GFP标记的迟缓爱德华菌感染诸氏鲻虾虎鱼后组织分布研究

Infection kinetics of GFP-Labeled Edwardsiella tarda in Mugilogobius chulae

为探讨迟缓爱德华菌(Edwarsiellatarda)入侵途径,建立感染模型,作者通过电转化法构建GFP标记的迟缓爱德华菌EtMc1512(质粒PMDpp-EGFP),实验设立浸泡组、腹腔注射组和肌肉注射组,感染后采集各组实验诸氏鲻虾虎鱼(Mugilogobius chulae)血液、鳃、肝脏、肠、肌肉,培养法统计分析各组织中的荧光细菌数;浸泡组取样时间为0、2、4、6、8、12、24 h,腹腔注射组和肌肉注射组取样时间为6、12、24、48、72、96h。结果显示,构建的EtMc1512-GFP具有较强荧光,GFP标记前后菌株毒力基因(citC、mukF、esrB、katB、fimA、gadB)检测结果均为阳性。浸泡感染后实验鱼各组织内的荧光菌随时间表现为先升后降的趋势,最高菌量出现在肠道(2.51×106CFU/g),其次为鳃(4.19×104CFU/g)、血液(1.65×104CFU/g),肠道荧光菌显著高于其他组织(P<0.05);腹腔注射感染后肝脏(4.55×106CFU/g)和血液(4.65×106CFU/g)菌量最高;肌肉注射感染后肌肉在48h首先检出荧光菌,血液(2.93×104 CFU/g)菌量最高。结果表明,肠道、肝脏和肌肉分别是迟缓爱德华菌浸泡感染、腹腔注射感染和肌肉注射感染诸氏鲻虾虎鱼的主要组织器官,在自然条件下迟缓爱德华菌经口感染诸氏鲻虾虎鱼风险较高。

Edwardsiella tarda is an important fish pathogen that causes septicemia in many marine and freshwater fish.In order to study the invasion pathway of E.tarda in Mugilogobius chulae and provide basic data on patho-genesis of E.tarda.The infection kinetics of E.tarda were investigated in vivo in M.chulae using a virulent strain,E.tarda EtMc1512,that was transformed with a plasmid encoding green fluorescent protein(PMDpp-EGFP)by electroporation.Fish were divided into three groups:bacteria bath-challenged fish,bacteria intraperitoneally-challenged fish,bacteria intramuscularly-challenged fish.The number of bacteria from blood,gills,liver,intestine and muscle were detected at 0,2,4,6,12,24 and 48 h after immersion challenge,which were also detected at 6,12,24,48,72 and 96 h after intraperitoneal and intramuscular challenge.The results showed that EtMc1512-GFP strain had the similar virulence characteristics as the parent strain and could be easily identified as bright green fluoresc-ing colonies.The virulence-related genes(citC,mukF,esrB,katB,fimA,gadB)have been detected simultaneity in strain EtMc1512 and EtMc1512-GFP.High numbers of bacteria were observed in the intestine(2.51×10^6 CFU/g),followed by the gills(4.19×10^4 CFU/g)and blood(1.65×10^4 CFU/g)of fish after immersion challenge.The number of bacteria was significantly higher in the intestine than other tissues(P<0.05).The highest numbers of bacteria were observed in the blood(4.65×10^6 CFU/g)and liver(4.55×10^6 CFU/g)of fish after intraperitoneal challenge.Bacteria was first detected in muscle(48 h)and the highest numbers of bacteria were observed in the blood(2.93×10^4 CFU/g)of fish after intramuscular challenge.These results indicate that the intestine,liver and muscle serve as an important infectious route of E.tarda in M.chulae after immersion,intraperitoneal and intramuscular challenge respectively.It would be high infection risk through the ingestion of contaminated food and water for M.chulae under natural conditions.