首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献，搜索用时 546 毫秒
1.
The outer membrane proteins of the marine aquatic animal pathogen, Vibrio alginolyticus, play an important role in the virulence of the bacterium and are potential candidates for vaccine development. In this study, the gene encoding an outer membrane protein‐OmpU was cloned and expressed in Escherichia coli. Polyclonal antibodies were raised in rabbits against the purified recombinant OmpU, and the reaction of the antibody was confirmed by Western blotting using the isolated OmpU and the recombinant OmpU of V. alginolyticus. To analyze the immunogenicity of the recombinant OmpU, crimson snapper, Lutjanus erythropterus Bloch, were immunized by intraperitoneal injection, and antibody response was assessed by the enzyme‐linked immunosorbent assay (ELISA). The results demonstrated that the recombinant OmpU produced an observable antibody response in all sera of the vaccinated fish. The vaccinated fish were challenged by virulent V. alginolyticus and observed to have high resistance to infection. These results indicate that the recombinant OmpU is an effective vaccine candidate against V. alginolyticus in L. erythropterus.  相似文献   

2.
为研究溶藻弧菌鞭毛蛋白flaC基因DNA疫苗对红笛鲷的免疫保护作用,实验构建了重组真核表达质粒pcDNA-flaC并将该质粒肌肉注射红笛鲷,采用PCR、RT-PCR、ELISA和攻毒试验等方法检测了该真核表达质粒在红笛鲷组织内的分布、表达和对红笛鲷的免疫保护.PCR结果显示,免疫接种7和28 d,注射点周围肌肉、鳃、肾脏、肝脏和脾脏都存在质粒分布;RT-PCR结果显示,免疫接种后第7天、14天和28天,红笛鲷不同组织内均有目的基因表达.ELISA结果表明,鱼血清内产生了抗FlaC蛋白的抗体,表明DNA疫苗免疫后鱼体表达了目的蛋白,并诱导产生了相应抗体.攻毒实验表明,免疫后的红笛鲷能较好地抵抗致病性溶藻弧菌的感染.结果表明,质粒pcDNA-flaC可能是抵抗溶藻弧菌感染的有效的疫苗候选物.  相似文献   

3.
Vibrio alginolyticus is a leading aquatic pathogen, causing huge losses to aquaculture. rpoS has been proven to play a variety of important roles in stress response and virulence in several bacteria. In our previous study, upon treatment with Cu2+, Pb2+, Hg2+ and low pH, the expression levels of rpoS were downregulated as assessed by RNA‐seq, while impaired adhesion ability was observed, indicating that rpoS might play roles in the regulation of adhesion. In the present study, the RNAi technology was used to knockdown rpoS in V. alginolyticus. In comparison with wild‐type V. alginolyticus, RNAi‐treated bacteria showed significantly impaired abilities of adhesion, growth, haemolytic, biofilm production, movement and virulence. Meanwhile, alterations of temperature, salinity, pH and starvation starkly affected rpoS expression. The present data suggested that rpoS is a critical regulator of virulence in V. alginolyticus; in addition, rpoS regulates bacterial adhesion in response to temperature, pH and nutrient content changes. These are helpful to explore its pathogenic mechanism and provide reference for disease control.  相似文献   

4.
参照GenBank上登录的弧菌鞭毛蛋白flaB基因序列设计引物,PCR扩增溶藻弧菌HY9901株的flaB全长基因,序列分析结果显示该基因为1134bp,编码377个氨基酸。与GenBank中其它弧菌的同源基因序列比对显示,溶藻弧菌flaB基因与副溶血弧菌flaB基因的同源性最高(92%)。将该基因定向克隆到原核表达载体pET-32a(+)中,在大肠杆菌BL21(DE3)中成功表达出带His-tag的融合蛋白,分子量大小与预期一致。优化的表达条件为28℃,0.4mmol/LIPTG浓度诱导10h。用纯化后的融合蛋白免疫SPF级小鼠,制备了多克隆抗体。Western-blotting结果表明鼠抗FlaB血清不仅能与诱导后的重组蛋白发生反应,而且能与天然的溶藻弧菌全菌蛋白发生反应,提示鞭毛蛋白FlaB可能是溶藻弧菌的重要保护性抗原之一,为下一步进行FlaB蛋白免疫原性的研究以及疫苗的制备奠定了基础。  相似文献   

5.
Vibriosis is a severe infection occurring in many commercially important marine fish species. In this study, vaccines containing Vibrio harveyi recombinant outer membrane protein K (rOmpK), outer membrane protein U (rOmpU) and rOmpK‐OmpU fusion protein in addition to the metabolizable MontanideTM ISA 763 A VG adjuvant were developed and evaluated in the orange‐spotted grouper. The results indicate that recombinant V. harveyi protein‐based vaccines resulted in a remarkably higher expression of IL‐1β and IL‐8 at 24 hr, and greater antibody production, as early as 2 weeks postimmunization. Notably, enhanced immune responses and significant protective efficacy against V. harveyi infections were observed in the fusion protein vaccine‐injected fishes with relative per cent survival value of 81.8%. Additionally, the rOmpK‐OmpU antisera presented a high bactericidal effect on not only V. harveyi, but also Vibrio parahaermolyticus and Vibrio alginolyticus. Our results demonstrated that the fusion protein rOmpK‐OmpU was an effective vaccine candidate that exhibited potentially great versatility for controlling vibrio infections.  相似文献   

6.
As the major opportunistic pathogen to both marine animals and humans, Vibrio alginolyticus (V. alginolyticus) has caused heavy economic losses to mariculture. ssDNA aptamer VA2 targeting live V. alginolyticus was generated by systematic evolution of ligands by exponential enrichment (SELEX) technology in our previous study. In this study, we first developed aptamer (VA2)‐based enzyme‐linked apta‐sorbent assay (VA2‐ELASA) for rapid detection of mariculture pathogen V. alginolyticus. The VA2‐ELASA could achieve the rapid detection for V. alginolyticus infection with high specificity and sensitivity. The VA2‐ELASA could specifically identify V. alginolyticus, but not other non‐target bacterial strains. VA2‐ELASA could detect V. alginolyticus at the concentration of 5 × 104/ml, the incubation time short to 1 min and the incubation temperature as high as 45°C, which proved sensitivity and stability of the novel VA2‐ELASA in this study. It took less than one hour to accomplish the detection process by VA2‐ELASA. The characteristics of specificity, sensitivity and easy operation make VA2‐ELASA a novel useful technology for the rapid diagnosis of pathogen V. alginolyticus in mariculture.  相似文献   

7.
The protective effect in rainbow trout (Oncorhynchus mykiss) of an experimental subunit vaccine targeting antigens in the parasite Ichthyophthirius multifiliis has been evaluated and compared to effects elicited by a classical parasite homogenate vaccine. Three recombinant parasite proteins (two produced in E. coli and one in insect cells) were combined and injected i.p., and subsequently, protection and antibody responses were analysed. Both the experimental and the benchmark vaccine induced partial but significant protection against I. multifiliis when compared to control fish. Specific antibody responses of vaccinated trout (subunit vaccine) were raised against one neurohypophysial n‐terminal domain protein #10 of three recombinant proteins, whereas the benchmark vaccine group showed specific antibody production against all three recombinant proteins. The immunogenic parasite protein #10 may be a potential vaccine candidate supplementing the protective I‐antigen in future vaccine trials.  相似文献   

8.
Vibrio alginolyticus (V. alginolyticus) is a major opportunistic pathogen to both marine animals and humans, which has also caused heavy economic losses to mariculture. The aim of this study was to develop highly specific aptamers for V. alginolyticus. Single‐stranded DNA (ssDNA) aptamers with high binding affinity to viable V. alginolyticus were generated by Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and identified by flow cytometric analysis in this study. The selected aptamers showed high specificity for V. alginolyticus and low apparent binding for other bacteria. The aptamers formed distinct stem‐loop structures, which could form the basis of aptamers’ specific binding to the target V. alginolyticus. Aptamer VA2 and VA8 showed particularly high binding affinity constant (Kd) of 14.31 ± 4.26 and 90.00 ± 13.51 nM, respectively. The aptamers produced no cytotoxic effects in vitro and in vivo. ssDNA aptamers were successfully selected against the viable bacteria pathogen V. alginolyticus by SELEX. The aptamers selected in this study could be not only applied as specific chemical molecular probes for studying V. alginolyticus pathogenesis to Trachinotus ovatus, but also developing rapid convenient diagnosis assay for V. alginolyticus infection, even when applied to the complex sample matrix, such as food and environment samples.  相似文献   

9.
The effect of dietary supplementation of probiotic bacterium Bacillus licheniformis on the histopathological changes in Macrobrachium rosenbergii juveniles (4.0 ± 0.02 g) challenged with known pathogenic strain of Vibrio alginolyticus are reported. Two isocaloric basal diets supplemented with probiotic bacteria B. licheniformis (1.0 × 109 cfu/g feed) and other without probiotic supplementation were fed to the M. rosenbergii juveniles for 45 days. The histological observations revealed no significant changes in the hepatopancreas and gut tissues of both the experimental and the control groups which indicate that the present bacterium is a safe candidate probiont for the host. Prawns were challenged with V. alginolyticus after 45 days of feeding with probiotic diet. The histopathological studies of the hepatopancreas revealed that M. rosenbergii fed with probiotic‐supplemented diet showed less changes as compared to the prawns fed with control diet on second and fourth day of post‐experimental challenge with V. alginolyticus. Histopathological observations revealed that the gills of the prawns fed with control diet were severely affected in comparison to the prawns fed with probiotic‐supplemented diet after challenging with V. alginolyticus. Results from this study revealed the improved protection by dietary incorporation of B. licheniformis in reducing the histopathological manifestations due to V. alginolyticus infection in freshwater prawn.  相似文献   

10.
Vibrio alginolyticus as an important pathogen in aquaculture can encounter the oxidative stress produced by the immune system during infection. Previous studies showed that sRNAs have important functions in response to oxidative stress in bacteria; however, less of sRNAs related to oxidative stress response were identified in V. alginolyticus. In this study, a total of 749 novel sRNAs were identified by RNA sequencing; among them, 128 sRNAs were up‐ or downregulated in response to oxidative stress. In addition, 1,870 genes exhibited variation on mRNA levels in oxidative stress response. By analysing the target genes of the sRNAs, we concluded that these sRNAs could regulate expressions of genes responsible for iron transport, catalase, GSH‐dependent defence system, electron transferred and stress response. Moreover, the functions of the sRNAs are also seemed related to the pathogenicity in V. alginolyticus. Based on the results, we constructed the oxidative stress model in V. alginolyticus. This study provides us the first outlook of sRNAs function in oxidative stress response in V. alginolyticus. Furthermore, this study can help us to prevent and control this important opportunistic pathogen in aquaculture.  相似文献   

11.
Vibrio alginolyticus is one of the most serious causative agents of diseases in cultured marine fish and shellfish. However, the characteristics of virulence factors in pathogenic V. alginolyticus are poorly known. To gain insight into fish diseases caused by V. alginolyticus, we carried out two-dimensional gel electrophoresis (2-DE) combined with MALDI-TOF mass spectrometry to identify uniquely expressed proteins in the disease-causing V. alginolyticus. V. alginolyticus strains were isolated from marine environments and diseased fish obtained from southern Thailand. We identified seven unique proteins in the disease-causing V. alginolyticus strain. Among those, the outer membrane protein A (OmpA) had the strongest expression. Therefore, the function of this protein was further analysed. To investigate the role of OmpA protein, an in-frame deletion mutant of ompA was constructed using the homologous recombination method. Although the ompA mutant V. alginolyticus strain (ΔompA) grew normally, the mutant exhibited a significant defect in the swarming ability and the biofilm formation. Furthermore, Galleria mellonella larvae injected with the mutant bacteria had a significantly greater survival percentage than those injected with the wild-type strain, demonstrating that OmpA protein is required for the pathogenicity of V. alginolyticus. Together, this study suggests a potential target for vaccine development against pathogenic V. alginolyticus strain.  相似文献   

12.
The gram‐negative bacterium, Vibrio alginolyticus, has frequently been identified as the pathogen responsible for the infectious disease called vibriosis. This disease is one of the major challenges facing brown‐marbled grouper aquaculture, causing fish farmers globally to suffer substantial economic losses. The objective of this study was to investigate the proteins involved in the immune response of brown‐marbled grouper fingerlings during their initial encounter with pathogenic organisms. To achieve this objective, a challenge experiment was performed, in which healthy brown‐marbled grouper fingerlings were divided into two groups. Fish in the treated group were subjected to intraperitoneal injection with an infectious dose of V. alginolyticus suspended in phosphate‐buffered saline (PBS), and those in the control group were injected with an equal volume of PBS. Blood samples were collected from a replicate number of fish from both groups at 4 h post‐challenge and analysed for immune response‐related serum proteins via two‐dimensional gel electrophoresis. The results showed that 14 protein spots were altered between the treated and control groups; these protein spots were further analysed to determine the identity of each protein via MALDI‐TOF/TOF. Among the altered proteins, three were clearly overexpressed in the treated group compared with the control; these were identified as putative apolipoprotein A‐I, natural killer cell enhancement factor and lysozyme g. Based on these results, these three highly expressed proteins participate in immune response‐related reactions during the initial exposure (4 h) of brown‐marbled grouper fingerling to V. alginolyticus infection.  相似文献   

13.
The autochthonous microbiota in the foregut, midgut and hindgut of juvenile grouper Epinephelus coioides following the dietary administration of probiotic Bacillus clausii for 60 days were assessed using polymerase chain reaction‐denaturing gradient gel electrophoresis (PCR‐DGGE). A complex and generally similar bacterial composition along the digestive tract of E. coioides was detected in the DGGE profiles, while several bacteria showed regional specialization. Similarity dendrogram revealed that the bacterial composition of the foregut was more similar to the midgut than the hindgut. Samples collected from the probiotic group and the control group showed generally similar DGGE patterns, while no significant difference in the total number of bands and Shannon index were observed between the probiotic group and the control group, suggested that probiotic B. clausii exerted no significant effect on the gut microbiota of E. coioides. However, various potentially beneficial bacteria, such as Enterococcus sp.‐like and Bacillus pumilus‐like bacterium were selectively stimulated by probiotic B. clausii, while some potential harmful species, like Staphylococcus sp.‐like and Vibrio ponticus‐like bacterium were depressed. These indicated that the gut microbiota was modified to some degree by probiotic B. clausii. Sequences analysis showed that the autochthonous gut bacteria of E. coioides could be classified into four groups, i.e. Proteobacteria, Firmicutes, Actinobacteria and unclassified bacteria.  相似文献   

14.
溶藻弧菌外膜蛋白（Va-OMP）的免疫原性及免疫保护性   总被引：10，自引：0，他引：10  
黄志坚 《水产学报》2006,30(4):538-543
利用初步制备的溶藻弧菌外膜蛋白（outer membrane protein of Vibrio alginolyticus, Va-OMP）、溶藻弧菌蛋白分子量58 kD外膜蛋白（Va-OMP58）和溶藻弧菌（Vibrio alginolyticus, Va）灭活菌苗、溶藻弧菌脂多糖（lipopolysaccharides of Vibrio alginolyticus, Va-LPS）菌苗等进行主动保护性和被动保护性的实验比较，VaOMP、Va-OMP58免疫组小鼠免疫后用活菌攻击，免疫保护率为62.5%～86.7%,而Va-LPS组和Va灭活菌苗组的免疫保护率为50%～62.5%，对照组的免疫保护率仅为6.7%。用不同抗血清免疫小鼠，活菌攻击后，Va-OMP组存活率达到53.3%和66.7%，VaOMP58组次之，存活率为40%和50%，Va灭活菌组的存活率为33.3%和46.7%，对照组为13.3%。与对照组相比较，Va-OMP组差异较显著，保护效果较好。溶藻弧菌外膜蛋白和蛋白分子量58 kD外膜蛋白的保护性效果高于溶藻弧菌灭活菌苗和溶藻弧菌脂多糖的保护性效果，证明外膜蛋白（outer membrane protein, OMP）具有较强的免疫原性。迟发性超敏反应试验也证明，OMP能诱发变态反应，间接证明OMP具有较强的引起细胞介导免疫反应的能力。因此实验证明，Va-OMP、Va-OMP58是能在小鼠产生体液免疫和细胞介导免疫的保护性抗原。  相似文献   

15.
Despite significant improvements in aquaculture to compensate wild catch, disease organisms have thrived in limiting its national and global potential. Using antibiotics, in a bid to remedy the havoc, has given rise to complications, attracting attention to disease prevention by immune enhancement against diseases. Grouper production has been inhibited for the threats of bacterial infection, particularly of Vibrio origin. Considering the rise in vibriosis cases, improved vaccines are necessary; moreover, recombinant vaccines, the choice for trial in the present experiment have been effective and more specific in improving immunity. The current work deals with grouper immune system enhancement with a recombinant vaccine developed from VirB11 gene in Vibrio harveyi. VirB11 was cloned in V. harveyi for recombinant vaccine development against vibriosis in orange‐spotted grouper (Epinephelus coioides). As indicated by the results, recombinant VirB11 protein showed effectiveness in conferring protection against vibriosis with observable specific antibody response in enzyme‐linked immunosorbent assay (ELISA) analysis; a significant increase (p < 0.05) in antibody levels was observed after a week and after 8 weeks post‐vaccination. From the weeks post‐vaccination, log2 (antibody titres) in the sera of vaccinated groups reached a peak of 14.2 at week 5 in the vaccinated group in comparison with a peak of approximately 5 and 2 in adjuvant and PBS controls. As indicated by the challenge results, 90% relative survival was observed in vaccinated group and 13% relative survival in control group I (adjuvant control). The cumulative performance of protein concludes VirB11 commendable for recombinant vaccine development.  相似文献   

16.
Lumpfish (Cyclopterus lumpus), a native fish of the North Atlantic Ocean, is utilized as cleaner fish to biocontrol sea lice infestations in Atlantic salmon aquaculture. However, bacterial infections are affecting cleaner fish performance. Vibrio anguillarum, the aetiological agent of vibriosis, is one of the most frequent bacterial infections in lumpfish, and effective vaccine programmes against this pathogen have been identified as a high priority for lumpfish. Vibrogen‐2 is a commercial polyvalent bath vaccine that contains formalin‐inactivated cultures of V. anguillarum serotypes O1 and O2, and Vibrio ordalii. In this study, we evaluated Vibrogen‐2 efficacy in lumpfish against a local isolated V. anguillarum strain. Two groups of 125 lumpfish were bath‐immunized, bath‐boost‐immunized at four weeks post‐primary immunization, and intraperitoneally (i.p.) boost‐immunized at eight weeks post‐primary immunization. The control groups were i.p. mock‐immunized with PBS. Twenty‐seven weeks post‐primary immunization, the fish were i.p. challenged with 10 or 100 times the V. anguillarum J360 LD50 dose. After the challenge, survival was monitored daily, and samples of tissues were collected at ten days post‐challenge. Commercial vaccine Vibrogen‐2 reduced V. anguillarum tissue colonization and delayed mortality but did not confer immune protection to C. lumpus against the V. anguillarum i.p. challenge.  相似文献   

17.
To evaluate the possible dietary application of live and heat‐inactivated probiotic Bacillus pumilus SE5 in grouper Epinephelus coioides, juveniles (14.6 ± 0.2 g) were fed either a basal control diet (without probiotic) or the basal diet supplemented with 1.0 × 108 CFU g?1 live (T1) and heat‐inactivated B. pumilus SE5 (T2). The heat‐inactivated probiotic significantly improved the final weight, weight gain (WG) and specific growth rate (SGR) at day 60 and significantly decreased the feed conversion ratio (FCR) at day 30 and 60, while the viable probiotic significantly decreased the FCR at day 60 (P < 0.05). Phagocytic activity, serum complement C3 and IgM levels as well as SOD activity elevated significantly in fish fed the heat‐inactivated probiotic for 60 days (P < 0.05). Furthermore, the heat‐inactivated probiotic remarkably up‐regulated expression of TLR2 and pro‐inflammatory cytokines (IL‐8 and IL‐1β) in head kidney (P < 0.05), but the viable probiotic failed to do so. These results indicated that heat‐inactivated B. pumilus SE5 can effectively improve the growth performance and immune responses of E. coioides.  相似文献   

18.
With the development of gene technology, expressing heterologous antigens in attenuated bacteria has become an important strategy to design multivalent vaccines. In our previous work, an attenuated Vibrio anguillarum named MVAV6203 was developed and proven to be an efficient live vaccine candidate. In this research, we aimed to express protective antigen glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) of Edwardsiella tarda in attenuated Vibrio anguillarum to establish a multivalent V. anguillarum vector vaccine. Several strategies were compared between low‐ vs. high‐copy plasmid‐mediated antigen expression, in vivo‐inducible vs. constitutive antigen expression and intracellular vs. surface‐displaying antigen expression. Zebrafish, Danio rerio (Hamilton), was applied as the fish model to evaluate the immune protection of the V. anguillarum vector vaccine candidates. Our results demonstrated that V. anguillarum MVAV6203 (pUTatLNG40), which harbours a low‐copy plasmid‐loaded antigen surface display system under the control of a constitutive promoter, presented the best protective efficacy against the infection of Vibrio anguillarum (relative per cent survival, RPS = 85%) and Edwardsiella tarda (RPS = 70%).  相似文献   

19.
Bacterial coldwater disease, caused by Flavobacterium psychrophilum, remains one of the most significant bacterial diseases of salmonids worldwide. A previously developed and reported live‐attenuated immersion vaccine (F. psychrophilum; B.17‐ILM) has been shown to confer significant protection to salmonids. To further characterize this vaccine, a series of experiments were carried out to determine the cross‐protective efficacy of this B.17‐ILM vaccine against 9 F. psychrophilum isolates (representing seven sequence types/three clonal complexes as determined by multilocus sequence typing) in comparison with a wild‐type virulent strain, CSF‐259‐93. To assess protection, 28‐day experimental challenges of rainbow trout (Oncorhynchus mykiss) fry were conducted following immersion vaccinations with the B.17‐ILM vaccine. F. psychrophilum strains used in challenge trials were isolated from several fish species across the globe; however, all were found to be virulent in rainbow trout. The B.17‐ILM vaccine provided significant protection against all strains, with relative percent survival values ranging from 51% to 72%. All vaccinated fish developed an adaptive immune response (as measured by F. psychrophilum‐specific antibodies) that increased out to the time of challenge (8 weeks postimmunization). Previous studies have confirmed that antibody plays an important role in protection against F. psychrophilum challenge; therefore, specific antibodies to the B.17‐ILM vaccine strain appear to contribute to the cross‐protection observed to heterologous strain. The ability of such antibodies to bind to similar antigenic regions for all strains was confirmed by western blot analyses. Results presented here support the practical application of this live‐attenuated vaccine, and suggest that it will be efficacious even in aquaculture operations affected by diverse strains of F. psychrophilum.  相似文献   

20.
Rainbow trout, Oncorhynchus mykiss (Walbaum), are able to raise a protective immune response against Aeromonas salmonicida subsp. salmonicida (AS) following injection vaccination with commercial vaccines containing formalin‐killed bacteria, but the protection is often suboptimal under Danish mariculture conditions. We elucidated whether protection can be improved by increasing the concentration of antigen (formalin‐killed bacteria) in the vaccine. Rainbow trout juveniles were vaccinated by intraperitoneal (i.p.) injection with a bacterin of Aeromonas salmonicida subsp. salmonicida strain 090710‐1/23 in combination with Vibrio anguillarum serotypes O1 and O2a supplemented with an oil adjuvant. Three concentrations of AS antigens were applied. Fish were subsequently challenged with the homologous bacterial strain administered by perforation of the tail fin epidermis and 60‐s contact with live A. salmonicida bacteria. The infection method proved to be efficient and could differentiate efficacies of different vaccines. It was shown that protection and antibody production in exposed fish were positively correlated to the AS antigen concentration in the vaccine.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号