Effects of citral and linalool on blood neutrophil toxicity and oxidative response in dairy cows

Abstract Text: Alternative therapies to controlling and treating mastitis are being pursued to reduce potential antibacterial resistance. Certain bioactive phytochemicals extracted from plants, such as citral and linalool, have demonstrated antimicrobial activity and may serve as acceptable alternatives for conventional mastitis treatments. It is unknown how these phytochemicals may interact with bovine polymorphonuclear cells (PMN), the predominant cell type recruited during mastitis. The objective of this study was to evaluate the effects of citral and linalool on cytotoxicity and oxidative response of bovine blood PMN in vitro. Blood was sampled from four healthy, primiparous Holstein dairy cows in mid-lactation (DIM>90). Polymorphonuclear cells were isolated and incubated for 2 hours with various concentrations of citral (0, 0.1, 0.2, 0.4, 0.8 and 10 µl/mL) and linalool (0, 0.1, 1.2, 2.4, and 10 µl/mL). Cytotoxicity was measured by non-radioactive, colorimetric assay to quantify lactate dehydrogenase production. Oxidative burst response for the PMN was measured by relative chemiluminescence of reactive oxidative species production after exposure to 1.6 µg/mL phorbol 12-myristate-13-acetate in addition to citral or linalool during incubation. Data were analyzed by ANOVA using the MIXED procedure of SAS 9.2. Each phytochemical was analyzed separately. Differences among treatments were determined using the PDIFF statements and significance was declared at P≤0.05. Citral concentrations of 0.8 and 10 µl/mL increased PMNL cytotoxicity to 33.0% and 68.3%, respectively (P<0.01), relative to the control. Oxidative burst response increased at 0.01 and 0.02 µl/mL concentrations of citral, whereas 0.4 and 0.8 µl/mL concentrations decreased oxidative burst (P<0.01). Linalool concentrations equal to or less than 2.4 µl/mL did not alter PMN cytotoxicity relative to the control and 10 µl/mL increased cytotoxicity to 81.2% (P<0.01). Oxidative response of PMN increased for 1.2, 2.4, and 10 µl/mL concentrations of linalool (P=0.02). In summary, citral and linalool do affect in vitro bovine blood PMN cytotoxicity and oxidative burst response. Concentrations less than 0.4 µl/mL of citral and 2.4 µl/mL of linalool were non-toxic to bovine blood PMN and concentrations between 0.1-0.2 µl/mL citral and 1.2-10 µl/mL linalool increased oxidative burst response. The use of citral and linalool as an alternative therapy for mastitis is promising as they may not interfere with the immune response during mastitis.

Keywords: mastitis, polymorphonuclear cell, alternative therapy