Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium are antigenically and genetically very similar organisms; however, they differ markedly in their virulence for cattle. We evaluated the capacity of bovine macrophages infected with M. avium subsp. paratuberculosis or M. avium subsp. avium to express major histocompatibility complex (MHC) class I and class II antigens on their surface and to interact with primed autologous lymphocytes. Our results indicate that infection of bovine macrophages with M. avium subsp. paratuberculosis promoted the downregulation of MHC class I and class II molecules on the macrophage surface within 24 and 12 h, respectively. Alternatively, MHC class II expression by M. avium subsp. aviuminfected macrophages was not detected until 24 h after infection, and the magnitude of the decrease was smaller. Decreased MHC class I expression by M. avium subsp. avium-infected macrophages was not detected. Unlike M. avium subsp. paratuberculosis-infected macrophages, M. avium subsp. avium-infected macrophages upregulated MHC class I and class II expression after activation by gamma interferon or tumor necrosis factor alpha. Further, M. avium subsp. avium-infected macrophages were lysed by primed autologous lymphocytes, whereas M. avium subsp. paratuberculosis-infected macrophages were not. Overall, the results support the hypothesis that the difference in the virulence of M. avium subsp. paratuberculosis and M. avium subsp. avium for cattle is dependent on a difference in the capacity of the organisms to suppress mycobacterial antigen presentation to T lymphocytes.

Three flow cytometric techniques were evaluated for determination of differential cell counts on canine clinical bone marrow specimens. Techniques included staining bone marrow specimens with 2'7'-dichlorofluorescein (DCF) or 3,3'-dihexyloxacarbocyanine iodide (DiOC6) and evaluation of forward-angle light scatter vs. side-angle light scatter plots. Flow cytometric evaluation of bone marrow cells stained with DCF failed to separate bone marrow cells into distinct cell populations. Staining with DiOC6 resulted in separation of bone marrow cells into populations of mature and immature erythroid cells, mature and immature myeloid cells, and lymphocytes. The scatter plot method resulted in identification of mature and immature erythroid cells, immature myeloid cells, metamyelocytes, and bands and segmenters. Lymphocytes could not be differentiated from mature erythroid cells by the scatter plot method. When the results of the DiOC6 method and the scatter plot method were compared with manual bone marrow differential cell counts, the scatter plot method had more similar mean values and higher correlation coefficients. The scatter plot method has the potential of providing rapid semiquantitative assessment of bone marrow differential cell counts in dogs for specimens that contain low numbers of lymphocytes.

Exotoxins which belong to the family containing the RTX toxins (repeats in toxin) contribute to a variety of important human and animal diseases. One example of such a toxin is the potent leukotoxin (LKT) produced by the bovine respiratory pathogen Mannheimia haemolytica. LKT binds to CD18, resulting in the death of bovine leukocytes. In this study, we showed that internalized LKT binds to the outer mitochondrial membrane, which results in the release of cytochrome c and collapse of the mitochondrial membrane potential ( M. haemolytica is the principal bacterial pathogen in the bovine respiratory disease complex (19, 58). The most important virulence factor of M. haemolytica is the LKT, which has potent cytotoxic effects on ruminant leukocytes but not on leukocytes from other species (38). M. haemolytica LKT is a member of the RTX toxin (repeats in toxin) family, which includes LKTs and hemolysins produced by a number of gramnegative bacteria. It was originally thought that M. haemolytica LKT damages cells by inserting into the cell membrane, resulting in pore formation and necrosis (53). At lower LKT concentrations, however, susceptible cells die via caspase-dependent apoptotic pathways (8,10,45,46). LKT was first reported to bind to the ␤ 2 integrin CD18/CD11a, also known as leukocyte functional antigen 1 (LFA-1) (1,24,28). Recent studies, however, have shown that Mac-1 (CD18/CD11b) also binds LKT and that CD18 is the functional receptor for LKT (9, 30). After binding to LFA-1, LKT induces apoptosis of bovine leukocytes as a result of activation of caspase 1, caspase 3, and caspase 9 (4, 10, 31, 46). The LFA-1-dependent cytotoxicity of LKT is a conundrum, because signaling through LFA-1 generally results in cell adhesion and promotes cell survival (34,44,51).In this study we hypothesized that mitochondrial damage and caspase 9 activation could be due to direct binding of LKT to the mitochondrial outer membrane (MOM). In this paper we show that LKT directly targets mitochondria in BL-3 cells, creating lesions in the MOM that can lead to release of proapoptotic proteins, culminating in cell death. We also show that LKT targeting to mitochondria is dynamin-2 dependent and that mitochondria appear to be the principal source of dynamin-2 in BL-3 cells. Treating BL-3 cells with cyclosporine (CSA) depleted mitochondrial dynamin-2, thereby inhibiting LKT transport to mitochondria and cell death. MATERIALS AND METHODSLKT production and purification. Crude LKT was prepared and purified as described previously and was stored at Ϫ80°C until it was used in experiments (4). Inactive toxin from an lktC mutant of M. haemolytica strain A1 (SH 1562), which produces an LKT protein with no biological activity (LKT inact ) (generously provided by S. K. Highlander, Baylor College of Medicine, Houston, TX), was prepared in a similar manner.Cell lines, cell cultures, and antibodies. Nonadherent bovine lymphoblastoid cells (BL-3 cells) and adherent murine macrophages (RAW 264.7 cells) (kindly provided by Ronald Schultz, University of...

Bovine respiratory disease resulting from infection with Mannheimia haemolytica commonly results in extensive vascular leakage into the alveoli. M. haemolytica produces two substances, lipopolysaccharide (LPS) and leukotoxin (LKT), that are known to be important in inducing some of the pathological changes. In the present study, we examined bovine pulmonary epithelial (BPE) cell and bovine lung microvascular endothelial cell monolayer permeability, as measured by trans-well endothelial and epithelial cell electrical resistance (TEER), after incubation with LPS, LKT, or LPS-activated neutrophils. Endothelial cell monolayers exposed to LPS exhibited significant decreases in TEER that corresponded with increased levels of proinflammatory cytokines, apoptosis, and morphological changes. In contrast, BPE cells exposed to LPS increased the levels of production of inflammatory cytokines but displayed no changes in TEER, apoptosis, or visible morphological changes. Both cell types appeared to express relatively equal levels of the LPS ligand Toll-like receptor 4. However, TEER in BPE cell monolayers was decreased when the cells were incubated with LPS-activated neutrophils. Although the incubation of BPE cells with LKT decreased TEER, this was not reduced by the incubation of LKT with a neutralizing antibody and was reversed when LKT was preincubated with the LPS-neutralizing compound polymyxin B. Because BPE cells did not express the LKT receptor CD11a/CD18, we infer that contaminating LPS was responsible for the decreased TEER. In conclusion, LPS triggered changes in endothelial cells that would be consistent with vascular leakage, but neither LPS nor LKT caused similar changes in epithelial cells, unless neutrophils were also present.

Inflammatory mediators tested had minimal effects on neutrophil oxygen radical production or degranulation but did prime neutrophils for oxygen radical production. Incubation with PAF and TNF-alpha caused a decrease in neutrophil deformability and altered neutrophil shape and size. Results of our study indicate that PAF- and TNF-alpha-induced changes in neutrophil deformability and size may cause integrin- and selectin-independent trapping of neutrophils in the lungs of cattle with pneumonic pasteurellosis.