Evaluation of L-selectin expression and assessment of protein tyrosine phosphorylation in bovine polymorphonuclear neutrophil leukocytes around parturition.

Impaired polymorphonuclear neutrophil leukocyte (PMN) function around parturition has been associated with increased clinical mastitis in dairy cows. Rolling and attachment of PMN to the endothelium is the first step in the recruitment process and is accomplished by interaction between L-selectin on PMN and its ligand on endothelial cells. Furthermore, tyrosine phosphorylation is involved in the initiation of many PMN functions. The objective of this work was to determine changes in expression of L-selectin and tyrosine phosphorylation in the perinatal period. Eight clinically healthy Holstein cows were used as PMN donors at d-21, -14, -7,0 (calving), +1, +2, +7, +14, +28. Evaluation of L-selectin expression was carried out on activated and resting PMN. Anti-bovine L-selectin monoclonal antibody (MAB) and flow cytometric analysis were used to measure the percentage of PMN fluorescing and receptor expression (log mean fluorescent channel, LMFC). Activated and resting PMN showed similar trends in % PMN fluorescence and LMFC. The percentage of PMN fluorescing tended to decrease at parturition, followed by a significant increase at d +14 and +28 (P <0.02). For LMFC a decrease was observed on d +1 followed by an increase through d +28 (P < 0.01). Protein tyrosine phosphorylation of lysates prepared from PMN isolated throughout the study was detected by electrophoresis and western blotting using anti-phosphotyrosine MAB. Several protein bands were tyrosine phosphorylated. Two of these bands (42-44 kDa and 90 kDa) varied in intensity over time. The intensity of the 42-44 kDa band gradually increased from d -7, peaked at d +7 (P < 0.03), and steadily decreased to d +28 (P < 0.02). Antibody to activated mitogen protein kinase reacted with the 42-44 kDa band. Reduced PMN function during the periparturient period could be related to reduced L-selectin adhesion molecules on the cell surface, and to modulation in the phosphorylation of functionally important molecules

L-selectin and chemotaxis throughout bone marrow granulocyte maturation in the bovine

Polymorphonuclear neutrophilic leukocytes (PMNL) play a pivotal role during inflammation. Bone marrow ( BM) reserves are depleted as cells are released into circulation for recruitment to infection sites. Expression of L-selectin on the cell membrane allows neutrophils to roll along the activated endothelium. Whereas mechanisms leading to recruitment to infection sites are well established, expression of BM adhesion molecules in cows is limited. In this study, we assessed L-selectin expression and chemotactic response to zymosan-activated serum (ZAS) in bovine BM cells and in circulating neutrophils. Isolated blood PMNL and BM cells were used from 9 dairy cows, for quantifying L-selectin expression using flow cytometry, and from 12 dairy cows for chemotaxis studies. All granulocytic maturation stages expressed L-selectin. The percentage of cells fluorescing increased significantly in BM band and mature granulocytes and reached maximal expression on circulating neutrophils. Bone marrow band and segmented cells showed the highest L-selectin density. Chemotaxis through micropore filters in response to zymosan-activated fetal bovine serum was first observed in the myelocytic and metamyelocytic stages, and it increased with maturation and release into the blood stream. From these results, we conclude that L-selectin expression varies among stages of granulocytic maturation within the BM and differs from circulating PMNL. Further, BM cells are capable of migration starting at the metamyelocytic stage, and compared with BM cells, circulating neutrophils are more chemotactively active

Treatment of acute Escherichia coli mastitis in cows with enrofloxacin: effect on clinical signs and chemiluminescence of circulating neutrophils.

We have studied the effect of treatment with enrofloxacin on local and general clinical signs and chemiluminescence of circulating polymorphonuclear leucocytes during experimentally induced Escherichia coli mastitis in cows immediately after parturition. Twelve cows were infected with 10(4) cfu Esch. coli P4:O32 into both left quarters. Six cows received an intravenous injection of 5 mg enrofloxacin/kg at 10 h after infection and a second enrofloxacin treatment administered subcutaneously at 30 h post infection. The other six cows were controls that received no treatment. General clinical signs (fever, tachycardia, loss of appetite, reduced rumen motility and depression) were similar in both groups. Local clinical signs, such as swelling, pain and firmness of the inflamed mammary quarters, were less severe in the treated cows. We saw no difference in the appearance of the milk: fleck and watery or purulent milk were observed in both groups. The beneficial effects of treatment with enrofloxacin were mainly on milk production and composition. The decline in milk production and the changes in milk concentrations of lactose, Na+ and bovine serum albumin were less pronounced in the treated cows. Treatment with enrofloxacin accelerated the clearance of bacteria from the infected quarters, but had no effect on the chemiluminescence response of isolated polymorphonuclear leucocytes. The changes in the number of circulating leucocytes and the appearance of immature neutrophils in the circulation of the treated cows indicated possible beneficial effects on migration of neutrophils into the inflamed glands. Higher milk somatic cell counts in the treated cows supported this hypothesis. The results of this study indicated that treating cows that have been experimentally infected with Esch. coli mastitis after parturition with enrofloxacin reduced the severity of the disease, especially the decline in milk production and the changes in milk composition

Chemiluminescence of bovine polymorphonuclear leucocytes during the periparturient period and relation with metabolic markers and bovine pregnancy-associated glycoprotein.

The respiratory burst activity of polymorphonuclear leucocytes (PMN) was evaluated in eight Holstein cows from 8 weeks before until 6 weeks after calving by chemiluminescence (CL). The CL response started to decrease 1 week before parturition, reaching a minimum during the first 2 weeks after calving. From week 3 of lactation, CL increased again and returned to original levels by week 6 of lactation. Plasma concentrations of 3-hydroxybutyric acid, total bilirubin and bovine pregnancy-associated glycoprotein started to increase before parturition to reach a maximum during the first or second week of lactation. The concentrations of glutamic-oxaloacetic transaminase, lactate dehydrogenase, non-esterified fatty acids and bilirubin increased after calving, reaching a maximum during the second week. A small decrease in plasma cholesterol during the week before and after calving was followed by an increase. The CL response of the PMN showed significant temporal relationships with the plasma concentrations of 5-hydroxybutyric acid, bovine pregnancy-associated glycoprotein, bilirubin, glutamic-oxaloacetic transaminase, non-esterified fatty acids. that with cholesterol was nearly significant. This means tl-lat the change in the CL response with time coincided with the changes in plasma concentrations of these substances with time and that these changes were significantly related with each other. The results: of this study show that the decreased respiratory burst activity of bovine PMN around parturition may be related to the extent of the metabolic and hormonal changes, although the causative relationships are not proven, these results support earlier results suggesting that S-hydroxybutyric acid and bovine pregnancy-associated glycoprotein may directly affect neutrophil function, whereas non-esterified fatty acids, cholesterol, bilirubin, and liver enzymes map have potential as diagnostic markers of impaired neutrophil function and consequently increased disease risk around parturition