leukotoxin and Escherichia-coli-Infections

To evaluate the role of leukotoxin (LKT) of Mannheimia haemolytica and lipopolysaccharide (LPS) of E. coli 055:B5 in pathogenesis of bovine respiratory disease (BRD) we investigated their in vitro effects on cultured bovine neutrophils. Functional parameters of neutrophils including degranulation, generation of superoxide, and nitric oxide were distorted in response to both toxins. The most essential reaction of neutrophils was found in respect to release of elastase after addition of LKT as well as LPS at concentration of 300 microg/ml. Moreover, we observed an increased release of myeloperoxidase (MPO) and alkaline phosphatase (ALK-P) from polymorphonuclear cells (PMN) after addition of LKT and LPS. We also found enhanced superoxide generation by bovine neutrophils after exposure to different concentrations of LKT and LPS. In cultures of PMN treated with LKT, concentration of nitrite increased with growing concentrations of LKT. Lower values of nitrite were obtained in cultures exposed to LPS. Partial lysis of PMN, determined by LDH (lactate dehydrogenase) leakage, started at concentration of 300 microg/ml for both toxins, meanwhile LKT concentration above 300 microg/ml was lethal. Our study has revealed that neutrophils in response to both toxins exaggerate release of analysed substances, which participate in worsening the course of the disease and play a role in lung injury during BRD. Toxins introduced to the cultural medium stimulate release of studied constituents from neutrophils by combined activation and lysis of neutrophils.

Topics: Alkaline Phosphatase; Animals; Bacterial Toxins; Cattle; Cattle Diseases; Escherichia coli; Escherichia coli Infections; Exotoxins; Lipopolysaccharides; Mannheimia haemolytica; Neutrophils; Pasteurellosis, Pneumonic; Peroxidase; Respiratory Tract Infections

Hemoglobin, but not albumin, has long been recognized as an infection potentiating factor in experimental Escherichia coli peritonitis, but the mechanism has defied definition. We have shown previously that stroma-free hemoglobin is not toxic to polymorphonuclear neutrophils. To test the hypothesis that hemoglobin provides a nutritional boost to the growth of E. coli in vivo, we inoculated E. coli into dialysis bags containing equivalent amounts of stroma-free hemoglobin or albumin. These bags were implanted into the peritoneal cavity of rats and at intervals the fluid was removed and the bacteria enumerated. This technique allows for intraperitoneal bacterial growth but eliminates the variables of lymphatic clearance and phagocytic ingestion. The growth rate of E. coli was the same irrespective of the nutritional supplement in the bag. Thus there is no experimental support for the notion that hemoglobin directly accelerates E. coli proliferation under in vivo conditions. To test the hypothesis that a leukocyte toxin may result from E. coli growth in hemoglobin, we exposed normal human neutrophils to the sterilized contents of the peritoneal dialysis bags. In vitro function of the neutrophils (viability, random migration, chemotaxis, phagocytosis, bacterial killing, and chemiluminescence) was significantly depressed by prior exposure to hemoglobin supernatants that had supported E. coli proliferation in vivo. Stroma-free hemoglobin had minimal adverse effects. Albumin supernatants that had supported E. coli proliferation in vivo had significantly less effect on neutrophil function even though the endotoxin levels were identical to the hemoglobin E. coli solutions. We must conclude that leukotoxins result from E. coli growth in solutions of pure hemoglobin. The data support the idea that the infection potentiating effect of hemoglobin in vivo is due to such leukotoxins.

Topics: Animals; Bacterial Toxins; Blood Bactericidal Activity; Cell Division; Cell Movement; Cell Survival; Chemotaxis, Leukocyte; Dialysis; Escherichia coli; Escherichia coli Infections; Exotoxins; Hemoglobins; Humans; Luminescent Measurements; Male; Neutrophils; Peritonitis; Phagocytosis; Rats; Rats, Inbred Strains; Serum Albumin, Bovine