Infection routes of Aeromonas salmonicida in rainbow trout monitored in vivo by real‐time bioluminescence imaging

Recent development of imaging tools has facilitated studies of pathogen infections in vivo in real time. This trend can be exemplified by advances in bioluminescence imaging (BLI), an approach that helps to visualize dissemination of pathogens within the same animal over several time points. Here, we employ bacterial BLI for examining routes of entry and spread of Aeromonas salmonicida susbp. salmonicida in rainbow trout. A virulent Danish A. salmonicida strain was tagged with pAKgfplux1, a dual‐labelled plasmid vector containing the mutated gfpmut3a gene from Aequorea victoria and the luxCDABE genes from the bacterium Photorhabdus luminescens. The resulting A. salmonicida transformant exhibited growth properties and virulence identical to the wild‐type A. salmonicida, which made it suitable for an experimental infection, mimicking natural conditions. Fish were infected with pAKgfplux1 tagged A. salmonicida via immersion bath. Colonization and subsequent tissue dissemination was followed over a 24‐h period using the IVIS spectrum imaging workstation. Results suggest the pathogen's colonization sites are the dorsal and pectoral fin and the gills, followed by a progression through the internal organs and an ensuing exit via the anal opening. This study provides a tool for visualizing colonization of A. salmonicida and other bacterial pathogens in fish.     

Probiotic efficiency of Bacillus sp. in Labeo rohita challenged by Aeromonas hydrophila: assessment of stress profile,haemato‐biochemical parameters and immune responses

Fish are always susceptible to a wide variety of deadly pathogens which cause a huge loss in aquaculture industries. In this investigation, we have demonstrated the in vivo probiotic efficiency of Bacillus sp. MVF1 (GenBank Acc. No. KP256503) in Labeo rohita challenged with pathogenic strain of Aeromonas hydrophila (MTCC 1739). To check the probiotic potential of the selected bacterial strain, fish were divided into four groups: control, D1, D2 and D3. A total of 100 days (70 days probiotic feeding + 71th day sampling and 28 days challenged test + 29th day sampling) of feeding trial was conducted. To establish the probiotic potential of Bacillus sp. MVF1, certain haematological parameters (haemoglobin, total erythrocyte and leucocyte count), serum biochemical parameters (total protein, albumin and globulin), immune parameters (serum lysozyme and total IgM levels) and hepatic stress profile (malondialdehyde production, superoxide dismutase and catalase activity) have been measured. Our results demonstrated that red blood cell number, white blood cell number and haemoglobin content were much higher in D2 group fish compared to other groups and control fish. Similarly, total protein contents, albumin concentration, globulin concentration, lysozyme activity and IgM production were also recorded to be highest in D2 group fish. This finding clearly indicated the probiotic potential of Bacillus sp. MVF1 in L. rohita. Furthermore, our results also demonstrated that 1 × 10⁷ CFU g^?1 feed (D2) provides better immunity compared to 1 × 10⁵ (D1) and 1 × 10⁹ (D3). Due to beneficial effects, the bacterium Bacillus sp. MVF1 might be useful in aquaculture industries to reduce the disease susceptibility.     

温和气单胞菌毒力基因的检测及其对鲫鱼致病性试验

为探究温和气单胞菌菌株A.S1的致病性,采用PCR方法扩增溶血素基因(hly)、细胞兴奋性肠毒素基因(alt)、黏附素基因(aha1)和气溶素基因(aerA)4种毒力基因,并运用动物回归试验探究菌株A.S1对鲫鱼的致病性。结果表明,菌株A.S1可扩增出hly、aha1和alt 3种毒力基因,未扩增出aerA基因。动物回归试验显示,试验组鲫鱼在腹腔注射温和气单胞菌0.3mL(浓度为1.5×108 CFU/mL)后,5d内死亡率达100%。表明毒力基因型为hly+、aha1+、alt+和aerA-的菌株A.S1为高毒力菌株,且菌株的毒力基因型与致病性呈正相关。     

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.