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.

Organic Selenium Ameliorates Staphylococcus aureus-Induced Mastitis in Rats by Inhibiting the Activation of NF-κB and MAPK Signaling Pathways

“…MPO activity was used as a biomarker for the infiltration of neutrophils and macrophages, which plays important roles in evaluating the development of mastitis ( 27 ). The activity of MPO was detected in mammary tissues ( Figure 3 ).…”

Sensitive and rapid lateral-flow assay for early detection of subclinical mammary infection in dairy cows

“…ecules released by activated neutrophils and have antimicrobial properties essential for the innate defence mechanism against the invading pathogens. Moreover, it is essential for the transformation of hydrogen peroxide (H 2 O 2 ) into hypochlorous acid (HOCl) during the neutrophil's respiratory burst which is highly efficient in killing the invading microorganisms, and also causes huge cytotoxic damage to the host tissue 15,34 . The MPO www.nature.com/scientificreports/ enzyme is recognised as an inflammation biomarker in human medicine and is used as an important diagnostic tool for bacterial infections 35 , diagnosis of the wound 36 and the urinary tract infections 37 .…”

“…Neutrophils, the first line of cellular defense, form around 25% of the total leucocytes in normal milk and their number increase dramatically whenever there is an infection in the mammary tissues 8,13 . Therefore, diagnostic tests specifically detecting neutrophils related molecules can provide a more specific and reliable indicator of SCM than total SCC 7,14,15 .…”

The Peroxisome Proliferator-Activated Receptor γ Agonist Pioglitazone Protects Vascular Endothelial Function in Hypercholesterolemic Rats by Inhibiting Myeloperoxidase

“…Myeloperoxidase (MPO) is an oxidase that is stored in azurophilic granules of neutrophils and monocytes, which is released extracellularly during inflammation [6]. MPO plays an important role in the formation and development of many diseases, including atherosclerosis [7].…”