摘要
为明确湖南某养殖基地中华倒刺鲃大量死亡的原因,本研究采用常规方法从患病中华倒刺鲃肝脏、肾脏和脾脏等组织及腹水中分离出一株优势菌AH32,并对其进行形态学、生理生化特性、16S rRNA基因测序鉴定;利用K-B法检测菌株AH32对30种药物的耐药性;利用PCR分析该菌的多位点序列分型及检测该菌携带的毒力基因;通过动物人工感染实验分析菌株致病性。结果显示,AH32株为革兰氏阴性短杆菌;生化鉴定结果显示,AH32株对葡萄糖、麦芽糖、七叶苷等反应均为阳性,对乳糖、尿素、蜜二糖等反应均为阴性;16S rRNA基因序列与嗜水气单胞菌ATCC7966的同源性为99.78%。综合菌株理化和分子特征,确定该分离菌为嗜水气单胞菌。多位点序列分型(MLST)分析表明,AH32株的序列型为ST251。药敏试验显示该分离菌对复方新诺明、氟苯尼考等19种抗菌药物敏感,对链霉素、呋喃唑酮、氨苄西林等11种抗菌药物中度敏感或耐药。经鉴定,该菌株的毒力基因型为alt+aer-aha+ahp+lip+ela+。人工感染试验结果显示,AH32株对中华倒刺鲃具有较强的致病性,人工感染实验鱼的临床症状与自然发病症状相同,且从实验中华倒刺鲃的肝脏、肾脏、脾脏和腹水中可重新分离到该嗜水气单胞菌。本研究首次报道了嗜水气单胞菌对中华倒刺鲃具有致病性,可选用恩诺沙星、氟苯尼考和多西环素防治该菌的感染,为中华倒刺鲃的病害防治和健康养殖提供参考依据。
To identify the cause of abnormal massive mortality of Spinibarbus sinensis in a farm of Hunan province,a dominant bacterial strain AH32 was isolated from liver,kidney,spleen and ascites of diseased S.sinensis using a routine method.This isolated strain was identified by morphology,biochemical properties and polymerase chain reaction(PCR)amplification analysis based on 16S rRNA gene sequencing.Resistance of the strain AH32 to 30 drugs was detected by Kirby-Bauer method.PCR method was used for multilocus sequence typing analysis and virulence gene detection.The pathogenicity of the strain AH32was analyzed by artificial infection experiment.The results showed that the strain AH32 was gram negative,short-rod-shaped bacilli.Physiological and biochemical tests showed AH32 strain was positive for glucose,maltose and aesculin,while was negative for lactose,urea and melibiose.AH32 strain 16S r RNA gene sequence shared 99.78% homology with that of Aeromonas hydrophila ATCC7966.Based on its morphological and molecular characteristics,AH32 strain was identified as A.hydrophila.The results of chemosensitivity test showed that AH32 strain is susceptible to 19 kinds of antibiotics including sulfamethoxazole and florfenicol,and is moderately susceptible or resisted to 11 kinds of drugs including streptomycin,furazolidone and ampicillin.The sequence type of AH32 strain was ST251,and its virulence genotype was alt+aer-aha+ahp+lip+ela+.The results of artificial infection test showed that AH32 strain has strong pathogenicity to S.sinensis.S.sinensis displayed similar symptoms as the natural infection,and the same bacterium could be recovered from liver,kidney,spleen and ascites of infected S.sinensis.This study for the first time reported the pathogenicity of A.hydrophila on farmed S.sinensis.Sulfam-ethoxazole,florfenicol and doxycycline can be applied for prevention and treatment of this bacterium-induced infections.Our study would provide a theoretical reference for the disease control and healthy aquaculture of S.sinensis.
作者
高金伟
田兴
谢敏
李绍明
袁希平
邓时铭
宋锐
GAO Jin-wei;TIAN Xing;XIE Min;LI Shao-ming;YUAN Xi-ping;DENG Shi-ming;SONG Rui(Hunan Fisheries Science Institute,Changsha 410153,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2022年第2期208-213,共6页
Chinese Journal of Preventive Veterinary Medicine
基金
财政部和农业农村部:国家现代农业产业技术体系资助(CARS-45-48)
农业部淡水养殖病害防治重点实验室开放课题。
关键词
中华倒刺鲃
嗜水气单胞菌
毒力基因
多位点序列分型
耐药性
Spinibarbus sinensis
Aeromonas hydrophila
virulence genes
multilocus sequence typing
drug resistance
