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1.
In this study, various parameters affecting the ability of bovine neutrophils to ingest and kill a virulent strain of Pasteurella haemolytica A1 in vitro were examined. Ingestion of P. haemolytica was serum dependent (optimal serum concentration 10%) and was mediated principally by heat-stable opsonins, presumably antibodies, that could be removed by absorption with formalin-killed P. haemolytica. Ingested P. haemolytica were killed by neutrophils within 1-4 h incubation; the magnitude of killing being directly dependent on the number of neutrophils present. The number of viable P. haemolytica was reduced by approximately 1.5 log at bacterial concentrations of 0.01-100 P. haemolytica per neutrophil; a concomitant reduction in neutrophil viability was observed at the highest bacterial concentration (100:1). Bovine neutrophils underwent a vigorous luminol-enhanced chemiluminescence response after ingesting opsonized P. haemolytica, thus indicating that reactive oxygen intermediates were being formed that could have contributed to the intracellular killing of P. haemolytica.  相似文献   

2.
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   

3.
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   

4.
In this study we have addressed the in vitro effects of recombinant bovine interferon-gamma (rBoIFN-gamma) and interferon-alpha (rBoIFN-alpha 1) on oxidative functions of bovine neutrophils. Treatment with rBoIFN-gamma, but not rBoIFN-alpha 1, enhanced the luminol-dependent chemiluminescence (LDCL) response of bovine neutrophils to both opsonized zymosan particles and phorbol myristate acetate. Pre-incubation of neutrophils for 2 h at 39 degrees C with rBoIFN-gamma resulted in a 40% increase in both LDCL and release of hydrogen peroxide by neutrophils stimulated with opsonized zymosan. This enhancement was observed at doses ranging from 0.2 to 2000 units of rBoIFN-gamma per ml. In contrast to the results observed in the LDCL and hydrogen peroxide assays, preincubation of neutrophils with rBoIFN-gamma had no effect on the levels of superoxide anion released in response to opsonized zymosan. Pre-incubation with rBoIFN-gamma increased phorbol myristate acetate (PMA)-stimulated LDCL by 30%, although it had no effect on either superoxide anion or hydrogen peroxide release in response to PMA stimulation. Neither recombinant interferon directly elicited an oxidative burst from neutrophils in the absence of zymosan or PMA stimulation.  相似文献   

5.
To further define the role of Pasteurella haemolytica A1 leukotoxin in the pathogenesis of bovine pneumonic pasteurellosis, its in vitro effects on bovine neutrophils were investigated. Leukotoxin-containing culture supernatant, from P. haemolytica, stimulated a neutrophil respiratory burst as measured by the generation of oxygen-derived free radicals O2- and H2O2. This effect was immediate because preincubation of neutrophils with the culture supernatant for 5 min or longer substantially suppressed this respiratory burst. This suppression was due to cytolysis of the neutrophils. Prolonged incubation of neutrophils with the same culture supernatant caused further cytolysis and degranulation. Heat-inactivated P. haemolytica culture supernatant that had lost its cytotoxic properties failed to stimulate respiratory burst by neutrophils. Furthermore, the respiratory burst, cytolysis and degranulation were abrogated only by leukotoxin-neutralizing monoclonal and polyclonal antibodies, but not by antibodies against the lipopolysaccharide. These studies show that the leukotoxin component in the culture supernatant was responsible for the generation of oxygen-derived free radicals and proteolytic enzymes from neutrophils which may participate in direct lung injury.  相似文献   

6.
Pasteurella haemolytica A1 leukotoxic culture supernatant was evaluated for its ability to cause aggregation of bovine peripheral neutrophils. Neutrophils were isolated by a hypotonic lysis method and incubated with zymosan-activated plasma (ZAP), leukotoxic culture supernatant, antileukotoxin serum, calcium and magnesium-free media, p-bromophenacyl bromide and protein kinase C inhibitors. Aggregation was evaluated by changes in infrared light transmittance. Leukotoxic culture supernatant caused neutrophils to aggregate, and this effect was significantly removed by preincubation with antileukotoxin serum. Aggregation to ZAP and leukotoxin was dependent on the presence of extra-cellular calcium. Activation of protein kinase C by phorbol myristate acetate induced aggregation which was reduced by staurosporine; however, aggregation to leukotoxin did not involve protein kinase C activation. Phospholipase A2 inhibition did not alter the aggregation response to ZAP or to leukotoxin. The in vitro measurement of neutrophil aggregation induced by the leukotoxin of P. haemolytica reflects cytoskeletal and other activation events that may contribute to the intense inflammatory process which this organism induces in the lungs of cattle.  相似文献   

7.
In vitro interactions of bovine pulmonary lavage cells (PLC) and pathogenic isolates of Pasteurella haemolytica biotype A, serotype 1, were examined, using a luminol-dependent chemiluminescence (LDCL) assay. The PLC containing high concentrations of bovine alveolar macrophages were incubated with living and heat-killed P haemolytica at bacteria to PLC ratio of approximately 1:1. Kinetics of the mean LDCL response of bovine PLC to heat-killed P haemolytica cells were characterized by a gradual increase in the amount of light emitted over 150 minutes followed by a slight decrease at 180 minutes. In contrast, the LDCL responses of reaction mixtures containing living P haemolytica were characterized by the development of a maximal response at 60 minutes followed by a continued precipitous decrease in light emission to background values by 150 minutes. Differences were not noticed in the LDCL response of PLC suspensions from the same cow to 3 P haemolytica isolates. In each instance, reaction mixtures containing heat-killed bacteria had a similar LDCL profile that was characterized by continuous production of light over 180 minutes, whereas all reaction mixtures containing living bacteria underwent a precipitous decrease in light emission, which eventually resulted in a complete cessation of chemiluminescence. The PLC suspensions from different cattle did not respond to bacterial stimuli uniformly, with respect to the amplitude or detailed nature of the LDCL profile. The time that lapsed between the addition of living P haemolytica to PLC suspensions and the complete cessation of chemiluminescence varied for different cows.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

8.
Respiratory infection of cattle with bovine herpesvirus type 1 (BHV-1) predisposes cattle to secondary pneumonia with Mannheimia haemolytica as part of the bovine respiratory disease complex (BRD). One cell type that has received limited investigation for its role in the inflammation that accompanies BRD is the respiratory epithelial cell. In the present study we investigated mechanisms by which BHV-1 infection of respiratory epithelial cells contributes to the recruitment and activation of bovine polymorphonuclear neutrophils (PMNs) in vitro. Primary cultures of bovine bronchial epithelial (BBE) cells were infected with BHV-1 and assessed for cytokine expression by real-time PCR. We found that BHV-1 infection elicits a rapid IL-1, IL-8 and TNF-α mRNA response by BBE cells. Bovine PMNs exhibited greater adherence to BHV-1 infected BBE cells than uninfected cells. The increased adherence was significantly reduced by the addition of an anti-IL-1β antibody or human soluble TNF-α receptor (sTNF-αR). Pre-incubation of bovine PMNs with conditioned media from BHV-1 infected BBE cells increased PMN migration, which was inhibited by addition of an anti-IL-1β antibody, sTNF-αR, or an IL-8 peptide inhibitor. Conditioned media from BHV-1 infected BBE cells activated bovine PMNs in vitro as demonstrated by PMN shape change, production of reactive oxygen species and degranulation. PMNs also exhibited increased LFA-1 expression and susceptibility to M. haemolytica LKT following incubation with BHV-1 infected BBE cell conditioned media. Our results suggest that BHV-1 infection of BBE cells triggers cytokine expression that contributes to the recruitment and activation of neutrophils, and amplifies the detrimental effects of M. haemolytica LKT.  相似文献   

9.
Active infection with bovine herpesvirus-1 (BHV-1) increases the susceptibility of cattle to secondary bacterial pneumonia with Mannheimia (Pasteurella) haemolytica A1. In the present study we found that bovine PMNs incubated with conditioned media from BHV-1 infected peripheral blood mononuclear cells (PBMCs) exhibited increased LFA-1 expression, enhanced LKT binding and increased LKT cytotoxicity. These effects were abrogated when the conditioned medium was pre-incubated with an anti-IL-1beta Mab before being added to the PMNs. These findings suggest that BHV-1 infection, and the resulting release of IL-1beta and perhaps other inflammatory cytokines, can stimulate activation of LFA-1 in bystander bovine PMNs, thus enhancing the binding and biological effects of LKT.  相似文献   

10.
The adherence of viable and heat-treated Mycoplasma bovis to bovine peripheral blood neutrophils was studied by specific immunofluorescence staining and flow cytometry. Viable and heat-treated M. bovis cells, adhered to bovine neutrophils in dose-dependent fashion within a 30 min incubation. Fluorescence quenching using crystal violet indicated that unopsonized M. bovis cells remained on the surface of bovine neutrophils without experiencing significant ingestion. The effect of M. bovis adherence on neutrophil microbicidal function was examined by measuring luminol enhanced chemiluminescence (CL). Adherent M. bovis cells did not elicit a bovine neutrophil CL response over a 75 min incubation period. M. bovis inhibited the capacity of bovine neutrophils to mount a CL response. Inhibition occurred whether viable or heat-treated M. bovis cells were used and it occurred when neutrophils were stimulated with opsonized zymosan (OZ) or phorbol myristate acetate (PMA). Inhibition of the PMA stimulated neutrophil CL response required cytadherence by M. bovis cells. These findings suggest that activation of the bovine neutrophil respiratory burst was inhibited at or distal in the pathway to the activation of protein kinase C (PKC), the site of PMA stimulation, and that it was mediated by a direct interaction between the adhering M. bovis cells and the bovine neutrophil membrane.  相似文献   

11.
Calves, 90 to 130 days old, were inoculated with bovine herpesvirus-1 (BHV-1) or parainfluenza-3 (PI-3) virus. Pulmonary lavage specimens obtained from calves before virus inoculation contained 98% alveolar macrophages (AM) and 1% neutrophils. Six days after inoculation, the mean percentage of neutrophils in lavage specimens had significantly increased to 7.9 +/- 6.0% in BHV-1-inoculated calves and to 18.3 +/- 9.9% in PI-3 virus-inoculated calves, reflecting viral-induced pulmonary inflammation that was confirmed histologically. Approximately 75% of AM obtained before virus inoculation had Fc surface receptors, and 60% had C3b receptors. Six days after inoculation, the percentage of AM with Fc and C3b receptors was significantly reduced to 69.7 +/- 8.6% and 27.1 +/- 19.8%, respectively, in BHV-1-inoculated calves and to 67.8 +/- 15.4% and 38.8 +/- 23.2%, respectively, in PI-3 virus-inoculated calves. Alveolar macrophages obtained after virus inoculation were significantly impaired in their ability to phagocytize opsonized Staphylococcus epidermidis, but were able to kill ingested bacteria. Alveolar macrophage dysfunctions caused by BHV-1 or PI-3 respiratory infection did not differ appreciably.  相似文献   

12.
The purpose of these studies was to determine mechanisms of pulmonary tissue damage mediated by Pasteurella haemolytica and interaction with bovine neutrophils. Bovine pulmonary artery endothelial cell monolayers were treated with various combinations of P. haemolytica factors including bacterial culture supernatant (CS) and purified LPS, with and without bovine neutrophils. Damage to endothelial cells was monitored by 51Cr release, cell detachment rate, and morphological changes. At 5 h post-treatment (PT) bacterial factors produced very little toxic change in cells, however, by 22 h PT both crude leukotoxin and LPS caused high levels of cytotoxicity and detachment. Neutrophils did not augment toxicity mediated by LPS, but actually protected endothelial cells from low levels of LPS. When the LPS component of CS was neutralized with polymyxin B, leukotoxin mediated neutrophil killing resulted in extensive endothelial cell damage. These results suggest that LPS may directly injure endothelial cells and this toxic effect may be reduced by neutrophils. However, neutrophil killing by leukotoxin may also contribute to endothelial cell damage in the absence of LPS.  相似文献   

13.
The effects of opsonized zymosan, phorbal myristate acetate, and live Pasteurella haemolytica on superoxide anion production by bovine pulmonary macrophages were determined. The anion responses were dose-dependent for all stimuli, except for unopsonized P haemolytica. The effect of viable P haemolytica on macrophage viability was related to bacterial dosage and the presence of opsonizing antibody. Superoxide responses varied directly with the dose of opsonized live P haemolytica, but indirectly with macrophage viability.  相似文献   

14.
Groups of caesarean-derived, colostrum-deprived lambs were inoculated by the intratracheal route with Pasteurella haemolytica, either alone or 4 or 6 days after the inoculation of parainfluenza virus type 3 (PI3). Other groups were inoculated with PI3 followed by veal infusion broth, or with uninfected cell culture fluid followed by veal infusion broth (controls). All lambs were killed 24 h after the second inoculation. Pulmonary phagocytic cells were recovered by lavage and separated into alveolar macrophage (AM) and neutrophil fractions by density gradient centrifugation. Bacterial proliferation was detected in the lungs of all five lambs inoculated with P. haemolytica 6 days after PI3 but in only one of five inoculated with P. haemolytica 4 days after PI3 and one of five inoculated with P. haemolytica alone. The number of phagocytic cells recovered from the lungs was highest in animals inoculated with P. haemolytica 6 days after PI3 and, overall, a greater number of both AM and neutrophils was recovered from the lungs of animals where bacterial proliferation occurred (greater than 10(5.0) P. haemolytica 100 g-1 lung) than from those that controlled the bacterial infection. Oxygen-dependent bactericidal activity of AM and neutrophils was measured by chemiluminescence. Infection with PI3 and P. haemolytica increased the chemiluminescence responses. The highest responses were recorded from lambs inoculated with P. haemolytica 6 days after PI3, the group where pulmonary clearance was poorest. Overall, responses were higher in lambs in which bacterial proliferation occurred than in those that controlled the infection. On the other hand, oxygen-independent bactericidal activity, measured by the direct effects of neutrophil lysates on Escherichia coli, was lowest in lambs inoculated with P. haemolytica 6 days after PI3 and was lower in lambs where bacterial proliferation occurred.  相似文献   

15.
A luminol-dependent chemiluminescence (LDCL) assay was used to evaluate the response of bovine polymorphonuclear leukocytes; (neutrophils [PMN]) to living and heat-killed Escherichia coli, Pasteurella multocida (type A, serotype 3), and P haemolytica (biotype A, serotype 1), and to heat-killed P haemolytica and sterile culture supernatant from living P haemolytica. Control cultures containing PMN that had not been phagocytically stimulated with bacteria had a modest increase in LDCL during the initial 10 minutes of incubation, followed by a gradual decline throughout the 120-minute incubation period. Bovine PMN emitted LDCL more efficiently when the cells were exposed to living E coli or P multocida than when they were exposed to the same bacteria killed by heat. The mean LDCL values for reaction mixtures containing living E coli or P multocida peaked at 30 minutes of incubation and remained above values for mixtures containing the same heat-killed bacteria. Kinetics of the LDCL response of bovine PMN to heat-killed P haemolytica were similar (although reduced in amplitude) to that observed with killed E coli or P multocida. The LDCL response of bovine PMN to living P haemolytica was not like that for E coli or P multocida, and was characterized by the development of a peak response at 10 minutes followed by a precipitous decrease in responsiveness and a subsequent complete cessation of LDCL. Addition of sterile culture supernatant from living P haemolytica to test samples containing heat-killed P haemolytica induced a response similar to that obtained with the living microorganism.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

16.
A luminol-dependent chemiluminescence (LDCL) assay was used to assess the response of polymorphonuclear leukocyte (PMN) preparations from 4 species of ruminants (ie, cattle, sheep, goats, and antelopes) and 6 species of nonruminants (ie, swine, dogs, cats, rabbits, horses, and persons) to both opsonized and nonopsonized preparations of living and heat-killed Pasteurella haemolytica and Staphylococcus aureus and to opsonized and nonopsonized heat-killed strains of each bacterium in the presence of sterile culture supernatant (leukotoxin) from P haemolytica. The LDCL responses of PMN preparations from each of the species studied were greater for living than for heat-killed S aureus. The most efficient LDCL emission was observed with reaction mixtures containing opsonized living S aureus. Regardless whether they contained killed or living bacteria, the opsonized S aureus preparations elicited LDCL emissions more efficiently than did the corresponding nonopsonized preparations. Living P haemolytica cells and their sterile culture supernatant inhibited the LDCL emissions of phagocytically stimulated PMN preparations from ruminants, but not those from nonruminants. The LDCL response of ruminant PMN to nonopsonized living P haemolytica was characterized by the development of a peak response at 10 minutes of incubation followed by a precipitous decrease and a subsequent complete cessation of chemiluminescence. The peak LDCL response was higher for opsonized living P haemolytica than for nonopsonized living bacteria, and the increased response lasted longer. However, opsonization of living P haemolytica with the serum samples tested only temporarily spared the ruminant PMN preparations from the detrimental effects of leukotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

17.
Calves were exposed to an aerosol of infectious bovine rhinotracheitis (IBR) virus followed five days later by an aerosol of Pasteurella haemolytica. The animals were subjected to bronchoalveolar lavage before IBR and four days after, and again at 0, 4, 24, and 48 hours following Pasteurella haemolytica challenge. The results of these experiments suggest that neutrophil infiltration into the lung, in response to the presence of the bacteria was delayed thereby allowing the bacteria to become established in the lung. Neutrophils in infected animals displayed little random migration in vitro and did not respond to a chemotactic stimulus. It was also found that alveolar macrophages from virus-infected animals were not able to produce neutrophil chemotactic factors. These data suggest that the decrease in neutrophil chemotaxis and the lack of chemotactic factor production by the alveolar macrophage following infection with infectious bovine rhinotracheitis virus may predispose infected cattle to a secondary bacterial infection.  相似文献   

18.
Live Pasteurella haemolytica biotype A, serotype 1 isolates (n = 3) and Escherichia coli K-12, strain W3110, were reacted with bovine pulmonary lavage cell (PLC) suspensions. The comparative effects of the different bacteria on the functional and metabolic activity of alveolar macrophages (AMO) in the PLC suspensions were assessed simultaneously by use of 51Cr release, luminol-dependent chemiluminescence (LDCL), and AMO bactericidal assays. The bovine PLC responded differently to E coli, than to the 3 P haemolytica isolates in each of the 3 experimental test systems; however, responses to each of the P haemolytica isolates were not found to be significantly different. Unopsonized live P haemolytica cells adversely affected the functional and metabolic response of PLC, whereas there was no evidence of a cytotoxic (cytocidal) influence of E coli. A difference in 51Cr release for reaction mixtures containing E coli and P haemolytica was not detected at zero time; however, at each subsequent time, reaction mixtures phagocytically stimulated with P haemolytica had significantly increased amount of 51Cr release (P less than 0.05), compared with those mixtures containing E coli. Bovine AMO in the PLC suspensions were able to effectively kill E coli in vitro, but were unable to prevent survival and subsequent growth of P haemolytica. The luminol-dependent chemiluminescence profiles for reaction mixtures phagocytically stimulated with E coli provided evidence of sustained production of oxygen radicals with antimicrobial capabilities by bovine AMO in the PLC. Production of these highly reactive antimicrobial oxidants appeared initially in cultures containing P haemolytica but, subsequently, their production declined precipitously and ceased altogether.  相似文献   

19.
A new method for the isolation of bovine neutrophilic granulocytes from peripheral blood based on centrifugation in a discontinuous metrizamide gradient has been developed. The procedure is rapid, taking only about 2 h, and gives highly purified (greater than 90%) neutrophils in a high yield (approximately 85%). The function of the cells, as measured by chemiluminescence and migration assays, is not significantly influenced by the isolation procedure. Eosinophils can also be isolated by a slight variation of the method. Initial applications of the separation procedure indicate its usefulness in clinical studies of bovine neutrophil function. A variation between individuals in the function of the cells was thus demonstrated. Moreover, the chemiluminescence of neutrophils from infected animals was found to be greatly increased, and neutrophil migration was shown to be stimulated by in vivo ACTH treatment.  相似文献   

20.
Three, 5, or 7 days after inoculation with bovine viral diarrhea (BVD) virus (n = 12) or Mycoplasma bovis (n = 12), groups of calves were exposed to aerosols of Pasteurella haemolytica and were euthanatized 4 hours later. Histologic lesions in the lungs and the ratios of neutrophils to alveolar macrophages, collected by bronchoalveolar lavage, were compared with those of clinically healthy calves (n = 8) and calves inoculated with BVD virus only (n = 4), M bovis only (n = 4), or P haemolytica only (n = 2). Inoculation with BVD virus or M bovis did not have a significant (P greater than 0.05) effect on the neutrophil/macrophage ratio in the bronchoalveolar lavage. Aerosol exposure to P haemolytica induced a marked and significant (P less than 0.01) change in the neutrophil/macrophage ratio (from less than 1:9 to greater than 9:1). The reversed neutrophil/macrophage ratio in calves exposed to P haemolytica correlated well with the histologic changes in which small bronchi and bronchioles were plugged with purulent exudate. Inoculation with BVD virus did not induce gross or microscopic lesions in the lungs. Inoculation with M bovis resulted in a severe peribronchial lymphoid hyperplasia with mild exudation of neutrophils and macrophages into the cranioventral parts of the lungs.  相似文献   

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