Flow cytometric assessment of myeloperoxidase in bovine blood neutrophils and monocytes

Abstract

Myeloperoxidase (MPO) is a lysosomal peroxidase enzyme mainly stored in the azurophilic granules of neutrophils playing an important role in innate immunity for first-line protection against microorganisms in many species including cattle. As such, determination of MPO has become of great interest for the diagnosis of infectious and inflammatory diseases in multiple species such as humans. In cattle, MPO determination is rarely done because methods to assess MPO in this species are limited: functional assays have been described earlier, but so far, the quantification of MPO at the single cell level has not been done yet. In the present paper, an innovative flow cytometric method to assess MPO in blood leukocytes of dairy cattle is described. A commercial anti-bovine MPO was used following density gradient separation to isolate polymorphonuclear (PMN) and mononuclear (MN) leukocytes from blood. Identification of PMN and MN, subdivided in monocytes and lymphocytes, was based on the expression of the surface markers CH138A and CD172A. The optimized protocol was subsequently evaluated on blood samples of 17 Holstein Friesian heifers. Myeloperoxidase expression was measured flow cytometrically and visualized by fluorescence microscopic imaging of sorted PMN and MN populations. We suggest this innovative method to be useful in the field for early detection of cows at higher risk for inflammatory diseases such as mastitis and metritis during the transition period.