The chemiluminescent response of bovine polymorphonuclear leucocytes isolated from milk and blood

Polymorphonuclear leucocytes (PMN) were isolated from milk and blood of healthy cows, and the generation of reactive oxygen by the two cell populations was compared by measuring chemiluminescence (CL) after stimulation with zymosan. The ratio of milk to blood PMN CL was relatively constant in a given animal, but varied widely between different cows, ranging from 0.3 to 1.3.The relative contributions of various oxygen species to CL was studied by measuring quenching using different oxygen scavengers. While the relative contributions of H₂O₂, ^?O₂ and ′O₂ seemed to be similar in both milk and blood PMN, the OH· radical was clearly more prominent in PMN isolated from milk than from blood. In addition, blood PMN CL was more dependent on the presence of glucose in the reaction medium than milk PMN CL. Furthermore, the CL response to phorbol myristate acetate, to the Ca ionophore A23187 and to Sendai virus was different in the two cell types. The results suggest that CL generation in milk PMN differs from that in blood PMN in quantitative as well as qualitative aspects.     

A rapid microassay for measuring the luminol-dependent chemiluminescent response in canine whole blood

A microassay for the luminol-dependent chemiluminescence (CL) response in canine whole blood was developed to measure indirectly the oxidative metabolism of peripheral blood leukocytes. Fifty microliters of blood were mixed with 705 microliters of Hank's balanced salt solution containing 25 mM Hepes and 1.3 x 10(-4) M luminol. This mixture was allowed to equilibrate for 5 min after which 60 microliters of latex beads (0.801 microns diameter) were added as a stimulant, and the CL response was monitored continuously for 5 min at 37 degrees C using a luminometer. The whole blood CL response was significantly correlated (r = 0.784, P less than 0.01, n = 14) with the number of neutrophils in the peripheral blood. Further, the whole blood CL response was abolished by the depletion of neutrophils after passing the blood through an adherence column and by the addition of sodium azide. The relative chemiluminescent light unit (RCLU) was a reliable marker for comparing each peak value in different samples. The coefficient of variation (CV) of repetitive samples was 9.87%, and the CV of 14 normal dogs was 15.7%. This method is useful and applicable for screening the CL response in canine whole blood.     

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.     

Optimization of conditions for in vitro production of radical oxygen species and expression of tissue factor by canine mononuclear cells and granulocytes for use in high-throughput assays

The purpose of this study was to optimize conditions for high throughput measurement of radical oxygen species (ROS) production and expression of tissue factor, also termed procoagulant activity, by canine leukocytes. Granulocytes and mononuclear cells were separated by density gradient centrifugation from peripheral blood collected on several occasions from three healthy large breed dogs. To determine optimal conditions for ROS production, granulocytes were incubated for 1 or 3h in PBG (PBS containing 0.5% BSA and 5mM glucose) or RPMI containing 10% fetal bovine serum (FBS); lipopolysaccharide (LPS), zymosan, peptidoglycan (PGN) and phorbol myristate acetate (PMA) were used as stimuli. ROS was assessed by conversion of the nonfluorescent dye dihydrorhodamine 123 to fluorescent rhodamine 123 by radical species released into the media. To identify optimal conditions for expression of tissue factor, mononuclear cells were incubated for 5h in RPMI containing different concentrations of heat-inactivated FBS (HI-FBS), and LPS, zymosan, PGN or PMA as stimuli. Expression of tissue factor was determined using a one-stage recalcification assay performed in an automated nephelometric coagulation analyzer. Neither LPS nor zymosan increased ROS production by granulocytes incubated in PBG media. In contrast, granulocytes incubated in RPMI had dose-dependent increases in ROS production in response to zymosan and PGN. ROS production was significantly increased by incubation with concentrations of LPS of 0.01microg/ml or greater, and by zymosan concentrations of 0.1microg/ml or greater. ROS production in response to incubation with PMA was significantly increased starting at 10(-7)M, and was significantly greater for cells incubated in RPMI than cells incubated in PBG. LPS-, zymosan- and PGN-stimulated procoagulant activity increased in a dose-dependent manner, whereas PMA-stimulated procoagulant activity peaked at 10(-7)M. Increasing concentrations of HI-FBS significantly increased LPS-, zymosan- and PGN-induced procoagulant activity of mononuclear cells. Results obtained in this study indicate production of ROS by canine granulocytes is optimal when these cells are incubated for 3h in RPMI with LPS (0.1microg/ml), zymosan (10 microg/ml), PGN (10 microg/ml), and PMA (10(-7)M). Furthermore, canine mononuclear cells express procoagulant activity in response to LPS, zymosan, PGN, and PMA, and responses to LPS, zymosan and PGN are enhanced by the addition of HI-FBS. These findings suggest that HI-FBS retains important serum proteins that facilitate interactions between each of these bacterial or yeast derived products and the mononuclear cells. Consequently, future studies regarding the regulation of procoagulant activity by canine mononuclear cells should be performed in the presence of HI-FBS. Both assays utilized in this study allow high throughput of samples, and therefore are appropriate choices for rapid screening of conditions and/or therapeutic interventions affecting the canine inflammatory system.     

Lack of Hemocyte Chemiluminescence Stimulation by Perkinsus marinus in Eastern Oysters Crassostrea virginica with Dermo Disease

Abstract

Phagocytosis of zymosan A particles by hemocytes of eastern oyster Crassostrea virginica stimulates the production of reactive oxygen species (ROS), which are easily quantified by luminol-augmented chemiluminescence (CL). Antimicrobial defense mechanisms of hemocytes may involve the activity of cytotoxic ROS. The CL response to phagocytosis of zymosan by hemocytes from C. virginica with advanced Perkinsus marinus infections is more elevated than that produced by zymosan in cells from uninfected oysters. This effect is perhaps akin to macrophage activation. Phagocytosis of P. marinus cells by hemocytes withdrawn from uninfected oysters produced no detectable CL response. Hemocytes withdrawn from oysters with P. marinus infections ranging from light to heavy were evaluated for CL responses after phagocytosis of zymosan or P. marinus. Increases in CL stimulation by zymosan were seen as the intensity of infection increased. Despite avid phagocytosis of P. marinus, CL activity of the hemocytes was not seen, regardless of the level of infection of the host. Lack of hemocytic ROS stimulation by ingestion of P. marinus cells may contribute to in vivo survival of this parasite.     

Local and systemic effects of endotoxin mastitis on the chemiluminescence of milk and blood neutrophils in dairy cows

The local and systemic effects of intramammary lipopolysaccharide (LPS) injection on the chemiluminescence (CL) of milk and blood polymorphonuclear leukocytes (PMN) were investigated in six healthy early lactation cows. Clinical signs of acute mastitis such as fever, increased heart rate and a decreased milk production were observed in all cows. Before LPS challenge, the CL activity of milk PMN was significantly lower than that of blood PMN (P < 0.01). A significant negative correlation was found between pre-challenge milk and blood PMN CL and, the decreased milk production in unchallenged quarters. The CL activity of milk PMN from LPS-injected quarters increased following LPS challenge, whereas it remained unchanged in control quarters. The CL activity of blood PMN showed a biphasic increase, with two peaks and a valley below pre-challenge CL activity (P < 0.01). At post-challenge hours (PCH) 6 and 12, the CL activity of milk PMN from LPS-injected quarters exceeded that of blood PMN (P < 0.05 and P < 0.001, respectively). The decreased CL activity of blood PMN and the enhanced CL activity of milk PMN during endotoxin-induced mastitis was reflected by changes in the shape of the CL curve. In blood PMN, a decrease of the second peak of the CL curve suggests that the myeloperoxidase (MPO)-H2O2 system is impaired during endotoxin-induced mastitis. In contrast, the MPO-H2O2 system was enhanced in milk PMN from challenged quarters. The highest duration and intensity of reactive oxygen intermediate (ROI) production was observed in milk PMN from LPS-injected quarters at PCH 12. The increased viability of PMN in LPS-injected quarters and to a lesser extent in control quarters suggests possible effects of both facilitated diapedesis and inflammatory mediators on milk PMN survival. In conclusion, our results suggest that a combination of local and systemic action of E. coli endotoxin is involved in the priming of milk PMN during mastitis.     

High milk neutrophil chemiluminescence limits the severity of bovine coliform mastitis

Polymorphonuclear neutrophil (PMN) function changes during mastitis. To investigate the contribution of milk PMN to the severity of Escherichia coli (E. coli) mastitis, chemiluminescence (CL) of blood and milk PMN and their efficiency to destroy coliform bacteria in the mammary gland were examined following the induction of E. coli mastitis in early lactating cows. To better assess and define the degree of mastitis severity, cows were classified as moderate and severe responders according to milk production loss in the non-infected quarters at post-infection hour (PIH) 48. There was an inverse relationship between pre-infection milk PMN CL and colony-forming units at PIH 6. In moderate cows, the pre-infection blood and milk PMN CL was approximately 2-fold higher than that of severe cows. The probability of severe response increased with decreasing pre-infection PMN CL. At the beginning of the infection blood and milk PMN CL was consistently higher, and milk PMN CL increased faster after infection in moderate cows. At PIH > 48 milk PMN CL in severe cows exceeded that of moderate cows. The somatic cell count (SCC) in moderate cows increased faster than colony-forming units, whereas in severe cows the results were reversed. The kinetics of CL activity for blood and milk PMN before and during the early phase of infection confirmed an impairment in PMN CL activity for severe responding cows. High pre-infection blood and milk PMN CL and the immediate increase of milk PMN CL and SCC after infection limited bacterial growth thereby facilitating the recovery of E. coli mastitis in moderate cows. Our study strengthens the idea that pre-existing milk PMN (a static part of the udder's immune defense) functions as a "cellular antibiotic" before and during infection, and low milk PMN CL is a risk factor for bovine coliform mastitis.     

Relationship between Ultrasonographic Assessment of the Corpus Luteum and Plasma Progesterone Concentration during the Oestrous Cycle in Monovular Ewes

Ultrasonographic observations of the corpus luteum (CL) and collection of blood samples for progesterone radioimmunoassay were performed daily during 15 oestrous cycles in Spanish Merino ewes, a consistently monovular breed. Ultrasonographic image of the CL changed during the oestrous cycle, increasing its echogenic pattern from ovulation to luteolysis. The size of the CL and mean progesterone levels were significantly affected by day of cycle (p < 0.05 and p < 0.001, respectively). Both increased their values from day 1 to day 12 (from 49.6 ± 7.4 to 154.6 ± 11.8 mm² and from 0.2 ± 0.0 to 2.8 ± 0.5 ng/ml, respectively) and then declined sharply until day 0 (28.2 ± 5.3 mm² and 0.1 ± 0.0 ng/ml, respectively). There was a significant correlation between CL area and plasma progesterone concentrations during the entire oestrous cycle, taking the developing and regressing phases of the CL separately (p < 0.05). A central cavity was observed in 33.3% of the CL studied. The presence of this cavity had no effect in total luteal‐tissue area of the CL nor on oestrous cycle length or on progesterone concentrations. Likewise, the cavity did not affect the correlations observed between CL size and progesterone levels, CL size and day of cycle and progesterone levels and day of cycle. It is concluded that ultrasonographic assessment of CL area is a reliable method for estimating peripheral plasma progesterone levels, regardless to the presence or absence of a cavity in the CL.     

Effects of extracellular lactate on production of reactive oxygen species by equine polymorphonuclear leukocytes in vitro

Objective-To evaluate effects of extracellular lactate on viability, shape change, lactate metabolism, and reactive oxygen species (ROS) production in equine polymorphonuclear leukocytes (PMNs). Sample-PMNs isolated from equine venous blood samples. Procedures-PMNs were incubated with 0 to 300mM lactate for 30 minutes before each experiment. Viability was assessed via trypan blue exclusion. Shape change was assessed via flow cytometry and light microscopy. Relative quantification of monocarboxylic acid transporter and lactate dehydrogenase lactate dehydrogenase (LDH) isotype mRNAs was performed with a real-time PCR assay. Effects of lactate at a pH of 7.4 to 6.0 on ROS production in response to phorbol 12-myristate 13-acetate, opsonized zymosan, or N-formyl-methionyl-leucyl-phenylalanine was assessed by luminol-dependent chemiluminescence. Results-Lactate had no effect on viability of PMNs but did alter their size and density. Monocarboxylic acid transporter 1 and lactate dehydrogenase B mRNA values were not altered. Monocarboxylic acid transporter 4 and lactate dehydrogenase A mRNA values were significantly decreased. Lactate incubation of cells significantly decreased PMN-derived luminol-dependent chemiluminescence and induced different sensitivities to stimulants (phorbol 12-myristate 13-acetate, opsonized zymosan, and N-formyl-methionyl-leucyl-phenylalanine). The response ratio to N-formyl-methionyl-leucyl-phenylalanine revealed that PMNs were primed by incubation with up to 50mM lactate, significantly increasing the production of ROS. Incubation with lactate and acidic pH caused a synergistic effect on ROS production. Conclusions and Clinical Relevance-Extracellular lactate potentially has a direct effect on the capacity to produce ROS by equine PMNs, which may be associated with alterations in innate immune functions within a short period after high-intensity exercise.     

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.     

Effects of ovariohysterectomy on canine blood neutrophil respiratory burst: a chemiluminescence study

OBJECTIVE: To examine blood neutrophil counts and luminol-enhanced chemiluminescence (CL) responses in dogs undergoing ovariohysterectomy (OH), premedicated with 2 different drugs. STUDY DESIGN: Randomized clinical study. ANIMALS: Forty-two healthy client-owned bitches. METHODS: Dogs had OH under isoflurane anesthesia with either acepromazine or medetomidine, both in combination with butorphanol, administered as preanesthetic medication. Blood samples were collected when the dog was admitted, at the end of surgery, and the next day (approximately 20 hours after surgery). Blood neutrophils were counted automatically, and neutrophil oxidative activity was assessed by measuring blood CL responses (induced by opsonized zymosan and enhanced by luminol) at 37 degrees C for 40 minutes. RESULTS: Number of circulating neutrophils was significantly increased the day after surgery reflected by enhanced blood CL responses. Neutrophil CL, however, was not significantly altered. No significant differences were detected for perioperative Polymorphonuclear neutrophil (PMN) characteristics between the 2 preanesthetic regimens. CONCLUSIONS: In conclusion, despite clearly increasing the number of circulating neutrophils, OH did not significantly affect neutrophil respiratory burst, as measured by whole-blood CL responses. CLINICAL RELEVANCE: Surgical operation of moderate intensity (e.g., OH) did not significantly alter one of the important immune functions, neutrophil oxidative activity. Further studies are warranted to confirm the significance of this finding, and to assess the value of following this variable in different animal patient populations.     

Effect of fetal bovine serum and heat-inactivated fetal bovine serum on microbial cell wall-induced expression of procoagulant activity by equine and canine mononuclear cells in vitro

OBJECTIVE: To determine the effect of fetal bovine serum (FBS) and heat-inactivated FBS (HI-FBS) on lipopolysaccharide (LPS)- and zymosan-induced procoagulant activity of equine and canine mononuclear cells. SAMPLE POPULATION: Mononuclear cells from 18 horses and 3 dogs. PROCEDURES: Cells were incubated with various concentrations of FBS, HI-FBS, LPS, zymosan, polymyxin B, and anti-LPS-binding protein monoclonal antibody or combinations of these constituents. A 1 stage recalcification assay was used to determine procoagulant activity. RESULTS: Addition of FBS to media significantly increased procoagulant activity; equine and canine cells were stimulated by 1% and 10% FBS, respectively. Coincubation of cells with FBS and polymyxin B did not reduce this effect, suggesting that the response was not attributable to LPS contamination. Addition of HI-FBS to media did not stimulate procoagulant activity of equine or canine cells, and the sensitivity of the equine cells to LPS was significantly increased by HI-FBS. This increased LPS sensitivity was reduced 40% with monoclonal antibody directed against human recombinant LPS-binding protein. Increasing concentrations of HIFBS significantly increased LPS- and zymosan-induced procoagulant activity of canine cells. CONCLUSION AND CLINICAL RELEVANCE: Procoagulant activity production in equine and canine mononuclear cells was significantly increased by addition of FBS, whereas heat inactivation of FBS eliminated this effect. Heat inactivation did not eliminate the function of serum proteins involved in enhancement of LPS and zymosan-induced procoagulant activity. Results suggest that HI-FBS can be used as a source of serum proteins that increase the sensitivity of mononuclear cells to bacterial and yeast cell wall components.     

Chemiluminescence and chemotaxis assay of canine granulocytes: a methodological study

Chemiluminescence (CL) of isolated granulocytes and of whole blood from dogs was evaluated. Chemiluminescence of whole blood samples created an undesired quenching effect by the red blood cells which makes the assay difficult to apply in pathological cases with low formation of oxygen metabolites. This problem was avoided when chemiluminescence was determined, using isolated granulocytes. A cell concentration of 5 x 10(9)/l was needed to create optimal conditions. The Boyden chamber technique was used for study of random migration and chemotaxis. Casein (0.1%), zymosan activated serum with and without epsilon-amino-n-caproic-acid and homologous serum were effective chemoattractants for canine granulocytes, while FMLP (formyl-methionyl-leucyl-phenylalanin) did not attract canine granulocytes.     

Determination of the oxidative burst chemiluminescent response of avian and murine-derived macrophages versus corresponding cell lines in relation to stimulation with Salmonella serotypes

In contrast to mammalian systems, avian species lack a resident or harvestable macrophage population in the abdominal exudate. Peritoneal macrophages in the chicken can be elicited if an inflammatory agent such as sephadex is injected. This study examines the kinetics of different macrophage populations, derived by different methods of isolation and from different hosts, with respect to the elicited oxidative burst upon infection with host-adapted Salmonella serotypes.The nature of the oxidative burst elicited by murine and avian-derived and cell line macrophages was determined after stimulation with phorbol myristate (PMA), zymosan A, and Salmonella serotypes. Both murine and chicken peritoneal macrophages, chicken blood monocytes and corresponding cell lines, J774A.1 and HD-11, were unable to produce a detectable chemiluminescent (CL) response after interaction with Salmonella using the luminescent probe luminol. However, both PMA and zymosan A induced a CL response in all cell types, with PMA eliciting a higher and earlier peak response (pkH) than zymosan A. Lucigenin-enhanced CL in both murine and chicken macrophages was achieved with PMA, zymosan A and Salmonella serotypes. In this case, zymosan A induced higher responses than PMA. In the peritoneal macrophages of both hosts, there were no significant differences in the oxidative burst induced by the different Salmonella serotypes. However, the J774A.1 (murine) cells demonstrated significant differences, with S. enterica serotype Choleraesuis (S. choleraesuis and S. gallinarum producing the highest response. In the HD-11 (chicken) cells, S. choleraesuis and S. dublin elicited the higher CL. With both cell lines, S. abortusovis failed to induce an appreciable CL response.In these experiments it was demonstrated that oxidative burst was not detectable in monocytes/macrophage populations using luminol, which suggests a link to the lack of a myeloperoxidase system in these cells. Lucigenin-enhanced CL appeared independent from the myeloperoxidase system, indicating production of another oxidative species compared with luminol. No discernable effect of host specificity with regard to Salmonella serotype and respective host was seen in host-derived or cell line macrophages, and cell line macrophages displayed altered functional characteristics with regard to oxidative burst in comparison with their primary counterparts.     

Generation and functional characterization of ovine bone marrow-derived macrophages.

A method for the culturing and propagation of ovine bone marrow-derived macrophages (BMM) in vitro is described. Bone marrow cells from sterna of freshly slaughtered sheep were cultured in hydrophobic (teflon foil) bags in the presence of high serum concentrations (20% autologous serum and 20% fetal calf serum). During an 18 day culture period in the absence of added conditioned medium, and without medium change, a strong enrichment of mononuclear phagocytes was achieved. Whereas the number of macrophages increased four to fivefold during this time, granulocytes, lymphoid cells, stem cells and undifferentiated progenitor cells were reduced to less than 3% of their numbers at Day 0. This resulted in BMM populations of 94 +/- 3% purity. These cells had morphological and histochemical characteristics of differentiated macrophages, and they performed functions similar to those of non-activated, unprimed human monocyte-derived macrophages. Thus, they avidly ingested erythrocytes coated with IgG of heterologous or homologous origin. They expressed a modest level of procoagulant activity, but upon triggering with lipopolysaccharide (LPS), a marked increase in cell-associated procoagulant activity was observed. LPS triggering promoted the secretion of interleukin-1, as evidenced by measurement of murine thymocyte costimulatory activity, and transforming growth factor-beta. Using the mouse L929 cell cytotoxicity assay as an indication of tumor necrosis factor (TNF) activity, no TNF activity was detected in the same supernatants, a result possibly due to species restriction. BMM generated low levels of O2- upon triggering with phorbol 12-myristate 13-acetate (PMA). On the other hand, no O2- production was observed upon stimulation with zymosan opsonized with ovine or human serum. Using luminol-enhanced chemiluminescence (CL) as a more sensitive indicator of an oxidative burst, both PMA or zymosan were able to trigger CL, but the response was subject to partial inhibition by sodium azide, an inhibitor of myeloperoxidase. This points to non-macrophage cells contributing also to the CL response, and is consistent with the view that unprimed BMM elicit a low oxidative burst upon triggering with strong inducers of a burst. Our functional characterization now allows us to apply priming and activation protocols and to relate their effect to functional alterations.     

山羊外周全血中性粒细胞化学发光检测条件

应用全血细胞化学发光（ＣＬ）技术，测定了山羊外周全血中性粒细胞（ＰＭＮ）活性氧自由基（ＯＦＲ）的化学发光，对试验要素包括鲁米诺浓度、酵母多糖浓度、测定时间，以及化学发光测定仪的额定电压、工作温度作了试验筛选，比较了其对山羊ＰＭＮ－ＣＬ的发光本底、峰值及峰时变化的影响。结果表明，测定山羊全血ＰＭＮ－ＣＬ的最优条件：鲁米诺浓度为２×１０－３ｍｏｌ／Ｌ，酵母多糖浓度为５ｇ／Ｌ，测量时间为２ｓ；ＳＨＧ－Ⅰ型生物化学发光测定仪额定电压及工作温度设置分别为４．０Ｖ和（３３±１．５）℃。     

Granulocyte function in dogs experimentally infected with a Swedish granulocytic Ehrlichia species

Granulocyte function was studied in six dogs inoculated with a Swedish granulocytic Ehrlichia species and in four control dogs. Whole blood chemiluminescence (CL) was enhanced in the dogs with granulocytic ehrlichiosis. Both CL after stimulation with zymosan and spontaneous CL was significantly increased at peak of infection compared with pre-infection levels. Ingestion of FITC-labelled serum-opsonized yeast cells was high and stable in both groups. The ingestion was lower when the yeast cells were opsonized with anti-yeast IgG. However, there was no difference between groups. The labelling intensity of anti-human CD11b, CD18 and CD32 mAb on the granulocytes in dogs with ehrlichiosis was similar to that in control dogs. The opsonic activity in serum collected at the peak of infection was not different from serum drawn prior to inoculation. Opsonic activity was investigated both by yeast cell ingestion and by chemiluminescence after stimulation with zymosan. The serum from infected dogs enhanced the respiratory burst without stimulation with zymosan of leukocytes from healthy dogs. This suggests that serum at the peak of infection contains granulocyte activators. In this study we found normal phagocytosis together with evidence of enhanced oxidative metabolism in the granulocytes from dogs with granulocytic ehrlichiosis.     

Variations among unbred heifers in the activities of polymorphonuclear leucocytes from the mammary gland and blood

The phenotypic characteristics are described for the activity of polymorphonuclear leucocytes NMN) obtained by either lavage of the cavity system of juvenile mammary glands stimulated with a synthetic muramyl dipeptide analogue or isolation from the peripheral blood. Attention was paid to the variability of characteristics and its sources, and to correlations among them. The following characteristics were investigated in 27 clinically healthy, unbred Bohemian Red Pied x Holstein heifers: migration activity in situ, number of phagocytosing PMN, phagocytotic index, bactericidal activity of PMN and unstimulated and zymosan-stimulated luminol-dependent chemiluminescence. Considerable individual variation was found in the characteristics. Significant differences between blood PMN and PMN from lavages after influx induction were found for bactericidal activity (P < 0.05) and chemiluminescence (P < 0.01). A significant correlation between blood PMN and mammary gland PMN was found only for the number of phagocytosing cells (r = 0.329; P < 0.01). Highly significant positive correlations (P < 0.01) were demonstrated between the number of phagocytosing PMN [a], phagocytotic index [b], and bactericidal activity [c] in both blood PMN (r(ab) = 0.602; r(ac) = 0.565; r(bc) = 0.529) and mammary gland PMN (r(ab) = 0.730, r(ac) = 0.618, r(bc) = 0.589). No significant correlation was demonstrated for non-stimulated (NS), zymosan-stimulated (ZS), or opsonized zymosan-stimulated (OZS) chemiluminescence with any of the other characteristics of phagocytotic activity, in either blood PMN or mammary gland PMN (P > 0.05). The animal was a highly significant source of variability for all the phagocytotic activity characteristics (P < 0.01). Udder quarter was a non-significant source of variability for all the characteristics of phagocytotic activity except for NS chemiluminescence (P < 0.05) and ZS or OZS chemiluminescence (P < 0.01). However, udder quarter was a non-significant source of variability of chemiluminescence indices ZS/NS and OZS/NS (P > 0.05). It has been demonstrated that in situ migration activity, the number of phagocytosing PMN, phagocytotic index, bactericidal activity of PMN and chemiluminescence indices of PMN collected from juvenile mammary glands of unbred heifers after influx induction can be regarded as candidate early markers of resistance to mammary infections.     

Effect of mammary secretions on functions of polymorphonuclear leukocytes in pigs

OBJECTIVE: To determine the effects of porcine mammary secretions on polymorphonuclear (PMN) leukocyte function and to relate concentrations of estradiol-17beta and cortisol in mammary secretions to PMN cell function. SAMPLE POPULATION: Mammary secretions from 10 healthy sows and blood PMN leukocytes from 27 healthy sows. PROCEDURE: Mammary secretions were collected within 24 hours after parturition (colostrum) and 12 to 13 days later (milk). Chemoattractant properties were assessed by use of a cell migration assay. Phagocytic capacity of PMN cells in colostrum and milk was assessed by recording chemiluminescence following phagocytosis of Escherichia coli or zymosan. Estradiol-17beta and cortisol concentrations were determined by use of radioimmunoassays. RESULTS: Chemoattractant properties of colostrum and milk were significantly greater than that of zymosan-activated serum. However, chemoattractant properties did not differ significantly between the 2 types of secretions. The capacity of PMN cells in colostrum to phagocytose either zymosan or E. coli was less, compared with cells in milk, and the ability of cells in either type of mammary secretion to phagocytose E. coli was greater than the ability to phagocytose zymosan. Concentrations of estradiol-17beta and cortisol were greater in colostrum, compared with milk. No clear relation was evident between PMN cell activity and hormone concentrations in mammary secretions. CONCLUSIONS AND CLINICAL RELEVANCE: Although chemoattractant properties of colostrum and milk did not differ, the phagocytic capacity of PMN cells in colostrum was significantly less than that of cells in milk. This may predispose sows to coliform mastitis during the early postparturient period.     

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.