Two outer membrane proteins are bovine lactoferrin-binding proteins in Mannheimia haemolytica A1.

Mannheimia haemolytica is a Gram negative bacterium that is part of the bovine respiratory disease, which causes important economic losses in the livestock industry. In the present work, the interaction between M. haemolytica A1 and bovine lactoferrin (BLf) was studied. This iron-chelating glycoprotein is part of the mammalian innate-immune system and is present in milk and mucosal secretions; Lf is also contained in neutrophils secondary granules, which release this glycoprotein at infection sites. It was evidenced that M. haemolytica was not able to use iron-charged BLf (BholoLf) as a sole iron source; nevertheless, iron-lacked BLf (BapoLf) showed a bactericidal effect against M. haemolytica with MIC of 4.88 ± 1.88 and 7.31 ± 1.62 µM for M. haemolytica strain F (field isolate) and M. haemolytica strain R (reference strain), respectively. Through overlay assays and 2-D electrophoresis, two OMP of 32.9 and 34.2 kDa with estimated IP of 8.18 and 9.35, respectively, were observed to bind both BapoLf and BholoLf; these OMP were identified by Maldi-Tof as OmpA (heat-modifiable OMP) and a membrane protein (porin). These M. haemolytica BLf binding proteins could be interacting in vivo with both forms of BLf depending on the iron state of the bovine.

Localization of annexins A1 and A2 in the respiratory tract of healthy calves and those experimentally infected with Mannheimia haemolytica.

Annexins A1 and A2 are proteins known to function in the stress response, dampening inflammatory responses and mediating fibrinolysis. We found, in healthy cattle recently arrived to a feedlot, that lower levels of these proteins correlated with later development of pneumonia. Here we determine the localization of annexin A1 and A2 proteins in the respiratory tract and in leukocytes, in healthy calves and those with Mannheimia haemolytica pneumonia. In healthy calves, immunohistochemistry revealed cytoplasmic expression of annexin A1 in the surface epithelium of large airways, tracheobronchial glands and goblet cells, to a lesser degree in small airways, but not in alveolar epithelium. Immunocytochemistry labeled annexin A1 in the cytoplasm of neutrophils from blood and bronchoalveolar lavage fluid, while minimal surface expression was detected by flow cytometry in monocytes, macrophages and lymphocytes. Annexin A2 expression was detected in surface epithelium of small airways, some mucosal lymphocytes, and endothelium, with weak expression in large airways, tracheobronchial glands and alveolar septa. For both proteins, the level of expression was similar in tissues collected five days after intrabronchial challenge with M. haemolytica compared to that from sham-inoculated calves. Annexins A1 and A2 were both detected in leukocytes around foci of coagulative necrosis, and in necrotic cells in the center of these foci, as well as in areas outlined above. Thus, annexins A1 and A2 are proteins produced by airway epithelial cells that may prevent inflammation in the healthy lung and be relevant to development of pneumonia in stressed cattle.

Neutrophil gelatinase-associated lipocalin (NGAL) and insulin-like growth factor (IGF)-1 association with a Mannheimia haemolytica infection in sheep.

This study was aimed at mapping the tissue distribution of some inflammatory parameters associated with a Mannheimia haemolytica (M. haemolytica) infection in sheep. The M. haemolytica was isolated and characterized from the affected lungs of slaughtered animals. Cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-10, insulin-like growth factor (IGF)-1, as well as the acute-phase protein, neutrophil gelatinase-associated lipocalin (NGAL), were identified in the lung tissues, the serum, and the lymph nodes of M. haemolytica infected sheep, by enzyme-linked immunosorbent assay (ELISA). NGAL and IGF-1 pointed to an innate immune response, and epithelial cell repairing, respectively. The adaptive immune response was identified through the type of cytokines present in the affected sheep, as TNF-α represents the pro-inflammatory cytokines, and IL-10 represents the anti-inflammatory cytokines. M. haemolytica isolates were confirmed by polymerase chain reaction (PCR) and DNA sequences. There was a significant difference in the concentrations of NGAL, IGF-1, TNF-α, and IL-10, as observed in the affected sheep when compared to the healthy sheep. This study, for the first time, closely describes the distribution of some key and new inflammatory parameters in the tissue homogenate of affected lungs.

Comparative nitric oxide production by LPS-stimulated monocyte-derived macrophages from Ovis canadensis and Ovis aries.

Bighorn sheep are more susceptible to respiratory infection by Mannheimia haemolytica than are domestic sheep. In response to bacterial challenge, macrophages produce a number of molecules that play key roles in the inflammatory response, including highly reactive nitrogen intermediates such as nitric oxide (NO). Supernatants from monocyte-derived macrophages cultured with M. haemolytica LPS were assayed for nitric oxide activity via measurement of the NO metabolite, nitrite. In response to LPS stimulation, bighorn sheep macrophages secreted significantly higher levels of NO compared to levels for non-stimulated macrophages. In contrast, levels of NO produced by domestic sheep macrophages in response to M. haemolytica LPS did not differ from levels detected in non-stimulated cell cultures. Nitrite levels detected in supernatants of LPS-stimulated bighorn macrophage cultures treated with an inducible nitric oxide synthase (INOS) inhibitor, N(G)-monomethyl-L-arginine, were similar to that observed in non-stimulated cultures indicating a role for the iNOS pathway.

Comparison of pharmacokinetics of danofloxacin and enrofloxacin in calves challenged with Mannheimia haemolytica.

OBJECTIVE: To compare concentrations of danofloxacin, enrofloxacin, and ciprofloxacin in plasma and respiratory tissues of calves treated after challenge with Mannheimia haemolytica. ANIMALS: 75 calves. PROCEDURE: 24 hours after challenge with M. haemolytica, 72 calves with clinical signs of respiratory tract disease were randomly assigned to 1 of 12 equal treatment groups. Three nonchallenged, nontreated calves formed a control group. Challenged calves were treated with danofloxacin (6 and 8 mg/kg, SC) and enrofloxacin (8 mg/kg, SC) once. At 1, 2, 6, and 12 hours after treatment, 6 calves from each treatment group were euthanatized. Antimicrobial drug concentrations were assayed in various specimens. Peak plasma concentration (Cmax)-to-minimum inhibitory concentration (MIC; Cmax-to-MIC) ratios and the area under the concentration versus time curve over a 12-hour period-to-MIC ratios (AUC(12h)-to-MIC) were calculat-ed. RESULTS: Danofloxacin and enrofloxacin had MICs of 0.03 microg/mL for the M. haemolytica challenge isolate. Danofloxacin administered at doses of 6 and 8 mg/kg resulted in numerically higher geometric mean concentrations of danofloxacin in plasma and all respiratory tissues than geometric mean concentrations of enrofloxacin after treatment with enrofloxacin. Geometric mean concentrations of enrofloxacin were numerically higher than geometric mean concentrations of ciprofloxacin metabolite in plasma and almost all respiratory tissues. Danofloxacin and enrofloxacin achieved Cmax-to-MIC ratios >10 and AUC(12h)-to-MIC ratios >125 hours. CONCLUSIONS AND CLINICAL RELEVANCE: When used to treat pneumonic pasteurellosis in calves, danofloxacin and enrofloxacin can be expected to deliver concentration-dependent bactericidal activity against M. haemolytica, the bacteria most commonly associated with bovine respiratory tract disease.

Surfactant protein D expression in normal and pneumonic ovine lung.

Surfactant protein D (SP-D) is a collagenous calcium-dependent lectin constitutively expressed by alveolar type II pneumocytes and non-ciliated bronchiolar epithelial (Clara) cells. It binds to surface glycoconjugates expressed by a wide variety of microorganisms such as Gram-negative bacteria, influenza A virus, and various fungi, leading to pathogen inactivation or enhanced neutrophil and macrophage activity. Since a hallmark of bronchopneumonia is the initiation of inflammation in the bronchi and bronchoalveolar junction, we chose a classic ruminant model of bronchopneumonia caused by Mannheimia haemolytica to study the expression of SP-D within the bronchioles of infected lambs. Healthy weaned lambs were inoculated with either pyrogen-free saline (controls) or M. haemolytica intrabronchially using a fiber-optic bronchoscope. SP-D protein and mRNA expression in lung was detected by immunohistochemistry (IHC) and fluorogenic real-time relative quantitative reverse transcriptase polymerase chain reaction (real-time RT-PCR), respectively, during acute (1 day), subacute (15 days), and chronic (45 days) bronchopneumonia. At 15 and 45 days post-inoculation, areas of lung had peribronchiolar inflammatory cell infiltrate, epithelial cell hyperplasia, tortuosity of the airway lumens, and decreased intensity of SP-D protein staining and number of positive cells. The levels of SP-D mRNA were not increased or significantly altered by M. haemolytica infection when compared to control animals. In conclusion, cell-associated SP-D protein expression significantly decreases within hyperplastic epithelium of lungs from infected animals during chronic bronchopneumonia. Exhaustion of SP-D protein reserves and absence of SP-D gene upregulation during the progression of bacterial pneumonia into chronicity may result in failure to clear the pathogen from the lung and/or cause animals to be more susceptible to re-infection.

Mannheimia haemolytica leukotoxin-induced increase in leukotriene B4 production by bovine neutrophils is mediated by a sustained and excessive increase in intracellular calcium concentration.

Isolated bovine neutrophils were used to study the relationship between the duration and magnitude of the Mannheimia haemolytica leukotoxin-induced increase in intracellular calcium concentration and leukotriene B4 synthesis. In contrast to recombinant human C5a, which caused a transient, small increase in intracellular calcium concentration and no effects on leukotriene B4 synthesis, exposure of neutrophils to leukotoxin resulted in a rapid, sustained, large increase in intracellular calcium concentration, followed by leukotriene B4 synthesis. This leukotoxin-induced response was similar to those produced by the calcium ionophore, A23187, and phorbol myristate acetate, which also caused significant leukotriene B4 production. Manipulation of the duration and magnitude of leukotoxin- and A23187-induced intracellular calcium concentration increase confirmed that a high and sustained intracellular calcium concentration was necessary to stimulate production of leukotriene B4, which is believed to play an important role in the pathogenesis of pulmonary M. haemolytica infection.

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.

Role of tissue factor in intra-alveolar fibrin deposition and coagulopathy associated with pneumonic pasteurellosis in cattle.

OBJECTIVE: To determine the role of tissue factor (TF) in the coagulation events leading to intra-alveolar fibrin deposition and intravascular thrombosis associated with pneumonic pasteurellosis in cattle. ANIMALS: Healthy 2- to 4-week-old male Holstein calves. PROCEDURES: Blood and bronchoalveolar lavage samples were collected before and at 1, 2, 4, and 6 hours after inoculation of saline solution or Pasteurella haemolytica. Total leukocyte count, platelet count, plasma total protein concentration, prothrombin time, and partial thromboplastin time were measured in blood samples. Total nucleated cell count, total protein concentration, and procoagulant activity were measured in bronchoalveolar lavage samples. Additionally, platelet survival in blood platelet accumulation in affected lung tissue, and gross and microscopic lung lesions were determined. RESULTS: Administration of TF monoclonal antibodies (MAB) TF1-1F7 prevented the decrease in platelet survival and the increase in bronchoalveolar lavage fluid TF-dependent procoagulant activity observed in calves not treated with MAB TF1-1F7 antibody, but did not attenuate the increase in lavage fluid neutrophil numbers and total protein concentration, MAB TF1-1F7 administration reduced the percentage of lung affected by pneumonic lesions from 51.81% to 10.40% and attenuated intra-alveolar deposition of fibrin, neutrophils, and erythrocytes. CONCLUSION: Intra-alveolar fibrin deposition and activation of coagulation in cattle with pneumonic pasteurellosis is, at least in part, mediated by TF. CLINICAL RELEVANCE: Treatments that neutralize TF activity may attenuate lung injury in cattle with pneumonic pasteurellosis.

Oxytetracycline concentrations in plasma and lung of healthy and pneumonic calves, using two oxytetracycline preparations.

Drug concentrations in lung tissue and plasma pharmacokinetics of a long-acting preparation of oxytetracycline were compared with those of a standard preparation of oxytetracycline given to healthy and pneumonic calves. Both preparations achieved higher concentrations in the pneumonic lung when compared with those in the normal-appearing lung of the same group. A difference in concentrations in lung tissue was not found between preparations at either 24 or 48 hours. The plasma pharmacokinetic values did differ between preparations. Comparatively, the long-acting oxytetracycline had a smaller rate constant for disposition, a larger volume of distribution divided by bioavailability constant, and a longer half-life. A difference in pharmacokinetic values was not observed between healthy and pneumonic calves.