key: cord-0765857-2gewib47
authors: Leite, F.; Sylte, M.J.; O’Brien, S.; Schultz, R.; Peek, S.; van Reeth, K.; Czuprynski, C.J.
title: Effect of experimental infection of cattle with bovine herpesvirus-1 (BHV-1) on the ex vivo interaction of bovine leukocytes with Mannheimia (Pasteurella) haemolytica leukotoxin
date: 2002-01-01
journal: Vet Immunol Immunopathol
DOI: 10.1016/s0165-2427(01)00397-x
sha: ab52dcab06a5175319e7fa36f117e883056c91ce
doc_id: 765857
cord_uid: 2gewib47

Mannheimia (Pasteurella) haemolytica A1 produces an extracellular leukotoxin (LKT) that is reported to bind the β(2)-integrin CD11a/CD18 (LEA-1) on ruminant leukocytes. LKT binding induces activation, and subsequent cytolysis, of these cells. It is well known that active viral infection greatly increases the susceptibility of cattle to pasteurellosis. To better understand the mechanism by which this occurs, we investigated the effects of experimental in vivo infection of cattle with bovine herpes virus-1 (BHV-1) on the ex vivo interaction of bovine leukocytes with the M. haemolytica LKT. In this study, we demonstrated that active BHV-1 infection increased the expression of the β(2)-integrin CD11a/CD18 (as defined by the mAb BAT75) on bovine peripheral blood neutrophils, enhanced the binding of LKT to bronchoalveolar lavage (BAL) leukocytes and peripheral blood neutrophils, and increased the killing of BAL leukocytes and peripheral blood leukocytes by LKT. In addition, BHV-1 greatly increased the number of BAL, resulting in many more LKT-responsive cells being present in the lungs. These findings might explain in part the increased susceptibility of BHV-1 infected cattle to pneumonic pasteurellosis.

The bovine respiratory disease (BRD) complex remains a major economic problem for both beef and dairy cattle industries in North America, and throughout the world (Frank, 1989; Wittum et al., 1996) . One of the clinically severest components of BRD is pneumonic pasteurellosis (shipping fever), which is characterized by acute lobar ®bronecrotizing pleuropneumonia with extensive polymorphonuclear leukocyte in¯ux and death within pulmonary alveoli (Slocombe et al., 1985; Leite et al., 1999) . Mannheimia haemolytica A1 is the primary bacterial pathogen responsible for shipping fever (Yates, 1982; Gonzalez and Maheswaran, 1993) . M. haemolytica produces several virulence factors, foremost of which is a leukotoxin (LKT), whose cytotoxic activity is speci®c for ruminant leukocytes and platelets (Gonzalez and Maheswaran, 1993) . The LKT is a Ca 2dependent pore-forming cytolysin, that is a member of the family of bacterial toxins designated RTX (repeats in toxin) (Welch, 1991) . It has been reported that RTX toxins bind to b 2 -integrins on target cells (Lally et al., 1997) . M. haemolytica LKT was reported to bind to the b 2 -integrin CD11a/CD18 (LFA-1), or CD18 alone, on bovine leukocytes (Ambagala et al., 1999; Li et al., 1999; Jeyaseelan et al., 1999; Leite et al., 2000) . This binding induces formation of pore-like structures in the plasma membrane, resulting in both activation of leukocytes, and death by apoptosis and necrosis Czuprynski, 1995, 1996; Wang et al., 1998; Sun et al., 1999; Clinkenbeard et al., 1994) .

Co-infection with a variety of respiratory viruses, including bovine herpesvirus-1, bovine virus diarrhea virus (BVDV), bovine parain¯uenza-3, bovine respiratory syncytial virus and bovine coronavirus (Yates, 1982; Ohmann and Babiuk, 1985; Liu et al., 1999; Storz et al., 2000) increases the susceptibility of cattle to M. haemolytica LKT infection. The mechanism by which viral infection impairs resistance of cattle to pulmonary pasteurellosis has not been clearly elucidated. One mechanism that might be involved is the release of in¯ammatory cytokines during viral infection (Ohmann and Babiuk, 1985; Gonzalez and Maheswaran, 1993) . These cytokines (i.e. IL-1b, TNF-a) can activate bovine leukocytes and modulate the migration, and functional activation of b 2 -integrins on bovine leukocytes (Nagahata et al., 1995) .

We hypothesized that BHV-1 infection might result in the release of IL-1b and other cytokines that enhance the expression of the putative LKT receptor CD11a/CD18 (i.e. LFA-1) on bovine leukocytes, thus amplifying their interaction with M. haemolytica LKT. The purpose of the present study was to determine whether BHV-1 infection increased CD11a/CD18 expression, LKT binding and LKT killing of bronchoalveolar lavage (BAL) and peripheral blood leukocytes in vitro. The results we obtained suggest that active BHV-1 infection enhances the biological response of bovine leukocytes to M. haemolytica LKT ex vivo.

Ten 12±14 month-old, crossbreed, beef calves weight; 320 AE 20 kg were used in this experiment. The 10 animals were maintained in a herd that was free of BHV-1 and BVDV. All animals in the herd were tested monthly for antibody to BVH-1 and BVDV to ensure their viral-free status. One week before initiation of the experiment, all animals were tested and found to be free of detectable serum neutralizing antibodies to BHV-1. The animals were randomly assigned to two groups. Experimental infection of six calves was accomplished by intranasal inoculation of 10 6 plaque forming units (PFU) of BHV-1 (Cooper strain) suspended in 10 ml of RPMI 1640 (Mediatech, Cellgro, Herndon, VA). The four control animals were inoculated intranasally with 10 ml of RPMI 1640 alone.

The animals were restrained in a chute and sedated with a combination of intravenous xylazine (0.03 mg/kg) and butorphanol (0.03 g/kg). The nostrils and external nares were wiped clean with sterile cotton gauze. BAL was then performed on each individual by holding the head and neck in extension and passing a 33 French semi-¯exible tipped lavage catheter nasotracheally (Bivona Veterinary Products, Gary, IN). The catheter was wedged into a distal bronchus and held in place with an in¯ated cuff, after which approximately 120 ml of sterile physiologic saline was injected via the catheter. Rapid aspiration permitted harvest of greater than 60% of the injected¯uid volume in each case. The lavage¯uid was kept in an ice bucket until future assay were performed. At that time, the number of nucleated cells per milliliter was counted using a hemacytometer. Cytocentrifuge smears were prepared, stained with Diff-Quik, and examined microscopically to determine a differential cell count.

Brie¯y, peripheral blood was collected from the jugular vein of infected and control cattle using a 19 g needle attached to Vacutainer tubes (Becton-Dickinson, Rutherford, NJ) containing sodium citrate (0.38% ®nal volume) as anticoagulant. Blood was collected on days 0 (1 h before virus inoculation), 3, 5, and 7 post-infection. The blood was centrifuged (250Âg for 20 min), and the platelet-rich plasma was removed. Leukocytes were obtained by rapid hypotonic lysis, washed twice in Hank's balance salt solution (HBSS), and resuspended at 10 6 cells/ml in RPMI 1640, with 2% fetal bovine serum. The leukocyte population was greater than 95% viable as estimated by Trypan blue exclusion.

A strain of M. haemolytica A1 (generously provided by Dr. R.E. Briggs, National Animal Disease Center USDA/ARS, Ames, IA) was used in this study as described previously (Tatum et al., 1998; Leite et al., 2000) . Brie¯y, M. haemolytica A1 was inoculated onto blood agar and incubated overnight (Remel, Lenexa, KS) at 37 8C. The bacteria were washed from the agar surface with 10 ml of brain heart infusion broth that contained 0.5% yeast extract (BHI/YE; Difco, Detroit, MI) and incubated at 37 8C for 1 h while rotating (16 rpm) in 15 ml polypropylene tubes. A 10 ml aliquot of this suspension was then used to inoculate 200 ml of BHI/YE in a 500 ml Erlenmeyer¯ask. The¯ask was then incubated for 2 h at 37 8C with gentle shaking. The bacteria were collected by centrifugation (1600Âg for 15 min), resuspended in 200 ml of RPMI 1640 supplemented with L-glutamine (4.0 mM), and incubated on a shaker apparatus for 4 h at 37 8C. The bacteria were harvested by centrifugation (1600Âg for 20 min), and the crude leukotoxincontaining supernatant was collected and passed through a 0.45 mm pore size bottle top ®lter (Nalgen, Rochester, NY) to remove any residual bacteria. Aliquots (20 ml) of crude leukotoxin were concentrated over an Amicon ultra ®ltration unit equipped with a 62 mm diameter XM-50 ultra ®ltration membrane. The volume was then reduced to 10±20 ml over a 1±2 h period by applying 60 psi transmembrane pressure with nitrogen gas. The partially puri®ed leukotoxin preparation that remained was collected and stored as 5 ml aliquots at À70 8C. Leukotoxic activity was con®rmed by Trypan blue exclusion, using bovine peripheral blood leukocytes incubated with serial dilutions of toxin for 30 min at 37 8C. One unit of LKT activity was de®ned as the dilution causing 50% killing of bovine peripheral blood leukocytes as determined by Trypan blue exclusion. Partially puri®ed LKT was stored at À70 8C until used in an experiment.

Biotinylated LKT was prepared as described previously (Brown et al., 1997) . Brie¯y, an 80:1 molar ratio of NHS-LC-biotin (Pierce Chemical, Rockford, IL) to partially puri®ed LKT was incubated in an ice bath for 20 min. The mixture was then concentrated to 1 ml in a prechilled Amicon Centricon tube (50 kDa cutoff). The reaction was stopped by the addition of crystalline bovine serum albumin (30 mg), and incubated at 4 8C for 30 min. Unbound biotin was eliminated by buffer exchange over a Sephadex G-25 column (1 Â 25 cm 2 ) using phosphate-buffered saline (PBS, pH 7.2) as elution buffer. The LKT eluted in the void volume in 7±8 min, as monitored by absorbance at 280 nm. Total protein in the eluted LKT was determined using the microplate Bradford assay. Leukotoxin activity was con®rmed by incubating biotinylated LKT with bovine peripheral leukocytes for 30 min at 37 8C followed by Trypan blue exclusion.

Flow cytometry analysis of LKT binding was performed as described previously (Brown et al., 1997) . Brie¯y, leukocytes 1 Â 10 6 were incubated with biotinylated LKT (10 units) for 10 min at 4 8C. The cells were washed and resuspended in HBSS. Extra-avidinuorescein isothiocyanate (FITC) (4 ml) (Sigma, St. Louis, MO), was added, and the cells were incubated for 30 min at 4 8C. The cells were washed twice with HBSS and resuspended in 0.5 ml of HBSS. The stained cells were ®xed with 0.4% paraformaldehyde (®nal concentration), washed and resuspended in HBSS. BAL and peripheral blood leukocytes were analyzed by¯ow cytometry with a Coulter Epics-Pro®le II¯ow cytometer (10,000 cells were scored for green¯uorescence on a log scale). The peripheral blood leukocytes were gated into granulocyte (PMN) and mononuclear cell (PBMC) populations and analyzed by¯ow cytometry.

Peripheral blood leukocytes and BAL from infected and control animals were incubated at 4 8C for 45 min, with an anti-CD11a/CD18 (BAT 75A). The BAT75 mAb is reported by the supplier (VMRD, Pullman, WA) to bind both CD11a/CD18 (i.e. LFA-1), although the precise binding sites have not been reported. Following this, the cells were washed and resuspended in 50 ml of HBSS. A biotinylated (Fab H ) 2 rabbit anti-mouse IgG antibody (50 mg/ml, Sigma, St. Louis, MO) was added, and the cells incubated at 4 8C for 30 min. The cells were washed twice with HBSS, resuspended in 0.5 ml of HBSS, and ®xed with 0.4% paraformaldehyde (®nal concentration). The cells were then washed and analyzed by¯ow cytometry with a Coulter Epics-Pro®le II¯ow cytometer (10,000 cells were scored for green uorescence on a log scale). The peripheral blood leukocytes were gated into granulocyte (PMN) and mononuclear cell (PBMC) populations and analyzed by¯ow cytometry.

Trypan blue exclusion was used to evaluate LKT cytotoxicity for bovine leukocytes. Because of the number of samples and time constraints, peripheral blood leukocytes were not separated into PMN and PBMC enriched populations. Brie¯y, BAL and peripheral blood leukocytes 1  10 6 were incubated with LKT (1 unit) for 1 h at 37 8C on a rotating platform. A 1:10 dilution of the cell suspension was prepared, an equal volume of 0.4% Trypan blue was added, and the mixture was incubated for 3 min at room temperature. Using a hemacytometer, at least 200 cells were counted and the percent cytotoxicity was calculated as total number of dead blue cells=total number of cells  100.

The¯ow cytometry data were analyzed using the WInMDI version 2.8 software. Other data were analyzed for statistical signi®cance using a one-way analysis of variance followed by an unpaired T-test, using the Instat software program (GraphPad, San Diego, CA). Statistical signi®cance was set at P < 0:05.

3.1. Total number of BAL cells are greatly increased in cattle experimentally infected with BHV-1 A 2.3-fold increase in cell numbers was observed P < 0:05 in BHV-1 infected cattle as compared with the control group on day 4 post-infection. The cell populations were primarily mononuclear cells; there was no difference in the number of PMNs between the groups (Fig. 1) .

The expression of CD11a/CD18 on BAL ( Fig. 2A) , and PMNs and PBMCs from infected and control animals was examined by¯ow cytometry. Essentially all BAL, from both BHV-1 infected and control animals, stained positive for CD11a/CD18 (data not shown). There was no signi®cant difference in mean channel¯uorescence for CD11a/CD18 on BAL (day 4 post-infection) from BHV-1 infected and control cattle P > 0:05 ( Fig. 2A) . We observed a slight increase in expression of CD11a/CD18 on days 3, 5 and 7 post-infection, however; these values were not different P > 0:05 from control PBMCs (Fig. 2B) . A signi®cant increase P > 0:05 in CD11a/CD18 expression was observed on PMNs at 5 and 7 days post-infection from BHV-1 infected animals, as compared to the PMNs from control animals (Fig. 2C ).

As illustrated in Fig. 3A , BAL from BHV-1 infected cattle exhibited a 1.9-fold increase in LKT-binding cells as compared with BAL from control cattle P < 0:05. PBMCs from BHV-1 infected cattle exhibited a 1.4-fold P < 0:05 increase in LKT-binding to the cells on day 5 post-infection (Fig. 3B) . PMNs from infected cattle exhibited 1.8-and 2-fold increases P < 0:05 in LKT binding on days 5 and 7 post-infection, respectively (Fig. 3C) .

We observed an 11% increase in the cytotoxicity (based on Trypan blue exclusion) of LKT for BAL from BHV-1 infected cattle, compared with BAL from the control group, Fig. 1 . Increased number of BAL cells recovered from BHV-1 infected cattle at day 4 post-infection. BAL cells were suspended in 1 ml of PBS, and counted using a hemacytometer. Cytocentrifuge smears were prepared, stained, and 100 cells from each sample were counted to determine the number of mononuclear and polymorphonuclear cells. The data represent the mean AE S:E: number of cells recovered from BHV-1 infected (six animals) and control cattle (four animals). Asterisks indicate statistically significant differences between BHV-1 infected and control animals P < 0:05. Fig. 2 . CD11a/CD18 expression on BAL (A), PBMCs (B), and peripheral blood PMNs (C) from cattle at various times after experimental infection with BHV-1. Cell preparations from BHV-1 infected n 6 and control n 4 animals were incubated (40 min at 4 8C) with anti-CD11a/CD18a mAb antibody (BAT75A, 50 mg/ml final concentration). The cells were then washed, incubated with an FITC-labeled secondary antibody, and analyzed by flow cytometry (10,000 cells were scored for green fluorescence). The data represent the mean AE S:E: channel fluorescence of BHV-1 infected (six animals) and control animals (four animals). Asterisks indicate statistically significant differences between BHV-1 infected and control animals P < 0:05. Fig. 3 . M. haemolytica LKT binding to BAL (A), PBMCs (B), and peripheral blood PMNs (C) from cattle experimentally infected with BHV-1. Cells from infected and control animals were incubated with biotinylated M. haemolytica LKT (10 units) for 10 min on ice. The cells were washed, extra-avidin-FITC was added and the cells incubated on ice for 20 min. The stained cells were washed, fixed with 4% paraformaldehyde, and analyzed by flow cytometry (10,000 cells were scored for green fluorescence). The data represent the mean AE S:E: channel fluorescence of cells from BHV-1 infected (six animals) and control animals (four cattle). Asterisks indicate statistically significant differences between BHV-1 infected and control animals P < 0:05. at day 4 post-infection (Fig. 4A) . Because of the number of samples and assays being performed, we did not separate peripheral blood leukocytes into PMN and PBMC enriched populations. We observed a 42 and 18% increase in the cytotoxicity of LKT for PBL from BHV-1 infected cattle at days 5 and 7, respectively, as compared with peripheral blood leukocytes from control cattle (Fig. 4B) . 

In this study, we demonstrated that BHV-1 infection increases the ex vivo interaction of M. haemolytica leukotoxin with bovine leukocytes. We recovered greatly increased number of BAL from the BHV-1 infected cattle, with alveolar macrophages and other mononuclear cells being the most prominent cell populations as reported previously (Foreman et al., 1981) . The presence of more LKT-responsive cells in the lungs of BHV-1 infected cattle could play an important role in the pathogenesis of pasteurellosis. LKTmediated activation and death of increased number of BAL would corroborate with the intense lung in¯ammation and damage noted in pasteurellosis (Slocombe et al., 1985; Wittum et al., 1996) . It is presumed that the in¯ammatory process elicited by BHV-1 infection results in secretion of cytokines locally and into the systemic circulation (Ohmann and Babiuk, 1985; Yoo et al., 1995) . These in¯ammatory cytokines would induce increased expression of adhesion molecules on leukocytes and endothelial cells resulting in increased migration and functional activation of b 2 -integrins on leukocytes in the lung (Arnaout, 1990; Williams and Solomkin, 1999) .

In this study, we observed increased cytotoxicity of LKT for PBLs at 5 and 7 days postinfection. Peripheral blood mononuclear cells exhibited a slight increase in LKT binding at day 5 post-infection P > 0:05, whereas PMNs exhibited increased CD11a/CD18 expression and increased LKT binding at 5 and 7 days post-infection P < 0:05. These observations support the previously reported association between LFA-1 expression, and the binding and cytotoxic activity of LKT for bovine peripheral blood leukocytes (Ambagala et al., 1999; Li et al., 1999; Jeyaseelan et al., 1999; Leite et al., 2000) . We believe it is likely that the increased CD11a/CD18 expression by PMNs from BHV-1 infected cattle is mediated by in¯ammatory cytokines secreted during viral infection, rather than the direct effects of the virus. This supposition is based on reports that productive infection by BHV-1 does not occur in bovine peripheral blood leukocytes (Foreman et al., 1982a,b) . We have previously reported that bovine PMNs stimulated with IL-1b in vitro increased CD11a/CD18 expression, LKT binding and cytotoxicity (Leite et al., 2000) . These latter effects were inhibited by addition of the same BAT75 anti-CD11a/CD18 mAb used in the present study. The BAT75 mAb is reported to bind to both CD11a and CD18, although the precise binding site has not been determined. As a result, our results do not allow us to exclude the possible contributions of other b 2 -integrins (i.e. CD11b/CD18) in our¯ow cytometry studies. Recently, we have obtained preliminary data indicating that incubation of bovine PMNs with other cytokines (TNF-a, IFN-g) also upregulates CD11a/CD18, and LKT binding and killing (unpublished observations).

Bovine lung leukocytes constitutively expressed b 2 -integrins; the mAb (BAT75A) used in this study recognized nearly all BAL cells from both BHV-1 infected and uninfected cattle (Leite et al., 2000) . Nor did we observe differences in the intensity of CD11a/CD18 expression by BAL cells from BHV-1 infected or control cattle ( Fig. 2A) . In contrast, we observed a signi®cant increase in LKT binding (Fig. 3) , and LKT mediated killing (Fig. 4) , of BAL from BHV-1 infected cattle. Stimulation of leukocytes can lead to conformational changes in the b 2 -integrin molecules that result in increased receptor af®nity, rather than increased receptor number (Hibbs et al., 1991; Barnett et al., 1998) . Thus, one possible explanation for the apparent discrepancy between increased LKT binding to BAL, without increased CD11a/CD18 expression, is that the in¯ammatory process in the BHV-1 infected lung stimulated a conformational change in CD11a/CD18 that increased its af®nity for LKT. We have previously reported that RGD peptides block the binding of LKT to resting, but not IL-1b stimulated, bovine PMNs (Leite et al., 2000) . This prior observation is also consistent with the inference that CD11a/CD18 on stimulated bovine leukocytes has an increased af®nity for LKT. However, we cannot have the possibility that the mAb (BAT75A) recognize other b 2 -integrins on leukocytes that do not play a role as a receptor to LKT, or that BAL cells possess an additional LKT receptor, besides CD11a/CD18.

There are some limitations in our study. First, we used only one mAb (BAT75A) to evaluate CD11a/CD18 expression. The binding site for this mAb has not been fully characterized. Thus, it is possible it might bind to other members of the b 2 -integrin family. A second limitation is that we did not distinguish which peripheral blood cell population was most susceptible to the action of LKT, since we did not separate these cells into PMN and PBMC enriched populations before assessing cytotoxicity. Although the method used to assess cytotoxicity (Trypan blue exclusion) is tedious and more subjective than some assays, it has been used by our laboratory and others to determine cell viability, and offers relatively rapid results as compared to other methods (i.e. color development in MTT or XTT assays). One ®nal limitation of our study was the day that we chose to perform the BAL (day 4 post-infection). This time was chosen because some previous reports suggested that it fell within the window of increased susceptibility to pasteurellosis (Yates, 1982) . Fig. 5 . Proposed model for the mechanism of viral±bacterial synergism in BRD. Viral infection induces the release of inflammatory cytokines that stimulate CD11a/CD18 expression on leukocytes. This will enhance M. haemolytica LKT binding to bovine leukocytes. LKT binding will activate leukocytes to release inflammatory mediators (e.g. reactive oxygen intermediates, enzymes, etc.) that will damage lung tissue. Eventually, the LKT kills the leukocytes and impairs the clearance of bacteria from the lungs. Together, these effects will contribute to the severe pneumonia that characterizes BRD. However, we observed an increase in CD11a/CD18 expression and LKT binding to PMNs on day 5 and 7 post-infection, suggesting that, ideally, BAL would be performed more than once after infection (i.e. 3±7 days post-infection). However, we were concerned that performing BAL more than once on the same animal would confound our analyses. Financial constraints prevented us from adding more animals that would have allowed us to sample BAL, at more than one time point, without lavaging any animal more than once.

Based on our observations, we suggested that one possible explanation for the viral± bacterial synergism observed in BRD is that BHV-1 infection results in the release of cytokines, which in turn attract lung leukocytes, and increase leukocyte expression of CD11a/CD18. When M. haemolytica infects the lower airways of a BHV-1 infected cow, it ®nds a favorable environment with increased number of lung leukocytes. These may have activated CD11a/CD18, which facilitates LKT binding and killing of these cells.

Once established in the lungs, the continued release of LKT and other bacterial virulence factors (i.e. LPS) by M. haemolytica would stimulate the continued release of in¯ammatory cytokines (Yoo et al., 1995; Morsey et al., 1999) that would sustain CD11a/CD18 expression and exacerbate the effects of M. haemolytica LKT. Based on our observations, we have devised a working model for the mechanism by which in¯ammatory cytokine stimulation of LFA-1 on leukocytes of BHV-1 infected cattle would predispose cattle to pasteurellosis (Fig. 5) .

The leukotoxin of Pasteurella haemolytica binds to b 2 integrin on bovine leukocytes

Structure and function of the leukocyte adhesion molecules CD11/CD18 (Review)

ICAM-1-CD18 interaction mediates neutrophil cytotoxicity through protease release

Binding of Pasteurella haemolytica leukotoxin to bovine leukocytes

Pasteurella haemolytica leukotoxin-induced synthesis of eicosanoids by bovine neutrophils in vitro

Effect of infectious bovine rhinotracheitis virus infection of calves on cell population recovered by lung lavage

Effect of infectious bovine rhinotracheitis virus infection on bovine alveolar macrophage function

Pasteurellosis in cattle

The role of induced virulence factors produced by Pasteurella haemolytica in the pathogenesis of bovine pulmonary pasteurellosis. Review and Hypothesis

Regulation of adhesion to ICAM by the cytoplasmic domain of LFA-1 integrin b subunit

Lymphocyte function-associated antigen 1 is a receptor for Pasteurella haemolytica leukotoxin in bovine leukocytes

RTX toxins recognize a b 2 integrin on the surface of human target cells

Use of TUNEL staining to detect apoptotic cells in lungs of cattle experimentally infected with Pasteurella haemolytica

Recombinant bovine interleukin-1b amplifies the effect of partially purified Pasteurella haemolytica leukotoxin on bovine neutrophils in a b 2 -integrin-dependent manner

Bovine CD18 identified as a species specific receptor for Pasteurella haemolytica leukotoxin

Synergistic effects of bovine respiratory syncytial virus and noncytopathic bovine viral diarrhea virus infection on selected bovine alveolar macrophage functions

Cytokine profiles following interaction between bovine alveolar macrophages and Pasteurella haemolytica

Expression and role of adhesion molecule CD18 on bovine neutrophils

Viral±bacterial pneumonia in calves: effect of bovine herpesvirus-1 on immunologic functions

Importance of neutrophils in the pathogenesis of acute pneumonic pasteurellosis in calves

Dissociation of cytolysis and monokine release by bovine mononuclear phagocytes incubated with Pasteurella haemolytica partially purified leukotoxin and lipopolysaccharide

Pasteurella haemolytica leukotoxin induces bovine leukocytes to undergo morphologic changes consistent with apoptosis in vitro

Coronavirus and Pasteurella infections in bovine shipping fever pneumonia and Evan's criteria for causation

Correlation of Pasteurella haemolytica leukotoxin binding with susceptibility to intoxication of lymphoid cells from various species

Construction of an isogenic leukotoxin deletion mutant of Pasteurella haemolytica serotype 1: characterization and virulence

Molecular and biochemical mechanisms of Pasteurella haemolytica leukotoxin-induced cell death

Integrin-mediated signaling in human neutrophil functioning

Relationship among treatment for respiratory tract disease, pulmonary lesions evident at slaughter and rate of weight gain in feedlot cattle

A review of infectious bovine rhinotracheitis, shipping fever pneumonia and viral± bacterial synergism in respiratory disease in cattle

Purified Pasteurella haemolytica leukotoxin induces expression of inflammatory cytokines from bovine alveolar macrophages

We thank Steve Giles for assistance in preparation of the illustrations. This work was supported by funds from the Wisconsin Agricultural Experiment Station, the University of Wisconsin-Madison Industrial and Economic Development Research Program, and the USDA National Research Initiative. F. Leite was supported by CAPES-Ministe Ârio da Educac Ëa Äo, Brazil.