鸭源绿色气球菌的分离鉴定及16S rRNA基因序列测定与遗传进化分析  

Isolation,Identification,16S rRNA Gene Sequencing and Genetic EvolutionAnalysis of Aeromonas aeruginosa from Duck

作　　者：乔宏兴[1,2] 丁勇 张立恒 宋予震[1,2] 董青 边传周[1,2] 赵俊强 QIAO Hongxing;DING Yong;ZHANG Liheng;SONG Yuzhen;DONG Qing;BIAN Chuanzhou;ZHAO Junqiang(College of Veterinary Medicine, Henan University of Animal Husbandry and Economy,Zhengzhou 450046,China;Henan Province International Joint Laboratory of Animal Intestinal Diseases in Henan Province, Zhengzhou 450046,China;College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450002,China;Henan Rongruixiang Pharmaceutical Co.,Ltd., Shangqiu 476200,China)

机构地区：[1]河南牧业经济学院动物医药学院,郑州450046 [2]河南省动物肠道疫病国际联合实验室,郑州450046 [3]河南农业大学动物医学院,郑州450002 [4]河南融瑞祥制药有限公司,商丘476200

出　　处：《中国畜牧兽医》2022年第2期435-442,共8页China Animal Husbandry & Veterinary Medicine

基　　金：河南省自然科学基金项目(202300410189);河南省科技攻关项目(212102110372);河南牧业经济学院预防兽医重点学科项目(mxk2016102);河南牧业经济学院校科技创新团队项目(2018KYTD13);河南牧业经济学院博士科研启动资金资助项目(2021HNUAHEDF004)。

摘　　要：【目的】研究河南某鸭场雏鸭发生急性死亡的致病菌及其系统发育地位。【方法】采集发病鸭场病死鸭肝脏、脾脏,进行细菌形态学观察、革兰氏染色、生化特性鉴定、16S rRNA基因的序列分析、药敏试验及致病性试验。【结果】分离菌在鲜血琼脂培养基上长出表面光滑,凸起,乳白色,呈α-溶血的革兰氏阳性球菌。生化试验结果表明,分离菌对麦芽糖、蔗糖、棉籽糖、硝酸盐还原反应、MP-VP试验、尿素、赖氨酸脱羧酶、鸟氨酸脱羧酶、七叶苷均为阳性,对木糖、乳糖、葡萄糖、山梨醇、硫化氢、枸橼酸盐、蛋白胨、甘露醇、苯丙氨酸、甲基红试验等均为阴性。16S rRNA基因测序及BLAST比对结果显示,分离菌16S rRNA基因序列与NCBI上已发布的绿色气球菌相似性性在94.8%~99.9%之间,其中与菌株Mnlv1、Mnlv2、W66等相似性最高,达99.9%;与菌株GXBL-1相似性最低,为94.8%。系统发育树分析表明,分离菌与绿色气球菌FL0915MS、Mnlv2等绿色气球菌在同一分支上,属于同一属群,其中与FL09亲缘关系最近。药敏试验结果显示,分离菌对磷霉素、氨苄西林、阿莫西林敏感;对青霉素、新生霉素、阿米卡星等中度敏感;对利福平、新霉素、杆菌肽、红霉素、恩诺沙星等耐药。致病性试验结果表明该菌具有致死性,并随着接种浓度增大而增强。【结论】本研究确定了河南某鸭场引起雏鸭急性死亡的病原菌是绿色气球菌,为该菌引起疾病的临床诊断和治疗提供了参考依据。【Objective】The experiment was aimed to study the pathogenic bacteria of acute death of ducklings in a duck farm in Henan province and its phylogenetic status.【Method】The liver and spleen of dead ducks in the diseased duck farm were collected.The bacterial morphology,Gram-staining,biochemical characteristics,sequence analysis of 16S rRNA gene,drug sensitivity test and pathogenicity test were carried out.【Results】The isolated bacteria grew on blood agar medium with smooth,convex,milky white andα-hemolytic Gram-positive cocci.Biochemical test results showed that the isolated bacteria were positive for maltose,sucrose,raffinose,nitrate reduction reaction,MP-VP test,urea,lysine decarboxylase,ornithine decarboxylase and aescin,and negative for xylose,lactose,glucose,sorbitol,hydrogen sulfide,citrate,peptone,mannitol,phenylalanine and methyl red.The results of 16S rRNA gene sequencing and BLAST comparison results showed that the 16S rRNA gene sequence of the isolated strain was 94.8%-99.9%similar to that of Aerococcus viridans published on NCBI,and the similarity with strains Mnlv1,Mnlv2 and W66 was the highest,up to 99.9%.The similarity with strain GXBl-1 was the lowest,which was 94.8%.Phylogenetic tree analysis showed that the isolated bacteria belonged to the same genus and group on the same branch as strains of Aerococcus viridans FL09,15MS and Mnlv2 etc.,and had the closest genetic relationship with FL09.The results of drug sensitivity showed that the isolated strain was sensitive to fosfomycin,ampicillin and amoxicillin,moderately sensitive to penicillin,neomycin and amikacin,it was resistant to rifampicin,neomycin,bacitracin,erythromycin and enrofloxacin.The results of pathogenicity test showed that the strain was lethal and increased with the increase of inoculation concentration.【Conclusion】This study determined that the pathogen causing the acute death of ducklings in a duck farm in Henan province was Aerococcus viridans,which provided a reference basis for clinical diagnosis and treatment of

关 键 词：鸭 绿色气球菌 分离鉴定 16S rRNA 遗传进化分析 

分 类 号：S852.611[农业科学—基础兽医学]