Assessment of the in vitro acaricidal activity of Bravecto® (fluralaner) and a proposed orange oil-based formulation vehicle for the treatment of Sarcoptes scabiei.

BACKGROUND: Sarcoptic mange is a serious animal welfare concern in bare-nosed wombats (Vombatus ursinus). Fluralaner (Bravecto®) is a novel acaricide that has recently been utilised for treating mange in wombats. The topical 'spot-on' formulation of fluralaner can limit treatment delivery options in situ, but dilution to a volume for 'pour-on' delivery is one practicable solution. This study investigated the in vitro acaricidal activity of Bravecto, a proposed essential oil-based diluent (Orange Power®), and two of its active constituents, limonene and citral, against Sarcoptes scabiei. METHODS: Sarcoptes scabiei were sourced from experimentally infested pigs. In vitro assays were performed to determine the lethal concentration (LC50) and survival time of the mites when exposed to varying concentrations of the test solutions. RESULTS: All compounds were highly effective at killing mites in vitro. The LC50 values of Bravecto, Orange Power, limonene and citral at 1 h were 14.61 mg/ml, 4.50%, 26.53% and 0.76%, respectively. The median survival times of mites exposed to undiluted Bravecto, Orange Power and their combination were 15, 5 and 10 min, respectively. A pilot survival assay of mites collected from a mange-affected wombat showed survival times of < 10 min when exposed to Bravecto and Orange Power and 20 min when exposed to moxidectin. CONCLUSIONS: These results confirm the acaricidal properties of Bravecto, demonstrate acaricidal properties of Orange Power and support the potential suitability of Orange Power and its active constituents as a diluent for Bravecto. As well as killing mites via direct exposure, Orange Power could potentially enhance the topical delivery of Bravecto to wombats by increasing drug penetration in hyperkeratotic crusts. Further research evaluating the physiochemical properties and modes of action of Orange Power and its constituents as a formulation vehicle would be of value.

Impact of Oral Phytozen EQ Supplementation on Plasma Cortisol and Behavior Responses of Young Horses Exposed to Stressful Stimuli.

Calming supplements are common in the equine industry. This study tested the hypothesis that Phytozen EQ, a blend of citrus botanical oils, magnesium, and yeast would reduce startle response as well as reduce behavioral and physiological signs of stress in young (1.5-6 years of age) horses (n = 14) when tied in isolation and when trailered in isolation. During the 59-day trial, horses were assigned to either the control (CON; n = 7) or treatment (PZEN; n = 7) group that received 56 g of Phytozen EQ daily. Horses underwent a 10-minute isolation test on d 30 and a 15 minute individual trailering test on day 52 or 55. For both tests, blood samples were obtained pre, immediately after, and 1-hour post for analysis of plasma cortisol concentrations, which were analyzed by repeated measures ANOVA. On day 59, horses underwent a startle test, for which time to travel 3 m and total distance traveled were recorded. These data were analyzed using a T-test. During trailering, PZEN horses tended to have lower overall geometric mean (lower, upper 95% confidence interval) cortisol concentrations than CON (81 [67, 98] vs. 61 [48, 78] ng/mL; P = .071). For the startle test, PZEN horses tended to have longer geometric mean times to travel 3 m than CON horses (1.35 [0.39, 4.70] vs. 0.26 [0.07, 0.91 seconds, P = 0.064). Other data points were not different between treatments (P > .1). It is possible that this dietary supplement could have beneficial calming effects on horses undergoing trailering or in novel situations.

Unveiling the Potential Ways to Apply Citrus Oil to Control Causative Agents of Pullorum Disease and Fowl Typhoid in Floor Materials.

This study investigates the potential role of Cold-pressed Valencia Terpeneless citrus oil (CO), as a natural antimicrobial, in controlling causative agents of pullorum disease and fowl typhoid in floor materials for poultry farming, specifically wooden chips. The study addresses the issues that have arisen as a result of the reduction in antibiotic use in poultry farming, which has resulted in the re-emergence of bacterial diseases including salmonellosis. CO efficiently inhibits the growth of pathogens including various serovars of Salmonella enterica (SE), including SE serovar Gallinarum (S. Gallinarum) and SE serovar Pullorum (S. Pullorum), in a dose-dependent manner. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of CO showed potential for controlling diverse S. Gallinarum and S. Pullorum isolates. Growth inhibition assays demonstrated that 0.4% (v/w) CO eliminated S. Pullorum and S. Gallinarum from 24 h onwards, also impacting poultry gut microbiota and probiotic strains. Floor material simulation, specifically wooden chips treated with 0.4% CO, confirmed CO's effectiveness in preventing S. Gallinarum and S. Pullorum growth on poultry house floors. This study also investigated the effect of CO on the expression of virulence genes in S. Gallinarum and S. Pullorum. Specifically, the study revealed that the application of CO resulted in a downregulation trend in virulence genes, including spiA, invA, spaN, sitC, and sifA, in both S. Pullorum and S. Gallinarum, implying that CO may alter the pathogenicity of these bacterial pathogens. Overall, this study reveals that CO has the potential to be used as a natural antimicrobial in the prevention and management of Salmonella-related infections in chicken production, offering a viable alternative to control these re-emerging diseases.

Effects of Molecular Distillation on the Chemical Components, Cleaning, and Antibacterial Abilities of Four Different Citrus Oils.

Essential oils (EOs) from citrus fruits are excellent aromatic resources that are used in food, cosmetics, perfume, and cleaning products. EOs extracted from four citrus varieties, sweet orange, grapefruit, mandarin, and lemon, were separated into two fractions by molecular distillation. The composition, physicochemical properties, cleaning ability, and antimicrobial activity of each EO were then systematically evaluated. The relationships between each of the aforementioned characteristics are also discussed. In keeping with the principle of "like dissolves like," most citrus EOs show better cleaning ability than acetone and all tend to dissolve the fat-soluble pigment. The key components of citrus EOs are 1-Decanol, α-terpineol, geraniol, and linalool for the inhibition of Staphylococcus aureus, Escherichia coli, Candida albicans, and Vibrio parahaemolyticus, respectively. The findings of this study will be of significant importance for the effective utilization of citrus peel resources and in the development of future applications for citrus EOs. Chemical Compounds Studied in This Article: (+)-α-Pinene (PubChem CID: 6654); ß-Phellandrene (PubChem CID: 11142); 3-Carene (PubChem CID: 26049); ß-Myrcene (PubChem CID: 31253); D-Limonene (PubChem CID: 440917); γ-Terpinene (PubChem CID: 7461); Octanal (PubChem CID: 454); Decanal (PubChem CID: 8175); Linalool (PubChem CID: 6549); 1-Octanol (PubChem CID: 957); ß-Citral (PubChem CID: 643779); α-Terpineol (PubChem CID: 17100); Hedycaryol (PubChem CID: 5365392); α-Citral (PubChem CID: 638011); 1-Decanol (PubChem CID: 8174); Geraniol (PubChem CID: 637566).

The stability of three different citrus oil-in-water emulsions fabricated by spontaneous emulsification.

In this study, emulsions were prepared through spontaneous emulsification, using three different citrus oils as the oil phase and Tween 80 as the surfactant. Utilizing 4% Tween 80, three types of citrus oil emulsions were prepared with small particle size, monomodal distribution and high transmission. After 24 h, each emulsion exhibited different degrees of gravitational separation. Mandarin oil emulsions were the most unstable, showing coalescence of small droplets with an obvious cream layer formed at 9 h. Bergamot oil emulsions possessed small droplets with the best stability over 24 h, due to their relatively polar components (e.g. linalyl acetate) and water-insoluble constituents (e.g. γ-terpinene). These results suggest that the emulsifying properties and instability mechanism of citrus oil emulsions are strongly dependent on the inherent properties and composition of citrus oils. This study is significant for the development of an effective strategy to improve the stability of citrus oil-based colloidal systems.

Physicochemical and Sensory Properties of Cold Pressed Oils from Florida Hamlin and Valencia Oranges Affected by Huanglongbing.

Cold pressed oils from huanglongbing (HLB) symptomatic (SY) and asymptomatic (AS) Hamlin and Valencia oranges were assessed for 2 y (2014 to 2015 and 2015 to 2016 seasons) with 2 harvest dates for each orange variety per year. Physicochemical properties (optical rotation, aldehyde content, ultraviolet [UV] absorbance, refractive index, and specific gravity) mandated by the United States Pharmacopeia (USP) for orange oil quality were assessed. Hamlin and Valencia oils showed minor differences in physicochemical properties based upon disease stage. However, all Hamlin oils had aldehyde contents below the USP minimum and Valencia oil from late season SY oranges had specific gravities above the USP maximum. Significant differences based on harvest year were seen for aldehyde content, refractive index, optical rotation, and UV absorbance. While none of these changes led to an oil being out of USP specifications, they indicate a need to monitor the quality of oil every year to ensure a consistent product. Flavor taste panels were performed both years by adding 0.035% oil samples to a uniform orange juice base. Aroma panels were done by smelling pure oil. There were no significant differences between SY and AS oils for flavor, although panelist race was a significant factor in several of the panels. There were significant differences between the aroma of SY and AS oils for both 2015 to 2016 Hamlin Early and Valencia Late samples. Overall, these results show HLB can have an effect on the aroma and USP mandated physicochemical properties of Florida orange oils, although flavor may be unaffected by this plant disease.

Encapsulation of Polymethoxyflavones in Citrus Oil Emulsion-Based Delivery Systems.

The purpose of this work was to elucidate the effects of citrus oil type on polymethoxyflavone (PMF) solubility and on the physicochemical properties of PMF-loaded emulsion-based delivery systems. Citrus oils were extracted from mandarin, orange, sweet orange, and bergamot. The major constituents were determined by GC/MS: sweet orange oil (97.4% d-limonene); mandarin oil (72.4% d-limonene); orange oil (67.2% d-limonene); and bergamot oil (34.6% linalyl acetate and 25.3% d-limonene). PMF-loaded emulsions were fabricated using 10% oil phase (containing 0.1% w/v nobiletin or tangeretin) and 90% aqueous phase (containing 1% w/v Tween 80) using high-pressure homogenization. Delivery systems prepared using mandarin oil had the largest mean droplet diameters (386 or 400 nm), followed by orange oil (338 or 390 nm), bergamot oil (129 or 133 nm), and sweet orange oil (122 or 126 nm) for nobiletin- or tangeretin-loaded emulsions, respectively. The optical clarity of the emulsions increased with decreasing droplet size due to reduced light scattering. The viscosities of the emulsions (with or without PMFs) were similar (1.3 to 1.4 mPa·s), despite appreciable differences in oil phase viscosity. The loading capacity and encapsulation efficiency of the emulsions depended on carrier oil type, with bergamot oil giving the highest loading capacity. In summary, differences in the composition and physical characteristics of citrus oils led to PMF-loaded emulsions with different encapsulation and physicochemical characteristics. These results will facilitate the rational design of emulsion-based delivery systems for encapsulation of PMFs and other nutraceuticals in functional foods and beverages.

Citrus-derived oil inhibits Staphylococcus aureus growth and alters its interactions with bovine mammary cells.

This experiment examined the effects of cold-pressed, terpeneless citrus-derived oil (CDO) on growth of Staphylococcus aureus, which a major cause of contagious bovine mastitis, and invasion of bovine mammary cells (MAC-T). To determine minimum inhibitory concentration, we used the broth dilution method, using CDO concentrations range from 0.0125 to 0.4% with 2-fold dilutions. Growth inhibition was examined by adding 0.00, 0.05, 0.025, 0.0125, and 0.00625% CDO to 10(5) cfu/mL S. aureus in nutrient broth and enumerating colonies after serial dilution. In a 96-well plate, S. aureus (10(7) cfu/mL) was allowed to form a biofilm, treated with 0, 0.025, 0.5, or 1% CDO, and then was measured using a spectrophotometer. Cytotoxic effect on immortalized MAC-T cells was also examined at various concentrations of CDO using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. We observed that the minimum inhibitory concentration of CDO to inhibit the growth of S. aureus in vitro was 0.025% CDO. A time kill curve for CDO action on S. aureus over 4h was generated. The CDO completely eliminated S. aureus after 3h of incubation at a concentration of 0.25%, or after 2h of incubation at concentrations of 0.05%. It was also observed that CDO had no effect on preformed biofilms except at a concentration of 0.05%, in which a significant reduction in the measured absorbance was noted. In addition, the association and invasion of S. aureus to MAC-T cells were significantly inhibited after 1h of treatment with CDO. Citrus-derived oil was also able to increase cellular proliferation of MAC-T cells at concentrations up 0.05% and had no effect at a concentration of 0.1% after 1 h. Our data suggests that CDO should be considered for further research as a preventive and therapeutic against bovine mastitis.

The effect of citrus-derived oil on bovine blood neutrophil function and gene expression in vitro.

Research on the use of natural products to treat or prevent microbial invasion as alternatives to antibiotic use is growing. Polymorphonuclear leukocytes (PMNL) play a vital role with regard to the innate immune response that affects severity or duration of mastitis. To our knowledge, effect of cold-pressed terpeneless Valencia orange oil (TCO) on bovine PMNL function has not been elucidated. Therefore, the objective of this study was to investigate the effect of TCO on bovine blood 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 120min at 37°C and 5% CO2, and phagocytosis (2×10(6) PMNL) and chemotaxis (6×10(6) PMNL) assays were then performed in vitro. For gene expression, RNA was extracted from incubated PMNL (6×10(6) PMNL), and gene expression was analyzed using quantitative PCR. The supernatant was stored at -80°C for analysis of tumor necrosis factor-α. Data were analyzed using a general linear mixed model with cow and treatment (i.e., control or TCO) in the model statement. In vitro supplementation of 0.01% of TCO increased the chemotactic ability to IL-8 by 47%; however, migration of PMNL to complement 5a was not altered. Treatment did not affect the production of tumor necrosis factor-α by PMNL. Expression of proinflammatory genes (i.e., SELL, TLR4, IRAK1, TRAF6, and LYZ) coding for proteins was not altered by incubation of PMNL with TCO. However, downregulation of TLR2 [fold change (FC=treatment/control)=-2.14], NFKBIA (FC=1.82), IL1B (FC=-2.16), TNFA (FC=-9.43), and SOD2 (FC=-1.57) was observed for PMNL incubated with TCO when compared with controls. Interestingly, expression of IL10, a well-known antiinflammatory cytokine, was also downregulated (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 upregulated in PMNL incubated with TCO. Incubation of PMNL with TCO enhanced PMNL chemotaxis in vitro. The expression of genes involved in the inflammatory response was primarily downregulated. Results showed that 0.01% TCO did not impair the function of PMNL 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.

Authenticity analysis of citrus essential oils by HPLC-UV-MS on oxygenated heterocyclic components.

Citrus essential oils are widely applied in food industry as the backbone of citrus flavors. Unfortunately, due to relatively simple chemical composition and tremendous price differences among citrus species, adulteration has been plaguing the industry since its inception. Skilled blenders are capable of making blends that are almost indistinguishable from authentic oils through conventional gas chromatography analysis. A reversed-phase high performance liquid chromatography (HPLC) method was developed for compositional study of nonvolatile constituents in essential oils from major citrus species. The nonvolatile oxygenated heterocyclic components identified in citrus oils were proved to be more effective as markers in adulteration detection than the volatile components. Authors are hoping such an analysis procedure can be served as a routine quality control test for authenticity evaluation in citrus essential oils.

Inhibition of growth and alteration of host cell interactions of Pasteurella multocida with natural byproducts.

Pasteurella multocida is a leading cause of fowl cholera in both free-range pasture and conventional/commercially raised poultry. Its infection is a serious threat to poultry health and overall flock viability. Organic poultry is comparatively more vulnerable to this pathogen. It is a significant cause of production loss and price increase of poultry products, specifically organic poultry products. Some plant products are well documented as sources of natural antimicrobials such as polyphenols found in different berry pomaces and citrus oil. Pomace, a byproduct (primarily of seeds and skins) of fruits used for juice and wine production, and citrus oil, the byproduct of citrus juice production, show promising antimicrobial activity against various pathogens. Here, we showed for the first time that blackberry and blueberry pomace extracts and citrus oil inhibited P. multocida growth. Minimum bactericidal concentrations were determined as 0.3 and 0.4 mg/mL gallic acid equivalent for blackberry and blueberry pomace extracts, respectively. Similarly, only 0.05% citrus oil (vol/vol) completely inhibited P. multocida growth. Under shaking conditions, the antimicrobial activity of both pomace extracts and citrus oil was more intensive. Even citrus oil vapor also significantly reduced the growth of P. multocida. In addition, cell surface hydrophobicity of P. multocida was increased by 2- to 3-fold and its adherence to chicken fibroblast (DF1) and bovine mammary gland (MacT) cells was reduced significantly in the presence of pomace extracts only. This study indicates that these natural products might be good alternatives to conventional antimicrobial agents, and hence, may be used as feed or water supplements to control fowl cholera and reduce production loss caused by P. multocida.