Bovine pulmonary endothelial cell damage mediated by Pasteurella haemolytica pathogenic factors

Abstract

The in vitro effects of Pasteurella haemolytica components on bovine pulmonary endothelial monolayers were investigated to determine the relative role of individual bacterial factors in the pathogenesis of bovine pulmonary pasteurellosis. Bovine pulmonary endothelial monolayers were treated with P. haemolytica bacterial culture supernatant (CS) and P. haemolytica lipopolysaccharide. At 22 h postinoculation, the CS produced severe damage to the endothelial cells, indicated by high 5'Cr release, extensive cellular detachment, and morphologic changes characterized by cell contraction, cytoplasmic blebbing, and loss of monolayer confluency. The neutralization of leukotoxin activity of the CS by heat inactivation was ineffective in decreasing the damage to endothelial cells; however, leukotoxin-neutralizing monoclonal antibody slightly diminished the toxic effect. P. haemolytica lipopolysaccharide by itself or as a supplement to CS produced endothelial cell damage similar to that of CS. The preincubation of CS dilutions (10-1 and 10-2) or P. haemolytica lipopolysaccharide with polymyxin B almost completely eliminated cell toxicity. These studies show that P. haemolytica produces a soluble factor that is consistent with bacterial lipopolysaccharide and that is directly toxic to bovine pulmonary endothelial cells in vitro.

Protective role of bovine neutrophils in Pasteurella haemolytica-mediated endothelial cell damage

“…A field isolate of P. haemolytica (serotype 1, biotype A) was used in all experiments. The bacteria were grown from frozen stocks as previously described (6). Briefly, reconstituted bacteria were plated on enriched blood agar (EBA) for 18 h at 37°C, and then 7 to 10 bacterial colonies were transferred to brain heart infusion broth with 5% fetal bovine serum (FBS).…”

“…Several P. haemolytica virulence factors have been identified, including capsular polysaccharide (9, 11), leukotoxin (1,2,20,35), and lipopolysaccharide (LPS) (10,18,30,37). Although leukotoxin rapidly kills and lyses bovine neutrophils (PMN), it does not directly damage bovine endothelial cells (EC) in vitro (6). However, bovine EC in vitro are very sensitive to the toxic effects of P. haemolytica LPS, whether in purified form or as a component of a bacterial culture preparation (6,27).…”

“…Although leukotoxin rapidly kills and lyses bovine neutrophils (PMN), it does not directly damage bovine endothelial cells (EC) in vitro (6). However, bovine EC in vitro are very sensitive to the toxic effects of P. haemolytica LPS, whether in purified form or as a component of a bacterial culture preparation (6,27). LPS is probably important in the natural disease because it has been demonstrated near EC in pulmonary lesions caused by P. haemolytica (42) and intratracheal LPS inoculation in cattle results in significant pulmonary lesions (37).…”

Characterization of lipopolysaccharides from four Pasteurella haemolytica serotype strains: evidence for presence of sialic acid in serotypes 1 and 5

“…In addition, it has been reported that P. haemolytica-dcrived endotoxin is associated with pulmonary hemorrhage, edema and acute inflammation [8]. it has also been reported that the cndotoxin is directly toxic to bovine pulmonary endothelial cells in vitro [9]. In an effort to obtain a better understanding of the pathogenesis and immunology of P. haemolytica disease, we have undertaken to isolate and purify LPS from four strains representative of four different serotypes, to acquire an indication of the overall structural elements of these LPS and to analyze the sugar and fatty acid compositions of these LPS.…”

Cloning and characterization of the galE locus of Pasteurella haemolytica A1

“…LPS is involved in inflammation and pulmonary damage during the course of the disease (6,40,44). LPS is also involved in indirect (30) and direct (4,31) damage to bovine pulmonary endothelial cells. The LPS of P. haemolytica A1 possesses biological properties typical of gram-negative endotoxin (18,34), but unlike Escherichia coli endotoxin, the LPS from P. haemolytica A1 causes pathological changes in calves' lungs (39).…”