Spray-drying-microencapsulated Minthostachys verticillata essential oil and limonene as innovative adjuvant strategy to bovine mastitis vaccines.

Design of innovative adjuvant strategies with an appropriate safety profile is relevant to developed subunit or inactivated microorganism vaccines for bovine mastitis. Minthostachys verticillata essential oil (EO) has demonstrated ability to stimulate the innate immune response and adjuvant effect similar to Al(OH)3. Here we evaluated the adjuvant effect of EO and its metabolite, limonene (L) alone and microencapsulated by spray-drying, using an inactivated Enterococcus faecium strain bovine-mastitis inducer. The gas chromatography-mass spectrometry analysis showed that microencapsulation process did not alter the EO or L chemistry. Microencapsulated EO (McEO) or L (McL) (2.0, 2.5 and 5.0 mg/ml) decreased the viability of bovine mammary gland epithelial cells in a dose-dependent way. Balb/c mice (n = 32) were subcutaneously inoculated (day 0) and revaccinated (day 14 and 28) with saline solution, inactivated bacteria alone or combined with Incomplete Freund's Adjuvant; EO or L (2.5 mg/ml); McEO or McL (5.0 mg/ml); or microcapsule wall material (Mc) alone (2.5 mg/ml). EO, L, McEO and McL stimulated E. faecium-specific IgG (IgG1 or IgG2a) with opsonizing capacity and increased the proportion of CD4+ and CD8+ T cells producers of IFN-γ. Microencapsulation was an effective strategy to increase the adjuvant potential of EO or L. These new adjuvants deserve further study to evaluate their incorporation into vaccines for bovine mastitis.

Minthostachys verticillata essential oil activates macrophage phagocytosis and modulates the innate immune response in a murine model of Enterococcus faecium mastitis.

Minthostachys verticillata essential oil (EO) is a natural product that reports immunomodulatory effects on human T cells as well as anti-inflammatory activity. Bovine mastitis is a worldwide disease, mainly caused by bacteria, affecting milk quality and yield, leading to high economic losses. Environmental pathogens, as Enterococcus faecium, are implicated in the disease. Antibiotic therapy is adequate, although it can leave residues in milk, causing problems in human health. The search of immunomodulatory substances for bovine mastitis treatment is a promising alternative strategy. The aim of this study was to characterize the effect of M. verticillata EO on macrophage phagocytosis and evaluate its immunomodulatory and protective effects in mice challenged with E. faecium. The results showed that EO activated macrophage phagocytosis mechanisms inducing reactive oxygen species production. Moreover, EO modulated the innate immune response in mammary glands of female Balb/c mice challenged with E. faecium decreasing the infiltration of polymorphonuclear neutrophils and IL-1ß and TNF-α mRNA expression. In addition, EO increased the expression of IL-10 in the last hours of infection. Treatment with EO did not increase the number of activated CD4+ or CD8+ T cells or the production of specific antibodies. These results suggest that EO play an important role in helping to resolve the infection in the first hours without activating adaptive immunity. In addition, a marked decrease of the bacterial count in the glands of mice treated with EO was observed. A natural product such as M. verticillata EO could have a potential use to control bovine mastitis.

Effect of the Essential Oil of Minthostachys verticillata (Griseb.) Epling and Limonene on Biofilm Production in Pathogens Causing Bovine Mastitis.

Bovine mastitis causes large annual economic losses around the world. Different microorganisms are associated with the disease. The capacity of pathogens to adhere to bovine mammary epithelial cells is associated with biofilm production which leads to antibiotic resistance. Research is now leading to search alternative control methods and medicinal plants constitute a natural, safe, effective and inexpensive option. Minthostachys verticillata is an autochthonous medicinal plant of Argentina with multiple ethnobotanical properties. In a previous study, we demonstrated that the essential oil (EO) of this species and limonene, one of its compounds, inhibited the growth of mastitis pathogens. The objective of the present work was to determine the inhibitory effect of the essential oil of M. verticillata and limonene, on biofilm formation and on mature biofilm produced by pathogens isolated from bovine mastitis. Time kill assay and bacterial lysis were also determined. Furthermore, RAPD-PCR assays were performed to determine changes in bacterial DNA after EO and limonene exposition. Bacterial isolates were identified as Escherichia coli (EC3 and EC9), Bacillus pumilus (BP5, BP6, and BP7) and Enterococcus faecium (EF1) by rRNA 16S sequencing and MALDI-TOF MS. All the strains were able to form biofilm. Addition of both lactose and sucrose did not affect biofilm production. MIC values for EO were 3.6 mg/ml for E. faecium; 0.9 mg/ml for E. coli (EC3), 14.5 mg/ml for E. coli (EC9), 1.8 mg/ml for B. pumilus (BP7), 3.63 mg/ml for B. pumilus (BP6) and 29.0 mg/ml for B. pumilus (BP7). MIC values for limonene were 6.6 mg/ml for B. pumilus (BP6) and 105 mg/ml for B. pumilus (BP5). These results demonstrated that EO was more effective than limonene, showing also bactericidal action against E. faecium (minimal inhibitory concentration (MBC) = 29.0 mg/ml). This result was corroborated by time of death assay, observing a cell decrease after at 6 h, and then by bacterial lysis assay. Both EO and limonene affected mature biofilm of isolated strains. The results contribute to the study of EO and limonene which may serve as a therapy against bovine mastitis pathogens inhibiting the development of pathogenic bacteria.