The effect of citrus-derived oil on bovine blood neutrophil response in vitro

Authors: GARCIS, M - University Of Maryland; Elsasser, Theodore; BISWAS, D - University Of Maryland; MOYES, K - University Of Maryland

Submitted to: Joint Abstracts of the American Dairy Science and Society of Animal Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/18/2014
Publication Date: 7/18/2014
Citation: Garcis, M., Elsasser, T.H., Biswas, D., Moyes, K. 2014. The effect of citrus-derived oil on bovine blood neutrophil response in vitro. Joint Abstracts of the American Dairy Science and Society of Animal Science. 64.

Interpretive Summary: Concerns have emerged regarding the potential for antimicrobial drugs to lose effectiveness against infectious pathogens because of the capabilities of some microbes that become resistant to the actions of the drug to proliferate and spread. Consequently there is a large thrust in research towards identifying new compounds that can either replace traditional antibiotics or complement the present use of antibiotic drugs and preserve their efficacy. Oils derived from rind of citrus fruit have been demonstrated to be effective in decontaminating microbes from solid surfaces like countertops. Unrefined citrus oils, however, contain terpenes, a class of compound relatively injurious to delicate cells. If a compound is to be effective as an alternative to a traditional antibiotic drug, not only must it have some level of antimicrobial activity but it also must not be harmful to the immune cells of the host that are also needed to control infection. We determined whether a citrus oil product derived from the rind of Valencia oranges and processed to eliminate terpenes altered the functional capacity of immune cells in the blood (called polymorphonuclear leukocytes). Testing a very low dose (0.01%) in cultured cells, we established that the orange oil did not harm the functions of the cells and, in fact, increased their capability to be called to sites of infection, a process called chemotaxis. The data suggest that low concentrations of Valencia orange oil might be an effective strategy to use in animal diseases like mammary gland infection as an alternative to antibiotic use.

Technical Abstract: Research on the use of products with germicidal activity to treat or prevent microbial invasion as alternatives to antibiotic drug use is expanding. To our knowledge, potential for cold-pressed terpeneless Valencia orange oil (TCO) to control mastitis in dairy cows as an alternative therapy has not been explored. Therefore, the objective of this study was to investigate the effect of TCO on bovine blood polymorphonuclear leukocyte (PMNL) chemotaxis and phagocytosis capabilities and the expression of genes involved in inflammatory response in vitro. Polymorphonuclear leukocytes were isolated from jugular blood of 12 Holstein cows in mid-lactation and were incubated with 0.0 or 0.01% TCO for 120 min at 37ºC and 5% CO2 before performing the in vitro phagocytosis (2 × 106 PMNL) and chemotaxis (6 × 106 PMNL) assays. For TCO effects on gene expression, RNA was extracted from incubated PMNL (6 × 106 PMNL) and was analyzed using qPCR. The supernanant was stored at -80ºC for quantification of tumor necrosis factor-a (TNF- a). Supplementation of cells with 0.01% of TCO did not change the capacity of PMNL to phagocytize fluorescent microspheres. The TCO treatment selectively increased the chemotactic movement responses to interleukin-8 by 47% but not that stimulated by complement C5a. Media concentrations of TNF- a were not altered by TCO treatment. Expression of pro-inflammatory genes (i.e. SELL, TLR4, IRAK1, TRAF6, and LYZ) coding for proteins known to aid PMNL to fight against pathogen invasions were not altered by incubation of PMNL with TCO. However, down-regulation of TLR2 (fold change (FC; treatment/control) = -2.14), NFKBIA (FC = 1.82), IL1B (FC = -2.16), TNFA (FC = -9.43), and SOD2 (-1.57) was observed for PMNL incubated with TCO when compared to controls. Interestingly, expression of IL10, a well known anti-inflammatory cytokine was also down-regulated (-FC = -3.78) whereas expression of IL8 (FC = 1.93), a gene coding for the cytokine IL-8 known for its chemotactic function, tended to be up-regulated in PMNL incubated with TCO. Results show that 0.01% TCO does not impair or compromise the function of PMNL as tested in vitro. Future studies investigating the use of TCO as an alternative therapy for treatment of mastitis, including dose and duration, for cows during lactation are warranted.