Changes in selected cytokines, acute-phase proteins, gonadal hormones and reproductive organs of non-pregnant does challenged with Mannheimia haemolytica serotype A2 and its LPS endotoxin.

Previous investigations have revealed that lipopolysaccharide (LPS) endotoxin from certain Gram-negative bacteria could adversely affect the reproductive system of female animals. However, it is unknown whether LPS endotoxin of Mannheimia haemolytica serotype A2, the principal causative bacteria that cause pneumonic mannheimiosis in small ruminants, may also induce similar insidious effects. Therefore, this study aimed to investigate the effects of M. haemolytica serotype A2 and its LPS endotoxin on the responses of female gonadal hormones (progesterone and oestrogen), pro-inflammatory cytokines (interleukin-1ß, interleukin-6), acute-phase proteins (haptoglobin and serum amyloid A) and cellular changes via histopathology study of female reproductive organs of the treatment does. Twelve clinically healthy, non-pregnant, crossbred does were randomly allocated into three equal groups. Group 1 was administered intranasally with 2 ml of sterile phosphate-buffered saline (PBS) and served as a negative control group. Group 2 was challenged intranasally with 2 ml of bacterial inoculum containing 109 colony-forming units (CFU)/ml of M. haemolytica serotype A2, while Group 3 was challenged intravenously with 2 ml of LPS endotoxin extracted from 109 CFU/ml of M. haemolytica serotype A2. Following that, blood samples were collected serially at pre-determined intervals for serological analyses. All does were euthanised 60 days post-challenges, and tissue samples from the ovaries, oviducts, uterine horns, uterine body, cervix and vagina were collected for histopathological study. The serological result revealed a significant increase (p < 0.05) in the mean concentrations of progesterone, oestrogen, interleukin-1ß, interleukin-6, haptoglobin and serum amyloid A for both challenged groups. Histopathologically, all reproductive organs (except the cervix and vagina) from both challenged groups displayed significant cellular alterations (p < 0.05) characterised by haemorrhage and congestion, necrosis and degeneration, inflammatory cell infiltration and oedema. This study provides new information that elucidates the potential role of pneumonic mannheimiosis in the pathogenesis of female infertility amongst small ruminants.

Engineering and characterization of human ß-defensin-3 and its analogues and microcin J25 peptides against Mannheimia haemolytica and bovine neutrophils.

Mannheimia haemolytica-induced bovine respiratory disease causes loss of millions of dollars to Canadian cattle industry. Current antimicrobials are proving to be ineffective and leave residues in meat. Antimicrobial peptides (AMPs) may be effective against M. haemolytica while minimizing the risk of drug residues. Cationic AMPs can kill bacteria through interactions with the anionic bacterial membrane. Human ß-Defensin 3 (HBD3) and microcin J25 (MccJ25) are AMPs with potent activity against many Gram-negative bacteria. We tested the microbicidal activity of wild-type HBD3, three HBD3 peptide analogues (28 amino acid, 20AA, and 10AA) derived from the sequence of natural HBD3, and MccJ25 in vitro against M. haemolytica. Three C-terminal analogues of HBD3 with all cysteines replaced with valines were manually synthesized using solid phase peptide synthesis. Since AMPs can act as chemoattractant we tested the chemotactic effect of HBD3, 28AA, 20AA, and 10AA peptides on bovine neutrophils in Boyden chamber. Minimum bactericidal concentration (MBC) assay showed that M. haemolytica was intermediately sensitive to HBD3, 28AA and 20AA analogues with an MBC of 50 µg/mL. The 10AA analogue had MBC 6.3 µg/mL which is likely a result of lower final inoculum size. MccJ25 didn't have significant bactericidal effect below an MBC < 100 µg/mL. Bovine neutrophils showed chemotaxis towards HBD3 and 20AA peptides (P < 0.05) but not towards 28AA analogue. Co-incubation of neutrophils with any of the peptides did not affect their chemotaxis towards N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP). The data show that these peptides are effective against M. haemolytica and are chemotactic for neutrophils in vitro.

Differentiated ovine tracheal epithelial cells support the colonisation of pathogenic and non-pathogenic strains of Mannheimia haemolytica.

Mannheimia haemolytica is the primary bacterial species associated with respiratory disease of ruminants. A lack of cost-effective, reproducible models for the study of M. haemolytica pathogenesis has hampered efforts to better understand the molecular interactions governing disease progression. We employed a highly optimised ovine tracheal epithelial cell model to assess the colonisation of various pathogenic and non-pathogenic M. haemolytica isolates of bovine and ovine origin. Comparison of single representative pathogenic and non-pathogenic ovine isolates over ten time-points by enumeration of tissue-associated bacteria, histology, immunofluorescence microscopy and scanning electron microscopy revealed temporal differences in adhesion, proliferation, bacterial cell physiology and host cell responses. Comparison of eight isolates of bovine and ovine origin at three key time-points (2 h, 48 h and 72 h), revealed that colonisation was not strictly pathogen or serotype specific, with isolates of serotype A1, A2, A6 and A12 being capable of colonising the cell layer regardless of host species or disease status of the host. A trend towards increased proliferative capacity by pathogenic ovine isolates was observed. These results indicate that the host-specific nature of M. haemolytica infection may result at least partially from the colonisation-related processes of adhesion, invasion and proliferation at the epithelial interface.

Pathogenic Mannheimia haemolytica Invades Differentiated Bovine Airway Epithelial Cells.

The Gram-negative bacterium Mannheimia haemolytica is the primary bacterial species associated with bovine respiratory disease (BRD) and is responsible for significant economic losses to livestock industries worldwide. Healthy cattle are frequently colonized by commensal serotype A2 strains, but disease is usually caused by pathogenic strains of serotype A1. For reasons that are poorly understood, a transition occurs within the respiratory tract and a sudden explosive proliferation of serotype A1 bacteria leads to the onset of pneumonic disease. Very little is known about the interactions of M. haemolytica with airway epithelial cells of the respiratory mucosa which might explain the different abilities of serotype A1 and A2 strains to cause disease. In the present study, host-pathogen interactions in the bovine respiratory tract were mimicked using a novel differentiated bovine bronchial epithelial cell (BBEC) infection model. In this model, differentiated BBECs were inoculated with serotype A1 or A2 strains of M. haemolytica and the course of infection followed over a 5-day period by microscopic assessment and measurement of key proinflammatory mediators. We have demonstrated that serotype A1, but not A2, M. haemolytica invades differentiated BBECs by transcytosis and subsequently undergoes rapid intracellular replication before spreading to adjacent cells and causing extensive cellular damage. Our findings suggest that the explosive proliferation of serotype A1 M. haemolytica that occurs within the bovine respiratory tract prior to the onset of pneumonic disease is potentially due to bacterial invasion of, and rapid proliferation within, the mucosal epithelium. The discovery of this previously unrecognized mechanism of pathogenesis is important because it will allow the serotype A1-specific virulence determinants responsible for invasion to be identified and thereby provide opportunities for the development of new strategies for combatting BRD aimed at preventing early colonization and infection of the bovine respiratory tract.

Mutual antagonism between Mannheimia haemolytica and Pasteurella multocida when forming a biofilm on bovine bronchial epithelial cells in vitro.

Mannheimia haemolytica and Pasteurella multocida are two bacterial species implicated in the bovine respiratory disease complex (BRDC) that is costly to the beef and dairy cattle industries. Both bacterial species are thought to occupy a similar niche as commensals in the upper respiratory tract. Many bacteria are thought to exist as biofilms in their hosts, perhaps in close proximity with other bacterial species. We previously showed that M. haemolytica forms biofilm on bovine respiratory epithelial cells in vitro. We are interested in the possibility that M. haemolytica and P. multocida co-exist as biofilms in the upper respiratory tract of cattle. In this study, we begin to explore this possibility by assessing the ability of M. haemolytica and P. multocida to form a biofilm on bovine respiratory epithelial cells in vitro. We found that M. haemolytica and P. multocida are separately able to form biofilms on bovine respiratory epithelial cells, but mutually inhibit one another when incubated together as a biofilm. Both the biofilm matrix (crystal violet stain) and bacterial numbers (CFU and PCR) were reduced when M. haemolytica and P. multocida were incubated together on fixed epithelial cells. This inhibition does not appear to result from a soluble factor, as neither conditioned medium nor separation of the two species by a transwell filter membrane reproduced the effect. We infer that when located in close proximity on the epithelial surface, M. haemolytica and P. multocida mutually regulate one another.

Mannheimia haemolytica biofilm formation on bovine respiratory epithelial cells.

Mannheimia haemolytica is the most important bacterial agent associated with the bovine respiratory disease complex (BRDC), which causes worldwide economic losses to the cattle industry. M. haemolytica cells initially colonize the tonsillar crypts in the upper respiratory tract of cattle, from where they can subsequently descend into the lungs to cause disease. Many bacteria exist as biofilms inside their hosts. We hypothesize that M. haemolytica colonization of cattle during its commensal state may include biofilm formation. To begin to assess this possibility, we developed an in vitro system to study biofilm formation directly on bovine respiratory epithelial cells. Using fixed primary bovine bronchial epithelial cells, we observed M. haemolytica biofilm formation after a 48h incubation period at 37°C. Addition of mucin, the main component of mucus present in the upper respiratory tract, decreased M. haemolytica biofilm formation on bovine epithelial cells. We investigated the effects of prior viral infection of the epithelial cells on subsequent biofilm formation by M. haemolytica and found negligible effects. Utilization of this model system will provide new insights into the potential role of biofilm formation by M. haemolytica in the pathogenesis of BRDC.

Mannheimia haemolytica and its leukotoxin cause macrophage extracellular trap formation by bovine macrophages.

Human and bovine neutrophils release neutrophil extracellular traps (NETs), which are protein-studded DNA matrices capable of extracellular trapping and killing of pathogens. Recently, we reported that bovine neutrophils release NETs in response to the important respiratory pathogen Mannheimia haemolytica and its leukotoxin (LKT). Here, we demonstrate macrophage extracellular trap (MET) formation by bovine monocyte-derived macrophages exposed to M. haemolytica or its LKT. Both native fully active LKT and noncytolytic pro-LKT (produced by an lktC mutant of M. haemolytica) stimulated MET formation. Confocal and scanning electron microscopy revealed a network of DNA fibrils with colocalized histones in extracellular traps released from bovine macrophages. Formation of METs required NADPH oxidase activity, as previously demonstrated for NET formation. METs formed in response to LKT trapped and killed a portion of the M. haemolytica cells. Bovine alveolar macrophages, but not peripheral blood monocytes, also formed METs in response to M. haemolytica cells. MET formation was not restricted to bovine macrophages. We also observed MET formation by the mouse macrophage cell line RAW 264.7 and by human THP-1 cell-derived macrophages, in response to Escherichia coli hemolysin. The latter is a member of the repeats-in-toxin (RTX) toxin family related to the M. haemolytica leukotoxin. This study demonstrates that macrophages, like neutrophils, can form extracellular traps in response to bacterial pathogens and their exotoxins.

The role of Mannheimia species in ovine mastitis.

Mannheimia haemolytica is known to be an important cause of intramammary infection in sheep. It usually causes severe clinical mastitis, followed by toxaemia and gangrenous necrosis of the udder. However there are limited data available on the epidemiology and pathogenesis of mastitis associated with Mannheimia species. These organisms can be more significant as a cause of mastitis than Staphylococcus aureus in some flocks. Some data suggest the possibility of horizontal transmission of Mannheimia species between ewes via lamb sucking. There is no vaccine available for prevention, and the sudden onset of mastitis and its peracute nature renders most treatments unsuccessful. This review examines the significance of the species within this genus in sheep mastitis.

Identification of Mannheimia haemolytica adhesins involved in binding to bovine bronchial epithelial cells.

Mannheimia haemolytica, a commensal organism of the upper respiratory tract in cattle, is the principal bacterial pathogen associated with the bovine respiratory disease complex. Adherence to the respiratory mucosa is a crucial event in its pathogenesis. However, the bacterial components that contribute to this process are not fully characterized. In this study, we demonstrated that M. haemolytica adhered to bovine bronchial epithelial cells (BBEC) in vitro and that adherence was inhibited by anti-M. haemolytica antibody. Western blot analysis of M. haemolytica proteins that bind to BBEC showed a dominant protein band with an apparent molecular mass of approximately 30 kDa. Peptide sequences for the 30-kDa BBEC-binding proteins, as determined by liquid chromatography-tandem mass spectrometry, matched two M. haemolytica surface proteins: heat-modifiable outer membrane protein A (OmpA) and lipoprotein 1 (Lpp1). Western blotting showed that the 30-kDa protein band is recognized by both anti-M. haemolytica OmpA and anti-Lpp1 antibodies. Furthermore, incubation with anti-OmpA and anti-Lpp1 antibodies significantly inhibited M. haemolytica binding to BBEC monolayers. In summary, these results suggest that OmpA and Lpp1 contribute to adherence of M. haemolytica to bovine respiratory epithelial cells.

Effects of lipopolysaccharide and Mannheimia haemolytica leukotoxin on bovine lung microvascular endothelial cells and alveolar epithelial cells.

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.

Mechanisms underlying Mannheimia haemolytica leukotoxin-induced oncosis and apoptosis of bovine alveolar macrophages.

Mannheimia (Pasteurella) haemolytica leukotoxin (LktA) binds to the bovine beta2 integrins (such as LFA-1-CD11a/CD18) and leads to subsequent cellular effects in a dose dependent manner. The objectives of this study were to delineate the mechanisms that underlie LktA-induced oncosis and apoptosis and to examine the role of LktA/LFA-1 interaction in these events. The results demonstrate that LktA-induced oncosis proceeds through a LFA-1 and caspase-1 dependent pathway referred to as 'pyrotosis', as well as through a LFA-1- and caspase-1-independent pathway. LktA-induced apoptosis in alveolar macrophages involves activation of caspase-3 and engages the extrinsic and intrinsic pathways of apoptosis, with the extrinsic pathway being dependent on LFA-1 signaling and TNFalpha.

Tilmicosin induces apoptosis in bovine peripheral neutrophils in the presence or in the absence of Pasteurella haemolytica and promotes neutrophil phagocytosis by macrophages.

Pathogen virulence factors and inflammation are responsible for tissue injury associated with respiratory failure in bacterial pneumonia, as seen in the bovine lung infected with Pasteurella haemolytica. Tilmicosin is a macrolide antibiotic used for the treatment of bovine bacterial pneumonia. Recent evidence suggests that tilmicosin-induced neutrophil apoptosis may have anti-inflammatory effects. Using bovine leukocytes, we sought to define whether live P. haemolytica affected tilmicosin-induced neutrophil apoptosis, assessed the proapoptotic effects of tilmicosin in comparison with other drugs, and characterized its impact on phagocytic uptake of neutrophils by macrophages. Induction of apoptosis in the presence or absence of P. haemolytica was assessed by using an enzyme-linked immunosorbent assay for apoptotic nucleosomes. In addition, fluorescent annexin-V staining identified externalized phosphatidylserine in neutrophils treated with tilmicosin, penicillin, ceftiofur, oxytetracycline, or dexamethasone. Neutrophil membrane integrity was assessed by using propidium iodide and trypan blue exclusion. As phagocytic clearance of apoptotic neutrophils by macrophages contributes to the resolution of inflammation, phagocytosis of tilmicosin-treated neutrophils by esterase-positive cultured bovine macrophages was assessed with light microscopy and transmission electron microscopy. Unlike bovine neutrophils treated with penicillin, ceftiofur, oxytetracycline, or dexamethasone, neutrophils exposed to tilmicosin became apoptotic, regardless of the presence or absence of P. haemolytica. Tilmicosin-treated apoptotic neutrophils were phagocytosed at a significantly greater rate by bovine macrophages than were control neutrophils. In conclusion, tilmicosin-induced neutrophil apoptosis occurs regardless of the presence or absence of live P. haemolytica, exhibits at least some degree of drug specificity, and promotes phagocytic clearance of the dying inflammatory cells.

Pasteurella haemolytica leukotoxin and endotoxin induced cytokine gene expression in bovine alveolar macrophages requires NF-kappaB activation and calcium elevation.

In bovine alveolar macrophages (BAMs), exposure to leukotoxin (Lkt) and endotoxin (LPS) from Pasteurella haemolytica results in expression of inflammatory cytokine genes and intracellular calcium ([Ca2+]i) elevation. Leukotoxin from P. haemolytica interacts only with leukocytes and platelets from ruminant species. Upregulation of cytokine genes in different cells by LPS involves activation of the transcription factor NF-kappaB (NF-kappaB), resulting in its translocation from the cytoplasm to the nucleus. Using immunocytochemical staining and confocal imaging, we studied whether NF-kappaB activation represents a common mechanism for the expression of multiple cytokine genes in BAMs (Lkt-susceptible cells) stimulated with Lkt and LPS. Bovine pulmonary artery endothelial cells and porcine alveolar macrophages were used as nonsusceptible cells. The role of Ca2+ and tyrosine kinases in NF-kappaB activation and inflammatory cytokine gene expression was studied, since an inhibitor of tyrosine kinases attenuates LPS-induced [Ca2+]i elevation in BAMs. The results are summarized as follows: (a) Lkt induced NF-kappaB activation and [Ca2+]i elevation only in BAMs, while LPS effects were demonstrable in all cell types; (b) chelation of [Ca2+]i blocked NF-kappaB activation and IL-1beta, TNFalpha, and IL-8 mRNA expression; and (c) tyrosine kinase inhibitor herbimycin A blocked expression of all three cytokine genes in BAMs stimulated with Lkt, while only the expression of IL-1beta was blocked in BAMs stimulated with LPS. We conclude that cytokine gene expression in BAMs requires NF-kappaB activation and [Ca2+]i elevation, and Lkt effects exhibit cell type- and species specificity.

Effects of copper deficiency and copper deficiency coupled with high dietary iron or molybdenum on phagocytic cell function and response of calves to a respiratory disease challenge.

A study was conducted to determine the effects of supplementing a diet marginally deficient in copper (Cu) with iron (Fe), molybdenum (Mo), or Cu on phagocytic cell function and disease resistance of calves. Thirty-one calves were born to heifers fed a corn silage-based diet containing 4.5 mg of Cu/kg. Treatments consisted of 1) control (CON; no supplemental Cu, Fe, or Mo), 2) 600 mg of Fe added/kg (FE), 3) 5 mg of Mo added/kg (MO), or 4) 10 mg of Cu added/kg of DM (CU). Activity of superoxide dismutase was lower (P < .06) in neutrophils from MO vs CON or CU calves at 170 d of age. bactericidal activity of neutrophils from MO calves tended (P = .15) to be lower compared with those from CU calves at 70 d of age. Calves were inoculated intranasally with live infectious bovine rhinotracheitis virus (IBRV) 2 d after weaning, followed by intratracheal administration of Pasteurella hemolytica 5 d later. Iron- and Cu-supplemented calves exhibited higher (P < .01) body temperatures and lower (P < .06) feed intakes following IBRV inoculation compared with CON and MO calves. Copper-supplemented calves had higher levels of plasma tumor necrosis factor (TNF) than MO calves at weaning (P < .05) and tended to have higher plasma TNF (P = .11) than FE and MO calves 5 d after IBRV inoculation. These data indicate that dietary levels of Mo and Cu can affect body temperature and feed intake responses to disease by affecting TNF and perhaps other cytokines.

Pneumonic pasteurellosis associated with Pasteurella haemolytica in chipmunks (Tamias sibiricus).

A laboratory study was performed in order to identify the possible cause of death in chipmunks (Tamias sibiricus) imported from China with respiratory disease. Severe congestion, alveolar oedema and fibrinous pleuritis were observed. Biochemical analyses identified the causative organism as Pasteurella haemolytica. An in vitro susceptibility test using various antimicrobial agents revealed sensitivity to beta-lactams (ampicillin and amoxicillin) and streptomycin.

Pulmonary ventilation, mechanics, gas exchange and haemodynamics in calves following intratracheal inoculation of Pasteurella haemolytica.

A Pasteurella haemolytica A1 broth was injected intratracheally in eight calves and measurements of pulmonary function values (PFV) were made once before and hourly post inoculation (p.i.). Changes in PFVs, included increased respiratory rate and minute ventilation (up to 158% of baseline 2 h p.i.) and decreased tidal volume and lung dynamic compliance (up to 33% of baseline 3 h p.i.). Total pulmonary resistance was not affected. At and after 3 h p.i. there was a progressive impairement of gas exchange, as judged from arterial O2 tension which decreased up to 65% of baseline. In contrast, arterial CO2 tension was not affected. Pulmonary hypertension was observed during the 3 last h of the study and was attributable to an increased pulmonary vascular resistance. Severe neutropenia was observed at 3 h p.i. and post-mortem histological findings were consistent with an acute fibrinohemorragic bronchopneumonia. In conclusion, P. haemolytica airway challenge unequiovocally resulted in acute pneumonia, providing a reproducible pathophysiological model for investigations regarding new therapeutic strategies.

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

The bovine respiratory pathogen Pasteurella haemolytica secretes an exotoxin that is specific for ruminant leukocytes (leukotoxin). Previous studies have shown that subcytolytic concentrations of the leukotoxin stimulate bovine neutrophils to undergo a respiratory burst and degranulate. Relatively little is known about the stimulatory effects of the leukotoxin on bovine mononuclear phagocytes. In this study, we compared the relative cytolytic effects of partially purified leukotoxin on bovine peripheral blood monocytes and alveolar macrophages. We found monocytes to be approximately 8- to 10-fold more sensitive than alveolar macrophages to the cytolytic effect of leukotoxin. In addition, incubation of monocytes and alveolar macrophages with sublethal doses of leukotoxin stimulated release of IL-1 and TNF activities in a dose-dependent manner. Addition of an antileukotoxin MAb neutralized the cytolytic effects of leukotoxin, but potentiated TNF release. Heat inactivation also blocked the cytolytic activity of LKT, but only slightly reduced its ability to stimulate TNF release. Although the leukotoxin preparations were estimated to have only small amounts of lipopolysaccharide (LPS) contamination, as determined by a standard Limulus amebocyte lysate coagulation assay, a chromogenic Limulus assay indicated much greater amounts of LPS were present. Adding equivalent doses of P. haemolytica LPS largely duplicated the monokine release stimulated by leukotoxin. These results suggest that the stimulatory effects of the P. haemolytica leukotoxin on bovine mononuclear phagocytes may principally involve LPS, perhaps complexed with leukotoxin.

Alterations in pulmonary morphology and peripheral coagulation profiles caused by intratracheal inoculation of live and ultraviolet light-killed Pasteurella haemolytica A1 in calves.

Eighteen male Holstein calves were divided into groups of three and inoculated intratracheally with 5 x 10(9) logarithmic phase or ultraviolet light-killed Pasteurella haemolytica biotype A serotype 1. Serial coagulation profiles were done on one calf from each group during the first 24 hours after inoculation. One calf from each group was necropsied at 4, 12, and 24 hours after inoculation and lesions were characterized with light and transmission electron microscopy. We found that 1) the pulmonary intravascular macrophage may have an important role in the early intravascular inflammatory events; 2) there was morphologic evidence for local initiation of the coagulation cascade in the lung early in the disease process but it was not a consumptive process; and 3) killed-bacteria were capable of causing fibrin exudation, platelet aggregation and alveolar epithelial damage similar to live bacteria, but the degenerative changes in neutrophils, endothelial cells and intravascular fibrin formation that occur with live bacteria were not seen.