Specific antibodies induced by inactivated parapoxvirus ovis potently enhance oxidative burst in canine blood polymorphonuclear leukocytes and monocytes

We have recently shown that inactivated parapoxvirus ovis (iPPVO) effectively stimulates canine blood phagocytes. However, a potential link between innate and adaptive immunity induced by iPPVO remained open. The objective of this study was to define the effects of repeated iPPVO treatment of dogs to evaluate (i) iPPVO-specific antibody production, and (ii) modulation of iPPVO-induced oxidative burst by anti-iPPVO antibodies. Serum analysis of dogs treated repeatedly with iPPVO (Zylexis^®) showed transient production of non-neutralising iPPVO-specific IgG. There was a correlation between iPPVO-specific IgG levels and enhanced oxidative burst rates in vitro upon transfer of immune sera. Even four years after Zylexis^® treatment considerably stronger oxidative burst rates in response to iPPVO were observed in monocytes and PMN, whereas only moderate burst rates were detected in monocytes, but not in PMN, from dogs treated with a placebo. Depletion of serum IgG by protein A-sepharose or by parapoxvirus ovis coupled to sepharose abolished the increase of oxidative burst responses and resulted in burst rates similar to blood leukocytes from control dogs. However, uptake of viral particles was found to be independent of iPPVO-specific IgG and restricted to cells with dendritic and monocytic morphology. These data demonstrate that non-neutralising iPPVO-specific IgG is produced during treatment with Zylexis^®. Moreover, for the first time the interaction of iPPVO with antibodies is shown to enhance oxidative burst.     

Interaction between Salmonella typhimurium and phagocytic cells in pigs. Phagocytosis, oxidative burst and killing in polymorphonuclear leukocytes and monocytes

Interactions between Salmonella typhimurium and peripheral blood leucocytes from healthy, Salmonella-free pigs were investigated in vitro. Both granulocytes and monocytes phagocytized FITC-labelled heat-killed Salmonella bacteria as shown by flow cytometry. Phagocytosis in whole blood and isolated leucocytes was measured as acquired fluorescence in the leukocytes and was both time and dose related. Living, serum-opsonized Salmonella bacteria induced a dose-dependent oxidative burst in PMNs and monocytes as measured by luminol-enhanced chemiluminescence (LC). When opsonized in normal serum the Salmonella bacteria, in the range of 2-5 x 10(7) cfu, induced a LC response in monocytes comparable to the level of responses induced by phorbol myristate acetate (PMA) and opsonized zymosan, and the Salmonella-induced response was only marginally reduced by superoxide dismutase (SOD). Intracellular killing of Salmonella by monocytes was assessed from plate colony counts of lysed monocytes and showed that Salmonella typhimurium was able to survive and proliferate in adherent monocytes in vitro despite a reduction in intracellular cfu during the first hour's incubation in cells from some pigs. Experiments with the exhaustion of oxidative burst in non-adherent monocytes were performed by prestimulation with PMA, heat-killed Salmonella or buffer. Prestimulation with PMA led to a strong reduction in oxidative burst induced by living opsonized Salmonella bacteria, whereas prestimulation with heat-killed bacteria gave rise to an enhanced response. In these experiments intracellular killing of the added living Salmonella gave variable results, in that monocytes from two out of three pigs showed no essential change in intracellular bactericidal activity, but with cells from one pig a less pronounced bactericidal activity was found after prestimulation with PMA.     

Repeatability of flow cytometric and classical measurement of phagocytosis and respiratory burst in bovine polymorphonuclear leukocytes

Five methods for measurement of phagocytosis and respiratory burst activity of bovine blood polymorphonuclear leukocytes (PMNs) were evaluated. Eight cows were repeatedly sampled over a two week period and parallel samples tested in all five assays to assess the repeatability and stability of the methods. In the flow cytometric phagocytosis assay, ingestion of fluorescein labeled bacteria was measured, and in the flow cytometric assay for respiratory burst, oxidation of a dye by reactive oxygen species was recorded. In the classical assays, bactericidal effect on opsonized, live bacteria was quantified by the conversion of an indicator substance, superoxide anion production was assayed by the reduction of cytochrome c, whereas myeloperoxidase activity was determined with a radioactive iodination assay. The results showed that the Phagotest, Bursttest, cytochrome c and iodination assays gave repeatable results when samples were run in the same setup on the same day. Although day-to-day variability was significant in all assays, the described methods comprise a panel of useful tests for the evaluation of phagocytosis and respiratory burst activity in bovine PMNs. The flow cytometric methods represent a convenient alternative to the classical methods for measurement of phagocytosis and respiratory burst in bovine blood PMNs.     

Phagocytosis of bovine blood and milk polymorphonuclear leukocytes after ozone gas administration in vitro

To determine the effects of ozone on the phagocytosis of bovine polymorphonuclear leukocytes (PMNs), ozone gas was administered in vitro on the blood and milk of healthy lactating cows, cows with acute mastitis, and cows with milk fever. In the blood of healthy dairy cattle, although there was no significant effect of ozone gas on the viability of the leukocytes, phagocytosis of PMNs significantly decreased. In contrast, ozone gas administration in vitro significantly increased phagocytosis of PMNs from the blood of cows with acute mastitis and milk fever, and from mastitic milk. These findings showed that ozone administration in vitro has positive and negative effects on bovine PMN phagocytosis, depending on the health status of the animal.     

Flow cytometric study of oxidative burst activity in bovine neutrophils

A flow cytometric procedure was evaluated to measure the oxidative burst activity (hydrogen peroxide formation) of bovine neutrophils. The method involves measuring the oxidation of intracellular dichlorofluorescein to fluorescent dichlorofluorescein (DCF). Phorbol myristate acetate (PMA) was used to perturb the neutrophil plasma membrane. The sources of variation introduced into the DCF assay were also examined. The sources of variation were attributable to the isolation of neutrophils from blood, variation between duplicate assays and duplicate flow cytometric determinations of oxidative product formation, variation in neutrophil oxidative product formation among cows, and the variation (over repeated daily and weekly neutrophil isolations) in neutrophil oxidative product formation. A final objective was to determine effects of dexamethasone on oxidative product formation, and whether differences existed between blood and mammary neutrophils in oxidative product formation. There was an increasing trend in the formation of DCF with increasing time of incubation and with increasing PMA concentration. Increasing the concentration of PMA decreased lag time and increased the rate of oxidative product formation. The increase in DCF formation was statistically significant up to a PMA concentration of 10 ng/ml. This concentration was considered optimal for bovine neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)     

A simple method for the in vitro determination of phagocytic activity of bovine polymorphonuclear leukocytes

A simple method for the in vitro determination of the phagocytic activity of bovine polymorphonuclear leukocytes (PMNL) is described. An enriched PMNL population was obtained from peripheral blood of healthy cattle and mixed with opsonized Candida utilis or with opsonized bovine red blood cells on BSA treated coverglass slides. The phagocytosis and killing of C. utilis was determined simultaneously on the coverglass slides stained with acridine orange. Phagocytosis of bovine erythrocytes was determined by microscopic examination of the Wright-Giemsa stained slides. This method is appropriate under limited laboratory conditions because it does not require highly sophisticated equipment and materials, it is easy and rapid to perform, reproducible and inexpensive. Therefore, it could be used as a first evaluation of the phagocytic activity of bovine PMNL.     

Apoptosis and necrosis of blood and milk polymorphonuclear leukocytes in early and midlactating healthy cows.

Increased milk somatic cell counts (SCC) are used as an indicator for bovine mastitis. During mastitis, polymorphonuclear leukocytes (PMN) become the predominant cell type. Shortly after parturition, the severity of mastitis is increased and several PMN functions are downregulated. Apoptotic and necrotic processes of PMN could influence SCC and PMN functions. In this study, the percentages of apoptotic and necrotic PMN in blood and milk from early and midlactating healthy cows were compared. Apoptosis and necrosis of PMN were quantified using a dual-color flow cytometric procedure with fluorescein labeled annexin-V (green) and propidium iodide (red). Using this technique three different subpopulations of bovine PMN could be detected: apoptotic cells (high intensive green fluorescence), necrotic cells (high intensive green and high intensive red fluorescence) and viable cells (low intensive green and low intensive red fluorescence). Following a 4 h incubation of blood from both groups of cows at 37 degrees C to induce apoptosis, the mean percentage of apoptotic blood PMN was significantly higher (P < 0.01) in early lactating cows (15.1%, n = 9) compared with midlactating cows (5.3%, n = 10). The mean percentage of necrotic PMN remained lower than 5% in all cows. In contrast to blood, no significant difference was found between the percentage of apoptotic PMN in milk from early (41.2%, n = 7) and midlactating cows (34.0%, n = 8). The percentage of necrotic PMN in milk from early lactating cows (25.9%, n = 7) was significantly higher than that in midlactating cows (14.2%, n = 8) (P < 0.05). Higher percentages of apoptotic as well as necrotic PMN were consistently found in milk compared to blood in all cows. From these results, it can be concluded that spontaneously induced apoptosis was higher in blood PMN from early lactating cows than in blood PMN from midlactating cows. The higher percentage of necrotic milk PMN in early lactating cows than in midlactating cows could be explained by the induction of secondary necrosis.     

The effect of pasture turnout on milk somatic cell count,polymorphonuclear leukocytes and milk composition in cows housed in tie stalls

Abstract

This study investigated milk somatic cell count (SCC), polymorphonuclear leukocytes (PMNs) and milk composition in dairy cows, which were kept in tie stalls, during the first days after pasture turnout. Thirty-five cows of the Swedish Red Breed, free of clinical signs of mastitis and with a geometric mean of SCC 67 × 10³ cells/mL, were turned out to pasture after the morning milking on day 0 and then monitored for the next five days on pasture. Samples of cow composite milk were taken at every milking and analysed for SCC, PMN percentage of the total SCC and milk composition. There was a marked increase in both PMN proportion and SCC with the highest SCC value during the study recorded at the evening milking on day 0. The highest value in morning milk was observed on day 1. Milk SCC values in evening and morning milk declined after day 0 and day 1, respectively, but remained on a higher level compared with before turnout to pasture. Milk composition was only slightly altered. Since the changes in SCC and milk composition were of low magnitude, although statistically significant, these effects of pasture turnout can be considered as of minor importance for the milk quality.     

The effect of low levels of dietary cobalt on the chemiluminescence response of polymorphonuclear leukocytes of goats

Twenty ten-week-old newly weaned male Batinah goats were randomly assigned to a control (n = 10) and a treated (n = 10) group and were fed a diet containing 0.1 mg/kg DM cobalt (Co). Goats in the treated group received bi-monthly subcutaneous injections of 2000 μg of hydroxycobalamin. The phagocytic function of the polymorphonuclear leukocytes (PMN) were tested using a luminol-dependent chemiluminescence assay with opsonized zymosan as the phagocytic target. One month after the onset of the experiment PMN from the control group exhibited a significantly (p < 0.05) lower CL response, which continued for the second month. The results of the present study demonstrated that low levels of dietary cobalt leads to an early impairment of phagocytic function. This may at least in part, be an explanation as to why at the field level in Oman young goats fed diets containing low levels of Co appear to be more susceptible to infections.     

Separation of mononuclear leukocytes and polymorphonuclear leukocytes from equine blood.

The present study describes a two step technique for the separation of mononuclear leukocytes or mononuclear and polymorphonuclear leukocytes from whole equine blood. First, the leukocyte rich plasma was obtained by sedimentation of erythrocytes in the undiluted blood. Subsequently, separation of the different populations of white blood cells was performed by centrifugation with different gradients overlaid with the leukocyte rich plasma. The optimal separation of the mononuclear cells was obtained by the centrifugation of the leukocyte rich plasma overlaying the gradient containing 24 parts of 9.5% ficoll and ten parts of 34% isopaque. The mononuclear leukocytes (95% lymphocytes and 5% monocytes) formed a monolayer band at the plasma-ficoll-isopaque interface and other blood cells migrated to the bottom of the tube. For the separation of mononuclear and granular leukocytes from the blood, the gradient containing 24 parts of 10% ficoll and ten parts of 34% isopaque was used. The separated monuclear leukocytes responded to stimulation with phytohemagglutin and viability of both mononuclear and polymorphonuclear leukocytes was not affected by ficoll-isopaque separation.     

The influence of mononuclear leukocytes on the chemotactic responsiveness of polymorphonuclear leukocytes of bovines

Polymorphonuclear leukocytes (PMN) isolated from the blood of bovines which do not react with an influx of cells into the milk upon intramammary administration of low doses of endotoxin (Group II), show a diminished chemotactic activity in vitro as compared to PMN of bovines which do respond to intramammary endotoxin administration (Group I; 8). The purpose of the present study was to determine whether other immune cells affected the chemotactic activity of PMN of Group I and II bovines. Mononuclear leukocyte suspensions of Group I animals had no effect on the migration of PMN of Group I animals. In contrast, the distance migrated by Group II PMN was diminished in the presence of Group II mononuclear leukocytes. As a consequence, this phenomenon resulted in a much larger difference in chemotactic activity of PMN in vitro between Group I and Group II bovines. It can be concluded that the chemotactic activity of PMN of animals which are more susceptible for mastitis as indicated by non-responsiveness to intramammary endotoxin-infusions, was further down-regulated by mononuclear leukocytes.     

The involvement of polymorphonuclear leukocytes in the pathogenesis of bronchopneumonia in calves. IV. Myeloperoxidase activity.

Stable oxidant and hypochlorous acid production in neutrophils of bronchopneumonic calves was investigated. The production of these compounds was much more intense in neutrophils of diseased calves than in the control cells. The production of stable oxidants and hypochlorous acid was restrained by free radical scavengers. Functional response of neutrophils was inhibited by cyclooxygenase products and stimulated by lipoxygenase products of the arachidonic acid cascade. Superoxide anion production is not connected with the degranulation process (beta-glucuronidase release), and the two events are under different control mechanisms.     

Adhesion molecule and homing receptor expression on blood and milk polymorphonuclear leukocytes during the periparturient period of dairy cattle

Adhesion molecule and homing receptor expression on blood and milk polymorphonuclear leukocytes (PMN) from periparturient dairy cattle was studied. Both percentages and the mean fluorescence intensity (MFI) of PMN expressing CD11a, CD44, CD62L, and LPAM-1 (alpha4 beta7) were evaluated at seven time points during the twenty-one day period post calving. CD11a and CD62L were expressed on 94-100% of PMN in both blood and milk and there were no significant differences in these percentages at any time point. LPAM-1 was expressed on 3-10% of the PMN in the blood and 13-45% in the milk and the percentage of cells expressing LPAM-1 in milk was significantly (P<0.05) greater than in blood at 0, 4, 10, 14, 18 and 21 days after calving. CD44 was expressed on 11-39% of the PMN in blood and 33-69% in the milk and the percentage of cells expressing CD44 in milk was significantly (P<0.05) greater than in blood at all time points. The MFI of CD11a on milk PMN was consistently higher than that of blood PMN throughout the study period and significantly (P<0.05) higher at days 4, 10 and 18 after calving.     

Assessment of bovine mammary gland macrophage oxidative burst activity in a chemiluminescence assay

A major bactericidal mechanism of neutrophils and macrophages is the generation of toxic oxygen-free radicals upon phagocytosis of microbes. Studies were conducted to assess the oxidative metabolism of bovine mammary gland macrophages. Bovine mammary gland macrophages were challenge exposed with a variety of phagocytic stimuli in an in vitro, luminol-assisted chemiluminescence assay. A measurable oxidative burst was observed when macrophages were challenge exposed with heat-aggregated bovine immunoglobulin, opsonified zymosan, and nonosponified zymosan. Addition of superoxide dismutase decreased mammary gland macrophage chemiluminescence in a dose-dependent manner. Brucella abortus, when opsonified with antiserum, lacteal antibody, or normal serum, produced an oxidative event, whereas nonopsonified B abortus did not. When challenge exposed with phagocytic stimuli, mammary gland macrophages produced an oxidative burst similar to that produced by other phagocytes for which an oxidative event is known to be bactericidal.     

Functional deficiency of polymorphonuclear leukocytes in simian acquired immunodeficiency syndrome

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 AIDS, 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.     

Secretory activity of equine polymorphonuclear leukocytes: stimulus specificity and priming effects of bacterial lipopolysaccharide.

Neutrophil (PMN) contributions to the acute inflammatory process and host defense include generation of bioreactive oxygen metabolites and secretion of granule enzymes. We assessed equine PMN secretion using several PMN stimuli, singly and in combination with bacterial lipopolysaccharide (LPS). LPS avidly associated with equine PMN, as shown by strong PMN labeling with FITC-conjugated LPS. LPS alone (1 or 10 micrograms ml-1) was a weak stimulus for PMN superoxide anion (O2-) generation, but preincubation with LPS followed by phorbol ester (PMA, 10 ng ml-1) significantly augmented (P less than 0.01) secretion of O2- (19.38 nmol O2- per 2 x 10(6) PMN per 5 min) over the amount generated by PMA stimulation alone (13.75 nmol O2-). A qualitatively similar, but smaller O2(-)-generation response occurred when either opsonized zymosan or recombinant human C5a was used as the PMN stimulus. Arachidonic acid (ArA; 50-200 microM) was a potent stimulus, with secreted O2- levels similar to those from PMA-stimulated PMN. Preincubation of PMN with either the formyl peptide, fMLP, or platelet-activating factor before stimulation with ArA did not significantly increase O2- generation over levels obtained using ArA alone. Release of PMN granule enzymes was also quantitated. A small amount of lysozyme secretion resulted when PMN were exposed to LPS alone (8.20% of total cell content), and PMA stimulation caused marked release of PMN lysozyme (44.45%). Non-specific proteolytic activity in PMN supernatants, assessed by cleavage of a collagen-rich substrate, was minimal with LPS as a sole stimulus (5.08%). There was significant proteolytic activity (P less than 0.01) in supernatants from PMA-stimulated PMN (27.21%), and preincubation with LPS followed by PMA stimulation slightly enhanced (P less than 0.05) the release of PMN proteases (34.62%). The activities of beta-glucuronidase, acid phosphatase, and alkaline phosphatase were minimal in PMN supernatants when using LPS and PMA as stimuli. The activity of PMN granule enzymes was found to be sensitive to the presence of normal equine serum, and proteolytic activity was markedly reduced (80.13% reduction) in the presence of 10% pooled serum.     

Changes in peripheral leukocytes enzymes activity and plasma metabolite concentrations in growing Holstein calves

Plasma metabolite and immunoreactive insulin concentrations and activities of enzymes related to energy metabolism in peripheral leukocytes were measured in growing Holstein calves. A ratio of girth of abdomen divided by girth of thorax (A/T ratio) of calves was significantly elevated after weaning, and the A/T ratio maybe a good indicator to evaluate rumen development. Plasma glucose and free fatty acid concentrations were changed in calves accompanying change in feeding. Activities of lactate dehydrogenase with pyruvate as substrate (LDH-P) and hexokinase (HK) in cytosolic fractions of peripheral leukocytes decreased significantly after weaning the calves reflecting the change of energy source from milk replacer with high percentages of fat and glucose and lactose as absorbable carbohydrate to pelleted feed containing starch as less absorbable carbohydrate and roughage. Some peripheral leukocyte enzymes such as LDH and HK may be good indicators to evaluate changes in energy metabolism of growing calves.     

Bovine milk fat globules do not inhibit C5a chemotactic activity

The C5a complement fragment is a potent inflammatory molecule but its contribution to the inflammatory response of the mammary gland remains uncertain. One of the unresolved questions is the possible interference of whole milk with C5a. In this study, the chemotactic activity ofpurifled bovine C5a was tested in the presence of whole or skimmed milk. Milk from healthy glands acted as a chemoattractant, which could mask any inhibitory effect on C5a activity. To circumvent milk activity, washed milk fat globules were incubated with optimal (1 nM) or suboptimal (0.1 nM) concentrations of C5a, and the eventual chemoattractant activity was measured. There was no reduction in C5a-induced migration through a polycarbonate filter or shape-change of neutrophils. The concentrations of C5a determined by ELISA in the fluid phase were not reduced after incubation with the fat globules. It can be concluded that bovine milk fat globules do not trap or degrade C5a. Although these results do not explain the inhibitory effect of whole milk in the C5a-induced recruitment of neutrophils in milk, they suggest that milk does not inhibit the second major activity of C5a (apart from chemotaxis), i.e. the stimulation of phagocytic and bactericidal activities of neutrophils.     

Inhibition by suramin of the NADPH oxidase from horse polymorphonuclear leukocytes

Suramin strongly inhibits the NADPH dependent oxidative activity of the plasma membrane fraction from activated horse polymorphonuclear leukocytes. The kinetics of inhibition reveals the existence of one effective binding site located on the inner surface of the plasma membrane with an apparent K_i value for suramin of about 1 mM. Although administration of suramin to intact cells does not affect the immediate respiratory burst, prolonged preincubation in vitro with the drug results in a decrease of H₂O₂ production without lowering the phagocytic activity of the leukocytes. Possible implications in vivo are briefly discussed.     

Effects of extracellular Ca2+ on phagocytosis and intracellular Ca2+ concentrations in polymorphonuclear leukocytes of postpartum dairy cows

The aim of this study was to determine the effects of extracellular Ca(2+) concentration ([Ca(2+)](e)) on phagocytosis and intracellular Ca(2+) concentration ([Ca(2+)](i)) in bovine polymorphonuclear leukocytes (PMNs). The experiments were performed by using blood samples from parturient paretic and clinically normal parturient cows and manipulating the [Ca(2+)](e) in vitro. Phagocytosis by PMNs (with and without stimulation with phorbol myristate acetate and inhibition with cytochalasin B) and resting [Ca(2+)](i) were significantly lower in parturient paretic cows. Repletion of Ca(2+) in the extracellular media for the samples from these animals increased phagocytosis and resting [Ca(2+)](i). In the blood of clinically normal parturient cows, decreasing the [Ca(2+)](e) decreased phagocytosis and resting [Ca(2+)](i) in PMNs, but increasing the [Ca(2+)](e) did not affect phagocytosis. These results suggest that the hypocalcemic condition of parturient paretic cows in vivo causes decreased phagocytosis and resting [Ca(2+)](i) in PMNs, which may partly contribute to greater susceptibility to infection.