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1.
Flow cytometric study of oxidative burst activity in bovine neutrophils   总被引：2，自引：0，他引：2  
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)  相似文献   

2.
A procedure to measure phagocytosis by blood and milk neutrophils was developed. One milliliter of heat-killed 32P-labeled Staphylococcus aureus ([32P]SA) (180-200 X 10(6) CFU), 1 ml of phosphate-buffered saline solution (PBSS), and 2 ml of serum, whole milk, skimmed milk, whey, or PBSS were incubated in duplicate for 60 minutes at 37 C. Isolated blood or milk nuetrophils (polymorphonuclear leukocytes (PMN), 25 X 10(6) cells/ml; 1 ml) were added and incubated at 37 C for 30 minutes. Unphagocytosed [32P]SA organisms were lysed by incubation with 5 ml of lysostaphin (10 U) at 37 C for 30 minutes, and the PMN and phagocytosed T2P]SA were removed by centrifugation. Radioactivity of the supernatant was determined in a scintillation spectrometer and was used in estimate the percentage of [32P]SA phogocytosed. With this procedure, 25 assays in duplicate could be conducted each day with an expected coefficient of variation between duplicates of 5.6%. Blood PMN phagocytosed 80, 44, 74, 72, and 11% of the [32P]SA when incubated in serum, whole milk, skimmed milk, whey, and PBSS, respectively. Mik PMN phagocytosed 78, 44, 72, 74, and 22%, respectively. The addition of cream to either skimmed milk or serum reduced phagocytosis of [32P]SA by both blood and milk PMN. The inhibitory effect of cream was verified by the microscopic observation that PMN containing large quantities of ingested fat contained fewer S aureus. Seemingly, PMN upon entering the alveoli of the mammary gland become less efficiently phagocytic for bacteria, because of the presence of milk fat globules. This phef intramammary infection by invading mastitic pathogens.  相似文献   

3.
A procedure for the measurement of phagocytosis by bovine polymorphonuclear leukocytes (PMN) of 32P-labeled Staphylococcus aureus was modified so that a larger number of samples could be compared in a single run, and smaller volumes of sample, PMN, and 32P-labeled S aureus could be used. Results were highly reproducible, with a coefficient of variation between duplicate determinations of less than or equal to 2%. Lysostaphin was prepared from the supernatant of S staphylolyticus and was compared with a commercially available preparation. Effects of lysostaphin on PMN and influence of incubation media on release of 32P from 32P-labeled S aureus by lysostaphin were examined.  相似文献   

4.
Sixty-three drugs, belonging to 10 chemical classes, were tested in vitro to determine effects on phagocytosis of 32P-labeled Staphylococcus aureus by neutrophils isolated from milk. Within each class, the number of antibiotics tested were: nonsteroidal anti-inflammatory drugs (NSAID; 8), peptolids (2), aminoglycosides (8), tetracyclines and fusidic acid (4), beta-lactam antibiotics (25), secretolytic agents (2), macrolides (5), polypeptides (2), and antibacterial quinolones (8). Percentage of phagocytosis was determined after incubating (2 hours at 37 C) 12.5 x 10(6) viable neutrophils, 200 x 10(6) 32P-labeled S aureus with antibiotics and 5% skimmed milk. Concentrations of antibiotics tested were 1,000, 500, and 10 micrograms/ml of incubation media. When compared with nonantibiotic controls at the highest drug concentration, the NSAID acetylsalicylic acid and centrophenoxine increased phagocytosis 23.2 and 8.8%, respectively, and benzydamine, indomethacin, phenylbutazone, ibuprofen, and acetominophen decreased phagocytosis 22.8, 14.2, 9.8, 27.0, and 18.2%, respectively. The peptolids novobiocin and pristinamycin decreased phagocytosis 24.5 and 22.0%, respectively. The aminoglycosides tobramycin, amikacin, and gentamicin decreased phagocytosis 21.1, 15.4, and 19.2%, respectively. For the tetracyclines and fusidic acid, minocycline and doxycycline decreased phagocytosis 39.8 and 54.2%, respectively. The beta-lactam antibiotics carfecillin, cephapirin sodium, and cephacetrile sodium decreased phagocytosis 11.2, 12.8, and 23.8%, respectively. The secretolytic agent, bromhexin, increased phagocytosis 10.8%. These data indicate that the potential for enhanced phagocytosis exists through use of some NSAID, and for depressed phagocytosis through use of aminoglycosides, peptolids, tetracyclines, and beta-lactams, as well as certain other NSAID.  相似文献   

5.
Neutrophils were isolated from the blood and mammary gland of 3 multiparous lactating cows and 3 nulliparous heifers. Neutrophil function was evaluated by phagocytosis and luminol-dependent chemiluminescence. Peroxidase activity was detected by use of transmission electron microscopy. Compared with that for blood neutrophils, percentage of phagocytosis was 9.6% lower for neutrophils isolated from the mammary gland of lactating cows, but this difference was not observed between neutrophils isolated from the mammary gland and from the blood heifers. Similarly, after subtraction of chemiluminescence values in the absence of zymosan, phagocytosing neutrophils from the mammary gland of lactating cows had lower chemiluminescence than did those from the blood of such cows. For heifers, however, chemiluminescent activity by phagocytosing neutrophils obtained from the mammary gland was similar to that of blood neutrophils. Chemiluminescent activity of resting neutrophils from the mammary gland of lactating cows pretreated with cytochalasin B was not inhibited, compared with that of nontreated resting neutrophils (controls). This was attributed to xanthine oxidase activity. Transmission electron microscopy of mammary gland neutrophils from lactating cows revealed peroxidase-positive material associated with milk-fat globule membranes and with phagosomes containing zymosan. Results indicated that ingestion of fat and casein by neutrophils isolated from milk caused a decrease in phagocytic and chemiluminescent activity. Also, luminol-dependent chemiluminescence was not a reliable measure of milk neutrophil function, because of interference by xanthine oxidase.  相似文献   

6.
To examine the effect of parity on polymorphonuclear neutrophils (PMN) function, phagocytic and bactericidal activity of the PMN isolated from blood and milk against Staphylococcus aureus was compared between groups of 6 primiparous and 6 multiparous healthy dairy cows during early lactation using bacteriological and PMN-pathogen interaction assays. Latex-stimulated luminol-amplified chemiluminescence (CL) and viability of these PMN were also investigated. The phagocytosis and killing of S. aureus by blood were remarkably higher than those of milk PMN. Similarly, the CL and viability in blood PMN were markedly higher than in milk PMN. Both in blood and in milk the phagocytosis of S. aureus by PMN in primiparous cows was substantially higher than in multiparous cows. The killing activity of blood PMN against S. aureus was 42.3+/-3.4% and 23.2+/-1.7% in primiparous and multiparous, respectively. Milk PMN killed only 20.7+/-2% S. aureus in primiparous and 10.2+/-1.3% in multiparous cows. Blood and milk PMN CL and milk PMN viability were significantly higher in primiparous cows. The pronounced reduction in phagocytic and bactericidal activity in blood and milk-resident PMN from multiparous cows, in part, resulted from the pronounced decrease of PMN viability and free radicals production capacity; this suggests that heifers' udders could be more protected against S. aureus, which remains to be tested in the field.  相似文献   

7.
Six primiparous Holstein cows were fed a Se-deficient diet, beginning at least 90 days before their first calving, and 6 other primiparous cows were given the same diet plus a supplement of 2 mg of Se/cow/d as sodium selenite. All cows were fed their diets for the duration of the experimental period. One uninfected quarter of each cow was injected with 25 micrograms of Escherichia coli endotoxin at postpartum week 5. Leukocytes were isolated by centrifugation from milk collected at postinjection hour 16. Isolated cells were 92 +/- 3% neutrophils and were incubated with Staphylococcus aureus or E coli in a 1:300 ratio. Phagocytosis and intracellular killing by neutrophils were assessed after 0, 30, 60, and 90 minutes by a fluorochrome assay, using acridine orange. Viability of neutrophils was assessed by use of trypan blue. Superoxide anion production and hydrogen peroxide production by neutrophils also were determined. Cows fed Se-deficient diets had significantly (P less than 0.05) lower blood Se concentration and blood glutathione peroxidase activity than cows fed Se-supplemented diets. Selenium status had no effect on the phagocytic capacity of neutrophils. Neutrophils obtained from cows fed Se-supplemented diets killed a significantly (P less than 0.05) higher percentage of ingested bacteria than did neutrophils from cows fed the Se-deficient diet. Viability was significantly (P less than 0.05) reduced by incubation with S aureus in neutrophils from both groups of cows, with neutrophils from Se-deficient cows having lower viability.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

8.
A study was conducted to determine relationships among plasma and milk corticosteroids, milk immunoglobulins, and phagocytosis of Staphylococcus aureus by polymorphonuclear leukocytes (PMN) isolated from milk of cows given injections of 0 (saline solution only), 100, and 200 IU of ACTH. Also determined were the effects of ACTH on mobilization of PMN into milk from the mammary gland irritated by infusion of 100 ml of saline solution containing 0.1% oyster glycogen. Cows (n = 10) were injected 6 times within a 48-hour period with 0, 100, or 200 IU of ACTH. Immediately before cows were given the 5th injection, 2 mammary quarters were infused with the saline-glycogen solution. Five hours after the 6th injection, milk from infused quarters was collected, and PMN were isolated, washed, and resuspended in autologous skimmed milk collected 5 hours after the 4th injection and before the udder was infused. Isolated PMN were incubated with S aureus and percentage of phagocytosis was determined. Concentrations of total corticosteroids in plasma and milk increased after cows were given injections of 100 and 200 IU of ACTH. The concentrations of IgA, IgG1, IgG2, IgM, and bovine serum albumin in milk after 4 injections of 0, 100, and 200 IU of ACTH were similar to preinjection (base line) concentrations. Injections of 100 and 200 IU of ACTH significantly increased the concentrations of total circulating leukocytes. Concentrations of leukocytes in milk tended to increased with increasing doses of ACTH, but the differences were not significant. Injection of 100 and 200 IU of ACTH reduced phagocytosis of S aureus by PMN. After 60 minutes of incubation, phagocytosis averaged 57% and 54%, respectively, for the ACTH treatments and 70% for controls (saline only). Results indicate that injections of ACTH that result in physiologic and pharmacologic plasma concentrations of corticosteroids inhibit phagocytosis. Impairment of phagocytosis appeared to be a direct effect of corticosteroid concentration o PMN and was not due to reduced concentrations of immunolobulins. These data indicate that reduced phagocytosis by PMN could be important in increased susceptibility to disease during stress in lactating dairy cows.  相似文献   

9.
A Flow Cytometric method for the evaluation of the phagocytic capacity of bovine blood neutrophils is described. The neutrophils were isolated from bovine blood by a one step discontinuous gradient of Percoll. By this technique of isolation, 90 ± 2.8 % (mean ± s) of the granulocytes in the whole blood were recovered.Isolated neutrophils were incubated with FITC labeled S. aureus or zymosan particles in a ratio of 1:20 and 1:10, respectively, and a final serum concentration of 10 %. Phagocytosis was terminated after 15 min and the number of extracellular bacteria or zymosan particles and the percentage of phagocytic granulocytes were registered by Flow Cytometry (FCM). FCM and microscopic studies revealed that eosinophils play a minor role in the phagocytosis of bacteria. The neutrophils were the main population of the granulocytes which were actively phagocytic. Variation among cows in the ability of their blood neutrophils to phagocytize bacteria was evident.  相似文献   

10.
Cows with subclinical mastitis caused by Staphylococcus aureus were subjected to subcutaneous injections with either an extract from the root of Panax ginseng CA Meyer at a dose of 8 mg/kg body weight per day for 6 days, or with saline as a control. The injection areas were checked for adverse reactions. The daily milk production was measured before and after treatment. Blood was collected for total and differential leucocyte counts, identification of lymphocyte subpopulations using flow cytometry, lymphocyte proliferation test, and neutrophil phagocytosis and oxidative burst assay. Quarter milk samples were collected for bacteriological analysis and somatic cell counts (SCC). After the end of treatment, the numbers of S. aureus-infected quarters and milk SCC tended to decrease in ginseng-treated cows. Phagocytosis and oxidative burst activity of blood neutrophils were significantly increased 1 week after ginseng treatment, but the proliferative response of blood lymphocytes did not change significantly. The number of monocytes in ginseng-injected cows was significantly higher 1 week post-treatment than pre-treatment, and the number of lymphocytes was significantly higher than pre-infusion at 2 and 3 weeks after ginseng treatment. Similar changes were not observed in the control group. The present findings indicate that ginseng treatment can activate the innate immunity of cows and may contribute to the cow's recovery from mastitis. It is therefore suggested that ginseng has a potential as a stimulator of the immune system of dairy cows.  相似文献   

11.
Phagocytic and nitroblue tetrazolium (NBT) reductive activities of blood neutrophils from 19 Holstein heifers were measured by light microscopic and spectrophotometric methods, respectively. These functional properties of neutrophils correlated well (r = 0.64) and varied significantly (P less than 0.05) among animals studied. Variations in phagocytosis and NBT reductive activities attributable to the source of sera were determined in experiments in which cells from the same cows and zymogen particles opsonized with heat-inactivated autologous or homologous sera were used. Variations attributable to the source of cells were determined in experiments in which cells from different cows and particles opsonized with pooled sera from all the cows were used. Most of the variation in phagocytic properties and NBT reductive activities was attributable to the source of cells (ie, each cow). The source of sera contributed slightly to the variation in NBT reductive activities, but not to the phagocytic properties. These results support the concept of functional heterogeneity of neutrophils among cows.  相似文献   

12.
The association between Staphylococcus aureus chronic mammary gland infection and the resulting immune response expressed by the production of specific IgG and IgA antibodies in blood and milk was studied in Israeli Holstein cows. Specific antibodies of the IgG class were detected in sera of 82.6 per cent of the cows chronically infected by S aureus, while in 17.4 per cent no such antibodies could be detected. Specific IgG antibodies to S aureus were neither detected in sera of cows free of mammary infection nor in those infected with different coagulase-negative staphylococci (CNS) such as S intermedius, S chromogenes or S haemolyticus. In milk, specific IgG antibodies to S aureus were detected only in cows with positive serology. The end point dilutions in the milk were 5 to 30 per cent of that of blood from the same cow. No significant difference in IgG titres was found in the same cow if the quarter was infected with S aureus or not. Specific antibodies to S aureus of the IgA class could not be detected in the sera of any of the cows included in this study. In milk, a specific IgA antibody was detected only in the samples from the S aureus infected quarters in which S aureus was isolated at the time of the experiment. In the same cow, quarters infected by S aureus were found to have a significantly higher IgA titre (P < 0.0001) than that of the non-infected ones.  相似文献   

13.
A rapid method is described for parallel isolation of white blood cells, granulocytes and purified neutrophils from peripheral blood of normal cattle. The mean recovery (+/- S.D.) of white blood cells, granulocytes and purified neutrophils isolated from peripheral blood of 13 cows was 66.4 +/- 12.6%, 68.7 +/- 20.0% and 38.0 +/- 20.9%, respectively. The mean purity of the isolated granulocyte and neutrophil suspensions was 94.0 +/- 3.8% and 95.0 +/- 6.0%, respectively. Viability of isolated cells was more than 97%.  相似文献   

14.
Biofilm production by 221 Staphylococcus aureus isolates from 45 dairy herds was evaluated. Isolates were from composite milk of 117 cows, from teat skin of 70 cows, and from 34 milking machine unit liners. Of S. aureus from milk samples, 41.4% were biofilm producers, as compared to 24.7 and 14.7% of the isolates collected from skin and liners. Pulsed field gel electrophoresis (PFGE) best categorized S. aureus biofilm producers as compared to phage typing and binary typing. PFGE types that were significantly associated with isolation from milk as opposed to teat skin or liners, had isolates that were more likely to produce biofilm than PFGE types that were isolated from milk, skin and liners at similar frequencies. By contrast, PFGE type A was significantly associated with isolation from teat skin and had few biofilm producers. PFGE type Q, which is exclusively a milk, isolate produced more biofilm as evidenced by absorbance values. Given S. aureus that are associated with milk are more likely to produce biofilm as compared to extramammary sources (teat skin and milking unit liners), suggests that biofilm production is a risk factor for infection.  相似文献   

15.
Leukocidin toxin from a bovine strain of Staphylococcus aureus was partially purified by ion exchange chromatography. An enzyme-linked immunosorbent assay was developed to quantitate antibodies specific for leukocidin in bovine milk. This was used to assay quarter samples from 88 cows in a S aureus-infected herd for antibody levels to the toxin. Milk samples from 65 cows with S aureus infections in at least one quarter produced a mean optical density of 1.054, whereas milk samples from 23 cows that were free of bacteria on cultural examination had a mean optical density of 0.584. There was a significant difference (P less than 0.001) in milk anti-leukocidin levels between these 2 groups. Evaluation of serum samples from 40 of these cows indicated that the milk anti-leukocidin concentrations were reflective of systemic anti-leukocidin values. The capability of 57 milk samples to neutralize the cytolytic effect of minimal amounts of leukocidin on bovine peripheral blood neutrophils was examined. Good correlation existed between the enzyme-linked immunosorbent assay antibody concentration and toxin-neutralizing capability of individual milk samples.  相似文献   

16.
The aim of the research was to test whether exogenic virulence factors secreted by Staphylococcus aureus isolates are involved in mechanisms that allow the bacteria to modulate and evade phagocytosis by bovine polymorphonuclear neutrophils. The research was based on the comparison of the effects of supernatants, prepared from cultures of 30 S. aureus isolates, on the functional properties of bovine neutrophils in vitro. S. aureus isolates were collected from milk samples from cows with clinical mastitis. Supernatants, which were used to treat leukocytes, were prepared from 18 h S. aureus cultures. Exogenic virulence factors secreted by S. aureus isolates significantly influenced the phagocytosis parameters evaluated. Depending on their leukotoxic or superantigenic properties, supernatants could affect the ingestion process, and also showed an influence on the digestion efficiency and phagocytosis carried out by bovine polymorphonuclear neutrophils in vitro.  相似文献   

17.
Using bovine neutrophils and radio-labelled Staphylococcus aureus, skim milk samples taken at 4 stages of lactation from the 4 mammary quarters of 48 cows were used in an in vitro phagocytosis assay. Immunoglobulin (Ig) isotype concentrations in the milk samples were estimated by use of an ELISA procedure. To determine associations of Ig concentrations with phagocytosis, correlations, simple regressions, and partial regressions were calculated. Simple correlations were computed between each Ig isotype and phagocytosis percentage for each lactation number stage of lactation category. Ranges of these correlations for IgM, IgG1, IgG2, and IgA were 0.33 to 0.60; -0.16 to 0.43; 0.04 to 0.46; and -0.30 to 0.36, respectively. Correlations for concentrations of IgG2 and IgM with percentage of phagocytosis tended to be slightly higher for samples from older cows, in contrast to the correlations calculated for IgA and IgG1. Multiple regression of percentage of phagocytosis calculated simultaneously on concentrations of the 4 Ig isotypes in the sample indicated that IgM, followed by IgG2 and IgA, was most closely associated with phagocytosis. Partial regression calculated on concentration of IgG1 was not significant. Addition of bacteriologic status of the quarter and somatic cell concentration in the milk sample did not increase accuracy of predicting percentage of phagocytosis, compared with use of Ig concentrations alone. These results supported the attribution of unique modes of action to IgM and IgG2 in promoting phagocytosis by neutrophils.  相似文献   

18.
Nine dairy herds (mean size, 149 cows) with bulk-tank milk somatic cell counts of less than 300,000 cells/ml and greater than 80% of cows with Dairy Herd Improvement Association linear somatic cell counts less than or equal to 4 were selected for study. Each herd was monitored for 12 consecutive months. Duplicate quarter-milk specimens were collected from each cow for bacteriologic culturing at beginning of lactation, cessation of lactation, and at the time of each clinical episode of mastitis. Streptococcus agalactiae was never isolated and Staphylococcus aureus was isolated from less than 1% of all quarters. There were 554 episodes of clinical mastitis. During the year of study, the incidence rate of clinical mastitis varied from 15.6 to 63.7% of cows among the 9 herds. Mean costs per cow per year in herd for mastitis prevention were: $10 for paper towels, $3 for nonlactating cow treatment, and $10 for teat disinfectants. Mean cost associated with clinical mastitis was $107/episode. Approximately 84% ($90) of the costs attributed to a clinical episode were associated with decreased milk production and nonsalable milk. Costs of medication and professional veterinary fees per clinical episode varied significantly among the 9 herds. Three of the herds did not have a veterinarian treat a clinical episode of mastitis during the year of study even though 2 of these herds had the first and third highest incidence rates of clinical mastitis. When calculated on a per cow in herd basis, mean costs of $40/cow/year were attributed to clinical mastitis.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

19.
The influence of the pH of suspending medium on bovine neutrophil (PMN) function was assessed in tests of phagocytosis and killing of Staphylococcus aureus. Intracellular killing was markedly inhibited by moderate extracellular acidification whereas phagocytosis was little affected, except at the lowest pH level (pH 5.0). The killing of S. aureus by extracts of isolated PMN lysosomal granules showed a similar pH dependence and was optimal at pH levels above neutrality. Survival of S. aureus within PMN from different cows varied significantly and the relative differences in PMN bactericidal efficiency were maintained at all pH levels. The acidification of extracellular medium during incubation which resulted from metabolic activity of the PMN themselves, increased with increasing ratios of bacteria:PMN and varied significantly among cows. Addition of methylamine (10 mM) to elevate phagolysosomal pH inhibited phagocytosis and had no effect on intracellular survival of S. aureus. However, a lower concentration (1.5 mM) did not affect phagocytosis, but reduced bacterial survival without altering the relative differences in efficiency of PMN from different cows. It is suggested that the acidity of the extracellular medium may both reflect and influence the pH changes occurring within PMN phagosomes and, thereby, modulate the efficiency of intracellular destruction of S. aureus.  相似文献   

20.
In a study involving 56 quarters (14 cows), it was seen that standing estrus did not affect circulating neutrophil and milk somatic cell concentrations, neutrophil phagocytosis, milk production, or occurrence of clinical mastitis. The subcutaneous injection of 17beta-estradiol (E2) (0.05 mg/kg of body weight 2 times daily for 2 consecutive days) did not affect neutrophil phagocytosis or milk somatic cell values. However, a significant increase in circulating neutrophils and a significant decrease in milk production were observed. The injection of E2 was followed by clinical mastitis in 4 uninfected quarters, in 7 quarters infected with Corynebacterium bovis, and in 1 quarter infected with Streptococcus uberis and Escherichia coli.  相似文献   

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