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Ultrastructural characterization of apoptosis in bovine lymphocytes exposed to Pasteurella haemolytica leukotoxin

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  • 1 Department of Anatomy, Pathology and Pharmacology, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078.
  • | 2 Present address is the Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, Denver, CO 80262.
  • | 3 Department of Anatomy, Pathology and Pharmacology, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078.
  • | 4 Department of Anatomy, Pathology and Pharmacology, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078.
  • | 5 Department of Anatomy, Pathology and Pharmacology, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078.
  • | 6 Department of Anatomy, Pathology and Pharmacology, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078.
  • | 7 Department of Microbiology and Immunology, Baylor College of Medicine, Houston, TX 77030.

Abstract

Objective—To characterize ultrastructural changes of bovine lymphocytes exposed to Pasteurella haemolytica leukotoxin (LKT).

Sample Population—Partially purified LKT from a wild type P haemolytica A1 strain and inactive pro-LKT from an isogeneic mutant P haemolytica strain. Isolated bovine lymphocytes were obtained from 2 healthy calves.

Procedure—Isolated bovine lymphocytes were incubated with various concentrations of LKT and pro-LKT for 3 hours at 37 C and examined by use of transmission electron microscopy. A cytochemical Klenow DNA fragmentation assay was used to examine lymphocytes for DNA fragmentation.

Results—Lymphocytes incubated with LKT at a high concentration (1.0 toxic U/ml) had ultrastructural evidence of cytoplasmic and nuclear membrane rupture and swelling or lysis of mitochondria. Low concentrations of leukotoxin (0.1 toxic U/ml) induced DNA fragmentation in 80% of lymphocytes. Ultrastructurally, these cells had nuclear membrane blebbing, cytoplasmic vaculation, chromatin condensation, nuclear fragmentation, and membrane-bound apoptotic bodies. Incubation of lymphocytes with LKT at extremely low concentrations (0.001 toxic U/ml) or with pro-LKT did not alter their ultrastructure. Inclusion of 0.5 mM ZnCl2 in the medium blocked leukotoxin-induced ultrastructural changes in bovine lymphocytes.

Conclusions and Clinical Relevance—Low concentrations of LKT induce apoptosis and high concentrations induce oncotic cell lysis in bovine lymphocytes. The ability of low LKT concentrations to induce apoptosis in host leukocytes may allow bacteria to escape host immune surveillance and colonize the host. (Am J Vet Res 2000;61:51–56)

Abstract

Objective—To characterize ultrastructural changes of bovine lymphocytes exposed to Pasteurella haemolytica leukotoxin (LKT).

Sample Population—Partially purified LKT from a wild type P haemolytica A1 strain and inactive pro-LKT from an isogeneic mutant P haemolytica strain. Isolated bovine lymphocytes were obtained from 2 healthy calves.

Procedure—Isolated bovine lymphocytes were incubated with various concentrations of LKT and pro-LKT for 3 hours at 37 C and examined by use of transmission electron microscopy. A cytochemical Klenow DNA fragmentation assay was used to examine lymphocytes for DNA fragmentation.

Results—Lymphocytes incubated with LKT at a high concentration (1.0 toxic U/ml) had ultrastructural evidence of cytoplasmic and nuclear membrane rupture and swelling or lysis of mitochondria. Low concentrations of leukotoxin (0.1 toxic U/ml) induced DNA fragmentation in 80% of lymphocytes. Ultrastructurally, these cells had nuclear membrane blebbing, cytoplasmic vaculation, chromatin condensation, nuclear fragmentation, and membrane-bound apoptotic bodies. Incubation of lymphocytes with LKT at extremely low concentrations (0.001 toxic U/ml) or with pro-LKT did not alter their ultrastructure. Inclusion of 0.5 mM ZnCl2 in the medium blocked leukotoxin-induced ultrastructural changes in bovine lymphocytes.

Conclusions and Clinical Relevance—Low concentrations of LKT induce apoptosis and high concentrations induce oncotic cell lysis in bovine lymphocytes. The ability of low LKT concentrations to induce apoptosis in host leukocytes may allow bacteria to escape host immune surveillance and colonize the host. (Am J Vet Res 2000;61:51–56)