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.     

猪肺疫多杀性巴氏杆菌的分离鉴定

为了研究四川乐山某猪场爆发的保育猪严重呼吸道疾病的主要病原,试验采用病理剖检、病毒检测、细菌分离鉴定、生化鉴定、动物回归试验以及多杀性巴氏杆菌种特异性KMT基因的PCR检测对病原进行鉴定。结果表明:发病猪有严重肺炎症状,支气管内部有少量泡沫,腹股沟淋巴结出血、水肿,其他实质器官未见明显病理变化;病毒检测结果显示为蓝耳病病毒阳性,猪瘟病毒、伪狂犬病毒、圆环病毒2型阴性;从病猪肺脏中分离获得大肠杆菌和1株蓝耳病病毒继发的高致病性多杀性巴氏杆菌;该分离菌株对多种药物均高度敏感。     

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 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.     

Bovine pneumonic pasteurellosis: model for Pasteurella haemolytica- and Pasteurella multocida-induced pneumonia in cattle

Pneumonic lesions in calves were induced with Pasteurella haemolytica or P multocida. The inoculum, consisting of a suspension of either organism, was administered by transthoracic intrapulmonic injection to 23 calves. Three died of septicemia; the 20 remaining, killed 96 hours after inoculation, had an expanding unifocal pneumonia qualitatively comparable with that of acute pneumonic pasteurellosis (shipping fever). The concentration of bacteria that consistently produced a lesion was a 5-ml volume containing 10(9) colony-forming units of bacteria; a concentration of 10(6) colony-forming units inconsistently produced lesions. Bacteria, except in calves that developed septicemia and died, remained localized at the injection site. The inflammatory process spread within the lungs, not only through airways, but through the interlobular and interalveolar septa as well.     

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.     

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.     

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.     

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.     

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.     

Serotypes and electrophoretic protein profiles of Pasteurella haemolytica isolated from pneumonic ovine lungs.

Serotypes and SDS-PAGE protein profiles of P. haemolytica isolated from pneumonic ovine lungs were investigated. Of 268 P. haemolytica isolates, 232 (86.6%) were serotypable. A total of 12 serotypes were recognized in 20 different geographic origins of central Turkey. The most common serotype was A2, followed by A7, A1 and T4. Serotypes A13, A14, A16 and T15 could not be detected. In SDS-PAGE, marked differences between major bands of biotype A and T strains were found. In numerical analysis of protein profiles, biotype A and T strains were separated at 58% similarity level. Biotype A isolates produced a cluster at 80% similarity level, and biotype T isolates at 92% similarity level. No single cut off level was able to discriminate between each serotype studied and isolates could not be clustered on the basis of their geographic origins.     

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 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.     

Immunoperoxidase evaluation of pneumonic lesions induced by Pasteurella haemolytica in calves

Calves inoculated with Pasteurella haemolytica serovar 1 developed lesions of coagulation necrosis in the lungs that were sharply demarcated by leukocytes. The P haemolytica antigen was detected in the area of coagulation necrosis in histologic sections, using an immunoperoxidase technique. In the central area of the necrotic tissue, the bacterial antigen was diffusely presented in the necrotic alveolar wall, fibrin, serous exudate, and degenerated leukocyte. The bacterial antigen also was found in some groups of degenerating leukocytes around the necrotic tissue. The bacterial colonies among these leukocytes had strong specific reactions against P haemolytica. The bacterial antigen was observed in the cytoplasm of macrophages in alveoli around the necrotic lesion. These findings confirmed that coagulation necrosis is an important lesion in calves with pneumonia caused by P haemolytica.     

Interactions of cold stress and Pasteurella haemolytica in the pathogenesis of pneumonic pasteurellosis in calves: changes in pulmonary function

Thirteen healthy neonatal Holstein calves were cold stressed twice by hosing with cold water for 20 minutes, 12 hours between hosings. Measurements of the pattern of ventilation [tidal volume (VT), respiratory frequency (f), minute ventilation (VMIN), and functional residual capacity (FRC)], gas exchange properties of the lungs [alveolar ventilation (VA), oxygen uptake (VO2), CO2 production (VCO2), dead space ventilation (VD), dead space/tidal volume ratio (VD/VT), arterial oxygen tension (PaO2), arterial CO2 tension (PaCO2) and alveolar-arterial oxygen difference (AaDO2)] and of the mechanical properties of the pulmonary system [dynamic compliance (Cdyn), pulmonary resistance (RL), and total respiratory system resistance (RRS)] were taken. Calves responded to chilling by increasing VO2 and VCO2 necessitating an increase in VA. This was accomplished by increasing VT with reciprocal decreases in f so that VMIN remained constant. There was no change in Cdyn, RL, or AaDO2. Seven of these 13 calves were then exposed to intratracheal inoculation of 2 X 10(9) organisms of Pasteurella haemolytica, the remaining calves serving as controls. Within 1 hour, calves exposed to P haemolytica had increased VMIN, f, VD/VT, and VD. There was a decrease in PaO2 associated with increased AaDO2, but no change in PaCO2, Cdyn or RL. By 3 hours after inoculation, there were pronounced changes in PaO2 and AaDO2, and Cdyn was reduced below base-line values. By 12 hours after inoculation, calves infected with P haemolytica had increased RL and RRS and PaCO2, in addition to the previously mentioned changes. Data from Pasteurella-exposed calves indicate that gas exchange impairment and peripheral lung injury occur rapidly and that increases in airway resistance develop relatively late in the disease.(ABSTRACT TRUNCATED AT 250 WORDS)