The purpose of this in vitro study was to determine whether Pasteurella haemolytica capsular extract (CE) damages bovine pulmonary endothelial cells (EC) directly or through neutrophil-mediated mechanisms. Chromium 51-labeled EC were treated with the following variables: CE (1, 10, and 100 ng of protein/ml), CE and bovine neutrophils (10(6) cells/well), and CE and polymyxin B (500 U/ml). Although only minimal damage to EC occurred by 5 hours after treatment, by 22 hours after treatment, the 10-ng and 100-ng CE dose produced severe damage to EC, as indicated by 51Cr release, cellular detachment, and loss of monolayer confluency. The component in the CE that was toxic to the EC was lipopolysaccharide, evidenced by effective neutralization of the toxic effect with polymyxin B. Neutrophils inhibited the CE-mediated EC toxicity and were activated, as indicated by shape change and adhesion to EC monolayers. We concluded that the lipopolysaccharide component of CE causes direct damage to EC, which can be attenuated by neutrophils and polymyxin B.

Cows naturally infected with Brucella abortus developed antibody (Ab) responses to a nonlipopolysaccharide antigen (NLA) purified from B abortus strain 1119-3. Sera from strain 19-vaccinated cows did not have detectable amounts of Ab. Weak lymphoproliferative responses to NLA were observed in blood mononuclear cell suspensions obtained from infected cows. There was no evidence of NLA-specific lymphoproliferation in cell suspensions from healthy cows. Nonlipopolysaccharide antigen binding to bovine blood mononuclear cells was observed by antigen-consumption assays and direct binding of radiolabeled antigen. Cells from infected cows bound less NLA than did cells from healthy cows when assays were conducted with intact blood mononuclear cell preparations (monocytes plus lymphocytes). Monocytes obtained from any group did not bind NLA. Purified B lymphocytes from infected and healthy vaccinated cows bound about 3 times more NLA than did T lymphocytes, but there were no apparent differences between the 2 groups in extent of binding. Results of the study indicate that bovine lymphocytes have binding sites for a NLA purified from B abortus strain 1119-3.

Lipopolysaccharide (LPS) fractions were obtained from smooth cultures of Brucella abortus strains 2308 and S-19 by butanol extraction procedures. The LPS from the initial butanol extraction contained 10 to 15% protein and was reduced to less than 1% protein by treatment with proteinase K. The LPS fractions were identified and characterized on the basis of the chemical analysis, sodium dodecyl sulfate gel electrophoresis, cesium chloride gradients, electron microscopy, and gel immunodiffusion. Results indicated that the butanol procedure is a reliable method in the extraction of LPS from Brucella abortus cells. Proteinase K-treated LPS containing less than 1% protein from strain 2308 was used to vaccinate BALB/cByJ mice. Immune and protective criteria for vaccinated and nonvaccinated mice were increased immunoglobulin (IgG and IgM) titers in sera of prechallenge-exposed mice, reduced colony-forming units/spleen, and splenomegaly in post-challenge-exposed mice. Results indicated that proteinase K-treated LPS was immunuogenic as well as protective for mice.

Bovine pulmonary artery cells in cell culture were exposed to lipopolysaccharide (LPS) purified from Pasteurella haemolytica serotype A1. This resulted in severe membrane damage, which caused a time- and dose-dependent release of lactate dehydrogenase that was first detected 4 hours after exposure and reached a maximal mean release of 67% after 24 hours of exposure to 1 microgram of LPS/ml. Mean release of 51chromium followed by a similar pattern and reached a maximum of 61% following 24 hours of exposure to 10 micrograms of LPS/ml. Morphologically, endothelial cells responded to LPS by marked cell membrane retraction, the formation of numerous cytoplasmic blebs, and ruffling of the cell membrane. Subsequently, the cells became round and detached. Cell detachment reached a mean of 95% following 8 hours of exposure to 1 microgram of LPS/ml. These studies demonstrated that P haemolytica LPS is capable of causing direct damage to bovine pulmonary arterial endothelial cells, which may be important in the pathogenesis of bovine pneumonic pasteurellosis.

Monoclonal antibodies recognizing the O-polysaccharide portion of Brucella abortus strain 2308 provided BALB/c mice with passive protection against challenge exposure with the homologous strain. Numbers of colony-forming organisms in the spleen were reduced by IgM and IgG monoclonal antibodies. Active immunization of mice, using B abortus 2308S lipopolysaccharide, resulted in production of IgM antibody at 14 days. Clearance of organisms in the actively immunized mice after challenge exposure at 14 days was nearly identical to that in passively immunized mice. Mice either passively or actively immunized were effectively protected from 0 to 28 days. Bacterial colonization of the spleen was observed to increase in both groups of mice at 56 days and indicated that humoral responses were effective in eliminating the organism in the early stages of infection, but other immune mechanisms were necessary for protection of mice in the later stage of infection with virulent strains of B abortus.

The effects of prolonged plasmapheresis of cattle on total and antigen-specific immunoglobulin production were evaluated. Five adult cows were hyperimmunized by repeated IV administration of live, logarithmic-phase Pasteurella haemolytica A1 organisms. Three of the cows underwent plasmapheresis daily for 3 weeks. From 2 cows, serum was only obtained periodically. Anti-P haemolytica antibody was assayed by indirect hemagglutination and a kinetic-augmented, antigen-capture ELISA for capsular polysaccharide and lipopolysaccharide/outer membrane protein antigens. Total serum immunoglobulin concentration was determined for IgM, IgG1, and IgG2 by primary radial immunodiffusion. Anti-P haemolytica A1 activity increased rapidly after immunization. After beginning plasmapheresis, the antigen-specific antibody activities remained nearly constant. In general, antilipopolysaccharide/outer membrane protein activity (in terms of concentration) was higher than anti-capsular polysaccharide activity and was not affected as much by the plasmapheresis. Total serum Ig concentration decreased transiently by a small amount after beginning plasmapheresis.

Purified lipopolysaccharides (LPS) from 16 serotypes of Pasteurella multocida were complexed with Aspergillus fumigatus ribosomes. The complexes were used as inocula to prepare antisera, in chickens, for somatic antigen typing by the gel diffusion precipitin test (GDPT). Antisera made against 15 of 16 LPS reacted with their respective specific heat-stable antigens in the GDPT and homologous LPS in the passive hemagglutination test. Antisera could not be made against serotype 15 LPS. Correlation was not observed between intensity of the precipitin reaction in the GDPT and titer to homologous LPS in the passive hemagglutination test. Most antisera cross-related with other heat-stable antigens of other serotypes in the GDPT. Many of these cross-reactions were eliminated by dilution. Cross-reactions that occurred in the GDPT with antisera made against LPS of serotypes 2, 5, 7 and 8 could not be eliminated by dilution.

Three antigens were prepared from a type-3 avian strain of Pasteurella multocida, and their chemical and immunologic characteristics were studied. An antigen, designated 2.5S, was extracted with 2.5% NaCl solution and purified by chromatography. Lipopolysaccharide (LPS) was extracted with phenol-water, and a third antigen, designated FS, was extracted in 0.3% formalin solution containing 0.85% NaCl and purified by differential centrifugation. The 2.5S and the FS antigens consisted of 40% protein and 15% carbohydrate, whereas LPS did not contain a substantial amount of protein. A major protein component with a molecular weight of 44,000 was detected in the 2.5S antigen, as well as in the FS antigen. Of the 3 antigens, LPS had thehighest activity in mouse lethality and Limulus lysate tests. Antigenic cross-reactions among the 3 antigens were demonstrated by immunodiffusion tests. The 2.5S antigen was indistinguishable from the FS antigen, as both antigens contained the LPS component of approximately 45%. Treatments with various reagents indicated that the 2.5S and FS antigens contained at least 2 antigenic determinants. The first was a heat-stable protein sensitive to protease or phenol-water, and the second was a periodate-sensitive carbohydrate, which was an major antigenic determinant on the LPS antigen.

OBJECTIVE To determine the in vitro effects of epinephrine, norepinephrine, and dobutamine on lipopolysaccharide (LPS)-stimulated production of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-10 (IL-10) in blood from healthy dogs. SAMPLES Blood samples from 9 healthy dogs. PROCEDURES Blood samples were incubated with LPS from Escherichia coli O127:B8 or PBSS (control) for 1 hour. Afterward, the samples were incubated with 10μM epinephrine, norepinephrine, or dobutamine or with saline (0.9% NaCl) solution (control) for 23 hours. Leukocyte viability was assessed by use of trypan-blue exclusion in blood from 2 dogs to ensure cell viability was not altered by the catecholamines. Tumor necrosis factor-α, IL-6, and IL-10 concentrations were measured in the supernatant in duplicate with a canine-specific multiplex bead-based assay. Blood samples from 2 dogs were used to create dose-response curves to evaluate whether the observed cytokine modulation was dependent on catecholamine concentration. RESULTS Incubation of blood with epinephrine and norepinephrine significantly increased LPS-stimulated production of IL-10, compared with the control. Epinephrine and norepinephrine significantly decreased LPS-stimulated production of TNF-α, compared with the control. Epinephrine and norepinephrine did not significantly alter LPS-stimulated production of IL-6. Dobutamine did not alter catecholamine production. CONCLUSIONS AND CLINICAL RELEVANCE Epinephrine and norepinephrine, but not dobutamine, had immunomodulatory effects on LPS-stimulated TNF-α and IL-10 production in blood from healthy dogs in this in vitro model of sepsis. Data suggested that dobutamine may have immune system-sparing effects in dogs with sepsis.

Achyranthes japonica Nakai (A. japonica) is a medicinal herb found widely distributed throughout Korea. The biological activities of A. japonica are well-documented and include anti-fungal, anti-inflammatory, and immunity enhancement. The objective of the present study was to investigate the immune-related activities of A. japonica extract in dogs. The extract was acquired by ethanol extraction and purified by filtration. To examine the effect of A. japonica extract on immune cell viability, human lymphocytes, such as Jurkat T-cells and Ramos B-cells, were exposed to the extract. After treatment with the extract, the number of Ramos B-cells was increased, whereas Jurkat T-cells remained unaffected. Griess assay revealed decreased nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated mouse macrophage Raw 264.7 cells after exposure to A. japonica extract. To evaluate the in-vivo effect in dogs, feed containing A. japonica extract was provided to 8 dogs for 2 months. Blood samples were collected before, during, and after consumption of the feed. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood samples and the number of T-cells and B-cells were assessed using flow cytometry with anti-dog fluorescein isothiocyanate (FITC)-conjugated CD3 and anti-dog phycoerythrin (PE)-conjugated CD21 antibodies, respectively. We observed a significant increase in the average number of B-cells in the PBMCs during ingestion of the feed containing A. japonica. In addition, enzyme-linked immunosorbent assay (ELISA) revealed a decrease in the levels of tumor necrosis factor-alpha (TNF-α), a pro-inflammatory cytokine, in 3 out of 8 dogs and increased levels of interleukin-10 (IL-10), an anti-inflammatory cytokine, in 4 out of 8 dogs. Taken together, we believe that these changes indicate that A. japonica extract is beneficial in improving the immunity of dogs by stimulating B-cells and inducing production of anti-inflammatory responses.