The N-acetyl-D-glucosamine specific adhesin from Mannheimia haemolytica activates bovine neutrophils oxidative burst

In this work we identified specific bovine leukocytes that were bound by the Mannheimia haemolytica adhesin molecule (MhA) and the biological effect on the leukocytes. Histochemical staining and flow cytometry showed that MhA bind neutrophils (90%) and monocytes (5%). MhA induced an oxidative response in purified neutrophils; this effect was 1.5-fold higher than the effect observed with control cells activated with Zymosan. Cellular binding by MhA was inhibited with GlcNAc and its oligomers, as well as by glycoproteins containing tri- and tetra-antennary N-glycosydically linked glycans. MhA-induced oxidative burst was significantly inhibited by GlcNAc, iodoacetamide, superoxide dismutase, and piroxicam (p<0.05). Our findings suggest that among bovine leukocytes, neutrophils are the main target for MhA, inducing production of oxidative radicals by non-opsonic mechanism that seem to play an important role in tissue damage during mannheimiosis.     

Diapedesis across mammary epithelium reduces phagocytic and oxidative burst of bovine neutrophils.

The effect of diapedesis on the phagocytic and oxidative burst activity of polymorphonuclear neutrophil (PMN) was examined, using an in vitro cell culture model consisting of a monolayer of primary mammary epithelial cells. Isolated blood PMN from 10 cows were added to the basal side of the epithelial cell monolayer. Diapedesis was induced by the addition of complement factor C5a to the apical side of the monolayer. PMN phagocytosis of Staphylococcus aureus and oxidative burst were measured before diapedesis on PMN that were non-activated and activated by incubation with C5a and on PMN after diapedesis, using flow cytometry. The percentages of PMN fluorescing due to phagocytosis of S. aureus and oxidative burst were reduced by 21.2 and 14.4%, respectively, after diapedesis. Pre-incubation in the presence of C5a had no effect on percentage PMN fluorescing due to phagocytosis or oxidative burst. The capacity for individual migrated PMN to phagocytose S. aureus and to produce an oxidative burst, as measured by the intensity of fluorescence, decreased by 34.2 and 30.3%. Activation of PMN with C5a increased intensity due to the oxidative burst, but had no effect on intensity due to phagocytosis. These data show that PMN diapedesis across mammary epithelium results in decreased phagocytosis and oxidative burst of the PMN.     

Decreased respiratory burst activity in neonatal bovine neutrophils stimulated by protein kinase C agonists.

Newborn calves have a high susceptibility to bacterial infections, which may be related to the impaired neutrophil defense functions in newborns. The oxygen-dependent production of the free radical superoxide anion (O2-) represents an important part of the leukocyte respiratory burst central to neutrophil-directed defenses against bacterial infection. Because protein kinase C (PKC) activation is considered to be an important step in the signal transduction pathway for the O2- generating system, we compared O2- production by newborn and adult bovine neutrophils stimulated with 3 different PKC agonists. When the phorbol ester phorbol 12-myristate 13-acetate (PMA) was used, PKC-dependent O2- generation from newborn neutrophils was significantly reduced (P less than 0.01) for all concentrations of PMA tested (10, 100, and 500 ng/ml). In addition, newborn neutrophils had a significantly (P less than 0.01) reduced lag time for O2- generation. Similar significantly (P less than 0.01) reduced O2- generation from newborn neutrophils was observed with an additional phorbol ester (phorbol 12,13-dibutyrate); lag times were not calculated for phorbol 12,13-dibutyrate. When O2- generation was stimulated with a synthetic diacylglycerol analogue (1,2-dioctanoyl-sn-glycerol), less O2- was generated from both adult and newborn neutrophils than was obtained with the phorbol esters, and newborn neutrophils produced significantly (P less than 0.01) less O2- only at 50 microM 1,2-dioctanoyl-sn-glycerol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impairment of oxidative burst in porcine neutrophils induced by pseudorabies virus

Industrial swine production is affected by several serious viral diseases, such as pseudorabies, hog cholera, porcine reproductive and respiratory syndrome, which are frequently complicated with the increased incidence of bacterial complications such as Actinobacillus pleuropneumoniae (APP). This clinical observation is suggestive of a virus-bacteria synergism on the pathogenesis. One hypothesis is that viruses induce polymorphonuclear cell (PMNs, primarily neutrophils) dysfunction resulting in defective antibacterial resistance. The purpose of this study was to use the pseudorabies virus (PrV) as a model to explore the possibility of virus-induced PMN dysfunctions in pigs. The goals were to evaluate, in ex vivo settings, the oxidative burst (OB) function of pig PMNs, and to evaluate whether PrV could affect these responses to APP. We found that PrV served as a mild OB stimulant (2-fold) to pig PMNs, which also launched a significant burst to phorbol 12-myristate 13-diacetate (PMA; 61-fold), to non-opsonized, heat-killed and formaldehyde-fixed APP (8-fold), and to normal pig serum-opsonized APP (34-fold). Interestingly, the PMA-induced OB could be reduced 50-70% by preincubating PMNs with PrV, and the critical target was not likely the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase itself. Inactivated PrV was as efficient as viable PrV at exerting the inhibitory effect. On the other hand, PrV exerted a primarily additive effect on APP-induced OB, when the cytotoxic effect of APP on PMNs was avoided. The current finding suggests the possibility that activated PMNs are susceptible to PrV-induced dysfunction, and that the PrV-APP synergism may require upstream stimuli of PMNs to be initiated.     

Measurement of phagocytosis and oxidative burst of canine neutrophils: high variation in healthy dogs

Quantitative analysis of phagocytosis and oxidative burst in canine polymorphonuclear (PMN) cells was performed by flow cytometry techniques. Different concentrations of phorbol myristate acetate (PMA) were used to modulate PMN phagocytosis. A low concentration of PMA (3 nmol) resulted in increased phagocytic activity of canine PMN, which could not be enhanced by higher dosages. Experiments with a reference cell population showed high losses of PMN, most probably by adherence to plastic material. It was possible to avoid this loss by layering all ingredients on cushions of Histopaque. However, Histopaque had a negative influence on the phagocytic activity of canine PMN. The use of PMA led to a dosage-dependent increase in the oxidative burst measured by the production of reactive oxygen species (ROS). Cushions of Histopaque were used to avoid cell loss. There was no negative influence of Histopaque on ROS formation. Storage of canine PMN for 24 h at room temperature had no negative influence on phagocytosis or oxidative burst measurements. Variations in the ROS assays conducted by two different examiners could be eliminated by use of a Histopaque-cushion.     

Bb fragment of bovine complement factor B: stimulation of the oxidative burst in bovine monocytes.

In a previous study, we reported that fragment Bb of bovine complement factor B activated bovine monocytes, as demonstrated by the uptake of 3H-deoxyglucose. In the present study, the effects of Bb on the production of superoxide anion and hydrogen peroxide by bovine monocytes was investigated. The production of superoxide was measured by the superoxide dismutase inhibitable reduction of cytochrome c. The change in absorbance was determined by a spectrophotometer at a wavelength of 550 nm. Hydrogen peroxide production was measured by the horse-radish peroxidase-dependent oxidation of phenol red. The resulting color change was measured by a spectrophotometer at a wavelength of 620 nm. Fragment Bb (20 micrograms/mL) induced the generation of 0.96 +/- 0.2 (mean +/- SEM) nanomoles of superoxide/2.5 x 10(5) monocytes at 5 min. The production of superoxide peaked at 15 min (1.24 +/- 0.3 nanomoles). The production of hydrogen peroxide was also rapid: 0.195 +/- 0.05 nanomoles/2.5 x 10(5) monocytes at 5 min with a peak at 15 min (0.250 +/- 0.04 nanomoles). These observations show that fragment Bb, which has serine protease activity, induces bovine monocytes to generate reactive oxygen intermediates such as superoxide and hydrogen peroxide.     

Flow cytometric detection of myeloperoxidase in horse neutrophils: a novel technique in equine diagnostic research

Myeloperoxidase (MPO) is a protein of interest due to its involvement in equine pathologies. Until now, results in equine diagnostic research were achieved through extracellular MPO detection. However, studying the cellular MPO content in neutrophils has revealed important insights in human diseases. This study aimed to develop a technique for the specific detection of MPO on the single cell level defining a flow cytometric protocol for the detection of both equine surface-bound and cellular MPO. Both indirect and direct labeling techniques are described which include the comparison of two secondary antibodies and two linking-fluorochromes, respectively.     

The respiratory burst activity of bottlenose dolphin neutrophils elicited by several stimulants

To investigate the early host defense function in aquatic animals, the respiratory burst activity of bottlenose dolphin neutrophils against soluble and particulate stimulants was measured by luminol-dependent chemiluminescence assays and compared with those of bovine and human. Dolphin neutrophils generated the respiratory burst in response to phorbol 12-myristate 13-acetate (PMA), concanavalinA (ConA), heated-plasma (HP), and homologous-plasma opsonized zymosan except N-formyl-Met-Leu-Phe (fMLP). However, the respiratory burst of dolphin neutrophils stimulated by lipopolysaccharide and Staphylococcus aureus was inferior to those of bovine and human. Furthermore, DP-OZ also induced the respiratory burst of bovine and human neutrophils. In conclusion, dolphin neutrophils responded to several soluble and particulate stimulants as well as human neutrophils, but were refractory or slightly responded to bacterial agents.     

Flow cytometric analysis of the DNA content of bovine and human bone marrow cells

The defence against infection in high-yielding dairy cows is correlated with the number and function of circulating neutrophils and depends on their production in bone marrow. Therefore, the DNA content of isolated bone marrow cell suspensions from 7 calves, 7 cows and 14 humans was assayed by flow cytometry. Bovine sternal bone marrow samples were collected within 30 min of death, and human marrow samples were collected by sternal puncture and aspiration. Mononucleated cells were isolated by gradient centrifugation. In the bone marrow samples from calves and cows, 35 +/- 2.6% and 31.8 +/- 1.5% of the isolated bone marrow cells respectively were in the S/G2/M-phase. The difference between calves and cows was not significant. In the human samples, only 12 +/- 0.8% of the cells were in the S/G2/M-phase. A significant (P < 0.001) difference was observed between the two species. These results indicated that the proliferative, in activity of haematopoietic cells is significantly higher in cattle than in humans.     

Bovine milk enhances the oxidative burst activity of polymorphonuclear leukocytes in low concentrations

Bovine milk contains various immunoreactive components, and the activation of polymorphonuclear leukocytes (PMNLs) function in breast-fed infants has been reported. In this study, the effect of milk on the oxidative burst of bovine PMNLs was investigated in vitro. When PMNLs were incubated with 0.1% colostrum or normal milk, the oxidative burst induced by serum-opsonized Staphylococcus aureus was enhanced, and the enhancement declined dose-dependently. The enhancement of the oxidative burst by milk was not due to opsonins but the priming activities. Also, the phorbol 12-myristate 13-acetate (PMA)-induced oxidative burst increased after incubation with 0.1% colostrum, but the colostral enhancement of the oxidative burst was unaffected by the incubation time. These results suggest that bovine milk contains oxidative burst promoting factor(s).     

Proteomic survey of bovine neutrophils

Mastitis is a major economic concern for the dairy industry. Conditions such as parturition cause a transient immunosuppression that leads to increased incidence of mastitis. One facet of periparturient immunosuppression is a functional impairment of the blood and milk neutrophils in dairy cows. To better understand the biology of the bovine neutrophil we report the first proteomic analysis of the bovine neutrophil. We have identified over 250 proteins using one-dimensional electrophoresis followed by reverse-phase chromatography in line with electrospray tandem mass spectrometry. A large number of metabolic proteins were identified, including most of the enzymes required for generation of NADPH and ATP. In addition, many proteins were identified that participate in cell mobility and phagocytosis. All the bovine members of the cathelicidin family were identified, as well as other proteins with immunological functions. Proteins important for cell signaling, vesicular transport, control of apoptosis and other functions were identified giving an overview of the bovine neutrophil proteome.     

Effects of dietary yeast extract on turkey stress response and heterophil oxidative burst activity

1. Effective nutritional approaches to counteract the negative effects of stress may provide food animal producers with useful alternatives to antibiotics. In this study, turkeys were fed on a standard diet, or the same diet supplemented with yeast extract (YE), to determine if YE would improve disease resistance in a stress model.

2. At 16 weeks of age, half of the birds were exposed to a bacterial challenge using a coarse spray of the pen environment. A subset of control and challenged birds was also treated with dexamethasone (Dex) prior to challenge (Dex/challenge). At 18 weeks, another subset was subjected to a 12?h transport stress protocol (Challenge/transport). All birds were bled and necropsied the morning after transport. The numbers and proportions of blood cells and the heterophil oxidative burst activity (OBA) were determined. Serum corticosterone (Cort) levels of male birds were measured using a commercial ELISA kit. Body weight and gain were increased by YE during week 1.

3. YE decreased mortality and bacterial isolation following Dex/challenge only in females. Cort levels in male turkeys were decreased by YE and Dex treatment. OBA was higher in males and in birds given YE and was reduced by challenge and transport.

4. These results suggest there may be gender differences in the turkey stress response and that dietary YE has potential for modulating the impact of stress on innate immunity of turkeys.     

The effect of ex vivo refrigerated storage and cell preservation solution (Cyto-Chex II) on CD11b expression and oxidative burst activity of dog neutrophils

The expression of CD11b and oxidative burst activity of dog neutrophils undergoing ex vivo refrigerated storage was studied using flow-cytometry . Additionally, the effect of a proprietary cell stabilization reagent (Cyto-Chex) on the expression of CD11b and oxidative burst activity was studied. Expression of CD11b was very high (>90% positive) on dog neutr ophils isolated from peripheral blood. Dog neutrophils showed a rapid and sustained increase in CD11b antigen density (P<0.01) during refrigerated storage, this increase was prevented by treatment with Cyto-Chex but was not completely blocked on the first day. There were no significant differences in mean antigen density between any days in the non-preserved group or between Days 1 to 4 in the Cyto-Chex treated group. The non-treated group showed significantly greater mean antigen density at all time points when compared to the preservative treated group (P<0.0001). Treatment with Cyto-Chex did not interfere with measurement of oxidative burst function on the first 2 days. Alterations of both resting oxidative activity and stimulated response were observed over time in both treated and untreated blood samples. Cyto-Chex treated samples showed a dramatic, significant decline in stimulated response after the third day of storage (P<0.001), while non-treated cells showed steadily increasing, but non-significant differences in stimulated response. Cyto-Chex was demonstrated to be a useful reagent for stabilization of dog neutrophil membrane antigens during storage, however this reagent is not recommended for preservation of cells for functional assays.     

Antibacterial activity of bovine lactoferrin hydrolysate against mastitis pathogens and its effect on superoxide production of bovine neutrophils

Antibacterial activity of bovine lactoferrin hydrolysates (LFH) on microorganisms isolated from bovine mastitis, and superoxide (O(2)(-)) production of bovine neutrophils were evaluated. Antibacterial effects of LFH were measured in vitro against Staphylococcus aureus, coagulase-negative staphylococci, Streptococci, Enterococci, Escherichia coli, Klebsiella pneumoniae, yeast-like fungi and Prototheca zopfii isolated from clinical cases of bovine mastitis. To compare susceptibilities against LFH, minimal inhibitory concentration (MIC) values were determined by a micro-plate assay method. Most organisms were sensitive to LFH. Prototheca zopfii was highly sensitive to LFH; the growth of the microorganism was inhibited completely even at 1 mug/ml. Staphylococcus aureus and Escherichia coli were resistant to LFH. The production of O(2)(-) by bovine neutrophils was used to evaluate the effect of LFH administration on functional activity. Increase in O(2)(-) production by bovine neutrophils occurred upon addition of LFH to neutrophils. These results demonstrate that LFH possesses antibacterial activity against pathogens that cause mastitis and activates neutrophil superoxide production.     

Flow cytometric analysis of feline reticulocytes.

Hemolytic anemia was induced in five Domestic Shorthair cats (four adult males and one spayed female obtained from a breeding colony at Colorado State University, CO), and blood samples were analyzed from five other cats (two castrated male Domestic Shorthairs, one castrated male Domestic Longhair, one castrated male Persian, and one spayed female Siamese presented to the Veterinary Teaching Hospital at Colorado State University for miscellaneous problems). Blood samples taken from these cats had percentages of aggregate reticulocytes that ranged from 0% to 14.5% as determined by manual counting and were used to identify the best technique for staining cat reticulocytes for flow cytometric analysis. The best technique was mixing a blood sample (1/2,000 dilution) with 0.2 micrograms thiazole orange in 1 ml of diluent and incubating the mixture in the dark at room temperature for 30 to 60 minutes. The percentage of reticulocytes determined by flow cytometry correlated well (r = 0.88) with manually determined aggregate reticulocyte percentages; no significant differences were observed between the two techniques (P > 0.05). For the conditions used, punctate reticulocytes were not detected by flow cytometry. Samples with very high platelet numbers and very low packed cell volumes may show falsely elevated percentages of reticulocytes as determined by flow cytometry. The reproducibility of the flow cytometric technique was good; the coefficient of variation ranged from 4.8% to 17.9% in two samples with two different times of incubation. Staining of cat aggregate reticulocytes with thiazole orange and use of flow cytometric quantification is a reproducible technique that has a good correlation with the manual reticulocyte counting method.     

Flow cytometric analysis of T-lymphocyte subsets in cats.

We report a rapid, reliable method for the immunophenotype analysis of feline lymphocytes. Fluorescein isothiocyanate (FITC) conjugated to murine monoclonal antibodies f43, Fel 7 and fCD8 was used to identify phenotypes corresponding to feline T-cells, CD4+ T cells and CD8+ T cells. For isolation of white blood cells, whole blood lysis was faster, less variable and required much less sample than density gradient separation. To identify feline CD4+ and CD8+ cells simultaneously, directly conjugated FITC-fCD8 and phycoerythrin (PE) fCD4 (Fel 7) were used in two-color analysis. The two T cell sub-populations were non-overlapping. Dual-label and single-label values were not significantly different. Mean lymphocyte subset percentages in conventional and specific-pathogen-free (SPF) cats did not differ significantly. These values were: pan T lymphocytes (f43), 54.8%, CD4+ cells (Fel 7), 33.9%, and CD8+ cells (fCD8), 19.1%. Mean CD4/CD8 ratio was 1.9 in normal cats; the range was 1.2-2.6.     

Combined phagocytosis-nitroblue tetrazolium reduction test in bovine neutrophils

An assay was developed for the simultaneous evaluation of phagocytosis and oxidative metabolism of bovine blood neutrophils. Phagocytosis was evaluated by using opsonized zymosan, and oxidative metabolism was evaluated by nitroblue tetrazolium (NBT) reduction. Normal bovine neutrophils exhibited moderate variation in ability to phagocytize zymosan, but little variation in ability to reduce NBT. The subcellular location of NBT reduction to formazan was determined by electron microscopy. Electron dense formazan precipitate was observed along the inner membrane of phagosomes enclosing zymosan particles and radiating from the membrane toward the center of the phagosome.     

Flow cytometric testing of immunological effects of a phytomedicinal combination (equimun) and its compounds on bovine leucocytes

One of the major goals of this study was to establish fast, reliable and sensitive assays for the quality control of immunomodulating phytopreparations and to determine whether pharmacological compounds or phytopreparations have effects on bovine immune cells. Flow cytometric methods were chosen because they are very sensitive in the detection of even subtle effects on cells. In this study, we addressed the question of whether these methods are useful in monitoring the effects of EquiMun and its compounds on bovine leucocytes in vitro. EquiMun is a fixed combination of Echinacea purpurea (Ec), Thuja occidentalis (Th) and elemental phosphorus (Ph) in different starting concentrations. Separated blood mononuclear cells (MNC) and polymorphonuclear cells (PMN, mainly neutrophils) were cultured for up to 44 h in vitro in the presence or absence of the tested substances. Whereas MNC were not affected by any of the compounds, EquiMun, Ec, Th and Ph significantly reduced the forward scatter (size) of cultured PMN without affecting their side scatter (granularity). The size effects were paralleled by a significantly enhanced viability of PMN after 20 h in culture. The observed effects were constant over wide concentration ranges and indicate a very similar reaction of leucocytes from individual cows. Whereas spontaneous generation of reactive oxygen species (ROS) by neutrophils was up-regulated by Ph and EquiMun, EquiMun down-regulated the phorbol ester-stimulated ROS production. However, ROS generation by neutrophils displayed a large inter-individual variation with less apparent, down-regulatory effects of EquiMun. The ability of PMN to kill target cells via antibody-independent cellular cytotoxicity showed small inter-individual variations and was enhanced by Ec and Th but not by Ph and EquiMun, probably due to dose-dependent effects. In summary, the flow cytometric characterization of cellular viability and shape changes of neutrophils seem to be a suitable and reliable approach for the quality test of immunomodulating phytomedicines based on bioassays.     

Flow cytometric analysis of chicken NK activity and its use on the effect of restraint stress

Immune system is organized by the influence of both neural and endocrine systems. NK activity plays an important role in the innate immunity. In this study, we observed the effects of restraint stress on chicken peripheral blood NK activity. Viability of FITC-labeled RP9 was measured with PI after treatment with the effector cells. Chicken peripheral blood CD8alpha+ cells expressed strong cytotoxic activity, in contrast to thrombocytes, while peripheral blood CD3+ CD8alpha+ cells and CD4+ cells had little cytotoxic activity. Con A supernatant enhanced the cytotoxic activity of CD8alpha+ cells. Therefore, it is considered that these cytotoxic activities measured by flow cytometry (FCM) analysis are NK activity. When chickens were exposed to restraint stress, the levels of serum corticosterone increased transiently over a short period of time while the NK activity decreased. The decreased NK activity, however, did not recover to the intact levels for a long time, even once the serum corticosterone levels had recovered. These data indicate that chicken NK activity is able to be measured by flow cytometric analysis and that restraint stress causes severe damage to the chicken NK activity.     

Activation-induced mobilization of secretory vesicles in bovine neutrophils.

OBJECTIVE: To characterize mobilization of secretory granules in bovine neutrophils. SAMPLE POPULATION: Neutrophils obtained from four 6- to 18-month-old Holstein cattle. PROCEDURE: Mobilization of secretory granules in bovine neutrophils was determined by measuring changes in cell-surface alkaline phosphatase activity on cells treated with various inflammatory mediators. Subcellular distribution of the alkaline phosphatase activity was determined by analysis of bovine neutrophil homogenates fractionated on density gradients. RESULTS: Alkaline phosphatase-containing secretory granules of bovine neutrophils were readily mobilized by a number of inflammatory agents, including platelet-activating factor, interleukin-8, tumor necrosis factor-alpha, lipopolysaccharide, leukotriene B4, and zymosan-activated plasma. In contrast, N-formyl-methionyl-leucyl-phenylalanine did not have a significant effect. Phorbol myristate acetate induced a biphasic response with up-regulation of cell-surface alkaline phosphatase at low doses and a return to baseline or even a reduction in cell-surface alkaline phosphatase at higher doses (> or = 10 ng/ml). Subcellular fractionation of bovine neutrophil homogenates revealed that alkaline phosphatase activity resided in light-density membrane vesicles (ie, location of secretory granules), which were distinct from specific, azurophil, and large granules. CONCLUSIONS AND CLINICAL RELEVANCE: Bovine neutrophils respond to various inflammatory mediators by mobilizing alkaline phosphatase-containing secretory granules. This suggests that the process is an important early step in the host-defense response of bovine neutrophils.