Pathogenesis Of Bovine Pneumonic Pasteurellosis Research Articles

Prior exposure to Mannheimia haemolytica leukotoxin or LPS enhances β 2-integrin expression by bovine neutrophils and augments LKT cytotoxicity

Mannheimia ( Pasteurella) haemolytica serotype1 produces a variety of virulence factors that play an important role during the pathogenesis of bovine pneumonic pasteurellosis. Among these, a leukotoxin (LKT) and lipopolysaccharide (LPS) are thought to be the primary virulence factors that contribute to the characteristic pathology of pasteurellosis. Recent evidence suggests that M. haemolytica LKT binding to bovine leukocytes is mediated by the β 2-integrin CD11a/CD18 (LFA-1), which subsequently induces activation and death of these cells. Exposure of bovine peripheral blood neutrophils (PMNs) to LKT or LPS induces expression of inflammatory cytokines, which in turn can increase LFA-1 expression and conformational activation. In this study we demonstrated, by flow cytometry and Western blot, that bovine PMNs increased their LFA-1 expression following in vitro exposure to M. haemolytica LKT and LPS. Increased LFA-1 expression by PMNs exposed to LKT and LPS was associated with increased LKT binding and cell death. The results of this study suggest that M. haemolytica LKT and LPS might cooperatively increase LFA-1 expression, and by so doing amplify the lung inflammation that characterizes bovine pasteurellosis.

Inflammatory cytokines enhance the interaction of Mannheimia haemolytica leukotoxin with bovine peripheral blood neutrophils in vitro.

Mannheimia (Pasteurella) haemolytica A1 produces several virulence factors that play an important role in the pathogenesis of bovine pneumonic pasteurellosis. Foremost among these is a leukotoxin (LKT) that specifically kills ruminant leukocytes. Recent evidence suggests that M. haemolytica LKT binding to bovine leukocytes is mediated by the beta(2)-integrin CD11a/CD18 (lymphocyte function-associated antigen 1 [LFA-1]), which subsequently induces activation and cytolysis of these cells. Inflammatory cytokines, which are released during viral and bacterial infection, are reported to increase LFA-1 expression and conformational activation. We investigated the effects of the inflammatory cytokines interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma) on the interaction of M. haemolytica LKT with bovine peripheral blood neutrophils (PMNs). In this study we demonstrated, by flow cytometry, that bovine PMNs increased their binding to an anti-bovine LFA-1 monoclonal antibody (BAT75A) following in vitro incubation with IL-1beta, TNF-alpha, or IFN-gamma. Incubation with cytokines also increased CD18 expression, as assessed by real-time PCR and by Western blotting. Increased LFA-1 expression by PMNs exposed to cytokines was associated with increased LKT binding and cytotoxicity. The latter represented, at least in part, enhanced PMN apoptosis, as assessed by propidium iodine staining and caspase-3 activation. The results of this study suggest that inflammatory cytokines may play an important role in enhancing the biological response of bovine PMNs to M. haemolytica LKT.

Pulmonary expression of tumor necrosis factor alpha, interleukin-1 beta, and interleukin-8 in the acute phase of bovine pneumonic pasteurellosis.

Inflammatory cytokines are suspected to contribute to the pathogenesis of bovine pneumonic pasteurellosis (BPP) through neutrophil recruitment, leukocyte activation, and the induction of a broad array of soluble inflammatory mediators. An in vivo experimental model of BPP was used to characterize the pulmonary expression kinetics of tumor necrosis factor alpha (TNFalpha), interleukin-1 beta (IL-1beta), and interleukin-8 (IL-8) genes and proteins during the acute phase of disease development. Cytokine expression in bronchoalveolar lavage (BAL) fluid, BAL cells, and pneumonic lung parenchyma was quantitated by northern blot analysis, enzyme-linked immunosorbent assay (ELISA), and in situ hybridization at 2, 4, 8, 16, and 24 hours after endobronchial inoculation of Pasteurella (Mannheimia) haemolytica. Expression of TNFalpha, IL-1beta, and IL-8 was significantly increased in the airways and lung lesions of infected calves as compared with mock-infected controls. Although kinetic patterns varied, peak levels of cytokine mRNA occured within 8 hours postinfection (PI), and peak cytokine concentrations occurred within 16 hours PI. In all samples, IL-8 was expressed to the greatest extent and TNFalpha was least expressed. Expression of TNFalpha was restricted to alveolar macrophages. Alveolar and interstitial macrophages produced IL-1beta and IL-8 in the first 4 hours; bronchial and bronchiolar epithelial cells were also significant sources of IL-8 during this period. By 8 hours PI, neutrophils were the dominant source of both IL-1beta and IL-8. These findings demonstrate a spatial and temporal association between pulmonary expression of inflammatory cytokines and acute lung pathology, supporting the hypothesis that cytokines contribute to inflammatory lung injury in BPP.

Evidence ofPasteurella haemolyticalinked immune complex disease in natural and experimental models

The pathogenesis of bovine pneumonic pasteurellosis is not completely understood, and studies have not established thatPasteurella haemolyticaA1 (Ph1) virulence is exclusively responsible for the development of acute pulmonary lesions. The purpose of this investigation was to determine if immune complex disease is involved in the pathogenesis of bovine pneumonic pasteurellosis. A retrospective immunohistologic study of lung tissue from natural cases of bovine pneumonic pasteurellosis (44) as performed, and immune complexes were observed in alveloar spaces and walls in 88% of these cases. To study this pathologic mechanism experimentally, groups of mice were immunized with purified Ph1 outer membranes (OMs) or sham immunized on days 0 and 14. Mice were challenged intratracheally on day 24 with either live Ph1 or Ph1 OMs, and pulmonary lesions were assessed 24 h after challenge. Placebo immunized mice developed focal infiltrates of neutrophils and macrophages centered around large caliber bronchi. Mice immunized with Ph1 OMs and challenged with live Ph1 or OMs developed severe bronchointerstitial pneumonia with diffuse neutrophilic infiltration, focal necrosis, hemorrhage and edema, that is histologically similar to bovine pneumonic pasteurellosis. Immunohistology revealed flocculent aggregates of IgG and complement positive material within alveolar spaces and walls from mice challenged with live Ph1, and fine granular deposits of IgG and complement positive material were observed lining the alveolar walls from mice challenged with Ph1 OMs. Immunized mice exhibited high serum IgG antibody titers to Ph1 outer membrane proteins (OMPs). Results of this study suggest that immune complex disease plays a role in the pathogenesis of bovine pneumonic pasteurellosis.

Lectin Histochemistry of Normal and Herpesvirus-infected Bovine Nasal Mucosa

Proliferation of Pasteurella haemolytica serotype 1 in the nasal cavity following stress or viral infection is an important event in the pathogenesis of bovine pneumonic pasteurellosis. Enhanced adhesion of P. haemolytica to nasal mucosa could be one factor that predisposes animals to this proliferation. Nasal mucosa from normal and bovine herpesvirus-1 (BHV1)-infected cattle were examined histochemically for their glycoconjugate composition. Twenty lectins were screened, six of which were chosen for subsequent study. Three of these were specific for N-acetylgalactosamine (NAGal) (Dolichos biflorus, Glycine max, and Vicia villosa), and one each was specific for N-acetylgalactosamine/galactose (Griffonia simplicifolia-I), mannose/glucose (Canavalia ensiformis), and N-acetylglucosamine (Triticum vulgaris). For the surface mucosa and submucosal glands, there was greater reactivity in samples from BHV1-infected than from normal cattle for all six lectins. Reactivity was most prominent for the NAGal-specific lectins. Neuraminidase treatment of samples from normal and BHV1-infected cattle tended to result in greater lectin reactivity. Lectin reactivity was generally more intense in focally inflamed areas, but diffuse reactivity was not substantially affected by inflammation. BHV1-induced alteration of nasal mucosal glycoconjugates could enhance adhesion and colonization of P. haemolytica to nasal surfaces and may be one factor responsible for the increased number of P. haemolytica serotype 1 in the nasal cavity following viral infection.

Construction of isogenic mutants of Pasteurella haemolytica by allelic replacement

We describe methods for the mutagenesis of cloned Pasteurella haemolytica (Gram −) genes and for the construction of P. haemolytica mutants by allelic exchange. We used these methods to construct isogenic mutants of P. haemolytica which no longer synthesize three membrane lipoproteins (Lpp). A single genetic locus, consisting of three tandemly arranged genes encoding 28–30-kDa membrane Lpp, was replaced with a mutated locus which carries the β-lactamaseencoding Ap R gene from a 4.2-kb P. haemolytica plasmid. The inactivated locus was introduced into P. haemolytica by electroporation of a plasmid which carries the mutated locus, but is incapable of replicating in P. haemolytica. Southern and Western blot analyses indicate that the wild-type locus was replaced by the mutated locus through a doublecrossover recombination event and that the membrane Lpp were no longer produced by the mutant strain. These methods should be useful in constructing mutant loci which can be used to analyze the roles for various P. haemolytica proteins in the pathogenesis of bovine pneumonic pasteurellosis.

Enhancement of neutrophil-mediated injury to bovine pulmonary endothelial cells by Pasteurella haemolytica leukotoxin.

In this study, we used an in vitro coculture system to determine which virulence factor from Pasteurella haemolytica A1 was responsible for augmenting bovine polymorphonuclear neutrophil (PMN)-mediated killing of bovine pulmonary artery endothelial cells (BPAEC). A 51Cr release cytotoxicity assay was used as a measure of BPAEC killing. The mechanisms associated with this BPAEC killing were also studied. Our results demonstrated that the leukotoxin and not the lipopolysaccharide from P. haemolytica was responsible for augmenting the PMN-mediated killing of BPAEC. Furthermore, this augmented killing was related to the stimulation of PMNs by the leukotoxin. Killing of BPAEC by leukotoxin-stimulated PMNs was diminished in the presence of the H2O2 inactivator, catalase. The membrane-permeant H2O2, hydroxyl radical (HO.) scavenger 1,3-dimethyl-2 thiourea, and the HO. scavenger dimethyl sulfoxide but not the myeloperoxidase inhibitor sodium azide attenuated this BPAEC killing. Pretreatment of BPAEC with a 21-aminosteroid (U74500A), a potent iron chelator-antioxidant, provided the most effective protection against BPAEC killing induced by leukotoxin-stimulated PMNs. These data were compatible with the concept that the H2O2 generated by leukotoxin-stimulated PMNs interacts with intracellular iron in the endothelial cell to form highly reactive HO.. We suggest that HO. may be a key factor in BPAEC killing. Furthermore, since the elastase-specific inhibitor N-methoxy-succinyl-Ala-Ala-Pro-Val-chloromethyl ketone (CMK) also attenuated BPAEC killing and both CMK and 1,3-dimethyl-2 thiourea functioned additively in protecting against BPAEC killing, we conclude that both HO. and elastase may jointly contribute to BPAEC killing induced by leukotoxin-stimulated PMNs. This study broadens our understanding of how leukotoxin-stimulated PMNs injure lung endothelial cells and provides new insight into the pathogenesis of bovine pneumonic pasteurellosis.

The role of induced virulence factors produced by Pasteurella haemolytica in the pathogenesis of bovine pneumonic pasteurellosis: Review and hypotheses

In the pathogenesis of pneumonic pasteurellosis, there is an abrupt commensal to pathogen shift from a predominance of P. haemolytica serotype 2 (ST2) to serotype 1 (ST1) in the bovine upper respiratory tract (URT) microfloral population. This occurs following periods of stress associated with development of this disease. Data are reviewed from recent publications supporting the contention that surface-expressed ST1-specific factor(s) could be critical in mediating URT adhesion and colonization. Such factors may promote an increase in the number of ST1 organisms deposited through infective droplets into the lungs, beyond that efficiently cleared by normal lung defences. The seeding of these organisms into the lungs may provide numerous foci of infection that eventually progress into characteristic pneumonic lesions seen in the disease.

Serum levels of tumor necrosis factor-α in calves experimentally infected with Pasteurella haemolytica Al

The purpose of this study was to document the levels of tumor necrosis factor-α (TNF) in serum of calves experimentally infected intratracheally with Pasteurella haemolytica Al and to determine if elevated TNF levels correlate with development of pneumonic pasteurellosis in the bovine. Serum samples were collected at sequential time periods from 0 h to 72 h post inoculation with P. haemolytica. TNF levels in those sera were measured by a cytotoxicity assay utilizing the TNF-sensitive WEHI 164 mouse fibrosarcoma cell line. Serum TNF levels in infected cattle began to rise at 2 h post inoculation, peaked at approximately 8 h, and decreased to near control levels by 72 h. There was extreme variability in serum TNF among the inoculated animals with levels varying from 120 pg ml −1 to 5000 pg ml −1 at 8 h post inoculation. These levels did not correspond with the degree of lung involvement. All inoculated calves developed lesions of pneumonic pasteurellosis characterized by fibrinous pleuritis with necrotizing, hemorrhagic pneumonia. These results suggest that TNF is probably a significant inflammatory mediator involved in the pathogenesis of bovine pneumonic pasteurellosis.

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 O 2 − and H 2O 2. 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.

Evidence that blood-borne infection is involved in the pathogenesis of bovine pneumonic pasteurellosis.

Five calves were inoculated intravenously with 10(8) colony forming units (cfu) of Pasteurella haemolytica A1; the mean score for pneumonic consolidation 3 days post-inoculation was 28%, and the mean clinical score was 7.8. Five calves inoculated intratracheally with 10(9) cfu of the same strain of P. haemolytica had comparable scores (34% and 8.8). Histological lesions of fibrinous pneumonia were similar in all calves. P. haemolytica was recovered from all but one of the affected lungs. From one calf killed in extremis 3 hours after intravenous inoculation, numbers of bacteria recovered from lung were 1,000-fold greater than from liver and spleen. A similar difference in bacterial numbers was also obtained from a gnotobiotic calf killed in extremis, 12 hours after intravenous inoculation of 10(8) cfu P. haemolytica. Evidence from these experiments supports the hypothesis that the blood-borne route is important in the pathogenesis of bovine pneumonic pasteurellosis.

Cytotoxin of Pasteurella haemolytica acting on bovine leukocytes.

The toxicity of Pasteurella haemolytica culture supernatant for bovine and porcine cells was evaluated by 51Cr release. Sterile bacterial culture supernatant was toxic for bovine pulmonary lavage cells, peripheral blood lymphocytes and neutrophils, and cultured peripheral blood mononuclear cells, resulting in marked 51Cr release. Only slight release was induced from cultured Madin-Darby bovine kidney cells, porcine pulmonary lavage cells, peripheral blood mononuclear cells, lymphocytes, or neutrophils. No release was detected with primary bovine spleen cell cultures, bovine erythrocytes, or porcine erythrocytes. The demonstrated specificity of this cytotoxin for bovine leukocytic cells may be important in the pathogenesis of bovine pneumonic pasteurellosis.