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.

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.

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.

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.

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)

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.

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.

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.

A comparison of pyrogenicity and related properties seen in a suspension of Corynebacterium parvum and a gramnegative organism.

When the pyrogenic properties of C. parvum and a gramnegative organism, S. typhimurium, were compared it was found that the onset of the pyrogenic response to C. parvum was delayed relative to that of S. typhimurium and that secondary responses rarely occurred. In further experiments the interaction of C. parvum and S. typhimurium was studied. Although synergism was not demonstrated, the kinetics of the response to mixtures of the two vaccines was anomalous while pre-treatment of rabbits with C. parvum resulted in the elimination of the secondary pyrogenic response to S. typhimurium suspension. The implications of these results are discussed in terms of the known characteristics of endogenous pyrogen, lipopolysaccharide and peptidoglycan.

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.

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.