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.     

Evaluation of Mannheimia haemolytica leukotoxin prepared in nonsupplemented and BSA or FBS supplemented RPMI 1640 medium

Mannheimia haemolytica leukotoxin (Lkt) is the major factor that contributes to lung injury in bovine pneumonic pasteurellosis. Supernatant preparations containing Lkt produced by M. haemolytica serotype 1, grown in RPMI 1640 medium supplemented with BSA or FBS and without supplements were evaluated during this study. Analysis of obtained Lkt showed presence of 105 kDa antigen (SDS-PAGE electrophoresis). The obtained bacterial protein fraction estimated as Lkt was detected by Western blotting with mouse monoclonal (Mab 605 and Mab 601) anti-Lkt antibodies. No significant differences were found in obtained leukotoxin between wildtype and reference M. haemolytica strains. Our studies showed that growth in media supplemented with BSA or FBS had no significant influence on leukotoxin production. When BSA or FBS supplements were used, additional protein fractions in electrophoregrams SDS-PAGE were observed. These protein bands did not react with Mab 605 and/or Mab 601 in Western blotting analysis. Lkt immunogenicity was detected by immunoblotting with sera from Lkt immunized rabbits and calves.     

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.     

Novel protease produced by a Pasteurella trehalosi serotype 10 isolate from a pneumonic bighorn sheep: characteristics and potential relevance to protection

A strain of Pasteurella trehalosi serotype 10, E(CO)-100, isolated from a bighorn sheep that had succumbed to pneumonic pasteurellosis during an epizootic, was compared to well-characterized strains of P. trehalosi serotype 10 and Mannheimia haemolytica serotype 1. The gene for leukotoxin A (lktA) from E(CO)-100 was sequenced and found to be identical on an amino acid basis to a published sequence for lktA from P. trehalosi serotype 10. However, the toxic activity in culture supernatant measured over time for E(CO)-100 was quite different from reference strains. Typically, the ability of the supernatant to lyse target cells increases over time corresponding to the logarithmic growth of the organism, peaks at mid to late phase, then declines gradually. Supernatant from E(CO)-100 exhibited a sharp decline in toxicity after mid-logarithmic growth to undetectable levels. Investigation of this anomaly using a commercial kit with a porcine gelatin/bovine albumin substrate matrix revealed high protease activity in the supernatant of this strain compared to another P. trehalosi serotype 10 and to a M. haemolytica serotype 1. Protease activity was also visualized using gelatin based zymogram gels. This protease was not substrate specific as it was shown to degrade leukotoxin. Activity was neutralized by bighorn sera in a titratable manner. There was an association between the ability to neutralize protease and low pneumonic lung scores in bighorn sheep experimentally challenged with E(CO)-100 (r=0.5, P=0.1). This previously unidentified protease may be an important protective antigen in vaccines designed to prevent pneumonic pasteurellosis resulting from P. trehalosi in bighorn sheep.     

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.     

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.     

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.     

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)     

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.     

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)     

Maternally and naturally acquired antibodies to Mannheimia haemolytica and Pasteurella multocida in beef calves

The dynamics and duration of maternally derived antibodies as well as the onset of acquired immunity against Mannheimia haemolytica and Pasteurella multocida in range-pastured beef calves were investigated. Two groups of unvaccinated cattle were used in this study. Serum antibody responses were measured by enzyme-linked immunoassay for antibodies of the IgG1, IgG2 and IgM isotypes binding M. haemolytica whole cells (WC) or leukotoxin (LKT) and P. multocida outer membrane proteins (OMPs). Comparisons of mean antibody responses to M. haemolytica LKT and WC and P. multocida OMPs were made within each group. Maternally derived antibodies against M. haemolytica and P. multocida reached lowest levels at 30-90 days after birth. Calves began production of antibodies against M. haemolytica and P. multocida between 60 and 90 days of age in both groups. Based on the results of this study, in beef herds vaccinated against M. haemolytica and/or P. multocida, it may be best to vaccinate calves around 3 months of age. In contrast, beef calves from unvaccinated herds might benefit from vaccination at 4 months of age.     

Characterization of Pasteurella spp isolated from healthy domestic pack goats and evaluation of the effects of a commercial Pasteurella vaccine

OBJECTIVE: To characterize Pasteurella spp isolated from healthy pack goats and evaluate the effects of administration of a commercial Pasteurella vaccine. ANIMALS: 45 goats. PROCEDURE: Pharyngeal swab specimens and blood samples were collected on day 0 before vaccination with a Pasteurella (Mannheimia) haemolytica serotype A1 bacterin. Samples were also collected from 17 goats on days 21 and 35. Isolated Pasteurella spp were assigned to biovariant groups on the basis of results of biochemical utilization tests and serotyped. Serum antibody titers were determined. RESULTS: Multiple strains of Pasteurella spp were isolated from swab specimens and assigned to 30 nonhemolytic and 14 beta-hemolytic biovariant groups. The most common biovariant isolated was nonhemolytic P trehalosi belonging to group 2. This strain was isolated from 41 goats. Nonhemolytic P haemolytica strains were isolated from 31 goats, whereas beta-hemolytic strains of P trehalosi and P haemolytica were isolated from 8 and 35 goats, respectively. Vaccination with the A1 serotype did not affect the proportion of goats from which we isolated each biovariant group or the number of biovariant groups isolated. CONCLUSIONS AND CLINICAL RELEVANCE: Multiple strains of P haemolytica and P trehalosi belonging to nonhemolytic and beta-hemolytic biovariant groups were isolated from the pharynx of healthy domestic pack goats. Because hemolytic activity correlates with leukotoxin production, beta-hemolytic strains may have a greater potential to cause disease in naive populations of wild ruminants. However, vaccination with an A1 serotype bacterin did not decrease the proportion of culture-positive goats.     

Role of Mannheimia haemolytica leukotoxin in the pathogenesis of bovine pneumonic pasteurellosis

Bovine pneumonic pasteurellosis continues to be a major respiratory disease in feedlot cattle despite the recent advances in our understanding of the underlying complexities of causation. The etiological agent, Mannheimia haemolytica, possesses several virulence factors, including capsule, outer membrane proteins, adhesins, neuraminidase, endotoxin and exotoxic leukotoxin. Accumulating scientific evidence implicates leukotoxin as the primary factor contributing to clinical presentation and lung injury associated with this disease. Unlike other virulence factors, leukotoxin shows cell-type- and species-specific effects on bovine leukocytes. Recent investigations have delineated the mechanisms underlying the target-cell-specificity of leukotoxin and how this contributes to the pathogenesis of lung damage. This review summarizes current understanding of the secretion, regulation, mechanisms of action and evolutionary diversity of leukotoxin of M. haemolytica. Understanding the precise molecular mechanisms of leukotoxin is critical for the development of more effective prophylactic and therapeutic strategies to control this complex disease.     

Survey of restriction-modification systems and transformation in Mannheimia haemolytica and Pasteurella trehalosi

A significant obstacle to molecular studies of Mannheimia (Pasteurella) haemolytica, has been its resistance to genetic transformation. The lack of competence of many M. haemolytica strains has been attributed to the presence of restriction modification systems. In this study, representative strains of 12 M. haemolytica serotypes and four Pasteurella trehalosi serotypes were successfully transformed by electroporation using a recombinant vector derived from the native M. haemolytica A1 serotype plasmid pNSF2176. Transformation was achieved despite PCR-based evidence for the presence of genes encoding a type I restriction enzyme, phaI, and a type II restriction enzyme hsdM, in each of the M. haemolytica strains.     

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.     

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.     

Complexities of the pathogenesis of Mannheimia haemolytica and Haemophilus somnus infections: challenges and potential opportunities for prevention?

Progress in producing improved vaccines against bacterial diseases of cattle is limited by an incomplete understanding of the pathogenesis of these agents. Our group has been involved in investigations of two members of the family Pasteurellaceae, Mannheimia haemolytica and Haemophilus somnus, which illustrate some of the complexities that must be confronted. Susceptibility to M. haemolytica is greatly increased during active viral respiratory infection, resulting in rapid onset of a severe and even lethal pleuropneumonia. Despite years of investigation, understanding of the mechanisms underlying this viral-bacterial synergism is incomplete. We have investigated the hypothesis that active viral infection increases the susceptibility of bovine leukocytes to the M. haemolytica leukotoxin by increasing the expression of or activating the beta2 integrin CD11a/CD18 (LFA-1) on the leukocyte surface. In vitro exposure to proinflammatory cytokines (i.e. interleukin-1beta, tumor necrosis factor-alpha and interferon-gamma) increases LFA-1 expression on bovine leukocytes, which in turn correlates with increased binding and responsiveness to the leukotoxin. Alveolar macrophages and peripheral blood leukocytes from cattle with active bovine herpesvirus-1 (BVH-1) infection are more susceptible to the lethal effects of the leukotoxin ex vivo than leukocytes from uninfected cattle. Likewise, in vitro incubation of bovine leukocytes with bovine herpesvirus 1 (BHV-1) potentiates LFA-1 expression and makes the cells more responsive to leukotoxin. A striking characteristic of H. somnus infection is its propensity to cause vasculitis. We have shown that H. somnus and its lipo-oligosaccharide (LOS) trigger caspase activation and apoptosis in bovine endothelial cells in vitro. This effect is associated with the production of reactive oxygen and nitrogen intermediates, and is amplified in the presence of platelets. The adverse effects of H. somnus LOS are mediated in part by activation of endothelial cell purinergic receptors such as P2X7. Further dissection of the pathways that lead to endothelial cell damage in response to H. somnus might help in the development of new preventive or therapeutic regimens. A more thorough understanding of M. haemolytica and H. somnus virulence factors and their interactions with the host might identify new targets for prevention of bovine respiratory disease.     

RPMI 1640培养基体外培养结肠小袋纤毛虫的研究

试验旨在建立结肠小袋纤毛虫RPMI 1640培养方法,为进一步研究其生活史及致病机制奠定基础。研究不同淀粉含量、不同血清含量、不同初始pH及不同接种量下RPMI 1640培养结肠小袋纤毛虫的培养效果。结果发现,用RPMI 1640培养结肠小袋纤毛虫,最适宜的淀粉含量为30 mg/安瓿,血清含量为20%,pH为7.0;种群自然增长率和种群所达到的最高种群密度随接种量的不同而明显改变。RPMI 1640培养基可用于结肠小袋纤毛虫的体外培养。     

Effect of bovine granulocyte colony-stimulating factor on the development of pneumonia caused by Mannheimia haemolytica

The role of recruited neutrophils in Mannheimia haemolytica infection is controversial. We hypothesized that the neutrophilia induced by recombinant bovine granulocyte colony-stimulating factor (GCSF) would lead to rapid bacterial clearance and less severe lesions after infection with M. haemolytica. Two experiments (A and B) were conducted in which four calves per experiment were treated daily with 5 microg/kg GCSF and four calves per experiment were treated with saline. All 16 calves were challenged with 5 x 10(9) colony-forming units (cfu)/ml (experiment A) or 4.5 x 10(8) cfu/ml (experiment B) of M. haemolytica bacteria, into the right bronchus by bronchoscope-placed catheter. The mean maximal blood neutrophil counts in non-GCSF-treated and GCSF-treated calves before bacterial challenge were 5.6 +/- 0.7 x 10(9)/liter and 25.4 +/- 2.7 x 10(9)/liter, respectively. Two untreated calves became neutropenic and were euthanatized 2 days after infection because of severe respiratory distress. GCSF-treated calves had a 37% reduction in lung lesions compared with nontreated calves, and this difference was significant (P=0.04) when the effect of previous antibody titre to leukotoxin was considered. The effect of GCSF treatment on the severity of clinical signs seemed to be influenced by the antibody titre to M. haemolytica leukotoxin, although this effect could not be conclusively addressed. In conclusion, GCSF induced neutrophilia and partially protected calves against experimental infection with M. haemolytica. These results imply that increased numbers of neutrophils may, under some circumstances, protect against severe pneumonia caused by M. haemolytica.     

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.