Immune response in nonspecific mastitis: What can it tell us?

Abstract

We analyzed a large number of immune response parameters from quarter milk samples with distinct bacteriological and quarter somatic cell count (qSCC) statuses. Furthermore, we sought to explore and identify displayed immune response patterns in milk samples from mammary glands with nonspecific mastitis. Thus, 92 quarter milk samples from 28 cows were stratified into 4 groups, as follows: (1) 49 culture-negative control quarters with a low qSCC (<1 × 10 5 cells/mL) from 19 dairy cows (so-called healthy quarters); (2) 15 culture-negative quarters with high qSCC (>2 × 10 5 cells/mL; so-called quarters with nonspecific mastitis) from 10 dairy cows; (3) 8 culture-positive quarters with low qSCC (noninflammatory quarters with low qSCC) from 5 dairy cows; and (4) 20 culture-positive quarters with high qSCC (so-called truly infected quarters) from 8 dairy cows. Using flow cytometry, we evaluated the percentage of milk neutrophils and their viability, intracellular reactive oxygen species production, phagocytosis, and the expression of CD62L, CD11b, and CD44 for each of the 4 quarter strata. Furthermore, the percentage of monocyte/macrophages, B cells, and T lymphocyte subsets were evaluated by flow cytometry. Milk samples from bacteriologically negative quarters (both with a low and elevated qSCC) had a lower qSCC than those with bacteriologically positive outcomes (both with a low and elevated qSCC). As expected, the healthy quarters showed the lowest percentage of neutrophils and also showed a higher percentage of milk monocytes/macrophages and lower percentage of T lymphocytes than truly infected quarters. The most prominent result of the present study is that quarters with nonspecific mastitis showed the highest percentage of milk CD4 + T lymphocytes. The healthy quarters had a lower percentage of apoptotic neutrophils than noninflammatory and truly infected quarters, although it did not differ from those from the quarters with nonspecific mastitis. Our study supports the role of differential cell counting in the diagnosis of mastitis, as the milk leukocyte populations markedly fluctuate under healthy and inflammatory conditions. Furthermore, an increase in milk CD4 + T cells was associated with nonspecific mastitis, suggesting an increase in this leukocyte subpopulation is correlated with low bacterial shedding. Our study allows us to go further in our understanding of mammary gland immunity, providing further insights on potential protective mammary gland immunity, which we hypothesize can open new avenues for the development of novel targets that can promote bovine udder health.

Milk Lymphocyte Profile and Macrophage Functions: New Insights Into the Immunity of the Mammary Gland in Quarters Infected With Corynebacterium Bovis

“…In addition to our data regarding the lymphocytic and functional pro le of milk macrophages, other factors may be related to the potential protective effect of intramammary infection by C. bovis, such as inhibition of growth by competition, bacterial antagonism, increased activity of milk neutrophils, and the presence of plasmocytes in the parenchymal tissue and the teat apices of quarters colonized with C. bovis [6,16,21,40]. Furthermore, macrophages represent the rst line of defense of the mammary gland against invasive pathogens because they make up the predominant leukocyte population in healthy udder quarters [27,30,41]. Although the proportion of macrophages usually decreases during intramammary infections due to the rapid and massive in ux of neutrophils into the in ammatory site [19,42], macrophages still represented the predominant leukocyte population in quarters infected by C. bovis in the present study.…”

Milk Macrophage Function in Bovine Leukemia Virus-Infected Dairy Cows

“…Apoptosis of milk macrophages was determined by double staining with fluorescein isothiocyanate (FITC)-conjugated annexin-V and propidium iodide (PI) by flow cytometry analysis using a commercial kit (cat. n. K2350, APOPTEST-FITC, Dako Cytometrion, Netherlands), as previously described by Della Libera et al ( 4) and Souza et al (32). Initially, 2 × 10 5 milk cells were resuspended in 100 µL of binding buffer (10 mM HEPES, 150 mM NaCl, 1 mM MgCl 2 , and 1.8 mM CaCl 2 ) containing anti-annexin-V FITC antibody and incubated at room temperature for 20 min in the dark.…”

“…The phagocytosis assay was performed by flow cytometry using PI-conjugated S. aureus, as previously described (4,32,36). Briefly, 2 × 10 5 viable milk cells were incubated with 100 µL of PI-conjugated S. aureus for 30 min at 37 • C and 900 µL of PBS.…”

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.…”