Pulmonary immunity in calves following stimulation of the gut-associated lymphatic tissue by bacterial exotoxin.

Antibodies in serum and pulmonary lavage fluids were measured in calves following stimulation of the gut-associated lymphatic tissue (GALT) by inoculation of crude leukotoxin of Pasteurella haemolytica into the duodenum through a surgically placed catheter. Nine calves free of P. haemolytica were divided into two groups. Group 1 received an intraduodenal (ID) inoculation of leukotoxin and group 2 received an ID inoculation of phosphate buffered saline. Serum and pulmonary lavage fluids were collected weekly and assayed for antibodies specific to P. haemolytica including immunoglobulin (Ig)G, leukotoxin neutralizing antibodies (LNA), and IgA (lavage fluids only). The multiplicative increase (over baseline) in each class of antibody titer following ID inoculation of leukotoxin, the composite geometric mean increase of all antibodies together, and the composite number of the five antibody titers which increased at least fourfold were computed. Results showed that the geometric mean of each antibody titer and the two composite indices was higher in the GALT-primed groups than in the sham-primed group. The differences were statistically significant (p less than 0.05) for serum IgG and for the two composite indices. This experiment demonstrates for the first time that GALT stimulation by bacterial exotoxins results in increased pulmonary antibody levels in calves.     

Antileukotoxin antibody produced in the bovine lung after aerosol exposure to viable Pasteurella haemolytica

Experiments were performed to determine the in vivo immunogenicity of Pasteurella haemolytica leukotoxin. Calves were exposed twice to aerosol mists of viable P haemolytica, using a treatment regimen previously shown to induce a resistant state. Pulmonary lavage fluids and serum samples from these calves were assayed for leukotoxin-neutralizing antibodies. Before aerosol exposure, neutralizing antibody titers were routinely found in serum samples, but were not detectable in pulmonary lavage concentrates before exposure. After aerosol exposure, titers of toxin-neutralizing immunoglobulin (Ig)A and IgG antibodies were found in pulmonary lavage concentrates and were accompanied by increased serum toxin neutralization titers.     

Hematological changes in calves exposed to a mixture of lipopolysaccharide and crude leukotoxin of Pasteurella haemolytica.

The purpose of this investigation was to determine if culture supernatants of Pasteurella haemolytica containing crude leukotoxin and lipopolysaccharide (CLCL) causes disseminated intravascular coagulopathy (DIC) when injected into calves. The effect of intraduodenal (ID) exposure followed by a subsequent subcutaneous (SC) inoculation of either heat-treated or untreated CLCL was evaluated. The relative contribution of the crude leukotoxin and lipopolysaccharide (LPS) to the virulence of P. haemolytica was evaluated. One group of calves received an ID inoculation of CLCL followed two weeks later by a SC inoculation of CLCL; one group received an ID inoculation of tissue culture medium followed two weeks later by a SC inoculation of CLCL; and a third group received an ID inoculation of CLCL followed two weeks later by a SC inoculation of heat-treated CLCL. Hematological parameters used to evaluate DIC included white cell count, platelet count, neutrophil number, fibrinogen, fibrin degradation products, one stage prothrombin time (OSPT), activated partial thromboplastin time, body temperature and clinical signs. Each parameter was measured in calves at 0, 2, 4, 6, 12 and 24 h following the SC inoculation of CLCL. Each group had significant changes over time in all parameters except body temperature. Calves that received a SC inoculation of heat-treated CLCL had smaller changes in all parameters except OSPT compared to the other groups. Results suggest that the LPS and leukotoxin of P. haemolytica exert additive effects on the coagulation cascade and number of peripheral leukocytes, and that the ID inoculation of CLCL does not affect the response of calves to a SC inoculation of toxin.     

Systemic and pulmonary antibody responses of calves to Pasteurella haemolytica after intrapulmonary inoculation.

Systemic and pulmonary antibody responses of calves to Pasteurella haemolytica were evaluated by measuring immunoglobulin production in blood for 9 days and in pulmonary lavage fluid for 7 days after intrapulmonary inoculation. Clinical signs, pulmonary lesions, pulmonary and systemic inflammatory response, and amount of antigen in lavage fluid were used to evaluate the response of calves to challenge with P haemolytica. The pulmonary response consisted of production of IgG, IgE, and IgM antibodies to P haemolytica antigens and a 17- to 68-fold increase of cells in lavage fluid 8 hours after inoculation, with a gradual decrease toward normal. Antibodies of the IgM isotype to P haemolytica were demonstrated as early as 8 hours through 7 days after inoculation in 3 of 3 calves. Of the anti-P haemolytica isotypes, IgM was found in the highest concentration. In all of the inoculated calves, IgE was found 1 to 2 days after inoculation, and IgG was found in 2 of 3 inoculated calves from day 1 through 7 after inoculation. Detection of IgG correlated with smaller pulmonary lesions. Immunoglobulin A was not detected in lavage fluid. Serum was evaluated for IgG and IgM antibody response to P haemolytica. Specific IgM was detectable 5 days after inoculation, and IgG was detectable 7 days after inoculation. Pasteurella haemolytica antigens were not detected in serum or plasma. A transient increase in neutrophil count was found 8 hours after inoculation, with return to baseline values by 24 hours after inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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 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.     

The Immune Response of Calves given Mycoplasma bovis Antigens

Seven calves seven to 30 days of age were given Mycoplasma bovis antigen by different routes. Immunization was in two phases. The first consisted of single or multiple SC, IV or oral doses of antigen for two to four weeks. The second phase consisted of multiple SC or ID injections given from the eighth to the 19th week. The experiment was terminated at 26 weeks. Antibody titers were followed by indirect hemagglutination, growth inhibition and tetrazolium reduction inhibition. Total serum protein, protein fractions and IgG and IgM concentrations were determined in serums of one calf and the distribution of indirect hemagglutination antibodies in IgG and IgM classes were determined in serums of two of the calves.     

Response of Pasteurella haemolytica to erythromycin and dexamethasone in calves with established infection.

A subcutaneous soft tissue infection model in calves was used to study the in vivo response of Pasteurella haemolytica to erythromycin and dexamethasone. Two tissue chambers were implanted SC in each of 12 calves. At 45 days after implantation, all tissue chambers were inoculated with an erythromycin-sensitive strain of P haemolytica. Starting 24 hours after inoculation, calves were allotted to 4 groups of equal size and a 2 x 2-factorial arrangement of treatments was applied: 3 calves were given erythromycin (30 mg/kg of body weight, IM, for 5 days), 3 calves were given dexamethasone (0.05 mg/kg, IM, for 2 days), 3 calves were given erythromycin and dexamethasone, and the remaining calves served as nontreated controls. Chamber fluids were tested daily, and the response to treatment was measured. Neither erythromycin nor dexamethasone affected viability or growth of bacteria within tissue chambers. Dexamethasone had no effect on the influx of neutrophils into infected chambers. Despite repeated administration of a high dose of erythromycin and attainment of adequate concentration in serum, erythromycin concentration in chamber fluids did not exceed the minimal inhibitory concentration established in vitro. These results indicate that the clinical efficacy of erythromycin against P haemolytica sequestered in consolidated pneumonic lesions may not be well correlated with predictions based on serum pharmacokinetic and in vitro susceptibility data.     

Susceptibility of Pasteurella haemolytica to the bactericidal effects of serum, nasal secretions and bronchoalveolar washings from cattle

Several isolates of logarithmic-phase organisms of Pasteurella haemolytica were shown to be sensitive to an antibody and complement-mediated killing mechanism in adult bovine serum. Data suggested that the classical complement pathway was important in the induction of bactericidal activity of serum. Sera from calves after colostrum feedings (post-colostral sera) killed only 30% of the bacteria in spite of the presence of high levels of antibodies against P. haemolytica. Addition of post-colostral serum to heat-inactivated adult bovine serum decreased the bactericidal capacity of the latter. It was speculated that this inhibition may have been caused by the presence of blocking antibodies (IgA) found in the post-colostral serum. Undiluted nasal secretions collected from adult cattle were not bactericidal to P. haemolytica. The results also suggest that the bronchoalveolar washings (BAW) from vaccinated calves, in spite of having a high antibody titer, were less bactericidal to P. haemolytica than BAW from sham-vaccinated calves (71.12% vs. 83.12%). The bactericidal factor(s) present in BAW from sham-vaccinated calves was heat stable, not complement dependent, and was not related to lysozyme concentration.     

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)     

Changes in leukocyte populations in pulmonary lavage fluids of calves after inhalation of Pasteurella haemolytica

The distribution of leukocytes in bovine bronchoalveolar lavage fluids was determined in 15 calves at various times after aerosol exposure to Pasteurella haemolytica. For comparison, 10 calves were exposed to aerosols of phosphate-buffered saline solution; 15 calves, to Staphylococcus epidermidis; and 10 calves, to Salmonella typhimurium endotoxin. At 10 minutes after inhalation exposure for each group, the predominant cell type was the macrophage. Macrophages remained the predominant cell type throughout each lavage interval for calves exposed to phosphate-buffered saline solution and Staph epidermidis. For calves exposed to P haemolytica, there was a decrease in the percentage of macrophages detectable by 30 minutes after exposure, with a corresponding increase in the percentage of neutrophils. Sixty minutes after the inhalation exposure to P haemolytica, the percentages of macrophages and neutrophils in the lavage fluid were equal. By 240 minutes after exposure to P haemolytica, greater than 90% of the cells in the lavage fluids was neutrophils. The increase in the percentage of neutrophils in lavage fluids from calves exposed to S typhimurium endotoxin was similar to that seen for the calves exposed to P haemolytica.     

Bovine serum and nasal secretion immunoglobulins against Pasteurella haemolytica serotype 1 antigens

Experimental intranasal inoculation of cattle with Pasteurella haemolytica serotype 1 resulted in a group that shed the bacteria in their nasal secretions (colonized) and a group that did not shed (uncolonized). After inoculation, antibody titers in serum and nasal secretions against the total P haemolytica increased significantly, and the proportions of total antibody against specific P haemolytica antigens changed so that the proportion directed against the 94- and 62-kD antigens increased. Prior to inoculation, the proportion of total antibody in the serum against 94- and 62-kD antigens of P haemolytica was higher in calves that remained uncolonized than in those that became colonized with P haemolytica after exposure. Antibody specificity of serum and nasal secretions differed in the relative amounts directed against each P haemolytica antigen. The specificity against P haemolytica antigens differed between IgG and IgA isotypes of serum and nasal secretions, with IgA being directed against fewer antigens than was IgG.     

Effects of various vaccination protocols on passive and active immunity to Pasteurella haemolytica and Haemophilus somnus in beef calves.

Two field trials were conducted in a beef cow herd in Saskatchewan to determine the effectiveness of a combined Pasteurella haemolytica and Haemophilus somnus vaccine in increasing passively and actively acquired antibodies in beef calves. Vaccination of dams at 4 and/or 7 weeks prepartum was associated with increased antibody titers to P. haemolytica and H. somnus in their serum (P < 0.05), colostrum (P < 0.05), and serum of their calves at 3 days and 1 month of age (P < 0.05). There was no significant (P > 0.05) difference in antibody titers in the colostrum and serum of calves from single or double vaccinated dams. Calves vaccinated at 1 and 2 months of age in the face of maternal antibodies to P. haemolytica and H. somnus had significantly (P < 0.05) higher antibodies to P. haemolytica and H. somnus at 4 and 6 months of age than did unvaccinated calves. Calves vaccinated at 3 and 4 months of age in the face of low levels of preexisting antibodies had significantly (P < 0.05) higher antibodies to P. haemolytica at 5 months of age and to H. somnus at 5 and 6 months of age than did unvaccinated calves. Calves vaccinated once at 4 months of age had significantly (P < 0.05) higher antibody titers to P. haemolytica and H. somnus at 4.5 months of age than did unvaccinated calves, but this difference was not apparent at 6 months of age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of Pasteurella haemolytica and correlation with serum antibody response in clinically normal beef calves

Bacteria from the nasal cavity and trachea were cultured, and serum antibody titers determined for Pasteurella haemolytica serotype 1 in 164 beef calves obtained from a closed herd on range pasture. At the first sampling, P. haemolytica serotype 1 was cultured from 16.4% of the calves. Antibody titers were determined by a quantitative fluorimetric method and the mean titer was 9.5 +/- 5.8. Fifty-seven randomly selected calves were used to study the correlation of serum antibody response and positive culture of P. haemolytica under natural conditions. Clinical signs of respiratory disease were not observed in those calves. During the observation periods, there was a two-fold increase in the percentage of calves that were culture positive. There was no significant difference between mean serum antibody titers or frequency distribution of antibody titers from the two samplings. Comparisons between serum antibody titers, rise in titers, and P. haemolytica isolation failed to reveal any significant correlation. Of the 9 calves that had a decline in antibody titer to P. haemolytica, none was culture positive. Seroconversion to respiratory viruses did not correlate with P. haemolytica related variables.     

Sequential titration of bovine lung and serum antibodies after parenteral or pulmonary inoculation with Pasteurella haemolytica

Calves were vaccinated by intrabronchial or subcutaneous injection of formalinized Pasteurella haemolytica. Antibody in serum, nasal washings, and bronchoalveolar washings was titrated sequentially before and after calves were vaccinated and then challenge exposed with live homologous bacteria. Bronchoalveolar washings were collected by fiberoptics bronchoscopy, and antibody was titrated by indirect (antiglobulin) bacterial agglutination. Responsiveness to vaccination was related in initial serum antibody concentrations. Calves with serum antibody titers of 1:20 or more were nonresponsive, whereas with few exceptions, calves having titers of less than 1:20 responded to vaccination. Results indicated that serum and lung antibody were induced by subcutaneous or by intrabronchial inoculation of formalinized P haemolytica. By either route of immunization, serum antibody was more persistent than was lung antibody, and pulmonary challenge exposure with live P haemolytica did not alter existing titers.     

Vaccination of neonatal colostrum-deprived calves against Pasteurella haemolytica A1.

Colostrum-deprived Holstein calves were vaccinated at 2 and 4 wk of age with a Pasteurella haemolytica A1 culture supernatant vaccine to determine whether active immune responses and protection could be induced in this age group in the absence of maternal antibodies. All calves responded to vaccination with high titers of IgM antibodies to capsular polysaccharide within 1 wk of primary vaccination. Mean titers of IgG1 and IgG2 antibodies to this antigen increased significantly by 2 wk after secondary vaccination, but peak antibody titers were low. All of the vaccinated calves seroconverted with production of leukotoxin-neutralizing antibodies, but peak antibody titers were low. Vaccinated calves experienced considerable lung damage after experimental challenge, but survival rate, clinical scores, and percent lung involvement were significantly better than those of control (placebo-injected) calves.     

Development and evaluation of an enzyme-linked immunosorbent assay for bovine antibody (IgG) to Pasteurella haemolytica

The sensitivity of an indirect enzyme-linked immunosorbent assay (ELISA) for bovine IgG serum antibody to Pasteurella haemolytica was compared with that of an indirect hemagglutination (IHA) test. Pasteurella haemolytica serotypes were grown in a chemically defined cell culture medium, and soluble antigens released into the growth medium were used in the ELISA and IHA test. An ELISA with serotype-1 antigen consistently detected antibody in sera that were positive by IHA test (correlation, 99%). Sera reacting with serotype-1 ELISA antigens also reacted with ELISA antigens prepared from other serotypes. Although ELISA titers averaged 5 log2 units higher than IHA titers, plots of titers determined by the 2 methods were approximately linear. Titer increases detected in paired serum samples by either test were similar. The ELISA was more sensitive than was the IHA in detecting colostral IgG antibody in serum of newborn calves. The ELISA uses a simple, stable antigen preparation and detects antibody to P haemolytica serotypes that commonly infect cattle.     

The pulmonary clearance of Pasteurella haemolytica in calves given Corynebacterium parvum and infected with parainfluenza-3 virus.

Four control calves were aerosolized with parainfluenza-3 and one week later with Pasteurella haemolytica. Three calves were given Corynebacterium parvum at a dose of 15 mg/m2 body surface area, infected with parainfluenza-3 virus one week later, and aerosolized with P. haemolytica two weeks after C. parvum injection. All calves were killed four hours after P. haemolytica exposure and the bacterial retention in the lung was determined. Parainfluenza-3 viral infection did not exert any suppressive effect on pulmonary clearance of P. haemolytica in six out of seven calves used. However, the bacterial colony counts in the lungs of control calves were higher (P less than 0.05) than those in calves given C. parvum. Hence, C. parvum appeared to enhance bacterial clearance. Despite the marked influx of neutrophils into the lungs after the bacterial inoculation, the neutrophil:macrophage ratio in lavage samples was less in calves given C. parvum than in the control calves. The alveolar macrophages in C. parvum treated calves were generally larger but did not differ significantly (P less than 0.05) from those in the controls. There was no significant (P less than 0.05) correlation between the percentages of alveolar macrophages and the bacterial clearance. In calves given C. parvum, bacterial clearance was enhanced in those calves which had larger macrophages.     

Comparison of protection of experimentally challenged cattle vaccinated once or twice with a Pasteurella haemolytica bacterial extract vaccine.

Three groups of calves (15-18 per group) were injected twice at a 3-week interval with 2 doses of phosphate buffered saline (PBS, CONTROL group), 2 doses of PRESPONSE, a Pasteurella haemolytica A1 bacterial extract vaccine (PRESPONSE-2 group) or 1 dose of PBS followed by a 2nd vaccination with 1 dose of PRESPONSE (PRESPONSE-1 group). Three weeks after the 2nd vaccination, the calves were challenged intratracheally with P. haemolytica A1. Calves were evaluated clinically for 3 days prior to challenge and for 5 days after challenge. Six days postchallenge, calves were either euthanized or sent to slaughter and the lungs were evaluated for percent pneumonic tissue. There was a significant effect of single or double application of vaccine on clinical scores (P = 0.0409). Percent pneumonic tissue at necropsy was significantly affected by vaccine group (P = 0.014). Calves in the CONTROL group had significantly higher percent pneumonic tissue after arcsine transformation (45.30%) than calves in any group receiving PRESPONSE, regardless of vaccination frequency (25.18% and 25.78%, for calves receiving 2 doses or 1 dose of PRESPONSE, respectively). Both serum toxin neutralizing and direct agglutinating titers were negatively correlated with percent pneumonic tissue. Most importantly, 1 dose of PRESPONSE was as efficient as 2 doses at eliciting a protective immune response. It is concluded that the presence of P. haemolytica as a natural commensal in the upper respiratory tract of the calf can effectively prime the animal, and allow the animal to respond in an anamnestic nature to only 1 dose of this vaccine.