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.     

Effect of leukotoxin of Mannheimia haemolytica and LPS of E. coli on secretory response of bovine neutrophils in vitro

To evaluate the role of leukotoxin (LKT) of Mannheimia haemolytica and lipopolysaccharide (LPS) of E. coli 055:B5 in pathogenesis of bovine respiratory disease (BRD) we investigated their in vitro effects on cultured bovine neutrophils. Functional parameters of neutrophils including degranulation, generation of superoxide, and nitric oxide were distorted in response to both toxins. The most essential reaction of neutrophils was found in respect to release of elastase after addition of LKT as well as LPS at concentration of 300 microg/ml. Moreover, we observed an increased release of myeloperoxidase (MPO) and alkaline phosphatase (ALK-P) from polymorphonuclear cells (PMN) after addition of LKT and LPS. We also found enhanced superoxide generation by bovine neutrophils after exposure to different concentrations of LKT and LPS. In cultures of PMN treated with LKT, concentration of nitrite increased with growing concentrations of LKT. Lower values of nitrite were obtained in cultures exposed to LPS. Partial lysis of PMN, determined by LDH (lactate dehydrogenase) leakage, started at concentration of 300 microg/ml for both toxins, meanwhile LKT concentration above 300 microg/ml was lethal. Our study has revealed that neutrophils in response to both toxins exaggerate release of analysed substances, which participate in worsening the course of the disease and play a role in lung injury during BRD. Toxins introduced to the cultural medium stimulate release of studied constituents from neutrophils by combined activation and lysis of neutrophils.     

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.     

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.     

Actin polymerization enhances Pasteurella haemolytica leukotoxicity

Pasteurella haemolytica leukotoxin is cytotoxic to bovine leukocytes, causing increased cell membrane permeability, osmotic swelling, release of cytosolic proteins and cell lysis. These studies were designed to test if leukotoxin causes release of the cytoskeletal protein, actin, from bovine leukemia cells and if purified actin-influenced bacterial growth or leukotoxin production. Culture supernatants caused a 7-fold decrease in viability of bovine leukemia cells and increased cell permeability that was accompanied by release of beta-actin into the cell culture supernatant. Exposing P. haemolytica to purified actin solutions induced the conversion of monomeric G-actin to polymerized F-actin. This conversion was partially inhibited by bovine P. haemolytica immune, but not pre-immune, serum. Loss of streptomycin resistance following treatment of the organism with acridine orange ablated the polymerizing activity. Incubation of P. haemolytica in the presence of purified F-actin did not affect growth but resulted in culture supernatant that had 3.0-3.9-fold greater leukotoxicity compared to medium alone or medium containing G-actin, heat-denatured actin or albumin. The effect of actin on leukotoxicity was concentration-dependent and directly associated with increases in secreted leukotoxin. The interaction between P. haemolytica and actin is potentially detrimental to the host by inducing polymerization of actin into insoluble filaments and by enhancing leukotoxicity.     

BHV-1 infection and inflammatory cytokines amplify the interaction of Mannheimia haemolytica leukotoxin with bovine peripheral blood mononuclear cells in vitro

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.     

Effect of experimental infection of cattle with bovine herpesvirus-1 (BHV-1) on the ex vivo interaction of bovine leukocytes with Mannheimia (Pasteurella) haemolytica leukotoxin

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.     

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.     

Pneumonic pasteurellosis: examination of typable and untypable Pasteurella haemolytica strains for leukotoxin production, plasmid content, and antimicrobial susceptibility

Plasmid DNA screening experiments were conducted to determine whether a relationship existed between the presence of plasmids and antibiotic resistance in Pasteurella haemolytica or the capability to produce hemolysin or leukotoxin (cytotoxin). Regardless of plasmid content, all P haemolytica isolates produced characteristic hemolysis on blood agar plates. Similarly, standardized suspensions of living bacteria and sterile concentrated (approx 200:1) culture supernatant from strains representing each of the 15 recognized P haemolytica serotypes and 7 field strains of P haemolytica (biotype A, serotype 1) produced leukotoxin, which was detected by their capability to cause inhibition of the luminol-dependent chemiluminescence response of bovine neutrophils. However, neither living bacterial suspensions nor concentrated culture supernatant from 4 untypable P haemolytica strains or a P multocida strain caused an inhibition of the luminol-dependent chemiluminescence response. The production of neither hemolysin nor leukotoxin by P haemolytica seemed to be plasmid mediated. Leukotoxin production is apparently a stable phenotypic characteristic of pathogenic P haemolytica strains, and the gene(s) coding for this activity is probably located on the bacterial host chromosome. Antibiotic susceptibility profiles were determined for the different bacterial strains. Studies of ampicillin and penicillin resistance in 8 P haemolytica (biotype A, serotype 1) strains provided evidence that the plasmid, with size of approximately 5,200 base pairs, may code for their resistance to these compounds.     

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.     

The effect of Pasteurella haemolytica A1 leukotoxic culture supernate on the in vitro proliferative response of bovine lymphocytes

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.     

Comparison of the toxic and antigenic properties of single bovine isolates of Pasteurella haemolytica representing five serotypes and an untypable strain

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.     

Cytotoxic activity of Mannheimia haemolytica strains in relation to diversity of the leukotoxin structural gene lktA

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.     

Sequential development of antigens and toxins of Pasteurella haemolytica serotype 1 grown in cell culture medium.

Pasteurella haemolytica was grown in nonsupplemented cell culture medium, or in medium supplemented with bovine serum albumin (BSA) for 24 hours. The production of leukotoxin (LKT) and endotoxin was sequentially evaluated, as were bacterial antigens associated with bacterial cell lysates and culture supernates. Supplementation of medium with BSA had no effect on bacterial growth curves; however, LKT activity was detected earlier and was greater in culture supernates from BSA-supplemented media than from nonsupplemented medium. Leukotoxin antigen (105 kDa) was detected in culture supernates, using a monoclonal antibody, immunoblot analysis, and densitometry. The relative concentrations of LKT antigen were proportional to LKT activity. Endotoxin activity was initially lowest in the culture supernates from nonsupplemented medium, but increased during the incubation period, whereas endotoxin activity in BSA-supplemented culture supernates decreased with time in culture. In culture supernates from nonsupplemented medium, the number of antigenic bands identified by immunoblot analysis with hyperimmune anti-P haemolytica and densitometry was greater than in culture supernates from supplemented media. In bacterial lysates, a 95-kDa antigen was the major antigen detected, using the anti-LKT monoclonal antibody. The concentration of that antigen varied among lysates from nonsupplemented medium and BSA-supplemented media. Using hyperimmune anti-P haemolytica serum, minor differences were seen in the relative quantities of lysate-associated antigens dependent on time in culture and medium used. Among the major antigens seen, differences were most apparent for 150-, 100-, and 87-kDa antigens, whereas differences were not obvious for 42- 40-, and 30-kDa antigens.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Granulocyte plasma membrane damage by leukotoxic supernatant from Pasteurella haemolytica A1 and protection by immune serum.

Bovine respiratory disease caused by Pasteurella haemolytica may be partially mediated by a leukotoxin secreted by the microorganism. We examined the effect of leukotoxic Pasteurella supernatants on leakage of the cytosol enzyme lactate dehydrogenase and the lysosomal enzyme arylsulfatase from bovine granulocytes. Lactate dehydrogenase release (94%) was much higher than arylsulfatase release (38%) over 30 minutes of incubation. The Pasteurella supernatants inhibited superoxide production by stimulated granulocytes at concentrations which also caused substantial cell death as measured by failure to exclude trypan blue. Both toxic effects were prevented by serum from aerosol-immunized calves, and protection appeared to be antibody-specific by comparison with fetal bovine serum or with serum absorbed against intact P. haemolytica. These findings suggest that the leukotoxin may selectively disrupt the granulocyte plasma membrane, and that antibody directed against a surface component of the microorganism is also capable of protecting against the leukotoxin effect.     

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.     

Quantification of Mannheimia haemolytica leukotoxin by indirect ELISA

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of extracellular leukotoxin (LKT) produced in chemostat culture of Mannheimia haemolytica in a serum-free culture medium. Leukotoxin purified with preparative SDS-PAGE was used for the production of chicken polyclonal antibodies (PAb) that served as the primary detecting antibody. Excising the LKT protein from an analytical SDS-PAGE gel proved an efficient technique for the purification of the toxin. Consequently, the 102 kDa LKT polypeptide purified in this manner served as reference toxin and the resulting calibration curve was modelled using a four parameter logistic fit to relate absorbance to LKT protein concentration. The lower detection limit corresponded to an LKT concentration of 14.5 ng ml(-1). The presence of SDS, serum albumin and the coating pH had a distinct effect on the absorbance values of the indirect ELISA.     

Incubation of bovine PMNs with conditioned medium from BHV-1 infected peripheral blood mononuclear cells increases their susceptibility to Mannheimia haemolytica leukotoxin

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.     

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.