Merlin L. Kaeberle

Evaluation of bovine polymorphonuclear leukocyte function.

Bovine polymorphonuclear leukocytes (PMNs) were isolated from the peripheral blood of cattle. Five in vitro procedures were utilized to evaluate PMN function: 1) Random migration under agarose, 2) Ingestion of 125I-iododeoxyuridine labeled Staphylococcus aureus, 3) Quantitative nitroblue tetrazolium reduction, 4) Chemiluminescence and 5) Iodination. Normal values for bovine PMNs are reported and interpretation of results is discussed. The PMN function tests were designed so that all 5 procedures may be performed in a short period of time on the same cell preparation. This allows for the detection and partial characterization of a potential PMN dysfunction.

Activation of bovine neutrophils by recombinant interferon-γ

Abstract The effect of recombinant bovine interferon-γ (r-IFN-γ) on neutrophil functions was investigated and compared to the effects of an unpurined lymphokine preparation. Incubation of purified bovine neutrophils with r-IFN-γ or antigen-induced lymphokine for 2.5 hr at 37 °C resulted in impairment of the ability of neutrophils to migrate under agarose, and an enhancement of their ability to mediate antibody-dependent and antibody-independent cell-mediated cytotoxicity against chicken erythrocytes. Neither the lymphokine preparation nor the r-IFN-γ had any influence on Staphylococcus aureus ingestion, or iodination by neutrophils. The lymphokine preparation enhanced cytochrome c reduction by neutrophils and was weakly chemotactic, whereas the r-IFN-γ had neither of these effects. Only 5 min of r-IFN-γ preincubation with neutrophils were needed to trigger protein synthesis by the neutrophils resulting in inhibition of random migration. Therefore, recombinant interferon-γ acts as a neutrophil migration inhibition factor and a neutrophil activation factor resulting in enhanced neutrophil-mediated antibody-dependent and -independent cell-mediated cytotoxicity. Many, but not all, of the in vitro effects of an unpurified lymphokine preparation on neutrophil function can be attributed to the interferon-γ contained in the lymphokine.

Effect of Rhodococcus equi on equine polymorphonuclear leukocyte function

A procedure was developed for isolating large numbers of purified polymorphonuclear leukocytes (PMNs) from the peripheral blood of horses. Equine PMN function was evaluated by three procedures: 1) Staphylococcus aureus ingestion, 2) nitroblue tetrazolium reduction, and 3) iodination. Four preparations of R. equi were added to polymorphonuclear leukocytes (PMNs) in each test system. Live bacteria, heat-killed bacteria, the washed pellet from heat-killed bacteria, and the supernatant fluid from heat-killed bacteria were evaluated for effects on equine PMN function. None of the R. equi preparations had an effect on S. aureus ingestion by equine PMNs. Nitroblue tetrazolium reduction by PMNs, a measure of oxidative metabolism, was suppressed by pellet and supernatant fractions. Values for the iodination reaction were depressed by all R. equi preparations, indicating decreased activity of the myeloperoxidase-H2O2-halide system of the PMN. Further evaluation of the supernatant from heat-killed R. equi showed that it retained its inhibitory effect on iodination following autoclaving and/or passage through a 10,000 MW filter. R. equi fractions did not alter the enzymatic conversion of 125I to a protein-bound form in a PMN-free assay developed to evaluate this reaction. The presence of a surface component capable of inhibiting bactericidal mechanisms of the PMN may play an important role in intracellular survival of R. equi.

Attempts to use thiabendazole to improve the immune response in dexamethasone-treated or stressed cattle

Thiabendazole was evaluated in two separate experiments for its ability to enhance the immune response in dexamethasone-treated or stressed cattle. In the first experiment the cattle received either no drug treatment (controls), dexamethasone intramuscularly (IM), or dexamethasone IM plus thiabendazole orally. All animals were inoculated with heat-killed Brucella abortus strain 19, equine ferritin, tetanus toxoid, and live Corynebacterium equi at the time dexamethasone therapy was initiated. Dexamethasone (0.04 mg/kg/day IM for 3 days) significantly (p less than 0.05) inhibited the lymphocyte blastogenic response to mitogens and the antibody response to ferritin and tetanus toxoid. Thiabendazole given orally (16 mg/kg/day) beginning 24 h prior to antigen and dexamethasone administration and continued for 6 days failed to prevent the dexamethasone-induced suppression of the lymphocyte blastogenic or antibody responses. In the second experiment 51 cattle were divided into a control group and a thiabendazole-treated group. The animals were stressed by weaning, injection of antigen (equine ferritin, tetanus toxoid, B. abortus strain 19 and killed bovine viral diarrhea virus) and castration of the bulls on the day that thiabendazole therapy was started. Thiabendazole administered orally for 5 days at a dosage of 20 mg/kg did not enhance the antibody response to any of the antigens, and was associated with a significantly lower antibody response to B. abortus.

Haemophilus somnus-induced IgE in calves vaccinated with commercial monovalent H. somnus bacterins.

The ability of commercially available Haemophilus somnus bacterins to elicit an immunoglobulin E (IgE) response was examined in healthy calves using enzyme-linked immunosorbent assay (ELISA) and western blotting techniques. Thirty five calves were utilized in this study. Calves in Group 1 (n=7) did not receive any H. somnus vaccination and served as negative controls. Calves in each of Groups 2-5 (n=7 each) were vaccinated on days 0 (primary) and 14 (booster) with one of four commercially available H. somnus bacterins. Sera were harvested on days 0 and 14 and at weekly intervals for a total of 45 days. Sera were tested for the presence of IgE antibodies using a bovine IgE-specific ELISA. Low levels of H. somnus-specific IgE were detected by ELISA in all animals prior to the initiation of the study. All bacterins induced IgE levels that were significantly higher than control levels. Two bacterins elicited higher IgE levels at all time points. Sera were adsorbed against washed whole cells of either Salmonella typhimurium, P. multocida, or H. somnus or extracts of H. somnus. ELISA absorbance values were significantly decreased by adsorption with washed whole cells or extracts of H. somnus, whereas adsorption with other gram-negative bacteria only minimally decreased ELISA absorbance values. These results indicate that commercially available H. somnus bacterins can induce IgE antibody as early as 14 days post-vaccination. This IgE can be detected 45 days after the primary vaccination. Results also indicate that H. somnus-specific IgE antibodies can be found in serum of some cattle, possibly induced by existing or previous sensitization.

Haemophilus somnus—Induced interference with bovine neutrophil functions

The effect of Haemophilus somnus on bovine polymorphonuclear leukocyte (PMN) function was examined in vitro with whole cells and fractions extracted from the surface of this bacterium. The ability of PMNs to iodinate protein and ingest Staphylococcus aureus was significantly inhibited in the presence of live cells, heat-killed whole cells or supernatant fluid from heat-killed cells, but not in the presence of washed, heat-killed cells. None of the fractions inhibited nitroblue tetrazolium (NBT) reduction by PMNs. The PMN inhibitory factors were further characterized. The material that inhibited S. aureus ingestion was found to be a heat-stable cell surface material of greater than 300 000 MW. The fraction inhibiting iodination of protein was found to be less than 10 000 MW.

Histamine production by Haemophilus somnus.

Ten Haemophilus somnus isolates were grown on blood agar plates under a 5% CO2 atmosphere for 48 h. Harvested whole cells were washed and evaluated for the presence of histamine by ELISA. All H. somnus isolates had cell-associated histamine concentrations of between 18.5 and 200 ng/ml. In a separate study, the ability of H. somnus to secrete histamine into BHI growth medium was evaluated using H. somnus strains 8025 and 156A as well as a recent 156A respiratory isolate. Each strain or isolate was grown under various concentrations of CO2 to approximate the CO2 concentration in the bronchi. The histamine content of washed whole cells and medium supernatant were determined at various stages of incubation. Highest histamine concentrations were detected in the recent respiratory isolate; whole cells (225 ng/ml) after 120 h incubation in 15% CO2 and supernatant (1721 ng/ml) after incubation for 41 h in 25% CO2. This study indicates that different H. somnus isolates can produce and secrete histamine which may be enhanced by CO2 concentrations which approximate those in the bronchial tree. Results of this study may partially explain some of the post-vaccination reactions occasionally observed with H. somnus bacterins. Additional studies are needed to determine the actual role of H. somnus-derived histamine in the pathogenesis of bovine respiratory disease and airway hyperresponsiveness.

The effect of some common inactivation procedures on the antigens of bovine herpesvirus 1

Bovine embryonic kidney cells were infected with bovine herpesvirus 1 (BHV1) or were sham-inoculated. When cytopathic effect was apparent, the cells were treated with beta-propiolactone, formalin, heat (56 degrees C), or ultraviolet irradiation until the virus was inactivated. Infected-treated, infected-untreated (IU) and sham-inoculated cultures were solubilized using Triton X-100 detergent. Resulting preparations were tested by 2-dimensional- and fused rocket-immunoelectrophoresis and were evaluated for their ability to inhibit virus neutralization by BHV1 antiserum. Eleven viral antigens were detected consistently in IU preparations, which strongly inhibited virus neutralization. Eight or more IU antigens were detected in beta-propiolactone-treated, formalin-treated and heat-treated preparations; these inhibited virus neutralization less strongly than the IU preparations. No IU antigens were detected in ultraviolet-treated preparations, nor did this material inhibit virus neutralization. One of the IU antigens was reduced preferentially by all treatments. The selective destruction of antigens by the various treatments might allow antigen-specific serological testing to distinguish vaccinated from naturally-exposed cattle.

Differential Serologic Response to Mycoplasma Ovipneumoniae and Mycoplasma Arginini in Lambs Affected with Chronic Respiratory Disease

An enzyme-linked immunoabsorbent assay (ELISA) was used to evaluate the levels of antibodies to Mycoplasma ovipneumoniae and M. arginini in lambs with chronic respiratory disease. Sera were obtained from lambs in several flocks at various stages of the clinical disease and tested with sodium dodecyl sulfate (SDS)-treated M. ovipneumoniae and M. arginini whole cells and a crude capsular extract of M. ovipneumoniae as the antigens. There were low levels of antibody to M. ovipneumoniae in flocks sampled at the early stages of infection, whereas increased levels of antibody were present in lambs from flocks that had apparently recovered from the clinical disease. Slowly rising titers of circulating antibodies to M. ovipneumoniae were confirmed by sequential bleeding of lambs during the course of the clinical disease. However, antibody levels of M. arginini were more likely to increase earlier in the disease process. There was significant cross-reactivity between the 2 SDS-treated antigens in both the ELISA test and western immunoblotting. In contrast, the crude capsular extract was specific for detecting antibodies to M. ovipneumoniae.

Effects of thiabendazole on dexamethasone-induced suppression of lymphocyte and neutrophil function in cattle

Seven dosage levels of thiabendazole were evaluated for their ability to normalize lymphocyte or neutrophil function in dexamethasone-treated cattle. Dexamethasone administration (0.04 mg/kg intramuscularly daily for 3 days) significantly suppressed the lymphocyte blastogenic response to phytohemagglutinin, concanavalin A, and pokeweed mitogen. The lymphocyte blastogenic response to allogeneic lymphocytes was also suppressed in dexamethasone-treated cattle, but this effect was not statistically significant. Neutrophils from dexamethasone-treated cattle had a significantly enhanced ability to migrate under agarose, and significantly suppressed ability to reduce nitroblue tetrazolium, iodinate protein, and mediate antibody-dependent cell-mediated cytotoxicity. Thiabendazole when administered concurrently with dexamethasone in the dosage range from 1.0 to 25.0 mg/kg (orally) did significantly enhance lymphocyte blastogenic responsiveness to phytohemagglutinin, concanavalin A, pokeweed mitogen and allogeneic cells (in dexamethasone-treated cattle). This immunonormalizing effect was not observed at the 50 or 100 mg/kg dosage levels. Thiabendazole did not produce a consistent significant enhancement of any parameter of neutrophil function in dexamethasone-treated cattle.