Response of the ruminant respiratory tract to Mannheimia (Pasteurella) haemolytica

Pneumonia is a leading cause of loss to the sheep and cattle industry throughout the world. Mannheimia (Pasteurella) haemolytica is one of the most important respiratory pathogens of domestic ruminants and causes serious outbreaks of acute pneumonia in neonatal, weaned and growing lambs, calves, and goats.M. haemolytica is also an important cause of pneumonia in adult animals. Transportation, viral infections with agents such as infectious bovine rhinotracheitis virus, parainfluenza-3 virus or bovine respiratory syncytial virus, overcrowding, housing of neonates and weaned animals together and other stressful conditions predispose animals to M. haemolytica infection. This review assimilates some of the findings key to cellular and molecular responses of the lung from a pathologist′s perspective. It includes some of what is known and underscores areas that are not fully understood

Small, anionic, and charge-neutralizing propeptide fragments of zymogens are antimicrobial

Some inactive precursor proteins, or zymogens, contain small, amino terminus, homopolymeric regions of Asp that neutralize the cationic charge of the active protein during synthesis. After posttranslational cleavage, the anionic propeptide fragment may exhibit antimicrobial activity. To demonstrate this, ovine trypsinogen activation peptide, and frog (Xenopus laevis) PYL activation peptide, both containing homopolymeric regions of Asp, were synthesized and tested against previously described surfactant-associated anionic peptide. Peptides inhibited the growth of both gram-negative (MIC, 0.08 to 3.00 mM) and gram-positive (MIC, 0.94 to 2.67 mM) bacteria. Small, anionic, and charge-neutralizing propeptide fragments of zymogens form a new class of host-derived antimicrobial peptides important in innate defense

Antimicrobial Peptides and Surfactant Proteins: Potential New Factors Against Respiratory Tract Infection

Although some vaccines and antibiotics have been very effective in preventing and treating respiratory disease, they have not been fully satisfactory. Recently, components of the innate immune system have been increasingly appreciated for their role in host defense against microbial pathogens. These molecules include lysozyme and lactoferrin, but recent work in cattle, sheep, man and other species have identified new classes of peptides expressed by respiratory epithelial cells that have potent microcidal activity in nanogram quantities. These peptides, termed antimicrobial peptides (AMP), include defensins, cathelicidins and anionic peptides. Some are expressed continuously whereas expression of others is stimulated by infection/inflammation. In calves, we have found that two AMP from the defensin family, tracheal antimicrobial peptide (TAP) and lingual antimicrobial peptide (LAP), are expressed in the newborn and increased in response to Mannheimia (Pasteurella) haemolytica infection. In contrast, sheep beta defensin-1 (SBD-1) is not induced by infection and, in fact, appears decreased during viral infection with paramyxovirus-3 (Pl-3). Decreased SBD-1 by Pl-3 may increase the lung\u27s susceptibility to secondary infections or re-infections. Other innate defense molecules include proteins released with lung surfactant. Surfactant protein A and D (SAD) can opsonize and aggregate respiratory syncytial virus (RSV) and activate alveolar macrophages. Preliminary work suggests that chronic bacterial infections result in reduced SpA and SpD expression and we are currently assessing SAD expression in response to PI-3 and RSV. A long-term goal of our work is to identify ways to up-regulate expression of AMP and/or surfactant proteins in the neonate and at times of stress in older animals in order to reduce microbial colonization. Other investigators are seeking ways to utilize AMP\u27s as a new class of antibiotics

Antimicrobial activity of cathelicidins BMAP28, SMAP28, SMAP29, and PMAP23 against Pasteurella multocida is more broad-spectrum than host species specific

The antimicrobial activity of linear, cationic α-helical peptides from cattle (BMAP28), sheep (SMAP28 and SMAP29), and pigs (PMAP23) were assessed to determine if activity was selective for Pasteurella multocida from a particular animal species or broad-spectrum against all P. multocida tested. The antimicrobial activities of synthetic peptides were determined for P. multocida isolated from cattle (10 isolates), sheep (10 isolates), and pigs (10 isolates) in a broth microdilution assay. All thirty isolates of P. multocida were susceptible to BMAP28 (MICs and MBCs, 1.0–1.9 μM); SMAP28 and SMAP29 (MICs and MBCs, 0.2–0.7 μM); and PMAP23 (MICs and MBCs, 4.3 to ≥6.8 μM). Overall, the results of this study suggest that synthesized cathelicidins from cattle, sheep, and pigs had broad-spectrum antimicrobial activity against all P. multocida

A selectin inhibitor decreases neutrophil infiltration during acute Mannheimia haemolytica pneumonia

The degree to which the selectin inhibitor TBC1269 reduces neutrophil infiltration in specific microscopic locations of the lung during acute pneumonia of neonates was determined. Neonatal calves were inoculated intrabronchially with Mannheimia (Pasteurella) haemolytica or saline, and lung tissue was collected at 2 and 6 hours postinoculation (PI). One 6-hour group inoculated with M. haemolytica received TBC1269 intravenously before and after inoculation with M. haemolytica. Infiltrates of neutrophils were significantly higher in the alveolar lumen and septae but lower in the bronchial lumen and epithelium at 6 hours PI than at 2 hours PI. Significantly fewer neutrophils (P \u3e 0.05) were present in the alveolar lumen and septae, and the bronchiolar lumen and lamina propria in the lungs of TBC1269-treated calves compared with untreated calves at 6 hours PI. TBC1269 did not alter the infiltration into bronchi and blood vessels or the expression of the selectin-independent adhesion molecule, ICAM-1. This work suggests that during acute pneumonia of neonates 1) neutrophil infiltrates progressively increase in the alveolar lumens and septae but decrease in the bronchial lumen and epithelium with time, 2) TBC1269 reduces neutrophil infiltration into specific regions of alveoli and bronchioles rather than uniformly throughout the lung, and 3) selectin inhibition does not affect the location and intensity of ICAM-1 expression

Antimicrobial peptides in animals and their role in host defences

Domesticated animals have a large variety of antimicrobial peptides that serve as natural innate barriers limiting microbial infection or, in some instances, act as an integral component in response to inflammation or microbial infection. These peptides differ in size, composition, mechanisms of activity and range of antimicrobial specificities. They are expressed in many tissues, polymorphonuclear leukocytes, macrophages and mucosal epithelial cells. There is a small group of anionic antimicrobial peptides found in ruminants and a much larger group of cationic antimicrobial peptides found in all domesticated animals. The cationic peptides include linear, helical peptides, linear peptides rich in proline and cysteine-stabilized peptides with a b-sheet and are commonly referred to as cathelicidins and defensins. These peptides are generally broad-spectrum for Gram-positive bacteria, Gram-negative bacteria and fungi (e.g. myeloid antimicrobial peptides, a-, b-defensins, and protegrins) or are specific to one of these groups (e.g. porcine cecropin P1, Bac5, Bac7, PR-39 and prophenin)

Induction of CD18-Mediated Passage of Neutrophils by Pasteurella haemolytica in Pulmonary Bronchi and Bronchioles

Pasteurella haemolytica is an important respiratory pathogen of cattle that incites extensive infiltrates of neutrophils into the lung. In addition to the parenchymal damage caused by factors released by P. haemolytica, neutrophils contribute to the pathologic changes in the lungs. Molecules which mediate neutrophil infiltration into the lungs duringP. haemolytica pneumonia are poorly characterized. To determine whether the CD18 family (β2-integrin) of leukocyte adhesion molecules mediates initial passage of neutrophils into the pulmonary bronchi and bronchioles of lungs infected withP. haemolytica, three Holstein calves homozygous for bovine leukocyte adhesion deficiency (BLAD) (CD18-deficient neutrophils), and three age- and breed-matched control calves (normal CD18 expression) were inoculated with P. haemolytica A1 via a fiberoptic bronchoscope and euthanized at 2 h postinoculation. Sections of lung were stained for neutrophils, and the intensity of neutrophilic infiltration was determined by computerized image analysis. Significantly fewer (P \u3c 0.05) neutrophils infiltrated the lumen, epithelium, and adventitia of bronchioles and bronchi in lungs of calves with BLAD compared to normal calves, which had dense infiltrates within these sites at 2 h postinoculation. The reduced infiltration in calves with BLAD occurred despite the presence of an extremely large number of neutrophils in peripheral blood that is typical for these calves. The large number of neutrophils in the blood of calves with BLAD is probably a physiologic response that can occur without microbial colonization, since one calf with BLAD that was raised under germ-free conditions had large numbers of neutrophils in the blood that were similar to those in a calf with BLAD that was raised conventionally. Neutrophil counts in the germ-free and conventionally reared calves with BLAD were much higher than those in the three normal calves raised under germ-free conditions. The work in this study demonstrates that during the initial inflammatory response, neutrophils with normal CD18 expression pass more readily than CD18-deficient neutrophils into the walls and lumen of bronchi and bronchioles. It suggests that CD18 is needed for initial passage through the extensive extracellular matrix of the bronchi and bronchioles. This has potential importance for the development of therapies to direct or inhibit neutrophil infiltration into conducting airways rather than alveolar spaces

Differences in the Concentrations of Small, Anionic, Antimicrobial Peptides in Bronchoalveolar Lavage Fluid and in Respiratory Epithelia of Patients with and without Cystic Fibrosis

Affinity-purified rabbit polyclonal (PAB96-1) and mouse monoclonal (1G9-1C2) antibodies to synthetic H-DDDDDDD-OH, an antimicrobial anionic peptide (AP) originally isolated from ovine pulmonary surfactant, were prepared and used to assess the concentrations of AP-like molecules in human respiratory tract samples. In bronchoalveolar lavage fluids, concentrations of AP-like molecules measured by enzyme-linked immunosorbent assay were significantly lower in 13 patients with cystic fibrosis (CF) (mean ± standard deviation [SD], 0.78 ± 0.46 mM) than in 34 patients without CF (1.30 ± 0.66 mM) (P = 0.01). In pulmonary tissues of three patients without CF, very little antigen was stained in the apical cytoplasm of the bronchial and bronchiolar epithelium yet robust staining was seen in the alveolar epithelium. In pulmonary tissues of three patients with CF, robust staining of antigen was seen in the apical cytoplasm of the bronchial and bronchiolar epithelium yet no staining was seen in the alveolar epithelium. These results show that AP-like molecules are present in healthy human respiratory tract samples and differ in concentration and location of expression in patients with and without CF

Cellular Distribution of Anionic Antimicrobial Peptide in Normal Lung and during Acute Pulmonary Inflammation

Anionic peptides (APs) are small antimicrobial peptides present in human and ovine lung. In this study APs were also detected in bovine lung, and production of APs in lungs with acute inflammation induced by various stimuli was determined. The distribution and intensity of APs were determined by immunohistochemistry in lungs of 1) neonatal calves (1-3 days of age) inoculated with Mannheimia (Pasteurella) haemolytica, a known inducer of the bovine β-defensin lingual antimicrobial peptide (LAP) or pyrogen-free saline (PFS), and 2) growing calves (3 months of age) similarly inoculated with M. haemolytica, a lipopolysaccharide (LPS) from M. haemolytica, an LPS-associated protein from M. haemolytica, or PFS. APs were also detected by western blots with the same antibody in lungs of the calves above, as well as in calves inoculated with Pseudomonas aeruginosa, and an adult cow. Anionic peptide (AP) immunoreactivity was detected in bands (approximate weights) in the western blots of lung at 28-30 (strongest signal), 31, 45, and 52-60 kd regardless of inoculum. The adult cow lacked bands at 45 kd, but it had additional bands at 64 (inconsistently) and 35-38 kd. All these band sizes are consistent with those of the western blots of human and ovine lung. The cellular distribution of APs in lung of neonatal and growing cattle was similar to that in lung of human and sheep. In lungs with acute inflammation induced by live bacteria, LPS, or protein, AP distribution and intensity were similar to those in control (PFS-inoculated) lungs and slightly decreased in bronchioles. This work demonstrates that AP is present in lung of cattle and is thereby conserved among two ruminant species and man. Distribution and intensity of AP production are not enhanced by infection or acute inflammation and are decreased in bronchioles, which suggests that AP is not induced like β-defensins such as LAP, but, instead, is produced constitutively

In Situ Expression of Intercellular Adhesion Molecule-1 (ICAM-1) mRNA in Calves with Acute Pasteurella haemolytica Pneumonia

The in situ expression of intercellular adhesion molecule-1 (ICAM-1) mRNA in normal and pneumonic lung tissues of Holstein calves with bovine leukocyte adhesion deficiency (BLAD) was compared with that of age-matched non-BLAD Holstein calves by in situ hybridization. Twenty-four Holstein calves (both BLAD and non-BLAD) were randomly assigned to one of two experimental groups and inoculated intrabronchially with Pasteurella haemolytica or pyrogen-free saline. Lung tissues were collected and fixed in 10% neutral formalin at 2 or 4 hours postinoculation (PI). The expression and distribution of ICAM-1 mRNA in the different cell types of the lung tissue was detected by in situ hybridization with a 307-base-pair bovine ICAM-1 riboprobe. In lungs of both non-BLAD and BLAD saline-inoculated calves, ICAM-1 expression was present in epithelial cells but occurred in 60% of cells) in bronchiolar and alveolar epithelial cells and pulmonary endothelial cells of arteries and veins in both BLAD and non-BLAD calves inoculated with P. haemolytica. Bronchiolar epithelium had the highest intensity of mRNA expression and highest percentage of cells that were stained, whereas bronchial epithelium had the lowest intensity and percentage of cells stained. Most alveolar macrophages and neutrophils in infected lungs also expressed ICAM-1. ICAM-1 expression was generally increased in infected BLAD calves at 2 hours PI as compared with non-BLAD calves but not at 4 hours PI. The increased expression of ICAM-1 during acuteP. haemolytica pneumonia in calves suggests that ICAM-1 is upregulated and may play a role in leukocyte infiltration. The extent of ICAM-1 expression in P. haemolytica-inoculated calves with BLAD was initially enhanced but otherwise similar to that in non-BLAD calves