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: 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)     

Vaccination of calves with leukotoxic culture supernatant from Pasteurella haemolytica.

In three experiments subcutaneous vaccination of calves with adjuvanted bacteria-free leukotoxic culture supernatant from log phase cultures of Pasteurella haemolytica A1 (toxin 1) was shown to induce some protection against intrabronchial challenge with live P. haemolytica A1. This toxin 1 vaccine was as effective as a whole cell bacterin in stimulating agglutinating antibody to P. haemolytica. Induction of leukotoxin neutralizing activity was variable; in some cases vaccination only primed the animal to produce an anamnestic response after challenge, whereas in other instances antitoxic activity increased in response to immunization. Two doses of vaccine were shown to be more effective than a single immunization. Vaccination with leukotoxic culture supernatant from the nonpathogenic P. haemolytica serotype 11 was as effective as vaccination with toxin 1 in stimulating antitoxic activity but was not protective. This implies that both serospecific agglutinating activity and an antitoxic response are needed for immunity.     

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.     

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.     

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.     

Chemotactic response of bovine neutrophils to Pasteurella haemolytica culture fluid

Bovine neutrophil chemotactic activity was detected in the supernatant fluid of logarithmic phase cultures of P. haemolytica serotype 1. The chemoattractant was produced under culture conditions suitable for P. haemolytica leukotoxin production. An inverse correlation existed between the leukotoxin LC50 and the chemotactic activity in the culture fluid. Elimination of leukotoxin activity by heating, dilution or ultrafiltration, exposed the chemotactic activity in the culture fluid. The chemoattractant was partially resistant to heating (60 degrees C, 30 min), and had an apparent molecular weight greater than 100,000. Detection of chemotactic activity in both the concentrate and filtrate after XM300 filtration suggested that there might be more than one component with chemotactic activity or else that polymerization was occurring. Production of a potent neutrophil chemoattractant by P. haemolytica may explain the rapid infiltration of neutrophils that occurs during the early stages of bovine pneumonic pasteurellosis.     

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.     

Association among filamentous actin content, CD11b expression, and membrane deformability in stimulated and unstimulated bovine neutrophils

OBJECTIVE: To investigate rheologic properties of bovine neutrophils that may result in adhesion molecule-independent sequestration of neutrophils in inflamed lungs of cattle. ANIMALS: Healthy 2- to 4-week-old male Holstein calves. PROCEDURES: Neutrophil deformability, filamentous actin (F-actin) content, and CD11b expression was determined for unstimulated bovine neutrophils and bovine neutrophils incubated with the inflammatory mediators tumor necrosis factor-alpha (TNF), platelet-activating factor (PAF), interleukin-8 (IL-8), zymosan-activated plasma (ZAP), Pasteurella haemolytica-derived lipopolysaccharide (LPS), and P haemolytica leukotoxin. Neutrophils were separated into 3 subpopulations on the basis of size. The Factin content and CD11 b expression were evaluated by use of flow cytometry. Leukocyte deformability was evaluated by filtration of dilute whole blood. RESULTS: The subpopulation of the smallest-sized neutrophils (>90% of neutrophils) contained little F-actin. A subpopulation of slightly larger neutrophils had a profound increase in F-actin content and CD11 b expression. The subpopulation of the largest neutrophils had increased F-actin content and CD11b expression, compared with those for both subpopulations of smaller neutrophils. Incubation of neutrophils with PAF and ZAP but not TNF, IL-8, LPS, or leukotoxin, resulted in decreased neutrophil deformability and increased F-actin content. Incubation with PAF and TNF induced an increase in size of neutrophils. CONCLUSIONS AND CLINICAL RELEVANCE: Size can be used to identify subpopulations of large and rigid neutrophils in blood samples from healthy calves. Platelet-activating factor and activated complement fragments are potent inducers of F-actin formation and neutrophil rigidity. Physical changes in neutrophils may impede their transit through lung microvasculature and result in leukocyte trapping independent of adhesion molecule interactions with endothelial cells.     

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.     

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.     

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.     

Interaction of bovine neutrophils in Pasteurella haemolytica mediated damage to pulmonary endothelial cells

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.     

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.     

Evidence for the Pasteurella haemolytica cytotoxin as a product of actively growing bacteria

The release of soluble cytotoxin from Pasteurella haemolytica type 1 cultured at 37 C in RPMI 1640 medium containing 7% bovine fetal serum was evaluated, using bovine alveolar macrophages in a microplate 51Cr-release assay. Heat-labile leukotoxic activity was detected in culture supernatant during the lag and logarithmic-growth phases, but not in stationary-phase culture; toxic activity could not be demonstrated in sonicates of logarithmic (1 hour) or stationary (18 hour) phase organisms. This pattern of toxin release, typical of a bacterial exotoxin or metabolic enzyme, is unique among bacterial leukotoxins, which are typically cell-associated and released after autolysis or sonication from outgrown cultures.     

Effects of Pasteurella haemolytica A1 culture supernatant on mechanisms controlling bovine alveolar macrophage oxygen radical production.

Pasteurella haemolytica A1 culture supernatant containing leukotoxin, and modifiers of cyclic nucleotide and arachidonate metabolism, were evaluated for their ability to alter oxygen radical production by pulmonary alveolar macrophages obtained from seven Holstein calves. Calves were sedated, and underwent bronchoalveolar lavage to harvest macrophages, which were then incubated with culture supernatant and/or the drugs and toxins under study, and challenged with opsonized zymosan to induce oxygen radical generation. This was measured by a chemiluminescence technique. Pasteurella haemolytica A1 culture supernatant alone delayed the time to maximum oxygen radical production, although total production was increased. The cyclooxygenase inhibitor indomethacin and the phospholipase inhibitor p-bromophenacyl bromide significantly reduced maximum oxygen radical production, but their effects were diminished in the presence of culture supernatant. Although forskolin markedly inhibited oxygen radical generation, this effect was not altered by culture supernatant. Incubation of macrophages with pertussis toxin had no effect on oxygen radical production, while incubation with cholera toxin did inhibit production. This inhibitory effect was significantly lessened by concurrent incubation with P. haemolytica A1 culture supernatant.     

Pasteurella haemolytica leukotoxin: comparison of 51chromium-release, trypan blue dye exclusion, and luminol-dependent chemiluminescence-inhibition assays for sensitivity in detecting leukotoxin activity

Dilutions of concentrated, dialyzed Pasteurella haemolytica culture supernatant were caused to react with bovine neutrophil (PMN) suspensions, and then the trypan blue dye exclusion (TBDE), 51chromium (51Cr)-release, and luminol-dependent chemiluminescence-inhibition (LDCLI) assays were done to compare their relative sensitivities in detecting biological activity of P haemolytica leukotoxin (cytotoxin). The culture supernatant was concentrated approximately 200:1, and when caused to react as an undiluted preparation with bovine PMN, it was cytotoxic for 38.6% and 80.4% of PMN as determined by TBDE and 51Cr-release assays, respectively. This undiluted leukotoxin preparation caused 100% inhibition of the luminol-dependent chemiluminescence responses of bovine PMN. The LDCLI assay was the most sensitive of the 3 in vitro assays for P haemolytica leukotoxin activity--being approximately 17 times and 2,480 times more sensitive than the 51Cr-release and TBDE assays, respectively. The relative advantages and disadvantages of the 3 assays as in vitro systems for detecting and titrating leukotoxin activity and investigating the role of leukotoxin in disease pathogenesis and immunity are discussed. Because of its sensitivity, specificity, economy, technical ease, and potential for adaptation to automation, the LDCLI assay would seem to be the in vitro assay of choice for quantitating P haemolytica leukotoxin activity. To aid standardization of studies of leukotoxin between different laboratories, it is suggested that P haemolytica leukotoxin be quantitated and expressed as chemiluminescence inhibitory units.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Pasteurella haemolytica leukotoxin: chemiluminescent responses of peripheral blood leukocytes from several different mammalian species to leukotoxin- and opsonin-treated living and killed Pasteurella haemolytica and Staphylococcus aureus

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)     

The influence of Mannheimia haemolytica A1 seed culture inoculum cell density on the production of leukotoxin in submerged culture supernatant

Mannheimia haemolytica leukotoxin is produced during the logarithmic growth phase in submerged culture in RPMI 1640 medium with and without the addition of foetal calf serum or albumin. In order to establish a pattern of optimal leukotoxin production in small volumes in submerged cultures and to define some parameters involved, two high leukotoxin producing Mannheimia haemolytica strains were grown in RPMI 1640 medium containing either FCS or BSA. The cell growth and leukotoxin production abilities of each strain were determined concomitantly every hour in RPMI 1640 medium containing each of the additives over a time period of 6 h. The growth performance of three dilutions of a standardized seed culture inoculum prepared with each of the cultures and additives were simultaneously compared with each other using the above parameters. The different seed culture inoculum dilutions had a definite effect on the time and quantity of leukotoxin production. Both strains demonstrated peak leukotoxin production after 4 h of active growth. The addition of albumin to both isolates gave slightly increased leukotoxin levels, and both showed that the peak leukotoxin was not associated with peak cell concentration. Obvious quantitative differences in the ability of different M. haemolytica strains to produce leukotoxin were noted. Strain 12296 produced optimal leukotoxin concentration from the medium (1/25) dilution of the seed culture inoculum after 4 h, whereas strain 1/10 produced the same concentration with the low (1/5) dilution seed culture inoculum, possibly reflecting the superior production ability of the first strain. However, each strain of M. haemolytica appeared to have its own specific logarithmic cell growth and leukotoxin production pattern. The peak cell density of M. haemolytica grown in submerged RPMI 1640 culture medium cannot be used as an indication of optimal leukotoxin levels.