Veterinary Clinical Immunology Research Group, Departamento de Clínica Médica, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo 05508-270, Brazil; Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo 05508-270, Brazil; M-team and Mastitis and Milk Quality Research Unit, Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, Merelbeke 9820, Belgium; Programa de Pós-Graduação em Ciência Animal, Universidade Federal da Paraíba, Areia 58397-000, Brazil.
Veterinary Clinical Immunology Research Group, Departamento de Clínica Médica, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo 05508-270, Brazil; Programa de Pós-Graduação em Saúde, Bem-estar e Produção Animal Sustentável na Fronteira Sul, Universidade Federal da Fronteira Sul, Realeza 85770-000, Brazil.
J Dairy Sci. 2020 Jun;103(6):5376-5386. doi: 10.3168/jds.2019-17022. Epub 2020 Mar 27.
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 cells/mL) from 19 dairy cows (so-called healthy quarters); (2) 15 culture-negative quarters with high qSCC (>2 × 10 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.
我们分析了大量具有不同细菌学和乳房体细胞计数（qSCC）状态的季度奶样中的免疫反应参数。此外，我们还试图探索和识别非特异性乳腺炎乳房牛奶样本中显示的免疫反应模式。因此，将 28 头奶牛的 92 个乳区奶样分为 4 组：（1）49 个培养阴性对照乳区，qSCC（白细胞介素-1受体拮抗剂）低（<1×10 个细胞/mL），来自 19 头奶牛（所谓的健康乳区）；（2）15 个培养阴性乳区，qSCC 高（>2×10 个细胞/mL；所谓的非特异性乳腺炎乳区），来自 10 头奶牛；（3）5 头奶牛 8 个培养阳性低 qSCC（非炎症性低 qSCC）乳区；（4）8 头奶牛 20 个培养阳性高 qSCC（真正感染的乳区）。使用流式细胞术评估了每个 4 个乳区的牛奶中性粒细胞及其活力、细胞内活性氧产生、吞噬作用以及 CD62L、CD11b 和 CD44 的表达。此外，还通过流式细胞术评估了单核细胞/巨噬细胞、B 细胞和 T 淋巴细胞亚群的百分比。细菌学阴性（qSCC 低和高）的乳区的 qSCC 低于细菌学阳性（qSCC 低和高）的乳区。正如预期的那样，健康的乳区中性粒细胞百分比最低，而牛奶单核细胞/巨噬细胞百分比较高，T 淋巴细胞百分比较低，与真正感染的乳区相比。本研究最突出的结果是，非特异性乳腺炎乳区的牛奶 CD4+T 淋巴细胞百分比最高。与非炎症性和真正感染性乳区相比，健康乳区的凋亡中性粒细胞百分比较低，但与非特异性乳腺炎乳区无差异。我们的研究支持在乳腺炎诊断中使用差异细胞计数的作用，因为在健康和炎症状态下，牛奶白细胞群明显波动。此外，牛奶 CD4+T 细胞的增加与非特异性乳腺炎有关，表明这种白细胞亚群的增加与低细菌脱落有关。我们的研究使我们能够进一步了解乳腺免疫，为潜在的保护性乳腺免疫提供了更多的见解，我们假设这可以为开发新的目标开辟新的途径，以促进奶牛乳房健康。
