宽体金线蛭水肿病病原的分离鉴定与病理学研究

Isolation,identification and histopathological study on oedema pathogen from cultured leech(Whitmania pigra)

为明确引起宽体金线蛭(Whitmania pigra)水肿病爆发的原因,并为鱼药研究提供新思路,从患病水蛭的体腔液分离得到优势株CQ1808,通过形态学观察、生理生化特性测定和16S rDNA基因序列分析鉴定CQ1808菌株,采用K-B纸片扩散法对该菌进行药物敏感性试验,通过人工感染和病理组织切片观察病蛭组织的病理学变化。结果表明,CQ1808菌株为革兰氏阴性短杆菌。通过BLAST检索,发现该菌株与弗氏柠檬酸杆菌(Citrobacter freundii)的16S rDNA序列自然聚类。系统发育树表明,CQ1808菌株为弗氏柠檬酸杆菌。药敏试验结果显示,该菌对氨曲南、头孢曲松、头孢哌酮等21种药物高度敏感,对头孢他啶、头孢唑啉、卡那霉素等7种药物中度敏感,对青霉素、头孢呋辛、麦迪霉素等9种药物表现耐药。人工感染结果显示,CQ1808菌株可使水蛭出现与自然病蛭相似症状。组织病理学研究表明,病蛭组织结构整体异常,消化系统尤其是嗉囊受损严重。研究结果证明,引起宽体金线蛭水肿病的病原为弗氏柠檬酸杆菌。

To identify the cause of the Whitmania pigra oedema bacteria disease outbreak and to provide new ideas for fishery medicine research,in the present study,the pathogenic bacterial strain(named CQ1808)was isolated from the body cavity fluid of the diseased leeches and bacterial examination,histopathology and antimicrobial drugs test were performed.The results demonstrated that CQ1808 strain was a Gram-negative short bacillus,and was naturally clustered with the 16S rDNA sequence of Citrobacter freundii by the BLAST search.The phylogenetic tree suggested that the strain was Citrobacter freundii.The antidrug profiles of the isolate showed that CQ1808 strain was highly sensitive to 21 antibiotics including aztreonam,ceftriaxone and cefoperazone;intermediately susceptible to 7 antibiotics including ceftazidime,cefazolin and kanamycin;resistant to 9 antibiotics including penicillin,cefuroxime and midecamycin.The symptoms of W.pigra artificially infected with CQ1808 strain were similar to naturally infected leeches.Histopathological analysis showed that the overall organizational structure of the naturally diseased leeches was abnormal and the most distinct pathological changes occurred in the crop.This study indicated that C.freundii was the causative agent of oedema in farmed leeches.