中华鳖爱德华菌病病原菌的分离鉴定及致病因子研究

Identification and pathogenic facts studying for Edwardsiella tardafrom Edwardsiellosis of Trionyx sinensis

采用API 20E系列生化鉴定及16S rDNA和gyrB基因序列同源性分析方法,对从患病中华鳖(Trionyx sinen-sis)肝脏中分离到的一株细菌TL5m进行了鉴定,并通过人工感染试验,对该菌株进行了毒力检测;此外分别提取该TL5m株的主要致病因子外膜蛋白、脂多糖和胞外产物,对中华鳖进行毒力和免疫保护率试验。结果显示:菌株TL5m的API 20E鉴定编码为4544000,99.9%为迟钝爱德华氏菌(Edwardsiella tarda);其16SrDNA序列和gyrB基因序列(GenBank登录号分别:EF121756和GU563803)与迟钝爱德华氏菌的同源性最高(分别为94%和98%);菌株TL5m对中华鳖的半数致死量LD50为2.45×106 CFU/ind。药敏感结果显示菌株对磷霉素、菌必治、头孢孟多、头孢噻吩、壮观霉素高度敏感。外膜蛋白攻毒剂量60μg/ind和脂多糖攻毒剂量400μg/ind时,对中华鳖的致死率都为33.3%,胞外产物对中华鳖的LD50为31.73μg/ind;全菌灭活苗、胞外产物、外膜蛋白和脂多糖的免疫保护率分别为75%、62.5%、25%和87.5%。结果表明,发病中华鳖的病原菌为迟钝爱德华氏菌,其分泌的胞外产物对中华鳖具有较高毒力;提取的脂多糖对中华鳖遭受迟钝爱德华氏菌攻击具有较高免疫保护率。

By means of API 20E biochemical identification and 16S rDNA gyrB gene sequences analysis method,a pathogenic bacterial strain TL5m was isolated from the liver of diseased Chinese soft-shelled turtle and identified,then the virulence of this strain were tested by experimental infection.In addition,the main pathogenic factor outer membrane protein（OMP）,lipopolysaccharide（LPS）,and extracellular products of strain TL5m were extracted,to conduct virulence and immune protection rate test for Chinese soft-shelled turtle.The result shows： strain TL5m is identified as API 20E identification code 4544000,and its 99.9% are Edwardsiella tarda,its 16S rDNA sequences and gyrB gene sequences（GenBank accession numbers are EF121756 and GU563803） have the highest homologous（94% and 98% respectively） compared to E.tarda,the median lethal dose LD50 of strain TL5m to Chinese soft-shelled turtle is 2.45×106 CFU/ind.Drug sensitivity results shows： strain TL5m is highly sensitive to fosfomycin,ceftriaxone,cephalosporins,cefalotin and spectinomycin.When the infection dosage for OMP is 60 μg/ind and for LPS 400 μg/ind the death rate of Chinese soft-shelled turtles are 33.3%,the LD50 of extracellular products to the Chinese soft-shelled turtle is 31.73 μg/ind,the immune protection rate for whole-cell destruction live vaccine,extracellular products,OMP and LPS are 75%,62.5%,25% and 87.5%.The results show the pathogen of Chinese soft-shelled turtle disease is E.tarda.Its secretion of extracellular products has a high virulence to the Chinese soft-shelled turtle,extraction of LPS has a higher immune protection rate against E.tarda which infecting Chinese soft-shelled turtles.This provided a basis for developing the subunit vaccine against E.tarda.