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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
余露军, 李建军, 魏远征, 蔡 磊, 苗宗余, 黄 韧
广东省实验动物监测所 广东省实验动物重点实验室
摘要:
为探讨迟缓爱德华菌(Edwarsiella tarda)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、96 h。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(2.51×106 CFU/g), 其次为鳃(4.19×104 CFU/g)、血液(1.65×104 CFU/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(4.55×106 CFU/g)和血液(4.65×106 CFU/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。
关键词:  诸氏鲻虾虎鱼(Mugilogobius chulae)  迟缓爱德华菌(Edwarsiella tarda)  绿色荧光蛋白  入侵途径  组织分布
DOI:10.11759/hykx20180315001
分类号:
基金项目:国家科技支撑计划项目(2015BAI09B05); 广东省科技计划项目(2017B030314171, 2017A070702001)
Infection kinetics of GFP-Labeled Edwarsiella tarda in Mugilogobius chulae
YU Lu-jun, LI Jian-jun, WEI Yuan-zheng, CAI Lei, MIAO Zong-yu, HUANG Ren
Abstract:
Edwarsiella 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 pathogenesis 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 fluorescing 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 (2.51×106CFU/g), followed by the gills (4.19×104CFU/g) and blood (1.65×104CFU/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 (4.65×106CFU/g) and liver (4.55×106CFU/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 (2.93×104 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 E. tarda 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.
Key words:  Mugilogobius chulae  Edwarsiella tarda  green fluorescent protein (GFP)  invasion pathway  tissue distribution
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