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1.
The immune response to mixed whole cell antigens of Aeromonas hydrophila, Edwardsiella tarda and Pseudomonas fluorescens, the common Gram negative bacterial pathogens associated with diseases of Indian major carps were evaluated for their efficacy in triggering antibody responses in rohu, Labeo rohita (Ham.). The rohu yearlings were either immunized with antigens from single bacterial strain, A. hydrophila, E. tarda and P. fluorescens or a combination of all three. An antibody response was detected at 1st week post immunization that rose significantly (p<0.05) at 4th week post immunization in all the immunized groups. The antibody level started declining after 8th week but persisted up to 10th week post immunization in all the immunized groups. Similarly, no significant difference (p>0.05) in the antibody level was found between groups immunized with single and mixed bacterial antigens. Moreover, the use of mixed bacterial antigens did not jeopardize the specific immune response to the vaccine components. Upon challenge with single pathogen, a high relative percent survival was recorded in the group immunized with mixed bacterial antigens and was comparable to those fish immunized with the single bacteria. 相似文献
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Recombinant bovine soluble CD14 reduces severity of experimental Escherichia coli mastitis in mice 总被引：3，自引：0，他引：3
Endotoxin, or lipopolysaccharide (LPS), is responsible for pathogenesis of infections induced by Gram-negative bacteria, such as E. coli. The cellular response to LPS is modulated by interactions among LPS, LPS-binding protein (LBP) and CD14. Accumulated evidence shows that the soluble form of CD14 (sCD14) competes with membrane-bound CD14 (mCD14) for LPS and plays a pivotal role in regulating bacterial infection and septic shock caused by Gram-negative bacteria. Recombinant bovine sCD14 (rbosCD14) was produced by transfected insect sf/9 cells and its biological function was evaluated in mice. Eighty-one 8-week old BALB/cj female mice were randomly assigned to two groups, and injected intraperitoneally with either LPS (8 microg/g of body weight, n = 41) or LPS plus rbosCD14 (6.8 microg/g of body weight, n = 40). Survival rate at 24 h after injection for mice injected with either LPS or LPS plus rbosCD14 was 30 and 72%, respectively (P < 0.01). At 48 h survival rate was 7 and 37%, respectively (P < 0.01). To investigate the protective effect of rbosCD14 on experimentally induced mastitis in mice, two abdominal contralateral mammary glands of 7 lactating BALB/cj mice were injected through the teat canal with 10-20 colony-forming units (CFU) of Escherichia coli. One gland simultaneously received rbosCD14 (6 microg) and the other saline. At 24 h after challenge, glands that received rbosCD14 had less swelling and hemorrhaging, significantly lower bacterial counts (P < 0.05) and lower concentrations of TNF-alpha (P < 0.05). Results indicate that rbosCD14 is biologically functional and reduces mortality in mice from endotoxin shock and severity of intramammary infection by E. coli. 相似文献
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Porcherie A Cunha P Trotereau A Roussel P Gilbert FB Rainard P Germon P 《Veterinary research》2012,43(1):14
ABSTRACT: Escherichia coli is a frequent cause of clinical mastitis in dairy cows. It has been shown that a prompt response of the mammary gland after E. coli entry into the lumen of the gland is required to control the infection, which means that the early detection of bacteria is of prime importance. Yet, apart from lipopolysaccharide (LPS), little is known of the bacterial components which are detected by the mammary innate immune system. We investigated the repertoire of potential bacterial agonists sensed by the udder and bovine mammary epithelial cells (bMEC) during E. coli mastitis by using purified or synthetic molecular surrogates of bacterial agonists of identified pattern-recognition receptors (PRRs). The production of CXCL8 and the influx of leucocytes in milk were the readouts of reactivity of stimulated cultured bMEC and challenged udders, respectively. Quantitative PCR revealed that bMEC in culture expressed the nucleotide oligomerization domain receptors NOD1 and NOD2, along with the Toll-like receptors TLR1, TLR2, TLR4, and TLR6, but hardly TLR5. In line with expression data, bMEC proved to react to the cognate agonists C12-iE-DAP (NOD1), Pam3CSK4 (TLR1/2), Pam2CSK4 (TLR2/6), pure LPS (TLR4), but not to flagellin (TLR5). As the udder reactivity to NOD1 and TLR5 agonists has never been reported, we tested whether the mammary gland reacted to intramammary infusion of C12-iE-DAP or flagellin. The udder reacted to C12-iE-DAP, but not to flagellin, in line with the reactivity of bMEC. These results extend our knowledge of the reactivity of the bovine mammary gland to bacterial agonists of the innate immune system, and suggest that E. coli can be recognized by several PRRs including NOD1, but unexpectedly not by TLR5. The way the mammary gland senses E. coli is likely to shape the innate immune response and finally the outcome of E. coli mastitis. 相似文献
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Bannerman DD Chockalingam A Paape MJ Hope JC 《Veterinary immunology and immunopathology》2005,107(3-4):201-215
Almost half of all clinical cases of mastitis are caused by Gram-negative bacteria. Among these bacteria, intramammary infection by Pseudomonas aeruginosa remains one of the most refractory to antibiotic therapy. The ability to recognize potentially harmful pathogens whether previously encountered or not, as well as the induction of an initial pro-inflammatory response to these pathogens, are critical components of host innate immunity. Although the innate immune response to another Gram-negative mastitis-causing pathogen, Escherichia coli, has been well-characterized, little is known about the response to other Gram-negative bacteria, including P. aeruginosa. The objective of the current study was to characterize the systemic and localized bovine innate immune response to intramammary infection with P. aeruginosa. The contralateral quarters of ten mid-lactating Holstein cows were challenged with either saline or P. aeruginosa. Following the establishment of infection, milk samples were collected and assayed for changes in cytokine and growth factor concentrations, complement activation, and changes in the levels of soluble CD14 (sCD14) and lipopolysaccharide (LPS)-binding protein (LBP), two accessory molecules involved in host recognition of Gram-negative bacteria. Initial increases in milk somatic cell counts were evident within 12h of experimental challenge and remained elevated for >or=3 weeks. Increased permeability of the mammary gland vasculature, as evidenced by elevated milk levels of BSA, was initially observed 20 h post-infection and persisted for 2 weeks. Within 32 h of challenge, increased levels of IL-8, TNF-alpha, IL-10, and IL-12 were detected, however, the elevated levels of these cytokines were not sustained for longer than a 24h period. In contrast, elevations in IL-1beta, IFN-gamma, TGF-alpha, TGF-beta1, TGF-beta2, sCD14, LBP, and activated complement factor 5 (C5a) were sustained for periods of >48 h. Systemic changes were characterized by elevated body temperature, induction of the acute phase protein synthesis of serum amyloid A and LBP, and a transient decrease in circulating neutrophils and lymphocytes. Together, these data demonstrate the capability of the mammary gland to mount a robust innate immune response to P. aeruginosa that is characterized by the induction of pro-inflammatory cytokines, complement activation, and increased levels of accessory molecules involved in Gram-negative bacterial recognition. 相似文献
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Sohn EJ Paape MJ Connor EE Bannerman DD Fetterer RH Peters RR 《Veterinary research》2007,38(6):809-818
After intramammary infection, polymorphonuclear neutrophil leukocytes (PMN) are the first cells recruited into the mammary gland. Rapid recruitment of and bacterial phagocytosis and killing by PMN are the most effective defenses against establishment of bacterial infection. In addition to their phagocytic and bactericidal properties, PMN may play a key supportive role through secretion of cytokines during the innate immune response. We sought to determine whether bovine PMN produce cytokines in response to stimulation by lipopolysaccharide (LPS). To investigate the effects of LPS on the expression of cytokines secreted by bovine PMN, we measured the expression of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-12, and interferon (IFN)-gamma by ELISA after stimulation with different concentrations of LPS, and secretion of IL-8 after co-stimulation with LPS and either TNF-alpha or IL-1beta. Bovine PMN were shown to secrete TNF-alpha , IL-1beta, IL-12, IL-8 and IFN-gamma in response to LPS. Co-incubation of PMN with LPS and TNF-alpha increased secretion of IL-8 when compared to LPS alone. It was concluded that LPS stimulation up-regulates the secretion of cytokines by bovine PMN, and that co-incubation of LPS with TNF-alpha had an additive effect on the secretion of IL-8. These data show that bovine PMN, in addition to their phagocytic and bactericidal properties, may play a supportive role in the innate immune response to infection by Gram-negative bacteria through their ability to produce immuno-regulating cytokines. 相似文献
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Hsp90 is a molecular chaperone that is involved in diverse cellular processes including protein folding/repairing and signal transduction. Edwardsiella tarda is a serious fish pathogen that affects fish aquaculture worldwide. The aim of this study was to investigate the potential importance of HtpG, the prokaryotic homologue of Hsp90, in the pathogenesis of E. tarda. E. tarda HtpG is 627-residue in length and contains domain structures that are conserved among Hsp90 family members. Quantitative real time RT-PCR analysis indicated that expression of htpG is induced by heat shock and oxidative stress. Recombinant HtpG (rHtpG) purified from Escherichia coli exhibits apparent ATPase activity, which is optimal at 40°C. Mutation of htpG (i) affects bacterial growth at elevated temperature and renders the cells more sensitive to stress induced by reactive oxygen species, (ii) causes dramatic reduction in blood dissemination and general bacterial virulence, (iii) weakens the ability of E. tarda to block head kidney macrophage activation and to resist against the bactericidal effect of macrophages, and (iv) upregulates the expression of pro-inflammatory cytokines in macrophages. Taken together, these results indicate that HtpG is a biologically active protein that is required for E. tarda to cope with various stress conditions especially that encountered in vivo the host system during infection. 相似文献
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Streptococcus uberis and Serratia marcescens are Gram-positive and Gram-negative bacteria, respectively, that induce clinical mastitis. Once initial host barrier systems have been breached by these pathogens, the innate immune system provides the next level of defense against these infectious agents. The innate immune response is characterized by the induction of pro-inflammatory cytokines, as well as increases in other accessory proteins that facilitate host recognition and elimination of the pathogens. The objective of the current study was to characterize the innate immune response during clinical mastitis elicited by these two important, yet less well-studied, Gram-positive and Gram-negative organisms. The pro-inflammatory cytokine response and changes in the levels of the innate immune accessory recognition proteins, soluble CD14 (sCD14) and lipopolysaccharide (LPS)-binding protein (LBP), were studied. Decreased milk output, induction of a febrile response, and increased acute phase synthesis of LBP were all characteristic of the systemic response to intramammary infection with either organism. Infection with either bacteria similarly resulted in increased milk levels of IL-1 beta, IL-8, IL-10, IL-12, IFN-gamma, TNF-alpha, sCD14, LBP, and the complement component, C5a. However, the duration of and/or maximal changes in the increased levels of these inflammatory markers were significantly different for several of the inflammatory parameters assayed. In particular, S. uberis infection was characterized by the sustained elevation of higher milk levels of IL-1 beta, IL-10, IL-12, IFN-gamma, and C5a, relative to S. marcescens infection. Together, these data demonstrate the variability of the innate immune response to two distinct mastitis pathogens. 相似文献
10.
To evaluate potential of an auxotrophic Edwardsiella tarda mutant (Δalr Δasd E. tarda) as a delivery vehicle for DNA vaccine in fish, olive flounder (Paralichthys olivaceus) were immunized with the E. tarda mutant harboring plasmids (pG02-ASD-CMV-eGFP) for eukaryotic expression of the enhanced green fluorescent protein (eGFP) gene through either intraperitoneal (i.p.) or oral route, and the expression of eGFP in the internal organs and generation of antibody against eGFP in fish were analyzed. In fish i.p. injected with 2×10(7)CFU/fish of Δalr Δasd E. tarda harboring pG02-ASD-CMV-eGFP, expression of eGFP was detected in liver, kidney, and spleen from 1 day to 28 days post-injection. In fish orally administered with 1×10(9)CFU/fish of the bacteria, the eGFP band was detected in liver, kidney, and spleen from 1 day to 14 days post-administration, whereas, in intestine, the band was detected only at 1 day post-administration. Either oral or i.p. immunization of olive flounder with recombinant E. tarda that carried eGFP-expressing eukaryotic plasmids was successful to induce humoral adaptive immunity against not only E. tarda that was used as a delivery vehicle but also eGFP that was used as the reporter protein of DNA vaccine, suggesting attenuated E. tarda-vectored DNA vaccine has a potential to be used as a combined vaccine against infectious diseases in fish. 相似文献
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Vilte DA Larzábal M Mayr UB Garbaccio S Gammella M Rabinovitz BC Delgado F Meikle V Cantet RJ Lubitz P Lubitz W Cataldi A Mercado EC 《Veterinary immunology and immunopathology》2012,146(2):169-176
Cattle are the main reservoir of enterohemorrhagic Escherichia coli O157:H7, a bacterium that, in humans, causes hemorrhagic colitis and hemolytic uremic syndrome (HUS), a life-threatening disease, especially in children and older people. Therefore, the development of vaccines preventing colonization of cattle by E. coli O157:H7 could be a main tool for an HUS control program. In the present study, we evaluated bacterial ghosts (BGs) of E. coli O157:H7 as an experimental vaccine against this pathogen. BGs are empty envelopes of Gram-negative bacteria, which retain the morphological surface make-up of their living counterparts and are produced by controlled expression of the cloned protein E, which causes loss of all the cytoplasm content. In this work, E. coli O157:H7 BGs were used for subcutaneous immunization of calves. The vaccinated animals elicited significant levels of BG-specific IgG but not IgA antibodies in serum. Low levels of IgA and IgG antibodies against BGs were detected in saliva from vaccinated animals. Following oral challenge with E. coli O157:H7, a significant reduction in both the duration and total bacterial shedding was observed in vaccinated calves compared to the nonimmunized group. We demonstrated that systemic vaccination with E. coli O157 BGs provides protection in a bovine experimental model. Further research is needed to reach a higher mucosal immune response leading to an optimal vaccine. 相似文献
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Merchant M Mills K Williams S Kleckley F Sims A Elsey RM Bushnell J 《Veterinary immunology and immunopathology》2006,111(3-4):315-320
Alligators were injected intraperitoneally with four different doses (10, 1.0, 0.1, and 0.01 mg/kg body weight) of a mixture of bacterial lipopolysaccharides (LPS) derived from three different types of bacteria (Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae). Injection of the alligators with the LPS mixture resulted in a dose- and time-dependent increase in total peripheral leukocytes Lymphocytes increased at days 3 and 4 post-injection, and decreased back to baseline levels at day 7 for all doses. Alligators that were not treated, and those injected with pyrogen-free saline, did not exhibit statistically significant changes in total leukocytes during the course of the study. Injection of alligators with 0.5 mg LPS/kg body weight derived from one of three bacterial species revealed that the leukocyte increases observed were not statistically different for all three types of LPS. The animals displayed the same increases in total counts and the levels of all circulating leukocyte types were not different between animals treated with a combination of LPS from all three bacterial species. 相似文献
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P K Sahoo S C Mukherjee 《Comparative immunology, microbiology and infectious diseases》2001,24(3):143-149
Aflatoxin B1 (AFB1) is a potent immunomodulator in endotherms. The experiment was carried out to study the immunosuppressive nature of AFB1 in one ectothermic species of Indian major carp. Graded levels (0, 1.25, 5.00 mg/kg of body weight) of purified AFB1 were intraperitoneally (i.p.) injected into rohu (Labeo rohita) fingerlings weighing 30-50 g, and the fish were observed for a period of 90 days. At the end of the trial, blood samples were collected from the control group as well as the AFB1 injected fish and were screened for nitroblue tetrazolium (NBT) assay, serum total protein, albumin, globulin, albumin-globulin ratio (A:G), serum bactericidal activity and bacterial agglutination titre against Edwardsiella tarda. The aflatoxin-treated fish revealed a reduction of total protein, globulin levels, bacterial agglutination titre, NBT and serum bactericidal activities, as well as an enhanced A:G ratio without change in albumin concentration, irrespective of dose levels of toxin treatment, when compared to the control group. Thus, AFB1 proved to be immunosuppressive in rohu even at the lowest dose (1.25 mg/kg body weight) of toxin treatment. This could be of economic significance in intensive culture systems of rohu. 相似文献
15.
Kumar G Rathore G Sengupta U Kapoor D Lakra WS 《Comparative immunology, microbiology and infectious diseases》2010,33(2):133-144
Edwardsiella tarda is an important cause for hemorrhagic septicemia in fish and gastro and extra-intestinal infections in humans. Monoclonal antibodies (MAbs) were produced against outer membrane proteins (OMPs) of E. tarda ET-7, isolated from diseased snakehead (Ophiocephalus punctatus). Two stable hybridoma clones, designated as 3F10 and 2C3 MAbs were found to be potentially specific for E. tarda by indirect enzyme linked immunosorbent assay (ELISA). These MAbs recognized major immunogenic OMP band at 44kDa in Western blotting. Both MAbs belonged to the IgG1 isotype and recognized different epitopes of OMP as seen by competitive ELISA. These MAbs strongly reacted with all 17 isolates of E. tarda used in our study by indirect ELISA and Western blotting. Interestingly, no reaction was observed with the reference strain of E. tarda (MTCC 2400). The sensitivity of 3F10 MAb to detect whole cells of E. tarda was up to a level of 1x10(4)CFU/ml in indirect ELISA. No cross-reactivity of MAbs were seen with Escherichia coli, Salmonella arizonae, Pseudomonas fluorescens, Aeromonas hydrophila, Vibrio cholerae, Flavobacterium ferrugineum and Mycobacterium tuberculosis. These MAbs could be used for specific detection of E. tarda infection in fish by immunoassays. 相似文献
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Edwardsiella tarda is an enteric fish pathogen that has caused significant economic losses in a range of fish species residing in diverse ecological conditions. Several molecular methods relying on DNA fingerprinting (RAPD, RFLP and ERIC-PCR) and the gyrB gene marker have been used to characterize E. tarda isolates. However, all had drawbacks in resolving power and reproducibility. The present study was aimed at developing a novel Multi-locus Sequence Analysis (MLSA) scheme for genetic characterization of E. tarda isolates originating from multiple sources. MLSA has been described as an effective molecular tool with superior discriminatory power and reproducibility for exploring intra-species genetic diversity of several bacterial species. Nucleotide sequence fragments of eight protein coding housekeeping genes (gyrB, mdh, adk, dnaK, phoR, metG, pyrG and aroE2) were obtained from 23 fish pathogenic E. tarda isolates of different geographical origins, one human isolate and 3 reference strains. The phylogenetic relationships between isolates in individual gene analyses were not consistent, although some common patterns were apparent. Phylogenetic analysis based on concatenated sequences of seven gene loci, however, buffered the conflicting phylogenetic signals and resolved isolates according to their geographical origin and/or fish host. The MLSA revealed two major genetically diverging clusters in E. tarda isolates examined, one cluster representing isolates from fish and the other representing (in the main) human isolates, with E. ictaluri cluster situated in between. The results suggest, therefore, that the fish pathogenic E. tarda isolates may have been previously misclassified and probably represent one or more as yet unrecognized taxa within the genus Edwardsiella. The MLSA described here was robust enough in discriminating E. tarda isolates not only with respect to their geographical origins but also within different hosts from the same geographical location, high-lighting its potential application in tracing the source of infection and understand the epidemiological relationships among isolates of environmental, fish, other domestic animals or human origins. 相似文献
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Combined effects of beta-glucan and lipopolysaccharide (LPS) on survival and immune response were studied in Cyprinus carpio that were challenged with the pathogen Aeromonas hydrophila. beta-Glucan from Saccharomyces cervisiae and LPS from a virulent strain of A. hydrophila were used in this study. Different concentrations of beta-glucan+LPS mixture were administered on days 1, 7, and 14 through different routes (intraperitoneal injection, bathing, and oral administration). Control and test fish were challenged by intraperitoneal injection of LD50 concentration of A. hydrophila on day 16 and subsequently, mortality and relative percent survival (RPS) were recorded. Intraperitoneal injection elicited 100% RPS even at the lowest concentration (100 microg beta-glucan+10 microg LPS); whereas, oral administration improved RPS rate of carps at higher concentration (1% beta-glucan+0.25% LPS). Bathing did not improve the RPS. Test animals injected with even the minimum dose of the immunomodulators (100 microg beta-glucan+10 microg LPS/fish) had a significant increase in total blood leukocyte counts and an increase in the proportion of neutrophils and monocytes. Superoxide anion production by macrophages was also elevated, which presumably aided the efficient killing of bacterial pathogen. Lower concentration of beta-glucan+LPS had an adjuvant effect on antibody production as pretreatment by injection of 100 microg beta-glucan+10 microg LPS/fish resulted in higher antibody titer against A. hydrophila following vaccination. RT-PCR analyses showed that the expression of interleukin-1beta mRNA did not increase in test fish when compared with the control. Classical and alternative complement pathways were not affected by either the dose or the route of administration of the compounds. It may be concluded that intraperitoneal injection and oral administration, and not the bathing, of beta-glucan+LPS mixture in carp could enhance resistance to challenge by A. hydrophila through changes in several non-specific and specific immune responses. 相似文献
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S. Axiak-Bechtel B. Fowler D.H. Yu J. Amorim K. Tsuruta A. DeClue 《Research in veterinary science》2014
Dogs with lymphoma have altered innate immunity and little is known about the effects of chemotherapy on innate immune function in dogs. Lipopolysaccharide (LPS), lipoteichoic acid (LTA), and peptidoglycan (PG) – induced leukocyte cytokine production capacity, and phagocytosis and respiratory burst were evaluated in dogs prior to and following 6 weeks of chemotherapy. Dogs had decreased TNF production following LPS stimulation and increased IL-10 production following PG stimulation, which did not improve following remission of lymphoma. Dogs also had reduced E. coli-induced respiratory burst function after chemotherapy induced complete or partial remission. Dogs with lymphoma have an imbalance in pro-and anti-inflammatory cytokine production which did not improve with remission, and, following treatment, a decrease in respiratory burst function. Altered immune responses following exposure to bacterial pathogen associated molecular pattern motifs and bacteria may have many implications in the management of canine lymphoma. 相似文献
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菌蜕是通过PhiX174噬菌体溶菌基因E的可调控表达而形成的、缺少细胞浆和核酸且无繁殖能力的革兰氏阴性细菌菌体。这种非变性的灭活方式使菌蜕完好保留了细菌的各种抗原成分和免疫黏附分子,具有优良的免疫原性和固有的免疫佐剂性质以及免疫系统靶向性质。菌蜕的这些生物学特点使其在作为新型非变性灭活菌苗、作为递呈异源抗原的重组疫苗以及作为DNA疫苗甚至药物的载体方面都显现出巨大的研究开发前景。本研究成功构建了热敏诱导、pR、pL串联双启动子表达E基因的、具有不同抗性标记和复制起点的系列溶菌载体,比国外用pR或pL单启动子的溶茵载体在K-12株大肠杆菌(如XL1-blue)的溶茵效率高一个指数级。本研究还成功制备出禽致病性大肠杆菌(Avian pathogenic E.coli,APEC)野毒株的菌蜕,为进一步进行菌蜕疫苗的开发研究奠定了技术基础。 相似文献