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 531 毫秒
1.
Survival in host macrophages is an effective strategy for pathogenic bacteria to spread. Aeromonas hydrophila has been found to survive in fish macrophages, but the mechanisms remain unknown. In this paper, the roles and possible mechanisms of IcmF in bacterial survival in fish macrophages were investigated. First, a stable silencing strain icmF‐RNAi was constructed by shRNA and RT‐qPCR confirmed the expression of icmF was down‐regulated by 94.42%. The expression of Hcp, DotU and VgrG was also decreased in icmF‐RNAi. The intracellular survival rate of the wild‐type strain was 92.3%, while the survival rate of icmF‐RNAi was only 20.58%. The escape rate of the wild‐type strain was 20%, while that of the icmF‐RNAi was only 7.5%. Further studies indicated that the expression of icmF can significantly affect the adhesion, biofilm formation, motility and acid resistance of A. hydrophila, but has no significant effect on the growth of A. hydrophila even under the stress of H2O2. The results indicated that IcmF of A. hydrophila not only acts as a structural protein which participates in virulence‐related characteristics such as bacterial motility, adhesion and biofilm formation, but also acts as a key functional protein which participates in the interaction between bacteria and host macrophages.  相似文献   

2.
Interrupting quorum sensing represents a novel anti‐infective strategy to combat bacterial pathogen, and biodegradation of quorum sensing signal AHLs has been proved to be an efficient way to control pathogenic Gram‐negative bacteria in aquaculture. In this study, the effect of Bacillus firmus sw40 as efficient AHL‐degrading strain on virulence of fish pathogen Aeromonas hydrophila and disease resistance of gibel carp Carassius auratus gibelio was investigated. The results demonstrated that in vitro the B. firmus sw40 extracellular production (ECP) was able to significantly decrease protease production, haemolytic activity and biofilm formation in A. hydrophila. Dietary administration of B. firmus sw40 (109 CFU/g) for 4 weeks significantly reduced the inflammatory cytokines TNF‐1a, TNF‐2a and IFN‐γ genes expression, antioxidant parameter MDA and GSH levels in serum and increased antioxidant enzyme SOD activity. Besides, B. firmus sw40 could significantly increase the survival of gibel carp with pathogenic A. hydrophila infection.  相似文献   

3.
以嗜水气单胞菌野生株B11和基因稳定沉默株sodA-RNAi、sodB-RNAi和KatGRNAi为研究对象,比较野生株和沉默株在不同氧化胁迫条件下sodA,sodB和KatG表达的相关性及与细菌生长和存活的相关性,以探讨抗氧化胁迫基因在细菌抵御氧化胁迫过程中的作用和机制。结果显示,无氧化胁迫条件下sodA、sodB和KatG中任何一个基因被沉默后,其余两个基因的表达均受到显著抑制,说明这些抗氧化胁迫基因在表达上密切相关。在外源H2O2胁迫下,sodA、sodB和KatG中任何一个基因被沉默后,其他两个抗氧化胁迫基因的表达均表现为显著上调;在甲基紫晶(MV)诱导的内源性活性氧(ROS)胁迫下,各沉默株中sodA或sodB的表达水平总体表达为上调,但是在KatG的表达却呈现下调,说明sods的表达在细菌抵御内源性ROS的损伤中更具重要性。在H_2O_2胁迫下,随着H_2O_2浓度的增加,野生株B11的存活率仍然保持在较高的水平,而沉默株sodA-RNAi、sodB-RNAi和KatG-RNAi的存活率则显著降低;此外,菌株生长曲线显示,sodA、sodB和KatG的表达可影响嗜水气单胞菌生长曲线延滞期的长短。在不同浓度MV诱导的ROS胁迫下,野生株B11和沉默株KatG-RNAi的存活率仍然保持在较高的水平,沉默株sodA-RNAi和sodB-RNAi的存活率则显著降低,但4株菌仍然能保持一定的生长能力。研究表明,在不同氧化胁迫条件下,细菌可通过不同抗氧化胁迫基因的协同作用,共同抵御ROS的损伤,在H_2O_2的胁迫下,KatG对嗜水气单胞菌的生存更为重要,而在MV诱导的内源性ROS胁迫下,sod的表达对嗜水气单胞菌生存的贡献更为突出。  相似文献   

4.
A candidate vaccine against Aeromonas hydrophila in rainbow trout, Oncorhynchus mykiss, was developed using a bacterial lysate. To test the strength of protection, A. hydrophila challenge models were compared using injection into both the intraperitoneal (IP) cavity and the dorsal sinus (DS) with selected doses of live bacteria washed in saline or left untreated. Unlike the IP route, injection into the DS with either saline washed or unwashed cells resulted in consistent cumulative mortality and a dose response that could be used to establish a standard challenge having an LD50 of approximately 3 × 107 colony forming units per fish. Survivors of the challenge suffered significantly lower mortality upon re‐challenge than naïve fish, suggesting a high level of acquired resistance was elicited by infection. Passive immunization using serum from hyper‐immunized fish also resulted in significantly reduced mortality indicating protection can be transferred and that some portion of resistance may be antibody mediated. Vaccination of groups of rainbow trout with A. hydrophila lysate resulted in significant protection against a high challenge dose but only when injected along with Freund’s complete adjuvant. At a low challenge dose, mortality in all groups was low, but the bacterial lysate alone appeared to offer some protection.  相似文献   

5.
Fish-derived antimicrobial peptides are an important part of the innate immune system due to their potent antimicrobial properties. Piscidins are a class of antimicrobial peptides first described in hybrid striped bass (Morone chrysops x Morone saxatilis) but have also been identified in many other fish species. Previous work demonstrated the broad antimicrobial activity of piscidins against Gram-negative and Gram-positive bacterial species. This study sought to determine the extent to which class I (striped bass piscidin 1, white bass piscidin 1 and striped bass/white bass piscidin 3) and class II (striped bass piscidin 4 and white bass piscidin 5) piscidins inhibit biofilm formation of different Gram-negative bacteria. In general, the class I and II piscidins demonstrate potent activity against Escherichia coli and Flavobacterium columnare biofilms. The class II piscidins showed more activity against E. coli and F. columnare isolates than did the class I piscidins. The piscidins in general were much less effective against inhibiting Aeromonas hydrophila and A. veronii biofilm growth. Only the class I piscidins showed significant growth inhibition among the Aeromonas spp. examined.  相似文献   

6.
Vibrio harveyi, a luminescent Gram‐negative motile marine bacterium, is an important pathogen responsible for causing severe diseases in shrimp, finfish and molluscs leading to severe economic losses. Non‐luminescent V. harveyi obtained by culturing luminescent strains under static and dark condition were reported to alter the levels of virulence factors and metalloprotease gene and luxR expression when compared to their luminescent variants. Presently, we conducted an in vitro study aiming at the characterization of virulence‐related phenotypic traits of the wild‐type V. harveyi BB120 strain and its isogenic quorum sensing mutants before and after switching to the non‐luminescent status. We measured the production of caseinase, haemolysin and elastase and examined swimming motility and biofilm formation. Our results showed that switching from the bioluminescent to the non‐luminescent state changed the phenotypic physiology or behaviour of V. harveyi resulting in alterations in caseinase and haemolytic activities, swimming motility and biofilm formation. The switching capacity was to a large extent independent from the quorum sensing status, in that quorum sensing mutants were equally capable of making the phenotypic switch.  相似文献   

7.
Members of the genus Aeromonas are emerging human pathogens, causing a variety of extra‐intestinal, systemic and gastrointestinal infections in both immunocompetent and immunocompromised persons. Aeromonas virulence is multifaceted and involves surface‐associated molecules, motility, biologically active extracellular products and biofilm formation. Aeromonads, isolated from diverse freshwater fish species as well as sea water, were screened for biofilm formation, with varying physicochemical parameters including temperature, agitation and nutrient availability. Motility, cell surface characteristics (auto‐aggregation, hydrophobicity and S layer), and extracellular virulence factor production (haemolysis, proteolysis, DNase production) were also assessed to identify potential associations with the biofilm phenotype. Biofilm formation was influenced by environmental conditions, with isolates preferentially forming biofilms in nutrient‐rich media at 30 °C, although strong biofilm formation also occurred at 37 °C. Strong biofilm formation was observed for Aeromonas culicicola isolates following exposure to nutrient‐rich conditions, while Aeromonas allosaccharophila isolates preferred nutrient‐poor conditions for biofilm formation. Source‐/species‐specific correlations, ranging from weak to strong, were observed between biofilm formation and motility, cell surface characteristics and/or extracellular virulence factor production. Understanding the specific mechanisms by which Aeromonas species adhere to abiotic surfaces may aid in preventing and/or treating disease outbreaks in aquaculture systems and could lead to effective eradication of these fish pathogens.  相似文献   

8.
为探讨生物被膜动态演替过程中如何影响海洋无脊椎动物附着,实验选取了对厚壳贻贝附着具有不同诱导活性的弧菌Vibrio cyclitrophicus、V. chagasii和Vibrio sp. 22形成单一生物被膜,观察弧菌动态演替中生物被膜细菌密度、膜厚度和胞外产物等生物学特性变化,探究其对厚壳贻贝稚贝附着的影响。结果显示,弧菌生物被膜动态演替过程中,被膜细菌随着时间推移出现聚集现象,细菌密度和膜厚度也随着时间变化呈先增多后减少。除了Vibrio sp. 22,V. cyclitrophicus和V. chagasii生物被膜细菌密度和膜厚度与稚贝附着均有不同程度的相关性。所测弧菌生物被膜胞外产物的显微激光共聚焦结果分析发现,胞外多糖随着时间先增多,然后开始下降。相对比而言,胞外蛋白和胞外脂质无显著性变化。因而,胞外多糖变化规律与稚贝在被膜上附着变化相一致,表明胞外多糖是生物被膜动态演替过程中调控厚壳贻贝附着的重要因素。本实验初步探讨了生物被膜动态演替特征及其对厚壳贻贝稚贝附着的影响,对于后续进一步在海区开展生物被膜的动态演替与海洋底栖动物附着相互关系研究具有重要的学术价值,同时对于人工鱼礁礁体生物附着机理的研究具有重要的实践价值。  相似文献   

9.
The outer membrane protein of Aeromonas hydrophila is a potential candidate for vaccine development. In this study, after cloning and expression of ompTS, 270 common carp, weighing 44 ± 5.7 g divided into five groups, were injected intraperitoneally twice with 3‐week intervals. Groups included the following: PBS, PBS plus Freund's adjuvant, recombinant protein, recombinant protein plus Freund's adjuvant and 20 fish as negative control. Two weeks after the second injection, 30 fish of each group were challenged with a dose of 2 × LD50 of Aeromonas hydrophila and RPS was measured. The antibody level was measured using ELISA test. The protection of recombinant protein in the immunized fish with and without adjuvant, respectively, was about 82.61% and 78.26% (the protection of recombinant protein electroeluted from an SDS–PAGE with and without adjuvant, respectively, was about 78.62% and 69.57%). The average of antibody level in recombinant protein with and without adjuvant was significantly higher than the PBS group (p < .05). The ability of recombinant ompTS to increase the antibody level and to protect the fish from challenge by A. hydrophila demonstrated that recombinant ompTS protein injection can be used to immunize common carp against A. hydrophila infection.  相似文献   

10.
This study investigated the effects of glutamate (Glu) in low‐phosphorus diets on growth performance, haematological indices, antioxidant enzyme activity, immune‐related gene expression and resistance to Aeromonas hydrophila in juvenile mirror carp (Cyprinus carpio) (5.07 ± 0.02 g). Fish were fed either graded levels of Glu (0 g/kg, 5 g/kg,  10 g/kg and 20 g/kg, named G0, G0.5, G1 and G2, respectively) in a low‐phosphorus diet (15 g/kg NaH2PO4, 0.49), or a normal phosphorus diet ( 20 g/kg NaH2PO4, 0.61) without added Glu (C), for 8 weeks. At the end of the feeding trial, the fish were challenged with A. hydrophila. Compared with G0 group, 10 g/kg and 20 g/kg Glu supplementation of the low‐phosphorus diet significantly improved the final weight, WGR, SGR and PER, and decreased FCR (p < .05). Glu supplementation of the low‐phosphorus diet significantly enhanced the T‐AOC, SOD activity and GSH content in intestine (p < .05). Glu supplementation significantly reduced MDA content in foregut and midgut and increased CAT activity in midgut and hindgut (p < .05). Regarding immune‐related gene expression, Glu supplementation significantly diminished the up‐regulation of intestinal TNF‐α, IL‐1β and IL‐8 mRNA levels induced by phosphorus deficiency (p < .05). The survival rate of the G1 group was significantly higher than that of the G0 group (p < .05). In conclusion, 10 g/kg Glu supplementation in low‐phosphorus diets can improve the growth performance, enhance the activity of intestinal antioxidant enzymes and strengthen the immune function of juvenile mirror carp.  相似文献   

11.
Aeromonas hydrophila and Flavobacterium columnare, the aetiological agents of motile aeromonas septicaemia (MAS) and columnaris disease respectively, have been recently causing crippling mortalities to the sunshine bass, Morone chrysops female ×Morone saxatilis male (Percichthyidae), industry in the United States. Isolates of A. hydrophila and F. columnare obtained from fish that died during farm outbreaks were subjected to in vitro evaluation of florfenicol (FFC), copper sulphate (CuSO4) and potassium permanganate (KMnO4). Florfenicol inhibited the growth of A. hydrophila and F. columnare more than CuSO4 and KMnO4. The minimum inhibition concentration (MIC) of FFC was 0.04 ± 0 and 0.2 ± 0.1 mg L?1 for A. hydrophila and F. columnare respectively, while the 50% inhibition concentration (IC50) for A. hydrophila and F. columnare was 0.23 ± 0.01 and 0.4 ± 0.2 mg L?1 respectively. Copper sulphate was more effective against A. hydrophila than KMnO4; CuSO4 had a MIC of 83.2 ± 0 mg L?1 compared to 158.0 ± 0 mg L?1 for KMnO4. Copper sulphate was also more effective against F. columnare than KMnO4. The IC50 values of CuSO4 and KMnO4 towards F. columnare were 4.8 ± 0.3 and 8.7 ± 1.6 mg L?1 respectively, and the minimum bactericidal concentration values of CuSO4 and KMnO4 towards F. columnare were 25.0 ± 0 and > 158.0 mg L?1 respectively. In addition, F. columnare was more sensitive to CuSO4 and KMnO4 than A. hydrophila.  相似文献   

12.
Quorum sensing interference is a novel strategy in bacterial disease control. Degradation of quorum sensing signal molecules, such as N‐acyl homoserine lactones (AHLs), by enzymes is a potential interfering method. In this study, the in vivo effect of an N‐acyl homoserine lactonase from Bacillus sp. B546, AiiAB546, on the protection of zebrafish, Danio rerio, against Aeromonas hydrophila infection was first investigated by co‐feeding. The short‐term immune responses and the long‐term effects on the gut microbiota of zebrafish were analyzed by quantitative polymerase chain reaction (qPCR) and denaturing gradient gel electrophoresis (DGGE), respectively. The results showed that the expression levels of some anti‐infectious immunity‐related factors, including interleukin 10 (IL‐10), toll‐like receptor 5b (TLR5b), and inducible nitric oxide synthase 2a (iNOS2a) that were related to A. hydrophila virulent factors under regulation of quorum sensing, were more susceptible to the addition of AiiAB546. It indicated that AiiAB546 may attenuate the infection of A. hydrophila by interfering with quorum sensing. A. hydrophila was not detected in the enzyme co‐feeding diet group, which might be due to its inability to adhere to the gut with enzyme interference. This study revealed that AiiAB546 as a feed additive might represent a novel biocontrol agent in aquaculture; however, the topic merits further investigations using fish species of commercial interest.  相似文献   

13.
  相似文献   

14.
Vibrio vulnificus, Edwardsiella anguillarum and Aeromonas hydrophila are three common bacterial pathogens in cultivated eels. To protect farming eels from infection by these pathogens, a trivalent outer membrane protein (OMP) containing partial sequences of OmpU from V. vulnificus, OmpA from E. anguillarum and OmpII from A. hydrophila was expressed and purified; then, the OMP was used as a vaccine to immunize Japanese eels (Anguilla japonica). Whole-blood cell proliferation, antibody titres and complement and lysozyme activities were detected at different days post-immunization (dpi), and the relative per cent survival (RPS) was determined after eels were infected with V. vulnificus, E. anguillarum or A. hydrophila at 28 dpi. The results showed that the OMP significantly stimulates the antibody titres. At 14 days after the challenge (i.e. at 28 dpi), the RPS of OMP against V. vulnificus, E. anguillarum and A. hydrophila was 20%, 70% and 11.1%, respectively. The construction, expression and immunogenicity of a trivalent Omp were reported for the first time, and this study will provide a valuable reference for the development of fish multiplex vaccines.  相似文献   

15.
Aeromonas hydrophila is known to be causative agent of an infection named as Bacterial haemorrhagic septicaemia or red pest in freshwater fish. The aim of this study was to develop and validate the glycoprotein‐based fish vaccine against Aeromonas hydrophila. For this aim, after identification and characterization of A. hydrophila isolates from fish farms, one A. hydrophila isolate was selected as vaccine strain. Antigenic glycoproteins of this vaccine strain were determined by Western blotting and glycan detection kit. The connection types of these glycoproteins were examined by glycoprotein differentiation kit. Two glycoproteins, molecular weights of 19 and 38 kDa, with SNA connection type were selected for use in vaccination trials. After their purification by SNA‐specific lectin and size‐exclusion chromatography, protection studies with purified proteins were performed. For challenge trials, four experimental fish groups were designated: Group I (with montanide), Group II (with montanide and ginseng), Group III [with Al(OH)3] and Group IV [with Al(OH)3 and ginseng]. The survival ratings of fish were determined, and protection was calculated as 21.56%, 29.41%, 69.83% and 78.88% in groups I, II, III and IV, respectively. In conclusion, A. hydrophila glycoproteins with Al(OH)3 and ginseng could be used as a safe and effective vaccine for fish.  相似文献   

16.
Aeromonas hydrophila, a bacterium that is widespread in aquatic environments, is responsible for causing haemorrhagic disease in both aquatic and terrestrial species. With the purpose of developing a live vaccine, herein we have investigated nine strains of A. hydrophila (Ah-01 to Ah-09) isolated from diseased fish. A study of virulence factors that contribute to pathogenicity and immunogenicity in the host Cyprinus carpio suggests that the presence of β-hly, act and fla genes contribute to pathogenesis: strains Ah-01, Ah-02 and Ah-03 (β-hly+/act+/fla+ genotype) were highly pathogenic to C. carpio, whereas Ah-05 and Ah-06 (β-hly−/act−/fla− genotype) showed weak pathogenicity. Accordingly, Ah-02 and Ah-03 were selected to prepare inactivated vaccines, whereas Ah-05 and Ah-06 were chosen as live vaccines. Ah-06 live vaccine was found to have the best protective efficacy, with a protective rate of about 85%, whereas rates of other vaccines were significantly lower, in the range 37%–59%. In addition, DNA vaccines based on genes altA, aha and omp showed immune protection rates of 25%, 37.5% and 75%, respectively. Our data demonstrate that the β-hly−/act−/fla−/altA+/aha+/omp+ genotype has weak pathogenicity and high immunogenicity, and provide a simple and effective way to screen for live A. hydrophila vaccines.  相似文献   

17.
Different antibiotic‐based drugs are being used for the treatment of Aeromonas hydrophila infection in rainbow trout, and several studies emphasize the use of medicinal plants as immunostimulants for prophylactic measure against Aeromoniasis disease. However, therapeutic effects of aqueous methanolic extracts of tetra (Cotinus coggygria) against A. hydrophila in rainbow trout were not investigated. Four different concentrations of tetra extract (0 [control], 4, 8 and 12 mg/100 µl) and also two different positive control groups (florfenicol and doxycycline antibiotics) were administered orally using feeding needles to individual rainbow trout, Oncorhynchus mykiss of all experimental groups twice a day after intramuscular inoculation of A. hydrophila. The study period was for 10 days. On 0th, 3rd, 7th and 10th day, blood and tissues were collected from the fish and changes in humoral immune responses, haematology and immune‐related gene expressions were determined. In the study, superoxide radical production was decreased generally in all experimental groups except in 12 mg tetra and florfenicol treatments compared to control (p < .05). Lysozyme activity was generally decreased (p < .05), or no differences were observed in all experimental groups compared to the control. Myeloperoxidase activity was significantly increased in florfenicol‐treated fish group on 7th day (p < .05). Generally, myeloperoxidase activity showed an increase in almost all tetra‐treated groups. Haematological parameters increased but were not significantly high enough in treatments. Almost all immune‐related gene expressions were significantly enhanced on 3rd and 10th day of the study. Survival rate of 53.33% was found in control group. There were no significant differences in survival between control and 4 mg tetra‐treated group (p > .05). All the other groups' survival rate was significantly increased compared to control. The highest survival rate was found in florfenicol group (80%). In 12 mg tetra‐, doxycycline‐ and 8 mg tetra‐treated groups, survival rate was recorded as 74.44%, 70% and 70%, respectively. Our results suggest that tetra methanolic extract is an effective therapeutic remedy against A. hydrophila infection in rainbow trout at the dose of 24 mg/32.34 g body weight/day.  相似文献   

18.
The present study was conducted to determine the effects of Bacillus subtilisKADR1 and its subcellular components on immunity and disease resistance in Labeo rohita against Aeromonas hydrophila infection. Fish were fed diet containing different concentrations of live bacterial cells (DI‐106, DII‐108 and DIII‐1010 CFU/g) and another group of fish were immunized intraperitoneally with cellular components (WCPs, CWPs and ECPs) of Bacillus subtilisKADR1. After 4 weeks of trial, fish were challenged intraperitoneally with Aeromonas hydrophila cell suspension and survival percentage was recorded. Significantly higher post‐challenge survivability was recorded in fish groups fed 108 CFU/g of KADR1 (80.24%; RPS = 75.76%) or immunized with WCPs (77.77%; RPS = 72.73%), compared with the control (18.51%). Analysis of immunological parameters viz. serum lysozyme, phagocytosis, serum total protein, respiratory burst, serum IgM levels, superoxide dismutase and alternative complement pathway activity reflected significant enhancement (p < .05) of immune response in fish fed 106, 108 and 1010 CFU/g of live cells, or immunized with cellular components of Bacillus subtilisKADR1, with the highest values were observed DII fed group, followed by the group immunized with WCPs. Our results suggest that dietary administration of Bacillus subtilisKADR1 at 108 CFU/g can effectively enhance the immune responses and disease resistance in Labeo rohita against Aeromonas hydrophila.  相似文献   

19.
The two main diseases in the pangasius catfish industry are bacillary necrosis of Pangasianodon (BNP) and motile aeromonas septicaemia (MAS), where the aetiological agents have been identified as Edwardsiella ictaluri and Aeromonas hydrophila, respectively. In this study, apparently healthy Pangasianodon hypophthalmus were exposed to E. ictaluri, A. hydrophila or both bacterial species by intraperitoneal injection or immersion. There were 20 fish per treatment group, and the bacterial isolates used for the study were recovered from natural infections of BNP or MAS in farmed Vietnamese P. hypophthalmus. The results of the experimental infections mimicked the natural disease outbreaks reported from these pathogens in P. hypophthalmus. Furthermore, it was clearly demonstrated that E. ictaluri was only recovered from the fish exposed to the bacterium and not recovered from the animals receiving A. hydrophila.  相似文献   

20.
A 60 days study was conducted to evaluate the efficacy of water extract of Ocimum sanctum Linn. leaf on the immune response and disease resistance of Labeo rohita fingerlings against the Aeromonas hydrophila infection. Ocimum sanctum extract was incorporated in the diets (at 0.0%, 0.05%, 0.1%, 0.2%, 0.5% and 1%) of Labeo rohita, rohu fingerlings (6.6 ± 0.013 g). After 42 days blood, plasma and serum were sampled to determine super oxide anion production, lysozyme activity, total immunoglobulin in plasma, blood glucose, serum total protein, albumin, globulin, albumin:globulin ratio, WBC, RBC, haemoglobin content. Fish were challenged with A. hydrophila after 42 days and mortalities were recorded over 18 days post infection. The results demonstrate enhanced super oxide anion production, lysozyme activity, total immunoglobulin in plasma, serum total protein, globulin, total RBC counts, total WBC counts and haemoglobin content (P < 0.05) in treatments group compared with control group. Dietary O. sanctum extracts of 0.2% showed significantly (P < 0.05) higher protection relative percentage survival (RPS 40.00 ± 5.773%) against A. hydrophila infection than control. These results indicate that O. sanctum leaf extract stimulates the immunity and makes L. rohita more resistant to bacterial infection (A. hydrophila).  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