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.

Pasteurella haemolytica A1 purified capsular polysaccharide does not stimulate interleukin-1 and tumor necrosis factor release by bovine monocytes and alveolar macrophages.

Purified capsular polysaccharide (CPS) stimulated significant release of interleukin-1 (IL-1) activity from bovine blood monocytes but not alveolar macrophages in vitro. The ability of CPS to induce IL-1 release was resistant to boiling and inhibited by the addition of polymyxin beta. Thus, it is likely that the IL-1 release stimulated by CPS resulted from the small amount of contaminating lipopolysaccharide (LPS) that was present (an estimated 5 pg LPS per microgram CPS) rather than to a direct effect of CPS. Tumor necrosis factor activity was not detected in the culture supernatants of bovine monocytes incubated with purified CPS for 1-18 h in vitro.

Interaction of bovine alveolar macrophages with Pasteurella haemolytica A1 in vitro: modulation by purified capsular polysaccharide.

Preincubation of bovine alveolar macrophages with Pasteurella haemolytica A1 purified capsular polysaccharide markedly reduced the phagocytosis of P. haemolytica A1 in vitro in a dose-dependent manner. Both the percentage of macrophages with intracellular P. haemolytica and the mean number of bacteria per ingesting macrophage were decreased by treatment with capsular polysaccharide. Untreated bovine alveolar macrophages had little ability to kill P. haemolytica A1 in vitro at bacteria to macrophages ratios of 1 to 1 or greater; at lower ratios of bacteria to macrophages (1 to 3 or less) modest killing was observed. Preincubation with capsular polysaccharide impaired phagocytosis and killing of P. haemolytica A1 by alveolar macrophages even when the macrophages outnumbered the bacteria. These data indicate that P. haemolytica capsular polysaccharide inhibits the ability of alveolar macrophages to defend against P. haemolytica, as has been reported previously for bovine neutrophils.

Characterization of envelope proteins from Pasteurella haemolytica and Pasteurella multocida.

A method was devised for the reproducible isolation of envelopes from Pasteurella haemolytica serotype A2. It was also possible to prepare envelopes from other serotypes of P. haemolytica and Pasteurella multocida using this methodology. Examination of these preparations by SDS-PAGE showed major differences between strains of P. haemolytica and strains of P. multocida which allowed the clear distinction of isolates of these species. Amongst the P. haemolytica serotypes it was possible to distinguish envelope preparations made from A biotype and T biotype organisms easily, but it was not possible to identify individual serotypes from each other. Envelope profiles were sufficiently different between the individual P. multocida serotypes examined to allow each to be identified by its polypeptide profile. Experiments using radiolabelling, antibody absorption, and susceptibility to protease digestion, together with heat modifiability and detergent solubility characteristics indicated that 13 of the envelope proteins were probably surface-located. A high molecular mass immunogenic envelope protein was shown, by immunoblotting, to be present in all strains of P. haemolytica and P. multocida examined.

Effect of Pasteurella haemolytica (A1) capsular polysaccharide on sheep lung in vivo and on pulmonary surfactant in vitro.

Capsular polysaccharide (CP) of Pasteurella haemolytica (type A1) was first deposited by fiberoptic bronchoscopy into the lungs of sheep to examine lesions and changes in bronchoalveolar lavage cell populations and, later, was mixed with pulmonary surfactant to investigate alterations in physical properties or surface tension. At 22 hours after deposition, minimal lesions were seen in the lungs only at and contiguous to the site of CP deposition in 2 of 4 sheep. Microscopically, alveoli and interlobular septa were filled with edema fluid. Terminal airways and alveoli contained a moderate amount of neutrophils that varied between sheep. Significant differences in number or type of bronchoalveolar lavage cells were not observed in the weekly lavages between each group or among sheep within each group, either before or after deposition of CP or physiologic saline solution. After 6 hours of incubation at 37 C, CP-surfactant mixtures were examined with a surface tensiometer and centrifuged in sucrose gradients. The CP bound to surfactant, resulting in formation of a precipitate with a surface tension of 31.6 +/- 0.1 dynes/cm and a density of 1.07 to 1.08 g/ml. Lipopolysaccharide of P haemolytica, used as a control, also bound to surfactant, resulting in a complex with a surface tension of 57.7 +/- 0.4 dynes/cm and a density of 1.06 to 1.10 g/ml. Surfactant alone had a surface tension of 32.6 +/- 0.2 dynes/cm and density of 1.05 to 1.06 g/ml. The CP appears by itself not to be a direct major factor in the lung damage that develops in cases of pneumonic pasteurellosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of bovine neutrophil antibacterial activities by Pasteurella haemolytica A1 purified capsular polysaccharide.

Preincubation of bovine neutrophils with Pasteurella haemolytica A1 purified capsular polysaccharide markedly diminished their ability to ingest and kill P. haemolytica, but not Escherichia coli, in vitro. Ingestion and killing were impaired even when the P. haemolytica were preopsonized, thus suggesting that the inhibitory effects of the polysaccharide included a direct effect on bovine neutrophils rather than simply competition for serum opsonins. Preincubation of neutrophils with purified polysaccharide did not elicit a chemiluminescence response, nor did it alter the chemiluminescence response of neutrophils to subsequent stimulation with opsonized P. haemolytica or opsonized zymosan. In addition, purified polysaccharide alone was neither chemotactic nor did it induce a shape change in bovine neutrophils. These data suggest that the deposition of capsular polysaccharide in the lung during the onset of pulmonary pasteurellosis might impair the ability of neutrophils to ingest and kill P. haemolytica. The capsular polysaccharide of P. haemolytica, therefore, may contribute in part to the fibrinous pleuropneumonia that characterizes acute pasteurellosis.

Purification, characterization and immunological properties of the serotype-specific capsular polysaccharide of Pasteurella haemolytica serotype A7 organisms.

The serotype-specific capsular polysaccharide from two strains of Pasteurella haemolytica serotype A7 organisms was purified and characterized by chemical analysis and by 1H and 13C NMR spectroscopy using one- and two-dimensional methods. The polymer has the repeating unit----3)-beta-2-acetamido-2-deoxygalactopyranose-(1----3)-alpha- 2-acetamido- 2-deoxy-6-O-acetyl-glucopyranose-(1-phosphate----. It was immunogenic (capable of eliciting antibodies) for sheep. Chemical removal of O-acetyl groups destroyed both the ability of the polymer to adhere to sheep erythrocytes at neutral pH and the ability to form immune precipitates with specific antisera. Studies using the protein A-gold technique in the electron microscope showed the polysaccharide to be peripherally localized on the bacterial surface.

Purification, characterization and immunological properties of the capsular polysaccharide of Pasteurella haemolytica serotype T15: its identity with the K62 (K2ab) capsular polysaccharide of Escherichia coli and the capsular polysaccharide of Neisseria meningitidis serogroup H.

Capsular polysaccharide from two strains of Pasteurella haemolytica serotype T15 was purified and characterized by chemical analysis and NMR spectroscopy. The polymer, a teichoic acid, proved to be very similar in structure to the capsular polysaccharide of P. haemolytica serotype T4 and identical to the previously described K62 (K2ab) capsular polysaccharide of Escherichia coli, and the capsular polysaccharide of Neisseria meningitidis serotype H, i.e. ----(2-glycerol-3)----(phosphate)----(4-alpha-D-galactopyranose -1)---- with partial O-acetylation on the galactose residues. Electron microscopy with Protein A-gold labelled antisera showed that the polysaccharide was peripherally located on the surface of all three organisms. Chemical removal of O-acetyl groups from the polysaccharide yielded a structure identical to that previously described for E. coli K2 (K2a). Both O-acetylated and de-O-acetylated P. haemolytica T15 polymers, when absorbed on to sheep erythrocytes in passive haemagglutination assays, yielded identical antibody titres with sera raised against P. haemolytica T15, E. coli K2 or N. meningitidis H whole cells. De-O-acetylation of the Pasteurella polysaccharide influenced its precipitability with immune sera, but this could not be related to the absence of O-acetyl groups because the non-acetylated E. coli K2 polymer readily precipitated with a line of 'identity' with the acetylated P. haemolytica T15 polymer.

Purification, characterization and immunological properties of the serotype-specific capsular polysaccharide of Pasteurella haemolytica (serotype T4) organisms.

The serotype-specific capsular polysaccharide from two strains of Pasteurella haemolytica serotype T4 organisms was purified and characterized by chemical analysis and NMR spectroscopy. The polymer, a teichoic acid, has the backbone structure ----(2-glycerol-l)----(phosphate)----(6-alpha-D-galactose-1)---- and is partially O-acetylated on the C2 and C3 galactose residues. Chemical removal of O-acetyl groups from the polysaccharide destroyed both its ability to precipitate with antiserum raised against killed whole serotype T4 organisms and its ability to adhere to sheep erythrocytes in passive haemagglutination experiments. Attempts to elicit antisera using the purified polymer were unsuccessful but a partially purified material was immunogenic.

Purification, characterization and immunological properties of the serotype-specific capsular polysaccharide of Pasteurella haemolytica (serotype A1) organisms.

The serotype-specific capsular polysaccharide from two strains of Pasteurella haemolytica serotype A1 organisms was purified and characterized by chemical analysis and NMR spectroscopy. The polymer has the structure----3)-O-(2-acetamido-2-deoxy-4-O-acetyl-beta-D-mannopyranos yluronic acid)-(1----4)-O-(2-acetamido-2-deoxy-beta-D-mannopyranose)-(1----. The polysaccharide was immunogenic (able to evoke production of antibodies) for sheep but not for rabbits. Immuno electron-microscopy studies using the Protein A-gold technique showed the polysaccharide to be peripherally located on the bacterial surface. Reduction, oxidation and de-O-acetylation of the polymer did not appear to alter its immunological precipitability with specific antiserum, but all three treatments destroyed its ability to adhere to sheep erythrocytes at neutral pH. De-N-acetylation of the polymer destroyed both immunological precipitability and erythrocyte adherence.

An evaluation of the conditions necessary for optimal protein A-gold labelling of capsular antigen in ultrathin methacrylate sections of the bacterium Pasteurella haemolytica.

The protein A-gold (PAG) probe is a particulate immunocytochemical probe that is eminently suitable for quantification. In order to obtain critical results from the technique, a specific and reproducible probe is needed. To this end, the concentration of probe, the variation of labelling on different sections within a single grid, the effect of washing procedures, the variation of labelling with time and temperature and the effect of different storage conditions on the probe have been investigated using PAG labelling of capsular antigen on ultrathin methacrylate sections of the bacterium Pasteurella haemolytica. The results indicate that in this antigen-antibody system, and using a 20 nm probe, optimal results are achieved with 2 X 10(12) particles/ml, a labelling time of 60 min at room temperature and the PAG probe, which will have been stored at 4 degrees C, should be between 1- and 5-weeks-old. The efficiency of the probe is tested by evaluating different primary antibody concentrations, by evaluating cross reactions of the primary antibody and by evaluating the relative amounts of antibody against internal components of the bacterium present in different antisera.

A comparison of immunoferritin, immuno-enzyme and gold-labelled protein A methods for the localization of capsular antigen on frozen thin sections of the bacterium, Pasteurella haemolytica.

The staphylococcal protein A-gold method was found to be superior to the enzyme-or ferritin-linked antibody techniques for locating capsular antigens on cryosections of Pasteurella haemolytica, and its sensitivity was similar to the enzyme-linked antibody method. The sensitivity of conventionally fixed and embedded material and cryosections of heavily fixed, lightly fixed and unfixed material were shown to be similar under routine laboratory conditions.

The effect of staphylocoagulase in the mammary gland of the mouse.

Purified coagulase from a strain of S. aureus was inoculated into the mammary glands of mice which were examined at 4-hourly intervals over 28 h. Coagulase induced a neutrophil response at 4 h which was sustained throughout the experiment, and was accompanied by hyperplasia of the alveolar epithelium. There was no evidence of intravascular clotting. The response of the gland to coagulase was compared with the response following inoculation of 3 different strains of viable staphylococci.

Chemical and biological properties of a purified lymphoreticular-stimulating fraction of Corynebacterium parvum (Propionibacterium acnes type I).

A method is described in which washed whole cells of Corynebacterium parvum were chemically and enzymatically extracted to remove cytoplasm and cell-wall lipids. The resultant insoluble cell-wall residue possessed lympho-reticular stimulating properties as measured by their ability to increase spleen weight and protect against tumour-cell challenge. Analysis of the final product by chromatography and infrared spectroscopy has shown it to consist of carbohydrate and peptidoglycan, both of which appear to be necessary for the activities measured.

Effect immunization with highly purified alpha- and beta-toxins on staphylococcal mastitis in rabbits.

Experiments were carried out to determine whether immunization of female rabbits with highly purified staphylococcal alpha- or beta-toxins would protect them against intramammary challenge with staphylococci. High circulating anti-alpha-toxin titers reduced the lethal hemorrhagic edematous form of the disease ("blue-breast") produced by strains BB and Compton 201 to a localized chronic abscess form. No such protection was afforded by high anti-beta-toxin titers. Immunization with alpha- or beta-toxins produced no change in the clinical picture of the disease produced by CN.6708, a strain of Staphylococcus responsible for a natural outbreak of abscess-type rabbit mastitis. From these experiments it would appear that alpha-toxin is a key antigen in the blue-breast form of rabbit mastitis. Since the abscess form of the disease was not prevented by immunization with either alpha- or beta-toxin, other virulence factors must be acting to produce this more localized disease.

Comparative studies on the cell wall composition of some anaerobic coryneforms of varying lympho-reticular stimulatory activity.

Anaerobic diphtheroids possessing lympho-reticular stimulatory properties may differ considerably in their peptidoglycan composition. Spleen weight-increasing activity of strains directly parallels their antitumour properties. P. granulosum strains, inactive in assays for lympho-reticular stimulation, appear to have a higher cell wall alanine content than most of the P. acnes and P. avidum strains tested. Two P. acnes strains, however, had equivalently high alanine ratios and were stimulatory. The presence of galactose does not appear to be required for activity since P. acnes II strains which lack this sugar can be fully stimulatory. The existence of the species P. lymphophilum (Torrey) is further supported by the finding of two more serologically identical strains which do not cross react serologically with the other species in the group. These organisms are fully stimulatory but have lysine rather than DAP as their cell wall diamino acid.

Antibody to Corynebacterium parvum in normal human and animal sera.

Using a microtiter bacterial agglutination test, we have estimated antibodies to Corynebacterium parvum in "normal" human and "normal" and immune animal sera. Widely differing levels of C. parvum antibodies were found in the normal human sera. The median titer for all 310 human sera was 1:128, whereas that for the 1- to 17-year and 18- to 50-year subgroups was 1:64 and 1:512, respectively. Antibody titers in the various animal species were generally much lower.