Pasteurella (Mannheimia) haemolytica leukotoxin (Lkt) causes cell type-and species-specific effects in ruminant leukocytes. Recent studies indicate that P. haemolytica Lkt binds to bovine CD18, the common subunit of all ␤2 integrins. We designed experiments with the following objectives: to identify which member of the ␤2 integrins is a receptor for Lkt; to determine whether Lkt binding to the receptor is target cell (bovine leukocytes) specific; to define the relationships between Lkt binding to the receptor, calcium elevation, and cytolysis; and to determine whether a correlation exists between Lkt receptor expression and the magnitude of target cell cytolysis. We compared Lkt-induced cytolysis in neutrophils from control calves and from calves with bovine leukocyte adhesion deficiency (BLAD), because neutrophils from BLAD-homozygous calves exhibit reduced ␤2 integrin expression. The results demonstrate for the first time that Lkt binds to bovine CD11a and CD18 (lymphocyte function-associated antigen 1 [LFA-1]). The binding was abolished by anti-CD11a or anti-CD18 monoclonal antibody (MAb). Lkt-induced calcium elevation in bovine alveolar macrophages (BAMs) was inhibited by anti-CD11a or anti-CD18 MAb (65 to 94% and 37 to 98%, respectively, at 5 and 50 Lkt units per ml; P < 0.05). Lkt-induced cytolysis in neutrophils and BAMs was also inhibited by anti-CD11a or anti-CD18 MAb in a concentration-dependent manner. Lkt bound to porcine LFA-1 but did not induce calcium elevation or cytolysis. In neutrophils from BLAD calves, Lkt-induced cytolysis was decreased by 44% compared to that of neutrophils from control calves (P < 0.05). These results indicate that LFA-1 is a Lkt receptor, Lkt binding to LFA-1 is not target cell specific, Lkt binding to bovine LFA-1 correlates with calcium elevation and cytolysis, and bovine LFA-1 expression correlates with the magnitude of Lkt-induced target cell cytolysis.Leukotoxin (Lkt) and lipopolysaccharide produced by Pasteurella (Mannheimia) haemolytica serotype 1 are considered to be the primary virulence factors contributing to lung injury in bovine pneumonic pasteurellosis (BPP) (33,36,38,40), a disease of substantial economic importance to the beef and dairy cattle industries in North America (7,28,39). Lkt is a member of a family of gram-negative bacterial exotoxins termed RTX (for repeats in toxin) cytolysins (3). Although most RTX cytolysins interact with a variety of cell types from many different species (6), cytolysins produced by Actinobacillus actinomycetemcomitans, Actinobacillus pleuropneumoniae (ApxIIIA), and P. haemolytica are known to have cell type-and species-specific effects. The leukotoxin (LtxA) of A. actinomycetemcomitans, a human pathogen, interacts only with cells of the lymphocytic and monomyelocytic lineages of humans and some nonhuman primates (23); the Lkt of P. haemolytica, a ruminant pathogen, interacts only with ruminant leukocytes causing activation and cytolysis (4,15,25,33,40). A study by Lally et al. (23) has determined that two RTX cytolysins, ...

The severe fibrinonecrotic pneumonia associated with pneumonic pasteurellosis usually results from colonization of the lower respiratory tract by Pusteurellu huemolyticu biotype A, serotype l(A1). Despite recent research efforts, the authors lack a detailed understanding of the interactions and host response to P. huemolyticu in the respiratory tract. The authors hypothesize that management and environmental stress factors or viral infection alters the upper respiratory tract (URT) epithelium allowing P. huemolyticu to colonize the epithelium. Once the URT is colonized, large numbers of organisms enter the lung where they interact with alveolar macrophages. Endotoxin, released from the bacteria, crosses the alveolar wall where it activates pulmonary intravascular macrophages, endothelium, neutrophils, lymphocytes, platelets, complement, and Hageman factor leading to complex interactions of cells and mediators. It is the progression of this inflammatory response with neutrophil influx that is ultimately responsible for the pulmonary injury. Leukotoxin is a major virulence factor of P. haemolyiicu that allows it to survive by destroying phagocytic cells. At subcytolytic concentrations it may also enhance the inflammatory response by activating cells to produce mediators and release reactive oxygen metabolites and proteases. has been reproduced experimentally in calves by transthoracic or intratracheal administration of P. huemolytica A 1 alone.*.'* Despite recent research efforts, we lack a detailed understanding of the interaction and host response to P.huernolyticu A1 in the ruminant respiratory tract. This article reviews the information presently available on the interaction of this bacteria with the respiratory tract and discusses possible pathogenic mechanisms. We will not attempt to review the extensive literature on viral-bacterial synergism as it relates to pulmonary bacterial infections. In addition, we will not discuss the effect of stress on immune function and its effect on the susceptibility of cattle to shipping fever. However, the reader is referred to several articles on these s~bjects.~,~~~*'~-'' Several observations point to the central role that P. haemolyticu A1 has in bovine shipping fever. In clinically healthy cattle, P. huemolyticu are present in low numbers in the nasal passages and those that are isolated are predominantly biotype A serotype 2 (A2) which is rarely associated with shipping Exposure of healthy cattle to stressful agents such as viral infection, change in management practices (marketing, transportation and processing), and change in environmental (heat, cold) conditions, leads to an explosive growth and selective colonization by P. huemolyticu A1 in the upper

Aggregatibacter (Actinobacillus) actinomycetemcomitans is the causative organism of localized aggressive periodontitis, a rapidly progressing degenerative disease of the gingival and periodontal ligaments, and is also implicated in causing subacute infective endocarditis in humans. The bacterium produces a variety of virulence factors, including an exotoxic leukotoxin (LtxA) that is a member of the repeats-in-toxin (RTX) family of bacterial cytolysins. LtxA exhibits a unique specificity to macrophages and polymorphonuclear cells of humans and other primates. Human lymphocyte function-associated antigen 1 (LFA-1) has been implicated as the putative receptor for LtxA. Human LFA-1 comprises the CD11a and CD18 subunits. It is not clear, however, which of its subunits serves as the functional receptor that confers species-specific susceptibility to LtxA. Here we demonstrate that the human CD18 is the receptor for LtxA based on experiments performed with chimeric ␤2-integrins recombinantly expressed in a cell line that is resistant to LtxA effects. In addition, we show that the cysteine-rich tandem repeats encompassing integrin-epidermal growth factor-like domains 2, 3, and 4 of the extracellular region of human CD18 are critical for conferring susceptibility to LtxA-induced biological effects.

Bovine pneumonic pasteurellosis continues to be a major respiratory disease in feedlot cattle despite the recent advances in our understanding of the underlying complexities of causation. The etiological agent, Mannheimia haemolytica, possesses several virulence factors, including capsule, outer membrane proteins, adhesins, neuraminidase, endotoxin and exotoxic leukotoxin. Accumulating scientific evidence implicates leukotoxin as the primary factor contributing to clinical presentation and lung injury associated with this disease. Unlike other virulence factors, leukotoxin shows cell-type- and species-specific effects on bovine leukocytes. Recent investigations have delineated the mechanisms underlying the target-cell-specificity of leukotoxin and how this contributes to the pathogenesis of lung damage. This review summarizes current understanding of the secretion, regulation, mechanisms of action and evolutionary diversity of leukotoxin of M. haemolytica. Understanding the precise molecular mechanisms of leukotoxin is critical for the development of more effective prophylactic and therapeutic strategies to control this complex disease.

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An Actinobacillus pleuropneumoniae infection model in swine was established to study the expression of inflammatory cytokines during acute respiratory disease. Lavage fluid, lavage cells consisting primarily of alveolar macrophages, and lung tissue were analyzed for the presence of various cytokines at 2, 4, 8, and 24 h following endotracheal inoculation of A. pleuropneumoniae. Interleukin-1 beta (IL-1) and IL-8 mRNA levels were elevated within 2 h in lavage cells of animals inoculated with A. pleuropneumonia but not in cells from controls treated with saline-bovine serum albumin, based on Northern (RNA blot) analysis. Tumor necrosis factor (TNF) mRNA was present at low levels in all animals, and the level was not increased at any time point. In situ hybridization was more sensitive than Northern blotting and revealed elevations of all three cytokines in lavage cells within 2 to 4 h of A. pleuropneumoniae inoculation. IL-6 was detected in lavage cells by in situ hybridization but not by Northern blotting. In lung tissue obtained 18 to 24 h after A. pleuropneumoniae instillation, all cytokine mRNAs, including that of IL-6, were detected by Northern blot analysis. The levels of bioactive IL-1 and IL-6 in lavage fluids increased approximately 1,000-fold following A. pleuropneumoniae inoculation, but TNF bioactivity was not detected. Morphological localization of cytokine mRNAs by in situ hybridization indicated markedly increased levels of TNF, IL-1, and IL-8 mRNAs at the periphery of focal lung lesions. These findings indicate that inflammatory cytokines, particularly IL-1 and IL-8, are associated with the development of pleuropneumonia and may contribute to disease severity.