Whole blood stimulation assay (WBA) with killed gram-positive and gram-negative udder pathogens were used to investigate the interference of the endotoxin-binding antibiotic polymyxin B (PMB) on the ex vivo TNF-α response. Blood samples were collected from first to third lactating dairy cows in their early lactation (<50 days in milk, n = 32) period. The WBA was stimulated with both inactivated bacteria (e.g., dead Escherichia coli, Staphylococcus aureus, Streptococcus dysgalactiae, Streptococcus uberis), at a concentration of 2.5 × 10⁶/mL; and pathogen-associated molecular pattern molecules, namely E. coli LPS (10 μg/mL), and S. aureus peptidoglycan (PG, 10 μg/mL). The PMB was added at a concentration of 0, 12.5, 25, 50, 100, and 200 μg/mL to each stimulant, respectively. All bacteria stimulants resulted in an increased TNF-α response compared to the negative control. The PMB affected the TNF-α responses of gram-positive (except S. dysgalactaie), gram-negative bacteria; and bacterial cell wall components at a PMB concentration of 25–50 μg/mL. The LPS and E. coli had similar TNF-α response but PG had a lower TNF-α response than gram-positive bacteria. The doses of PMB (≥ 25 μg/mL) should be used with caution when using different types of pathogens or should be avoided in ex vivo TNF-α studies.

使用杀死革兰氏阳性和革兰氏阴性乳房病原体的全血刺激测定 (WBA) 来研究内毒素结合抗生素多粘菌素 B (PMB) 对离体TNF-α 反应的干扰。在泌乳早期（牛奶<50 天，n = 32）期间从第一至第三泌乳奶牛收集血样。用浓度为 2.5 × 10 ⁶ /mL 的两种灭活细菌（例如，死大肠杆菌、金黄色葡萄球菌、停乳链球菌、乳房链球菌）刺激 WBA ；和病原体相关的分子模式分子，即大肠杆菌LPS (10 μg/mL) 和金黄色葡萄球菌肽聚糖（PG，10 μg/mL）。PMB 分别以 0、12.5、25、50、100 和 200 μg/mL 的浓度添加到每种兴奋剂中。与阴性对照相比，所有细菌刺激剂导致 TNF-α 反应增加。PMB影响革兰氏阳性菌（除S.dysgalactaie）、革兰氏阴性菌的TNF-α反应；和细菌细胞壁成分，PMB 浓度为 25–50 μg/mL。LPS 和大肠杆菌具有相似的 TNF-α 反应，但 PG 的 TNF-α 反应低于革兰氏阳性菌。在使用不同类型的病原体时应谨慎使用 PMB (≥ 25 μg/mL) 的剂量，或在体外TNF-α 研究中应避免使用。