Lysis of bovine platelets by Pasteurella haemolytica leukotoxin

Pasteurella haemolytica A1 culture supernatants caused rapid cytolysis (less than 5 minutes) of isolated bovine platelets as measured by leakage of the cytoplasmic enzyme lactate dehydrogenase (LD). The platelet lytic factor had several features similar to P haemolytica leukotoxin. Like P haemolytica leukotoxin, the platelet lytic factor was produced by P haemolytica during logarithmic growth phase, was heat-labile, and was active against target cells (platelets) from ruminant species (cattle and sheep), but not from non-ruminant species (horses, pigs, and human beings). Additionally, the platelet lytic factor was neutralized with antileukotoxin rabbit serum. The amount of LD leaked by a fixed concentration of bovine platelets was proportional to the amount of toxin added at low toxic doses and became maximal at 88 +/- 11% of the total platelet LD activity for high doses of toxin. When a fixed dose of toxin was used and the platelet concentration was varied, LD leakage was initially proportional to the platelet concentration, but plateaued at higher platelet concentrations. The platelet lytic factor required Ca2+ and was inhibited by addition of the Ca2+ chelator ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid. Toxin-mediated platelet damage may be important in thrombi formation and fibrin exudation typically associated with P haemolytica pleuropneumonia of cattle.     

Effects of Pasteurella haemolytica leukotoxin on cultured bovine lymphoma cells

Leukotoxin activity from culture supernatants of Pasteurella haemolytica serotype 1 in logarithmic growth phase caused rapid (less than 5 min) release of intracellular K+, uptake of extracellular Ca2+, and swelling of cultured bovine lymphoma cells (BL3 cells). Release of 51CrO4(2-) and lactate dehydrogenase (LDH) from BL3 cells began after 15 minutes of incubation with leukotoxin at 37 C and was completed between 60 and 120 minutes of incubation. In addition, leukotoxin exposure of BL3 cells resulted in cell aggregation and adherence to glass surfaces. Scanning electron microscopy indicated that after 10 minutes of leukotoxin exposure, BL3 cells increased in size, and large membrane defects developed between 20 and 60 minutes of exposure. The rate of release of LDH from leukotoxin-exposed BL3 cells was proportional to the amount of leukotoxin added. At high cell concentrations, the activity of LDH released at completion was directly proportional to the amount of leukotoxin added. Leukotoxin-induced release of LDH required a divalent cation, whereas K+ release and cell swelling did not. The addition of Ca2+, Mn2+, and Ba2+ resulted in increased leukotoxin-induced release of LDH. Divalent cation concentrations of 0.5 to 2.5 mM resulted in 50% of maximal stimulation. Ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid blocked increased release of LDH caused by Ca2+ addition, but had no effect on K+ release or cell swelling. Leukotoxin action on BL3 cells (K+ release, cell swelling, Ca2+ uptake, and release of LDH) was prevented by incubation at 4 C.     

Pasteurella haemolytica leukotoxin induces histamine release from bovine pulmonary mast cells.

Pasteurella haemolytica A1 leukotoxic culture supernatant was evaluated for its ability to induce histamine release from bovine pulmonary mast cells isolated by enzymatic dispersion of lung tissue. Histamine was measured by a radioimmunoassay technique. Leukotoxic culture supernatant of P. haemolytica significantly released histamine in a time and concentration-related manner. This effect was lost when culture supernatant was heat-inactivated or preincubated with leukotoxin neutralizing rabbit serum. Preincubation of the mast cells with propranolol or p-bromophenacyl bromide reduced the histamine-releasing effect of leukotoxin, while verapamil enhanced release. Experimental infection of calves with P. haemolytica A1 reduced the total histamine content of pulmonary mast cells recovered at postmortem. Histamine release induced by P. haemolytica leukotoxin is likely an important factor in the pathogenesis of bovine pneumonic pasteurellosis.     

Pasteurella haemolytica leukotoxin induced apoptosis of bovine lymphocytes involves DNA fragmentation

It has been reported that Pasteurella haemolytica leukotoxin (LKT) induces morphologic changes in bovine leukocytes consistent with apoptosis in vitro, but DNA fragmentation was not observed. We investigated whether bovine lymphocytes undergo DNA fragmentation during LKT-induced apoptosis. Bovine peripheral blood lymphocytes were isolated by density gradient centrifugation and exposed to LKT or inactive pro-LKT protein from a lktC- mutant strain. After exposure, DNA fragmentation in lymphocytes was quantified colorimetrically by diphenylamine assay and visualized by agarose gel electrophoresis. At high LKT concentrations, bovine lymphocytes were lysed, but at low concentrations, LKT caused DNA fragmentation characteristic of apoptosis. Maximal DNA fragmentation in bovine lymphocytes was induced by 0.1 TU ml(-1) LKT following 3 h exposure, but only background level of DNA fragmentation was observed with the inactive pro-LKT. Equine lymphocytes that are resistant to LKT intoxication did not show DNA fragmentation following exposure to LKT. Preincubation of LKT with a neutralizing anti-LKT monoclonal antibody inhibited LKT-induced DNA fragmentation. Electrophoresis of DNA from bovine lymphocytes treated with 0.1 TU ml(-1) LKT demonstrated the typical 'ladder' pattern of internucleosomal DNA cleavage, the hallmark of apoptosis associated with activation of endonucleases. LKT-induced DNA fragmentation was inhibited by 0.5 mM ZnCl2, an endonuclease inhibitor. The results indicated that LKT at low concentrations induced apoptotic cell death of bovine lymphocytes, which may play a role in initiation and persistence of P. haemolytica infection.     

Ultrastructural characterization of apoptosis in bovine lymphocytes exposed to Pasteurella haemolytica leukotoxin

OBJECTIVE: To characterize ultrastructural changes of bovine lymphocytes exposed to Pasteurella haemolytica leukotoxin (LKT). SAMPLE POPULATION: Partially purified LKT from a wild type P. haemolytica A1 strain and inactive pro-LKT from an isogeneic mutant Phaemolytica strain. Isolated bovine lymphocytes were obtained from 2 healthy calves. PROCEDURE: Isolated bovine lymphocytes were incubated with various concentrations of LKT and pro-LKT for 3 hours at 37 C and examined by use of transmission electron microscopy. A cytochemical Klenow DNA fragmentation assay was used to examine lymphocytes for DNA fragmentation. RESULTS: Lymphocytes incubated with LKT at a high concentration (1.0 toxic U/ml) had ultrastructural evidence of cytoplasmic and nuclear membrane rupture and swelling or lysis of mitochondria. Low concentrations of leukotoxin (0.1 toxic U/ml) induced DNA fragmentation in 80% of lymphocytes. Ultrastructurally, these cells had nuclear membrane blebbing, cytoplasmic vaculation, chromatin condensation, nuclear fragmentation, and membrane-bound apoptotic bodies. Incubation of lymphocytes with LKT at extremely low concentrations (0.001 toxic U/ml) or with pro-LKT did not alter their ultrastructure. Inclusion of 0.5 mM ZnCl2 in the medium blocked leukotoxin-induced ultrastructural changes in bovine lymphocytes. CONCLUSIONS AND CLINICAL RELEVANCE: Low concentrations of LKT induce apoptosis and high concentrations induce oncotic cell lysis in bovine lymphocytes. The ability of low LKT concentrations to induce apoptosis in host leukocytes may allow bacteria to escape host immune surveillance and colonize the host.     

Pasteurella haemolytica leukotoxin: physicochemical characteristics and susceptibility of leukotoxin to enzymatic treatment

Sterile, concentrated culture supernatant from Pasteurella haemolytica (biotype A, serotype 1) strain 630 was subjected to physical, chemical, and immunologic treatments to determine their influence on leukotoxin (cytotoxin) activity contained in the supernatant. Each treated sample contained approximately 8 chemiluminescence inhibitory units of leukotoxin. Treatment effects were evaluated for their ability to inactivate leukotoxin activity. Leukotoxin activity in treated samples was determined by inhibition of the luminol-dependent chemiluminescence response of bovine neutrophils. Optimal leukotoxin synthesis by P haemolytica occurred when the bacteria were at the logarithmic growth phase, whereas stationary phase cultures contained minimal amounts of leukotoxin activity in their culture supernatant. Leukotoxin activity was heat labile; activity was substantially decreased when concentrated culture supernatant samples containing leukotoxin activity were incubated at 37 C for several hours. When concentrated culture supernatant was incubated at progressively decreasing temperatures, there was a progressive increase in the length of time that the leukotoxin retained its biologic activity. Samples stored at -70 C retained activity for at least 2 months. Leukotoxin activity was nondialyzable and was able to withstand considerable extremes in hydrogen ion concentration. Leukotoxin activity could not be pelleted when subjected to forces of 100,000 X g for 1 hour. Chemical and enzymatic studies suggested that P haemolytica leukotoxin contained carbohydrate and protein moieties. Chemical treatment with 0.2% sodium lauryl sulfate, 0.5% sodium deoxycholate, 7.5 mM EDTA and 8M urea with 8 mM 2-mercaptoethanol and enzymatic treatment with lipase, ribonuclease, and deoxyribonuclease had no discernible effect on leukotoxin activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium ion involvement in the action of Pasteurella haemolytica leukotoxin

The influence of Ca2+ ions on the cytotoxic activity of Pasteurella haemolytica leukotoxin was investigated. The divalent cation influenced the cytotoxic effect of the leukotoxin for sensitive BL-3 target cells, but its absence did not eliminate cytotoxicity. In short-term 1-h assays using neutral red uptake as a measure of cell viability, depletion of Ca2+ either by exhaustive dialysis or by addition of the Ca2+ chelators EDTA and EGTA eliminated the cytolytic effect of low doses of the toxin. Addition of Ca2+ to target cell cultures depleted of the divalent cation restored the cytolytic effect of the leukotoxin. Prolonged exposure of the BL-3 cells to the toxin abrogated the protective effect of EDTA and EGTA. Cell death measured by uptake of neutral red, exclusion of trypan blue and 51Cr release indicated that protection observed in the absence of free Ca2+ was temporary. Toxin-induced cytolysis equivalent to that observed in the presence of Ca2+ occurred following the initial 2-h exposure. In addition, verapamil, a Ca2+ channel blocker, prevented cell death during 1-h cytotoxicity assays. The protection afforded by verapamil was dose-dependent and was influenced by the concentration of Ca2+ in the buffer medium. The results suggest that Ca2+ positively influences the rapid initial phase of cell death resulting from exposure to the toxin, but is not required for the entirety of the cytolytic process.     

Effects of Pasteurella haemolytica A1 leukotoxin on bovine neutrophils: degranulation and generation of oxygen-derived free radicals.

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.     

Immunohistochemical localization of Pasteurella haemolytica A1-derived endotoxin, leukotoxin, and capsular polysaccharide in experimental bovine Pasteurella pneumonia

Six, 5- to 10-week-old male Holstein calves were inoculated intratracheally with 5 x 10(9) logarithmic growth phase Pasteurella haemolytica biotype A serotype 1 (A1). Immunohistochemical techniques in conjunction with the use of monoclonal antibodies directed against P. haemolytica A1-derived lipopolysaccharide (LPS), capsular polysaccharide, and a polyclonal rabbit anti-leukotoxin antibody were used to localize their respective antigens in tissue sections of pneumonic lung at the light and electron microscopic levels. We found the following: 1) LPS, capsular polysaccharide, and leukotoxin were released into the inflammatory exudate; 2) LPS was found within the cytoplasm of neutrophils (located in the alveolus and alveolar wall), alveolar macrophages, endothelial cells, pulmonary intravascular macrophages, and on epithelial cell surfaces; 3) capsular polysaccharide was found in the alveolus and alveolar macrophages but not in cells of the alveolar wall; and 4) leukotoxin was associated with cell membranes of degenerating inflammatory cells located in the alveolus. This is the first study that demonstrates the presence of leukotoxin in the pulmonary inflammatory lesions caused by P. haemolytica A1 and implicates endotoxin as an important factor in the genesis of the pulmonary lesions.     

Preliminary investigation of the mechanism of inhibition of bovine lymphocyte proliferation by Pasteurella haemolytica A1 leukotoxin

Pasteurella haemolytica A1 leukotoxic culture supernate has been shown to inhibit bovine lymphocyte blastogenesis induced by concanavalin A (Con A), pokeweed mitogen (PWM) and purified protein derivative (PPD). The various mechanisms by which this inhibition could be overcome were investigated in an effort to determine at which stage of cell activation the leukotoxin exerted its inhibitory effect. For both Con A and PWM stimulated cultures, the addition of partially purified bovine interleukin 1 reduced the leukotoxin-induced inhibition. Recombinant interleukin 2 had a similar effect. Addition of the glycolipid, monosialoganglioside was also able partially to overcome the inhibition.     

Characterization of Mannheimia (Pasteurella) haemolytica leukotoxin interaction with bovine alveolar macrophage beta2 integrins

Mannheimia (Pasteurella) haemolytica, the etiologic agent of bovine pneumonic mannheimiosis, produces an exotoxic leukotoxin. The leukotoxin (LktA) is a member of the RTX (repeats in toxin) family of bacterial cytolysins and is distinguished from other toxins by its unique target cell specificity to ruminant leukocytes occurring through binding to a specific receptor. We have demonstrated previously that the beta2 integrin LFA-1 is a receptor for LktA in bovine leukocytes and is involved in leukotoxin-induced biological effects. However the subunits within LFA-1 involved in binding to LktA, and post-binding signaling leading to cellular activation have not been well characterized. The purpose of our study was to pinpoint these precise subunits on bovine alveolar macrophages and to characterize their interaction with LktA. The results in this study indicate that although LktA can efficiently bind to the CD18 subunit of both LFA-1 and Mac-1, post-binding signaling events including elevation of intracellular calcium and CD18 tail phosphorylation are only observed through LFA-1. Furthermore, LktA also binds to the CD11a subunit of LFA-1. LktA binding to CD11a could be inhibited by a small molecule inhibitor of the I(inserted)-domain, the major ligand binding interface on CD11a. I-domain inhibition significantly blunts LktA-induced intracellular calcium elevation and tyrosine phosphorylation of the CD18 tail. Based on our results we suggest that LFA-1 serves as the functional leukotoxin receptor on bovine alveolar macrophages.     

Dissociation of cytolysis and monokine release by bovine mononuclear phagocytes incubated with Pasteurella haemolytica partially purified leukotoxin and lipopolysaccharide.

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.     

Interaction of bovine respiratory syncytial virus and Pasteurella haemolytica in the ovine lung

The potential synergistic effect of bovine respiratory syncytial virus (RSV) and Pasteurella haemolytica in the production of pneumonia after aerosol/intranasal infection of conventionally reared lambs was evaluated. A mild clinical response was observed in lambs given virus and/or bacteria. Gross pulmonary lesions were seen in 3 of 6 lambs given RSV and then P haemolytica 3 or 6 days later, respectively (groups D and E), and in 1 lamb of 5 given virus and bacteria simultaneously (group G). Gross lesions were not seen in control sheep (group A), in lambs given virus or bacteria alone (groups B and C), or in lambs exposed to bacteria and then virus 3 days later (group F). Bovine RSV and P haemolytica were recovered from the lungs of 5 of 7 lambs with macroscopic lesions. Gross pulmonary lesions were cranioventral firm areas of red consolidation. Microscopically, the predominant lesion was a suppurative bronchopneumonia. Bovine RSV was recovered from the nasal cavity of 8 of 27 (30%) lambs given RSV during days 3 to 6 after viral inoculation, including 1 lamb in group B, 2 in groups D, E, and F, and 1 in group G. Pasteurella haemolytica was recovered from the nasal cavity of 9 of 28 (32%) inoculated lambs, including 2 lambs from groups C and E, 3 in group D, and 1 in groups F and G. Viral antigen, as determined by immunofluorescence, was concentrated mainly in individual cells in alveolar walls, some alveolar macrophages, and a few bronchiolar epithelial cells. In vitro alveolar macrophage assays indicated decreased numbers of Fc receptors on those macrophages collected from lambs given RSV 6 days before P haemolytica infection, as compared with that in the other groups. These cellular defects disappeared after 24 hours of culture. Seemingly, bovine RSV does facilitate P haemolytica pulmonary infection in conventional, immuno-competent lambs and provides evidence for decreased Fc receptors on alveolar macrophages.     

Effects of Mannheimia haemolytica leukotoxin on apoptosis and oncosis of bovine neutrophils

OBJECTIVE: To investigate the concentration-dependent effects of Mannheimia haemolytica (formerly Pasteurella haemolytica) leukotoxin (LKT) on apoptosis and oncosis in bovine neutrophils and to examine the role of calcium ions (Ca2+) in LKT-induced apoptosis. SAMPLE POPULATION: Neutrophils isolated from blood samples obtained from healthy calves. PROCEDURE: Neutrophil suspensions were exposed to lytic or sublytic dilutions of LKT and then examined by use of transmission electron microscopy (TEM) or gel electrophoresis. Contribution of extracellular Ca2+ to LKT-induced apoptosis was investigated by incubating neutrophils with LKT or control solutions in buffer containing 1 mM CaCl2 or in Ca2+-free buffer containing 1 mM ethylene glycol-bis (b-aminoethyl ether)-N,N-tetraacetic acid (EGTA) prior to diphenyl amine analysis. RESULTS: Examination by TEM revealed that bovine neutrophils exposed to lytic dilutions of LKT had changes consistent with oncosis, whereas neutrophils exposed to sublytic dilutions of LKT and staurosporin, an inducer of apoptosis, had changes consistent with apoptosis. Effects of sublytic dilutions of LKT on apoptosis were confirmed by gel electrophoresis. Replacement of extracellular Ca2+ with EGTA, a Ca2+ chelator, reduced apoptosis attributable to the calcium ionophore A23187, but it did not have significant effects on apoptosis induced by LKT or staurosporin. CONCLUSIONS AND CLINICAL RELEVANCE: The ability of LKT to cause apoptosis instead of oncosis is concentration-dependent, suggesting that both processes of cell death contribute to an ineffective host-defense response, depending on the LKT concentration in pneumonic lesions. Furthermore, although Ca2+ promotes A23187-induced apoptosis, it is apparently not an essential second messenger for LKT-induced apoptosis.     

The production and evaluation of Pasteurella haemolytica leukotoxin in the supernatant of submerged cultures in fermenters

The optimal production of P. haemolytica leukotoxin in the culture supernatant of a fluid medium is dependent on a number of factors. The leukotoxin has to be produced by using a strain that is known for its ability to produce high quantities of leukotoxin, inoculated into the most suitable type of medium at the correct culture density containing the necessary supplements and harvested after a certain growth period. The volume in which it is produced may also have an influence. Two different procedures are described to produce the leukotoxin in 5 to 15-l quantities in RPMI 1640 medium. The first method used to produce leukotoxin is one that has been repeatedly described since the presence of the leukotoxin was first established in 1978. Using this method seven batches of leukotoxin were produced in litre quantities with leukotoxin activity ranging from 23-67 u/ml. The seed culture inoculum is prepared in brain heart infusion broth, which is centrifuged before the organisms are inoculated into RPMI 1640 medium containing 3.5% foetal calf serum and incubated for only 1 h in a fermenter, after, which the leukotoxin is harvested. An improved alternative method was devised which yielded higher levels of leukotoxin activity by utilising the ability of the P. haemolytica organisms to grow and produce leukotoxin during the logarithmic growth phase in a fermenter. A seed culture harvested in the log phase was prepared in brain heart infusion broth by means of a series of cultures and inoculated into RPMI 1640 containing 3.5% foetal calf serum. Three hours of active growth were allowed during which the leukotoxin was measured by its biological activity and an ELISA assay, and the increase in cell mass by means of the optical density every 30 min. The average leukotoxin biological activity measured 260 u/ml and by means of the ELISA test the leukotoxin concentration measured 315 u/l which is a substantial increase in leukotoxin production. In comparison the average optical density only measured 0.469 at 650 nm. Previous findings were substantiated that the highest cell density was not reflected in the highest leukotoxin activity. It is possible to induce high levels of leukotoxin secretion in submerged cultures with RPMI 1640 medium containing foetal calf serum in the controlled environment of a fermenter in large enough quantities for use as a vaccine by the improved preparation of the seed culture inoculum.     

Bovine CD18 identified as a species specific receptor for Pasteurella haemolytica leukotoxin.

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.     

Antileukotoxin antibody produced in the bovine lung after aerosol exposure to viable Pasteurella haemolytica

Experiments were performed to determine the in vivo immunogenicity of Pasteurella haemolytica leukotoxin. Calves were exposed twice to aerosol mists of viable P haemolytica, using a treatment regimen previously shown to induce a resistant state. Pulmonary lavage fluids and serum samples from these calves were assayed for leukotoxin-neutralizing antibodies. Before aerosol exposure, neutralizing antibody titers were routinely found in serum samples, but were not detectable in pulmonary lavage concentrates before exposure. After aerosol exposure, titers of toxin-neutralizing immunoglobulin (Ig)A and IgG antibodies were found in pulmonary lavage concentrates and were accompanied by increased serum toxin neutralization titers.     

Effects of Pasteurella haemolytica leukotoxic culture supernatant on bovine neutrophil aggregation.

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.     

The effect of Pasteurella haemolytica cytotoxin on bovine polymorphonuclear leukocytes can be attenuated by beta-adrenoceptor antagonists

It was investigated whether beta-adrenoceptor antagonists could disturb the interaction between cytotoxin preparations isolated from Pasteurella haemolytica and bovine polymorphonuclear leukocytes (PMNs). The toxicity of the cytotoxin preparation was evaluated by measuring the chemiluminescence response and the viability of the cells after incubation with the cytotoxin. No effect on cell viability was detected when PMNs were incubated with 63 micrograms cytotoxin per ml while the chemiluminescence response was diminished by approximately 30%. The beta-adrenoceptor antagonists alprenolol (10(-5) M) and propranolol (5 X 10(-7) - 5 X 10(-6) M) were able to attenuate this effect of cytotoxin on the chemiluminescence response of PMNs. It seemed unlikely that propranolol and alprenolol diminished the effect of cytotoxin on the chemiluminescence response of PMNs by their beta-adrenoceptor blocking potency because other beta-adrenoceptor antagonists used were without effect. Also, the membrane stabilizing characteristics of the beta-adrenoceptor antagonists used were probably not responsible for the diminished interaction between PMNs and the cytotoxin. Whether beta-adrenoceptor antagonists could be used in vivo to prevent or treat P. haemolytica infections in bovines remains to be examined.