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1.
本研究以引起珍珠龙胆石斑鱼[Epinephelus fuscoguttatus(♀)×Epinephelus lanceolatus(♂)]幼鱼“皮肤溃疡病”的哈维氏弧菌(Vibrio harveyi) ML01株为研究对象,采用平板膜覆盖技术和柱层析技术,分离、纯化了ML01株的胞外产物及分泌性蛋白.应用毒性试验、质谱分析与分子克隆技术,对纯化的胞外产物和3种主要的分泌性蛋白进行了特性分析与鉴定.结果显示,哈维氏弧菌ML01株的胞外产物(Extracellular products,ECPs)具有酯酶、明胶酶、淀粉酶、酪蛋白酶活性,无脲酶活性.ECPs对羊红细胞无溶血性,对斑马鱼(Danio rerio)的半数致死剂量(LD50)为19.55μg/g鱼体重.从ML01株中分离到3种主要的分泌蛋白P42、P36、P31,其分子量分别为42、36、31 kDa.经质谱鉴定和分析,这3种蛋白分别为哈维氏弧菌的外膜蛋白OmpU和OmpN,以及一种功能未知的蛋白.利用同源克隆,成功地从ML01株基因组中扩增到了P42、P36、P31的基因.序列测定和比对结果显示,ML01株的这3个基因与哈维氏弧菌ATCC 33843(GenBank CP009467)的相应基因相比,其开放阅读框(Open reading frame,ORF)序列的相似性分别为97.08％、100％、99.67％,其编码的多肽序列的相似性分别为99.71％、100％和99.93％.本研究对进一步分析哈维氏弧菌ML01株的致病机理、研发该菌的亚单位疫苗具有重要的参考价值. 相似文献
2.
Bacterial subcellular components and probiotics were successful for the stimulation of immunity and the prevention of Vibrio harveyi infections in rainbow trout, Oncorhynchus mykiss (Walbaum). Rainbow trout were immunized with whole inactivated cells of V. harveyi to obtain polyclonal antibodies against specific antigens. Western blotting showed a unique reactive band (∼93 kDa) between serum and bacterial proteins from outer membrane proteins (OMP) and extracellular products (ECP). Probiotics were selected according to their capability to inhibit V. harveyi . Two of these bacteria, i.e. A3-47 and A3-51, showed cross-reactivity with V. harveyi antiserum. Their OMPs and ECPs were reactive with V. harveyi antiserum in bands of ∼93 kDa for A3-51 and higher for A3-47. In vivo tests determined that fish fed with A3-51 produced cross-reactive antibodies against V. harveyi and also, the survival of these fish infected with V. harveyi was high, being similar to the level achieved with vaccinated fish. Thus, the probiotics, when administered as live preparations, were capable of producing cross-reactive antibody against specific bacterial pathogens. 相似文献
3.
Evidence of exotoxin secretion of Piscirickettsia salmonis,the causative agent of piscirickettsiosis
M E Rojas M Galleguillos S Díaz A Machuca A Carbonero P A Smith 《Journal of fish diseases》2013,36(8):703-709
Piscirickettsia salmonis is the aetiological agent of piscirickettsiosis, a disease which affects a variety of teleost species and that is particularly severe in salmonid fish. Bacterial‐free supernatants, obtained from cultures of three isolates of Piscirickettsia salmonis, were inoculated in Atlantic salmon, Salmo salar L., and in three continuous cell lines in an effort to determine the presence of secretion of extracellular products (ECPs) by this microorganism. Although steatosis was found in some liver samples, no mortalities or clinical signs occurred in the inoculated fish. Clear cytotoxicity was observed after inoculation in the cell lines CHSE‐214 and ASK, derived from salmonid tissues, but not in MDBK, which is of mammalian origin. The degree of cytotoxicity of the ECPs was different among the P. salmonis isolates tested. The isolate that evidenced the highest cytotoxicity in its ECPs exhibited only an intermediate virulence level after challenging fish with bacterial suspensions of the three P. salmonis isolates. Almost complete inhibition of the cytotoxic activity of ECPs was seen after proteinase K treatment, indicating their peptidic nature, and a total preclusion of the cytotoxicity was shown after their incubation at 50 °C for 30 min. Results show that P. salmonis can produce ECPs and at least some of them are thermolabile exotoxins that probably play a role in the pathogenesis of piscirickettsiosis. 相似文献
4.
Chabrillón M Rico RM Arijo S Díaz-Rosales P Balebona MC Moriñigo MA 《Journal of fish diseases》2005,28(9):531-537
Four bacterial isolates from farmed gilthead sea bream, Sparus aurata, included in a previous study as members of the Vibrionaceae and Pseudomonodaceae and the genus Micrococcus, have been evaluated for their adhesive ability to skin and intestinal mucus of farmed Senegalese sole, Solea senegalensis, and their antagonistic effect on Vibrio harveyi, a pathogen of sole. These isolates showed higher adhesion to sole mucus than the pathogenic strains of V. harveyi assayed. Only two of the isolates showed antagonistic activity to V. harveyi. Interactions of the four isolates with V. harveyi in respect of adhesion to skin and intestinal mucus under exclusion, competition and displacement conditions were studied. Three isolates were able to reduce the attachment to skin and intestinal sole mucus of a pathogenic strain of V. harveyi under displacement and exclusion conditions, but not under competition conditions. The in vivo probiotic potential of isolate Pdp11 was assessed by oral administration followed by challenge with the pathogenic V. harveyi strain Lg14/00. A group of 50 Senegalese sole received a commercial diet supplemented with 10(8) cfu g(-1) of lyophilized Lg14/00 for 15 days. A second group of fish received a non-supplemented commercial diet. After challenge the mortality of the fish receiving the diet supplemented with the potential probiotic isolate was significantly lower than that in the fish receiving the non-supplemented commercial diet. This study has shown that the ability to interfere with attachment of pathogens, as well as the adhesion to host surfaces, are suitable criteria for selection of candidate probiotics for use in the culture of Senegalese sole. 相似文献
5.
哈氏弧菌胞外产物对红鳍东方鲀的致病性 总被引：1，自引：0，他引：1
采用平板玻璃纸覆盖技术提取哈氏弧菌(Vibrio harveyi)H-06091菌株的胞外产物(ECP),通过肌肉和腹腔注射2种方式研究其对红鳍东方鲀(Fugu obscurus)的致病性。采用双层纸片法对其主要酶成份进行初步研究;测定其对绵羊红细胞和红鳍东方鲀红细胞的溶血活性;将哈氏弧菌在不同温度下(15℃、20℃、25℃、30℃、35℃、40℃)培养36h和25℃下培养不同时间(12h、18h、24h、36h、48h、60h、72h)后提取其胞外产物并对其蛋白质含量进行测定;经SDS-PAGE分离粗提胞外产物中蛋白成分,并用Dot-ELISA和Western-blot方法对其成分进行分析。实验结果显示,该胞外产物对红鳍东方鲀具有致病性;具有淀粉酶和酪蛋白酶活性,不具有卵磷脂酶、脂肪酶和明胶酶活性;对红鳍东方鲀红细胞有溶血活性,对绵羊红细胞无溶血活性;胞外产物蛋白质总含量在35℃培养36h后出现高值,与温度变化无明显相关性;25℃下培养24h、36h和48h的粗提胞外产物经SDS-PAGE分离,有3条蛋白质条带最明显,分子量分别约为31kD、38kD、60kD;其中分子量约为60kD蛋白条带,经Dot-ELISA和Western-blot方法证实其为半胱氨酸蛋白酶-1。根据以上结果认为哈氏弧菌H-06091菌株胞外产物含有多种毒力因子,与该菌的致病性密切相关。 相似文献
6.
Characterization of extracellular products from an isolate of Vibrio harveyi recovered from diseased post-larval Penaeus vannamei (Bonne) 总被引：2，自引：0，他引：2
Vibrio harveyi recovered from diseased post-larval Penaeus vannamei produced a thermostable exotoxin, which was lethal to Dublin Bay prawns, Nephrops norvegicus L., when injected intramuscularly. The extracellular products (ECPs) concentrated from tryptone soya broth supplemented with 1% (w/v) sodium chloride or from cellophane overlays on marine 2216E agar with incubation at 15, 22 and 27 °C were toxic, with the lethal dose 50% of the crude ECPs estimated to be 4.4 μgprotein prawn−1. Proteolytic, haemolytic and cytotoxic activities were detected, although the occurrence and quantity of these activities were influenced by cultural conditions. The ECPs which had been heated (100 °C for 10 min) or digested with protease K produced the same pathology as crude, untreated ECPs. Western blotting demonstrated that all the ECP preparations contained low molecular weight lipopolysaccharides, which may constitute the lethal toxin of V. harveyi. 相似文献
7.
哈维氏弧菌的分子遗传多样性研究 总被引：1，自引：0，他引：1
运用RAPD分型和BOX分型分子方法研究患病的海水鱼类体内和海水环境的101株哈维氏弧菌的分子遗传多样性。结果显示,从10个RAPD引物中发现只有PM2引物的分辨率和重复性较好,出现15条不同RAPD条带,在相似度65%下,101株哈维氏弧菌可以分为18种不同型;BOX分型出现20条不同的条带,在相似度为40%的情况下,可以把101株哈维式弧菌分15种不同的型;综合RAPD PM2和BOX分型,发现在相似度50%下,101株哈维氏弧菌可分18种型。综合RAPD和BOX分型数据可以很好地克服各自的缺点,得到更加准确的分型结果。 相似文献
8.
矛尾复鰕虎鱼病原哈氏弧菌的鉴定及特异性检测方法的建立 总被引：1，自引：0，他引：1
取体表出血、肌肉溃疡的虾蟹养殖塘混养大量死亡的矛尾复鰕虎鱼的深层溃烂组织及肝脏进行细菌分离,2种被检组织中均分离到2种优势生长菌落;人工感染试验表明,其中的一株分离菌(S090801)浓度为106～108 cfu/ml可引起矛尾复鰕虎鱼全部死亡,该菌的形态特征、生理生化特性及基于16SrRNA和gyrB 2种基因的系统发育学分析确定为弧菌属的哈氏弧菌。同时基于gyrB基因序列设计1对特异性引物,建立了一种哈氏弧菌快速、敏感的PCR检测方法,扩增目的片段大小为363bp,该方法检测哈氏弧菌模板DNA最低质量浓度为0.046875ng/μl,可用于哈氏弧菌引起的水产动物疾病的快速诊断及分子流行病学的调查研究。 相似文献
9.
R Company A Sitj ;-Bobadilla M J Pujalte E Garay P Alvarez-Pellitero and J P . ;rez-S;nchez 《Journal of fish diseases》1999,22(4):299-309
A bacteriological and parasitological study on cultured common dentex, Dentex dentex L., was conducted in a 2-year survey. Two different groups of fish were studied: (1) sampled only when mortalities occurred; and (2) sampled periodi-cally from 10days post-hatch until the juvenile stage. In both groups, peaks of mortalities coincided with high water temperatures. The same parasites were detected in both groups, except Ichthyophonus sp., which was identified only in the trunk kidney of fish from group 1. Two myxosporeans, Ceratomyxa sp. and Lepthotheca sp., were found in the bile and the trunk kidney, respectively. Epitheliocystis infection was also very frequent in the gills. An unidentified microsporidium, which was found in the liver, occurred very infrequently. Vibrio harveyi was dominant in samples from diseased animals (79%) and clearly associated with mortalities. It was not detected in water or larval homogenates. A high percentage (60%) of the isolates of this species were sorbitol positive. Other less frequently occurring bacteria were V. alginolyticus and Photobacterium (V.) damselae. The disease conditions found in common dentex were considered to be multifactorial in origin. 相似文献
10.
Two replicated controlled trials were conducted to determine the efficacy of florfenicol against Aeromonas salmonicida and Vibrio salmonicida infections in Atlantic salmon, Salmo salar L., smolts kept in 25‰ salt water. Infection with A. salmonicida was treated with florfenicol, oxolinic acid, oxytetracycline, trimethoprim/sulphadiazine or flumequine, whereas the V. salmonicida infection was treated with florfenicol or oxolinic acid only. A. salmonicida infection was induced by the introduction of cohabitant fish previously inoculated intraperitoneally. Medication started simultaneously in all test tanks on the first day of specific mortality among test fish. V. salmonicida infection was induced by intraperitoneal inoculation of all test fish. Medication started 1 day after infection. Medicated feeds were produced by coating the antibacterials on standard feed pellets, and administered twice daily for 10 consecutive days. With the dose used in the present trials, florfenicol was highly effective in reducing specific mortalities due to both infections. It was slightly more effective than oxolinic acid and trimethoprim/sulphadiazine against A. salmonicida infection. There was no significant difference between florfenicol and oxolinic acid in reducing specific mortalities due to V. salmonicida. 相似文献
11.
Out of 19 Vibrio harveyi isolates obtained from a diversity of hosts and geographical locations, 14 were pathogenic to rainbow trout, Oncorhynchus mykiss (Walbaum), and Atlantic salmon, Salmo salar L., with mortalities of up to 100% following intraperitoneal injections of 106 cells fish?1. The extracellular products (ECPs) of only five pathogenic isolates were harmful to fish. Both pathogenic and non‐pathogenic cultures produced ECPs containing caseinase, gelatinase, phospholipase, lipase and haemolysins. Vibrio harveyi VIB 645, which was the most pathogenic isolate, produced ECPs with a maximal effect on salmonids from preparations obtained by using cellophane overlays on tryptone soya agar supplemented with 1% (w/v) sodium chloride with incubation at 28 °C for 24 h. This preparation contained the highest titre of haemolytic activity to Atlantic salmon (1:256) and rainbow trout (1:32) erythrocytes. 相似文献
12.
以水温、盐度、pH、COD、NH3－N为环境因子，哈维氏弧菌（Vibro harveyi)感染日本对虾（Penaeus japonicus）仔虾，试验进行3d。结果表明，水温、温度、pH和COD对发光细菌的生长和日本对虾仔虾的感染死亡率具有明显的促进作用。在水温30℃，盐度32．8，pH7．654和COD10．2mg／L时，发光细菌在试验结束时的数量比初始时分别增加了11．2倍，6．5倍和7．9倍；日本对虾仔虾的死亡率比对照组分别增加了60％，45％，43％和50％。NH3－N对发光细菌的促生长作用不明显，但其质量浓度1．2－1．6mg/L时，能增加日本对虾仔虾对发光病的感染率和死亡率。 相似文献
13.
Joseph A. Adeyemi 《Fish physiology and biochemistry》2014,40(2):347-354
The impacts of bacterial infection on cultivated fish species, African catfish, were investigated using oxidative stress biomarkers [lipid peroxidation (LPO) and protein carbonylation] and the activities of important antioxidant/detoxifying enzymes [catalase and glutathione S-transferase (GST)]. Fish were inoculated via oral gavage with one of the following treatments: 1 × 105 CFU/ml of Escherichia coli (EC1), 2 × 105 CFU/ml of E. coli (EC2), 1 × 105 CFU/ml of Vibrio fischeri (V1), 2 × 105 CFU/ml of V. fischeri (V2), gavaged with distilled water and not gavaged. Fish were maintained in the laboratory for 7 days after the bacterial inoculation, and the levels of LPO, protein carbonylation, GST, and catalase activities were determined in the muscle, gills, and liver of fish. Fish inoculated with bacteria (either E. coli or V. fischeri) had a significant higher levels of tissue LPO, protein carbonylation, and GST activities in a tissue-specific pattern (liver > muscle > gills). This appears to be related with the levels of bacterial inoculation, with effects more pronounced in fish inoculated with either EC2 or V2. The catalase activity did not differ significantly between the inoculated and fish that were not inoculated. The results of this study indicate that bacterial inoculation could result in oxidative stress in fish, and liver has a higher rate of oxidative stress per mg tissue compared to the gills and the muscle. 相似文献
14.
Characterization of Vibrio spp. Associated with Diseased Shrimp from Culture Ponds of Andhra Pradesh (India) 总被引：1，自引：0，他引：1
Abstract.— Surveys undertaken on diseases caused by Vibrio spp. in Penaeus monodon from culture ponds of coastal Andhra Pradesh recorded the occurrence of five types of diseases: tail necrosis, shell disease, red disease, loose shell syndrome (LSS), and white gut disease (WGD). Among these, LSS, WGD, and red disease caused mass mortalities in shrimp culture ponds. Six species of Vibrio—V. harveyi , V. parahaemolyticus, V. alginolyticus, V. anguillarum , V. vulnificus , and V. splendidus —are associated with the diseased shrimp. The number of Vibrio spp. associated with each disease ranged from two to five. Additionally, shrimp with red disease had concurrent infections with white spot syndrome virus. Vibrio harveyi in the case of LSS and WGD, V. parahaemolyticus for red disease, and V. alginolyticus for shell disease are the major etiologcal agents. Differences occur in the degree of virulence of different species of Vibrio and also different isolates of the same species. Vibrio harveyi isolated from LSS shrimp is the most virulent. In general, all the Vibrio isolates from LSS shrimp tend to be more virulent as compared to their counterparts from other diseased shrimp. It is apparent that the degree of virulence of various Vibrio isolates depends on its source and the pond environmental conditions. Most of the Vibrio isolates showed susceptibility to oxytetracycline, norfloxacin, and ciprofloxacin. The luminous V. harveyi exhibited resistance to many antibiotics and susceptibility to only three drugs. Considering the emergence of antimicrobial resistant strains of Vibrio , the need for using probiotics in place of antibiotics for disease control is stressed. 相似文献
15.
哈维氏弧菌外膜蛋白OmpK基因的克隆及原核表达 总被引：8，自引：0，他引：8
根据哈维氏弧菌外膜蛋白OmpK的基因序列设计一对引物，应用聚合酶链式反应（PCR）方法，从分离自患病大黄鱼的哈维氏弧菌基因组中扩增获得一段约800bp的序列。将其克隆到pGEM-Teasy载体，测序结果证明该序列是哈维氏弧菌外膜蛋白OmpK基因。用PCR方法去除其信号肽序列，定向克隆到原核表达载体pGEX-4T-2构建重组表达质粒pGEX-4T-OmpK。IPTG诱导后能够在大肠杆菌BL21中高效表达分子量约为53kD的GST-OmpK融合蛋白。用纯化后的融合蛋白免疫新西兰兔获得了高效价的抗血清。Western-blotting分析表明，它与从哈维氏弧菌中提取的约27kD的外膜蛋白能够发生特异反应，提示外膜蛋白OmpK可能是哈维氏弧菌的重要保护性抗原之一。 相似文献
16.
The antigenicity of extracellular products (ECPs) derived from Mycobacterium spp. isolated from snakehead, Channa striata (Bloch), and Siamese fighting fish, Betta splendens (Regan), were examined by immunoblotting and enzyme-linked immunosorbent assay (ELISA) using sera collected from immunized rabbits, mice and fish (rainbow trout). All three species responded to a 65-kDa protein present in both the ECPs and whole cell sonicates (WCSs) from a variety of Mycobacterium spp. Cross-reactivity of anti- M. tuberculosis and anti-human heat-shock protein monoclonal antibodies (MAbs), and the presence of fibronectin binding proteins secreted into ECPs of mycobacteria were also examined. The MAbs against human 60-kDa heat-shock protein cross- reacted with the band at 65 kDa in the ECPs of TB1 (isolated from snakehead fish) and the type strain M. marinum, while the anti- M. tuberculosis MAb F29–47 elicited a strong reaction with a band at 21 kDa with most of the ECPs from mycobacterial strains examined. The major fibronectin-binding proteins were located between 21 and 25 kDa. The 65-kDa protein from ECPs of Mycobacterium spp. proved strongly immunogenic to rabbits, mice and fish. Rabbit antiserum against the 65-kDa protein from strain TB267 reacted with many non- Mycobacterium WCSs, and therefore, the 65-kDa protein from Mycobacterium spp. is believed to be a common protein found in many fish bacterial pathogens. 相似文献
17.
Arijo S Rico R Chabrillon M Diaz-Rosales P Martínez-Manzanares E Balebona MC Magariños B Toranzo AE Moriñigo MA 《Journal of fish diseases》2005,28(1):33-38
The protection of cultured sole, Solea senegalensis, against Vibrio harveyi and Photobacterium damselae subsp. piscicida was evaluated following the use of a divalent vaccine prepared with formalized whole cells and extracellular products of virulent strains of both pathogenic microorganisms and administered by the immersion route. Two prolonged immersions of 5-10 g fish in the divalent bacterin at a 1-month interval gave high levels of protection similar to those obtained when the respective monovalent vaccines were administered by the intraperitoneal route [relative percentage of survival (RPS) values >70%], which indicates that the former procedure can be a useful strategy with small fish. The high protection afforded by the divalent vaccine in sole lasted for 4 months after which the RPS values against both pathogens decreased significantly. 相似文献
18.
Abstract. At 9°C juvenile sockeye salmon, Oncorhynchus nerka , inoculated intraperitoneally with 105 Cryptobia (= Trypanoplasma ) salmositica began to succumb about day 15, with mortalities exceeding 90% by day 28. Fish inoculated at 8°C and acclimatized to 13°C began to die at about the same time but total mortalities were only about 75%. At 5°C the infection progressed more slowly, with the first mortalities occurring about day 25, but the disease seemed equally as lethal as at 9°C. All fish inoculated at 8°C and acclimatized to 20°C within 11 days survived the infection. Changes in salinity from fresh to sea water (30%o salinity) at 9°C or 13°C had almost no effect on the course of infection either when presmolts were slowly acclimatized to sea water over a period of 6-8 days or when smolts were transferred to sea water quickly (within 1-2 days). However, there were differences in susceptibility to disease among three species of salmon of smoking age. The order of decreasing sensitivity was chinook salmon, Oncorhynchus tshawytscha , with 100% mortality, sockeye salmon with 56 to 74% mortality and coho salmon, Oncorhynchus kisutch , with no mortality. 相似文献
19.
20.
An aquabirnavirus (ABV) and a formalin-inactivated betanodavirus [redspotted grouper nervous necrosis virus (RGNNV)] were investigated for their potential to prevent RGNNV-induced viral nervous necrosis (VNN) in the sevenband grouper, Epinephelus septemfasciatus (Thunberg). Three groups of fish were injected intramuscularly with ABV, intraperitoneally with inactivated RGNNV (iRGNNV) or with both ABV and iRGNNV. At 3, 7, 14, 21 and 28 days post-injection (p.i.), fish were challenged by intramuscular injection of RGNNV. Control fish, which received neither ABV nor iRGNNV, showed high mortalities in all RGNNV challenges. Fish that received only ABV exhibited relative percent survival (RPS) of >60 against RGNNV challenges at 3, 7, 14 and 21 days p.i., but not at 28 days p.i., while fish that received only iRGNNV showed significantly higher protection against RGNNV challenges only at 21 and 28 days p.i. In contrast, fish that received both ABV and iRGNNV showed 60 or higher RPS against all RGNNV challenges. Fish inoculated with iRGNNV with or without ABV exhibited similar high titres of neutralizing antibodies to RGNNV at 14, 21 and 28 days p.i. These results indicate that combined inoculation with iRGNNV and ABV conferred both rapid non-specific and delayed specific protection against VNN. 相似文献