一例甲鱼急性死亡的病原菌分离、鉴定与药敏分析
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摘要
为了给甲鱼病害防控的合理用药提供技术支持,本研究自2018年4至5月采集了湖南常德某甲鱼养殖场急性死亡病样,进行了病原分离、鉴定与药敏分析。试验采用无菌穿刺接种法从濒临死亡的病鳖肝脏中分离出1株病原菌,命名为HTG。利用血琼脂平板对该病原菌进行毒力检测,结合形态观察与16S rDNA序列分析法进行鉴定,采用K-B纸片扩散法对病原菌进行药敏分析。毒力检测显示HTG在血琼脂平板上呈β溶血;16S rDNA序列分析表明HTG与Aeromonas hydrophila strain JCM 1027的16S rDNA基因的核苷酸序列同源,相似性为100%,结合形态特征将HTG鉴定为嗜水气单胞菌;药敏分析结果显示HTG对多西环素高度敏感(抑菌圈直径达30 mm)。根据药敏分析结果,建议该养殖场采用内服多西环素,辅以保肝护肝渔药对该病进行防控。
In order to provide technological data for the rational drug use for disease prevention and control of Trionyx sinensis,isolation,identification and drug sensitivity analysis of pathogenic bacteria from a Trionyx sinensis acute death in Changde were performed from April to May of 2018. A strain of pathogenic bacteria named HTG was isolated from the liver of the dying Trionyx sinensis by aseptic puncture inoculation. The virulence of the pathogenic bacteria was detected by blood agar plate. The pathogenic bacteria were identified based on morphology observation and 16 S rDNA gene sequence analysis. The drug sensitivity tests were performed by Kirby-Bauer method. Virulence test indicated that the strain submitted β hemolysis. Phylogenetic analysis based on 16 S rDNA sequence indicated that HTG were homology with Aeromonas hydrophila strain JCM 1027,the similarity was 100%. Combined with morphological characteristics and16 S rDNA sequence analysis,the HTG was identified as Aeromonas hydrophila. The results of drug sensitivity analysis showed that HTG was highly sensitive to doxycycline( the diameter of the inhibition zone was up to 30 mm). Based on the results of drug sensitivity analysis test,oral doxycycline and liver protective fishery drugs are recommended for the disease prevention and control.
In order to provide technological data for the rational drug use for disease prevention and control of Trionyx sinensis,isolation,identification and drug sensitivity analysis of pathogenic bacteria from a Trionyx sinensis acute death in Changde were performed from April to May of 2018. A strain of pathogenic bacteria named HTG was isolated from the liver of the dying Trionyx sinensis by aseptic puncture inoculation. The virulence of the pathogenic bacteria was detected by blood agar plate. The pathogenic bacteria were identified based on morphology observation and 16 S rDNA gene sequence analysis. The drug sensitivity tests were performed by Kirby-Bauer method. Virulence test indicated that the strain submitted β hemolysis. Phylogenetic analysis based on 16 S rDNA sequence indicated that HTG were homology with Aeromonas hydrophila strain JCM 1027,the similarity was 100%. Combined with morphological characteristics and16 S rDNA sequence analysis,the HTG was identified as Aeromonas hydrophila. The results of drug sensitivity analysis showed that HTG was highly sensitive to doxycycline( the diameter of the inhibition zone was up to 30 mm). Based on the results of drug sensitivity analysis test,oral doxycycline and liver protective fishery drugs are recommended for the disease prevention and control.
引文
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[14]朱凝瑜,郑晓叶,曹飞飞,等.鳖源嗜水气单胞菌的耐药性研究[J].浙江农业学报,2017,29(9):1445-1450.ZHU Ningyu,ZHENG Xiaoye,CAO Feifei,et al. Antimicrobial resistance of Aeromonas hydrophila isolated from soft-shelled turtle Pelodiscus sinensis[J]. Acta Agriculturae Zhejiangensis,2017,29(9):1445-1450.
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[16]王帅兵,熊良伟,朱善元,等.嗜水气单胞菌分离鉴定与药敏试验[J].江苏农业科学,2017,45(24):165-168.WANG Shuaibing,XIONG Liangwei,ZHU Shanyuan,et al. Isolation,identification and drug sensitive test of Aeromonas hydrophila[J]. Jiangsu Agricultural Sciences,2017,45(24):165-168.
[17]肖根辉,王萍,刘明超,等.中华鳖致病性嗜水气单胞菌分离鉴定与药敏试验[J].经济动物学报,2011,15(1):56-60.XIAO Genhui,WANG Ping,LIU Mingchao,et al. Isolation,identification and drug sensitive test of Aeromonas hydrophila from chinese soft-shelled turtle[J]. Journal of Economic Animal,2011,15(1):56-60.
[18]史艳伟,孟丽华,蒋帮铜,等.四环素类抗生素对渔业生态环境的影响研究[J].科学养鱼,2016,32(6):52-53.SHI Yanwei,MENG Lihua,JIANG Bangtong,et al. Effects of tetracycline antibiotics on fishery ecological environment[J].Scientific Fish Farming,2016,32(6):52-53.
[19]卢静,王振宁,陈锐,等.几种中药单体和抗生素对嗜水气单胞菌及温和气单胞菌的体外抑菌活性研究[J].水生生物学报,2013,37(6):1128-1132.LU Jing,WANG Zhenning,CHEN Rui,et al. In vitro antibacterial activity of several chinese medicine monomers and antibiotics on Aeromonas hydrophila and Aeromonas sobria[J]. Acta Hydrobiologica Sinica,2013,37(6):1128-1132.
[20]孔维光,吴志新,李思思,等.益生芽孢杆菌对草鱼肠上皮细胞的黏附及对嗜水气单胞菌的抑制[J].华中农业大学学报,2017,36(05):67-73.KONG Weiguang,WU Zhixin,LI Sisi,et al. Adherence of Bacillus subtilis to intestinal epithelial cells of grass carp and its inhibition to Aeromonas hydrophila[J]. Journal of Huazhong Agricultural University,2017,36(05):67-73.
[21]何夙旭,王全民,黄路,等.芽孢杆菌R1共培养对嗜水气单胞菌NJ-1毒力基因表达的影响[J].水产学报,2016,40(03):299-307.HE Suxu,WANG Quanmin,HUANG Lu,et al. Effects of coculture of Bacillus sp. R1 and Aeromonas hydrophila NJ-1 on the virulent factor gene expression of NJ-1[J]. Journal of Fisheries of China,2016,40(03):299-307.
[2] ZHU Z,JIANG X,DENG S,et al. Isolation,identification and in vitro antimicrobial susceptibility of pathogenic Aeromonas veronii from soft-shelled turtles[J]. Agricultural Science&Technology,2016,17(4):804-809.
[3]陈永亮,高晓华,李怡,等.鳖源摩氏摩根菌分离鉴定及药敏特性分析[J].南方农业学报,2015,46(11):2046-2052.CHEN Yongliang,GAO Xiaohua,LI Yi,et al. Isolation,identification and antibiotic sensitivity test of Morganella morganii from Trionyx sinensis[J]. Guangxi Agricultural Sciences,2015,46(11):2046-2052.
[4]田飞焱,欧阳敏,张晓燕,等.中华鳖弗氏柠檬酸杆菌的分离、鉴定及药物敏感性试验[J].水产学杂志,2013,26(4):42-46.TIAN Feiyan,OUYANG Min,ZHANG Xiaoyan,et al. Isolation,identification and antibiotic sensitivity of pathogenic bacterium Citrobacter freundii from soft shell turtle Trionyx sinensis[J]. Chinese Journal of Fisheries,2013,26(4):42-46.
[5]杨移斌,艾晓辉,曹海鹏,等.中华鳖源粘质沙雷氏菌分离、鉴定及药敏特性研究[J].淡水渔业,2018,48(1):41-46.YANG Yibin,AI Xiaohui,CAO Haipeng,et al. Isolation,identification and antibiotic sensitivity of Serratia marcescens from Trionyx sinensis[J]. Freshwater Fisheries,2018,48(1):41-46.
[6]姜艳丽,李丽丽.嗜水气单胞菌的研究进展[J].黑龙江农业科学,2015,41(2):142-146.JIANG Yanli, LI Lili. Research progresss on Aeromonas hydrophila[J]. Heilongjiang Agricultural Sciences,2015,41(2):142-146.
[7]郑杰夫,张一柳,钟蕾.中华鳖源嗜水气单胞菌的研究进展[J].水产科学,2016,35(2):191-198.ZHENG Jiefu,ZHANG Yiliu,ZHONG Lei. Research advances on diseases of Aeromonas hydrophilain chinese soft-shelled turtle Trionyx sinensis[J]. Fisheries Science, 2016, 35(2):191-198.
[8]柏爱旭,张科,张敬友,等.黄颡鱼病原嗜水气单胞菌分离鉴定及致病性研究[J].江苏农业科学,2018,46(7):175-178.BAI Aixu,ZHANG Ke,ZHANG Jingyou,et al. Identification and detection of pathogenic Aeromonas hydrophila isolated from yellow catfish(Pelteobagrus fulvidraco)[J]. Jiangsu Agricultural Sciences,2018,46(7):175-178.
[9]叶彩燕,汪开毓,刘韬,等.西伯利亚鲟致病性嗜水气单胞菌的分离鉴定[J].中国预防兽医学报,2018,40(3):195-199.YE Caiyan,WANG Kaiyu,LIU Tao,et al. Isolation and identification of pathogenic Aeromonas hydrophila isolated from Acipenser Baerii[J]. Chinese Journal of Preventive Veterinary Medicine,2018,40(3):195-199.
[10]高东民,宋亚娇,吕爱军,等.鱼源嗜水气单胞菌的分离鉴定及药敏试验[J].黑龙江畜牧兽医,2017,61(5):149-152.GAO Dongmin,SONG Yajiao,LV Aijun,et al. Isolation,identification and drug sensitivity test of fish-derived Aeromonas hydrophila[J]. Heilongjiang Animal Science and Veterinary Medicine,2017,61(5):149-152.
[11]杨移斌,董靖,胥宁,等.黄鳝源嗜水气单胞菌DB01分离鉴定及药敏特性研究[J].淡水渔业,2016,46(5):43-48.YANG Yibin,DONG Jing,XU Ning,et al. Isolation and identification of Aeromonas hydrophila DB01 from Monopterus albus and its antibiotic sensitivity[J]. Freshwater Fisheries,2016,46(5):43-48.
[12]李梦茜,张振旺.罗非鱼细菌性败血症病原菌嗜水气单胞菌拮抗菌株的筛选和鉴定[J].江苏农业科学,2017,45(3):129-131.LI Mengxi,ZHANG Zhenwang. Isolation and identification of an antagonistic bacteria against Aeromonas hydrophila causing bacterial septicaemia of Oreochromis niloticus[J]. Jiangsu Agricultural Sciences,2017,45(3):129-131.
[13]徐阳,姚洪,乔帼,等.养殖大菱鲆感染嗜水气单胞菌的分离、毒力分析及ERIC-PCR分型[J].大连海洋大学学报,2018,33(4):430-434.XU Yang,YAO Hong,QIAO Guo,et al. Isolation,virulence and ERIC-PCR genotyping of Aeromonas hydrophila in farmed turbot Scophthalmus maximus(L.)[J]. Journal of Dalian Fisheries University,2018,33(4):430-434.
[14]朱凝瑜,郑晓叶,曹飞飞,等.鳖源嗜水气单胞菌的耐药性研究[J].浙江农业学报,2017,29(9):1445-1450.ZHU Ningyu,ZHENG Xiaoye,CAO Feifei,et al. Antimicrobial resistance of Aeromonas hydrophila isolated from soft-shelled turtle Pelodiscus sinensis[J]. Acta Agriculturae Zhejiangensis,2017,29(9):1445-1450.
[15]温周瑞,卢伶俐,陈霞,等.湖北四个地区养殖池塘嗜水气单胞菌的分离鉴定及其耐药性比较[J].科学养鱼,2017,33(2):59-61.WEN Zhourui,LU Lingli,CHEN Xia,et al. Isolation,identification and drug resistance of Aeromonas hydrophila from four aquaculture ponds in Hubei[J]. Scientific Fish Farming,2017,33(2):59-61.
[16]王帅兵,熊良伟,朱善元,等.嗜水气单胞菌分离鉴定与药敏试验[J].江苏农业科学,2017,45(24):165-168.WANG Shuaibing,XIONG Liangwei,ZHU Shanyuan,et al. Isolation,identification and drug sensitive test of Aeromonas hydrophila[J]. Jiangsu Agricultural Sciences,2017,45(24):165-168.
[17]肖根辉,王萍,刘明超,等.中华鳖致病性嗜水气单胞菌分离鉴定与药敏试验[J].经济动物学报,2011,15(1):56-60.XIAO Genhui,WANG Ping,LIU Mingchao,et al. Isolation,identification and drug sensitive test of Aeromonas hydrophila from chinese soft-shelled turtle[J]. Journal of Economic Animal,2011,15(1):56-60.
[18]史艳伟,孟丽华,蒋帮铜,等.四环素类抗生素对渔业生态环境的影响研究[J].科学养鱼,2016,32(6):52-53.SHI Yanwei,MENG Lihua,JIANG Bangtong,et al. Effects of tetracycline antibiotics on fishery ecological environment[J].Scientific Fish Farming,2016,32(6):52-53.
[19]卢静,王振宁,陈锐,等.几种中药单体和抗生素对嗜水气单胞菌及温和气单胞菌的体外抑菌活性研究[J].水生生物学报,2013,37(6):1128-1132.LU Jing,WANG Zhenning,CHEN Rui,et al. In vitro antibacterial activity of several chinese medicine monomers and antibiotics on Aeromonas hydrophila and Aeromonas sobria[J]. Acta Hydrobiologica Sinica,2013,37(6):1128-1132.
[20]孔维光,吴志新,李思思,等.益生芽孢杆菌对草鱼肠上皮细胞的黏附及对嗜水气单胞菌的抑制[J].华中农业大学学报,2017,36(05):67-73.KONG Weiguang,WU Zhixin,LI Sisi,et al. Adherence of Bacillus subtilis to intestinal epithelial cells of grass carp and its inhibition to Aeromonas hydrophila[J]. Journal of Huazhong Agricultural University,2017,36(05):67-73.
[21]何夙旭,王全民,黄路,等.芽孢杆菌R1共培养对嗜水气单胞菌NJ-1毒力基因表达的影响[J].水产学报,2016,40(03):299-307.HE Suxu,WANG Quanmin,HUANG Lu,et al. Effects of coculture of Bacillus sp. R1 and Aeromonas hydrophila NJ-1 on the virulent factor gene expression of NJ-1[J]. Journal of Fisheries of China,2016,40(03):299-307.