental results are as follows:(1) The experimental results show that a high pathogenic bacterium named BH1206, which belongs to Yersinia ruckeri. This is the first report on the high mortality for Rainbow trout infected by Yersinia ruckeri systematically in China. According to the physical and chemical characteristics as well as Biolog system, it can be determined primarily the bacteria for Yersinia ruckeri. A phylogenetic tree was constructed by comparing the 16 S r RNA sequence of the strain BH1206(Gen Bank accession number KJ850444) with other relative bacteria species in the Gen Bank. In the phylogenetic tree, BH1206 and Yersinia ruckeri wereclustered in a branch.According to morphological, physical and chemical characteristicsand phylogentic analysis, strain BH1206 was identified as Yersiniaruckeri. The optimum growth condition of BH1206 was p H=6.5-7.5, salinity=0, and temperature=28-30℃.(2) Antimicrobial susceptibility tests showed that BH1206 was highly susceptible to 11 kinds of drugsincluding ofloxacin, ciprofloxacin, norfloxacin, enrofloxacin, levofloxacin, chloramphenicol, tetracycline, doxycycline, tetracycline, and trimethoprim-sulfamethoxazole carbenicillin, while completely resistant to amoxicillin, rifampin, polymyxin.(3) The trial infected normal rainbow trout and did the qualitative probability data analysis, determining the lethal concentration of 50%(LD50), the logarithmic concentration is 6.46×107 CFU/mL, in pathological tissue sections can be observed to the liver and spleen cell necrosis, produce large amounts of inflammatory cells, intestinal tissue withering deformation, there was a marked kidney disease.(4) The immune experiment shows that after 30 days, the challenge trial showed that the RPS of soak immunity can reach up to 75.0% and 71.16%, and the RPS of injection immunity can reach up to 89.09% and 85.45%, injection immunized group significantly improved their immune protection rate. In this experiment, for prevention and follow-up studies of Yersinia ruckeri infection of rainbow trout to provide a theoretical basis.The following conclusions:The test analysis BH1206 strains isolated from diseased rainbow trout identified is Yersinia ruckeri, which is a pathogens of Enteric redmouthin rainbow trout.Intraperitoneal injection of formalin inactivated vaccine for immunization of BH1206 have reached a certain immune effects, immune protection rate reached 89.09%. |
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