Flow cytometric analysis: Interdependence of healthy and infected udder quarters

Abstract

An important question about intramammary infections that is still debated in the literature is the independence or interdependence of the quarters of dairy cows. The present study sought to explore milk neutrophil function and the milk lymphocyte profile of uninfected quarters from uninfected and infected (one infected quarter per cow) udders to evaluate interdependence of the quarters. Thus, 32 (8 cows) and 18 (6 cows) uninfected quarters from uninfected and infected udders were used, respectively. Using flow cytometry, we evaluated the percentage of milk neutrophils and their expression of adhesion molecules L-selectin (CD62L), β2-integrin (CD11b), and an endothelial-selectin ligand (CD44); levels of intracellular reactive oxygen species (ROS); phagocytosis of Staphylococcus aureus by milk neutrophils; and neutrophil viability. Furthermore, we assessed the percentage of B-cell (CD21(+)) and T-lymphocyte subsets (CD3(+)/CD4(+)/CD8(-), CD3(+)/CD8(+)/CD4(-), CD3(+)/CD4(+)/CD25(-), CD3(+)/CD4(+)/CD25(+), and CD3(+)/CD4(-)/CD25(-)) using flow cytometry with monoclonal antibodies. The infected quarter did not affect somatic cell count or the percentage of neutrophils in the neighboring uninfected quarters. Furthermore, the infected quarter did not influence neutrophil viability, intracellular reactive oxygen species production, or phagocytosis of S. aureus by milk neutrophils. Conversely, the expression of adhesion molecules CD11b, CD62L, and CD44 by milk neutrophils differed between uninfected quarters from infected versus uninfected udders. The lymphocyte subsets did not differ between groups, except for a higher percentage of B cells in uninfected quarters from infected udders than in those from uninfected udders. Thus, our study strongly supports the hypothesis of interdependence of quarters based on the influence of infection on both the percentage of B cells and the expression of adhesion molecules by milk neutrophils in the neighboring uninfected quarters.

Normal milk microbiome is reestablished following experimental infection with Escherichia coli independent of intramammary antibiotic treatment with a third-generation cephalosporin in bovines

“…Jensen and colleagues [73] have evaluated the transcriptional response of uninfected quarters in animals challenged with two major mastitis pathogens and described that the response in non-affected quarters was greater in animals with E. coli -associated mastitis [73]. Although other immune parameters were not evaluated in the current study, the numerical increase in somatic cell count in unchallenged quarters after intramammary infection with E. coli observed here is in agreement with Jensen et al, [73] and Blagitz et al, [71], indicating that the immune response in the mammary gland is to some extent influenced by the status of adjacent quarters.…”

“…Due to the anatomic structure of the udder, it is generally assumed that infection in one quarter should not affect the immune status of neighboring quarters. Recent studies have contested this hypothesis, providing evidence of interdependence between infected and healthy quarters [71, 72]. Jensen and colleagues [73] have evaluated the transcriptional response of uninfected quarters in animals challenged with two major mastitis pathogens and described that the response in non-affected quarters was greater in animals with E. coli -associated mastitis [73].…”

Implications of Intranasal Polyvalent Vaccination for Pulmonary Alveolar Macrophage and Neutrophil Functions in Calves

“…Reactive oxygen species production by alveolar neutrophils, monocyte-like macrophages and vacuolized macrophages was evaluated by ow cytometry using 2',7'-dichloro uorescein diacetate (cat. n. D6883, Sigma Aldrich, St. Louis, MO, USA) as a probe [50][51][52][53][54]. For this assay, 10,000 to 15,000 BAL cells were examined per sample.…”

“…The phagocytosis assay of alveolar neutrophils, monocyte-like macrophages and vacuolized macrophages was performed by ow cytometry using propidium iodide-labeled Staphylococcus aureus as previously described [20,[50][51][52][53] at multiplicity of infection of 120. For this assay, 10,000 to 15,000 BAL cells were examined per sample.…”

<b>Etiological and molecular profile of pathogens causing clinical mastitis, and antimicrobial use in dairy herds</b>

“…Mammary quarters are considered anatomically independent from each other (Tucker, 1981), although recent studies support the hypothesis of the immunological interdependence of quarters based on the influence of infections on certain immune response parameters in contralateral uninfected quarters (BLAGITZ et al, 2015;PAIXAO et al, 2017).…”