First isolation of Vibrio tapetis and an atypical strain of Aeromonas salmonicida from skin ulcerations in common dab (Limanda limanda) in the North Sea

Skin ulcerations rank amongst the most prevalent lesions affecting wild common dab (Limanda limanda) with an increase in prevalence of up to 3.5% in the Belgian part of the North Sea. A complex aetiology of these ulcerations is suspected, and many questions remain on the exact factors contributing to these lesions. To construct the aetiological spectrum of skin ulcerations in flatfish, a one‐day monitoring campaign was undertaken in the North Sea. Fifteen fish presented with one or more ulcerations on the pigmented and/or non‐pigmented side. Pathological features revealed various stages of ulcerations with loss of epidermal and dermal tissue, inflammatory infiltrates and degeneration of the myofibers bordering the ulceration, albeit in varying degrees. Upon bacteriological examination, pure cultures of Vibrio tapetis were retrieved in high numbers from five fish and of Aeromonas salmonicida in one fish. The V. tapetis isolates showed cross‐reactivity with the sera against the representative strain of serotype O2 originating form a carpet‐shell clam (Ruditapes descussatus). Moreover, the A. salmonicida isolates displayed a previously undescribed vapA gene sequence (A‐layer type) with possible specificity towards common dab. Further research is necessary to pinpoint the exact role of these agents in the development of skin ulcerations in common dab.     

Comparative pathogenicity of Vibrio spp., Photobacterium damselae ssp. damselae and five isolates of Aeromonas salmonicida ssp. achromogenes in juvenile Atlantic halibut (Hippoglossus hippoglossus)

Juvenile Atlantic halibut (~100 mg, Hippoglossus hippoglossus) were exposed to Vibrio proteolyticus, a Vibrio spp. isolate, Photobacterium damselae ssp. damselae and five different isolates of Aeromonas salmonicida ssp. achromogenes via an hour‐long bath immersion to ascertain their variation in pathogenicity to this fish species. Results were analysed using Kaplan–Meier survival analysis. Analysis of the data from challenges using A. salmonicida ssp. achromogenes revealed three survival values of zero and a spread of values from 0 to 28.43. Challenges using a Vibrio spp isolate, V. proteolyticus and P. damselae resulted in Kaplan–Meier survival estimates of 31.21, 50.41 and 57.21, respectively. As all bacterial species tested could induce juvenile halibut mortalities, they must all be considered as potential pathogens. However, the degree of pathogenicity of A. salmonicida is isolate dependent.     

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.     

27株嗜水气单胞菌致病性及ERIC—PCR指纹图谱分析

从福建省淡水养殖鱼类体内分离到27株嗜水气单胞菌，根据嗜水气单胞菌16SrDNA基因和气溶素基因（aerolysin）的保守序列，设计2对引物，对所分离到的27株嗜水气单胞菌进行双重PCR扩增，扩增结果表明，其中18株为含有Aer毒力基因的潜在致病性嗜水气单胞菌，占总菌数的66．67％。应用ERIC-PCR分型技术对27株嗜水气单胞菌菌株进行分析，以相似度54．00％为限，所有菌株可分为Ⅰ和Ⅱ两大聚类，以76．00％相似度为界，27株嗜水气单胞菌可分为11个聚类，同一个聚类中菌株分离区域基本相同。分析结果表明，分离的嗜水气单胞菌基因型的多样性和分离地域具有一定的关联，也表明ERIC-PCR可以有效应用于嗜水气单胞茵分子流行病学调查。     

Immunostimulatory effect of water soluble fraction of Nyctanthes arbortristis leaves on the immune response in Oreochromis mossambicus (Peters)

Use of immunostimulants as a prophylactic measure against diseases in fish is considered as an effective alternative to antibiotic use. Plant‐derived immunostimulants have recently received more attention, as most of them are cost‐effective and eco‐friendly. This study was carried out with the objective of evaluating the possible immunostimulatory activity of intraperitoneally injected water soluble fraction of Nyctanthes arbortristis (an Indian medicinal plant) leaves on (i) specific immunity (antibody response), (ii) nonspecific immunity (Lysozyme activity, ROS production) and (iii) functional immunity in terms of disease resistance against Aeromonas hydrophila in Oreochromis mossambicus. The results of the study showed that all the tested doses (i.e. 3.2, 16, 80 and 400 mg kg^?1) of water soluble fraction of Nyctanthes arbortristis leaves significantly enhanced both primary and secondary antibody responses to heat killed A. hydrophila. Significant enhancement in serum lysozyme activity by all the doses of water soluble fraction was observed on day 10. The lower doses of 3.2 and 16 mg kg^?1 of water soluble fraction alone enhanced intracellular ROS production. Fish treated with both single and double dose of water soluble fraction showed maximum disease resistance. The highest dose (400 mg kg^?1) of water soluble fraction exhibited highest relative per cent survival in both single dose and double dose groups.     

Antimicrobial Susceptibility and Characterization of Outer Membrane Proteins of Aeromonas hydrophila Isolated in China

Aeromonas hydrophila isolates from clinical cases （n=43） were tested against 8 antimicrobial agents and typed by outer membrane protein （OMP） pattern by using sodium dodecyl sulfate gel electrophoresis. All isolates were resistant to ampicillin （MICs, ≥16 μg mL-1） and sulfamonomethoxine （MICs≥64 μg mLl）, but susceptible to norfloxacin （MICs,≤0.5 μg mL-1）. There was a high incidence of resistance to erythromycin （90.70%） and tylosin （93.02%）, while a low incidences of resistance to ciprofloxacin （2.33%）, enrofloxacin （2.33%） and florfenicol （4.65%）. Six different outer membrane protein patterns were found among 34 isolates by analyzing proteins in the range of 22 to 50 kDa, other than 9 isolates with their respective profiles. The strains with the similar OMP profiles had similar resistances. Compared with the other strains from the same OMP patterns, NB-1, A.Pun and MR-1 had lacked the proteins in the range of 30 to 45 kDa and their resistance to florfenicol substantially increased. It is speculated that the outer membrane protein changes might correlate with decreased susceptibility to florfenicol in the three strains. Some strains which showed completely identical OMP types had a little difference in their resistance to fluoroquinolones, indicating that there might be other factors that were involved in the antimicrobial resistance of A. hydrophila.     

单壁碳纳米管载嗜水气单胞菌外膜蛋白亚单位疫苗的研制及免疫效果评价

为了筛选细菌的疫苗候选蛋白,本实验根据课题组前期的研究,挑选了2个嗜水气单胞菌外膜蛋白(A0KLQ6和A0KH89),以A0KHR9(OmpAII)为对照,通过比较外膜蛋白与碳纳米管载外膜蛋白通过腹腔注射与浸泡免疫模式,比较和评价斑马鱼的免疫反应与免疫保护效果。结果显示,腹腔注射组和浸泡组与普通蛋白组对比,碳纳米管载蛋白能够显著提高斑马鱼免疫相关基因表达量。腹腔注射组在5μg剂量下免疫A0KHR9、A0KLQ6和A0KH89蛋白分别获得61.60%、 65.39%和84.96%相对免疫保护率(relative immune protection rate,RPS),在5μg剂量下免疫碳纳米管载蛋白SWCNTs-A0KHR9、SWCNTsA0KLQ6和SWCNTs-A0KH89分别获得68.10%、77.50%和90.43%的RPSs。浸泡组在40 mg/L剂量下免疫A0KHR9、A0KLQ6和A0KH89蛋白分别获得30.80%、25.85%和37.80%的RPSs,在40 mg/L最高剂量下免疫碳纳米管载蛋白疫苗SWCNTs-A0KHR9、SWCNTsA0KLQ6和SWCNTs-A0KH89分别获得63.60%、73.74%和68.49%的RPSs。研究表明,铁离子限制下嗜水气单胞菌外膜蛋白A0KLQ6和A0KH89等能够作为抗嗜水气单胞菌的亚单位疫苗候选蛋白,而单壁碳纳米管载蛋白通过注射免疫与浸泡免疫均能显著提升免疫疗效,是一种疫苗的适宜纳米载体。以上研究为寻找高效的嗜水气单胞菌亚单位疫苗候选蛋白及佐剂提供了理论依据。     

青虾源维氏气单胞菌双重PCR检测方法的建立

根据NCBI已发表的维氏气单胞菌(Aeromonas veronii)促旋酶B亚单位基因gyrB和编码细菌RNA聚合酶σ70因子基因rpoD的保守序列设计引物,建立了一种快速检测青虾源维氏气单胞菌的双重PCR检测方法。结果显示:青虾源维氏气单胞菌gyrB和rpoD基因PCR扩增产物大小分别为815 bp、554 bp,而对嗜水气单胞菌(Aeromonas hydrophila)、弗氏柠檬酸杆菌(Citrobacter freundii)、哈维弧菌(Vibrio harveyi)、副溶血性弧菌(Vibrio parahemolyticus)、需钠弧菌(Vibrio natriegens)、非O1霍乱弧菌(non-O1 Vibrio cholerae)、阴沟肠杆菌(Enterobacter cloacae)、产气肠杆菌(Enterobacter aerogenes)扩增结果皆为阴性;灵敏性试验结果显示该方法检测到的最低菌体DNA量为1.8×10-2 ng/μL,且10份送检样本检测结果与传统细菌分离鉴定结果相一致。结果表明,双重PCR检测方法特异性好、灵敏性高,适用于青虾源维氏气单胞菌的快速检测。     

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。