Bacterial Phagocytosis Monitored by Fluorescence and Extracellular Quenching: Use of Blood Clot-Derived PMNs

Musclow, C E; Farkas-Himsley, Hannah; L, Spragg; Braun, Adrian

doi:10.1093/labmed/18.8.527

Cited by 5 publications

(4 citation statements)

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“…The phagocytotic activity was assessed by fluorescence microscopy of phagocytosing cells adhering to a cover glass and stained with acridine orange (Smith and Romel, 1977) after quenching of extracellular bacteria with crystal violet (Musclow et al., 1987) using the following modified procedure. A broth culture of Staphylococcus aureus strain Newbould 305 (CCM 6275), obtained after 18 h of incubation at 37°C, was washed with PBS by centrifugation at 1000 g and its density was adjusted with HBSS to 10 6 cells per 1 ml.…”

Section: Methodsmentioning

confidence: 99%

Variations among Unbred Heifers in the Activities of Polymorphonuclear Leucocytes from the Mammary Gland and Blood

Rysanek

Babák

Sládek

et al. 2001

Journal of Veterinary Medicine, Series B

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The phenotypic characteristics are described for the activity of polymorphonuclear leucocytes (PMN) 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´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. Signi®cant differences between blood PMN and PMN from lavages after in¯ux induction were found for bactericidal activity (P < 0.05) and chemiluminescence (P < 0.01). A signi®cant correlation between blood PMN and mammary gland PMN was found only for the number of phagocytosing cells (r 0.329; P < 0.01). Highly signi®cant 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 signi®cant correlation was demonstrated for non-stimulated (NS), zymosanstimulated (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 signi®cant source of variability for all the phagocytotic activity characteristics (P < 0.01). Udder quarter was a non-signi®cant 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-signi®cant 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 in¯ux induction can be regarded as candidate early markers of resistance to mammary infections.

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Section: Methodsmentioning

confidence: 99%

Variations among Unbred Heifers in the Activities of Polymorphonuclear Leucocytes from the Mammary Gland and Blood

Rysanek

Babák

Sládek

et al. 2001

Journal of Veterinary Medicine, Series B

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“…The method of Pantazis and Kniker (1979) was modified to include the use of CV to quench extracellular bacterial fluorescence (Musclow et aL, 1987;Pruzanski and Saito, 1988). Fixed slides were immediately viewed using an ultraviolet microscope with an oil-immersion objective and an unfiltered halogen light source (Nikon Labo Phot Fluorescent Microscope, Nippon Kogeku K.K., Tokyo, Japan).…”

Section: Assay Of Phagocytosis and Microbial Killingmentioning

confidence: 99%

“…Phagocytosis and killing of bacteria are among the most important functions of PMNL. One method of evaluating PMNL function is the fluorochrome microassay (Pantazis and Kniker, 1979;Musclow et aL, 1987;Pruzanski and Saito, 1988). In one application of this method, glass-adherent PMNL suspensions exposed to a given number of bacteria pretreated with serum are subjected to staining with acridine orange (AO), which stains dead bacteria red and live bacteria green.…”

Section: Introductionmentioning

confidence: 99%

“…Various modifications of a fluorochrome microassay have been used in clinical and research investigations in humans (Absolom, 1986;Hed, 1986;Moore and Humbert, 1986), including the detection of PMNL bactericidal defects (Pantazis and Kniker, 1979;Goldner et al, 1983;Musclow et al, 1987;Pruzanski and Saito, 1988). Bovine leukocyte phagocytosis and bacterial killing of Escherichia coli and Staphylococcus aureus were evaluated using a fluorochrome microassay in 22 normal bulls of four Swiss dairy breeds (Zanetti et al, 1987).…”

Section: Introductionmentioning

confidence: 99%

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Polymorphonuclear neutrophil leukocyte function in clinical bovine patients and in cows with or withoutStaphylococcus aureus mastitis

et al. 1992

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A fluorochrome microassay was used to investigate peripheral blood polymorphonuclear leukocyte (PMNL) function in cattle. Glass-adherent PMNL were reacted with Staphylococcus aureus preincubated in 20% bovine serum for 30, 60 and 90 min. Coverslips were stained with acridine orange (AO) followed by crystal violet to quench extracellular bacterial fluorescence. PMNL function was evaluated by counting the number of dead (stained red with AO) and live (stained green with AO) S. aureus contained within 100 PMNL. A phagocytic index was calculated as the average number of bacteria contained within PMNL. The percentage killing of S. aureus was calculated from the average proportion of S. aureus within PMNL that were dead. Six clinically normal Holstein calves, 3-4 months of age, were sampled on 6 consecutive days. PMNL phagocytosis and killing did not vary significantly (p greater than 0.05) among repeated samplings per calf. PMNL function increased with increasing time of incubation of PMNL with S. aureus. Means (+/- SD) for percentage killing were 46.7 +/- 13.1, 57.4 +/- 11.6, and 62.1 +/- 9.8% for 30, 60 and 90 min of reaction, respectively. Means (+/- SD) for the phagocytic index were 2.9 +/- 0.8, 3.6 +/- 1.0, and 4.2 +/- 1.1 bacteria/PMNL for 30, 60 and 90 min of reaction, respectively. PMNL function was determined in 30 normal cattle of various breeds, age and sex, and these values were pooled to provide normal values for PMNL function. When values for bovine clinical patients (n = 25) with various diagnoses were compared with normal values (defined by the mean +/- 2SD for the 30 normal cattle) for PMNL function, only one patient was observed to exhibit PMNL hypofunction. A cow with disseminated intravascular coagulation in association with peracute coliform mastitis exhibited decreased PMNL killing capacity. Abnormal PMNL function was uncommon in the hospital population studied. Peripheral blood PMNL function was evaluated in lactating Holstein cows with (n = 15) or without (n = 15) chronic subclinical S. aureus mastitis. There was no significant (p greater than 0.05) difference in PMNL function among these cows.

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Immune and physiological responses of turkeys with green-liver osteomyelitis complex

et al. 1997

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A study of field turkeys was undertaken in order to determine the involvement of relative immunological differences in the etiology of turkey osteomyelitis complex (TOC). Lame and normal turkeys were sampled from commercial flocks just prior to processing in two separate trials. After testing for functions of both humoral and cellular immunity, the turkeys were necropsied and examined for lesions of TOC. There were significantly higher relative spleen and over weights and significantly lower body weights and relative bursal weights in birds with TOC. The birds with TOC had lower response to phytohemagglutinin-P in both in vivo and in vitro tests as well as lower circulating lymphocyte counts and higher monocyte, heterophil, and total white blood cell counts. There was a significantly higher antibody response to sheep red blood cells in turkeys with TOC, whereas antibody response to Salmonella pullorum antigen was not different. There were no significant differences in the percentages of mononuclear cells or heterophils able to phagocytize bacteria or latex particles, or kill bacteria; however, the heterophils from turkeys with TOC lesions did phagocytize significantly fewer latex particles per cell than did those of the healthy turkeys. Total serum protein, uric acid, and blood urea nitrogen levels were higher in birds with TOC, whereas hemoglobin, iron, alkaline phosphatase, and gamma-glutamyl-transferase levels were lower. Although many of the differences in birds with TOC could be caused by the normal host reaction to infection, further study may reveal innate differences that contribute to susceptibility to TOC.

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Bacterial Phagocytosis Monitored by Fluorescence and Extracellular Quenching: Use of Blood Clot-Derived PMNs

Cited by 5 publications

References 0 publications

Variations among Unbred Heifers in the Activities of Polymorphonuclear Leucocytes from the Mammary Gland and Blood

Variations among Unbred Heifers in the Activities of Polymorphonuclear Leucocytes from the Mammary Gland and Blood

Polymorphonuclear neutrophil leukocyte function in clinical bovine patients and in cows with or withoutStaphylococcus aureus mastitis

Immune and physiological responses of turkeys with green-liver osteomyelitis complex

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