Decreased neutrophil function following administration of chemotherapy has been reported in dogs with lymphoma. The first objective of our study was to determine if neutrophil oxidative burst and phagocytic activity are affected by chemotherapy 7 to 10 days following initiation of treatment in dogs with lymphoma and non-lymphoma malignancies. The second objective was to determine if there is a correlation between neutrophil numbers and neutrophil function before or after initiation of chemotherapy. Flow cytometric assessment of neutrophil oxidative burst and phagocytosis following stimulation with Escherichia coli was performed in 9 dogs diagnosed with lymphoma and 17 non-lymphoma tumor-bearing dogs pre- and post-chemotherapy, as well as 14 tumor-free control dogs. Spearman rank correlation was performed to determine if blood neutrophil numbers and neutrophil function were significantly correlated. Lymphoma patients showed significantly reduced percentage neutrophil oxidative burst post-chemotherapy compared to healthy controls as well as compared to pre-chemotherapy values (P = 0.0022 and P = 0.0020, respectively). Lymphoma patients also exhibited significantly reduced neutrophil phagocytosis activity post-chemotherapy compared to controls and pre-chemotherapy values (P = 0.0016 and P = 0.014, respectively). Dogs with non-lymphoma malignancies also showed a significant decrease in both percentage oxidative burst and phagocytosis post-chemotherapy compared to pre-chemotherapy values (P = 0.00040 and P = 0.029, respectively). Neutrophil numbers and function were not significantly correlated. The results of the study suggest that chemotherapeutic treatment decreases neutrophil oxidative burst and phagocytic activity 7 to 10 days post-treatment in dogs with various malignancies. Furthermore, neutrophil numbers cannot be used to predict neutrophil function.

Objective-To use in vitro assays to evaluate the effects of a novel immunosuppressive agent, FTY720, on biological functions (migration, phagocytosis, and production of reactive-oxygen species ROS) of feline peripheral neutrophils and determine the cytotoxic effects of FTY720 on feline peripheral neutrophils. Sample Population-Peripheral neutrophils obtained from 8 healthy cats. Procedure-Peripheral neutrophils were isolated from blood samples obtained from the 8 cats and exposed to the phosphorylated form of FTY720 (FTY720-P). A fluorescence-based in vitro evaluation of migration was performed. Phagocytosis of microbes and production of ROS were evaluated by use of a 2-color flow cytometry system. Samples of whole blood obtained from the cats were incubated with various concentrations of FTY720-P, fluorescein-labeled Staphylococcus aureus, and dihydroethidium. Cytotoxic effects were evaluated by use of propidium iodide staining. Results-Addition of FTY720-P caused a slight nonsignificant decrease in phagocytosis and production of ROS by feline peripheral neutrophils. Migration activity of feline peripheral neutrophils was significantly increased by the addition of FTY720-P. Addition of FTY720-P at concentrations considered for clinical use did not increase the death rate of feline peripheral neutrophils. Conclusions and Clinical Relevance-FTY720 does not inhibit critical functions of feline peripheral neutrophils in vitro.

Marked differences in bone marrow cellularity were observed between cattle affected with leukocyte adhesion deficiency (LAD) and control cattle. The number of nucleated cells in bone marrow was 2.9 to 8.8 times higher in cattle affected with LAD, compared with controls. The myeloid-to-erythroid ratio of bone marrow from 3 cattle affected with LAD ranged from 2.4 to 12. Deficient CD18 expression on neutrophils isolated from bone marrow of cattle with LAD was clearly detected by flow cytometric analysis. Neutrophils from bone marrow of cattle affected with LAD appeared round and not flat, after adherence to plastic wells under agarose, whereas neutrophils from bone marrow of clinically normal cattle were firmly spread on the surface of plastic wells. In the chemotaxis under-agarose assay, many pseudopodia were detected on bone marrow neutrophils from clinically normal cattle, but were not detected on bone marrow neutrophils from cattle with LAD. Activities of chemotactic movements and phagocytosis of neutrophils isolated from bone marrow of cattle affected with LAD were documented to be severely impaired.

Previously identified 39-, 50-, and 76-kd integral outer membrane proteins (IOMP) of Actinobacillus pleuropneumoniae, a respiratory tract pathogen, were separated by electroelution of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-obtained fragments and their role in opsonophagocytosis by porcine leukocytes was investigated by flow cytometry of fluorescein-labeled A pleuropneumoniae. Using specific antisera, immunoblot analysis indicated that the 3 proteins were antigenically distinct. Antibodies against each IOMP have an important role as opsonins for phagocytosis by porcine leukocytes. The effect of using a combination of an 3 of the specific antisera was minimal. Antiserum absorbed against intact A pleuropneumoniae and Escherichia coli organisms indicated that the antibodies to the 39-, 50-, and 76-kd IOMP were specific for A pleuropneumoniae antigens. Nonheat-treated antiserum did not increase phagocytosis, compared with heat-inactivated antiserum, indicating that complement may not have a major role in opsonization of A pleuropneumoniae.

Polymorphonuclear cells have a critical role in the pathogenesis of bovine mastitis. We have documented that experimentally induced Staphylococcus aureus mastitis is associated with cyclic increase and decrease in the quantity of viable bacteria shed in the milk. Concomitant with this cycling of bacteria is an inverse cycling of the hosts cells within the milk. Such somatic cells were determined to be greater than or equal to 95% polymorphonuclear cells. The quality of these cells was evaluated by measuring their relative efficiency of bacterial killing and phagocytosis at various times during an infection. Host polymorphonuclear cells had as much as 10,000-fold variation in the bactericidal failure rate for staphylococci during cell cycling. The most efficient bactericidal effect was observed at or near the peak of the somatic cell count (SCC). The ability of these cycling cells to ingest fluorescent beads was also quantitated by use of flow cytometry. The percentage of phagocytic polymorphonuclear cells that ingested fluorescent latex beads ranged from 15 to 80% of the total cell population during cell cycling, and tended to be optimal at or near peak SCC. In addition, the average number of beads ingested varied between 1 and 2 particles/polymorphonuclear cell, with as many as 17% of the phagocytic cells ingesting 4 or more beads at maximal efficiency. Polymorphonuclear cells from quarters infected with S aureus varied quantitatively (total SCC) and qualitatively (bactericidal activity and phagocytic ability) during the course of an infection. Not only is the quantity of host's phagocytic cells in the mammary gland central to the defense mechanism against infection, but the biological activation state appears to be equally important. The role of these cells in the pathogenesis of a cycling infection is presented in a model to explain the cyclic nature of mastitis.

The functional characteristics of polymorphonuclear leukocytes (PMN), considered to be the first line of host defense against infections, from rhesus macaques confirmed to have simian retrovirus (SRV)-induced simian acquired immunodeficiency syndrome (SAIDS), were evaluated. The PMN from SRV antibody-positive macaques without clinical signs were chemotactically responsive. Their phagocytic and killing capabilities were normal, and their cell membranes were highly deformable. However, PMN from SRV antibody-positive macaques and with persistent lymphadenopathy, as well as having at least 3 of the 11 common clinical signs of SAIDS, were chemotactically nonresponsive. Their phagocytic and killing capabilities were compromised, and their cell membranes were rigid and nondeformable. In general, PMN from macaques with clinically confirmed SAIDS were functionally deficient. The results are similar to those obtained in other retroviral infections and can be clinically significant, because the host defense deficiency may be responsible for the recurrent and opportunistic infections in SAIDS.

Equine neutrophil antibody was raised in rabbits inoculated with equine neutrophils isolated to purity > 99.0%, using Percoll density-gradient sedimentation. Neutrophil antibody was detected by use of agar gel diffusion, leukoagglutination, indirect immunofluorescence, staphylococcal protein A and streptococcal protein G binding, and phagocytic inhibition techniques. Precipitin lines and leukoagglutination were seen in antiserum dilutions of 1:4 and 1:64, respectively. The specific nature of leukoagglutination was characterized by the formation of rosette-like clumps of neutrophils. Specific bright membranous fluorescence was seen in neutrophils treated with the antiserum and exposed to fluorescein-conjugated goat anti-rabbit immunoglobulin, and staphylococcal protein A and streptococcal protein G. Whereas the indirect immunofluorescence and protein G-binding tests were equally sensitive and resulted in titer of 1:256, the protein A-binding test was less sensitive and resulted in titer of only 1:32. Nonspecific binding of protein A and protein G was noticed as uniform or patchy cellular fluorescence in a small number of neutrophils. Treatment of neutrophils with antiserum up to dilution of 1:8 resulted in a significant (P < 0.05) suppression of phagocytosis of opsonized zymosan particles. Thus, protein G-binding and indirect immunofluorescence tests are highly sensitive to detect neutrophil antibody and may be used to diagnose immune-mediated neutropenias in horses and, possibly, in other animal species.

Six primiparous Holstein cows were fed a Se-deficient diet, beginning at least 90 days before their first calving, and 6 other primiparous cows were given the same diet plus a supplement of 2 mg of Se/cow/d as sodium selenite. All cows were fed their diets for the duration of the experimental period. One uninfected quarter of each cow was injected with 25 microgram of Escherichia coli endotoxin at postpartum week 5. Leukocytes were isolated by centrifugation from milk collected at postinjection hour 16. Isolated cells were 92 +/- 3% neutrophils and were incubated with Staphylococcus aureus or E coli in a 1:300 ratio. Phagocytosis and intracellular killing by neutrophils were assessed after 0, 30, 60, and 90 minutes by a fluorochrome assay, using acridine orange. Viability of neutrophils was assessed by use of trypan blue. Superoxide anion production and hydrogen peroxide production by neutrophils also were determined. Cows fed Se-deficient diets had significantly (P < 0.05) lower blood Se concentration and blood glutathione peroxidase activity than cows fed Se-supplemented diets. Selenium status had no effect on the phagocytic capacity of neutrophils. Neutrophils obtained from cows fed Se-supplemented diets killed a significantly (P < 0.05) higher percentage of ingested bacteria than did neutrophils from cows fed the Se-deficient diet. Viability was significantly (P < 0.05) reduced by incubation with S aureus in neutrophils from both groups of cows, with neutrophils from Se-deficient cows having lower viability. Superoxide anion production did not differ significantly between neutrophils from the 2 groups, but extracellular hydrogen peroxide concentration was significantly (P < 0.05) higher in neutrophils harvested from milk of cows fed the Se-deficient diet.

The immune receptor-mediated functions of bovine alveolar macrophages (AM) inoculated in vitro with bovine herpesvirus-1 (BHV-1) or parainfluenza-3 (PI-3) virus were tested in the presence or absence of virus-specific antiserum or pulmonary lavage fluids collected from calves 6 days after inoculation with BHV-1 or PI-3 virus. The Fc and C3b phagocytic indices of noninoculated AM, collected from 6- to 16-week-old calves, ranged from 75 to 87 and 59 to 64, respectively, and the binding indices ranged from 5 to 8 and 22 to 28, respectively. Infection of AM with either BHV-1 or PI-3 virus had no significant effect on receptor-mediated phagocytosis or binding, with the exception of a significant (P < 0.05) decrease, from 64 to 46, of the C3b phagocytic index of PI-3 virus-infected AM. The addition of lavage fluids, collected after BHV-1 or PI-3 virus infection, to AM infected with the respective virus caused a significant (P < 0.05) decrease in phagocytic indices with values for the Fc and C3b indices in BHV-1-infected AM decreasing from 81 to 49 and from 47 to 8, respectively, and those for the PI-3 virus-infected AM from 79 to 51 and from 46 to 15, respectively. The binding indices of virus-infected AM increased with the addition of viral lavage fluids, but the only significant (P < 0.05) increase was for C3b binding in PI-3 virus-infected cells, which increased from 33 to 56. Virus-specific serum added to AM infected with the respective virus also caused significant (P < 0.05) decreases in the Fc and C3b phagocytic indices, with those for BHV-1-infected AM decreasing from 81 to 24 and from 47 to 5, respectively, and those for PI-3 virus-infected AM from 79 to 23 and from 46 to 3, respectively. The Fc binding index significantly (P < 0.05) increased with the addition of virus-specific serum from 8 to 34 and from 10 to 42 in BHV-1 and PI-3 virus-infected AM, respectively. The C3b binding index of these AM also increased, but not significantly. Infection of AM with either BHV-1 or PI-3 virus had no significant effect on the phagocytosis of opsonized (OPZ) or nonopsonized (nonOPZ) Staphylococcus epidermidis (SE). The addition of lavage fluids, obtained after BHV-1 infection, to AM infected with BHV-1, significantly (P < 0.05) decreased the percentage of phagocytosis of OPZ-SE from 28 to 21 and had a similar, but less substantial effect, on the phagocytosis of nonOPZ-SE. Lavage fluids collected after PI-3 virus inoculation, added to PI-3 virus-infected AM did not have a notable effect on the phagocytosis of OPZ-SE, but did cause a significant (P < 0.05) decrease in the percentage of phagocytosis of nonOPZ-SE from 25 to 17. The addition of virus-specific serum to infected AM caused significant (P < 0.05) decreases in the percentage of phagocytosis of OPZ-SE and nonOPZ-SE, with the values in the BHV-1-infected AM going from 28 to 11 and 16 to 9, respectively, and in the PI-3-infected AM from 36 to 12 and 25 to 13, respectively. Alveolar macrophages infected with either BHV-1 or PI-3 virus, in the presence or absence of lavage fluids from virus-infected calves or virus-specific serum, killed ingested SE as readily as noninfected AM. On the basis of the findings of this study, we suggest, as with other virus infections, that products of the host antiviral immune response interact with AM infected with BHV-1 or PI-3 virus or cause impaired internalization of receptor-bound particles, resulting in impaired AM antimicrobial functions.

Live Pasteurella haemolytica biotype A, serotype 1 isolates (n = 3) and Escherichia coli K-12, strain W3110, were reacted with bovine pulmonary lavage cell (PLC) suspensions. The comparative effects of the different bacteria on the functional and metabolic activity of alveolar macrophages (AMO) in the PLC suspensions were assessed simultaneously by use of 51Cr release, luminol-dependent chemiluminescence (LDCL), and AMO bactericidal assays. The bovine PLC reponsed differently to E coli, than to the 3 P haemolytica isolates in each of the 3 experimental test systems; however, responses to each of the P haemolytica isolates were not found to be significantly different. Unopsonized live P haemolytica cells adversely affected the functional and metabolic response of PLC, whereas there was no evidence of a cytotoxic (cytocidal) influence of E coli. A difference in 51Cr release for reaction mixtures containing E coli and P haemolytica was not detected at zero time; however, at each subsequent time, reaction mixtures phagocytically stimulated with P haemolytica had significantly increased amount of 51Cr release (P less than 0.05), compared with those mixtures containing E coli. Bovine AMO in the PLC suspensions were able to effectively kill E coli in vitro, but were unable to prevent survival and subsequent growth of P haemolytica. The luminol-dependent chemiluminescence profiles for reaction mixtures phagocytically stimulated with E coli provided evidence of sustained production of oxygen radicals with antimicrobial capabilities by bovine AMO in the PLC. Production of these highly reactive antimicrobial oxidants appeared initially in cultures containing P haemolytica but, subsequently, their production declined precipitously and ceased altogether.