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 × 105 cells/mL) from 19 dairy cows (so-called healthy quarters); (2) 15 culture-negative quarters with high qSCC (>2 × 105 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.

中文翻译：

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非特异性乳腺炎的免疫反应：它能告诉我们什么？

我们分析了具有不同细菌学和四分之一体细胞计数（qSCC）状态的四分之一牛奶样品的大量免疫反应参数。此外，我们试图探索和鉴定来自患有非特异性乳腺炎的乳腺的牛奶样品中显示的免疫反应模式。因此，将来自28头奶牛的92个季度的牛奶样品分为4组：（1）来自19头奶牛的49个培养阴性对照季度的qSCC（<1×105细胞/ mL）低（所谓的健康季度） ）; （2）来自10头奶牛的15个qSCC高的培养阴性季度（> 2×105细胞/ mL；所谓的非特异性乳腺炎季度）；（3）来自5头奶牛的8个qSCC较低的培养阳性季度（qSCC较低的非炎症季度）；（4）来自8头奶牛的20个具有较高qSCC的培养阳性季（所谓的真正感染季）。使用流式细胞仪，我们评估了四个四分之一层的乳中性粒细胞百分比及其生存力，细胞内活性氧的产生，吞噬作用以及CD62L，CD11b和CD44的表达。此外，通过流式细胞术评估单核细胞/巨噬细胞，B细胞和T淋巴细胞亚群的百分比。细菌学阴性季度（qSCC较低和升高）的牛奶样品的qSCC低于细菌学阳性结果（qSCC较低和升高的牛奶样品）。不出所料 健康区的中性粒细胞百分比最低，牛奶单核细胞/巨噬细胞的百分比较高，T淋巴细胞的百分比低于真正感染的区。本研究最突出的结果是非特异性乳腺炎患者的四分之一显示出牛奶CD4 + T淋巴细胞的百分比最高。健康区的细胞凋亡中性粒细胞比例低于非炎症区和真正感染区，尽管与非特异性乳腺炎区相比没有差异。我们的研究支持差异细胞计数在乳腺炎诊断中的作用，因为在健康和炎性条件下，乳白细胞的数量会明显波动。此外，牛奶CD4 + T细胞的增加与非特异性乳腺炎有关，提示这种白细胞亚群的增加与细菌散发率低有关。我们的研究使我们对乳腺免疫力有了更深入的了解，对潜在的保护性乳腺免疫力提供了进一步的见解，我们认为这可以为开发新的靶标提供新的途径，这些靶标可以促进乳腺健康。