半滑舌鳎源创伤弧菌分离鉴定与药敏试验
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摘要
为查明引起河北省昌黎县某海水养殖场半滑舌鳎患病及死亡的病原菌,本研究从半滑舌鳎肝、脾、腮、肠道分离纯化得到1株优势菌株,对其进行了理化鉴定和16S rDNA序列分析、致病性试验、毒力基因检测和中西药的药敏试验。结果显示,分离菌为创伤弧菌(V.vulnificus),序列比对显示与创伤弧菌同源性为100%,菌株命名为V.vulnificus QHD-1。动物回归试验证明该菌是半滑舌鳎的病原菌。小鼠半数致死量(LD_(50))为2.4×10~6 CFU/mL,携带毒力基因溶细胞素(vvhA)、细胞毒素(rtxA)、铁载体(viuB)、荚膜多糖(wza)等。西药药敏试验结果显示,分离菌株V.vulnificus QHD-1对氟苯尼考等7种药物敏感,对氧氟沙星等5种药物中敏;对头孢拉定、土霉素等6种药物耐药;中药药敏试验结果表明,苏木、诃子、乌梅、五倍子抑抑菌效果较好;最小抑菌浓度(MIC)和最小杀菌浓度(MBC)均为1.9～7.8 g/L;中药联合抑菌试验结果表明,黄芩和黄柏组合、五倍子和黄芩组合、乌梅和五倍子组合、艾叶和荆芥组合、板蓝根和黄芩组合对创伤弧菌表现协同作用。研究结果证明V.vulnificus QHD-1对鱼和小鼠具有较强的致病性,具有一定的公共卫生危害性;同时药敏试验结果为该地区鱼病的防治提供了依据。
In order to identify the pathogen caused a severe disease in aquaculture in Changli county of Hebei province,a dominant bacterium was isolated from the liver,spleen,gill,intestinal of Cynoglossus semilaevis,physical and chemical identification,16 SrDNA sequence analysis,pathogenicity tests and virulence gene tests were performed.The results showed that the bacterium was physicochemically identified as V.vulnificus,and the sequence alignment showed 100% homology with V.vulnificus and the strain was named V.vulnificus QHD-1.Animal pathogenicity tests showed that the bacterium could cause the death of Cynoglossus semilaevis,and the LD_(50 )of mice was 2.4×10~6 CFU/mL.The virulence gene test results showed that the isolates carried rtxA,vvhA,viuB,and wza virulence gene.Western medicine susceptibility test results showed that the isolated strain V.vulnificus QHD-1 was sensitive to 7 kinds of drugs such as florfenicol etc,and moderately sensitive to 5 kinds of drugs such as ofloxacin,etc;and it was resistant to 6 drugs such as cephradine and oxytetracycline,etc.The susceptibility test results of traditional Chinese medicines showed that the diameter of inhibition zones of Caesalpinia sappan,Terminalia chebula,Prunus mume,Galla chinensis had better antibacterial effect,MIC and MBC were from 1.9 to 7.8 g/L;the results of the combined antibacterial test showed that the combinations of Scutellaria baicalensisand Phellodendron amurense,the combinations of Galla chinensis and Scutellaria baicalensis,the combinations of Prunus mume and Galla chinensis,the combinations of Folium Artemisiae Argyi and Schizonepeta,the combinations of Radix isatidisand Scutellaria baicalensis showed synergy bacteriostasis effect on Vibrio vulnificus,and then the combinations of Ligustrum lucidum and Caesalpinia sappan showed additive bacteriostasis effects on Vibrio vulnificus.The results showed that V.vulnificus QHD-1 had strong pathogenicity to fish and mice,and certain public health significance.The results of drug sensitivity test provide the basis for the prevention and treatment of fish diseases in this area.
In order to identify the pathogen caused a severe disease in aquaculture in Changli county of Hebei province,a dominant bacterium was isolated from the liver,spleen,gill,intestinal of Cynoglossus semilaevis,physical and chemical identification,16 SrDNA sequence analysis,pathogenicity tests and virulence gene tests were performed.The results showed that the bacterium was physicochemically identified as V.vulnificus,and the sequence alignment showed 100% homology with V.vulnificus and the strain was named V.vulnificus QHD-1.Animal pathogenicity tests showed that the bacterium could cause the death of Cynoglossus semilaevis,and the LD_(50 )of mice was 2.4×10~6 CFU/mL.The virulence gene test results showed that the isolates carried rtxA,vvhA,viuB,and wza virulence gene.Western medicine susceptibility test results showed that the isolated strain V.vulnificus QHD-1 was sensitive to 7 kinds of drugs such as florfenicol etc,and moderately sensitive to 5 kinds of drugs such as ofloxacin,etc;and it was resistant to 6 drugs such as cephradine and oxytetracycline,etc.The susceptibility test results of traditional Chinese medicines showed that the diameter of inhibition zones of Caesalpinia sappan,Terminalia chebula,Prunus mume,Galla chinensis had better antibacterial effect,MIC and MBC were from 1.9 to 7.8 g/L;the results of the combined antibacterial test showed that the combinations of Scutellaria baicalensisand Phellodendron amurense,the combinations of Galla chinensis and Scutellaria baicalensis,the combinations of Prunus mume and Galla chinensis,the combinations of Folium Artemisiae Argyi and Schizonepeta,the combinations of Radix isatidisand Scutellaria baicalensis showed synergy bacteriostasis effect on Vibrio vulnificus,and then the combinations of Ligustrum lucidum and Caesalpinia sappan showed additive bacteriostasis effects on Vibrio vulnificus.The results showed that V.vulnificus QHD-1 had strong pathogenicity to fish and mice,and certain public health significance.The results of drug sensitivity test provide the basis for the prevention and treatment of fish diseases in this area.
引文
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[2] 王明义,胡成进.创伤弧菌致病性及其毒力因子研究进展[J].中国微生态学杂志,2017,29(12):1470-1473.
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[6] 高桂生,张艳英,张召兴,等.半滑舌鳎皮肤溃疡病的病原分离鉴定与药物敏感性分析[J].中国兽医学报,2016,36(4):595-599.
[7] 黎炯,叶星,卢迈新,等.罗非鱼创伤弧菌的分离鉴定和药敏试验[J].江西农业大学学报,2011,33(5):965-970.
[8] 王洪彬,李永慧,吴同垒,等.中草药对美人鱼发光杆菌的体外抑菌试验[J].黑龙江畜牧兽医,2018(6):155-157.
[9] 聂鲡蓉,郑曙明,仇登高.12种中药有效成分对3株水产致病菌体外抑菌试验[J].水生态学杂志,2010,3(6):17-20.
[10] 吕彪,宋志元,储君,等.20种中草药提取物对4种常见病原菌体外抑菌试验[J].中兽医学杂志,2016(4):4-6.
[11] 周文中,毕可东,邹本革,等.氟苯尼考与盐酸土霉素体外联合抑菌效果研究[J].中国畜牧兽医,2011,38(7):201-203.
[12] 王玉姣,刘冰璇,董佳琦,等.怀府胎菊等五种中草药不同组合体外抑菌效果比较[J].饲料博览,2016(10):26-30.
[13] 王明义,胡成进.创伤弧菌致病性及其毒力因子研究进展[J].中国微生态学杂志,2017(12):1470-1473.
[2] 王明义,胡成进.创伤弧菌致病性及其毒力因子研究进展[J].中国微生态学杂志,2017,29(12):1470-1473.
[3] ROLAND F P.Leg gangrene and endotoxin shock due to vibrio parahaemolyticus-an infection acquired in New England coastal waters[J].N Engl J Med,1970,282(23):1306-1306.
[4] HOLLIS D G,WEAVER R E,BAKER C N,et al.Halophilic vibrio species isolated from blood cultures[J].J Clin Microbiol,1976,3(4):425-431.
[5] FARMER J J.Vibrio (“Beneckea”) vulnificus,the bacterium associated with sepsis,septicaemia,and the sea[J].Lancet,1979,314(8148):903-903.
[6] 高桂生,张艳英,张召兴,等.半滑舌鳎皮肤溃疡病的病原分离鉴定与药物敏感性分析[J].中国兽医学报,2016,36(4):595-599.
[7] 黎炯,叶星,卢迈新,等.罗非鱼创伤弧菌的分离鉴定和药敏试验[J].江西农业大学学报,2011,33(5):965-970.
[8] 王洪彬,李永慧,吴同垒,等.中草药对美人鱼发光杆菌的体外抑菌试验[J].黑龙江畜牧兽医,2018(6):155-157.
[9] 聂鲡蓉,郑曙明,仇登高.12种中药有效成分对3株水产致病菌体外抑菌试验[J].水生态学杂志,2010,3(6):17-20.
[10] 吕彪,宋志元,储君,等.20种中草药提取物对4种常见病原菌体外抑菌试验[J].中兽医学杂志,2016(4):4-6.
[11] 周文中,毕可东,邹本革,等.氟苯尼考与盐酸土霉素体外联合抑菌效果研究[J].中国畜牧兽医,2011,38(7):201-203.
[12] 王玉姣,刘冰璇,董佳琦,等.怀府胎菊等五种中草药不同组合体外抑菌效果比较[J].饲料博览,2016(10):26-30.
[13] 王明义,胡成进.创伤弧菌致病性及其毒力因子研究进展[J].中国微生态学杂志,2017(12):1470-1473.
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