In Vitro Activity of 30 Essential Oils against Bovine Clinical Isolates of Prototheca zopfii and Prototheca blaschkeae

Protothecal mastitis poses an emergent animal health problem in dairy herds, with a high impact on dairy industries, causing heavy economic losses. Current methods of treating protothecal infections are ineffective, and no drug is licensed for use in cattle. The aim of the present study was to check the antialgal activity of 30 chemically defined essential oils (EOs) against Prototheca zopfii and Prototheca blaschkeae isolated from the milk of dairy cows with mastitis. A microdilution test was carried out to estimate the antialgal effectiveness of the selected chemically defined EOs. The microdilution test showed different degrees of inhibition among the examined Prototheca species. The activity of some of the examined EOs seem interesting. In particular, Citrus paradisi yielded the lowest minimal inhibitory concentration values (0.75%) for both algal species. P. zopfii appeared to be more sensitive to EOs in comparison to P. blaschkeae. The present study investigated the in vitro susceptibility of P. zopfii and P. blaschkeae to a wide range of EOs, obtained from different botanical families. Further investigations are necessary to evaluate the efficacy of EO-based formulations intended for the disinfection of both udder and milking products.


Results
All the identified compounds in the 30 EOs are listed in Table 1, while the main class of constituents are reported in Table 2. The total identified volatile fractions ranged from 86.2% in S. sclarea to 100.0% in C. aurantium, C. bergamia, C. lemon, C. aurantium var. dulcis, I. verum, L. hybrida, and S. album. The composition of all the EOs revealed more than 290 compounds; however, only those with a percentage of 5% or more are reported in Table 1. The main compounds were represented by monoterpene hydrocarbons and oxygenated monoterpenes, while oxygenated sesquiterpenes were abundant only in S. album (88.8%). I. verum, P. abies, and S. album showed high percentages of phenylpropanoids (above 90%) which are also the main class of compounds found in F. vulgare, although in a lower percentage (56.5%). Non-terpenes were present in high amounts in A. nobilis (esters, 75.6%) and C. zeylanicum (56.5%).
Statistical analysis was used to assess the high volume of information provided by the EOs' composition. The classes of compounds in the EOs were subjected to multivariate analysis. Hierarchical cluster analysis HCA showed the presence of two main groups ( Figure 1). The first group (A) includes the following oils: S. sclarea, T. capitatus, P. graveolens, A. graveolens, C. citratus, L. hybrida, H. italicum, O. basilicum, S. montana, T. vulgaris, O. majorana, L. cubeba, and O. vulgare. The second group (B) included all remaining oils, and it was further divided into two subgroups: B2 with EOs from C. zeylanicum, A. nobilis, S. album, S. aromaticum, P. anisum, I. verum, and F. vulgare, and B1 with the other EOs. running the assay in triplicate. Conventional, commonly employed anti-Prototheca drugs (i.e., amphotericin B, voriconazole, and posaconazole) were also used. In detail, the in vitro susceptibility of the two algal species was assessed by E-test [32].

Results
All the identified compounds in the 30 EOs are listed in Table 1, while the main class of constituents are reported in Table 2. The total identified volatile fractions ranged from 86.2% in S. sclarea to 100.0% in C. aurantium, C. bergamia, C. lemon, C. aurantium var. dulcis, I. verum, L. hybrida, and S. album. The composition of all the EOs revealed more than 290 compounds; however, only those with a percentage of 5% or more are reported in Table 1. The main compounds were represented by monoterpene hydrocarbons and oxygenated monoterpenes, while oxygenated sesquiterpenes were abundant only in S. album (88.8%). I. verum, P. abies, and S. album showed high percentages of phenylpropanoids (above 90%) which are also the main class of compounds found in F. vulgare, although in a lower percentage (56.5%). Non-terpenes were present in high amounts in A. nobilis (esters, 75.6%) and C. zeylanicum (56.5%).
Statistical analysis was used to assess the high volume of information provided by the EOs' composition. The classes of compounds in the EOs were subjected to multivariate analysis. Hierarchical cluster analysis HCA showed the presence of two main groups ( Figure 1).   Principal compound analysis (PCA), where the first axis (PC1) explained for 45.4% and the second axis (PC2) for 33.0% (which resumed 78.4% of the total variability, Figure 2), evidenced that oxygenated monoterpenes (OMs) was the major class of compounds in all the EOs belonging to group A in HCA, and its percentages ranged from 40.6% in H. italicum EO to 89.2% in T. capitatus EO. The EOs that showed high amounts of phenylpropanoids were located in the upper-right quadrant of the PCA and referred to the plants present in subgroup B2 of the HCA, where P. anisum, I. verum, and S. aromaticum were characterized by the highest percentage of phenylpropanoids (up to 90%), while C. zeylanicum and A. nobilis oils evidenced relevant amounts of non-terpene derivatives (57.9% and 75.6% respectively). S. album EO that belonged to the same subgroup B2 was characterized by the highest percentage of oxygenated sesquiterpenes (88.8%). The presence of monoterpene hydrocarbons was predominant in the EOs of the plants inserted in subgroup B1, especially for all Citrus spp. (up to 90%), except for C. bergamia, which was characterized by an equivalent amount of monoterpene hydrocarbons and oxygenated monoterpenes.      Single compounds that showed a percentage of less than 5% in at least one of the tested EOs are removed from this      The microdilution test showed different degrees of inhibition versus the examined Prototheca species. C. paradisi yielded the lowest MIC values (0.75%) for both algal species. P. zopfii appeared to be more sensitive to EOs, in comparison to P. blaschkeae. T. vulgaris, L. cubeba, and O. vulgare were effective at 0.75% versus P. zopfii, and at 1% versus P. blaschkeae. More detailed data are reported in Table 3.

Discussion
The present study is the first to investigate the in vitro susceptibility of P. zopfii and P. blaschkeae to a wide range of EOs, obtained from different botanical families.
C. paradisi appeared to possess the most effective EO against both algal species. The activity of this EO against Prototheca spp. had not previously been investigated, and for this reason, our results cannot be compared to already published data.
However, according to comparable results referring to P. zopfii's susceptibility to EOs, MIC values obtained from our microdilution assay seemed to be higher, when compared to those already recorded. In fact, Tortorano et al. [15] reported MICs ranging from 0.03% to 0.12% for Melaleuca alternifolia, while MIC values for C. bergamia were in agreement with data obtained in the present study.
Grzesiak et al. [26] described C. zeylanicum EO as the most effective against P. zopfii, followed by T. vulgaris, S. aromaticum, and S. sclarea, with MICs ranging from 0.02% to 0.25%. However, these authors performed the assay following a different method than that implemented in the present study and by Tortorano et al. [15], making a comparison among the different results obtained difficult.
As indicated, T. vulgaris, L. cubeba, and O. vulgare exhibited an inhibitory effect at the level of 0.75% and 1% against P. zopfii and P. blaschkeae, respectively.
The main component of C. paradisi is limonene. Monoterpenes such as limonene and thymol can exert damage on membrane-embedded enzymes, modify fatty acid composition [33], and affect the respiration and permeability of cell membranes [34]. Furthermore, these monoterpenes are inhibitors of pectin methyl esterase and cellulase, causing consequent damage to fungal cell walls [35]. Limonene, in particular, has also been proven to alter the structure and function of the cell wall in Saccharomyces cerevisiae [36].
The activity of some of the examined EOs seem interesting, considering that no drug is currently licensed for use in bovine protothecosis. These compounds could be evaluated for topical administration in a dense excipient for an endomammary treatment of affected cows. The MIC values obtained for the above-mentioned EOs may be employed to treat mammary glands without damaging epithelial cells. Moreover, EOs could be used as disinfectants during milk processing by ensuring a high hygiene level. Protothecal mastitis can in fact be a consequence of poor hygienic conditions, and the search for effective products against these algae is on the rise. The efficacy of some teat disinfectants containing iodine, quaternary ammonium compounds, and dodecylbenzene sulphonic acid has been reported [37], indicating iodine as the most suitable disinfectant. Guanidine is a further compound which is effective both as an antiseptic for human wounds and as a surface disinfectant yielding algicidal activity at low concentration on P. zopfii [8]. More recently, the in vitro effect of iodopropynyl butylcarbamate alone and in combination with amphotericin B was assessed on P. zopfii and P. blaschkeae isolates obtained from dairy herds of different European countries, showing a satisfactory anti-Prototheca activity [38].
All of the above-mentioned products are proven to be active versus P. zopfii, but to the best of our knowledge only Jagielski et al. [38] provided information on P. blaschkeae susceptibility. Although not frequently, this species is also reported as responsible for bovine mastitis outbreaks [39].
Further investigations are needed to evaluate the efficacy of EO-based formulations in the disinfection of both udder and milking products.

Conclusions
Some among the examined EOs-in particular C. paradisi EO-showed interesting antialgal activity against both P. zopfii and P. blaschkeae. This finding could contribute to broaden the informational outlook on the unconventional treatment of both mastitic cows and dairy environments.

Conflicts of Interest:
The authors declare no conflict of interest.