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.     

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.     

Exposure of calves to aerosols of parainfluenza-3 virus and Pasteurella haemolytica.

The present study was undertaken to investigate whether sequential exposure to aerosols of parainfluenza-3 virus followed by Pasteurella haemolytica, or P. haemolytica followed by parainfluenza-3 virus, could lead to the production of pulmonary lesions in conventionally-raised calves. Twenty male calves with low serum antibody titres to both organisms were placed in five equal groups. Synergism of parainfluenza-3 virus and P. haemolytica was not demonstrated in any of the sequentially infected groups and pulmonary lesions were mild in all challenged calves. Clinical signs of disease were not present after exposure to parainfluenza-3 virus although the virus was repeatedly isolated from nasal secretions of all inoculated calves. Exposure to P. haemolytica produced a transient response which consisted of increased rectal temperatures and respiratory rates, with a mild neutrophilic leukocytosis and a mild left shift present six hours postinoculation and returning to normal within 24 hours. Results from this study suggest, although do not confirm, that reduced pulmonary clearance of inhaled P. haemolytica in parainfluenza-3 virus infected calves does not necessarily lead to production of severe pulmonary lesions and that previous exposure to aerosols of P. haemolytica may not enhance secondary parainfluenza-3 virus infection.     

Colonization of the tonsils of calves with Pasteurella haemolytica.

Tonsils of 10 calves were inoculated with Pasteurella haemolytica (PH) and the degree of colonization was followed by collecting sequential tonsil. wash specimens. Tonsils were colonized for at least 3 weeks after instillation of PH into the tonsillar sinus. Calves with colonized tonsils responded with serum and nasal secretion antibody responses to PH and to leukotoxin. Pasteurella haemolytica was detected in nasal mucus specimens of 2 calves during the week after inoculation of the tonsils, but all other specimens were culture-negative. Infectious bovine rhinotracheitis virus-induced respiratory tract disease 25 days later did not elicit a population increase of PH in the tonsils, and did not elicit shedding of PH in nasal mucus.     

The immune response to experimental Pasteurella haemolytica infection in calves

Four male dairy calves, ages 1-9 months, were inoculated intratracheally (IT), with log dilutions (1.5 X 10(3)-1.5 X 10(6)) of an isolate of P. haemolytica A-1. Doses of bacteria varied according to ages of the calves, older calves receiving the larger doses. All four calves became severely ill within 24 h after inoculation and antibiotic treatment was considered essential. Two months later the four calves remained healthy after IT injection of P. haemolytica, again given in log dilution (2.8 X 10(2)-2.8 X 10(5)). The control calf, given a dilution of only 28 viable P. haemolytica (plate count), developed severe respiratory infection 9 days post inoculation. Antibiotic treatment was given to this calf for 7 days, at which time recovery was evident. All five calves developed direct bacterial agglutination titers to P. haemolytica. Persistent leukocyte migration inhibition indexes of all calves were decreased by greater than or equal to 20% compared to their controls. Although the initial doses administered were low, the calves became ill. Most reports refer to massive doses necessary to produce primary disease and significant agglutination titers.     

Alterations in pulmonary morphology and peripheral coagulation profiles caused by intratracheal inoculation of live and ultraviolet light-killed Pasteurella haemolytica A1 in calves

Eighteen male Holstein calves were divided into groups of three and inoculated intratracheally with 5 x 10(9) logarithmic phase or ultraviolet light-killed Pasteurella haemolytica biotype A serotype 1. Serial coagulation profiles were done on one calf from each group during the first 24 hours after inoculation. One calf from each group was necropsied at 4, 12, and 24 hours after inoculation and lesions were characterized with light and transmission electron microscopy. We found that 1) the pulmonary intravascular macrophage may have an important role in the early intravascular inflammatory events; 2) there was morphologic evidence for local initiation of the coagulation cascade in the lung early in the disease process but it was not a consumptive process; and 3) killed-bacteria were capable of causing fibrin exudation, platelet aggregation and alveolar epithelial damage similar to live bacteria, but the degenerative changes in neutrophils, endothelial cells and intravascular fibrin formation that occur with live bacteria were not seen.     

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.     

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.     

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.     

Vaccination of calves with leukotoxic culture supernatant from Pasteurella haemolytica.

In three experiments subcutaneous vaccination of calves with adjuvanted bacteria-free leukotoxic culture supernatant from log phase cultures of Pasteurella haemolytica A1 (toxin 1) was shown to induce some protection against intrabronchial challenge with live P. haemolytica A1. This toxin 1 vaccine was as effective as a whole cell bacterin in stimulating agglutinating antibody to P. haemolytica. Induction of leukotoxin neutralizing activity was variable; in some cases vaccination only primed the animal to produce an anamnestic response after challenge, whereas in other instances antitoxic activity increased in response to immunization. Two doses of vaccine were shown to be more effective than a single immunization. Vaccination with leukotoxic culture supernatant from the nonpathogenic P. haemolytica serotype 11 was as effective as vaccination with toxin 1 in stimulating antitoxic activity but was not protective. This implies that both serospecific agglutinating activity and an antitoxic response are needed for immunity.     

Comparison of serological techniques to measure antibody to Pasteurella haemolytica A1.

Analysis of 45 sera was performed employing five techniques which are currently in use in three laboratories to measure anti-Pasteurella haemolytica antibodies. The enzyme linked immunosorbent assay, passive hemagglutination, complement fixation and direct and indirect bacterial agglutination assays were employed and a relationship between tests in the measurement of anti-P. haemolytica antibodies was demonstrated. Regression analysis together with prediction and confidence intervals were tabulated also. The conclusion drawn from statistical analysis was that all five tests are similar in their ability to detect immune responses (antibody and antigen(s) interactions) to Pasteurella haemolytica.     

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.     

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

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

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.     

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.