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.

Capacity of adherence, invasion and intracellular survival of Streptococcus uberis biofilm-forming strains.

AIM: Nine Streptococcus uberis strains with different biofilm-forming profiles in relation to their capacity of adherence and invasion to MAC-T cell lines were examined. Additionally, virulence genes were also linked to adherence and invasion. METHODS AND RESULTS: All S. uberis were able to adhere and invade the cells at different levels. UB56 strain showed the highest percentage of internalization (3.65%) and presented a moderate level of adhesion (4.6 × 106 ). In contrast, UB152, the most adherent strain (8.7 × 106 ) showed a low capacity to internalize (0.65%). Eight strains were able to persist intracellularly over 96 h regardless of their adherence or invasion level. Statistical analysis between biofilm-forming ability and the adhesion capacity showed no significant differences. Presence of virulence genes involved in the adhesion process (gapC, hasABC, lbp, pauA and sua) showed that the strains harboured different genes and seven patterns could be observed. CONCLUSION: Statistical analysis showed no correlation between the virulence gene patterns and the adhesion capacity or the percentage of internalization. Biofilm-forming ability did not influence the invasion capacity. Likewise, adherence and invasion capacity may be strain dependent. SIGNIFICANCE AND IMPACT OF THE STUDY: Findings from this study provide new insights on biofilm and invasion capacity of S. uberis strains. Results could help to design adequate control strategies.

Screening of bacteriocin associated genes of Streptococcus uberis strains.

A wide variety of contagious and environmental bacteria can cause bovine mastitis worldwide. Antibiotic therapy is currently used for the treatment of the disease, although its intensive use leads to the emergence of resistant strains. Bacteriocins arise as potential antibacterial option for mastitis treatment. The aim of this work was to analyze bacteriocin associated genes as Streptococcus uberis ubericin A (ubaA), ubericin A immunity protein (ubaI), uberolysin A (ublA), Lantibiotic nisin-U (nsuA and nsuB) in 68 S uberis strains. Furthermore, the ability of the strains to inhibit important mastitis pathogens was assayed. Results showed that genes were present in combination and all the strains carried at least one gene. Seven bacteriocion associated gene patterns were identified. S. uberis strains were able to inhibit different mastitis pathogens and the greatest inhibition was observed in CNS strains. The results obtained provide new insights on antibacterial activity produced by S. uberis strains against different mastitis pathogens and could contribute to the development of strategies to treat intramammary infections.

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.

Evaluation of environmental and nutritional factors and sua gene on in vitro biofilm formation of Streptococcus uberis isolates.

The pathogenesis of Streptococcus uberis is attributed to a combination of extracellular factors and properties such as adherence and biofilm formation. The aim of this work was to evaluate the influence of different factors, additives and bovine milk compounds on S. uberis biofilm formation, as the presence of the sua gene by PCR. Additionally, extracellular DNA and the effect of DNaseI were evaluated in the biofilms yielded. Optimal biofilm development was observed when the pH was adjusted to 7.0 and 37 °C. Additives as glucose and lactose reduced biofilm formation as bovine milk compounds tested. PCR assay showed that not all the isolates yielded sua gene. Extrachromosomal ADN was found in cell-free supernatants, suggesting that DNA released spontaneously to the medium. The results contribute to a better understanding of the factors involved in biofilm production of this important pathogen associated with mastitis in order to promote the design of new therapeutic approaches.