Experimental Pneumonic Pasteurellosis Research Articles

Effects of dexamethasone on distribution and function of peripheral mononuclear blood cells in pneumonic calves

Experimental pneumonic pasteurellosis was used in 30 Holstein calves to compare the effects on the immune response of different treatments: antibiotics alone or antibiotics in combination with either steroidal (SAID) or non-steroidal anti-inflammatory drugs (NSAID). Clinical parameters and the effect of treatments on the mitogen-induced proliferation of peripheral mononuclear blood cells (PMBCs) and on their distribution were evaluated. Calves in all three treated groups showed rapid improvement, but clinical signs were less marked in both groups receiving anti-inflammatory drugs. Limited difference in the effect on the immune system of treatments was observed. No inhibition of lymphocytes proliferation was detected in these experimental conditions in the dexamethasone (DM)-treated group. There was no variation among groups for the CD4+, CD5+, CD8+ and CD21+ populations. The only noteworthy change was a transient increase in the percentage of the monocyte population (CD14+) in the DM-treated group compared to the group treated with NSAID.

Intranasal vaccination of rabbits with Pasteurella multocida A:3 outer membranes that express iron-regulated proteins.

To determine efficacy of intranasal vaccination of rabbits with Pasteurella multocida A:3 outer membrane proteins (OMP) expressing iron-regulated OMP (IROMP) in conferring protection against experimental challenge exposure. 52 male New Zealand White rabbits. Rabbits were vaccinated intranasally on days 0, 7, and 14; some vaccines included cholera toxin (CT) as an adjuvant. Concentrations of intranasal IgA and serum IgG antibodies against P multocida OMP were determined. In experiment A, rabbits were vaccinated with either phospate-buffered saline solution (PBSS), PBSS-CT, OMP-CT, or IROMP-CT, challenge-exposed intranasally on day 16, and euthanatized and necropsied on day 28. Rabbits were also vaccinated with OMP or IROMP without CT and were not challenge-exposed. In experiment B, rabbits were vaccinated with PBSS, PBSS-CT, IROMP, or IROMP-CT. On day 17, rabbits were challenge-exposed intranasally. Nasal bacteria and antibodies were determined on day 24. In experiment A, OMP-CT vaccination stimulated mucosal and systemic antibody responses to the bacterium and enhanced resistance against challenge exposure. Intranasal bacterial counts were not significantly reduced. Vaccination with IROMP-CT stimulated mucosal and systemic antibodies, enhanced resistance to challenge exposure, and significantly reduced nasal bacterial counts. In experiment B, natural infection was detected in several rabbits at challenge exposure; however, IROMP-CT-vaccinated rabbits had significantly higher serum and nasal antibody responses, compared with other rabbits IROMP-CT-vaccinated rabbits had significantly lower nasal bacterial counts compared to control rabbits. Intranasal vaccination of rabbits with P multocida outer membranes containing IROMP and CT stimulated immunity against experimental pneumonic pasteurellosis.

Evaluation of efficacy of three commercial vaccines against experimental bovine pneumonic pasteurellosis

The objective of this study was to evaluate the efficacy of three commercial vaccines against experimental pneumonic pasteurellosis in cattle. The three vaccines were: (a) One Shot (SmithK-line Beecham, West Chester, PA.), (b) Presponse (Langford Laboratories, Guelph, Ontario) and (c) Once PMH (BioCor, Omaha, NE.). Protective immunity was evaluated in terms of lower clinical and pneumonic lesion scores after endobronchial challenge with virulent P. haemolytica. The results indicate that One Shot elicited antibodies against leukotoxin (Lkt), capsular polysaccharide (CP) and surface antigens (SA), while Presponse and Once PMH elicited antibodies against CP and SA. There was significant correlation between lung and serum antibody levels against Lkt ( P < 0.0001), CP ( P ≤ 0.0001) and IROMPs ( P ≤ 0.035). Animals that received the One Shot had significantly ( P ≤ 0.05) lower mean pneumonic lesion score (36.6 ± 10.97) as compared to the control group (48.6 ± 25.92). A significant negative correlation (−0.41; P ≤ 0.008) existed between serum antibody levels against Lkt and pneumonic lesion score. High serum antibodies against SA did not correlate with reduction in pneumonic lesion score. In addition, high antibody levels against CP did not correlate consistently with reduced pneumonic lesion scores. The results from this study demonstrates that commercial vaccines evaluated in this trial did not confer optimal protection in vaccinated calves, against experimental pneumonic pasteurellosis. However, One Shot vaccinates showed a better protective immunity compared to the other two vaccine groups.

Evaluation of three experimental subunit vaccines against pneumonic pasteurellosis in cattle

The objective of this study was to evaluate the efficacy of three Pasteurella haemolytica A1 derived experimental subunit vaccines against pneumonic pasteurellosis in cattle. The three vaccines were: (a) culture supernatant (CS) containing leukotoxin (Lkt), lipopolysaccharide (LPS) and capsular polysaccharide (CP); (b) sodium salicylate extract (SSE) containing iron regulated outer membrane proteins (IROMPs), LPS and CP; (c) and a combination of the above two. Vaccine efficacy was defined in terms of reduction in clinical and pneumonic lesion scores after intrapulmonic challenge with live P. haemolytica. The results indicate that the CS vaccine elicited antibodies against both Lkt and CP, while the SSE vaccine elicited antibodies against IROMPs and CP. Animals inoculated with the combination vaccine showed increased levels of antibodies against IROMPs, Lkt and CP. There was significant correlation between lung and serum antibodies against Lkt, CP and IROMPs. Animals that received the combination vaccine had significantly lower mean pneumonic lung score as compared to SSE and control groups. The animals which received CS vaccine had mean pneumonic lung score significantly lower than that of control group. A strong negative correlation existed between serum antibody levels against Lkt, IROMPs, CP and pneumonic lung scores. The results from this study demonstrate the usefulness of CS vaccine alone or in combination with SSE vaccine in bringing about optimal protection in vaccinated calves, against experimental pneumonic pasteurellosis.

Increased tumor necrosis factor-α and interleukin-1β expression in the lungs of calves with experimental pneumonic pasteurellosis

We used a well characterized pneumonic pasteurellosis model in calves to determine whether increased proinflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) expression and secretion were associated with pneumonic lesions. Bronchoalveolar lavage fluids, lavage cells consisting of alveolar macrophages and neutrophils with degenerative changes, and lung tissues were analyzed for the presence of TNF-α and IL-1β approximately 48 h following endobronchial inoculation of logarithmic phase Pasteurella haemolytica 12296 organisms. Levels of TNF-α and IL-1β mRNA were significantly increased in lavage cells of P. haemolytica-infected animals but not in cells from phosphate buffered saline (PBS) inoculated controls based on in situ hybridization analysis. Significantly increased levels of TNF-α, and IL-1β mRNA were also expressed within the pneumonic lesions from P. haemolytica-infected calves. In contrast, lung tissues from PBS-inoculated control calves had cytokine mRNAs expressed at extremely low levels. Increased levels of bioactive IL-1 and immunoreactive (not bioactive) TNF-α were found in lavage fluids from P. haemolytica-infected calves compared with lavage fluids from PBS-inoculated calves. These findings indicate that the proinflammatory cytokines TNF-α and IL-1, may be associated with pathogenesis of lung injury in bovine pneumonic pasteurellosis.

Identification of a potentially important antigen of pasteurella haemolytica

To identify antigens which may be important for stimulating immunity to pneumonic pasteurellosis, a bovine antiserum to whole P. haemolytica was used to screen a recombinant lambda gt11/ P. haemolytica expression library. One of the recombinant bacteriophage clones identified with the bovine antiserum, SW20C, expressed a fusion protein which was also recognized by rabbit antiserum to partially purified P. haemolytica culture supernatant and was found to be immunogenic in guinea pigs. The guinea pig antibody recognized a 100 kDa protein in P. haemolytica cell lysates. Sequence analysis of the cloned DNA from SW20C identified a fragment of 1443 bp with a small open reading frame that was contiguous with the lacZ sequence. The 153 bp P. haemolytica-specific open reading frame encoded a polypeptide of approximately 6kDa. Homology searches of Genbank and the EMBL data bases revealed no homology of this open reading frame with any other bacterial sequences including P. haemolytica leukotoxin and Ssa1. Evaluation of sera from calves that were scored either susceptible or resistant to experimental pneumonic pasteurellosis demonstrated a significant ( P < 0.001) correlation between the intensity of the antibody response to the SW20C antigen and resistance to disease.

Administration of a bolus formulation of baquiloprim and sulphadimidine to calves: plasma concentration - time profiles and efficacy in suppressing experimental pneumonic pasteurellosis

After administration of a bolus containing 0.8 g baquiloprim and 7.2 g ulphadimidine to six calves, plasma concentrations of these compounds exceeded MICs for Pasteurella haemolytica from about 3 hours to at least 48 hours. Experimental infection of three calves with 2.8×10 10 P. haemolytica type A1 organisms by the endobronchial route resulted in extensive pneumonic lesions, and smaller, similar lesions were found post mortem in two other calves which had received the same inoculum. Nine similar calves were also infected in the same manner after dosing six hours previously with a bolus providing doses of 4 mg/kg baquiloprim and 36 mg/kg sulphadimidine. Four of these calves received a second bolus 48 hours later. In contrast to the undosed calves, the treated calves exhibited few symptoms of respiratory disease following infection, and their temperatures remained normal. The mean temperatures of the control calves were significantly elevated for several days after infection. At post mortem examinations 6 days after infection, lung lesions were insignificant in all the dosed calves. P. haemolytica was isolated from the lungs of all 5 control calves but not from any treated animal. The combined dose of 40 mg/kg baquiloprim and sulphadimidine was the minimum recommended for use of the bolus in the field, and the results therefore predict efficacy when used in outbreaks of respiratory infection in calves associated with this organism.

The effects of stressful exercise on leukocytes in cattle with experimental pneumonic pasteurellosis

Seven yearling bulls were treated with stressful exercise and intrabronchial Pasteurella haemolytica A1. Group 1 bulls (nos. 1-4) underwent treadmill exercise and, 24 days later, intrabronchial instillation of P. haemolytica A1. Group 2 bulls (nos. 5-7) underwent treadmill exercise, followed 30 min later by intrabronchial P. haemolytica A1. Blood lactic acid values were raised (p less than 0.05) by treadmill exercise only, but plasma cortisol was raised (p less than 0.05) by treadmill exercise and by P. haemolytica A1 infection. Neutrophils in bronchoalveolar lavage (BAL) differed from control values 24 h after treadmill exercise, and 1 h and 4 h after P. haemolytica A1 infection. Respiratory disease was more severe and the gross lung lesions were larger in group 2 bulls than in group 1 bulls. P. haemolytica A1 was recovered from the livers, spleens and mesenteric lymph nodes of group 2 but not group 1 bulls, suggesting that group 2 bulls had experienced bacteraemia. Decreased neutrophils in BAL fluid from group 2 bulls at 1 h and 4 h after infection suggests that exercise transiently inhibited neutrophil egress from the blood to the alveoli; BAL neutrophils peaked at 1 h and 4 h after infection in group 1 bulls but declined at 24 h. We conclude that group 2 bulls were made more susceptible to experimental pneumonic pasteurellosis by stressful exercise.

Serum IgG and IgM antibody response in cattle to antigens of Pasteurella haemolytica

The serum IgG and IgM antibody responses of 48 cattle vaccinated with live Pasteurella haemolytica (LIVE), formalin-killed P. haemolytica in Freund's incomplete adjuvant (FIA), or formalin-killed P. haemolytica in aluminum hydroxide adjuvant (ALH) to a variety of P. haemolytica antigens were evaluated. Enzyme-linked immunosorbent assays (ELISAs) were used to determine the sequential and day 21 IgG and IgM antibody responses to whole P. haemolytica (WB), a capsular carbohydrate-protein subunit (CPS) extracted from the organism, P. haemolytica capsular carbohydrate (CC), and P. haemolytica leukotoxin (LT). LIVE and FIA vaccinates developed generally higher IgG and IgM responses to all antigens compared to ALH vaccinates. LIVE vaccinates developed IgG responses to LT which were significantly higher ( P<0.05) than all other vaccinates. In contrast, FIA vaccinates developed significantly higher IgG responses to CPS than all other vaccinates. On the basis of the ELISA results, similar or cross reacting antigenic sites were present in preparations containing surface antigens (WB, CPS and CC), but not LT. Disease resistance, as determined by experimental lesions induced in the 48 calves by transthoracic challenge with P. haemolytica, was significantly greater in the LIVE and FIA vaccinates compared with ALH vaccinates. No significant difference in resistance was detected between LIVE and FIA vaccinates. Lesions in ALH vaccinates were not significantly different than those in phosphate-buffered saline (PBS) controls. Increased IgG responses to all antigens were significantly associated with resistance to experimental disease; however, IgG responses to CPS were most highly correlated with resistance. The only IgM response which was significantly correlated with resistance was the response to CPS. These studies indicate that serum IgG antibody responses to various surface antigens of P. haemolytica, as well as LT, can enhance resistance to experimental pneumonic pasteurellosis. Serum IgM responses, however, do not appear to play a major role in resistance to experimental disease.

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.

Pasteurella haemolytica antigens associated with resistance to pneumonic pasteurellosis

Antigens associated with whole Pasteurella haemolytica biotype A serotype 1, a capsular carbohydrate-protein extract of the organism, and P. haemolytica leukotoxin were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antigens of the electrophoresed preparations were detected by Western blotting (immunoblotting) with sera from cattle which were either nonvaccinated or vaccinated with live or killed P. haemolytica vaccines and had variable degrees of resistance to experimental pneumonic pasteurellosis. Distinct, easily recognizable antigens of these preparations were identified, and the antibody responses to these antigens were quantified by densitometry. To determine their importance to disease resistance, we then compared antibody responses with experimental lesion scores. Antibody reactivity to surface antigens which were significantly correlated with resistance and present in two or more of the preparations were detected at 86, 66, 51, 49, 34, 31, and 16 kilodaltons (kDa). Of these, antibody responses to antigens at 86, 49, and 31 kDa appeared most important based on their concentration and significance levels. Antibody reactivity to leukotoxin antigens which were significantly correlated with resistance and common with important surface antigens were detected at 86, 66, and 49 kDa. Antibody responses to unique leukotoxin antigens which were significantly correlated with resistance were present at 92 and 58 kDa.

Failure of a Pasteurella haemolytica extract vaccine to protect calves against experimental pneumonic pasteurellosis

Failure of a Pasteurella haemolytica extract vaccine to protect calves against experimental pneumonic pasteurellosis

Chloramphenicol, lincomycin and oxytetracycline disposition in calves with experimental pneumonic pasteurellosis.

The effects of pneumonia on the pharmacokinetics of chloramphenicol, lincomycin, and oxytetracycline were evaluated in two-month-old calves. Pneumonia was induced by injection of Pasteurella haemolytica cultures directly through the thoracic wall into each lung. Six days prior to induction of pneumonia, the antibiotics were administered in a single i.v. dose. The antibiotics were administered again 48 (i.v.), 60 and 72 h (i.m.) following injection of P. haemolytica. The pharmacokinetics of chloramphenicol (25 mg/kg) and lincomycin (10 mg/kg) were not significantly different in calves with pneumonia. The hybrid rate constant beta for oxytetracycline was increased in calves with pneumonia from 0.0034 +/- 0.0003/min to 0.0048 +/- 0.0007/min between 2 h and 8 h. Thus the elimination half-life in serum was shortened from 212.4 +/- 20.3 min to 149.3 +/- 19.5 min. In addition, there was an apparent but not statistically significant decrease in K12 with pneumonia. These findings accentuate the need for observance of 12-h dose intervals with oxytetracycline.

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 < 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.

Vaccination against experimental pneumonic pasteurellosis.

Vaccination against experimental pneumonic pasteurellosis.

Experimental pneumonic pasteurellosis in sheep and cattle.

Experimental pneumonic pasteurellosis in sheep and cattle.