Vaccination studies against experimental bovine Pasteurella pneumonia.

Vaccination-challenge experiments were conducted in colostrum-deprived calves to evaluate the efficacy of Pasteurella bacterins and vaccines against experimental pneumonic pasteurellosis. Calves were vaccinated with formalin-killed bacterins and live vaccines, then challenge exposed intratracheally with P. haemolytica or P. multocida. Infectious bovine rhinotracheitis virus was inoculated intranasally three to four days prior to P. haemolytica challenge-exposure. All calves were examined for macroscopic and microscopic lesions after being found dead or following euthanasia four to seven days after challenge exposure with the bacterial pathogen. Clinical, hematological, and pathological responses to challenge exposure in aluminum hydroxide absorbed P. haemolytica and P. multocida bacterin-treated calves were consistent with the pneumonic lesions of pulmonary pasteurellosis in the control calves. An oil-adjuvanted P. haemolytica bacterin limited clinical and pathological responses in the affected calves whereas a P. multocida oil-adjuvanted bacterin did not. Both clinical and pathological responses to challenge exposure in calves vaccinated with live Pasteurella vaccines were less severe than those of the control calves. Vaccine effectiveness appeared to be dose dependent.     

Characterization of a Pasteurella multocida (serotype B) bovine pneumonic pasteurellosis model and the effect of antimicrobials during peracute infection

A method to produce bovine pneumonic pasteurellosis for experimental purposes was studied and the clinical response of experimentally infected calves to selected antimicrobials was characterized. Male Holstein calves stressed with multiple hot and cold water applications followed by intratracheal inoculation of broth cultures of Pasteurella multocida serotype B developed acute clinical illness consistent with pneumonia. Infected, untreated calves consistently developed classic pneumonic pasteurellosis, infected calves treated with either oxytetracycline or sulfadimethoxine recovered from acute clinical disease, and the uninfected controls remained healthy. This disease model offers potential for use in pharmacokinetic and target tissue drug concentration studies and for dosage titration of drugs intended for treatment of bacterial pneumonias.     

Induction of immunity against pneumonic pasteurellosis following experimental infection in calves.

Immunity against pneumonic pasteurellosis was studied in calves after recovery from experimental respiratory disease with Pasteurella haemolytica. Nine calves were exposed to aerosols of parainfluenza-3 virus and Pasteurella haemolytica A1 six days apart to produce respiratory disease. After recovery from the disease, these nine principal and four control calves were challenged with aerosols of bovine herpesvirus 1 and P. haemolytica A1 four days apart. With this viral-bacterial challenge, the nine principal animals failed to develop clinical responses to this bacterial challenge and their lungs did not show the growth of P. haemolytica on cultures, whereas two of four control calves had elevated temperatures and developed necropurulent pneumonia with the isolation of P. haemolytica from the lungs. The principal calves had developed high levels of cytotoxin neutralizing antibodies in their sera following parainfluenza-3 virus-P. haemolytica infection. This demonstrated that immunity against pneumonic pasteurellosis can be achieved, with a suggestion that further search for an effective vaccine for P. haemolytica is warranted.     

Characteristics of live bovine herpesvirus-1 vaccines

The common disease caused by bovine herpes virus 1 infection is febrile rhinotracheitis (FRT) and under certain conditions the virus is strongly implicated in pre-disposing cattle to pneumonic pasteurellosis. These illnesses account for a significant economic loss in the cattle industry worldwide and vaccination is widely applied. In naive cattle, and for a short period of time, old and new vaccines significantly reduce clinical signs of FRT and also virus shedding. A single intranasal vaccination affords significant protection in face of maternally derived antibodies, and the protection can be significantly prolonged by a booster intramuscular vaccination. Current data recommend vaccination in face of an outbreak and vaccines appear safe for pregnant cattle.     

Use of an indwelling bronchial catheter model of bovine pneumonic pasteurellosis for evaluation of therapeutic efficacy of various compounds.

A model of bovine pneumonic pasteurellosis, using an indwelling bronchial catheter for inoculation and subsequent lavage of a single main stem bronchus of the lung, was evaluated in a preliminary efficacy trial of an experimental therapeutic compound. Inoculation of 10(7) Pasteurella haemolytica organisms into the bronchus consistently induced a focal pneumonic lesion with typical morphology of pneumonic pasteurellosis in the left or right caudal lung lobe. The experimental treatment caused significant (P less than 0.05) reduction in lung lesion volume, compared with that of a saline-treated control. It also caused significant (P less than 0.05) reduction in lavage fluid bacterial counts at 48 hours after inoculation, compared with counts in the controls. The inflammatory cell count and the percentage of neutrophils increased markedly in lavage fluids 8 hours after inoculation, but differences were not detected between treatments. Significant differences between treatments were not found in clinical signs, rectal temperature, or histologic changes. This model appears to be a sensitive indicator of treatment efficacy and has the advantage over previous models of pneumonic pasteurellosis of allowing sequential monitoring of the primary lesion site.     

Comparison of serologic and protective responses induced by two Pasteurella vaccines.

Vaccine development for the prevention of pneumonic pasteurellosis remains a critical issue for the feedlot industry. Most currently available Pasteurella vaccines are formulated to stimulate immunity by either providing an adequate antigenic mass in the administered dose, or by relying on subsequent production of antigens by in vivo growth of live organisms. The ability of these different types of vaccines to stimulate rapid and high titres to key antigens is a key factor that will influence subsequent resistance to disease. The serologic and protective responses to a streptomycin-dependent, modified-live vaccine and a killed (bacterin-toxoid) vaccine against experimental pneumonic pasteurellosis were compared. Calves were vaccinated with a single injection of either a test vaccine or phosphate-buffered saline, challenged 14 d later by transthoracic injection with Pasteurella haemolytica, and euthanized 3 d post-challenge to evaluate the severity of pneumonia. On days 0, 7, and 14, serologic responses to various P. haemolytica antigens, including cell-associated and soluble antigens, were determined by enzyme-linked immunosorbent assays, and anti-leukotoxin antibody levels were determined by leukotoxin neutralization. The bacterin-toxoid elicited significantly greater serologic responses compared to controls for all antigens. The modified-live vaccine elicited a significantly greater response compared to controls for a whole-cell antigen preparation. Lesion scores were significantly smaller (greater protection) in calves that received the bacterin-toxoid, but not the modified-live vaccine, compared to controls.     

Pasteurella multocida and bovine respiratory disease

Pasteurella multocida is a pathogenic Gram-negative bacterium that has been classified into three subspecies, five capsular serogroups and 16 serotypes. P. multocida serogroup A isolates are bovine nasopharyngeal commensals, bovine pathogens and common isolates from bovine respiratory disease (BRD), both enzootic calf pneumonia of young dairy calves and shipping fever of weaned, stressed beef cattle. P. multocida A:3 is the most common serotype isolated from BRD, and these isolates have limited heterogeneity based on outer membrane protein (OMP) profiles and ribotyping. Development of P. multocida-induced pneumonia is associated with environmental and stress factors such as shipping, co-mingling, and overcrowding as well as concurrent or predisposing viral or bacterial infections. Lung lesions consist of an acute to subacute bronchopneumonia that may or may not have an associated pleuritis. Numerous virulence or potential virulence factors have been described for bovine respiratory isolates including adherence and colonization factors, iron-regulated and acquisition proteins, extracellular enzymes such as neuraminidase, lipopolysaccharide, polysaccharide capsule and a variety of OMPs. Immunity of cattle against respiratory pasteurellosis is poorly understood; however, high serum antibodies to OMPs appear to be important for enhancing resistance to the bacterium. Currently available P. multocida vaccines for use in cattle are predominately traditional bacterins and a live streptomycin-dependent mutant. The field efficacy of these vaccines is not well documented in the literature.     

A field evaluation of the efficacy of two vaccines against ovine pneumonic pasteurellosis

Two vaccines against pneumonic pasteurellosis were evaluated for efficacy in lambs transported by sea from New Zealand to Saudi Arabia. One vaccine contained whole cell antigens of Pasteurella haemolytica A2 grown under iron restricted conditions. The other contained Pasteurella haemolytica A1 cell surface and leucotoxin antigens. There was no clear evidence of either vaccine leading to a lower pneumonia death or lesion rate than for the control group.     

Mannheimia haemolytica and the pathogenesis of enzootic bronchopneumonia

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.     

Clinical and pathological evaluation of sulbactam/ampicillin for treatment of experimental bovine pneumonic pasteurellosis

An experiment was conducted to evaluate the efficacy of sulbactam/ampicillin for treatment of bovine pneumonic pasteurellosis. Twenty-one Hereford calves were experimentally infected with bovine herpesvirus-1 and an ampicillin-resistant strain of Pasteurella haemolytica, then treated for three days with either sulbactam/ampicillin, chloramphenicol, or a placebo. The treatments were evaluated by comparing clinical illness scores, total sick days, weight changes, mortality rates, and postmortem lung scores between treatment groups. Both antibiotics were highly effective in reducing respiratory disease in the experimentally infected calves. The clinical response to sulbactam/ampicillin treatment was comparable with that of chloramphenicol and was significantly improved compared with the response to the placebo treatment. These findings suggest that the efficacy of sulbactam/ampicillin may be comparable to that of chloramphenicol for treatment of pneumonic pasteurellosis involving ampicillin-resistant strains of P. haemolytica.     

Evidence of bovine immunoglobulin G1 (IgG1) protease activity in partially purified culture supernate of Pasteurella haemolytica A1.

In the bovine respiratory tract, IgG1 is a major secretory immunoglobulin (Ig), and both IgG1 and IgG2 are believed to be important in defense against pneumonic pasteurellosis (shipping fever) in calves. Here we provide evidence for hydrolysis of IgG1 in the presence of partially purified culture supernate (ppCS) from the respiratory pathogen Pasteurella haemolytica A1. Bovine IgG1 was hydrolysed sequentially into three distinct bands (approximately 39, 12, and 7 kDa respectively). Furthermore, partial hydrolysis of bovine IgG2 was observed, but neither bovine IgA nor IgM were affected by incubation with ppCS. These findings suggest that the production of an IgG1-specific protease by P. haemolytica A1 may be a virulence mechanism contributing to the pathogenesis of bovine pneumonic pasteurellosis.     

Naturally occurring Mycoplasma bovis-associated pneumonia and polyarthritis in feedlot beef calves.

Mycoplasma bovis is perceived as an emerging cause of mortality in feedlot beef cattle. This study examined the lesions and infectious agents in naturally occurring M. bovis-associated bronchopneumonia and arthritis and the relationship of this condition with bovine viral diarrhea virus (BVDV) infection. Standardized pathologic, immunohistochemical, and microbiologic investigations were conducted on 99 calves that died or were euthanized within 60 days after arrival in 72 feedlots. Cranioventral bronchopneumonia with multiple foci of caseous necrosis was identified in 54 of 99 calves, including 30 with concurrent fibrinosuppurative bronchopneumonia typical of pneumonic pasteurellosis. Mycoplasma bovis was consistently identified in these lesions by culture and immunohistochemistry, but also commonly in healthy lungs and those with pneumonia of other causes. Focal lesions of coagulation necrosis, typical of pneumonic pasteurellosis, were often infected with both Mannheimia haemolytica and M. bovis. Arthritis was present in 25 of 54 (46%) calves with M. bovis pneumonia, and all calves with arthritis had pneumonia. BVDV infection was more common in calves with lesions of bacterial pneumonia than in those dying of other causes, but BVDV infection was not more common in calves with caseonecrotic bronchopneumonia than those with fibrinosuppurative bronchopneumonia. Retrospective analysis identified cases of M. bovis pneumonia in the early 1980s that had milder lesions than the current cases. The findings suggest that, in at least some calves, M. bovis induces caseonecrotic bronchopneumonia within the lesions of pneumonic pasteurellosis.     

The pulmonary clearance of Pasteurella haemolytica in calves infected with bovine virus diarrhea or Mycoplasma bovis.

Based on current literature which commonly associates bovine virus diarrhea virus and Mycoplasma bovis with "pneumonic pasteurellosis," an investigation was conducted into the effect of these two pathogens on the capacity of bovine lung to clear inhaled Pasteurella haemolytica. There was no significant effect (p less than 0.05) of either bovine virus diarrhea virus or M. bovis on the mean clearance rate of P. haemolytica, nor did the time interval of three, five or seven days between the first inoculation and exposure to P. haemolytica and adversely affect the lung clearance rates. However, it was found that the left lungs and a higher bacterial retention (p less than 0.05) than the right lungs.     

Pasteurella haemolytica lipopolysaccharide-induced arachidonic acid release from and neutrophil adherence to bovine pulmonary artery endothelial cells

Bovine pulmonary artery endothelial cells (BPAEC) were labeled with 3H-arachidonic acid. Exposure of the labeled BPAEC to Pasteurella haemolytica lipopolysaccharide (LPS) resulted in a time- and dose-dependent release of radioactivity. The release was inhibited by 5 mM indomethacin, but inhibition was not caused by less than or equal to 500 microM indomethacin or hydrocortisone, which suggests that the release was caused primarily by a mechanism other than cyclooxygenase or phospholipase A2 metabolism of arachidonic acid. Pasteurella haemolytica LPS also caused increased adherence of bovine neutrophils to BPAEC through independent effects on both cell types. The increased adherence was inhibited by treatment of either cell type with cycloheximide or actinomycin D prior to LPS exposure, indicating that de novo protein synthesis was required in both cell types to promote the LPS-induced adherence. Lipopolysaccharide may be an important factor in neutrophil-mediated effects in pneumonic pasteurellosis by causing increased neutrophil adherence and, thus, the vascular sequestration of neutrophils. Together, these experiments provide additional evidence for the involvement of LPS in pneumonic pasteurellosis. Moreover, they provide evidence of LPS-induced endothelial activation, which could have broad ramifications in the inflammatory and immune responses of pneumonic pasteurellosis.     

Expression of tissue factor in experimental bovine pneumonic pasteurellosis and endotoxemia

Tissue factor (TF), a cell surface-associated cofactor and activator of coagulation factor VII, has been implicated in the local and systemic activation of coagulation associated with sepsis. This study describes the pattern of TF expression in experimental bovine pneumonic pasteurellosis and endotoxemia. Immunohistochemical techniques were used to localize TF antigen in tissue sections. Tissue factor expression was not observed in tissues from control animals. In response to Pasteurella haemolytica challenge, TF was expressed within alveolar walls, by mononuclear inflammatory cells within alveoli, and in walls of arteries, arterioles, bronchi, and bronchioles. Tissue factor was not detected in unaffected lung, liver, spleen, lymph node or kidney tissue. Administration of Escherichia coli endotoxin intravenously resulted in tissue factor expression in lung, spleen, and lymph node tissue. Results of this study indicate that TF is expressed locally at sites of inflammation and systemically in endotoxemia. Therefore, TF may be involved in coagulation events associated with pneumonic pasteurellosis.     

Protection of lambs against experimental pneumonic pasteurellosis by transfer of immune serum

Passive protection of specific pathogen-free lambs against experimental pasteurellosis was achieved using antisera from conventionally reared sheep which were either convalescent from experimental pneumonia or inoculated with Pasteurella haemolytica A2 vaccines. The complete immune sera, or immunoglobulin-rich fractions prepared from them, when administered separately or together provided 94-100% protection of recipients compared to control lambs. Antibodies to P. haemolytica in donor sera were quantified by anti-sodium salicylate extract (SSE) and anti-lipopolysaccharide (LPS) ELISA, bactericidal assay, cytotoxin neutralization and indirect haemagglutination. The anti-SSE ELISA titres correlated best with protective efficacy and could be used to measure antibody in recipient lambs immediately before challenge. The degree of protection was unaffected by prior infection with parainfluenza virus Type 3, suggesting that such exposure did not enhance exudation of circulating immunoglobulin into the respiratory tract. It was concluded that systemic humoral immunity alone can prevent pasteurellosis.     

Prevention of experimental bovine pneumonic pasteurellosis with an extract of Pasteurella haemolytica.

A total of 36 calves were used in three experiments to test the efficacy of a potassium thiocyanate extract of Pasteurella haemolytica in protecting against experimental pneumonia. In each of experiments A and B, 12 calves were divided into three equal groups. The first group was vaccinated with an aerosol of a potassium thiocyanate extract twice, two weeks apart; the second group was vaccinated subcutaneously once only with the same extract. The third group of calves in both experiments remained as unvaccinated controls. In experiment C, six calves were vaccinated intramuscularly and six were left as controls. Approximately one month after vaccination all calves were challenged with an aerosol of bovine herpesvirus 1 (isolate 108) followed in 4 d by an aerosol of P. haemolytica type A1 (the same strain from which the potassium thiocyanate extract had been made). Varying degrees of protection against subsequent development of experimental pneumonic pasteurellosis in cattle were seen in vaccinated calves as compared to control calves in these experiments. The results indicate that protection of cattle against pneumonic pasteurellosis may prove possible with a sub-cellular extract of P. haemolytica.     

A microbiological study of pneumonic calf lungs.

BITSCH, V., N. F. FRIIS and H. V. KROGH: A microbiological study of pneumonic calf lungs. Acta vet. scand. 1976, 17, 32–42.–Fifty pneumonic calf lungs were subjected to microbiologic screening with regard to bacteria, mycoplasmas, and viruses.Of bacteria the species most commonly found were Pasteurella multocida (eight lungs), Pasteurella hemolytica (eight lungs), and Corynebacterium pyogenes (13 lungs). Of special interest was the demonstration of Neisseria spp. in five lungs. Mycoplasma dispar was found in 31 lungs, Mycoplasma bovirhinis in 16 lungs, and Urea-plasma in 26 lungs. Cytopathogenic agents were demonstrated in 14 lungs. Four isolates were found to be bovine respiratory syncytial virus, three were bovine viral diarrhea virus, and two were bovine parainfluenza 3 virus. The remaining five cytopathogenic agents were not identified.     

Pasteurella haemolytica of Cattle: Serotype, Production of Bete-galactosidase and Antibacterial Sensitivity

Two hundred and three isolates of Pasteurella haemolytica from cattle were studied. They originated from the nasal cavity of cattle in housed herds; the nasal cavity and pneumonic lungs of experimental feedlot calves and from pneumonic bovine lungs submitted for bacteriological diagnosis.

To determine whether a single characteristic or combination of characteristics might be a feature of isolates collected from animals with pneumonic pasteurellosis (Shipping Fever), the following tests were made. Cultures were serotyped by indirect haemagglutination; the ability to produce beta-galactosidase was examined in the ortho-nitrophenyl-beta-D-galactopyranoside (ONPG) test and antibacterial sensitivity tests were done. None of these factors could be directly related to the role of P. haemolytica in “Shipping Fever”.

     

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

Mannheimia (Pasteurella) haemolytica A1 produces an extracellular leukotoxin (LKT) that is reported to bind the beta(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 beta(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.