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1.
Novel preservation condition without ultra-low temperature is needed for the study of pathogen in marine fishes. Freeze-drying is such a method usually used for preservation of terrigenous bacteria. However, studies using freeze-drying method to preserving marine microorganisms remain very limited. In this study, we optimized the composition of protectants during the freeze-drying of Edwardsiella tarda, a fish pathogen that causes systemic infection in marine fishes. We found that the optimal composition of protectant mixture contained trehalose (8.0%), skim milk (12.0%), sodium citrate (2.0%), serum (12.0%) and PVP (2.0%). Orthogonal and interaction analyses demonstrated the interaction between serum and skim milk or sodium citrate. The highest survival rate of E. tarda was observed when the concentration of NaCl was 10.0, 30.0 and between 5.0 and 10.0 g L?1 for preparing TSB medium, E. tarda suspension and protectant mixture, respectively. When E. tarda was frozen at ?80°C or ?40°C for 6 h, its survival rate was higher than that under other tested conditions. Under the optimized conditions, when the protectant mixture was used during freeze-drying process, the survival rate (79.63%–82.30%) of E. tarda was significantly higher than that obtained using single protectant. Scanning electron microscopy (SEM) image indicated that E. tarda was embedded in thick matrix with detectable aggregation. In sum, the protectant mixture may be used as a novel cryoprotective additive for E. tarda.  相似文献   

2.
Vibrio anguillarum is an important bacterial pathogen of aquatic organisms and a significant problem in aquatic farming.The rapid detection and identification of V.anguillarum,and other pathogens that infect marine organisms,is crucial to effective disease management.In this study,we developed a loop-mediated amplification (LAMP) assay to detect V.anguillarum in an hour in a single tube without the need for thermal cycling.Conserved regions of the metalloproteinase (empA) gene of V.anguillarum served as the targets for primer design.A fragment of the empA gene was amplified at 65°C in the presence of the primer mixture and Bst DNA polymerase.In the optimized LAMP assay,6.7 pg of V.anguillarum DNA could be detected.Six strains of V.anguillarum and 17 strains of non-V.anguillarum bacteria were used in this study to evaluate the species specificity of the primers.The six V.anguillarum strains gave a positive result in the LAMP assay.This method was also validated in V.anguillarum-infected fish.This LAMP method is more sensitive than PCR in the detection of V.anguillarum and shows good species specificity.The LAMP assay is therefore an effective method for the quick detection of V.anguillarum both in the laboratory and in the field.  相似文献   

3.
Mutant J61321 with enhanced siderophore production of Alteromonas aurantia AI8 was obtained after a series of chemical-physical mutageneses. It was found that the antibacterial activity against Vibrio anguillarum W-1 and siderophore production of the mutant were higher than those by the original strain A 18 which had been used in mariculture. The results of the specific assay（Csaky and Arnow methods） of siderophore showed that the sidrophore with hydroxamate group was produced by mutant J61321 and the original strain A 18, respectively, while the siderophore with catechol group was yielded by strain W-1 （Aibrio anguillarum）. Meanwhile, the siderophore yield, antibacterial activity and anti-chelator activity of strain J61321 were higher than those of its parent strain A 18.  相似文献   

4.
Vibrio anguillarum is a common bacterial pathogen in fish.However,little is known about its pathogenic mechanism,in part,because the entire genome has not been completely sequenced.We constructed a fosmid library for V.anguillarum containing 960 clones with an average insert size of 37.7 kb and 8.6-fold genome coverage.We characterized the library by end-sequencing 50 randomly selected clones.This generated 93 sequences with a total length of 57 485 bp covering 1.4% of the whole genome.Of these sequences,58...  相似文献   

5.
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6.
EmpA is an extracellular metalloprotease secreted by Vibrio anguillarum.For better understanding its role in the patho-genicity of V.anguillarum strain M3,empA insertion mutant was constructed.In the mutant it decreased in extracellular proteolytic activity,swarming motility,hemolytic activity and virulence on turbot(Scophthalmus maximus).Significant decline(by 5-fold)of extracellular proteolytic activity and similar growth curve between mutant and wild type strains indicated that EmpA was the major extracellular protease of M3.LD50 of mutant increased by 38-fold compared with wild type.No pro-EmpA was detected in the su-pernatant of culture,indicating that EmpA autolyzed to mature protein after 24 h.Secretion of EmpA in M3 was similar to that in NB10 strain.Attenuated virulence of mutant was similar to that of M93Sm strain.It was demonstrated that specific operation of EmpA was different from that in previous studies and EmpA contributed to the swarming motility and hemolytic activity in V.an-guillarum strain M3.The results provides insight into understanding the function of EmpA and its potential application in vaccine development.  相似文献   

7.
Deyeuxia angustifolia wetlands were widely distributed in the Sanjiang Plain in Northeast China. Due to strong demand for food production, large-area wetlands were reclaimed to farmlands, which threatened regional ecological security greatly. Since the 21 th century, returning farmlands to wetlands was widely adopted for natural restoration in the Sangjiang Plain. As the first reflection of wetland restoration, vegetation succession of restored D. angustifolia wetlands should be fully assessed. In this study, vegetation investigation was carried out in three restored D. angustifolia wetlands with 5, 8 and 12 yr restoration, respectively. Meanwhile, a natural D. angustifolia wetland was selected as reference wetland. Results showed that community composition changed greatly and there was visible community succession. Community dominant species changed from composite to gramineae as restoration time increasing.At first, weeds community appeared in the restored wetlands, especially the xerophytes developed to the pioneer species rapidly. And then, mesophytes and wetland species became the dominant species in the restored wetlands. Finally, wetland species, especially D. angustifolia, occupied the dominant position of restored community. Shannon-wiener index(H) and Simpson index(D) both decreased to close to natural D. angustifolia wetlands. Compared with natural D. angustifolia wetland, species composition and diversity in restored wetlands were more complex and higher. As restoration time increasing, there were not significant differences between community characteristics of restored wetlands and natural wetland. All these suggested that vegetation in reclaimed D. angustifolia wetland could be restored naturally, but its restored period is 10 yr at least. From another angle, it is important to protect current natural wetlands.  相似文献   

8.
Heating the oil shale by current underground to accomplish in-situ transformation process, is a viable direction. The electrical properties of oil shale under different temperatures, especially the resistance, become important. Charging oil shale underground to heat oil shale till kerogen’s decomposition temperature, then crude oil and other gases can be generated. The resistance of the oil shale samples was measured by Direct Current (DC) quadripole method to find out the variation rules of resistance value. In the experiments, oil shale presented its semiconductor property. When heated till 350℃-450℃, its resistance changed greatly, optional for heating and cracking. The porosity, oil content, media and composition affected the resistance largely.  相似文献   

9.
We cloned and sequenced a prtV-like gene from Vibrio anguillarum M3 strain.This prtV gene encodes a putative protein of 918 amino acids,and is highly homologous to the V.cholerae prtV gene.We found that a prtV insertion mutant strain displayed lower gelatinase activity on gelatin agar,lower protease activity against azocasein,and lower activity for four glycosidases.This prtV mutant strain also had increased activity for two esterases in its extracellular products,as analyzed by the API ZYM system.In additi...  相似文献   

10.
Interleukin-17(IL-17), a prototype member of IL-17 family, plays an important role in defending against extracellular pathogens as a pro-inflammatory cytokine. The function and distribution of IL-17 have been extensively studied in many vertebrates. However, few study has focused on the role of IL-17 in invertebrates, especially in mollusks. In this study, an IL-17 homolog was identified in Octopus ocellatus, which was designated as OoIL-17. The phylogenetic analysis showed that OoIL-17 is clustered well with other invertebrate IL-17, indicating it is highly similar with the IL-17 of other invertebrates. The expression of OoIL-17 gene was analyzed with qRT-PCR in a variety of healthy tissues and the hemocytes infected with Vibro anguillarum or Micrococcus luteus. The mRNA of OoIL-17 gene is constitutively expressed at different levels in all examined tissues of healthy O. ocellatus, including mantle, stomach, hemocytes, muscle, gonad, hepatopancreas, systemic heart and gill. The lowest expression was observed in mantle while the highest was observed in hepatopancreas. The expression level of OoIL-17 gene is significantly up-regulated in O. ocellatus hemocytes upon infection with V. anguillarum and M. luteus, indicating its active involvement in the host immune response against bacterial pathogens. The results laid the foundation for further understanding the innate immune mechanisms of IL-17 in O. ocellatus and mollusks.  相似文献   

11.
Trumpet shell, Charonia sauliae, is an endangered and valuable species, but its artificial propagation protocol has not been successfully established. To estimate the possibility of cryopreservation for larvae of C. sauliae, which is a potential preparation for its artificial reproduction and further research, in this study a protocol for the cryopreservation of veliger larvae of trumpet shell was optimized. Through a two-step cryopreservation procedure, four kinds of cryoprotectants (ethylene glycol, 1, 2-propanediol,dimethyl sulfoxide and glycerol) were employed at three concentrations (1.0, 1.5 and 2.0 molL-1) respectively and survival rates of larvae were determined after a storage of 1h. The larvae frozen with these four cryoprotectants after 1h storage were cultured, and then survival rates were determined at 24, 72 and 120h after thawing. Dimethyl sulfoxide at a concentration of 1.5 molL-1 showed the best protective effect in all experiments (p<0.05). And survival rates of larvae frozen with dimethyl sulfoxide were determined after 1,7 and 15 d of storage. The survival rates of larvae frozen with 1.5 molL-1 dimethyl sulfoxide after 1h, 1d, 7 d and 15 d of storage were 80.77% ±7.51%, 80.34% ±11.28%, 83.10% ±9.14% and 77.23%±6.22% respectively. No significant differences in survival rates of larvae frozen with dimethyl sulfoxide were observed after various storage periods (p>0.05).  相似文献   

12.
Studies were conducted to determine the cause of the acute mortality of half-smooth tongue sole Cynoglossus semilaevis Günther juveniles in a fish farm in Jimo, Shandong Province, China, in June 2006. Gross signs of the diseased tongue sole included several petechiae and ecchymoses on the body and fin necrosis and hemorrhagic lesion at the base of the fin. Bacteria were isolated from kidney, liver and hemorrhagic lesions of the diseased tongue sole. Among14 strains, SJ060621 was proved to be highly virulent to juvenile tongue sole with LD50 value of 1.0×105 colony forming units (CFU)mL-1, while the remaining 13 were avirulent. Among the 16 antibiotics tested, SJ060621 was sensitive to gentamicin and nitrofurantoin. It was identified as Listonella anguillarum with conventional plate and tube tests in combination with API 20E analysis. 16S rRNA gene and partial HSP60 gene sequenceing analysis revealed that the strain was highly homologous with L. anguillarum. Examination of the infected musculature by electron microscopy indicated numerous bacteria and lots of macrophages containing phagocytosed bacteria. Histopathological investigations revealed severe necrotic degenerative changes in the infected organs. Indirect immunofluorescence assay (IFA) was employed to detect the location of occurrence of bacteria, and bacteria were found in aggregations in the inflammatory areas in musculature.  相似文献   

13.
Sea cucumber Apostichopus.japonicus is an important marine economic species in Asian countries due to its profound nutritional and medicinal value.So far,with the rapid development of intensified artificial aquaculture of sea cucumbers,the use of antibiotics is still an inexpensive and dispensable way to treat pathogenic infections,especially during the nursery phase.However,there is little information on the effects of antibiotics on the intestinal microbiota of sea cucumber.Therefore an Illumina based sequencing method was used to examine the intestinal bacterial composition of juvenile A.japonicas following diets with three typical antibiotics(tetracycline,erythromycin.,and norfloxacin)under 15,30,and 45 d.The findings reveal that different antibiotics have distinct effects on the growth performance of juvenile sea cucumbers.However,the richness and diversity of microbiota were barely affected by antibiotics but the community composition alterations indicated that the three antibiotics exhibited their respective patterns of reshaping the intestinal bacteria of juvenile sea cucumbers.In common,the abundance of some sensitive genera with helpful functions,such as Thalassotalea,Shewanella,Sulfitobacter,and Halomonas decreased significantly with exposure to antibiotics and the abundance of multiple potential pathogenic-and suspected antibiotic-resistant microorganisms like Arcobacter,Leucothrix,and Clostridium_sensu_stricto_1 was found increased significantly in the antibiotic groups.These results suggest that low doses of antibiotics could affect the composition of the intestinal microbiota of sea cucumbers and might increase the risk of infection of the hosts.This study could help us to explore how antibacterial compounds modify the gut microbiota of sea cucumbers and provide theoretical guidance in hatchery management by scientific antibiotic use in sea cucumber mariculture.  相似文献   

14.
Carbohydrases secreted by marine Vibrio sp. YKW-34 with strong Laminaria cell wall degrading ability were screened, and among them alginate lyase was found to be dominant. The effects of medium composition and culturing condition on the production of alginate lyase by marine Vibrio sp. YKW-34 in flask were investigated in this study. In the culture medium of marine broth, no alginate lyase was produced. The activity of the alginate lyase, after being induced, reached 5 UmL^-1. The best inoculum volume and inoculum age were 10% and 12h, respectively. The optimal temperature for alginate lyase production was 25℃. The fermentation medium was composed of 0.5% of Laminaria powder and 0.2% of KNO3 with an initial acidity of pH 8.0. Alginate could induce alginate lyase production but not as efficiently as Laminaria powder did. The addition of fucoidan, cellulose and glucose had negative effect on the alginate lyase production. Other kinds of nitrogen sources, such as yeast extract, beef extract and peptone, had positive effect on the growth of the microorganism and negative effect on alginate lyase production. In addition, the time course of alginate lyase production under the optimized condition was described. The optimal harvest time was 48 h.  相似文献   

15.
中国南海珊瑚岛礁拥有巨大的资源潜力和科研价值。珊瑚岛礁第四系覆盖层结构为珊瑚砂和珊瑚骨架的松散堆积物,无胶结,钻探取心、成井极端困难。通过南海诸岛礁数百口钻井的施工实践,总结出"套管连续跟进干钻取心技术",工艺要点为4个步骤循环操作,称为"四步法"。使用该技术:①能确保珊瑚岛礁第四系覆盖层岩心采取率达到95%以上,并基本保持岩心原状性;②能实现在珊瑚岛礁第四系覆盖层中不使用泥浆护壁情况下安全钻进成井,达到避免地层过水通道堵塞、满足抽水试验要求的效果。研究成果对于珊瑚岛礁钻探具有重要的借鉴作用。   相似文献   

16.
研究了湛江东海岛3种不同养殖模式虾池弧菌对抗菌药物的耐药性,并对虾池水域环境的水温、盐度、DO、pH、叶绿素a、DOC、无机氮、无机磷、硅酸盐、细菌总数和弧菌数量变化进行了监测和评价。结果表明,调查的三个虾池的弧菌对万古霉素产生耐药性,高位新池和传统精养池的弧菌对利福平产生耐药性,对其他抗菌药尚未形成耐性,3个虾池的弧菌对恩诺沙星和氯霉素高度敏感,传统精养池和天然虾池的弧菌对复方新诺明、庆大霉素、TMP和环丙沙星高度敏感;在水平差异上,高位新池的弧菌比传统精养池和天然虾池对12种抗菌药更具耐药性,同时多重耐药菌株在高位新池中出现,可能与虾苗来源和配合饲料中添加了抗菌药物有关。水质监测结果表明,养殖过程中,虾池水域环境逐渐富营养化,无机氮和Chla超过富营养化阈值,细菌总数增加;两个精养池的水体富营养化程度高于天然虾池;调查期间的弧菌数量在对虾发病的感染剂量阈值范围之内,尚无弧菌病害发生的潜在危险。  相似文献   

17.
The present study evaluated the effects of chilled storage and cryopreservation on sperm motion characteristics, antioxidant enzyme activities, and lipid peroxidation in the Pacific cod Gadus macrocephalus. Sperm motility and the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (Gr), and lipid peroxidation (measured via malondialdehyde (MDA) content) were determined after the milt was stored at 4°C for 12 h, cryopreserved without cryoprotectant in 12% propylene glycol (PG), cryopreserved in 12% PG+0.1 mol/L trehalose, or cryopreserved in 12% PG spermatozoa but centrifuged to decant the supernatant prior to cryopreservation (only sperm cells were cryopreserved). After chilled storage or cryopreservation, the SOD, CAT and GPx activities were reduced in sperm cells and increased in seminal plasma in almost all treatments; sperm motility parameters were also decreased. However, the addition of trehalose into the cryoprotectant could significantly improve the postthaw sperm quality as revealed by the sperm average path velocity. This improvement might be attributed to the function of trehalose in scavenging reactive oxygen species. Chilled storage and cryopreservation had significant effects on sperm motion characteristics, antioxidant enzyme activities, and lipid peroxidation in the Pacific cod.  相似文献   

18.
Trumpet shell, Charonia sauliae, is an endangered and valuable species, but its artificial propagation protocol has not been successfully established. To estimate the possibility of cryopreservation for larvae of C. sauliae, which is a potential preparation for its artificial reproduction and further research, in this study a protocol for the cryopreservation of veliger larvae of trumpet shell was optimized. Through a two-step cryopreservation procedure, four kinds of cryoprotectants (ethylene glycol, 1, 2-propanediol, dimethyl sulfoxide and glycerol) were employed at three concentrations (1.0, 1.5 and 2.0 molL−1) respectively and survival rates of larvae were determined after a storage of 1h. The larvae frozen with these four cryoprotectants after 1 h storage were cultured, and then survival rates were determined at 24, 72 and 120 h after thawing. Dimethyl sulfoxide at a concentration of 1.5 molL−1 showed the best protective effect in all experiments (p<0.05). And survival rates of larvae frozen with dimethyl sulfoxide were determined after 1, 7 and 15 d of storage. The survival rates of larvae frozen with 1.5 molL−1 dimethyl sulfoxide after 1 h, 1 d, 7 d and 15 d of storage were 80.77% ±7.51%, 80.34% ±11.28%, 83.10% ±9.14% and 77.23% ±6.22% respectively. No significant differences in survival rates of larvae frozen with dimethyl sulfoxide were observed after various storage periods (p>0.05).  相似文献   

19.
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20.
对汶川8.0级地震前后陕西地区地倾斜观测资料的分析发现,汶川地震前陕西地区地倾斜存在沿龙门山断裂带方向由东北向西南震中迁移,震后地倾斜反向迁移,迁移平均速度为3～4 km/天。进一步分析认为,震前陕西地区地倾斜向震源迁移可能对汶川8.0级地震起到了触发作用,而震后地倾斜向远场迁移有可能是诱发陕甘川交界的中强余震的主因。  相似文献   

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