Purification and partial characterization of a macrophage cytotoxin from Pasteurella haemolytica

A protein from Pasteurella haemolytica that was highly immunogenic and toxic toward bovine alveolar macrophages was partially purified. When isolated from culture supernatants of P haemolytica serotype 1 or serotype 6, the protein reacted on Ouchterlony immunodiffusion tests with antisera from 12 serotypes of P haemolytica, but did not cross-react with antisera to serotypes of P multocida. This indicated that the protein may be specific for P haemolytica. Bacteria were grown in dialysis culture in a brain-heart infusion and calf-serum growth medium. The protein was isolated from the medium by ultrafiltration and size-exclusion chromatography and has a molecular weight of approximately 150,000 daltons. The protein, which is highly immunogenic and has the characteristics of a virulence factor, is common to all serotypes of P haemolytica, and may be an effective agent for immunization against P haemolytica in cattle.     

Pasteurella haemolytica cytotoxin: relative susceptibility of bovine leucocytes

An in vitro 51Cr-release assay was used to compare the susceptibility of various leucocytes from normal cattle to Pasteurella haemolytica cytotoxin. Neutrophils were found to be more sensitive than mammary or bronchoalveolar macrophages. Neutrophils induced with lipopolysaccharide (LPS) and mammary macrophages activated in vitro with LPS were as sensitive as homologous untreated cells. Bronchoalveolar macrophages from adult cows were significantly more resistant than those from calves. Sub-cytolytic concentrations of cytotoxin did not impair killing of para-influenza-3 virus infected MDBK cells by mammary macrophages.     

Pulmonary lesions induced by a Pasteurella haemolytica cytotoxin preparation in calves

Intrabronchial instillation of a Pasteurella haemolytica type A1 crude cytotoxin preparation in calves resulted in pulmonary gross and microscopic lesions comparable to spontaneous and experimental pasteurellosis. In the acute stage of the lesion electronmicroscopy revealed intravascular accumulation, degeneration and fragmentation of leukocytes in the interalveolar septa. Secondary thrombus formation and increased vascular permeability resulted in alveolar flooding, fibrin deposition, extravasation of erythrocytes and loss of alveolar epithelium. No cytotoxicity was observed for the tracheal (in vitro) and bronchial epithelium (in vivo). The pathogenesis of the vascular lesions and their significance for the development of the typical lesions of pneumonic pasteurellosis is discussed.     

Pasteurella haemolytica cytotoxin neutralizing activity in sera from Ontario beef cattle.

A random sample of sera obtained from cattle necropsied as part of the Bruce County Beef Project in 1980-81 was assayed for the ability to neutralize the cytotoxin of Pasteurella haemolytica A1. Cattle dying of fibrinous pneumonia had significantly lower neutralizing activity in serum than cattle which died for reasons other than pneumonia. Activity in pneumonic cattle was also lower than the mean of twelve samples randomly chosen from sera of cattle bled on entry to feedlots in the fall of 1979. A role for the toxin neutralizing response in resistance to pneumonic pasteurellosis is proposed.     

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.     

Pasteurella haemolytica cytotoxin: production by recognized serotypes and neutralization by type-specific rabbit antisera

A sterile culture supernatant from each of the 12 recognized serotypes of Pasteurella haemolytica was toxic to bovine alveolar macrophages when assayed by 51Cr release. Types appeared to differ in their ability to liberate cytotoxin, although this may have reflected strain variation rather than serotype-related differences. Toxicity was partially neutralized by type-specific rabbit antisera, with neutralization of the homologous toxin being more effective than that of the heterologous serotype.     

Anticytotoxin activity of bovine sera and body fluids against Pasteurella haemolytica A1 cytotoxin.

Toxin neutralizing activity of bovine sera and body fluids against Pasteurella haemolytica type A1 cytotoxin was evaluated by 51Cr release assay using bovine peripheral blood mononuclear leukocytes as the target cells. Sera collected from precolostral calves did not exert anticytotoxin activity at 10(-1) or higher dilutions, whereas randomly selected complement fixing antibody-negative sera neutralized on average over 90% of cytotoxin activity at the 10(-1) dilution and less than 50% of the toxin activity at 10(-2) or higher serum dilutions. Nasal secretions and lung washings of some of the cattle tested also contained cytotoxin neutralizing activity. The antibody nature of the cytotoxin neutralizing activity was demonstrated by its neutralization with bovine immunoglobulin G2 purified from pooled seropositive sera. Sera from a group of cattle which were vaccinated with a potassium thiocyanate extract of P. haemolytica, but which subsequently developed fibrinous pneumonia after aerosol challenge with bovine herpesvirus 1 and P. haemolytica, had significantly lower anticytotoxin activity than sera from another group of cattle which did not develop the disease after similar vaccination and challenge. Cattle which survived a natural outbreak of shipping fever had higher anticytotoxin activity than those having fibrinous pneumonia in the aforementioned experimental group, although there was no statistical difference between them and a randomly selected CF seronegative group. It is probable that this cytotoxin neutralizing antibody exerts a beneficial effect in protection of cattle against pneumonic pasteurellosis.     

A crude cytotoxin vaccine protects sheep against experimental Pasteurella haemolytica serotype A2 infection

Three vaccines containing Pasteurella haemolytica serotype A2 antigens were tested for their ability to protect sheep against a homologous challenge. A crude cytotoxin preparation in combination with a sodium salicylate extract (SSE) or crude cytotoxin alone were highly protective (98 and 86%, respectively), whereas SSE alone was poorly (47%) protective. These findings indicated that the crude cytotoxin was an essential component of a protective vaccine. Protection correlated with serum cytotoxin-neutralising (CN) titres and bactericidal activity, which were stimulated by antigens in the crude cytotoxin preparation.     

Saline-extracted antigens of Pasteurella haemolytica: separation by chromatofocusing, preliminary characterization, and evaluation of immunogenicity

Saline extracts of logarithmic-phase Pasteurella haemolytica, serotype 1, were separated by chromatofocusing. The resulting fractions were analyzed by immunodiffusion and an enzyme-linked immunosorbant assay, and six antigen groups (AG's) were identified. AG 1 did not bind to the column, AG's 2, 3 and 4 were eluted with a decreasing pH gradient, and AG's 5 and 6 were eluted with an increasing NaC1 gradient. Fractions containing each AG were pooled and further purified by gel filtration. The AG's were subsequently characterized as to protein, carbohydrate and 2-keto-3-deoxyoctanate (KDO) content. AG's 1, 5, and 6 had higher carbohydrate contents than AG's 2, 3 and 4. Only AG 5 contained detectable levels of KDO. The AG's were also analyzed by SDS-PAGE and immunoblotting. Each AG produced a characteristic pattern of proteins and antigens, although two antigenic proteins were common to all AG's. AG 1 contained the greatest number of antigenic proteins. Immunization of mice with each AG in Freund's incomplete adjuvant resulted in a strong antibody response to the homologous AG for four of the six AG's. Limited protection against a P. haemolytica challenge was observed in mice that were immunized with AG 2 or 4.     

Purification and characterization of a 94-kDa Pasteurella haemolytica antigen

A 94-kDa antigen of Pasteurella haemolytica Serotype 1, which was previously shown to elicit serum and nasal secretion antibody response to the bacterium, was purified and characterized. The antigen was purified by high performance liquid chromatography utilizing ion exchange, then size exclusion columns. It was a membrane protein that was copurified with 6-7% lipopolysaccharide. It had an isoelectric point of 4.6. Most other serotypes of P. haemolytica possessed a similar antigen.     

Effect of intratracheal inoculation of Pasteurella haemolytica cytotoxin on the integrity of rat lung.

This study was conducted to investigate the in vivo effect of a single intratracheal inoculation of Pasteurella haemolytica cytotoxin on the rat lung. Changes in the biochemical and cytological composition of bronchoalveolar lavage fluid were used to estimate the magnitude of pulmonary cell injury, inflammatory response, vascular permeability and functional status of pulmonary alveolar macrophages. Effect of treatment was compared with rats intratracheally inoculated with supernatants of Pasteurella multocida or with sterile physiological saline solution (vehicle). Results indicated that Pasteurella haemolytica supernatants were not significantly toxic for the lungs of rats.     

Effects of Pasteurella haemolytica cytotoxin on ovine peripheral blood leucocytes and lymphocytes obtained from gastric lymph

Ovine peripheral blood leucocytes were separated on discontinuous Percoll density gradients into fractions rich in lymphocytes (PBLy) and polymorphonuclear leucocytes. Supernatant fluid from a dialysis sac culture of Pasteurella haemolytica biotype A serotype 1 (A1) was cytotoxic to these leucocytes in vitro. PBLy retained viability after storage in liquid nitrogen and could be employed in cytotoxicity assays. However, sheep cannulated via the common gastric lymph duct were an excellent source of large numbers of homogeneous lymphoid cells (GLy) which also stored well in liquid nitrogen. As both freshly collected and stored GLy were killed by culture supernatant fluid GLy offer advantages as target cells for further characterisation of the extracellular cytotoxin produced by P. haemolytica. From the results obtained, it is considered that all ovine leucocytes are susceptible to P. haemolytica cytotoxin.     

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.     

Serum neutralization of cytotoxin from Pasteurella haemolytica, serotype 1 and resistance to experimental bovine pneumonic pasteurellosis

Sera from several groups of experimental calves were tested for cytotoxin neutralizing capacity. The relationship between this capability and resistance of the animals to an experimental challenge was examined. All undiluted bovine sera tested, other than fetal bovine serum, neutralized cytotoxin. Preadsorption of selected sera with formalin-killed P. haemolytica did not reduce their neutralizing capacity. Crude IgG fractions extracted from bovine sera retained neutralizing capacity as well. Cytotoxin neutralization titers were determined by serial dilution of sera from cattle which were previously unexposed, naturally exposed, or exposed by vaccination to the organism. Both live and killed vaccines were used. Prior exposure to live organisms resulted in the production of antibodies to both cell surface antigens and cytotoxin, whereas exposure to the killed vaccine resulted in the production of antibodies primarily to cell surface antigens. Resistance to experimental challenge with the organism correlated directly with serum cytotoxin neutralizing titers.     

Deoxyribonucleic acids from Pasteurella multocida and Pasteurella haemolytica]

Pasteurella multocida and P. haemolytica strains contain between 1.5 and three per cent phosphorus, between nine and 14 per cent nitrogen, between two and four per cent DNA, and between five and 18 per cent RNA, the precise figures depending on culturing conditions. High-molecular DNA may be isolated by means of bacteriolysis, using deoxycholate or dodecylsulphate and the usual steps of purification, with yield and purity differing by strains. DNA with sufficient purity can be obtained from Sepharose 2 B by gel chromatography. The isolated DNA yields were characterised, base values being between 37 and 38 per cent GC for P. haemolytica and between 41 and 48 per cent GC for P. multocida. Highly suitable precursors to DNA synthesis for tritium labelling are 3H-thymidine, which is incorporated in excess of 3H-thymine by a factor of 255, as well as 3H-uracil, with its activity being recovered also from the pyrimidine bases of DNA via pyrimidine biosynthesis.     

The effect of Pasteurella haemolytica and the leukotoxin of Pasteurella haemolytica on bovine lung explants.

Bovine lung explants were used in a study designed to compare the pathogenic effects of Pasteurella haemolytica type 1, a nonpathogenic organism Neisseria subflava, or the crude leukotoxin of P. haemolytica on alveolar macrophages and lung parenchymal cells. Concentrated, purified peripheral blood neutrophil suspensions were added with the bacteria to some explants. Duplicate pairs of cultures from each treatment group were fixed at regular intervals up to 24 hours after seeding and morphological changes were assessed by light and electron microscopy. Pasteurella haemolytica caused deterioration of alveolar macrophages within one hour but did not affect parenchymal cells for more than 12 hours. Neisseria subflava did not affect alveolar macrophages initially, but caused an accelerated deterioration after four hours. After 24 hours, bacterial overgrowth caused similar deterioration of all cells in explants seeded with either bacterium. Alveolar macrophages phagocytosed large numbers of N. subflava but rarely ingested P. haemolytica. Added neutrophils did not have any discernible effect on any of the explants and did not potentiate bacterial effects. Addition of crude leukotoxin of P. haemolytica to the culture medium significantly accelerated alveolar macrophage deterioration without apparent effect on parenchymal cell survival. These results support the hypothesis that the severe tissue destruction of fulminant pneumonic pasteurellosis is not a direct result of bacterial 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 infections in sheep

Summary Pasteurella haemolytica causes two distinct disease syndromes in sheep. P. haemolytica biotype A causes septicaemia in young lambs and pneumonia in all ages of sheep. Biotype T produces an acute systemic disease affecting principally the upper alimentary tract and lungs in young adult sheep. The bacteriology, epidemiology and clinical and necropsy findings of the two diseases are described and the current situation regarding their experimental reproduction and immunology is reviewed.     

Pasteurella haemolytica infections in sheep

Summary

Pasteurella haemolytica causes two distinct disease syndromes in sheep. P. haemolytica biotype A causes septicaemia in young lambs and pneumonia in all ages of sheep. Biotype T produces an acute systemic disease affecting principally the upper alimentary tract and lungs in young adult sheep. The bacteriology, epidemiology and clinical and necropsy findings of the two diseases are described and the current situation regarding their experimental reproduction and immunology is reviewed.