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Flow Cytometric Measurement of Bovine Milk Neutrophil Phagocytosis

Flödescytometrisk bestämning av fagocytos hos neutrofila leukocyter isolerade från mjölk

Abstract

A flow cytometric method for the evaluation of the phagocytic capacity of bovine milk polymorphonuclear neutrophils (PMN) is described. Milk PMN were isolated from stripping milk collected from udder quarters fitted with abraded intramammary devices (AIMD). A significant increase in the milk somatic cell count was observed in the stripping milk after the insertion of AIMD (308×103 and 1447×l03 cells/ml milk before respectively after the insertion of the AIMD, p < 0.001).

PMN were also isolated from blood by a discontinous gradient of Percoli. Blood and milk PMN were incubated for 15 min with FITC-labeled bacteria in a ratio of 1 PMN:20 bacteria and a final serum concentration of 10 %. The number of extracellular bacteria and the percentage of phagocytic cells were measured by a flow cytometer.

Percentage of phagocytized bacteria by milk PMN was significantly lower than that by blood PMN (p < 0.05). A smaller number of active phagocytes was present among cells isolated from milk than among cells isolated from blood. The phagocytic capacity of milk PMN reflects that of blood PMN in the same animal. A large variation in the phagocytic capacity of blood and milk PMN among animals was observed.

Sammanfattning

I arbetet beskrivs en flödescytometrisk metod för bestämning av fagocytoskapaciteten hos neutrofila leukocyter (PMN) i komjölk. PMN isolerades från eftermjölksprov från juverfjärdedelar i vars juvercisterner spiraler gjorda av plasttråd med skrovlig yta (AIMD) hade placerats. Cellkoncentrationen i mjölken ökade från 308 × 103 til 1447 × 103 celler/ml efter införandet av plastspiralerna i juverfjärdedelarna. Ökningen var signifikant (p < 0,001).

För isolering av PMN från blod användes en diskontinuerlig Percollgradient.

PMN från blod och mjölk inkuberades i 10 % serum med FITC-märkta bakterier i proportionen 1 PMN:20 bakterier. Inkubationstid, 15 min. Antalet extracellulära bakterier och frekvensen fagocyterande celler mättes med en flödescytometer.

PMN isolerade från mjölk fagocyterade signifikant lägre andel av bakterierna än PMN isolerade från blod (p < 0,05). En lägre andel av fagocyterna i mjölken var aktiva jämfört med i blodet.

Fagocytoskapaciteten hos PMN isolerade från mjölken speglar den hos blodet från samma djur. Variationen mellen djur är stor.

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Acknowledgment

Prof. Göran Åström is cordially thanked for helpful discussion and advices. Mrs. Gudrun Mellberg-Wieslander is thanked for typewriting the manuscript. This study was supported by grants from the Swedish Council for Forestry and Agricultural Research.

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Saad, A.M. Flow Cytometric Measurement of Bovine Milk Neutrophil Phagocytosis. Acta Vet Scand 28, 333–342 (1987). https://doi.org/10.1186/BF03548601

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Keywords

  • intramammary device
  • milk cells
  • blood PMN