凡纳滨对虾黑鳃病病原的分离鉴定及耐药性分析

doi:10.13560/j.cnki.biotech.bull.1985.2019-1158

生物技术通报 ›› 2020, Vol. 36 ›› Issue (8): 120-128.doi: 10.13560/j.cnki.biotech.bull.1985.2019-1158

凡纳滨对虾黑鳃病病原的分离鉴定及耐药性分析

林嘉铭¹, 葛辉^1,2, 林克冰², 杨章武², 周宸², 吴建绍², 王国栋¹, 张哲², 杨求华², 王艺磊¹

1.集美大学水产学院农业农村部东海海水健康养殖重点实验室,厦门 361021;
2.福建水产研究所福建省海洋生物增养殖与高值化利用重点实验室,厦门 361000

收稿日期:2019-11-27 出版日期:2020-08-26 发布日期:2020-08-27
作者简介:林嘉铭,男,硕士研究生,研究方向：甲壳动物免疫学;E-mail：206558292@qq.com
基金资助:
福建省种业创新与产业化工程海洋与渔业项目（2017FJSCZY02）,厦门市科技计划项目2017产学研协同创新及科技合作项目（2016S0870）,2019年福建省海洋与渔业结构调整专项（2019HYJG02）,福建省省属公益类科研院所基本科研专项（2019R1013-2）

Isolation,Identification and Antibiotic Sensitivity Analysis of Bacterial Pathogen from Litopenaeus vannamei with Black Gill Disease

LIN Jia-ming¹, GE Hui^1,2, LIN Ke-bing², YANG Zhang-wu², ZHOU Chen², WU Jian-shao², WANG Guo-dong¹, ZHANG Zhe², YANG Qiu-hua², WANG Yi-lei¹

1. Key Laboratory of Healthy Mariculture for the East China Sea,Ministry of Agriculture,Fisheries College,Jimei University,Xiamen 361021;
2. Key Laboratory of Cultivation and High-value Utilization of Marine Organisms in Fujian Province,Fisheries Research Institute of Fujian,Xiamen 361012

Received:2019-11-27 Published:2020-08-26 Online:2020-08-27

摘要/Abstract

摘要： 旨在鉴定凡纳滨对虾（Litopenaeus vannamei）越冬亲虾出现黑鳃病的病原并对该病原进行耐药性分析。采用平板分离法从濒死的病虾病灶（鳃丝）分离到一株优势菌株,综合菌体的形态学鉴定以及真菌ITS-rDNA基因序列进行分析。结果显示,优势菌株与腐皮镰刀真菌（Fusarium solani）的序列同源性达99％以上,构建的系统发育树与腐皮镰刀真菌聚为一类,最终确定分离的优势菌株为腐皮镰刀真菌。进一步根据柯赫氏法则进行人工感染试验,证实其为病原菌且具有较强的致病性,病虾出现黑鳃,以5.0×10⁵ CFU/mL浓度感染的对虾（25 µL/尾）7 d致死率为53.33%。选取11种常见药物,采用琼脂稀释法和参照美国国家临床试验标准委员会（NCCLS）的M38-A2方案进行药物敏感试验,初步筛选后发现该菌株对克霉唑、益康唑、特比萘芬、制霉菌素、那他霉素、酮康唑等6种药物敏感,且益康唑的最小抑菌浓度（MIC）为1 μg/ mL,特比萘芬MIC为4 μg/ mL,对克霉唑、那他霉素的MIC为8 μg/mL,对咪康唑、伊曲康唑的MIC为16 μg/mL。本研究确定了分离到的优势菌株的致病性、耐药性和分类地位,为凡纳滨对虾越冬亲虾黑鳃病的防控提供了参考。

关键词: 凡纳滨对虾, 病原分离鉴定, 腐皮镰刀真菌, 药敏试验

Abstract: In order to identify the pathogen of black gill disease in Litopenaeus vannamei overwintering parent shrimp and analyze its drug resistance,a dominant strain was isolated from the focus in the gill of a dying diseased shrimp via plate method,and then analyzed by combining the morphology of the strain and its ITS-rDNA gene sequence. The results showed that the dominant strain had 99% sequence homology with Fusarium solani,and the constructed phylogenetic tree clustered as one branch with F. solani;therefore the isolated dominant strain was determined as F. solani. The artificial infection was conducted based on Koch’s rule,and the results confirmed that the pathogen had strong pathogenicity and disease shrimps had black gill. The shrimps infected with concentrations of 5.0×10⁵ CFU/mL（25 µL/shrimp）for 7 d died 53.33%. Agar dilution method and M38-A2 scheme of NCCLS were used for drug sensitivity test with selected 11 common drugs. After preliminary screening,it was found that the strain was sensitive to clotrimazole,econazole,terbinafine,nystatin,natamycin and ketoconazole. The MIC of econazole was 1 μg/mL,that of terbinafine（TBF）was 4 μg/mL,that of clotrimazole and natamycin was 8 μg/mL,and that of miconazole and itraconazole was 16 μg/mL. In conclusion,the pathogenicity,drug resistance and taxonomic status of the dominant strain were determined in this study,which may provide a reference for the prevention and control of black gill disease of overwintering parent shrimp.

Key words: Litopenaeus vannamei, isolation and identification of pathogen, Fusarium solani, drug sensitivity test

林嘉铭, 葛辉, 林克冰, 杨章武, 周宸, 吴建绍, 王国栋, 张哲, 杨求华, 王艺磊. 凡纳滨对虾黑鳃病病原的分离鉴定及耐药性分析[J]. 生物技术通报, 2020, 36(8): 120-128.

LIN Jia-ming, GE Hui, LIN Ke-bing, YANG Zhang-wu, ZHOU Chen, WU Jian-shao, WANG Guo-dong, ZHANG Zhe, YANG Qiu-hua, WANG Yi-lei. Isolation,Identification and Antibiotic Sensitivity Analysis of Bacterial Pathogen from Litopenaeus vannamei with Black Gill Disease[J]. Biotechnology Bulletin, 2020, 36(8): 120-128.

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凡纳滨对虾黑鳃病病原的分离鉴定及耐药性分析

Isolation,Identification and Antibiotic Sensitivity Analysis of Bacterial Pathogen from Litopenaeus vannamei with Black Gill Disease

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