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1.
Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, produces Apx toxins that are recognized as major virulence factors. Recently, we showed that ApxIIIA-cytotoxic activity specifically targets Sus scrofa leukocytes. Since both LtxA from Aggregatibacter actinomycetemcomitans (aggressive periodontitis in humans) and LktA from Mannheimia haemolytica (pneumonia in ruminants) share this characteristic, respectively towards human and ruminant leukocytes, and because both use the CD18 subunit to interact with their respective LFA-1, we hypothesized that ApxIIIA was likely to bind porcine CD18 to exercise its deleterious effects on pig leukocytes. A β 2−integrin-deficient ApxIIIA-resistant human erythroleukemic cell line was transfected either with homologous or heterologous CD11a/CD18 heterodimers using a set of plasmids coding for human (ApxIIIA-resistant), bovine (-resistant) and porcine (-susceptible) CD11a and CD18 subunits. Cell preparations that switched from ApxIIIA-resistance to -susceptibility were then sought to identify the LFA-1 subunit involved. The results showed that the ApxIIIA-resistant recipient cell line was rendered susceptible only if the CD18 partner within the LFA-1 heterodimer was that of the pig. It is concluded that porcine CD18 is necessary to mediate A. pleuropneumoniae ApxIIIA toxin-induced leukolysis.  相似文献   

2.
Bovine herpesvirus-1 (BHV-1) has been reported to increase the susceptibility of cattle to respiratory disease caused by Mannheimia (Pasteurella) haemolytica A1. The principal virulence factor of M. haemolytica is a leukotoxin (LKT) that can specifically kill ruminant leukocytes following its binding to the beta2-integrin CD11a/CD18 (lymphocyte function-associated antigen 1 (LFA-1)). In this study, we investigated the effects of experimental infection of bovine peripheral blood mononuclear cells (MNCs) with BHV-1 in vitro, on the subsequent interaction of these cells with the M. haemolytica LKT. We found that BHV-1 infection increased LFA-1 expression (as assessed by flow cytometry), and subsequently enhanced LKT binding and cytotoxicity to bovine MNCs. We also found that BHV-1 infection increased CD18, IL-1beta, and IFN-gamma mRNA expression by MNCs. As previously reported for bovine polymorphonuclear neutrophils (PMNs), MNCs increased their expression of LFA-1, and their LKT binding and cytotoxicity, following exposure to IL-1beta, TNF-alpha, and IFN-gamma. These findings suggest that BHV-1 infection, and the resulting release of inflammatory cytokines, can stimulate expression of LFA-1 in bovine MNCs, thus enhancing the binding and biological effects of LKT. If such a mechanism occurs in vivo it might explain, in part, the increased susceptibility of BHV-1 infected cattle to bovine pasteurellosis.  相似文献   

3.
Progress in producing improved vaccines against bacterial diseases of cattle is limited by an incomplete understanding of the pathogenesis of these agents. Our group has been involved in investigations of two members of the family Pasteurellaceae, Mannheimia haemolytica and Haemophilus somnus, which illustrate some of the complexities that must be confronted. Susceptibility to M. haemolytica is greatly increased during active viral respiratory infection, resulting in rapid onset of a severe and even lethal pleuropneumonia. Despite years of investigation, understanding of the mechanisms underlying this viral-bacterial synergism is incomplete. We have investigated the hypothesis that active viral infection increases the susceptibility of bovine leukocytes to the M. haemolytica leukotoxin by increasing the expression of or activating the beta2 integrin CD11a/CD18 (LFA-1) on the leukocyte surface. In vitro exposure to proinflammatory cytokines (i.e. interleukin-1beta, tumor necrosis factor-alpha and interferon-gamma) increases LFA-1 expression on bovine leukocytes, which in turn correlates with increased binding and responsiveness to the leukotoxin. Alveolar macrophages and peripheral blood leukocytes from cattle with active bovine herpesvirus-1 (BVH-1) infection are more susceptible to the lethal effects of the leukotoxin ex vivo than leukocytes from uninfected cattle. Likewise, in vitro incubation of bovine leukocytes with bovine herpesvirus 1 (BHV-1) potentiates LFA-1 expression and makes the cells more responsive to leukotoxin. A striking characteristic of H. somnus infection is its propensity to cause vasculitis. We have shown that H. somnus and its lipo-oligosaccharide (LOS) trigger caspase activation and apoptosis in bovine endothelial cells in vitro. This effect is associated with the production of reactive oxygen and nitrogen intermediates, and is amplified in the presence of platelets. The adverse effects of H. somnus LOS are mediated in part by activation of endothelial cell purinergic receptors such as P2X7. Further dissection of the pathways that lead to endothelial cell damage in response to H. somnus might help in the development of new preventive or therapeutic regimens. A more thorough understanding of M. haemolytica and H. somnus virulence factors and their interactions with the host might identify new targets for prevention of bovine respiratory disease.  相似文献   

4.
Mannheimia haemolytica is an important etiological agent of pneumonia in domestic sheep (DS, Ovis aries). Leukotoxin (Lkt) produced by this organism is the principal virulence factor responsible for the acute inflammation and lung injury characteristic of M. haemolytica caused disease. Previously, we have shown that the leukocyte-specific integrins, beta(2) integrins, serve as the receptor for Lkt. Although it is certain that CD18, the beta subunit of beta(2) integrins, mediates Lkt-induced cytolysis of leukocytes, it is not clear whether CD18 of all three beta(2) integrins, LFA-1, Mac-1 and CR4, mediates Lkt-induced cytolysis of DS leukocytes. Since polymorphonuclear leukocytes, which express all three beta(2) integrins, are the leukocyte subset that is most susceptible to Lkt, we hypothesized that all three beta(2) integrins serve as the receptor for Lkt. The objective of this study was to determine whether DS LFA-1 serves as a receptor for M. haemolytica Lkt. We cloned the cDNA for DS CD11a, the alpha subunit of LFA-1, and co-transfected it along with the previously cloned cDNA for DS CD18, into a Lkt-non-suceptible cell line. Transfectants stably expressing DS LFA-1 were bound by Lkt. More importantly, Lkt lysed the DS LFA-1 transfectants in a concentration-dependent manner. Pre-incubation of Lkt with a Lkt-neutralizing monoclonal antibody (MAb), or pre-incubation of transfectants with MAbs specific for DS CD11a or CD18, inhibited Lkt-induced cytolysis of the transfectants. Exposure of LFA-1 transfectants to low concentrations of Lkt resulted in elevation of intracellular [Ca(2+)](i). Taken together, these results indicate that DS LFA-1 serves as a receptor for M. haemolytica Lkt.  相似文献   

5.
CD18, the common β subunit of β2-integrins, associates with four distinct chains to give rise to four different β2-integrins: CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1), CD11c/CD18 (CR4), and CD11d/CD18. Previously, we and others showed that CD18 of LFA-1 serves as a receptor for Mannheimia haemolytica leukotoxin (Lkt). Level of expression of Mac-1 is higher than that of LFA-1 and other β2-integrins on polymorphonuclear leukocytes (PMNs), which constitute the leukocyte subset most susceptible to Lkt. Hence, it is likely that CD18 of Mac-1 also mediates Lkt-induced cytolysis. Co-expression of CD11b and CD18 of cattle on Lkt-resistant cells is necessary to irrefutably demonstrate the role of Mac-1 in Lkt-induced cytolysis. This approach is hindered by lack of availability of complete sequence of cattle CD11b. Therefore, in this study, we cloned and sequenced the full length cDNA encoding cattle CD11b. The 3459 bp cDNA of cattle CD11b encodes a polypeptide of 1152 amino acids. The deduced amino acid sequence of CD11b of cattle exhibits 75% identity to that of humans and chimpanzees, 74% identity to that of dogs, and 70% identity to that of mice and rats. Availability of cattle CD11b cDNA should facilitate the elucidation of Lkt-receptor interactions in cattle and other species.  相似文献   

6.
The leukotoxin of Mannheimia haemolytica has a very high degree of amino acid diversity because the lktA gene has a complex mosaic structure that has been derived by horizontal DNA transfer and intragenic recombination. The objective of the present study was to determine the effect of this amino acid diversity on leukotoxin cytotoxicity against bovine and ovine cell types. This was done by comparing the luminol-dependent chemiluminescence response of bovine and ovine neutrophils after pre-incubation with the various leukotoxin types. The LktA1.1-type leukotoxin associated with bovine serotype A1 and A6 strains differs from the LktA1.2-type leukotoxin produced by ovine serotype A1 and A6 strains at a single amino acid position and has enhanced activity against bovine but reduced activity against ovine neutrophils. These findings, together with the exclusive association of the LktA1.1-type leukotoxin with bovine strains, suggest that this leukotoxin type has an adaptive advantage in the bovine host. Leukotoxins LktA6-LktA10 are associated with ovine strains and have complex mosaic structures and diverse amino acid sequences but similar levels of cytotoxic activity against bovine and ovine neutrophils, respectively. However, ovine neutrophils were more sensitive to the cytotoxic activities of these leukotoxins than were bovine neutrophils. LktA8- and LktA10-type leukotoxins are associated with serotype A2 and A7 strains that are responsible for the majority of ovine disease cases, but LktA6-, LktA7- and LktA9-type leukotoxins are associated with less common serotypes. These findings contribute to the growing body of evidence suggesting that factors other than leukotoxin cytotoxicity are responsible for the full expression of virulence in M. haemolytica. Overall, the extensive recombinational exchanges within the lktA gene of M. haemolytica have had little effect on leukotoxin function which is highly conserved.  相似文献   

7.
Mannheimia (Pasteurella) haemolytica A1 produces an extracellular leukotoxin (LKT) that is reported to bind the beta(2)-integrin CD11a/CD18 (LEA-1) on ruminant leukocytes. LKT binding induces activation, and subsequent cytolysis, of these cells. It is well known that active viral infection greatly increases the susceptibility of cattle to pasteurellosis. To better understand the mechanism by which this occurs, we investigated the effects of experimental in vivo infection of cattle with bovine herpes virus-1 (BHV-1) on the ex vivo interaction of bovine leukocytes with the M. haemolytica LKT. In this study, we demonstrated that active BHV-1 infection increased the expression of the beta(2)-integrin CD11a/CD18 (as defined by the mAb BAT75) on bovine peripheral blood neutrophils, enhanced the binding of LKT to bronchoalveolar lavage (BAL) leukocytes and peripheral blood neutrophils, and increased the killing of BAL leukocytes and peripheral blood leukocytes by LKT. In addition, BHV-1 greatly increased the number of BAL, resulting in many more LKT-responsive cells being present in the lungs. These findings might explain in part the increased susceptibility of BHV-1 infected cattle to pneumonic pasteurellosis.  相似文献   

8.
Pneumonia caused by Mannheimia haemolytica is an important disease of cattle (BO), domestic sheep (DS, Ovis aries) and bighorn sheep (BHS, Ovis canadensis). Leukotoxin (Lkt) produced by M. haemolytica is cytolytic to all leukocyte subsets of these three species. Although it is certain that CD18, the beta subunit of beta(2) integrins, mediates Lkt-induced cytolysis of leukocytes, whether CD18 of all three beta(2) integrins, LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18) and CR4 (CD11c/CD18), mediates Lkt-induced cytolysis of BO, DS and BHS leukocytes remains a controversy. Based on antibody inhibition experiments, earlier studies suggested that LFA-1, but not Mac-1 and CR-4, serves as a receptor for M. haemolytica Lkt. PMNs express all three beta(2) integrins, and they are the leukocyte subset that is most susceptible to Lkt. Therefore we hypothesized that all three beta(2) integrins serve as the receptor for Lkt. The objective of this study was to determine whether Mac-1 of BO, DS and BHS serves as a receptor for Lkt. cDNAs for CD11b of BO, DS and BHS were transfected into a Lkt-non-susceptible cell line along with cDNAs for CD18 of BO, DS and BHS, respectively. Transfectants stably expressing BO, DS or BHS Mac-1 specifically bound Lkt. These transfectants were lysed by Lkt in a concentration-dependent manner. Increase in intracellular [Ca(2+)](i) was observed in transfectants following exposure to low concentrations of Lkt indicating signal transduction through secondary messengers. Collectively, these results indicate that Mac-1 from these three species serves as a receptor for M. haemolytica Lkt.  相似文献   

9.
Leukotoxin (Lkt) is the primary virulence factor secreted by Mannheimia haemolytica which causes pneumonia in ruminants. Previously, we have shown that CD18, the beta subunit of beta(2) integrins, mediates Lkt-induced cytolysis of ruminant leukocytes. CD18 associates with four distinct alpha subunits giving rise to four beta(2) integrins, CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1), CD11c/CD18 (CR4), and CD11d/CD18. It is not known whether all the beta(2) integrins serve as a receptor for Lkt. Since PMNs are the leukocyte subset that is most susceptible to Lkt, and Mac-1 expression on PMNs exceeds that of other beta(2) integrins, it is of interest to determine whether Mac-1 serves as a receptor for Lkt which necessitates the cloning of CD11b and CD18. In this study, we cloned and sequenced the cDNA encoding CD11b of Ovis canadensis (bighorn sheep) and Ovis aries (domestic sheep). CD11b cDNA is 3455 nucleotides long encoding a polypeptide of 1152 amino acids. CD11b polypeptides from these two species exhibit 99% identity with each other, and 92% with that of cattle, and 70-80% with that of the non-ruminants analyzed.  相似文献   

10.
Ligand blotting of Pasteurella haemolytica leukotoxin (LKT) susceptible BL3 bovine lymphoma cell membranes with LKT detected two putative receptors with Mr of 95 and 100 kDa, whereas no LKT binding to membrane proteins was detected for LKT non-susceptible human leukemic cells. Anti-bovine CD18 and CD11a/CD18 mAb recognized 95 and 100kDa bands from BL3 cell membranes. CD18 isolated from BL3 cell membranes bound LKT. Pre-incubation of BL3 cells with anti-bovine CD18 or CD11a/CD18 mAb caused partial inhibition of LKT-induced leukolysis. Therefore, we propose that bovine CD18 acts as a species-specific leukocyte receptor for P. haemolytica LKT.  相似文献   

11.
The bovine respiratory pathogen Pasteurella haemolytica secretes an exotoxin that is specific for ruminant leukocytes (leukotoxin). Previous studies have shown that subcytolytic concentrations of the leukotoxin stimulate bovine neutrophils to undergo a respiratory burst and degranulate. Relatively little is known about the stimulatory effects of the leukotoxin on bovine mononuclear phagocytes. In this study, we compared the relative cytolytic effects of partially purified leukotoxin on bovine peripheral blood monocytes and alveolar macrophages. We found monocytes to be approximately 8- to 10-fold more sensitive than alveolar macrophages to the cytolytic effect of leukotoxin. In addition, incubation of monocytes and alveolar macrophages with sublethal doses of leukotoxin stimulated release of IL-1 and TNF activities in a dose-dependent manner. Addition of an antileukotoxin MAb neutralized the cytolytic effects of leukotoxin, but potentiated TNF release. Heat inactivation also blocked the cytolytic activity of LKT, but only slightly reduced its ability to stimulate TNF release. Although the leukotoxin preparations were estimated to have only small amounts of lipopolysaccharide (LPS) contamination, as determined by a standard Limulus amebocyte lysate coagulation assay, a chromogenic Limulus assay indicated much greater amounts of LPS were present. Adding equivalent doses of P. haemolytica LPS largely duplicated the monokine release stimulated by leukotoxin. These results suggest that the stimulatory effects of the P. haemolytica leukotoxin on bovine mononuclear phagocytes may principally involve LPS, perhaps complexed with leukotoxin.  相似文献   

12.
Previously, we have shown that CD18, the beta-subunit of beta(2)-integrins, serves as a receptor for leukotoxin (Lkt) secreted by Mannheimia (Pasteurella) haemolytica on bovine leukocytes. Anti-CD18 monoclonal antibodies (mAbs) inhibit Lkt-induced cytolysis of bighorn sheep (Ovis canadensis) leukocytes suggesting that CD18 may serve as a receptor for Lkt on the leukocytes of this species as well. Confirmation of bighorn sheep CD18 as a receptor for Lkt, and elucidation of the enhanced Lkt-susceptibility of bighorn sheep polymorphonuclear leukocytes (PMNs), necessitates the cloning and sequencing of cDNA encoding bighorn sheep CD18. Hence, in this study we cloned and sequenced the cDNA encoding CD18 of bighorn sheep, and compared with that of other animal species. The cDNA of bighorn sheep CD18 has an open reading frame (ORF) of 2310bp. CD18 sequences obtained individually from peripheral blood mononuclear cells (PBMCs) and PMNs were identical to each other. Comparison of the deduced 770-amino acid sequence of CD18 of bighorn sheep with that of domestic sheep, goats, cattle, humans and mice revealed 99, 98, 95, 82 and 80% identity, respectively. Availability of cloned bighorn sheep CD18 cDNA should allow the molecular characterization of M. haemolytica Lkt-receptor interactions in bighorn sheep and other ruminants that are susceptible to this disease.  相似文献   

13.
Leukotoxin (Lkt) and LPS are the major virulence determinants of Mannheimia haemolytica that contribute to the pathogenesis of bovine and ovine pneumonic pasteurellosis. We have previously identified bovine and ovine CD18 as the functional receptor for Lkt. LPS complexes with Lkt resulting in increased thermal stability and enhanced cytotoxic activity of Lkt. Cellular recognition of LPS involves several different molecules including CD14. We hypothesized that expression of ovine CD14 together with LFA-1 or Mac-1 would enhance Lkt-induced cytotoxicity. Ovine cDNA for CD14 was amplified by PCR and cloned into mammalian expression vectors. The 1122 bp cDNAs for bighorn sheep (BHS) and domestic sheep (DS) CD14 encode 373 amino acids which exhibit 99% identity with each other. Ovine CD14 plasmids were transfected either into HEK-293 cells, or previous HEK-293 transfectants stably expressing ovine LFA-1 or Mac-1. Flow cytometric analysis of transfectants confirmed the cell surface expression of CD14. The transfectants expressing LFA-1 or Mac-1 and the transfectants co-expressing CD14 with LFA-1 or Mac-1 did not show any significant difference in Lkt-induced cytotoxicity when incubated with LPS complexed Lkt. In contrast, incubation of the LFA-1 or Mac-1 and LFA-1/CD14 or Mac-1/CD14 transfectants with Lkt which lacks LPS, resulted in reduced cytotoxicity. None of the above transfectants showed any difference in [Ca2+](i) elevation when incubated with both types of Lkt preparations. Lkt did not induce any cytotoxicity or [Ca2+](i) elevation in ovine CD14 transfectants or parent HEK-293 cells. Based on these findings, we conclude that expression of CD14 together with LFA-1 or Mac-1 does not enhance Lkt-induced cytotoxicity, whereas LPS enhances cytotoxicity by complexing with Lkt.  相似文献   

14.
Mannheimia haemolytica induced pneumonias are only observed in goats, sheep and cattle. The bacterium produces several virulence factors,whose principal ones are lipopolysaccharide and leukotoxin. The latter is cytotoxic only for ruminant leukocytes, a phenomenon that is correlated with its ability to bind and interact with the ruminant beta2-integrin Lymphocyte Function-associated Antigen 1. This paper globally reviews all the information available on host-pathogen interactions underlying respiratory mannheimiosis (formerly pasteurellosis), from the stable and the Petri dish to the biochemical cascade of events triggered by the leukotoxin inside ruminant leukocytes. One conclusion can be made: the most widespread cattle respiratory disease with the most important impact on beef production worldwide, is probably due to a tiny ruminant-specific focal variation in the CD18- and/or CD11a-expressing genes.  相似文献   

15.
The effect of sublethal concentrations of the Pasteurella haemolytica leukotoxic culture supernate on bovine lymphocyte blastogenesis was investigated. Blastogenesis in cultures stimulated with either concanavalin A (Con A) or pokeweed mitogen (PWM) was inhibited in the presence of the supernate, as was the response to purified protein derivative in lymphocytes from BCG-vaccinated cattle. Partially purified leukotoxin had a similar effect. Pre-incubation of the leukotoxic supernate with a polyclonal rabbit antiserum raised to the immunogenic molecule of recombinant leukotoxin (r LktA) abrogated this effect, implicating leukotoxin as the factor responsible for the inhibition. B cell enriched cultures tended to be more sensitive to leukotoxic effects than did T cell enriched cultures. Although only ruminant cells are susceptible to the lethal effects of P. haemolytica leukotoxin, the toxin did inhibit both Con A- and PWM-induced proliferation of human and dog lymphocytes. As well, at high leukotoxin doses, Con A-stimulated pig lymphocyte proliferation was reduced. Rabbit lymphocytes were not affected by leukotoxin in either Con A- or PWM-stimulated cultures.  相似文献   

16.
The hypothesis that an altered expression of CD11/CD18 on bovine circulating monocytes, polymorphonuclear leukocytes (PMN), or both, contributes to an increased mastitis susceptibility in periparturient cows was tested. Expression of CD18 and CD11a, -b, -c on bovine monocytes and PMN were assessed in 8 Friesian-Holstein cows by flow cytometry from 2 wk before calving to 5 wk after calving. Minor changes in adhesion molecule expression levels were detected throughout the experimental period. Compared with PMN, monocytes exhibited an expression level that was similar for CD18, higher for CD11a and CD11c, but lower for CD11b. Differences in density may reflect the relative importance of these adhesion molecules on both leukocyte types. In this study, the decreased number of milk resident macrophages and PMN observed during the periparturient period could not be attributed to changes of CD11/CD18 levels on circulating leukocytes.  相似文献   

17.
The monoclonal antibody (MAb), C5B6, recognizes the CD11c/CD18 molecule on the surface of bovine peripheral blood monocytes. C5B6 was reactive with 69-83% monocytes, all granulocytes, and less than 5% of lymphocytes from cattle. Of the lymphocyte series, the antibody had specificity for large lymphocytes and two lines expressing T cell markers, but was not reactive with small lymphocytes, thymocytes, a tumor cell line of B-cell lineage, an interleukin 2 (IL2)-dependent T cell line, fibroblasts, or human, sheep, goat or pig peripheral blood mononuclear cells. No dual fluorescence was seen using C5B6 and antibodies to bovine IgM, CD2, CD4 or CD8. Immunoprecipitation of 125I labeled peripheral blood mononuclear cells with C5B6 antibody defined two bands: 150,000 and 95,000 Da. Antibody to the beta chain (CD18) of the leukocyte adhesion receptor family precipitates the 95 kDa beta subunit and the three associated alpha subunits (180, 165 and 150). The bands obtained using MAb C5B6 correlated with the p150/95 bands observed using an antibody that precipitated the alpha and beta chains of the leukocyte adhesion receptor family. Functionally, the primary but not the secondary proliferative response to alloantigens was inhibited by C5B6 MAb. No effect was seen using C5B6 MAb in cytotoxicity assays or in the secondary proliferative response to Brucella abortus or bovine herpes virus type 1.  相似文献   

18.
坏死梭杆菌白细胞毒素(Lkt)是一组对反刍动物白细胞特别是多形性白细胞(PMNs)有特异性毒性作用的细胞外毒素,被认为是坏死梭杆菌感染动物的主要毒力因子。白细胞毒素的物理稳定性较低,高温或极端pH环境中都能使白细胞毒素活性丧失。研究发现,白细胞毒素开放阅读框(ORF)全长9 726bp,由3个基因(lktB、A和C)组成,结构基因是第2个基因(lktA)。白细胞毒素对白细胞的毒性作用有剂量依赖性,并且溶血活性较低,不能在豚鼠猪皮肤上形成皮肤坏死症状。  相似文献   

19.
Single strains of 5 different P. haemolytica serotypes (1, 2, 5, 6 and 9) and an untypable strain were compared in an attempt to detect differences which might be related to virulence. All but the untypable strain caused extensive lesions when injected into the lungs of healthy cattle. Each strain was found to be encapsulated and to be toxic in vitro for bovine leukocytes. Each strain also produced leukotoxin in vitro. The toxins varied, however, in total toxic activity and in the kinetics of leukotoxin production. Vaccination of cattle with each of the serotype strains elicited antibodies to organism somatic antigens and, to various degrees, the production of leukotoxin-neutralizing antibodies which showed no strain specificity in cross-neutralization studies. Although each of the serotype strains appeared to be a potential bovine pathogen, subtle differences were observed which may explain the importance of Serotype 1 strains in bovine pneumonic pasteurellosis.  相似文献   

20.
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