Detection and quantification of Aeromonas salmonicida in fish tissue by real‐time PCR

Furunculosis, a septicaemic infection caused by the bacterium Aeromonas salmonicida subsp. salmonicida, currently causes problems in Danish seawater rainbow trout production. Detection has mainly been achieved by bacterial culture, but more rapid and sensitive methods are needed. A previously developed real‐time PCR assay targeting the plasmid encoded aopP gene of A. salmonicida was, in parallel with culturing, used for the examination of five organs of 40 fish from Danish freshwater and seawater farms. Real‐time PCR showed overall a higher frequency of positives than culturing (65% of positive fish by real‐time PCR compared to 30% by a culture approach). Also, no real‐time PCR‐negative samples were found positive by culturing. A. salmonicida was detected by real‐time PCR, though not by culturing, in freshwater fish showing no signs of furunculosis, indicating possible presence of carrier fish. In seawater fish examined after an outbreak and antibiotics treatment, real‐time PCR showed the presence of the bacterium in all examined organs (1–482 genomic units mg^?1). With a limit of detection of 40 target copies (1–2 genomic units) per reaction, a high reproducibility and an excellent efficiency, the present real‐time PCR assay provides a sensitive tool for the detection of A. salmonicida.     

半滑舌鳎溃疡病原杀鲑气单胞菌的分离鉴定与药敏试验

半滑舌鳎(Cynoglossus semilaevis Günther)是广受我国消费者认可的名优鱼类,具有很高的营养价值和经济价值,然而,随着工厂化养殖模式的不断发展,疾病成为制约半滑舌鳎养殖业健康发展的关键因素,其中,体表溃疡病为其常见病症。从严重溃疡病半滑舌鳎体内分离到1株优势菌株HX0416,该菌可在TCBS培养基上生长,并可在绵羊血平板上形成清晰的β-溶血环;经过16S rRNA和rpo D基因序列同源性分析并结合生化特性鉴定为杀鲑气单胞菌(Aeromonas salmonicida)。健康半滑舌鳎幼鱼感染试验表明,该菌对半滑舌鳎具有较强致病性,感染72 h(1.04×106cfu·m L-1)对攻毒鱼的致死率达91.7%。药敏试验结果显示,该菌对环丙沙星、恩诺沙星、诺氟沙星等喹诺酮类和头孢曲松、头孢吡肟等头孢菌素类药物比较敏感,而对硫酸新霉素、土霉素等氨基糖苷类和四环素类药物多重耐药,尤其是对磺胺甲噁唑、氨苄西林等磺胺类和β-内酰胺类药物严重耐药。     

黄鳝人工感染温和气单胞菌的组织病理学研究

对黄鳝(Monopterus albus)人工感染温和气单胞菌(Aeromonas sobria)的临床症状和病理变化进行了观察。结果显示,感染后12~36h为死亡高峰期,其感染症状与自然发病的黄鳝相似。染病黄鳝的肝脏、肠道和肾脏的病理变化明显,肝细胞变性,肠绒毛脱落,肾小球结构消失并充满大量红细胞;感染后9~27h,肝细胞索排列紊乱,肝细胞肿大并空泡化;感染后3~30h,小肠绒毛脱落,固有层淋巴小结增生,粘膜下层、肌层、浆膜层红细胞增多;感染后3~21h,肾小管上皮细胞肿胀,严重颗粒变性,肾小球上皮细胞坏死;感染后21~30h,肌纤维细胞坏死,大量炎性细胞浸润。     

Effects of dietary supplementation with probiotic live yeast Debaryomyces hansenii on the immune and antioxidant systems of leopard grouper Mycteroperca rosacea infected with Aeromonas hydrophila

Two trials were conducted to determine the efficacy of fish fed live yeast Debaryomyces hansenii strain CBS 8339 on immune and antioxidant systems in leopard grouper Mycteroperca rosacea infected with Aeromonas hydrophila. Juveniles (12±0.5 g) were fed with a control diet or a D. hansenii‐supplemented diet (10⁶ colony‐forming units per gram) for 5 weeks. The live weight of fish was registered on a weekly basis. After 4 weeks, fish from each treatment were immunocompromised with pathogenic A. hydrophila and further fed for 1 week in order to evaluate the effect on immunological and antioxidant parameters. Generally, the results showed enhanced growth performance in fish fed the diet containing yeast compared with the control. Addition of live yeast had no significant effect on the immunological parameters after 4 weeks of feeding. However, post infection with A. hydrophila fish fed the yeast‐supplemented diet resulted in a significant increase in the levels of plasmatic immunoglobulin M. Superoxide dismutase and catalase (CAT) activities were significantly higher in the yeast group. In this fish, CAT and heat shock protein 70 genes were up‐regulated before and after infection of A. hydrophila. The present study is the first one reporting that yeast (D. hansenii) can enhance immunity and resistance against A. hydrophila.     

The Incidence of Aeromonas Species in the Feces of Nondiarrheic Horses

Aeromonas species have been isolated from the feces of diarrheic adult horses and foals. However, Aeromonas species have also been isolated from the feces of approximately 5% of clinically normal adult horses outside the United States. The objectives of this study were to determine whether Aeromonas species can be shed in the feces of nondiarrheic adult horses and identify any effects of season of year, transportation, and hospitalization on possible fecal shedding of Aeromonas species. Feces were cultured for Aeromonas and Salmonella species from 138 nondiarrheic adult horses examined in the hospital and by field services for complaints other than gastrointestinal disease. Fecal Aeromonas and Salmonella species were isolated from 6.5% and 2.2% of nondiarrheic horses, respectively. All Aeromonas isolates were identified as A caviae. There were no apparent effects on time of year, hospitalization, or transport on isolation of Aeromonas species from feces. Isolation of Aeromonas species from diarrheic horses in the absence of other possible enteric pathogens indicates intestinal disease as a result of Aeromonas infection, but Aeromonas species can also occur as copathogens with Salmonella infections.     

3种抗生素对3种常见鱼类病原菌的抑菌活性研究

为研究盐酸恩诺沙星、乳酸恩诺沙星和乳酸诺氟沙星对嗜水气单胞菌(Aeromonas hydrophila)、鳗弧菌(Vibrio anguillarum)和哈维氏弧菌(Vibrioharveyi)的体外抑菌活性,本实验通过测定抑菌圈直径、最小抑菌浓度(MIC)、最小杀菌浓度(MBC)和杀菌曲线,研究3种常用喹诺酮类抗生素药物对3种水产病原菌的体外药效学特性.结果表明:乳酸诺氟沙星对嗜水气单胞菌和哈维氏弧菌抑菌效果最好,MIC分别为0.244μg/mL和1.954μg/mL:盐酸恩诺沙星对鳗弧菌抑菌效果最好,MIC为0.122μg/mL;8 MIC的乳酸诺氟沙星药液在12 h内完全杀灭嗜水气单胞菌,在24 h内也能全部杀灭哈维氏弧菌,8 MIC的盐酸恩诺沙星药液在24 h内全部杀灭鳗弧菌.本研究为3种抗生素在水产养殖中的应用提供实验依据.     

50种中草药体外抑杀嗜水气单胞菌的药效研究

为筛选有效抑制嗜水气单胞菌(A.hydrophila)的中草药,以期为水产养殖业中防治该菌引起的疾病提供实验依据.本研究采用琼脂扩散法(打孔法)测定了地榆等50种中草药对A.hydrophila的抑制作用,并用二倍稀释法测定抑菌效果较强的25种中草药对A.hydrophila的最小抑菌浓度(MIC)和最小杀菌浓度(MBC).结果显示地榆的抑菌效果最好,其抑菌圈直径达20mm以上,MIC为3.91 mg/mL,MBC为7.81 mg/mL;黄连等24种中草药有一定的抑菌和杀菌效果,巴戟天等25种中草药无抑菌作用.结果表明地榆对A.hydrophila的体外抑制效果最好.     

Molecular characterization of tetracycline- and quinolone-resistant Aeromonas salmonicida isolated in Korea

The antibiotic resistance of 16 Aeromonas (A.) salmonicida strains isolated from diseased fish and environmental samples in Korea from 2006 to 2009 were investigated in this study. Tetracycline or quinolone resistance was observed in eight and 16 of the isolates, respectively, based on the measured minimal inhibitory concentrations. Among the tetracycline-resistant strains, seven of the isolates harbored tetA gene and one isolate harbored tetE gene. Additionally, quinolone-resistance determining regions (QRDRs) consisting of the gyrA and parC genes were amplified and sequenced. Among the quinolone-resistant A. salmonicida strains, 15 harbored point mutations in the gyrA codon 83 which were responsible for the corresponding amino acid substitutions of Ser⁸³→Arg⁸³ or Ser⁸³→Asn⁸³. We detected no point mutations in other QRDRs, such as gyrA codons 87 and 92, and parC codons 80 and 84. Genetic similarity was assessed via pulsed-field gel electrophoresis, and the results indicated high clonality among the Korean antibiotic-resistant strains of A. salmonicida.     

框镜鲤嗜水气单胞菌的分离鉴定

以患病濒死的框镜鲤为材料,得到1株细菌,动物回归试验证明其具有较强的致病性,然后对病原菌的形态学、培养特性、理化特性、16SrRNA序列分析等生物学特性进行了鉴定与分析。结果表明,该菌株为嗜水气单胞菌。     

18种中草药煎液对温和气单胞菌的抑、杀菌效果

采用琼脂扩散法(打孔法)测定了五倍子等18种中草药对2株病原温和气单胞菌(Aerom onas sobria)的抑制作用,并采用试管2倍稀释法测定抑菌效果较强的中草药对这2株温和气单胞菌的抑菌和杀菌浓度。结果显示:五倍子、苏木、地锦草、石榴皮、黄芩、虎杖和山楂对病原温和气单胞菌的抑菌效果较强;苏木、五倍子、地锦草和石榴皮4种中药对2株温和气单胞菌的最小抑菌浓度(M IC)为1.56～6.25 mg/mL之间,最小杀菌浓度(MBC)为3.12～12.5 mg/mL之间,其中五倍子的抑菌和杀菌浓度最低,分别为1.56 mg/mL和3.12 mg/mL;而金银花等11种中草药抑菌作用较小。结果表明,所选取的18种中草药中五倍子对防治由温和气单胞菌感染引起的水产动物疾病最为有效。