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棘胸蛙腐皮症病原菌的鉴定及疫苗免疫效果研究
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Abstract:
为确定棘胸蛙(Quasipaa spinosa)腐皮病的病原，并为生产中防治该病提供理论依据，本研究从病蛙病灶处分离病原菌，进行人工感染和病原菌的形态特征、理化特性及分子生物学分析，同时开展药敏试验和疫苗免疫防治试验。结果表明，从患病棘胸蛙病灶处分离得到的优势菌QS01，通过人工回归感染试验证实该菌为棘胸蛙腐皮病的病原菌，结合其表型特征及16S rRNA、gyrB基因序列分析判定为弗氏柠檬酸杆菌(Citrobacter freundii)。药敏试验结果显示，该菌对恩诺沙星、氯霉素和头孢他啶等8种抗生素敏感，而对其他抗生素敏感度低或具有一定的抗性。采用注射、浸泡和喷雾方式给蛙接种QS01甲醛灭活疫苗，三组的平均效价均于第20 d时达到峰值，血清抗体效价最高分别为1:64~128 (101.6)、1:16~32 (20.2)和1:16~32 (16)，明显高于对照组，经攻毒后免疫保护率分别达到100%、85.71%和71.43%。试验发现弗氏柠檬酸杆菌可感染棘胸蛙导致其腐皮病，使用疫苗对此病的防治是有效可行的。
In order to determine the pathogen of the skin rot disease of Rana spinosa and provide theoretical basis for the prevention and control of the disease in production, this study isolated the pathogenic bacteria from the diseased frog focus, carried out artificial infection and the morphological characteristics, physical and chemical characteristics and molecular biological analysis of the pathogenic bacteria, and carried out drug sensitivity test and vaccine immunity and control test. The results showed that the dominant bacteria QS01 isolated from the diseased lesions of Rana spinosa was confirmed to be the pathogen of skin rot disease of Rana spinosa by artificial regression infection test. Combined with its phenotypic characteristics and 16S rRNA, gyrB gene sequence analysis, it was determined to be Citrobacter freundii. The medicine sensitive test result showed that the bacteria is sensitive to eight kinds of antibiotics including Enrofloxacin, Chloramphenicol, Ceftazidimeetc, but lowly sensitive or resistant to other tested antimicrobial agents. Adopting these ways included injection, immersion and atomizing to inoculate frog with vaccine named QS01 inactivated by formaldehyde, average titer of three immunity groups reached the peak in the 20 d. The highest serum antibody titers were 1:64~128 (101.6), 1:16~32 (20.2) and 1:16~32 (16), which were significantly higher than those of the control group. The immune protection rate for Q. spinosa after injection, immersion and spray of QS01 inactivated vaccine can reach 100%, 85.71% and 71.43%. Our research shows that Citrobacter frederii is the pathogen of skin rot disease of Rana spinosa, and it is effective and feasible to use vaccine to control ulcerative skin disease.
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