Anticytotoxin activity of bovine sera and body fluids against Pasteurella haemolytica A1 cytotoxin.

Toxin neutralizing activity of bovine sera and body fluids against Pasteurella haemolytica type A1 cytotoxin was evaluated by 51Cr release assay using bovine peripheral blood mononuclear leukocytes as the target cells. Sera collected from precolostral calves did not exert anticytotoxin activity at 10(-1) or higher dilutions, whereas randomly selected complement fixing antibody-negative sera neutralized on average over 90% of cytotoxin activity at the 10(-1) dilution and less than 50% of the toxin activity at 10(-2) or higher serum dilutions. Nasal secretions and lung washings of some of the cattle tested also contained cytotoxin neutralizing activity. The antibody nature of the cytotoxin neutralizing activity was demonstrated by its neutralization with bovine immunoglobulin G2 purified from pooled seropositive sera. Sera from a group of cattle which were vaccinated with a potassium thiocyanate extract of P. haemolytica, but which subsequently developed fibrinous pneumonia after aerosol challenge with bovine herpesvirus 1 and P. haemolytica, had significantly lower anticytotoxin activity than sera from another group of cattle which did not develop the disease after similar vaccination and challenge. Cattle which survived a natural outbreak of shipping fever had higher anticytotoxin activity than those having fibrinous pneumonia in the aforementioned experimental group, although there was no statistical difference between them and a randomly selected CF seronegative group. It is probable that this cytotoxin neutralizing antibody exerts a beneficial effect in protection of cattle against pneumonic pasteurellosis.     

Immunogenicity of a soluble antigen against Pasteurella haemolytica-associated pneumonia in calves

Three experiments were performed to evaluate the immunogenic potency of a soluble fraction of Pasteurella haemolytica against pneumonic pasteurellosis in calves. A soluble antigen was extracted by a 2.5% saline solution from P. haemolytica. Weaned Holstein bull calves, seronegative for infectious bovine rhinotracheitis virus ( IBRV ) and the pasteurella antigen, were vaccinated either by repeated subcutaneous (SC) vaccination, or by exposure 3 times to the aerosol of P. haemolytica antigen. Challenge exposure to aerosol of P. haemolytica was preceded by infection with IBRV , or in experiments 2 and 3, the virus exposures were combined with a stress treatment. The lung lesions were examined at necropsy 3 to 8 days post infection. In the first experiment, all the vaccinated calves produced specific antibody response to the pasteurella antigen, and none of the calves including controls showed significant lesions in the lung. In the second experiment 2 aerogenically vaccinated calves had no lesions. One of the two SC-vaccinated calves had mild consolidated lesions. Two control calves, one of which died 3 days following the challenge, developed severe fibrinous pneumonia with consolidation of 50% or more of the lung surfaces. P. haemolytica was isolated only from the 2 control animals. In the third experiment, 2 of the 3 control calves developed moderate to severe consolidation, but P. haemolytica was isolated only from one of them. Two of the three aerosol-vaccinated calves also developed significant lesions and one of them yielded the bacteria from the lung. Three SC-vaccinated calves had slight lesions and the organism was not isolated from their lungs. The results did not consistently indicate an immunogenic potential of the soluble antigen against P. haemolytica-related pneumonia. The effect of stress on the pathogenesis of bovine viral pneumonia and correlation between pneumonic lesions and antibacterial resistance in situ are discussed.     

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.     

Aerosol vaccination of calves with pasteurella haemolytica against experimental respiratory disease.

Three experiments were conducted on calves in which the efficacy of vaccination with live Pasteurella haemolytica in aerosol was tested by challenge with sequential aerosol exposure to bovine herpesvirus 1 and P. haemolytica. Neither single nor multiple aerosol vaccinations protected against the experimental disease. Macroscopically recognizable rhinitis, tonsillitis, tracheitis and pneumonia occurred in both controls and vaccinates. In one experiment as many as three aerosol vaccinations with live P. haemolytica for up to 20 minutes failed to elicit clinical signs in exposed calves. Pasteurella haemolytica was isolated less frequently from tissues of vaccinated calves than from those of nonvaccinated calves. Pasteurella haemolytica was isolated from deep nasal swabs of 4/14 vaccinated calves five and six days after viral exposure. It was concluded that although bovine herpesvirus 1 vaccination has been shown previously to prevent the experimental disease produced by bovine herpesvirus 1-P. haemolytica, live P. haemolytica vaccination by aerosol will not provide the same protection.     

Bovine pneumonic pasteurellosis: experimental induction in vaccinated and nonvaccinated calves.

A study was undertaken to investigate the effects of the immune response induced by combined aerosol and parenteral vaccination on the lung lesions induced in calves by Pasteurella haemolytica AI. Twenty-four calves, twelve of which had been vaccinated with killed P. haemolytica by aerosol and subcutaneous injection in Freund's complete and incomplete adjuvant were challenged by intratracheal inoculation of live P. haemolytica. Serological response to vaccination was not marked but was best measured by the whole cell agglutination test or by indirect bacterial agglutination rather than by the passive haemagglutination test. Titres of vaccinates were positively correlated with the degree of pneumonic change following challenge while in nonvaccinated controls, titres were negatively correlated with lung lesions. These findings suggest the occurrence of an immunologically mediated hypersensitivity pneumonitis in the lungs of vaccinates and point to the potential efficacy of live bacterial aerosols for stimulation of protective immunity in pneumonic pasteurellosis.     

Experimental immunisation of lambs against pneumonic pasteurellosis

Methods of immunising lambs against pneumonic pasteurellosis, caused by several serotypes of Pasteurella haemolytica, were assessed in specific pathogen free lambs. Lambs were vaccinated intramuscularly with sodium salicylate extract (SSE) of P haemolytica, either alone or in combination with heat-killed organisms (HKO). SSE of P haemolytica type A1 protected vaccinated lambs against pneumonia resulting from challenge with the homologous serotype. SSE of type A2 also provided some protection but this was improved by vaccination with a combination of SSE and HKO.     

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.     

Experimental infection of lambs with an aerosol of Pasteurella haemolytica.

Specific pathogen free lambs were exposed to an aerosol of Pasteurella haemolytica. Ten lambs vaccinated with an adjuvanted killed P haemolytica vaccine and nine units of P haemolytica administered in an aerosol. Pneumonia histologically indistinguishable from natural pneumonic pasteurellosis occurred in one vaccinated lamb and in four control lambs.     

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.     

In vitro lymphocyte proliferative responses and gamma-interferon production as measures of cell-mediated immunity of cattle exposed to Pasteurella haemolytica.

Cell-mediated immune mechanisms may play a role in the pathogenesis and prevention of pneumonia in cattle caused by Pasteurella haemolytica serotype A1. To determine the circumstances required to stimulate and identify cell-mediated immune responses, calves were vaccinated with a commercial P. haemolytica bacterin or a live commercial P. haemolytica vaccine, or were infected intratracheally with virulent P. haemolytica. All calves were challenge-exposed intratracheally with P. haemolytica 31 d after vaccination or prior infection. Peripheral blood mononuclear cells and mediastinal and superficial cervical lymph node cells were stimulated with antigens prepared from P. haemolytica to evaluate in vitro proliferative responses and gamma-interferon production as measures of cell-mediated immunity. Strong proliferative responses and gamma-interferon production were detected in lymph node cells from calves vaccinated with the live vaccine and from infected calves, especially in response to stimulation with an outer membrane protein preparation from P. haemolytica. Greater proliferative responses and gamma-interferon production were associated with the lymph node nearer the site of bacterin administration (superficial cervical lymph node) or the site of infection (mediastinal lymph node), whereas greater proliferative responses and gamma-interferon production were associated with the more distant lymph node (mediastinal lymph node) in calves vaccinated with the live vaccine. Neither proliferative responses nor gamma-interferon production were detected in peripheral blood mononuclear cells from calves that were vaccinated for or infected with P. haemolytica. Antileukotoxin antibody titers were determined by a serum neutralization assay, and protection against pneumonic lesions was more closely correlated with antileukotoxin antibody responses than with lymphocyte proliferation or gamma-interferon responses.     

Pasteurella haemolytica: purification of saline-extractable proteins by isoelectrofocusing

A procedure was developed for separating antigens associated with a saline extract of Pasteurella haemolytica serotype 1. Seven antigens were identified by immunoelectrophoresis to be associated with the extract. The extract was subjected to preparative isoelectrofocusing in a pH range of 3-10. The majority of extracted proteins were found to have pI's of 4-6, whereas the carbohydrate antigen(s) were distributed over a pI range of 3.0-8.0. The fractions that were of interest were pooled and refocused in a narrower pH range to improve resolution of the protein antigens. Specific antigens from defined pH ranges were pooled to form 6 antigen groups. These antigen groups were examined further by immunoelectrophoresis, analytical isoelectrofocusing, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The molecular weights of the proteins found in the capsular extracts ranged from 33 k to greater than 80 k. Injection of mice with capsular extract or antigen Groups 1-6 in Freund's incomplete adjuvant resulted in a serum antibody response to the various antigens as detected by an enzyme-linked immunosorbent assay. Significant protection (P less than 0.05) against challenge with virulent P. haemolytica was seen in mice injected with antigen Groups 2 and 4. Six calves were immunized with saline extract. These calves had greater resistance to experimental pneumonic pasteurellosis than did 6 non-vaccinated calves. A serum antibody response to the crude extract and to each antigen group was detected in vaccinated calves by an enzyme-linked immunosorbant assay.     

The evolution of vaccines for bovine pneumonic pasteurellosis

Since the early 1900s bovine pneumonic pasteurellosis has been recognised as a major economic problem to European and North American cattle industries. Initial attempts to prevent the disease were complicated by incomplete knowledge of the causative organisms. Despite some early reports of vaccine-induced protection against disease, initial vaccines were of questionable protective value. From the late 1950s to the 1970s Pasteurella haemolytica and P multocida bacterins were the primary type of vaccine used commercially and experimentally. When viruses, most notably bovine herpesvirus 1 (infectious bovine rhinotracheitis virus) and parainfluenza-3 virus, were found to be associated with bovine respiratory disease, viral vaccines were used in attempts to prevent pneumonic pasteurellosis. Combinations of bacterins and viral vaccines were also developed and evaluated. Collectively, bacterins, viral vaccines and bacterin-virus combinations did not consistently reduce disease in experimental trials or field use. By the 1980s some studies using live vaccines were reportedly successful in reducing the incidence of pneumonic pasteurellosis. Current experimental studies revolve around the identification and incorporation of specific Pasteurella species antigen extracts into vaccines. The efficacy of these new extract vaccines is yet to be determined.     

Experimental production of bovine pneumonic pasteurellosis

Pneumonic pasteurellosis has been reproduced in conventional, weaned, Friesian-cross calves using a strain of Pasteurella haemolytica biotype A, serotype 1 (P haemolytica A1) isolated from a pathologically confirmed incident of bovine pneumonic pasteurellosis. The major clinical findings were pyrexia, hyperpnoea, tachypnoea, nasal discharge and reduced appetite. Fibrinous pneumonia was present in the lungs of animals at necropsy on days 2 and 3 after initial infection while by days 9 and 10 after initial infection many of the areas of fibrinous pneumonia were confined by a fibrous capsule forming well defined nodules. During the experiment natural transmission of the infecting strain of P haemolytica A1 occurred in two control calves which developed a condition identical to that in the artificially infected calves. P haemolytica A1 was repeatedly recovered from the nasopharynx of infected calves and at necropsy throughout the upper and lower respiratory tracts. Seroconversion, as measured by indirect haemagglutination, to the organism developed in all infected calves by days 9 and 10 after initial infection. The clinical, microbiological and pathological findings were identical to those seen in field incidents of bovine pneumonic pasteurellosis involving recently housed, weaned, single-suckled calves.     

Proliferation of Pasteurella haemolytica in the calf respiratory tract after an abrupt change in climate

Two groups of four calves were exposed to a poly-disperse aerosol of 1.3 X 10(4) to 16 X 10(4) colony forming units (CFU) of nalidixic acid resistant Pasteurella haemolytica type A1 litre-1 of air with a mass median aerodynamic diameter of 2.6 +/- 0.8 microns. One group of calves was kept at 5 degrees C, 72 per cent relative humidity (RH) and the second was subjected to an abrupt change in climate directly after aerosol exposure from 5 degrees C, 75 per cent RH to 13 degrees C, 84 per cent RH. Clearance of the organism from the respiratory tract of the calves was monitored over the subsequent 23 hours by a method of tracheal and nasopharyngeal swabbing. Clearance measured at the trachea in all calves in both groups was not a continuous, uninterrupted process but exhibited a temporary decline between eight and 14 hours. Calves subjected to an abrupt change in climate after aerosol challenge had raised respiratory rates eight to 14 hours later and P haemolytica proliferated in the nasopharynx over the entire 23 hours. There was no apparent effect of climate on P haemolytica in the trachea. It is suggested that rapid growth of P haemolytica accompanying a change in climate may be an important aetiological factor in pneumonic pasteurellosis of calves.     

Evidence that blood-borne infection is involved in the pathogenesis of bovine pneumonic pasteurellosis

Five calves were inoculated intravenously with 10(8) colony forming units (cfu) of Pasteurella haemolytica A1; the mean score for pneumonic consolidation 3 days post-inoculation was 28%, and the mean clinical score was 7.8. Five calves inoculated intratracheally with 10(9) cfu of the same strain of P. haemolytica had comparable scores (34% and 8.8). Histological lesions of fibrinous pneumonia were similar in all calves. P. haemolytica was recovered from all but one of the affected lungs. From one calf killed in extremis 3 hours after intravenous inoculation, numbers of bacteria recovered from lung were 1,000-fold greater than from liver and spleen. A similar difference in bacterial numbers was also obtained from a gnotobiotic calf killed in extremis, 12 hours after intravenous inoculation of 10(8) cfu P. haemolytica. Evidence from these experiments supports the hypothesis that the blood-borne route is important in the pathogenesis of bovine pneumonic pasteurellosis.     

Effect of prior natural exposure to Pasteurella haemolytica on resistance to experimental bovine pneumonic pasteurellosis

The effect of prior natural exposure to Pasteurella haemolytica, as determined serologically, was studied with respect to resistance to experimental pneumonic pasteurellosis in 20 calves from 3 experiments. Resistance to challenge exposure was measured using a lesion-scoring system. As measured by a quantitative fluorometric immunoassay, naturally acquired serum antibody titers to the organisms were 0 to 228. There was a significant correlation (P less than 0.05) between high naturally acquired antibody titers and resistance to transthoracic challenge exposure with P haemolytica.     

Evaluation of structural and functional alterations of circulating neutrophils in calves with experimentally induced pneumonic pasteurellosis

OBJECTIVES: To determine the structural and functional alterations in circulating neutrophils that may lead to sequestration in lung microvasculature and endothelial injury in calves with experimentally induced pneumonic pasteurellosis. ANIMALS: 10 healthy, 2- to 4-week-old male Holstein calves. PROCEDURES: Holstein calves were anesthetized and inoculated intrabronchially with Dulbecco phosphate buffered saline (0.9% NaCl) solution (DPBSS; 5 control calves) or 1 x 10(9) Pasteurella haemolytica organisms (5 infected calves). Blood samples were collected before and 1, 2, 4, and 6 hours after inoculation. Total and differential WBC count, dilute whole blood leukocyte deformability, neutrophil size distribution, and neutrophil surface CD11b expression were measured in blood samples. RESULTS: A progressive decrease in leukocyte deformability and increase in neutrophil size was detected 1, 2, 4, and 6 hours after inoculation of P haemolytica. Neutrophil surface CD11b expression was greater than baseline values at 6 hours after inoculation of P haemolytica. Two populations of neutrophils with an increase in size were detected in P haemolytica-infected calves. Both subpopulations had increased CD11b expression, compared with neutrophils that were typical in size. CONCLUSIONS AND CLINICAL RELEVANCE: Neutrophils circulate in an activated and nondeformable state in calves with experimentally induced pneumonic pasteurellosis. A decrease in neutrophil deformability and neutrophil aggregation may contribute to neutrophil trapping in the lung microvasculature during pneumonic pasteurellosis in calves.     

Effect of deferoxamine pretreatment on acute pneumonic pasteurellosis and neutrophil oxidative metabolism in calves.

Iron plays a central role in bacterial infections, influencing both bacterial virulence and host cellular defense mechanisms. We investigated whether iron chelation might be of benefit in the treatment of pneumonic pasteurellosis of calves. Neutrophils obtained from calves previously treated with the iron chelator, deferoxamine, were studied for their responses to latex and opsonized zymosan by luminol-enhanced chemiluminescence and to phorbol myristate acetate and opsonized zymosan by superoxide generation. Treatment with deferoxamine in vivo failed to influence these in vitro measures of neutrophil oxidative metabolism. Furthermore, iron depletion with deferoxamine failed to modify the pathophysiological derangements that occurred in calves following experimental induction of pneumonia by intratracheal inoculation with Pasteurella haemolytica. These data indicate that iron chelation using deferoxamine cannot be recommended as an adjunct to conventional therapy in the treatment of pneumonic pasteurellosis of cattle.     

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.     

Pasteurella species isolated from the bovine respiratory tract and their antimicrobial sensitivity patterns

Pasteurella haemolytica biotype A, serotype 1 (P haemolytica A1) was the most commonly isolated Pasteurella species from 80 calves examined at necropsy from 40 outbreaks of respiratory disease, the majority of which were pathologically confirmed as bovine pneumonic pasteurellosis (transit fever; shipping fever). Similarly, nasopharyngeal swabs from in-contact and apparently healthy calves indicated the widespread presence of P haemolytica A1. Pasteurella multocida and other serotypes of P haemolytica A1 were found including six isolations of P haemolytica T10, a fairly common pathogen in sheep. Approximately two-thirds of the isolates were tested for their antimicrobial sensitivity patterns and the degree of sensitivity for P haemolytica A1, the most frequently isolated serotype, was chloramphenicol (100 per cent), sulphamethoxazole trimethoprim (98 per cent), oxytetracycline (80 per cent), ampicillin (85 per cent), penicillin (82 per cent), streptomycin (3 per cent) and lincomycin (1 per cent).