Effect of vaccination with live or killed Pasteurella haemolytica on resistance to experimental bovine pneumonic pasteurellosis

Using 6- to 8-month-old beef calves, 3 experiments were conducted to compare the effect of vaccination with live or killed Pasteurella haemolytica on resistance to a transthoracic challenge exposure with the organism and to correlate serum antibody response with resistance. In each experiment, calves were vaccinated twice at 1-week intervals and were challenge exposed 21 days after the first inoculation. Lung lesions were evaluated by a system, such that higher scores indicated the more severe lesions. In each experiment, calves immunized with live P haemolytica had lower lesion scores than calves vaccinated with saline solution or bacterin. In 2 of the experiments, the differences were significant (P less than 0.05). In all experiments, calves vaccinated parenterally with a commercial P haemolytica/P multocida bacterin or with a formalin-killed P haemolytica bacterin had lesion scores that were not significantly different (P greater than 0.05) than for control calves vaccinated with saline solution. Live and killed bacterial preparations induced a significant serum antibody response to P haemolytica as measured by a quantitative fluorometric immunoassay. The antibody response to vaccination was not affected by preexisting titers to P haemolytica. Serum antibody titers were not consistently as high for calves vaccinated with bacterins as for calves vaccinated with live organisms. Although high antibody titers correlated with low lesion scores when calves vaccinated with saline solution or live organisms were analyzed collectively, there was not a significant correlation between the 2 variables when calves, vaccinated with saline solution or with bacterin, were analyzed collectively. These data indicate that, although bacterins may induce a detectable serum antibody response, they do not induce protection against transthoracic challenge exposure to P haemolytica.     

Serum antibodies to Pasteurella haemolytica lipopolysaccharide: relationship to experimental bovine pneumonic pasteurellosis

An enzyme-linked immunosorbent assay was used to determine the serum antibody response to Pasteurella haemolytica lipopolysaccharide (LPS) for calves vaccinated with saline solution, a formalin-killed P haemolytica bacterin, or live P haemolytica. Bacterin-vaccinated calves had a lower antibody response to LPS than did calves vaccinated with live P haemolytica. Calves vaccinated with either saline solution or the bacterin were more susceptible to intrapulmonic challenge exposure with P haemolytica than were calves vaccinated with liver organisms. Serum antibody responses to P haemolytica LPS did not seem important for resistance to challenge exposure, because there was no significant correlation (P greater than 0.05) between the lung lesion score and antibody response to P haemolytica LPS. There was a highly significant correlation (P less than 0.001) between antibody detected against P haemolytica LPS and that against formalin-killed P haemolytica. Competitive binding studies indicated that P haemolytica LPS is a major antigenic determinant on the surface of P haemolytica. There did not seem to be substantial cross-reaction between LPS from P haemolytica and that from Escherichia coli (serotype O26:B6).     

Serum neutralization of cytotoxin from Pasteurella haemolytica, serotype 1 and resistance to experimental bovine pneumonic pasteurellosis

Sera from several groups of experimental calves were tested for cytotoxin neutralizing capacity. The relationship between this capability and resistance of the animals to an experimental challenge was examined. All undiluted bovine sera tested, other than fetal bovine serum, neutralized cytotoxin. Preadsorption of selected sera with formalin-killed P. haemolytica did not reduce their neutralizing capacity. Crude IgG fractions extracted from bovine sera retained neutralizing capacity as well. Cytotoxin neutralization titers were determined by serial dilution of sera from cattle which were previously unexposed, naturally exposed, or exposed by vaccination to the organism. Both live and killed vaccines were used. Prior exposure to live organisms resulted in the production of antibodies to both cell surface antigens and cytotoxin, whereas exposure to the killed vaccine resulted in the production of antibodies primarily to cell surface antigens. Resistance to experimental challenge with the organism correlated directly with serum cytotoxin neutralizing titers.     

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.     

Immunologic response to Pasteurella haemolytica and resistance against experimental bovine pneumonic pasteurellosis, induced by bacterins in oil adjuvants

Immunogenicity of and protection afforded by Pasteurella haemolytica bacterins were studied in calves. Bacterins contained an aluminum hydroxide in gel (ALH) adjuvant or one of the following oil-in-water adjuvants: Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and trehalose dimycolate (TDM). On days 0 and 7, calves were vaccinated with phosphate-buffered saline solution (PBSS), a bacterin, or live P haemolytica. Transthoracic intrapulmonic challenge exposure was done on day 21. In 3 experiments, there were no significant (P greater than 0.05) differences between lung lesions induced in PBSS-or ALH bacterin-vaccinated calves. Both FCA and FIA bacterins significantly (P less than 0.05) enhanced resistance against challenge exposure. Resistance induced by FCA and FIA bacterins was comparable with that induced by vaccination with live P haemolytica. Calves vaccinated with FIA bacterin and challenge-exposed to P haemolytica at a concentration of 4.5 X 10(9) colony-forming units (4.5 times greater than used in the first 3 experiments) resisted challenge exposure similar to calves given live organisms. The TDM bacterin failed to enhance resistance. All bacterins caused a significant increase (P less than 0.05) in serum antibody to P haemolytica somatic antigens, as measured by a quantitative fluorometric immunoassay. Pasteurella haemolytica leukotoxin neutralizing antibody titers did not increase significantly (P greater than 0.05) in sera after vaccination with any bacterin. Vaccination with FCA and FIA bacterins resulted in a significant increase (P less than 0.001) in serum antibody to a carbohydrate-protein subunit of P haemolytica, as measured by an enzyme-linked immunosorbent assay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serum antibodies to antigens derived from a saline extract of Pasteurella haemolytica: correlation with resistance to experimental bovine pneumonic pasteurellosis

The serum antibody response was determined to 6 antigen groups (AG's) derived from a saline extract (SE) of Pasteurella haemolytica, serotype 1. Using an enzyme-linked immunosorbent assay, sera were analyzed from 65 calves that had been previously vaccinated with saline, the unfractionated SE, a bacterin, or live P. haemolytica. The serum antibody responses to the 6 AG's were correlated with resistance to an experimental transthoracic challenge with the organism. The antibody responses to AG's 1, 5, and 6 appeared to be potentially important in resistance to challenge. In the 3 experiments conducted, a significantly higher (p less than 0.05) increase in antibody was seen to AG's 1, 5, and 6 in calves vaccinated with live organisms compared to those vaccinated with the bacterin. A significant correlation (p less than 0.05) was seen between high antibody to AG 1 and resistance to challenge in all 3 experiments. In 2 of the 3 experiments, a significant correlation (p less than 0.05) was seen among high antibody titers to AG's 5 and 6 and resistance, whereas in 1 experiment the correlation was significant (p less than 0.05) between antibody to AG 4 and resistance. A rise in antibody to AG's 2 and 3 was seen only in calves vaccinated with SE. Because AG's 1, 5, and 6 are higher in carbohydrate than the other AG's, this suggests that antibody to polysaccharide antigens may be important to resistance. Other potentially protective antigens of P. haemolytica are discussed.     

Serum antibody response to carbohydrate antigens of Pasteurella haemolytica serotype 1: relation to experimentally induced bovine pneumonic pasteurellosis

The antibody responses to the capsular carbohydrate (CC) purified from Pasteurella haemolytica serotype 1 were determined by an ELISA, using 135 sera from 6 calves vaccinated with phosphate-buffered saline solution, formalin-killed P haemolytica bacterins, live P haemolytica, or an extract of P haemolytica referred to as carbohydrate-protein subunit (CPS). Calves vaccinated with live P haemolytica, bacterins, or CPS developed serum antibodies to CC. Bacterins containing Freund incomplete adjuvant or Freund complete adjuvant induced higher antibody responses than did bacterins containing aluminum hydroxide. In 4 of 6 experiments, high antibody responses to CC were significantly (P less than 0.05) correlated with resistance to transthoracic challenge exposure with P haemolytica. When calves were challenge exposed with a dose of P haemolytica that was 4.5 times greater than the standard challenge exposure dose or when calves that had been vaccinated with CPS were challenge exposed, antibody responses did not significantly (P greater than 0.05) correlate with resistance to challenge exposure. The amount of serum antibodies to CPS increased significantly (P less than 0.05) when calves were vaccinated with live or killed P haemolytica or with CPS, compared with that in calves given saline solution. In 5 of 6 experiments, correlation between high antibody responses and resistance to challenge exposure was significant (P less than 0.05). The correlation between those variables was not significant (P less than 0.07) for CPS-vaccinated calves. In the ELISA, treatment of CPS with sodium m-periodate, to oxidize periodate-sensitive carbohydrate epitopes, failed to markedly alter the antibody response to CPS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preliminary studies with a live streptomycin-dependent Pasteurella multocida and Pasteurella haemolytica vaccine for the prevention of bovine pneumonic pasteurellosis.

Twelve Pasteurella-free Holstein-Friesian calves were used in a study to test the efficacy of a live streptomycin-dependent Pasteurella multocida A:3 and streptomycin-dependent Pasteurella haemolytica A1 vaccine. The calves were inoculated intramuscularly twice at 14-day intervals with either the streptomycin-dependent vaccine, containing 1 X 10(6) colony forming units/mL P. multocida and 4 X 10(8) colony forming units/mL P. haemolytica, commercial bacterin, or phosphate buffered saline. Two weeks following the second vaccination, all calves were challenged by intranasal inoculation of 10(8) TCID50/4.0 mL infectious bovine rhinotracheitis virus followed three days later by intratracheal injection with 2.3 X 10(7) colony forming units/mL of a 16 hour culture of P. multocida A:3 and 2.6 X 10(8) colony forming units/mL of an 8 hour culture of P. haemolytica A1. Seven days after challenge with Pasteurella, calves were killed for collection of tissues at necropsy. Each calf was given a score based on macroscopic and microscopic lesions. The scores for the calves receiving live vaccines were significantly lower (p less than 0.025) than those for the controls. Also, the calves receiving live vaccines had a significant (p less than 0.05) increase in the level of serum antibody to P. haemolytica. The results of this preliminary study showed that the streptomycin-dependent vaccine offered better protection than the commercial bacterin against a virulent homologous challenge.     

Sequential lesions of experimental bovine pneumonic pasteurellosis

A strain of Pasteurella haemolytica biotype A serotype 1, which had been isolated from a pathologically-confirmed outbreak of bovine pneumonic pasteurellosis, was used successfully to reproduce the disease in conventional calves. The development of the various pathological features was studied at regular intervals following infection. The acute inflammatory reaction which had developed by day 2 after initial infection was characterised by flooding of the alveoli by oedema and neutrophils together with a mild degree of bronchiolar epithelial necrosis. This progressed to an acute exudative fibrinous pneumonia with extensive involvement of the interlobular septa and often with pleurisy. Subsequently, these pulmonary lesions became walled off by fibrous tissue which became infiltrated by plasma cells and lymphocytes. At this stage organisms could be demonstrated only within these nodules in the lung tissue.     

Immunologic response and resistance to experimentally induced pneumonic pasteurellosis in cattle vaccinated with various dosages of lyophilized Pasteurella haemolytica

Pasteurella haemolytica was lyophilized in an enriched soybean polypeptone broth. Lyophilization in this medium resulted in a mean 10-fold loss in P haemolytica viability, as opposed to up to a 10(4)-fold loss in viability when other media were used. Lyophilized P haemolytica was reconstituted and used as a live vaccine in 3 experiments. Calves were challenge exposed by transthoracic injection with virulent P haemolytica. In experiment 1, 2 subcutaneous injections (7-day interval between injections) with 5 ml of recently harvested (1 X 10(9) colony-forming units [CFU]/ml) or lyophilized (1 X 10(8) CFU/ml) P haemolytica significantly (P less than 0.001) enhanced resistance against challenge exposure, compared with resistance in calves given saline solution or sterile medium (control calves) or calves vaccinated with lyophilized organisms at a concentration of 1 X 10(6) CFU/ml. In experiment two, 1, 2, or 5 ml of lyophilized P haemolytica (1 X 10(8) CFU/ml) significantly (P less than 0.05) enhanced resistance, compared with resistance in calves given saline solution (control calves). In experiment three, 1 or 2 injections of lyophilized P haemolytica significantly (P less than 0.01) enhanced resistance against challenge exposure, compared with that of calves given saline solution. The mean lesion score for calves given 1 injection was not significantly higher than the mean lesion score for the group given 2 injections. Vaccination with lyophilized P haemolytica vaccine caused significant (P less than 0.05) increases in serum antibody to P haemolytica somatic antigens, to a carbohydrate-protein subunit of the organism, and to leukotoxin.     

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.     

Bovine pneumonic pasteurellosis: chemiluminescent response of bovine peripheral blood leukocytes to living and killed Pasteurella haemolytica, Pasteurella multocida, and Escherichia coli

A luminol-dependent chemiluminescence (LDCL) assay was used to evaluate the response of bovine polymorphonuclear leukocytes; (neutrophils [PMN]) to living and heat-killed Escherichia coli, Pasteurella multocida (type A, serotype 3), and P haemolytica (biotype A, serotype 1), and to heat-killed P haemolytica and sterile culture supernatant from living P haemolytica. Control cultures containing PMN that had not been phagocytically stimulated with bacteria had a modest increase in LDCL during the initial 10 minutes of incubation, followed by a gradual decline throughout the 120-minute incubation period. Bovine PMN emitted LDCL more efficiently when the cells were exposed to living E coli or P multocida than when they were exposed to the same bacteria killed by heat. The mean LDCL values for reaction mixtures containing living E coli or P multocida peaked at 30 minutes of incubation and remained above values for mixtures containing the same heat-killed bacteria. Kinetics of the LDCL response of bovine PMN to heat-killed P haemolytica were similar (although reduced in amplitude) to that observed with killed E coli or P multocida. The LDCL response of bovine PMN to living P haemolytica was not like that for E coli or P multocida, and was characterized by the development of a peak response at 10 minutes followed by a precipitous decrease in responsiveness and a subsequent complete cessation of LDCL. Addition of sterile culture supernatant from living P haemolytica to test samples containing heat-killed P haemolytica induced a response similar to that obtained with the living microorganism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presence of bacterial glycocalyx and fimbriae on Pasteurella haemolytica in feedlot cattle with pneumonic pasteurellosis.

This investigation was conducted to determine if Pasteurella haemolytica within feedlot cattle affected by pneumonic pasteurellosis express fimbriae (pili) and bacterial glycocalyx. Bacteriological culture of pulmonary tissue from three calves with fibrinous pneumonia resulted in heavy growth of P. haemolytica. Transmission electron microscopy of the lungs showed numerous microcolonies of gram-negative bacteria with morphology typical of Pasteurella haemolytica. The cells within these microcolonies possessed bacterial glycocalyces which stained with ruthenium red. Glycocalyx-encased microcolonies were also present in specimens examined by scanning electron microscopy. Typical P. haemolytica cells were evident in a tracheal specimen and these bacteria had radial glycocalyces consistent with polysaccharide and proteinaceous material condensed on linear structures suggestive of fimbriae. The pathogenetic importance of the bacterial glycocalyx and fimbriae in shipping fever pneumonia has yet to be established but their presence in clinical cases of Pasteurella pneumonia in feedlot cattle further supports a possible role in the initiation and progression of this disease as well as bacterial resistance to antimicrobial agents.     

Serum complement-fixing antibody to Pasteurella haemolytica A1 and its effect on experimentally induced pneumonic pasteurellosis in calves

Ninety-three calves comprising 16 experimental groups were exposed to viral (bovine herpesvirus-1 or parainfluenza-3 virus) and Pasteurella haemolytica aerosols. Serum samples from these calves were tested before and after exposure for antibodies to P haemolytica by a modified direct complement-fixation test. At slaughter of the calves, the extent of pneumonia produced was estimated for each calf and compared with the results of the modified direct complement-fixation tests. The extent of pneumonia was not related (P greater than 0.05) to the amount of anti-P haemolytica antibody produced by either naturally occurring or experimentally induced infection.     

Bovine pneumonic pasteurellosis: effect of culture age of Pasteurella haemolytica used as a live vaccine

Five experiments were conducted that compared aerosol immunization of calves with live Pasteurella haemolytica from logarithmic (6 hour) or stationary (20 to 22 hour) phase cultures. Calves were challenge exposed by transthoracic injection with P haemolytica. In 4 experiments, calves inoculated with 6-hour cultures had slightly lower mean lesion scores (indicating greater resistance to challenge exposure) than those inoculated with 20- to 22-hour cultures. High antibody titers, as detected by a quantitative fluorometric immunoassay or the indirect hemagglutination test, correlated directly with lung resistance (based on lesion scores) regardless of the age of the culture used as the immunogen.     

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.