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

Res Vet Sci. 2022 Dec;149:136-150. doi: 10.1016/j.rvsc.2022.04.014. Epub 2022 Jun 28.


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.

Keywords: Adjuvants; Bovine mastitis vaccines; Limonene; Microencapsulant polymers; Minthostachys verticillata essential oil.

MeSH terms

  • Adjuvants, Immunologic / pharmacology
  • Animals
  • CD8-Positive T-Lymphocytes
  • Cattle
  • Cattle Diseases*
  • Female
  • Immunoglobulin G
  • Lamiaceae* / chemistry
  • Limonene
  • Mastitis, Bovine* / microbiology
  • Mastitis, Bovine* / prevention & control
  • Mice
  • Oils, Volatile* / chemistry
  • Oils, Volatile* / pharmacology
  • Rodent Diseases*
  • Vaccines*


  • Adjuvants, Immunologic
  • Immunoglobulin G
  • Oils, Volatile
  • Vaccines
  • Limonene