Doctoral Dissertations

Gary L. Mason

Date of Award

12-1999

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Comparative and Experimental Medicine

Major Professor

Philip N. Bochsler

Committee Members

David Slauson, David Bemix, Roger Carroll

Abstract

Bovine respiratory disease complex (BRD) is the most costly disease problem of cattle in North America. BRD is multifactorial with contributions from management practices, environmental factors, and infectious agents including Pasteurella haemolytica A1, bovine herpes virus type 1 (BHV), parainfluenza type3 (PIS), and bovine virus diarrhea virus (BVD). Alveolar macrophages are intimately involved in defense of the lower respiratory tract against infectious agents. Expression of inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO ) is a key defensive response of rodent macrophages.Microbicidal activity of rodent macrophage derived-NO- has been demonstrated against many taxonomically diverse infectious agents in vitro and in vivo. The Work presented here was designed to characterize expression of iNOS and production of NO- by bovine alveolar macrophages and evaluate the microbicidal activity of NO- against important bovine pulmonary pathogens.Bovine alveolar macrophages recovered from the lungs of slaughter cattle were examined in cell culture. Recombinant human and bovine cytokines and endotoxin from Escherichia coli and Pasteurella haemolytica A1 were used to activate macrophages. NO- production was estimated by measuring nitrite accumulation in cell supernatants using the Greiss reaction. iNOS mRNA and protein expression were detected by Northern and Western blotting respectively.Bovine alveolar macrophages express INOS mRNA and protein and produceNO- in a time and dose-dependent fashion in vitro in response to stimuli known to be present in pneumonic tissue, including IFN-γ, IL-β, and endotoxin. Whole killed P. haemolytica A1 is also a potent inducer of NO- production. Competitive Inhibitors of iNOS restrict nitrite accumulation in cell culture media without affectingiNOS protein expression.Reactive nitrogen oxides chemically generated by S-Nitroso-N-acetyl-DL penicillamine(SNAP) and 3-morpholinosydnonimine (SIN-1) kill wild type P.haemolytica A1 in a dose-dependent fashion. Bovine alveolar macrophages kill the leukotoxin-deficient mutant P. Haemolytica A1 D153, but prior stimulation for NO production abrogates this effect. Wild type P. haemoiytica A1 readily kills macrophages .BHV, PI3, and BVD infection of macrophages depresses NO- production.This effect is due to loss of viability of macrophages infected with BHV and BVD,but is due to an alteration of cell function in PI3 infected macrophages. BVD andPI3 readily replicate in macrophages, but there was only minimal replication ofBHV in these cells. Prior stimulation of macrophages for NO- production did not significantly effect the replicative ability of any of these viruses in macrophages.Bovine alveolar macrophages express INOS and produce NO- in response to stimuli known to be present in pneumonic lung and in a fashion similar to that previously characterized in rodent macrophages. However unlike findings in rodent model systems, no microbicidal activity of macrophage derived NO- was demonstrated. The role and significance of NO- production by bovine alveolar macrophages in infectious pneumonia remains speculative.

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