Differential Leukocyte Count Method for Bovine Low Somatic Cell Count Milk

Dosogne, Hilde; Vangroenweghe, Frédéric; Mehrzad, Jalil; Massart-Leen, Am; Burvenich, Christian

doi:10.3168/jds.s0022-0302(03)73665-0

Cited by 81 publications

(98 citation statements)

References 19 publications

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“…Epithelial cells were reported to be the major cell type, on the basis of absence of labeling with anti-CD11a/CD18 antibodies, in the milk of healthy cows [97,98]. Others found a majority of lymphocytes in milk by flow cytometry, particularly in early lactation [47]. The differential leukocyte count in low SCC milk samples is difficult, and the methods used differ widely.…”

Section: Cellular Defensesmentioning

confidence: 99%

Innate immunity of the bovine mammary gland

2006

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-Understanding the immune defenses of the mammary gland is instrumental in devising and developing measures to control mastitis, the major illness of dairy ruminants. Innate immunity is an extremely broad field for investigation, and despite decades of research, our present knowledge of the innate defenses of the udder is incomplete. Yet, information is being gained on the recognition of pathogens by the mammary gland, and on several locally inducible defenses. The contribution of mammary epithelial cells to local defenses and to the mobilization of leucocytes is under growing scrutiny. Interactions of mastitis-causing bacteria such as Escherichia coli or Staphylococcus aureus and the mammary gland represents a suitable model for studies on innate immunity at an epithelium frontier. Powerful new research tools are radically modifying the prospects for the understanding of the interplay between the mammary gland innate defenses and mastitis-causing bacteria: genetic dissection of the immune response, microarray gene technology, transcriptomic methodologies and gene silencing by RNA interference will make possible the discovery of several of the key defense mechanisms which govern the susceptibility/resistance to mastitis at the molecular and genetic levels. It should then be possible to enhance the resistance of dairy ruminants to mastitis through immunomodulation and genetic improvement. mastitis / cattle / innate immunity / inflammation / milk

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Section: Cellular Defensesmentioning

confidence: 99%

Innate immunity of the bovine mammary gland

2006

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“…(ii) Staining with commercial SYTO 13 green fluorescent nucleic acid stain (Molecular Probes, Eugene, Oregon, USA) was used as described by Dosogne et al (2003) in a slight modification: 490 µl of cell suspension in RPMI 1640 was stained with 10 µl of diluted (1:40) SYTO 13 solution.…”

Section: Detection Of Pmn Apoptosismentioning

confidence: 99%

Expression of CD14 and CD44 on bovine polymorphonuclear leukocytes during resolution of mammary inflammatory response induced by muramyldipeptide and lipopolysaccharide

Langrova¹,

Sládek²,

Rysanek³

2008

Vet. Med. - Czech

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The aim of the study was to prove the effect of muramyldipeptide and lipopolysaccharide on the expression of CD14 and CD44 during an induced inflammatory response of the mammary gland and its resolution. The purpose was to clarify whether the CD14 and CD44 expression is controlled by the mechanisms of resolution. The CD44 had previously been judged to be an activation marker along with CD11b on polymorphonuclear leukocytes. The experimental inflammatory response was induced by muramyldipeptide and lipopolysaccharide, while phosphate buffered saline was used as a control. The course of the inflammatory response was monitored at four time points: 24 h and 48 h (initiation of inflammatory response), 72 h and 168 h (resolution of inflammatory response). The total number of cells was determined by a hemocytometer. Flow cytometry was used to determine differential counts of leukocytes, proportions of CD11b+ polymorphonuclear leukocytes, proportions of apoptotic and necrotic polymorphonuclear leukocytes, and proportions of CD14+ and CD44+ polymorphonuclear leukocytes. The proportion of CD11b+ polymorphonuclear leukocytes after induction of inflammation with muramyldipeptide was higher (P < 0.05) compared to that after induction by phosphate buffered saline, was highly significantly greater after lipopolysaccharide (P < 0.01), and remained at approximately the same level for the whole period of observation (168 h). A higher proportion of CD14+ polymorphonuclear leukocytes was observed 72 h after induction with phosphate buffered saline. A statistically highly significant lower proportion was observed after induction with muramyldipeptide (P < 0.01), and a statistically significant lower proportion was observed after induction with lipopolysaccharide (P < 0.05). Decrease in the proportion of CD14+ polymorphonuclear leukocytes followed. In the initial phase of the inflammatory response (24 to 72 h) there was a gradual increase in the proportion of CD44+ polymorphonuclear leukocytes, and more so after the phosphate buffered saline. A greatly lower proportion of CD44+ polymorphonuclear leukocytes was observed after administration of muramyldipeptide and lipopolysaccharide: 24 h (P < 0.01), 48 h (P < 0.05) and 72 h (P < 0.01). Compared with muramyldipeptide and lipopolysaccharide, there was a statistically highly significant (P < 0.01) lower proportion of CD44+ polymorphonuclear leukocytes observed 168 h after induction with phosphate buffered saline. Hence the proportion of CD44+ polymorphonuclear leukocytes is low in the initial phase of inflammation, and CD44, in contrast with CD11b, does not appear to be a polymorphonuclear marker of activation. The results of the study have shown that expression of CD14 and CD44 is controlled by the factors inducing inflammatory response as well as by the mechanisms of resolution.

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“…Apoptotic and secondary necrotic apoptotic neutrophils were enumerated by FCM (FACScan, Becton Dickinson Immunocytometry Systems, San Marcos, Ca, USA). Two different staining procedures were used: Simultaneous staining with Annexin V labelled with fluorescein isothiocyanate (FITC) and propidium iodide (PI) as described by Vermes et al (1995) and SYTO 13 staining as described by Dosogne et al (2003).…”

Section: Methodsmentioning

confidence: 99%

“…The commercial SYTO 13 green fluorescent nucleic acid stain (Molecular Probes, Eugene, OR, USA) was used as described by Dosogne et al (2003) in a slight modification: 490 µl of cell suspension in RPMI 1640 was stained with 10 µl of diluted (1:40) SYTO 13 solution. After incubation at laboratory temperature in the dark for 15 min, the specimens were placed on ice and stored up to the FCM measurement.…”

Section: Methodsmentioning

confidence: 99%

Spontaneous and induced cytolysis of leukocytes from bovine mammary gland in the course of cultivation in vitro - the correlation with neutrophil granulocytes apoptosis

Rysanek¹,

Sládek²,

Babak³

et al. 2006

Vet. Med.

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ABSTRACT:The process of leukocyte cytolysis and the manifestations of apoptosis and secondary necrosis of neutrophil granulocytes (hereafter only "neutrophils") were studied on four virgin heifers after the induction of leukocyte influx into the mammary gland and after their lavage in in vitro conditions. Phosphate buffered saline, muramyl dipeptide and a lipopolysaccharide were used for influx induction. Cytolysis and apoptosis were induced with heat stress, ultraviolet irradiation and spontaneous aging for 24 hours. The cytolysis was detected indirectly by determining the lactate dehydrogenase activity in the cultivation medium after the enzyme was released through cell lysis. The neutrophil apoptosis was detected using flow cytometry and two staining methods (i) simultaneous staining with Annexin V labelled FITC and propidium iodide and (ii) with SYTO 13. It was found that leukocytes of the mammary gland of virgin heifers undergo spontaneous aging during in vitro incubation. The fraction of lysed leukocytes rose in the course of the in vitro incubation and reached 21% up to 34% after 4 hours and 73% up to 79% after 24 hours, depending on the inductor of influx used. From among them, phosphate buffered saline resulted in the lowest incidence of cytolysis, the lipopolysaccharide in the highest incidence. The differences in the effect of influx inductors on leukocyte cytolysis became manifest during the first 4 hours of incubation in particular; the differences between inductors became insignificant after 24 hours. Heat stress, unlike ultraviolet irradiation, resulted in a significant increase in the fraction of lysed leukocytes. Ultraviolet radiation induced neutrophil apoptosis in a dominant way, while the effect of influx inducers and/or of the staining method used for flow cytometry had no effect. Heat stress also induced neutrophil apoptosis but to a lower extent than ultraviolet irradiation. Spontaneous leukocyte aging during the in vitro incubation resulted in an increasing share of apoptotic neutrophils depending on the duration of incubation. An increase in the share of necrotic neutrophils was only significant after influx induction with the lipopolysaccharide, but not after induction with buffered saline. Highly significant correlation between the percentage representation of apoptotic neutrophils and the percentage proportion of lysed leukocytes was shown, both after influx induction with phosphate buffered saline, and with the lipopolysaccharide and after both staining techniques (r = 0.767; 0.932; 0.966; 0.922). Statistically significant correlation was demonstrated between the proportion of necrotic neutrophils and the share of lysed leukocytes only after influx induction with the lipopolysaccharide (r = 0.579; 0.765). After the influx induction with phosphate buffered saline and staining with Annexin V and propidium iodide, statistically significant negative correlation between the percentage share of necrotic neutrophils and the percentage of lysed leukocytes (r = -0.653) was demonstrated. Thus ...

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Differential Leukocyte Count Method for Bovine Low Somatic Cell Count Milk

Cited by 81 publications

References 19 publications

Innate immunity of the bovine mammary gland

Innate immunity of the bovine mammary gland

Expression of CD14 and CD44 on bovine polymorphonuclear leukocytes during resolution of mammary inflammatory response induced by muramyldipeptide and lipopolysaccharide

Spontaneous and induced cytolysis of leukocytes from bovine mammary gland in the course of cultivation in vitro - the correlation with neutrophil granulocytes apoptosis

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