Effects of vitamins A and E on superoxide production and intracellular signaling of neutrophils in Holstein calves.

The effects of vitamin A and vitamin E treatment on superoxide (O2-) production of neutrophils and their intracellular signaling, including intracellular Ca2+, protein kinase C (PKC), and tyrosine kinase (TK), were examined in Holstein calves. After treatment with vitamin A, heat-aggregated IgG (H-agg.IgG)-induced O2- production in neutrophils ranged from 3.7+/-0.4 to 4.3+/-0.9 nmol (mean +/- SD), and it was significantly (P<0.05) higher than that in the control calves. Opsonized zymosan (OPZ)-induced O2- production was similar with that of control calves. Intracellular signaling of neutrophils in vitamin A-treated calves was enhanced compared with that of control calves when stimulated with H-agg. IgG, but not with OPZ. After treatment with vitamin E, H-agg.IgG- and OPZ-induced O2- production of neutrophils ranged from 4.2+/-0.8 nmol to 5.4+/-0.5 nmol and from 7.8+/-0.6 nmol to 8.1+/-0.4 nmol (mean +/- SD), respectively, and they were significantly (P<0.05) higher than those in control calves. Intracellular signaling was also enhanced compared with that of control calves. These results suggested that the effects of vitamin A and E treatment on O2- production were correlated to changes in intracellular signaling of neutrophils in Holstein calves.     

The involvement of polymorphonuclear leukocytes in the pathogenesis of bronchopneumonia in calves. V. Adherence to nylon fibres.

The adherence of neutrophils from bronchopneumonic calves to nylon fibres and the influence of this adherence on O2- and H2O2 production were studied. Polymorphonuclear leukocytes from bronchopneumonic calves were found to produce much more superoxide anion and hydrogen peroxide than neutrophils from healthy animals. A higher production of these compounds was observed in granulocytes adhering to nylon fibres than in cells in suspension. It is suggested that oxygen radicals production induced by the contact of granulocytes with a solid surface plays a role in the destruction of endothelium in vivo.     

The involvement of polymorphonuclear leukocytes in the pathogenesis of bronchopneumonia in calves. I. Activated granulocyte induced lipid peroxidation in red blood cells.

The influence of free radicals generated by polymorphonuclear leukocytes from bronchopneumonic calves on lipid peroxidation in red blood cells was studied. Incubation of granulocytes with red blood cells causes a rise in malondialdehyde production in these cells in linear dependence on the granulocyte number. Incubation of red blood cells with malondialdehyde causes a generation of adduct moiety of this compound with phosphatidylserine and phosphatidylethanolamine. This adduct is also present in erythrocytes which have been incubated with neutrophils of bronchopneumonic calves. Cytochrome c loaded erythrocyte ghosts showed a high reduction rate of this compound during incubation of ghosts with neutrophils of diseased calves. It is suggested that neutrophils of bronchopneumonic calves are in an activated state in the blood-stream, and free radicals generated by these cells are capable of peroxidizing the red blood cell membrane lipids and causing cross-links between phospholipid moieties in erythrocyte membranes.     

The involvement of polymorphonuclear leukocytes in the pathogenesis of bronchopneumonia in calves. III. Granulocyte activation by platelets.

The influence of platelets and platelet lysate on superoxide anion production and beta-glucuronidase release from neutrophils was investigated. Neither the suspension of unstimulated platelets from healthy calves nor its lysate had any influence on O2- production and enzyme release On the other hand, a great rise in superoxide anion production and beta-glucuronidase release was observed in the case of platelets from bronchopneumonic calves. It is suggested that platelets of bronchopneumonic calves can activate polymorphonuclear leukocytes, causing their degranulation and aggravating superoxide anion production.     

The involvement of polymorphonuclear leukocytes in the pathogenesis of bronchopneumonia in calves. II. Granulocyte-induced changes in erythrocyte membrane phospholipid topology.

Phospholipid topology in erythrocyte membranes of bronchopneumonic calves and changes in phospholipid asymmetry induced by incubation of erythrocytes with neutrophils of diseased calves were examined. Using aminophospholipid tracing by non-penetrating probe, trinitrobenzenesulfonic acid, and phospholipid hydrolysis by specific phospholipase A2 from Naja naja venom and sphingomyelinase from Staphylococcus aureus it was stated that in erythrocytes from diseased calves phosphatidylserine and, to some extent, phosphatidylethanolamine are externalized on the outer leaflet of the membrane. Similar results were obtained for erythrocytes of healthy calves after incubation with granulocytes of bronchopneumonic animals. It is suggested that granulocytes of bronchopneumonic calves cause perturbations in erythrocyte membrane phospholipid organization.     

Bovine recombinant granulocyte-macrophage colony-stimulating factor enhancement of bovine neutrophil functions in vitro

Neutrophils were purified from blood of dexamethasone-treated (0.04 mg/kg of body weight) and untreated calves. Cells were untreated (controls) or cultured in media containing 5 or 10 ng of bovine recombinant granulocyte-macrophage colony-stimulating factor (rbGM-CSF)/ml for 10 to 12 hours before being tested for various functions. Dexamethasone treatment of calves decreased luminol-dependent chemiluminescence, decreased phagocytosis of Pasteurella multocida and several Staphylococcus spp by various degrees, and decreased antibody-dependent cell-mediated cytotoxicity against bovine herpesvirus-infected cells by 26 to 32%. The percentage phagocytosis of coagulase-positive S aureus and S intermedius was higher than that of coagulase-negative S epidermidis for neutrophils from all calves. Culture of neutrophils with rbGM-CSF significantly increased (P less than 0.05) all of the aforementioned functions, compared with control neutrophils; however, rbGM-CSF-induced increases in function tended to be higher in neutrophils from dexamethasone-treated calves than in neutrophils from untreated calves.     

Oxygen radical production by avian leukocytes.

Oxygen radical production by heterophils of red-tailed hawks and chickens, and by neutrophils of calves, was evaluated in a chemiluminescence microassay. Leukocytes were isolated by centrifugation of blood in capillary tubes and then challenged with opsonized zymosan in the presence of luminol. Avian heterophils produced significantly fewer oxygen radicals than did bovine neutrophils.     

Pathogenesis of psoroptic scabies in Hereford heifer calves

Hereford heifer calves were experimentally infested with Psoroptes ovis. Histologic examination of skin specimens was conducted at weekly intervals before and after treatment with ivermectin on postinfestation week 7. Electron microscopy revealed numerous degranulating mast cells in the skin of infested but not in control calves. many active, as well as degenerate, neutrophils were in the scab on infested calves. Microscopic epidermal ulcers developed on infested calves when live mites were present but not after treatment. Numbers of dermal neutrophils and plasma cells decreased and numbers of circulating neutrophils increased 1 week after treatment. Numbers of dermal eosinophils and mast cells in calves with eosinophilia increased for several weeks after treatment. Statistical analysis indicated significant correlations (P less than 0.05) among numbers of dermal inflammatory cells, hemogram values, and changes in dermal thickness. Seemingly, mite-induced epidermal damage was the key pathogenic event in psoroptic scabies in calves. Mast cell degranulation contributed to the pathogenesis of the dermatitis, and neutropenia was caused by sustained, poorly compensated efflux of neutrophils into the scab through mite-induced breaks in the epidermis.     

Effect of vaccination on cell populations in lung washes from calves after infection with respiratory syncytial virus

The inflammatory response in the air-passages of the lungs of calves after intranasal inoculation with respiratory syncytial virus (RSV) was compared in RSV-vaccinated and control animals. Total cells recovered from lung washings remained the same; however, the fold by eight days after infection and the type of cells changed from a predominance (85 per cent) of macrophages to equal proportions of macrophages and neutrophils (45 per cent) during the course of infection. The absolute numbers of neutrophils rose by 15-fold. In contrast, when RSV-vaccinated calves were challenged, the total number of cells recovered from lung washings remained the same; however, the numbers of macrophages decreased and the numbers of neutrophils increased by fivefold. Cytological studies of the lung washings revealed no evidence of an exacerbated inflammatory response in RSV-vaccinated calves. Levels of virus replication were significantly reduced in RSV-vaccinated compared with control animals.     

Changes in leukocyte populations in pulmonary lavage fluids of calves after inhalation of Pasteurella haemolytica

The distribution of leukocytes in bovine bronchoalveolar lavage fluids was determined in 15 calves at various times after aerosol exposure to Pasteurella haemolytica. For comparison, 10 calves were exposed to aerosols of phosphate-buffered saline solution; 15 calves, to Staphylococcus epidermidis; and 10 calves, to Salmonella typhimurium endotoxin. At 10 minutes after inhalation exposure for each group, the predominant cell type was the macrophage. Macrophages remained the predominant cell type throughout each lavage interval for calves exposed to phosphate-buffered saline solution and Staph epidermidis. For calves exposed to P haemolytica, there was a decrease in the percentage of macrophages detectable by 30 minutes after exposure, with a corresponding increase in the percentage of neutrophils. Sixty minutes after the inhalation exposure to P haemolytica, the percentages of macrophages and neutrophils in the lavage fluid were equal. By 240 minutes after exposure to P haemolytica, greater than 90% of the cells in the lavage fluids was neutrophils. The increase in the percentage of neutrophils in lavage fluids from calves exposed to S typhimurium endotoxin was similar to that seen for the calves exposed to P haemolytica.     

Bone marrow response in cattle with chronic dermatitis caused by Psoroptes ovis

Six Hereford heifer calves were infested with Psoroptes ovis and compared to six uninfested control calves. Infested calves developed severe exudative dermatitis which covered 100% of the body surface by 7 weeks after infestation. They developed moderate nonregenerative anemia, decreased numbers of neutrophils and lymphocytes, and increased plasma protein and fibrinogen concentrations. Sternal marrow core biopsies taken 7 weeks after infestation were 75 to 90% cellular, while cores from uninfested calves were 10 to 20% cellular. Comparison of myelograms between the two groups of calves revealed increased M:E ratio, myelocyte and metamyelocyte hyperplasia with depletion of mature neutrophils and eosinophils in the P. ovis-infested calves. It was concluded that P. ovis-infested calves develop myeloid hyperplasia and that mange-related neutropenia is not due to bone marrow suppression by a mite or scab-associated toxin.     

The pulmonary clearance of Pasteurella haemolytica in calves given Corynebacterium parvum and infected with parainfluenza-3 virus.

Four control calves were aerosolized with parainfluenza-3 and one week later with Pasteurella haemolytica. Three calves were given Corynebacterium parvum at a dose of 15 mg/m2 body surface area, infected with parainfluenza-3 virus one week later, and aerosolized with P. haemolytica two weeks after C. parvum injection. All calves were killed four hours after P. haemolytica exposure and the bacterial retention in the lung was determined. Parainfluenza-3 viral infection did not exert any suppressive effect on pulmonary clearance of P. haemolytica in six out of seven calves used. However, the bacterial colony counts in the lungs of control calves were higher (P less than 0.05) than those in calves given C. parvum. Hence, C. parvum appeared to enhance bacterial clearance. Despite the marked influx of neutrophils into the lungs after the bacterial inoculation, the neutrophil:macrophage ratio in lavage samples was less in calves given C. parvum than in the control calves. The alveolar macrophages in C. parvum treated calves were generally larger but did not differ significantly (P less than 0.05) from those in the controls. There was no significant (P less than 0.05) correlation between the percentages of alveolar macrophages and the bacterial clearance. In calves given C. parvum, bacterial clearance was enhanced in those calves which had larger macrophages.     

Importance of neutrophils in the pathogenesis of acute pneumonic pasteurellosis in calves

Acute lung injury was induced in 24 calves by intratracheal inoculation with Pasteurella haemolytica. Calves in groups 1 and 2 were neutrophil depleted, using hydroxyurea given IV. Group 1 calves (n = 7) were inoculated intratracheally with saline solution, and group 2 calves (n = 7) were inoculated with P haemolytica. Group 3 calves (n = 7) had normal numbers of neutrophils and were inoculated with P haemolytica. Group 4 calves (n = 3) were treated acutely with hydroxyurea IV, had normal numbers of neutrophils, and were inoculated with P haemolytica. After inoculation, calves with normal numbers of neutrophils (groups 3 and 4) became hypoxemic 2 hours after inoculation, and hypoxemia persisted until necropsy (6 hours after inoculation). These calves also developed tachypnea, bradycardia, neutropenia, and lymphopenia. Lung lesions consisted of necrosis of the alveolar walls, intra-alveolar hemorrhage, and a severe exudative and necrotizing bronchopneumonia, with accumulation of proteinaceous fluid in alveoli and lymphatics. In neutrophil-depleted calves (groups 1 and 2), blood gas values, heart and respiratory rates, and numbers of circulating leukocytes did not change after inoculation with saline solution or with P haemolytica. At necropsy, the lungs of neutrophil-depleted calves were grossly normal. Therefore, neutrophils were required for the acute lung injury induced by P haemolytica. The protective effect of neutrophil depletion was a specific effect of hydroxyurea because calves with high circulating concentrations of hydroxyurea and calves with normal numbers of neutrophils (group 4) developed lung injury.     

The kinetics of mononuclear phagocytes in normal calves given Corynebacterium parvum.

Two groups of calves, three in each group, were used to determine the kinetics of mononuclear phagocytes in normal calves and in calves given Corynebacterium parvum intravenously, using tritiated thymidine as an in vivo deoxyribonucleic acid label. In normal calves, the mean production time of labelled monocytes in the bone marrow was 36.4 +/- 2.04 hours. The turnover rate of labelled monocytes from the bone marrow into the peripheral blood was 5.4 +/- 0.3% per hour and the disappearance rate of labelled monocytes from the circulation was 0.9 +/- 0.3%. The half lives of labelled blood monocytes were 22.5 hours for cells with 16-30 grains and 19.5 hours for cells with 31-50 grains. Alveolar macrophages were derived from peripheral blood monocytes. In calves given C. parvum, the production time, turnover rate and half lives of labelled monocytes did not differ significantly (p greater than 0.05) from the values in normal calves.     

Cytological findings in bronchoalveolar lavage fluid from feedlot calves: associations with pulmonary microbial flora.

Samples obtained by bronchoalveolar lavage (BAL) were used to evaluate pulmonary cytology in 59 feedlot calves with clinical signs of respiratory disease (cases) and 60 clinically normal comparison calves (controls). Many calves in both case and control groups had inflammatory changes in the lower respiratory tract, as determined by changes in proportions in the BAL differential cell count. Approximately 35% of cases and 40% of controls showed a normal differential cell count. It therefore appeared that the criteria used to select cases for treatment, which were similar to those often used in the field, were poor predictors of lower respiratory tract disease. A positive association was found between an increased proportion of neutrophils in BAL fluid and isolations of Pasteurella multocida and Mycoplasma bovis from BAL fluid.     

The Evaluation of Coagulation Profiles in Calves with Suspected Septic Shock

The purpose of the study reported here was to evaluate the haemostatic function in calves with suspected septic shock and to reflect the occurrence of disseminated intravascular coagulation (DIC). Twenty-six calves suspected of having septic shock (experimental group) and 10 clinically healthy calves (control group) were used. On admission, the experimental group of calves had been ill for an average of 2 days. Therapy was applied to the experimental group of calves. The packed cell volume (PCV), haemoglobin (Hb), white blood cell (WBC), red blood cell (RBC) and platelet (PLT) counts were determined. Blood smears for toxic neutrophil and schistocyte intensity were evaluated. For the coagulation profile, plasma activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen and fibrin/fibrinogen degradation products (FDPs) values were determined. Toxic neutrophils in blood smears were observed in 12 calves of the experimental group. APTT was prolonged in the experimental group compared with the control group (p < 0.05). Fibrinogen concentration was found to be higher in the experimental group than in the control group (p < 0.001). Total leukocyte counts were higher in the experimental group compared with the control group (p < 0.01). Platelet counts in the experimental group were lower than the control group (p < 0.001). However, when the individual values of coagulation profiles of each calf were evaluated, 8 calves had at least three abnormal coagulation profiles (APTT >72 s, PT >34.5 s, TT >33.7 s, FDPs >5 microg/ml, PLT < or = 150 x 10(3)/mm(3)) and abnormal erythrocyte morphology (schistocytes > or = 1). The most common abnormal tests in the coagulation profile were APTT and PT (7 cases), FDPs (6 cases), thrombocytopenia (4 cases), and schistocytes in blood smears (8 cases) in these 8 calves. The results of this study indicate that DIC might be a significant risk factor for mortality in calves with suspected septic shock.     

Anticoccidial efficacy of medium-chain triglycerides (MCT) in calves

Anticoccidial efficacy of dietary fat was evaluated in calves with coccidial infection (Eimeria spp., including E. bovis and E. zuernii). Medium-chain triglycerides (MCT)--natural edible fats composed of caprylic (C8), capric (C10), and lauric (C12) acids -- were given orally with milk to 5 calves and with 10% glucose solution to 3 older, weaned calves by using the reticular groove reflex. After 3 to 11 days of MCT feeding, all Eimeria spp. oocysts had disappeared from the feces of all calves. MCT had no adverse effects on appetite or on fecal pH, ammonia, lactic acid, or volatile fatty acid levels. MCT feeding for coccidial control in calves has minimal side-effects and has benefits in terms of residue-free food production.     

Evaluation of structural and functional alterations of circulating neutrophils in calves with experimentally induced pneumonic pasteurellosis

OBJECTIVES: To determine the structural and functional alterations in circulating neutrophils that may lead to sequestration in lung microvasculature and endothelial injury in calves with experimentally induced pneumonic pasteurellosis. ANIMALS: 10 healthy, 2- to 4-week-old male Holstein calves. PROCEDURES: Holstein calves were anesthetized and inoculated intrabronchially with Dulbecco phosphate buffered saline (0.9% NaCl) solution (DPBSS; 5 control calves) or 1 x 10(9) Pasteurella haemolytica organisms (5 infected calves). Blood samples were collected before and 1, 2, 4, and 6 hours after inoculation. Total and differential WBC count, dilute whole blood leukocyte deformability, neutrophil size distribution, and neutrophil surface CD11b expression were measured in blood samples. RESULTS: A progressive decrease in leukocyte deformability and increase in neutrophil size was detected 1, 2, 4, and 6 hours after inoculation of P haemolytica. Neutrophil surface CD11b expression was greater than baseline values at 6 hours after inoculation of P haemolytica. Two populations of neutrophils with an increase in size were detected in P haemolytica-infected calves. Both subpopulations had increased CD11b expression, compared with neutrophils that were typical in size. CONCLUSIONS AND CLINICAL RELEVANCE: Neutrophils circulate in an activated and nondeformable state in calves with experimentally induced pneumonic pasteurellosis. A decrease in neutrophil deformability and neutrophil aggregation may contribute to neutrophil trapping in the lung microvasculature during pneumonic pasteurellosis in calves.     

Effects of pasteurization of colostrum on subsequent serum lactoferrin concentration and neutrophil superoxide production in calves

OBJECTIVE: To determine the effects of pasteurization of colostrum on serum lactoferrin concentration and neutrophil oxidative function by comparing values from calves given pasteurized (76 C, 15 minutes) colostrum versus calves given fresh frozen colostrum. ANIMALS: 8 Holstein bull calves were used to study the effects of pasteurization of colostrum on the absorption of lactoferrin and neutrophil oxidative burst. Three additional calves were used to study the effect of exogenous lactoferrin on neutrophil oxidative burst. METHODS: Calves were fed fresh frozen or heat pasteurized colostrum (76 C for 15 minutes) via esophageal feeder within 4 hours of birth. Neutrophils were isolated from whole blood samples. Neutrophil oxidative burst was induced by phorbol ester (300 ng/ml) stimulation of cells (1 X 10(6) cells) at 37 C. Serum lactoferrin concentrations were compared, using immunoblot analysis. Serum IgG concentrations were determined by radial immunoassay. Comparisons were made between the use of the 2 types of colostrum in calves by measuring subsequent serum IgG and lactoferrin concentrations and neutrophil superoxide production. RESULTS: Serum IgG and lactoferrin concentrations increased more in calves receiving fresh frozen colostrum. Neutrophil superoxide production was higher in neutrophils prepared from calves receiving fresh frozen colostrum. Colostral lactoferrin addition to neutrophil incubations resulted in increased oxidative burst. CONCLUSIONS AND CLINICAL RELEVANCE: Compared with calves given fresh frozen colostrum, calves given pasteurized colostrum had decreased serum IgG and lactoferrin concentrations and neutrophil superoxide production 24 hours after administration. These results suggest that pasteurizing bovine colostrum at 76 C for 15 minutes has substantial effects on passive transfer of proteins and neutrophil function.     

Some characteristics of a natural infection by parainfluenza-3 virus in a group of calves.

Parainfluenza-3 (Pi3) virus infection in a group of 25 calves is described. The virus was isolated from the lungs of four calve at days 6, 7, 13 and 55 after they were housed together at birth. Intracytoplasmic inclusion bodies were seen by light microscopy in bronchial and bronchiolar epithelial cells of two of these calves. Virus infected cells were detected by electron microscopy in three of the four calves. Haemagglutination inhibition antibodies to Pi3 virus were found in the sera of the calves. Despite the virus being present in the group from one week, a significant increase in antibody titre was found in only two animals although all the calves were in contact with each other during the study period. The pulmonary lesions in the four infected calves consisted of a bronchitis and bronchiolitis with infiltration of the walls and lumena of these structures by neutrophils and an adjacent neutrophil infiltration of alveoli some of which were collapsed.