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.     

Improvements of virulence factor phenotypic tests for Aeromonas salmonicida subsp. salmonicida,a major fish pathogen

Aeromonas salmonicida subspecies salmonicida, a fish pathogen, expresses various virulence factors such as an A-layer, lipases and proteases during the infection process. Not all strains of this bacterium express the same virulence factors. It is important to be able to evaluate which factors are present when characterizing strains. The A-layer and secreted lipases and proteases are usually detected by agar-based tests that require long incubation (24 h and more) and may provide ambiguous results. In the present study, protocols have been optimized to determine the presence of these virulence factors using liquid tests. For A-layer detection, the optimized method stains the positive bacteria with Coomassie Brilliant Blue. The lipases are detected by a colorimetric biochemical reaction triggered by the degradation of p-nitrophenyl dodecanoate into a yellow product detectable by spectrophotometry, if the result is positive. Both of these tests show results in less than an hour. Finally, the protease activity is measured by clarification of a medium containing milk during an overnight bacterial growth. These new protocols provide opportunities for quicker characterization of A. salmonicida subsp. salmonicida strains and, particularly, provide more precise results.     

养殖刺参溃疡病杀鲑气单胞菌的分离、致病性及胞外产物特性分析

从患溃疡病的养殖刺参(Apostichopus japonicus)病灶处分离出1株优势菌H1,以浸浴、创伤浸浴、体腔注射和体壁肌肉注射等方式进行感染实验,证实菌株H1为养殖刺参溃疡病病原菌,并证明该菌通过体表创伤侵入的方式感染刺参,以创伤浸浴和体壁肌肉注射感染的LD50(半数致死量)分别为2.26×107CFU/尾和1.80×107CFU/尾。经形态学观察、生理生化特性分析和mini API系统鉴定,确定菌株H1为杀鲑气单胞菌杀日本鲑亚种(Aeromonas salmonicida ma-soucida)。提取菌株H1的胞外产物(ECP)进行致病性实验,结果表明ECP可导致刺参死亡,其对刺参的LD50为5.24μg蛋白/g体质量。H1-ECP具有酪蛋白酶、明胶蛋白酶、几丁质酶和淀粉酶活性,并具有溶血素活性;对底物偶氮酪蛋白(Azocasin)作用的酶比活力可达到674.5活力单位/mg蛋白,最适作用温度为50℃;对热不稳定,70℃作用30 min时,酪蛋白酶活性降到0;100℃作用30 min,ECP对刺参的毒性消失;ECP酶活可被10 mmol/L EDTA完全抑制,可被5 mmol/L PMSF抑制98.8%,Ca2 和Mg2 可使酶活性分别提高约9%和4%。结论认为,该病原菌通过体表创伤侵入方式感染宿主刺参,菌株H1胞外产物是其对刺参致病的因子之一。     

杀鲑气单胞菌杀鲑亚种和杀日本鲑亚种PCR检测方法的建立

杀鲑气单胞菌(Aeromonas salmonicida)是一种重要的鱼类致病菌,可以感染多种海淡水鱼类。杀鲑气单胞菌包括5个亚种,目前常用的生理生化特征和16S rDNA序列分析方法很难实现亚种的快速精确区分。为实现杀鲑气单胞菌亚种的快速鉴定和检测,针对我国常见的杀鲑气单胞菌杀鲑亚种(A. salmonicida subsp. salmonicida)和杀日本鲑亚种(A. salmonicida subsp. masoucida),本研究开发了其特异性的PCR检测方法。根据Gene Bank已公布的杀鲑气单胞菌基因组信息,选择杀鲑亚种phoB基因和杀日本鲑亚种LOC111476736基因作为目标基因,根据其序列设计特异性引物,进一步对PCR反应的退火温度、引物浓度、dNTPs浓度、Mg2+浓度和酶浓度5个方面进行了优化,并测试了该方法的特异性、敏感性和应用效果。结果显示,2对引物分别可以扩增出杀鲑气单胞菌杀鲑亚种522 bp的phoB特异性基因片段和杀日本鲑亚种515 bp的LOC111476736特异性基因片段。杀鲑亚种特异性引物最适退火温度为64 ℃,10 µmol/L引物、2 mmol/L dNTPs、25 mmol/L MgSO4和1 U/µL KOD酶的最适添加量分别为1.5、2、1.5和0.5 µL。杀日本鲑亚种特异性引物最适退火温度为64 ℃,10 µmol/L引物、2 mmol/L dNTPs、25 mmol/L MgSO4和1 U/µL KOD酶的最适添加量分别为0.75、1、1.5和0.5 µL。以鳗弧菌(Vibrio anguillarum)、美人鱼发光杆菌(Photobacterium damselae)、杀鱼爱德华氏菌(Edwardsiella piscicida)、杀鲑气单胞菌其他亚种等14种其他水产病原菌或常见环境菌为模板进行PCR检测,均无特异性条带。该方法对杀鲑气单胞菌杀鲑亚种的检测灵敏度为12.8 CFU/反应(菌体)或17.6 fg/反应(DNA),对杀鲑气单胞菌杀日本鲑亚种的检测灵敏度为23.8 CFU/反应(菌体)或27.2 fg/反应(DNA)。利用杀鲑气单胞菌杀鲑亚种和杀日本鲑亚种分别对大菱鲆(Scophthalmus maximus)进行人工感染实验,感染后取病鱼组织进行PCR检测,结果显示,本方法可以从感染后的大菱鲆中分别检测到相应病原。综上所述,本研究建立了杀鲑气单胞菌杀鲑亚种和杀日本鲑亚种的特异性PCR检     

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。     

养殖裸盖鱼(Anoplopoma fimbria)疥疮病病原菌的分离与鉴定

裸盖鱼(Anoplopoma fimbria)是一种名贵的冷水经济鱼类,已在我国北方地区开展小规模的工厂化繁育和养成.目前,我国关于裸盖鱼的病害研究仍是空白.本研究对山东烟台一养殖场自然发生疥疮病的裸盖鱼进行了病原分析,从发病鱼体内分离得到形态一致的优势菌株,命名为AF-1,并对其进行了致病性检测、菌种鉴定及药物敏感性研究.人工感染实验证明,AF-1对裸盖鱼有致病性,呈现症状与自然发病状态一致;结合形态观察、革兰氏染色、生理生化特征、16S rRNA和gyrB基因序列进化树分析,将AF-1鉴定为杀鲑气单胞菌(Aeromonas salmonicida);药物敏感性实验结果显示,AF-1对青霉素、阿莫西林、头孢噻吩等13种抗生素具有耐药性,对氟苯尼考、氟甲喹等16种抗生素敏感.综上所述,本研究首次报道了我国养殖裸盖鱼感染杀鲑气单胞菌病例,为裸盖鱼养殖过程中的疾病防控和疫苗开发提供了参考.     

杀鲑气单胞菌杀日本鲑亚种胞外产物毒性及免疫原性分析

用玻璃纸平板法提取了杀鲑气单胞菌杀日本鲑亚种Aeromonassal monicida masoucida的胞外产物（ECP）。毒性试验证实,ECP对剑尾鱼Xiphophorus helleri具有致死性,其半致死量（LD50）为4.72μg蛋白/g体重。SDS-PAGE表明,ECP由13条蛋白带组成。利用大鼠抗ECP血清进行的Western-blot印迹显示,组成ECP的13条蛋白带中有7条具有免疫原性,能够引发机体的免疫应答反应产生抗体,其分子量分别为88、70、42、39、36、22和15kDa。     

Temporal and spatial variability in growth and condition of dab (Limanda limanda) and sprat (Sprattus sprattus) larvae in the Irish Sea

Variability in the high mortality rate during early life stages is considered to be one of the principal determinants of year‐class variability in fish stocks. The influence of water column stability on the spatial distribution of fish larvae and their prey is widely acknowledged. Water column stability may also impact growth through the early life history of fishes, and consequently alter the probability of survival to maturity by limiting susceptibility to predation and starvation. As a test of this concept, the variability in condition and growth of dab (Limanda limanda) and sprat (Sprattus sprattus) larvae was investigated in relation to seasonal stratification of the water column in the north‐western Irish Sea. RNA/DNA ratios and otolith microincrement analysis were used to estimate nutritional status and recent growth rates of larvae captured on four cruises in May and June of 1998 and 1999. Dab and sprat larvae were less abundant in 1999 and were in poorer condition with lower growth rates than in 1998. Dab larvae of <13 mm also exhibited spatial variability with higher RNA/DNA ratios at the seasonal tidal‐mixing front compared with stratified and mixed water masses. However, the growth and nutritional status of sprat larvae was uncorrelated to water column stability, meaning the more favourable feeding conditions generally associated with the stratified pool and tidal‐mixing front in the Irish Sea were not reflected in the growth and condition of these larvae. This suggests that the link between stability, production and larval growth is more complicated than inferred by some previous studies. The existence of spatio‐temporal heterogeneity in the growth and condition of these larvae has implications for larval survival and the recruitment success of these species in the Irish Sea.     

vapA (A‐layer) typing differentiates Aeromonas salmonicida subspecies and identifies a number of previously undescribed subtypes

Sequence variation in a region of the virulence array protein gene (vapA; A‐layer) was assessed in 333 (‘typical’ and ‘atypical’) isolates of the fish pathogenic bacterium Aeromonas salmonicida. Resulting similarity dendrograms revealed extensive heterogeneity, with nearly all isolates belonging to either of 14 distinct clusters or A‐layer types. All acknowledged A. salmonicida subspecies (except ssp. pectinolytica, from which no vapA sequence could be obtained) were clearly separated, and notably, all isolates phenotypically identified as ssp. salmonicida formed a distinct and exclusive A‐layer type. Additionally, an array of un‐subspeciated atypical strains formed several equally prominent clusters, demonstrating that the concept of typical/atypical A. salmonicida is inappropriate for describing the high degree of diversity evidently occurring outside ssp. salmonicida. Most representatives assessed in this study were clinical isolates of spatiotemporally diverse origins, and were derived from a variety of hosts. We observed that from several fish species or families, isolates predominantly belonged to certain A‐layer types, possibly indicating a need for host‐/A‐layer type‐specific A. salmonicida vaccines. All in all, A‐layer typing shows promise as an inexpensive and rapid means of unambiguously distinguishing clinically relevant A. salmonicida subspecies, as well as presently un‐subspeciated atypical strains.     

Evaluation of an experimental Aeromonas salmonicida epidemic in chinook salmon, Oncorhynchus tshawytscha (Walbaum)

To determine the dynamics of the transmission of Aeromonas salmonicida ssp. salmonicida infection, chinook salmon, Oncorhynchus tshawytscha, were exposed to bacteria by cohabitation. The latent period (time between exposure and infectivity) was determined by exposing a group of chinook salmonid fingerlings to A. salmonicida by bath, then, at daily intervals, by holding five exposed (donor) fish with approximately 50 naive fish for 24 h. The latent period was 3 days post-infection and the time period between the initial exposure to bacteria and the beginning of bacterial shedding was 4.5 days for the same animals. The prevalence and intensity of infection in the donor fish, to which recipient fish were exposed, i.e. the level of exposure, was highly correlated with the development of disease in recipient (susceptible) chinook salmon (r2 = 0.57). An experiment was conducted to determine the daily progress of infection and development of a furunculosis epidemic among recipient fish by cohabiting a single exposed fish with 43 unexposed salmon. At daily intervals, all fish (in seven treatment tanks and one control tank daily) were sacrificed and tested for the presence of A. salmonicida in the kidney (n = 3520). Over 10 days, mean prevalence among recipient fish reached 75% and disease related mortality exceeded 50%. Bacterial concentrations in the water continued to increase over the duration of the experiment in concert with the number of infected animals present in the population.     

Atypical Aeromonas salmonicida infection in the black rockfish, Sebastes schlegeli Hilgendorf, in Korea

Cultured black rockfish, Sebastes schlegeli, suffered mass mortalities during winter 2008 and spring 2009 in Korea, showing clinical signs of ulcer lesions and haemorrhages over their body surface. The aetiological agent was identified as Aeromonas salmonicida (strains RFAS-1, -2 and -3), which is a non-pigmented, slow-growing bacterium. Phenotypes of RFAS strains showed variation, while 16S rRNA, gyrB, rpoD, dnaJ and recA gene sequences of all the strains were affiliated to A. salmonicida. In particular, vapA gene sequences of the strains were most closely related to one of the five subspecies of A. salmonicida subsp. masoucida (=KCCM 40239(T) ). LD(50) values of RFAS-1 for intraperitoneal and intramuscular injection were 1.5 × 10(5.25) and 1.5 × 10(6.4) cfu/rockfish, respectively. However, A. salmonicida strains KCCM 40239(T) and SAS-1, which originate from masou and chum salmon, respectively, were not pathogenic to black rockfish. RFAS strains, possessing A-layer protein on their surface, exhibited β-haemolytic activity against rockfish erythrocytes and capability to survive in rockfish serum, which seem to be associated with virulence.     

Antibody-coated gold nanoparticles immunoassay for direct detection of Aeromonas salmonicida in fish tissues

Aeromonas salmonicida is the causative agent of furunculosis, a disease that affects both salmonid and non‐salmonid fish. Detection of A. salmonicida can be labour intensive and time consuming because of the difficulties in distinguishing the bacterium from other species given the wide variety of existing biochemical profiles and the slow growth characteristics which allow other organisms to overgrow the A. salmonicida. Herein, we report the development of a specific immunoassay using gold‐conjugated polyclonal antibodies for the rapid detection of A. salmonicida in fish tissues. Monodispersible 13‐nm gold nanoparticles were coated with polyclonal antibodies specific to A. salmonicida. Reddish purple agglutination of gold particles indicated the presence of A. salmonicida in samples. Positive reactions were detected visually with the naked eye. No agglutination was observed when A. salmonicida antibody‐coated gold nanoparticles were tested with other common bacterial fish pathogens, thereby verifying the specificity of the assay. The assay could detect A. salmonicida in fish tissues down to 1 × 10⁴ CFU mL^?1, and results were obtained within 45 min. The antibody‐coated gold nanoparticles were stable for at least 2 months at 4°C. The immunoassay using antibody‐coated gold nanoparticles represents a promising tool for the rapid and specific detection of A. salmonicida in fish tissues.     

Histopathological changes in the livers of dab, Limanda limanda (L.)

Abstract. The dab, Limanda limanda (L.), is a common species of flatfish found in the shallow waters of the North Sea, particularly in inshore regions. Because of its distribution and habits, it is a useful species for investigations into the biological effects of marine pollution; it is also susceptible to certain, easily-detectable, gross diseases, including a variety of hepatic lesions, some of which are putative neoplasias. Investigations over the past 10 years on the histopathology of dab livers, have revealed that many lesions are associated with parasitic infections or trauma, and others include the presence of basophilic foci and putative adenoma as well as cell storage changes. The significance of these lesions is discussed in relation to other biological and physical data, and the necessity for histological evaluation is emphasized.     

Characterization of the microsomal epoxide hydrolase of hepatic microsomes of the common dab,Limanda limanda

Epoxide hydrolase of microsomal membranes of the common dab (Limanda limanda) has been characterized using p-nitrostyrene oxide as substrate. Under the conditions of assay used, the turnover number with this substrate was higher than found for the more frequently used styrene oxide and steady state kinetics were observed. The enzyme had a K_M of 0.12 mM and optima for pH and temperature between pH 8–10.2 and 50–60°C respectively. Enzyme activity was unaffected by low concentrations of ionic and non-ionic detergents but was inhibited by higher concentrations of Lubrol and Brij. The enzyme protein did not react with monospecific antibodies to rat or human microsomal epoxide hydrolase during Western blotting. Large inter-individual variation in enzyme activity was found but the enzyme does not appear to be expressed in a gender-specific way. Fish were administered a wide range of hydrocarbons which are known to alter the expression of cytochrome P450 1A but these had no effect other than benzothiophene which caused a small increase in enzyme activity.Abbreviations mEH microsomal epoxide hydrolase - pNSG p-nitrostyrene glycol - pNSO p-nitrostyrene oxide     

Translocation of viable Aeromonas salmonicida across the intestine of rainbow trout, Oncorhynchus mykiss (Walbaum)

The pathogenic bacterium Aeromonas salmonicida is the causative agent of the destructive disease furunculosis in salmonids. Horizontal transmission in salmonids has been suggested to occur via the skin, gills and/or intestine. Previous reports are contradictory regarding the role of the intestine as a route of infection. The present study therefore investigates the possibility of bacterial translocation across intestinal epithelia using Ussing chamber technology, in vitro. Intestinal segments were exposed for 90 min to fluorescein isothiocyanate-labelled pathogenic A. salmonicida. Sampling from the serosal side of the Ussing chambers showed that bacteria were able to translocate across the intestinal epithelium in both the proximal and distal regions. Plating and subsequent colony counting showed that the bacteria were viable after translocation. During the 90 min exposure to A. salmonicida, the intestinal segments maintained high viability as measured by electrical parameters. The distal region responded to bacterial exposure by increasing the electrical resistance, indicating an increased mucus secretion. This study thus demonstrates translocation of live A. salmonicida through the intestinal epithelium of rainbow trout, suggesting that the intestine is a possible route of infection in salmonids.     

杀鲑气单胞菌可视化重组酶聚合酶扩增检测技术的建立

本研究根据杀鲑气单胞菌(Aeromonas salmonicida)的铁载受体(fstA)基因的保守序列设计特异性引物和探针,建立了一种基于重组酶聚合酶扩增(recombinase polymerase amplification,RPA)与侧向流试纸条(lateral flow strips,LFS)相结合的可视化快...     

The impact of Aeromonas salmonicida infection on behaviour and physiology of Atlantic salmon (Salmo salar L.)

This study examined the effect of Aeromonas salmonicida infection on the swimming behaviour and physiology of Atlantic salmon (Salmo salar L.). Fish were injected in the dorsal muscle with either 100 μL bacterium solution (3.05 × 10⁷CFU mL^?1) Aeromonas salmonicida, or 100 μL 0.9% NaCl (as control group). Compared with the control group, the pathogen injected group significantly impaired the critical swimming speed (U_crit) and exhausting time (P < 0.05), which were reduced by 37% and 39% at severe time (day 6, when most of the fish challenged by Aeromonas salmonicida died) respectively. Furthermore, the blood parameters related to their swimming behaviour were also influenced significantly by pathogen injection (P < 0.05). The results showed that the high density lipoprotein (HDL), haemoglobin and total protein decreased by about 63%, 49% and 74% at the end, respectively, while lactate increased by about 29% on day 6. The results suggested that the swimming performance of Atlantic salmon might be a useful indicator of disease, and it was feasible to warn the outbreaks of acute disease by fish behaviour.