Role of platelet-activating factor in alveolar septal injury associated with experimentally induced pneumonic pasteurellosis in calves

OBJECTIVE: To determine whether platelet-activating factor (PAF) is involved in acute lung microvascular injury associated with pneumonic pasteurellosis in calves. ANIMALS: 15 healthy 2- to 4-week-old male Holstein calves. PROCEDURE: Calves were anesthetized and inoculated intrabronchially with saline (0.9% NaCl) solution (n = 5) or 1x10(9) Pasteurella haemolytica organisms (n = 10). Of the 10 calves inoculated with P haemolytica, 5 also were treated with WEB 2086, a potent inhibitor of PAF, and 5 were treated with vehicle. Blood and bronchoalveolar lavage samples were collected before and 1, 2, 4, and 6 hours after inoculation of P. haemolytica. Blood samples were analyzed to evaluate total number and differential counts of leukocytes, dilute whole-blood leukocyte deformability, size of neutrophils, and neutrophil CD11b expression. Bronchoalveolar lavage samples were analyzed for total number and differential counts of nucleated cells, total protein concentration, and hemoglobin concentration. Size and gross and histologic appearance of lung lesions also was determined. RESULTS: Treatment of calves with WEB 2086 reduced size of lung lesions, attenuated the increase in microvascular permeability, and reduced neutrophil infiltration in the first 4 hours after inoculation. Treatment with WEB 2086 also attenuated a decrease in leukocyte deformability, increase in size of neutrophils, and CD11b expression by circulating neutrophils. CONCLUSIONS AND CLINICAL RELEVANCE: It appears that PAF is a major mediator for altered lung microvascular permeability and activation of circulating neutrophils in the first 4 hours after onset of pneumonic pasteurellosis in calves.     

Evaluation of structural and functional alterations of circulating neutrophils in calves with experimentally induced pneumonic pasteurellosis

OBJECTIVES: To determine the structural and functional alterations in circulating neutrophils that may lead to sequestration in lung microvasculature and endothelial injury in calves with experimentally induced pneumonic pasteurellosis. ANIMALS: 10 healthy, 2- to 4-week-old male Holstein calves. PROCEDURES: Holstein calves were anesthetized and inoculated intrabronchially with Dulbecco phosphate buffered saline (0.9% NaCl) solution (DPBSS; 5 control calves) or 1 x 10(9) Pasteurella haemolytica organisms (5 infected calves). Blood samples were collected before and 1, 2, 4, and 6 hours after inoculation. Total and differential WBC count, dilute whole blood leukocyte deformability, neutrophil size distribution, and neutrophil surface CD11b expression were measured in blood samples. RESULTS: A progressive decrease in leukocyte deformability and increase in neutrophil size was detected 1, 2, 4, and 6 hours after inoculation of P haemolytica. Neutrophil surface CD11b expression was greater than baseline values at 6 hours after inoculation of P haemolytica. Two populations of neutrophils with an increase in size were detected in P haemolytica-infected calves. Both subpopulations had increased CD11b expression, compared with neutrophils that were typical in size. CONCLUSIONS AND CLINICAL RELEVANCE: Neutrophils circulate in an activated and nondeformable state in calves with experimentally induced pneumonic pasteurellosis. A decrease in neutrophil deformability and neutrophil aggregation may contribute to neutrophil trapping in the lung microvasculature during pneumonic pasteurellosis in calves.     

A guinea pig model of bovine pneumonic pasteurellosis.

The induction of pneumonic pasteurellosis in guinea pigs (Cavia porcellus) was examined. Specific pathogen free male guinea pigs were anesthetized and a tracheostomy performed to introduce 10(5), 10(4) or 10(3) Pasteurella haemolytica-A1 into the left principal bronchus. The surgical site was closed with tissue adhesive and staples and the animals were monitored for signs of respiratory tract infection. Within 24 hours after inoculation they became depressed, anorectic, pyretic and dyspneic. Fibrinous pleuropneumonia with prominent areas of necrosis and hemorrhage was present. Pericardial effusion was a frequent finding. There was infiltration of the pleura and alveoli with degenerate heterophils and macrophages, a hyperplastic mesothelium and fibrin exudation on the pleura and within alveoli. Hemorrhage, congestion, consolidation, edema and fibrin exudation were prominent in the hilar region of the lungs. Bacterial colonies were evident in all airways. More bacteria were recovered from infected lungs than were inoculated (p less than 0.05) indicating P. haemolytica was actively multiplying in the lungs. Hematological and clinical chemistry data were consistent with fibrinous pneumonia, however, blood cultures were positive for P. haemolytica in 61% (11/18) of animals sampled. Examination of pneumonic pasteurellosis in guinea pigs may be useful in studying pathogenetic and pathological features applicable to bovine pneumonic pasteurellosis (shipping fever pneumonia).     

Changes in blood and bronchoalveolar lavage fluid components in calves with experimentally induced pneumonic pasteurellosis

Pneumonic pasteurellosis was experimentally induced in calves by inoculation of 5 x 10(8) Pasteurella haemolytica organisms into the right diaphragmatic lung lobe. Blood and bronchoalveolar lavage fluid samples were obtained prior to inoculation and at postinoculation hour (PIH) 2, 4, and 6. Calves developed acute lung injury, characteristic of pneumonic pasteurellosis. Lesions were found only in the right diaphragmatic lobe. By PIH 4, significant (P less than 0.01) increases were detected in lavage fluid total cell count, neutrophil count, total protein and albumin concentrations, and alkaline phosphatase (ALP) and lactic dehydrogenase (LD) activities. Myeloperoxidase and elastase activities did not increase. Neutrophil depletion ameliorated the lung lesions and prevented the increase in lavage fluid cell count, total protein, and albumin concentrations and ALP and LD activities. Treatment with the iron chelator, deferoxamine mesylatehydroxyethyl starch, attenuated the increase in total protein and albumin concentrations and ALP and LD activities at PID 4, but not PIH 6. Treatment with a neutrophil function inhibitor, pentoxifylline, prevented the increase in lavage fluid neutrophil numbers, but accentuated the increase in total protein and albumin concentrations, and ALP, LD, myeloperoxidase, and elastase activities.     

Effect of Pasteurella haemolytica-derived endotoxin on pulmonary structure and function in calves

The role of endotoxin in the pathogenesis of acute pneumonic pasteurellosis is uncertain. Recently, we reported that Escherichia coli-derived endotoxin given by airway inoculation fails to induce lung injury in calves. Because Pasteurella haemolytica-derived endotoxin may differ substantially from E coli in its pathogenicity, we repeated these studies with Pasteurella endotoxin. Intratracheal inoculation of P haemolytica endotoxin caused hypoxemia and increased the alveolar-arterial oxygen differences without causing hypercarbia or changes in lung mechanical properties and volumes. In contrast, IV inoculation of endotoxin caused systemic hypotension, leukopenia, gas exchange impairment, increased total pulmonary resistance, and decreased dynamic compliance. Both routes of inoculation increased serum endotoxin concentrations and were associated with areas of pulmonary hemorrhage, edema, and acute inflammation. We concluded that P haemolytica-derived endotoxin is pathogenic by IV and airway routes of inoculation, and therefore differs from E coli endotoxin in its ability to induce lung lesions in calves.     

Clinical and pathological evaluation of sulbactam/ampicillin for treatment of experimental bovine pneumonic pasteurellosis

An experiment was conducted to evaluate the efficacy of sulbactam/ampicillin for treatment of bovine pneumonic pasteurellosis. Twenty-one Hereford calves were experimentally infected with bovine herpesvirus-1 and an ampicillin-resistant strain of Pasteurella haemolytica, then treated for three days with either sulbactam/ampicillin, chloramphenicol, or a placebo. The treatments were evaluated by comparing clinical illness scores, total sick days, weight changes, mortality rates, and postmortem lung scores between treatment groups. Both antibiotics were highly effective in reducing respiratory disease in the experimentally infected calves. The clinical response to sulbactam/ampicillin treatment was comparable with that of chloramphenicol and was significantly improved compared with the response to the placebo treatment. These findings suggest that the efficacy of sulbactam/ampicillin may be comparable to that of chloramphenicol for treatment of pneumonic pasteurellosis involving ampicillin-resistant strains of P. haemolytica.     

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 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.     

The pulmonary clearance of Pasteurella haemolytica in calves infected with bovine virus diarrhea or Mycoplasma bovis.

Based on current literature which commonly associates bovine virus diarrhea virus and Mycoplasma bovis with "pneumonic pasteurellosis," an investigation was conducted into the effect of these two pathogens on the capacity of bovine lung to clear inhaled Pasteurella haemolytica. There was no significant effect (p less than 0.05) of either bovine virus diarrhea virus or M. bovis on the mean clearance rate of P. haemolytica, nor did the time interval of three, five or seven days between the first inoculation and exposure to P. haemolytica and adversely affect the lung clearance rates. However, it was found that the left lungs and a higher bacterial retention (p less than 0.05) than the right lungs.     

Experimental model of swine pneumonic pasteurellosis using crude Actinobacillus pleuropneumoniae cytotoxin and Pasteurella multocida given endobronchially.

This study was designed to develop and characterize a swine pneumonic pasteurellosis model by concurrent introduction of Pasteurella multocida type A and Actinobacillus pleuropneumoniae crude cytotoxin. After a series of preliminary experiments, a combination of 4 x 10(9) P. multocida and 4,000 toxic units of A. pleuropneumoniae crude cytotoxin was determined to produce optimal results. A total of 48 pigs were divided into four groups of 12 pigs each. The control group received buffered saline only. Four pigs from each group were randomly selected for necropsy 3, 7 and 14 days postinoculation (PI). Inoculation of pigs with P. multocida and A. pleuropneumoniae cytotoxin (group 1) resulted in moderate to severe pneumonia. Pasteurella multocida was isolated from pneumonic lesions, grossly normal lung, and bronchial lymph nodes of all group 1 pigs throughout the 14 day experimental period. Pathological changes typical of field cases of swine pneumonic pasteurellosis were produced. Pigs inoculated with P. multocida alone (group 2) had pneumonic lesions and P. multocida was reisolated from lungs at three days PI. Pasteurella multocida was not isolated from these pigs at 7 and 14 days PI, except for one pig in which an abscess developed in the thorax. Pulmonary lesions induced by A. pleuropneumoniae crude cytotoxin alone (group 3) were transient and resolved by seven days PI. Group 1 pigs had significantly greater lung lesion volumes than group 2 and 3 pigs at 3, 7 and 14 days PI. Statistical analysis indicated a significant interactive effect of P. multocida and A. pleuropneumoniae cytotoxin on the development of lung lesion volumes at 7 and 14 days PI (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Bovine pneumonic pasteurellosis: experimental induction in vaccinated and nonvaccinated calves.

A study was undertaken to investigate the effects of the immune response induced by combined aerosol and parenteral vaccination on the lung lesions induced in calves by Pasteurella haemolytica AI. Twenty-four calves, twelve of which had been vaccinated with killed P. haemolytica by aerosol and subcutaneous injection in Freund's complete and incomplete adjuvant were challenged by intratracheal inoculation of live P. haemolytica. Serological response to vaccination was not marked but was best measured by the whole cell agglutination test or by indirect bacterial agglutination rather than by the passive haemagglutination test. Titres of vaccinates were positively correlated with the degree of pneumonic change following challenge while in nonvaccinated controls, titres were negatively correlated with lung lesions. These findings suggest the occurrence of an immunologically mediated hypersensitivity pneumonitis in the lungs of vaccinates and point to the potential efficacy of live bacterial aerosols for stimulation of protective immunity in pneumonic pasteurellosis.     

The importance of interleukin-8 as a neutrophil chemoattractant in the lungs of cattle with pneumonic pasteurellosis.

Interleukin-8 (IL-8), an in vitro and in vivo neutrophil chemoattractant, is expressed at high levels in the lesions observed in bovine pneumonic pasteurellosis. Because of the role of neutrophils in the pathogenesis of pneumonic pasteurellosis, we investigated the relative importance of IL-8 as a neutrophil chemoattractant in this disease. Bronchoalveolar lavage (BAL) fluid was harvested from calves experimentally infected with bovine herpesvirus-1 and challenged with Mannheimia haemolytica. Neutrophil chemotactic activity was measured in pneumonic BAL fluid samples treated with a neutralizing monoclonal antibody to ovine IL-8, and compared to the activity in samples treated with an isotype-matched control antibody. Bronchoalveolar lavage fluid was analyzed at a dilution which induced a half-maximal response, and the concentrations of antibody were optimized in a preliminary experiment. Following incubation of replicate samples of diluted pneumonic bovine BAL fluid with 70 microg/mL of IL-8-neutralizing antibody or control antibody, the neutrophil chemotactic activities of the samples were determined using an in vitro microchemotaxis assay. Overall, pretreatment of BAL fluid samples with neutralizing anti-IL-8 antibody reduced neutrophil chemotactic activity by 15% to 60%, compared to pretreatment with control antibody. This effect was highly significant (P < 0.001), and was present in 5 of 5 samples. These data indicate that IL-8 is an important neutrophil chemoattractant in calves with pneumonic pasteurellosis, but that mediators with actions redundant to those of IL-8 must also be present in the lesions.     

Characterization of a Pasteurella multocida (serotype B) bovine pneumonic pasteurellosis model and the effect of antimicrobials during peracute infection

A method to produce bovine pneumonic pasteurellosis for experimental purposes was studied and the clinical response of experimentally infected calves to selected antimicrobials was characterized. Male Holstein calves stressed with multiple hot and cold water applications followed by intratracheal inoculation of broth cultures of Pasteurella multocida serotype B developed acute clinical illness consistent with pneumonia. Infected, untreated calves consistently developed classic pneumonic pasteurellosis, infected calves treated with either oxytetracycline or sulfadimethoxine recovered from acute clinical disease, and the uninfected controls remained healthy. This disease model offers potential for use in pharmacokinetic and target tissue drug concentration studies and for dosage titration of drugs intended for treatment of bacterial pneumonias.     

Effects of experimentally induced Pasteurella haemolytica pneumonia on the pharmacokinetics of erythromycin in the calf

Pneumonic pasteurellosis was produced experimentally in 3- to 4-month-old Holstein bull calves by bilateral intrapulmonary administration of 5 X 10(7) to 10(9) colony-forming units of Pasteurella haemolytica. Of 8 calves, 4 developed minor pulmonary changes, 1 died of an apparent bacteremia within 24 hours, and 3 developed extensive pneumonic changes. At 1 week before (1 dose) and at 48, 60, and 72 hours (3 doses) after Pasteurella administration, the calves were given erythromycin at a dosage of 15 mg/kg, and the pharmacokinetic values were determined. There were statistically (P less than or equal to 0.05) significant increases in the distribution and elimination rates associated with pneumonia. The elimination half life decreased from 132.7 +/- 9.6 minutes in prepneumonic calves to 111.1 +/- 13.8 minutes and 99.7 +/- 2.6 minutes in calves with minor and with moderate pneumonic changes, respectively. There also was a decrease in apparent volume of distribution with pneumonia. Erythromycin tissue concentrations were determined 2 hours after the last dose was given to the calves with pneumonia. Tissue concentrations in the pneumonic lung areas were as high or higher than those in nonaffected lung tissues in the same animals. Because of the increased rate of elimination from serum in pneumonic calves, it may be advisable to use shorter dosage intervals in calves with severe respiratory tract disease.     

High pressure pulsatile lavage and high pressure syringe lavage in the treatment of contaminated wounds in dogs

The effectiveness of high pressure pulsatile lavage and syringe lavage in cleansing experimentally contaminated and infected wounds was studied. Such treatment significantly (P < 0.05) lowered the bacterial counts in contaminated wounds, but high pressure pulsatile lavage caused extensive damage to tissue and was considered to be unsuitable for treatment of wounds in the loose skin areas of the dog. Both pulsatile and syringe lavage caused significant (P < 0.05), but transient, reduction in the bacterial counts in infected wounds but both methods failed to lower the wound bacterial counts to 10⁵ or less. Successive wound biopsies for quantitative bacterial analysis showed that syringe lavage with povidone-iodine caused greater reduction in bacterial numbers than did pulsatile lavage with povidone-iodine. Combination of high pressure syringe lavage with povidone-iodine and systemic penicillin-streptomycin given at the time of lavage sufficiently reduced the bacterial numbers to allow for safe closure of the wounds.     

Efficacy of Sulbactam, a B-lactamase Inhibitor, Combined with Ampicillin in the Therapy of Ampicillin-resistant Pneumonic Pasteurellosis in Feedlot Calves

Two field efficacy studies, involving a total of 80 naturally infected feedlot calves, were conducted to compare the efficacy of sulbactam-ampicillin with that of penicillin-dihydrostreptomycin in the treatment of pneumonic pasteurellosis. Cultures from pretreatment nasal swabs were predominantly ampicillin/ penicillin resistant Pasteurella haemolytica. Clinical observations revealed that cattle treated with penicillin-dihydrostreptomycin responded poorly, whereas those treated with sulbactam-ampicillin responded promptly. Twenty-four hours after initiation of treatment, mean body temperatures of the calves in the sulbactam-ampicillin groups had decreased by 2.1°C, whereas, in the penicillin-dihydrostreptomycin calves there was little change. The difference between the two treatments was statistically significant (P≤0.01). The combined mortality for the two studies in the penicillin-dihydrostreptomycin treated groups was 18%. No mortality occurred in the sulbactam-ampicillin treated groups. Our data show that sulbactam-ampicillin was more effective than penicillin-dihydrostreptomycin in the treatment of pneumonia caused by ampicillin/penicillin resistant strains of Pasteurella in feedlot calves.     

Effect of deferoxamine pretreatment on acute pneumonic pasteurellosis and neutrophil oxidative metabolism in calves.

Iron plays a central role in bacterial infections, influencing both bacterial virulence and host cellular defense mechanisms. We investigated whether iron chelation might be of benefit in the treatment of pneumonic pasteurellosis of calves. Neutrophils obtained from calves previously treated with the iron chelator, deferoxamine, were studied for their responses to latex and opsonized zymosan by luminol-enhanced chemiluminescence and to phorbol myristate acetate and opsonized zymosan by superoxide generation. Treatment with deferoxamine in vivo failed to influence these in vitro measures of neutrophil oxidative metabolism. Furthermore, iron depletion with deferoxamine failed to modify the pathophysiological derangements that occurred in calves following experimental induction of pneumonia by intratracheal inoculation with Pasteurella haemolytica. These data indicate that iron chelation using deferoxamine cannot be recommended as an adjunct to conventional therapy in the treatment of pneumonic pasteurellosis of cattle.     

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.     

An outbreak of Pasteurella multocida pneumonia in lambs during a field trial of a vaccine against Pasteurella haemolytica

A vaccine against pneumonic pasteurellosis was evaluated for efficacy at two dilutions in lambs transported by sea from New Zealand to Saudi Arabia. The experimental vaccine was a killed Pasteurella haemolytica serotype A1 and A2 preparation. There was no evidence of either dilution of the vaccine leading to a lower pneumonia death or lesion rate than for the control group. However, bacteriological examinations to establish the causality of the pneumonia cases showed Pasteurella multocida to be the dominant organism, while P. haemolytica types A1 and/or A2 occurred at only a very low incidence.     

Bovine pneumonic pasteurellosis: model for Pasteurella haemolytica- and Pasteurella multocida-induced pneumonia in cattle

Pneumonic lesions in calves were induced with Pasteurella haemolytica or P multocida. The inoculum, consisting of a suspension of either organism, was administered by transthoracic intrapulmonic injection to 23 calves. Three died of septicemia; the 20 remaining, killed 96 hours after inoculation, had an expanding unifocal pneumonia qualitatively comparable with that of acute pneumonic pasteurellosis (shipping fever). The concentration of bacteria that consistently produced a lesion was a 5-ml volume containing 10(9) colony-forming units of bacteria; a concentration of 10(6) colony-forming units inconsistently produced lesions. Bacteria, except in calves that developed septicemia and died, remained localized at the injection site. The inflammatory process spread within the lungs, not only through airways, but through the interlobular and interalveolar septa as well.