Molecular cloning of CD18 of bison, deer and elk, and comparison with that of other ruminants and non-ruminants.

Pneumonia caused by Mannheimia haemolytica is an important disease of cattle, domestic sheep, bighorn sheep and goats. Leukotoxin (Lkt) produced by M. haemolytica is cytolytic to all leukocyte subsets of these species. Lkt utilizes CD18, the beta subunit of beta(2)-integrins, as its functional receptor on leukocytes of these species. Cytotoxicity assays revealed that leukocytes from bison, deer, and elk are also susceptible to Lkt-induced cytolysis. The availability of cDNA encoding CD18 of bison, deer and elk would facilitate the comparison of a greater number of ruminant CD18 cDNA with that of non-ruminants as a means of the elucidation of the molecular basis for the specificity of M. haemolytica Lkt for ruminant leukocytes. Herein, we report the cloning and characterization of bison, deer, and elk CD18. The full-length cDNA of bison and deer consists of 2310bp with an ORF encoding 769 amino acids while elk CD18 consists of 2313bp with an ORF encoding 770 amino acids. This gene is highly conserved among ruminants compared with non-ruminants. Phylogenetic analysis based on amino acid sequences showed that CD18 of bison is most closely related to that of cattle while CD18 of deer and elk are more closely related to each other.

Bovine herpesvirus type 1 infection of bovine bronchial epithelial cells increases neutrophil adhesion and activation.

Respiratory infection of cattle with bovine herpesvirus type 1 (BHV-1) predisposes cattle to secondary pneumonia with Mannheimia haemolytica as part of the bovine respiratory disease complex (BRD). One cell type that has received limited investigation for its role in the inflammation that accompanies BRD is the respiratory epithelial cell. In the present study we investigated mechanisms by which BHV-1 infection of respiratory epithelial cells contributes to the recruitment and activation of bovine polymorphonuclear neutrophils (PMNs) in vitro. Primary cultures of bovine bronchial epithelial (BBE) cells were infected with BHV-1 and assessed for cytokine expression by real-time PCR. We found that BHV-1 infection elicits a rapid IL-1, IL-8 and TNF-alpha mRNA response by BBE cells. Bovine PMNs exhibited greater adherence to BHV-1 infected BBE cells than uninfected cells. The increased adherence was significantly reduced by the addition of an anti-IL-1beta antibody or human soluble TNF-alpha receptor (sTNF-alphaR). Pre-incubation of bovine PMNs with conditioned media from BHV-1 infected BBE cells increased PMN migration, which was inhibited by addition of an anti-IL-1beta antibody, sTNF-alphaR, or an IL-8 peptide inhibitor. Conditioned media from BHV-1 infected BBE cells activated bovine PMNs in vitro as demonstrated by PMN shape change, production of reactive oxygen species and degranulation. PMNs also exhibited increased LFA-1 expression and susceptibility to M. haemolytica LKT following incubation with BHV-1 infected BBE cell conditioned media. Our results suggest that BHV-1 infection of BBE cells triggers cytokine expression that contributes to the recruitment and activation of neutrophils, and amplifies the detrimental effects of M. haemolytica LKT.

Fatal pneumonia epizootic in musk ox (Ovibos moschatus) in a period of extraordinary weather conditions.

The musk ox is adapted to extreme cold and regarded as vulnerable to the impacts of climate change. Population decline is proposed to occur due to changes in forage availability, insect harassment, parasite load, and habitat availability, while the possible role of infectious diseases has not been emphasized. The goal of the present article is to describe an outbreak of fatal pasteurellosis that occurred in the introduced musk ox population of Dovrefjell, Norway in 2006, causing the death of a large proportion of the animals. The epizootic coincided with extraordinary warm and humid weather, conditions that often are associated with outbreaks of pasteurellosis. The description is based on long series of data from the surveillance of the musk ox population, weather data from a closely located meteorological station, and pathoanatomical investigation of the diseased animals. It is concluded that the weather conditions likely were the decisive factors for the outbreak. It is suggested that such epizootics may occur increasingly among cold-adapted animals if global warming results in increased occurrence of heat waves and associated extreme weather events, thereby causing population declines and possibly extinctions.

Impairment of innate immune responses of airway epithelium by infection with bovine viral diarrhea virus.

Bovine viral diarrhea virus (BVDV) infection is an important risk factor for development of shipping fever pneumonia in feedlot cattle, and infects but does not cause morphologic evidence of damage to airway epithelial cells. We hypothesized that BVDV predisposes to bacterial pneumonia by impairing innate immune responses in airway epithelial cells. Primary cultures of bovine tracheal epithelial cells were infected with BVDV for 48 h, then stimulated with LPS for 16 h. Expression of tracheal antimicrobial peptide (TAP) and lingual antimicrobial peptide (LAP) mRNA was measured by quantitative RT-PCR, and lactoferrin concentrations were measured in culture supernatant by ELISA. BVDV infection had no detectable effect on the constitutive expression of TAP and LAP mRNA or lactoferrin concentration in culture supernatant. LPS treatment provoked a significant increase in TAP mRNA expression and lactoferrin concentration in the culture supernatant (p<0.01), and these effects were significantly (p<0.02, p<0.01) abrogated by prior infection of the tracheal epithelial cells with the type 2 ncp-BVDV isolate. In contrast, infection with the type 1 ncp-BVDV isolate had no effect on TAP mRNA expression or lactoferrin secretion. LPS treatment induced a significant (p<0.001) upregulation of LAP mRNA expression, which was not significantly affected by prior infection with BVDV. These data indicate that infection with a type 2 BVDV isolate inhibits the LPS-induced upregulation of TAP mRNA expression and lactoferrin secretion by tracheal epithelial cells, suggesting a novel mechanism by which this virus abrogates respiratory innate immune responses and predisposes to bacterial pneumonia in cattle.

Efficacy of tulathromycin compared with tilmicosin and florfenicol for the control of respiratory disease in cattle at high risk of developing bovine respiratory disease.

Three studies conducted at feedlots in Colorado, Idaho, and Texas examined the comparative efficacy of tulathromycin injectable solution for the treatment of cattle at high risk of developing undifferentiated bovine respiratory disease (BRD). Each study randomly allocated 250 calves to receive tulathromycin at 2.5 mg/kg and 250 calves to receive either tilmicosin at 10 mg/kg (Colorado site) or florfenicol at 40 mg/kg (Idaho and Texas sites) on arrival at the feedlot. Calves were housed by treatment group in pens with 50 calves/pen. Beginning 3 days after antimicrobial treatment, cattle were observed for signs of BRD daily until harvest. In all three studies, the treatment success rates at 28 days after treatment and at harvest were significantly higher (P < or = .013) for cattle treated with tulathromycin than for cattle treated with either tilmicosin or florfenicol. Fewer tulathromycin-treated cattle were removed from the group as "chronics" or "mortalities" at 28 days posttreatment (P < or = .014) in all three studies. Tulathromycin demonstrated superior efficacy compared with tilmicosin and florfenicol when treating groups of high-risk cattle before the onset of signs of BRD.

Clinical effectiveness of tulathromycin, a novel triamilide antimicrobial, for the control of respiratory disease in cattle at high risk for developing bovine respiratory disease.

The efficacy of tulathromycin in decreasing the incidence of morbidity and mortality due to bovine respiratory disease (BRD) in 1,239 high-risk cattle was investigated at four US feedlots. Calves not exhibiting clinical signs of BRD received one of three treatments administered subcutaneously in the neck: physiologic saline at 0.02 ml/kg, tulathromycin at 2.5 mg/kg, or tilmicosin at 10 mg/kg. Each treatment group consisted of 413 calves. Calves with clinical signs of BRD and rectal temperatures of 104 degrees F or higher on days 1 through 14 were considered treatment failures (BRD morbidity). Nasopharyngeal swabs from saline-treated morbidities were submitted for isolation and identification of BRD organisms. Respiratory disease morbidity was highest in calves treated with saline and significantly (P < or = .0001) lower in calves administered tulathromycin or tilmicosin. Morbidity from BRD was significantly (P < or = .0001) higher in calves treated with tilmicosin than in calves treated with tulathromycin. Under conditions of this study, tulathromycin, given to calves at high risk of developing BRD, was significantly more effective in reducing BRD morbidity when compared to both saline- and tilmicosin-treated calves.

[Mannheimia haemolytica and the pathogenesis of enzootic bronchopneumonia]. / Mannheimia haemolytica und die Pathogenese der Enzootischen Bronchopneumonie.

Mannheimia (M.) haemolytica (formerly Pasteurella [P.] haemolytica) is the primary aetiological agent of pneumonic pasteurellosis--one of the most important respiratory diseases in cattle and sheep. While bovine pneumonic pasteurellosis is regarded to be mainly caused by M. haemolytica serotype A1, and in Germany during the last years also by serotype A6, sheep can be infected by all serotypes although there is an increased prevalence of serotypes A2 and A5-7. The obligate pathogenicity of M. haemolytica is proven by isolation of pure cultures from pneumonic lungs as well as by infection studies. Knowledge about the virulence mechanisms of M. haemolytica and their molecular basis are fragmentary, most probably due to the complex gene regulation of virulence associated factors in lung tissues. This review summarizes the current literature covering virulence factors to substantiate a model of pathogenesis. After serotype A1 strains have colonized the bovine upper respiratory tract they replace other serotypes by mechanisms unknown to date. After fulminant proliferation in the upper respiratory tract the microorganisms colonize the lower respiratory tract, finally entering alveolar spaces. An inflammatory cascade is initiated by M. haemolytica LPS and Leukotoxin, causing activation of the complement system and release of cytokines. Pathognomonic for bovine pneumonic pasteurellosis is the strong influx of neutrophiles accompanied by accumulation of fibrin, finally causing necrosis of alveolar spaces. Depending on lesion size this fibronecrotizing pneumonia can result in death of the animals. In addition, possible protective antigens are discussed. There is still a great effort in the development of efficacious vaccines against pneumonic pasteurellosis in cattle and sheep caused by various M. haemolytica serotypes worldwide. The scarce knowledge concerning presence and distribution of virulence associated factors in M. haemolytica strains and their role in pathogenesis made it difficult to determine a suitable vaccine candidate in the past. In addition, there is lack of knowledge concerning the variability of virulence factors in individual isolates. Genome sequence analysis of M. haemolytica, enabling proteomics and transciptomics, hopefully will give new insight into the pathogenesis of pneumonic pasteurellosis.

Antibody responses of cattle with respiratory coronavirus infections during pathogenesis of shipping fever pneumonia are lower with antigens of enteric strains than with those of a respiratory strain.

The serum antibody responses of cattle with respiratory coronavirus infections during the pathogenesis of shipping fever pneumonia were analyzed with different bovine coronavirus antigens, including those from a wild-type respiratory bovine coronavirus (RBCV) strain (97TXSF-Lu 15-2) directly isolated from lung tissue from a fatally infected bovine, a wild-type enteropathogenic bovine coronavirus (EBCV) strain (Ly 138-3), and the highly cell culture-adapted, enteric prototype strain (EBCV L9-81). Infectivity-neutralizing (IN) and hemagglutinin-inhibiting (HAI) activities were tested. Sequential serum samples, collected during the onset of the respiratory coronavirus infection and at weekly intervals for 5 weeks thereafter, had significantly higher IN and HAI titers for antigens of RBCV strain 97TXSF-Lu15-2 than for the wild-type and the highly cell culture-adapted EBCV strains, with P values ranging from <0.0001 to 0.0483. The IN and HAI antibody responses against the two EBCV strains did not differ significantly, but the lowest titers were detected with EBCV strain L9-81.

Role of Mannheimia haemolytica leukotoxin in the pathogenesis of bovine pneumonic pasteurellosis.

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.

Ultrastructural study of ovine pulmonary pasteurellosis: involvement of neutrophils and macrophages.

Pasteurellosis is a common infectious disease characterised by fibrinous pneumonia and involving neutrophils and macrophages. This study aimed to determine the timing and extent of the pathogenic involvement of these cell elements in lesions induced in experimentally-infected lambs. A concentration of approximately 3x10(8) bacteria/ml. was inoculated into 30 two-month-old disease-free Merino lambs. Five lambs were assigned to each of five experimental batches, slaughtered on days 1, 3, 7, 11 and 15 following intratracheal inoculation, and to one control batch inoculated with a sterile solution. One control animal was slaughtered at the same time as each experimental batch. More characteristic lesions occur in bronchioles, peribronchial tissue and alveoli and are characterised by fibrinous processes. From the start of the experiment, epithelial-cell disruption and loss of microvilli were apparent; cell debris, desquamate cells and bacterial elements were observed in bronchiolar lumina, embedded in a fibrillar granular material. Alveolar structures displayed fewer neutrophils and macrophages, containing phagocytic vacuoles. Laminar bodies were apparent in type II pneumocytes. The interseptal area contained similar cell types, as well as abundant edema. In the course of the experiment, macrophage numbers increased in all the areas involved, with signs of intense phagocytic activity. The final phase of the experiment was characterised by a mild interseptal infiltrate and by clear alveolar lumina.

Mast cell density and substance P-like immunoreactivity during the initiation and progression of lung lesions in ovine Mannheimia (Pasteurella) haemolytica pneumonia.

To determine the density of mast cells (MCs) and the extent of substance P (SP) immunoreactivity during initiation and progression of pneumonic pasteurellosis (PP), 18 lambs were inoculated intrabronchially with Mannheimia (Pasteurella) haemolytica or saline, and lung tissue was collected at 1, 15 and 45 days post-inoculation (n=3, each group). Additionally, the left (non-inoculated) contralateral lungs in bacteria-inoculated animals were collected as controls. At 1 day after bacterial inoculation the lungs had typical M. haemolytica lesions. These pneumonic lesions had fewer numbers of MCs and reduced histamine content. Macrophages infiltrating some of the inflamed areas were strongly immunoreactive for SP. At 15 days, MCs remained scarce at sites where lung damage persisted, i.e. pyogranulomatous foci, but were increased in number in areas of interstitial damage. Pulmonary ganglion neurons were strongly immunoreactive for SP. By 45 days the fibrosing changes became more defined as pleural fibrosis, fibrosing alveolitis, alveolar epithelial hyperplasia and bronchiolitis obliterans. These lungs had increased numbers of MCs, but histamine content was not different from saline- and non-inoculated left lungs. Substance P immunoreactivity occurred only in nerves and was scarce and mild. This work demonstrates that MC density decreases initially with PP, but increases with progression of PP. SP fibres tend to be decreased during the initiation and at 45 days of PP, but other cells, such as macrophages and neuronal ganglion cells, produce substance P during progression of PP and thereby constitute an additional source of substance P.

Lipopolysaccharide enhances cytolysis and inflammatory cytokine induction in bovine alveolar macrophages exposed to Pasteurella (Mannheimia) haemolytica leukotoxin.

Pasteurella (Mannheimia) haemolytica leukotoxin (Lkt) and lipopolysaccharide (LPS) are the primary virulence factors contributing to the pathogenesis of lung injury in bovine pneumonic pasteurellosis. Previous studies have characterized in vitro responses of bovine alveolar macrophages (AMs) to Lkt and LPS. Activation of AMs with Lkt or LPS causes induction of proinflammatory cytokines, and Lkt causes cytolysis of AMs at higher concentrations. Since AMs are exposed to both of these bacterial virulence factors during disease, previous studies may have underestimated the possibility of functional interactions between Lkt and LPS. The purpose of this study was to characterize the effect of simultaneous exposure to both Lkt and LPS on AM cytolysis and proinflammatory cytokine expression. Using cellular leakage of lactate dehydrogenase as an indirect measure of cytolysis, we studied AM responses to Lkt alone, LPS alone and Lkt+LPS. We found that 80-200 pg/ml LPS, which does not itself cause cytolysis, synergistically enhanced the cytolysis induced by 2-5 Lkt units (LU)/ml Lkt. Northern blot analysis demonstrated that synergism between Lkt and LPS resulted in increased levels of IL-8 mRNA, and that the kinetic patterns of TNF-alpha and IL-8 mRNA expression induced by Lkt+LPS differed from those induced by each agent separately. Finally, the WEHI 164 (clone 13) bioassay was used to show that Lkt/LPS synergism resulted in enhanced secretion of biologically active TNF-alpha. These results provide direct evidence of synergism between Lkt and LPS in AM cytolysis and inflammatory cytokine expression. Additional studies to characterize the molecular basis of this phenomenon are indicated.

Conglutinin and immunoconglutinin titers in stressed calves in a feedlot.

OBJECTIVE: To determine whether increased conglutinin titers are evident in stressed calves that do not develop respiratory tract disease in feedlots, compared with respiratory tract disease, and to determine the increase in immunoconglutinin titers. ANIMALS: 101 mixed-breed beef calves. PROCEDURE: Calves were processed at 4 farms of origin and allowed to remain with their dams for another 100 days. Calves from each farm were brought to a centrally located order-buyer barn. In a feedlot, 101 calves were assigned to pens and observed daily for clinical signs of acute respiratory tract disease. When sick calves were detected, they were treated with antibiotics and isolated in a pen for 4 days. Conglutinin and immunoconglutinin titers were determined for all calves. RESULTS: During the 28-day study, 73 calves developed respiratory tract disease, whereas 28 calves remained healthy. Mean conglutinin titers differed significantly among calves from the 4 farms. Significant differences were not detected in conglutinin titers among calves on the basis of sex, morbidity, or vaccination status against Mannheimia haemolytica at each farm, the order-buyer barn, or the feedlot on days 8, 15, and 28 after arrival. Immunoconglutinin titers in calves differed significantly among farms and morbidity status. CONCLUSIONS AND CLINICAL RELEVANCE: Mean conglutinin titers in calves do not appear to be associated with the incidence of acute respiratory tract disease; however, increased immunoconglutinin titers appear to be associated with recovery of stressed calves from respiratory tract disease during the first 15 days after arrival in a feedlot.

The potential role of the Arthus and Shwartzman reactions in the pathogenesis of pneumonic pasteurellosis.

Pneumonic pasteurellosis (PP) is an economically important disease in cattle, sheep, and goats. Pasteurella haemolytica is commonly isolated from the severe fibrinopurulent pneumonia that characterize this respiratory syndrome. During infection, the bacteria produce leukotoxin (LKT) and lipopolysaccharide (LPS), both potent inducers of inflammation. Nonetheless, it has also been demonstrated that an exacerbated host's inflammatory response is responsible for the severe lung damage. Despite research in this field, the pathogenesis of PP is still incomplete. Two classical models of acute inflammatory response induced in laboratory animals, the Arthus and Shwartzman reactions, could explain the pathogenesis of the severe lung lesions that characterize PP.

A review of the road transport of cattle.

The transport of farm animals has been the subject of much research in recent years. This paper reviews the recent scientific literature pertinent to the road transport of cattle. The state of knowledge is summarised and recommendations for best practice based upon this are given. Areas which require further work are also identified.

Pasteurella haemolytica complicated respiratory infections in sheep and goats.

Respiratory infections which commonly occur in sheep and goats often result from adverse physical and physiological stress combined with viral and bacterial infections. Inevitably, Pasteurella haemolytica pneumonia occurs as a result of these interactions. In this review, we present recent advances in research on the complex etiology of pneumonia involving P. haemolytica. Initially stress, induced by factors such as heat, overcrowding, exposure to inclement weather, poor ventilation, handling and transport is a major predisposing factor. Respiratory viruses including parainfluenza 3 (PI-3) virus, adenovirus type 6 and respiratory syncytial virus (RSV), and to a lesser extent bovine adenovirus type 2, ovine adenovirus types 1 and 5, and reovirus type 1 cause respiratory infections and pneumonia. More importantly these viruses also dramatically increase the susceptibility of sheep and goats to secondary P. haemolytica infection. Primary infection of the lower respiratory tract, with Mycoplasma ovipneumoniae and Bordetella parapertussis can increase the susceptibility of sheep and goats to secondary P. haemolytica infection. It is possible that initial infections with viral or primary bacterial agents break down the antimicrobial barrier consisting of beta defensins and anionic peptides found in epithelial cells, resident and inflammatory cells, and serous and mucous secretions of the respiratory tract. Loss of barrier integrity may release P. haemolytica from its usual commensal status. Once in the lung, P. haemolytica becomes opportunistic. To grow and colonize, P. haemolytica uses extracellular products like O-sialoglycoprotein endopeptidase, neuraminidase and RTX leukotoxin, as well as cell-associated products such as capsular polysaccharide, lipopolysaccharide, outer membrane proteins, proteins involved in iron acquisition and a periplasmic superoxide dismutase. In lambs and kids, pneumonic pasteurellosis can be acute, characterized by fever, listlessness, poor appetite and sudden death. Sheep and goats that survive the acute stage may recover or become chronically affected showing reduced lung capacity and weight gain efficiency and sporadic deaths may occur. This infection is detrimental to sheep and goats throughout the world and flocks and herds of small ranches, dairy operations, or large feedlots are all affected.

Pasteurella haemolytica A1-derived leukotoxin and endotoxin induce intracellular calcium elevation in bovine alveolar macrophages by different signaling pathways.

Leukotoxin and endotoxin derived from Pasteurella haemolytica serotype 1 are the primary virulence factors contributing to the pathogenesis of lung injury in bovine pneumonic pasteurellosis. Activation of bovine alveolar macrophages with endotoxin or leukotoxin results in the induction of cytokine gene expression, with different kinetics (H. S. Yoo, S. K. Maheswaran, G. Lin, E. L. Townsend, and T. R. Ames, Infect. Immun. 63:381-388, 1995; H. S. Yoo, B. S. Rajagopal, S. K. Maheswaran, and T. R. Ames, Microb. Pathog. 18:237-252, 1995). Furthermore, extracellular Ca2+ is required for leukotoxin-induced cytokine gene expression. However, the involvement of Ca2+ in endotoxin effects and the precise signaling mechanisms in the regulation of intracellular Ca2+ by leukotoxin and endotoxin are not known. In fura-2-acetoxymethyl ester-loaded alveolar macrophages, intracellular Ca2+ regulation by leukotoxin and endotoxin was studied by video fluorescence microscopy. Leukotoxin induced a sustained elevation of intracellular Ca2+ in a concentration-dependent fashion by influx of extracellular Ca2+ through voltage-gated channels. In the presence of fetal bovine serum, endotoxin elevated intracellular Ca2+ even in the absence of extracellular Ca2+. Leukotoxin-induced intracellular Ca2+ elevation was inhibited by pertussis toxin, inhibitors of phospholipases A2 and C, and the arachidonic acid analog 5,8,11,14-eicosatetraynoic acid. Intracellular Ca2+ elevation by endotoxin was inhibited by inhibitors of phospholipase C and protein tyrosine kinase, but not by pertussis toxin, or the arachidonic acid analog. To the best of our knowledge, this is the first report of Ca2+ signaling by leukotoxin through a G-protein-coupled mechanism involving activation of phospholipases A2 and C and release of arachidonic acid in bovine alveolar macrophages. Ca2+ signaling by endotoxin, on the other hand, involves activation of phospholipase C and requires tyrosine phosphorylation. The differences in the Ca2+ signaling mechanisms may underlie the reported temporal differences in gene expression during leukotoxin and endotoxin activation.

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

Allelic replacement was used to generate two isogenic lktA deletion mutants of Pasteurella haemolytica serotype 1 that were incapable of synthesizing leukotoxin (Lkt). Southern blot data confirmed that lktA sequences were absent in the two P. haemolytica deletion mutants. Culture supernatants and whole cell lysates from the wild type P. haemolytica, D153 parent strain, but not the lktA deletion mutants, contained immunoreactive and bioactive leukotoxic protein. In addition, only the parent strain was haemolytic when grown on bovine and sheep blood agar plates. Virulence of the lktA deletion mutant, lktA 77, was compared with the parent in an experimentally infected calf model of pneumonic pasteurellosis. Results revealed significant reduction in virulence in the lktA mutant as measured by clinical and lung lesion scores. Notable differences in histological changes such as markedly reduced necrosis and lack of leukocyte degeneration occurred in calves infected with the lktA mutant in comparison with those infected with the parent wild-type strain. Thus, it appears that leukotoxin plays a important role in the pathogenesis of lung injury in bovine pneumonic pasteurellosis.

Treatment of experimentally induced pneumonic pasteurellosis of young calves with tilmicosin.

Twenty four (24) healthy male Holstein calves (< 70 kg) were each experimentally infected by intrabronchial inoculation of 4.0 x 10(9) viable cells of Pasteurella haemolytica-AI (B122) at Time = 0 h. At 1 h following inoculation animals received either: 1) Sham treatment with sterile 0.85% saline SC (n = 12); or 2) a single injection of 10 mg tilmicosin per kg body weight (n = 12). Calves that were non-infected and tilmicosin-treated were also included for determining tilmicosin concentrations in serum and lung tissue at 1, 2, 4, 6, 8, 24, 48, and 72 h (n = 3-per time). In the infected calves, response to therapy was monitored clinically. Serum samples were collected for determination of tilmicosin concentrations using HPLC. Any animal becoming seriously ill was humanely killed. Complete necropsy examinations were performed on all animals and included gross pathologic changes, bacteriologic analysis, histopathology, and determination of pulmonary concentrations of tilmicosin. Tilmicosin treated animals responded significantly better to therapy than saline-treated control calves. Clinical assessment of calves during the study indicated that tilmicosin-treated calves had significantly improved by T = 8 h compared to satine-treated animals (P < 0.05). At necropsy tilmicosin-treated calves had significantly less severe gross and histological lesions (P < 0.05) of the pulmonary tissue. Of the 12 saline-treated calves, 92% (11/12) had Pasteurella haemolytica-A1 in lung tissue, while of the tilmicosin-treated calves 0% (0/12) cultured positive for P. haemolytica. Mean (+/- standard error) serum tilmicosin concentrations in infected calves peaked at 1 h post-injection (1.10 +/- 0.06 micrograms/mL) and rapidly decreased to 0.20 +/- 0.03 microgram/mL, well below the MIC of 0.50 microgram/mL for P. haemolytica-A1 (B122), by 12 h. These serum concentrations were very similar to serum concentrations of tilmicosin in non-infected tilmicosin-treated calves. Lung tissue concentrations of the antibiotic were comparatively high, even at 72 h post-infection (6.50 +/- 0.75 ppm). Lung tissue concentrations at 72 h were significantly higher in experimentally infected calves than in non-infected tilmicosin-treated animals (P < 0.05). These data demonstrate that tilmicosin was effective in treating experimentally-induced pneumonic pasteurellosis as determined by alleviation of clinical signs, pathological findings at post mortem, and presence of viable bacteria from the lung. Concentrations substantially above MIC for P. haemolytica were present in lung tissue even at 72 h following a single subcutaneous injection of 10 mg tilmicosin per kg body weight.

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

Infection of the bovine lung with Pasteurella haemolytica results in an acute respiratory disorder known as pneumonic pasteurellosis. One of the key virulence determinants used by this bacterium is secretion of an exotoxin that is specific for ruminant leukocytes (leukotoxin). At low concentrations, the leukotoxin can activate ruminant leukocytes, whereas at higher concentrations, it inhibits leukocyte functions and is cytolytic, presumably as a result of pore formation and subsequent membrane permeabilization. We have investigated the possibility that the activation-inhibition paradox is explained in part by leukotoxin-mediated apoptosis (i.e., activation-induced cell death) of bovine leukocytes. Incubation of bovine leukocytes with P. haemolytica leukotoxin caused marked cytoplasmic membrane blebbing (zeiosis) and chromatin condensation and margination, both of which are hallmarks of apoptosis. The observed morphologic changes in bovine leukocytes were leukotoxin dependent, because they were significantly diminished in the presence of an anti-leukotoxin monoclonal antibody. In addition, bovine leukocytes incubated with culture supernatant from a mutant strain of P. haemolytica that does not produce any detectable leukotoxin failed to exhibit the morphologic changes characteristic of cells undergoing apoptosis. These observations may represent an important mechanism by which P. haemolytica overwhelms host defenses, contributing to the fibrinous pleuropneumonia characteristic of bovine pasteurellosis.