Anthelmintic Properties of Essential Oils to Control Gastrointestinal Nematodes in Sheep—In Vitro and In Vivo Studies

Herbal products such as essential oils may play a promising role in the treatment of infections caused by gastrointestinal nematodes (GINs). The aim of this study was to evaluate the in vitro potential of 11 essential oils (EOs) and one binary combination of isolated EO compounds, as well as the in vivo anthelmintic efficacy of two EO formulations. Four GIN genera were identified in the coproculture examination: Haemonchus, Trichostrongylus, Teladorsagia and Chabertia. The in vitro egg hatch test (EHT) was performed at six different concentrations (50, 12.5, 3.125, 0.781, 0.195 and 0.049 mg/mL) for each EO, whereas in the in vivo faecal egg count reduction test (FECRT), each EO sample was diluted in sunflower oil and orally administrated at a dose of 100 mg/kg to the different group of animals. In the EHT, the EOs of Origanum vulgare, Foeniculum vulgare, Satureja montana, Satureja hortensis and two types of Thymus vulgaris were the most effective. The dominant compounds of these EOs were carvacrol, thymol, anethol, p-cymene and γ-terpinene, indicating their importance for the anthelmintic activity. In the FECRT, both T. vulgaris EO type 1 and linalool:estragole combination show an anthelmintic potential with a mean effect on FECR of approximately 25%. The results suggest the possible role of tested EOs as anthelmintic agents in sheep farms, although further in vivo tests are needed.


Introduction
Gastrointestinal nematodes (GINs) are still the most prevalent parasites that cause disease in grazing ruminants worldwide, particularly in sheep and goats [1]. The prevalence of certain GIN genera depends on geographic and climate conditions. Generally, the most prevalent genera are Haemonchus, Trichostrongylus, Teladorsagia, Chabertia, Cooperia, study was also to evaluate the in vivo anthelmintic potential of T. vulgaris EO type 1 and linalool:estragole binary combination using the faecal egg count reduction test (FECRT) against GINs in sheep farms in order to estimate the possibility of their application in field conditions.
Qualitative and semi-quantitative chemical characterization of the essential oils was performed by gas chromatography-mass spectrometry (GC-MS) analyses, whereby the Agilent Technologies 6890 N gas chromatograph coupled with the Agilent Technologies 5975B electron ionization mass-selective detector were used. The analyses were done under the following technical conditions: injection volume of EO 1 µL; injector temperature 250 • C; split ratio 1:10; carrier gas helium; flow rate: 1 mL/min; capillary column: HP-5 (30 m × 0.25 mm, 0.25 µm); temperature program 50-270 • C; ion source temperature 230 • C; electron energy 70 eV; and quadrupole temperature 150 • C. Data were acquired in scan mode (m/z range 35-400) with a solvent delay of 2.30 min and processed using Agilent Technologies MSD ChemStation software (revision E01.01.335) combined with AMDIS (ver. 2.64) and NIST MS Search (ver. 2.0d) (Agilent Technologies, Inc., Santa Clara, CA, USA). The identification of compounds was done by comparison of mass spectra with data libraries (Wiley Registry of Mass Spectral Data, 7th ed. and NIST/EPA/NIH Mass Spectral Library 05) and confirmated by comparison of linear retention indices with literature data [34]. The relative amounts of components, expressed in percentages, were calculated by the normalization procedure according to the peak area in the total ion chromatogram.

Egg Hatch Test
The in vitro EHT was performed at the Regional Centre for Monitoring of Parasitosis (CREMOPAR), located in Eboli (Salerno), Italy in 2019. GIN eggs were recovered from faecal samples collected from the rectal ampulla of sheep with a natural-mixed infection on two farms located in that region. The samples were processed within 2 h of collection by using the recovery technique as described by Bosco et al. (2018) [35]. Firstly, faecal samples were homogenized and filtered under running water through meshes of different mesh sizes (1 mm, 250, 212 and 38 µm) to separate the nematode eggs from the faeces. Eggs retained on the last sieve were then washed and centrifuged for 3 min at 1500 RPM with distilled water, after which the supernatant was discarded. Following that, centrifugation was performed using a 40% sugar solution to float the eggs, which were then isolated into new tubes and mixed with distilled water. In the end, obtained solutions were centrifuged two more times to remove pellets and to obtain an aqueous solution with GIN eggs.
The EHT procedure was performed as described by Ferreira et al. (2018) [33], with some exceptions. Twenty-four-well plates containing aqueous solutions of approximately 150 eggs/well were used for this experiment as follows: six concentrations of each tested EO sample (50, 12.5, 3.125, 0.781, 0.195 and 0.049 mg/mL) were emulsified in Tween 80 (3%, v/v) and completed with distilled water in a final volume of 0.5 mL/well. The preparations were put on the incubator for 48 h at 27 • C, after which the number of eggs and firststage larvas (L 1 ) were counted under an inverted microscope. The obtained values were expressed as the mean percentage of the egg hatching inhibition. The positive control was thiabendazole at a concentration of 0.025 mg/mL, whilist the negative control was 3% Tween 80, v/v. The experiment was conducted twice with two replicates each, whereby obtained values were expressed as an arithmetic mean.

Faecal Egg Count Reduction Test
The in vivo FECRT was performed on the same two farms that were used for the EHT (Salerno).
The animals used for this study were mainly the Lacaune/Bagnolese mixedbreed dairy sheep, homogeneous in age (2 years ± 0.5), body weight (55 kg ± 0.5) and grazing season, without any prior anthelmintic treatments in at least 6 months.
After the confirmation of natural-mixed infection by GINs using the faecal egg count, on both examined farms a total of 48 sheep were divided into four groups (n = 12 per group) as follows: Both T. vulgaris type 1 EO and linalool:estragole were diluted in sunflower oil before administration to avoid their effects on mucosa of the gastrointestinal tract. Individual faecal samples were collected rectally on the day of treatment (D0) and after 7 and 14 days (D7 and D14), and stored shortly thereafter at 4 • C. The faecal samples were analysed individually using the Mini-FLOTAC technique with a detection limit of 5 eggs per gram (EPG) of faeces, using a sodium chloride flotation solution (specific gravity = 1.200), as described by Cringoli et al. (2017) [36]. The results were expressed as EPG in each sample with the calculation of averages within each group, and the reduction in GIN eggs in faeces after treatment was evaluated on D7 and D14 after treatment. Final results are presented as arithmetic means from two farms.

Coproculture
The equal quantity of faeces was collected from each sample before storage at the temperature of 4 • C, in order to create a pool for each faecal culture group at different time points (D0, D7 and D14). The procedure was done according to the protocol described by the Ministry of Agriculture, Fisheries and Food of UK in 1986 [37]. Developed third-stage larvae (L 3 ) were identified based on their morphological specifics, as suggested by van Wyk and Mayhew [38]. Identification and percentages of each nematode genera were conducted on 100 L 3 , whereby all larvae were identified if a sample had 100 or less L 3 present. In this way, of the total number of larvae identified, it was possible to obtain the percentage of each genus.

Statistical Analyses
The percentages of egg hatch inhibition were calculated using a formula proposed by For the comparation of values obtained for different concentrations and the controls within one EO, one-way Analysis of Variance (ANOVA) followed by Tukey's test (p < 0.05) was performed. On the other hand, for the comparation of the values of the same concentration between different EOs, two-way ANOVA followed by Tukey's test (p < 0.05) was used. Nonlinear regression/logarithmic distributions were applied for the calculation of half-maximal inhibitory concentrations (IC 50 ) [33].
The reduction in faecal egg counts (FECR) in animal's faeces was calculated by using a formula described by Macedo et al. (2010) [40]: where T 1 is EPG before treatment in treatment groups, T 2 is EPG after treatment (day 7 or 14) in treatment groups; C 1 is EPG before treatment in the negative control group; C 2 is EPG after treatment in the negative control group.
The obtained values were analyzed and compared by using Two-way ANOVA followed by Tukey's test (p < 0.05).
Two-way ANOVA (p < 0.05) was performed also in the anaylsis of the results of coproculture to evaluate the differences in the ratio between the percentage of GIN genera found pre-and post-treatment.
Statistical analyses were performed using the program GraphPad Prism 9.2.0. (Graph-Pad Holdings, LLC, San Antonio, CA, USA)

Egg Hatch Test
The obtained EHT results showed the great anthelmintic potential of tested EO samples ( Table 2)

Faecal Egg Count Reduction Test
Two tested samples, the T. vulgaris EO and the binary combination of linalool:estragole showed similar in vivo anthelmintic potential with a reduction in counted nematode eggs in faeces of 25.23% and 24.91% on Day 7 and 24.42% and 25.90% on Day 14, respectively (at flock level). However, EPG values do not statistically differ from sunflower oil (p > 0.05), although on Day 7 the EPG values of tested samples were similar to that of fenbendazole (p > 0.05). On Day 14, the counted EPG values of fenbendazole were significantly lower (p < 0.05) than that of tested samples (Table 3). Based on individual FECRT, the average percentage of individual EPG reduction in a groups of T. vulgaris and linalool:estragole reached 39.30% and 51.88% on Day 7, respectively, although it was less than 10% on Day 14. Individual FECRTs for fenbendazole were 84.73% and 87.32% on Days 7 and 14, respectively. Animals were observed for the presence of side effects after application of EOs, without any toxic effects observed.

Coproculture
Four genera of sheep GINs were identified on coproculture examination on both tested farms. In total, their representation on Day 0 was as follows: Haemonchus 53%, Trichostrongylus 29.5%, Teladorsagia 14.5% and Chabertia 3%. After treatment, their percentages changed somewhat differently depending on the treatment group, whereby in most groups the % of Haemonchus decreased and Trichostrongylus increased on days 7 and 14. The % of Teladorsagia increased in the control groups, while the % of Chabertia increased in the group treated by T. vulgaris and decreased in the group treated by linalool:estragole. However, neither these nor changes observed in the aspect before and after treatment were statistically significant (p > 0,05) in any of the tested groups. The percentages of each GIN genera in each in vivo treatment group after treatment (Day 7 and 14) are shown in Figure 1A-D.

Coproculture
Four genera of sheep GINs were identified on coproculture examination on both tested farms. In total, their representation on Day 0 was as follows: Haemonchus 53%, Trichostrongylus 29.5%, Teladorsagia 14.5% and Chabertia 3%. After treatment, their percentages changed somewhat differently depending on the treatment group, whereby in most groups the % of Haemonchus decreased and Trichostrongylus increased on days 7 and 14. The % of Teladorsagia increased in the control groups, while the % of Chabertia increased in the group treated by T. vulgaris and decreased in the group treated by linalool:estragole. However, neither these nor changes observed in the aspect before and after treatment were statistically significant (p > 0,05) in any of the tested groups. The percentages of each GIN genera in each in vivo treatment group after treatment (Day 7 and 14) are shown in Figure 1A-D.

Discussion
The use of plant products such as essential oils in anthelmintic treatments has many advantages. Firstly, EOs have a rich chemical composition of various bioactive compounds with huge pharmacological potential [14], which can lead to high activity against nematodes, as shown in our in vitro study. Furthermore, a large number of compounds in EOs that belong to different chemical classes may contribute to a reduced susceptibility to resistance [14,41]. Some reports state that botanical anthelmintics may be considered well tolerated by animals from the toxicology perspective and are related to the low amount of residues in meat and milk [33]. Although this statement is not proven and requires exact studies upon this, a large number of EOs, as well as their ingredients belonging to different chemical groups, offers an opportunity to find those who best meet these requirements, along with appropriate efficacy. This also refers to the price, which vary depending on the EO. Finally, their easy accessibility in regions with developed biodiversity allows easy acquisition [33] and, along with other mentioned factors, offers the possibility of a sustainable avenue for nematode control in ruminants.
Evaluation of the anthelmintic efficacy of EOs requires the application of appropriate methods. In vitro studies, wherein the EHT is one of the most frequently used and recommended tests [39], have an advantage due to their speed, low cost, high reproducibility, ease of application and lack of experimental animals [33]. Therefore, these tests are very useful for the initial evaluation of anthelmintic potential in new active substances, which in turn leads to further studies [41]. However, in vivo studies, whereby an FECRT is the method of choice for monitoring of anthelmintic efficacy [42], provide clearer data on efficacy and the possibilities for application in practice. Thus, the results of laboratory and field conditions testing do not necessarily coincide, whereby obtained efficacy is usually higher in laboratory testing [14]. This finding may be explained by many factors that can affect in vivo activity, which is especially true for EOs due to their unstable nature [20]. Therefore, both in vitro and in vivo tests are important in the process of developing new anthelmintic drugs [43].
In the present study, practically all tested samples demonstrated high ovicidal anthelmintic potential. The highest inhibitory effect on egg hatchability was shown by EOs of O. vulgare, F. vulgare, S. montana, S. hortensis and both T. vulgaris samples wherein all tested concentrations showed either a maximum effect or activity higher than 95%. The criteria for in vitro testing, whereby samples with efficacy greater than 90% can be considered effective for the control of nematodes including GINs [14,40,41], was set up by the World Association for the Advancement of Veterinary Parasitology (WAAVP). According to these criteria, mentioned EOs may present a very promising research subject for further studies. This also refers to EOs J. communis and M. piperita, since most of their concentrations were more than 90% efficient as well, although the activity of these EOs was dose-dependent. Two types of A. millefolium EOs and the binary combination of linalool:estragole also expressed dose-dependent activity with a higher efficacy at high concentrations (p > 0.05 compared to the thianbendazole) and medium or low activity at lower concentrations, which makes them suitable for further examinations as well.
The high obtained values of standard deviation for mean EPGs in FECRTs suggest large differences in worm burdens in animals. This can be explained by the fact that in small ruminants, gastrointestinal parasites are highly aggregated and over-dispersed within the host, whereby most of the parasite population (approximately 80%) is found in only 20-30% of hosts, while the majority of animals have low worm burdens [4,44]. In any case, two tested EO formulations showed a degree of anthelmintic potential with a reduction in the number of counted eggs in faecal samples of approximately 25% each day of testing (7 and 14 days after treatment). Although the positive control was clearly more effective, neither of them reach the needed efficacy of 90% recommended by the WAAVP for in vivo testing [42], which indicates developed resistance to benzimidazoles to some extent. The limited efficacy of the T. vulgaris EO type 1 and linalool:estragole may be explained by the fact that EOs and their active ingredients are prone to evaporation and destabilization [20,45], which makes them degradable in animal organisms as mentioned above. This particularly refers to ruminants keeping in mind the anatomical and physiological specifics of their gastrointestinal tract, which may affect perorally applied active substances [46]. Thus, active substances of tested EO formulations were most likely partially deactivated prior to reaching the targeted place of action in the abomasum and small intestine.
However, these obstacles may be circumvented by different encapsulation techniques that may protect the active substances from degradation and allow their better bioavailability and higher in vivo efficacy. Also, encapsulation may enable controlled release of active ingredients of EOs, as well as reduce their smell and taste that could be unpleasant for animals [20,24,44,47]. Another method is to change the means of application, such as to use lick blocks containing plant-based compounds that may provide long-term use. In some research, for example, these preparations effectively reduced coccidian invasion and had a beneficial effect on growth and body development of the lambs [48]. Nevertheless, the finding that there were no observed toxic effects on tested animals allows an opportunity for an increased dose in future trials to reach a higher efficacy. The obtained results and demonstrated efficacy should not be neglected since they suggest that these plant products, coupled with other methods, may still play a significant role in an integrated approach for nematode control in ruminants, in case they are proven ineffective as independent products [40,41]. Furthermore, our research group has already made new experimental efforts with the evaluation of the in vivo efficacy of other highly effective in vitro EOs from the present study (data not shown).
The results of coprocultre showed the presence of four GIN genera on the examined farms: Haemonchus, Trichostrongylus, Teladorsagia and Chabertia in different percentages. Although some changes in their percentages after treatment were observed while comparing different treatment groups, the results obtained from the cultures of all groups did not show any significant difference in the ratio between the percentage of genera found prior and post treatment. This result suggests that none of the employed treatments are specific for only a single genera.
The T. vulgaris EO was previously in vitro and in vivo tested for anthelmintic activity against sheep GINs. In a study by Ferreria et al. (2016), it also exhibited high ovicidal activity, where the concentrations ranging from 0.097-50 mg/mL inhibited the hatchability of H. contortus eggs by 49.4-100% with an IC 50 of 0.436 mg/mL [14]. However, in the same study, the tested EO failed to reduce the EPG at doses of 75, 150 and 300 mg/kg. The differences between these results and the results obtained in the present study may be explained by the differences in the chemical composition of used samples of T. vulgaris, as will be discussed later. On the other hand, the M. piperita EO was previously investigated in a study of Katiki et al. (2011), where the calculated IC 50 value was 0.26 mg/mL [49], similarly as in the present study. The rest of the samples tested in the present study are new, according to the best of our knowledge.
As discussed earlier, the anthelmintic activity of EOs originates from their rich chemical composition and many bioactive ingredients belonging to different chemical classes [50]. However, ingredients of EOs can significantly differ in their degree of anthelmintic activity [15], suggesting the importance of compounds present in EOs and their percentages. The GC-MS analyses performed in the present study showed that the presence of compounds such as carvacrol, anethole, thymol, p-cymen and y-terpinene was related to the heightened efficacy of tested samples, since the most effective EOs were composed of these ingredients. Indeed, the high activity of isolated anethole, carvacrol and thymol against H. contortus eggs was demonstrated in a study by Katiki et al. (2017) in EHT with obtained IC 50 values of 0.07, 0.11 and 0.13 mg/mL, respectively [15]. The ovicidal activity of thymol was also demonstrated in other studies, with obtained IC 50 values of 0.442 [14] and 0.08 mg/mL [51], as well as carvacrol and anethol with IC 50 values of 0.17 [52] and 0.69 mg/mL [53], respectively. According to the best of our knowledge, the activity of isolated p-cymen and y-terpinene against sheep GINs is still unconfirmed, although these ingredients were found in EOs that demonstrated high anthelmintic activity against these parasites, such as γ-terpinene in Melaleuca alternifolia (20.15%) [54].
The results of the conducted chemical analyses also suggest that the level of efficacy of EOs does not correspond with the number of identified compounds in tested samples. In contrast, the most efficient EOs such as O. vuglare, F. vulgare, S. montana and S. hortensis had the lowest number of identified compounds, while the least effective EOs, H. arenarium and two samples of A. millefolium, had the highest number of components. This indicates that not all components of EOs possess anthelmintic potential, but usually the main or two or three dominant compounds [55]. However, the presence of other compounds could contribute to the overall activity by synergistic action through a different mechanism; thus whole EOs usually express better efficacy than their isolated compounds when applied individually [14,53].
Finally, many factors can affect the chemical composition of EOs and thus their biological activities. These include various exogenous factors such as light, precipitation, growth site, soil properties (hydrology, pH and salinity), seasonal variation and endogenous factors such as the location of production and accumulation of the EOs in the plant, the age of the plant, as well as the genetic characteristics that regulate their secondary metabolism [56,57]. Additionally, biotic factors such as the presence of certain soil organisms and microorganisms may also be included [57]. All these factors lead to differences in the composition of EOs obtained from different plant species, but also in the same plant species as shown in the present study. Namely, the differences in chemical composition within two samples of T. vulgaris, as well as A. millefolium, EOs have led to the differences in their ovicidal activity, which were significant (p < 0.05) for some concentrations. Those differences are most likely derived from different plant varieties used for EO isolation by producers and, on the other hand, the different geographical origin of plants (in the case of T. vulgaris, samples were obtained from Serbia and France; A. millefolium samples were obtained from two different producers from Serbia).
All the previously mentioned findings related to the chemical composition of EOs should not be neglected and may be highly important to the pharmaceutical industry from the aspect of finding an appropriate EO-based formulation designed to control GINs in ruminants. This also indicates the importance of conducting GC-MS analyses in studies such as this before the selection of samples for testing and conduction of in vitro and in vivo tests.
Understanding the mechanism of action of EOs and their ingredients is important for their practical use in nematode control, given that it can ensure that useful information on the most appropriate formulation and delivery means is obtained [58]. However, current understanding is still inadequate and there are efforts in this field to improve upon this state. So far, numerous mechanisms of nematocidal activity of EOs are proven or suggested depending on the ingredients that make up their composition. These involve interruption of the nematode nervous system, the inhibition of AChE activity, interference with the neuromodulator octopamine or GABA-gated chloride channels, disruption of the cell membrane of the nematode thereby changing its permeability, membrane and ion channel perturbations modifying membrane-bound protein activity and the intracellular signalling pathways, etc. [58]. The activity of phenolic compounds, such as carvacrol and thymol, may be associated with damage caused to the cuticle and digestive apparatus on nematode larvae. Additionaly, these compounds are most likely related to the neurotoxic effect on the free-living nematodes, since they interact with SER-2 tyramine receptors. The cuticular changes and possible neurotoxicity may interfere with the permeability of the cuticle and motility, hindering the maintenance of homeostasis within these parasites [51,52]. Therefore, different ingredients of EOs induce different neurological and structural changes in nematodes, which may give rise to their paralysis and death.
The idea of the use of EOs for various purposes, including anthelmintic, is relatively new and most data relating to their use in animals are still based on anecdotal observations without scientific validation [59], which are not enough for their wider use in nematode control. However, although there is still a lack of data on the anthelmintic efficacy of EOs, there has been a steady increase in studies aiming to verify the anthelmintic potential of plants [59]. The lack of studies is especially noticable in relation to in vivo testing [18], whereby the results of trials conducted so far, including the present study, show the inferior activity of EOs compared to synthetic drugs [40]. However, finding an appropriate formulation (plant species, chemical composition, dose and method of application) is not a simple task, thus requiring a larger number of studies and works [60][61][62], whereby the comparison between results obtained for different EO samples is also important for the selection. Finally, novel encapsulation methods offer the possibility of overcoming these problems and achieving appropriate efficiency, as discussed earlier.

Conclusions
An efficient alternative to commercial anthelmintics is urgently needed due to the development of resistance and the economic losses it entails. Therefore, simultaneous testing of different EO samples may contribute to finding valuable anthelmintic agents. The present study demonstrated the high in vitro, but also, to some extent, in vivo, an-thelmintic potential of the tested natural-based formulations against sheep GINs. From this perspective, the highest potential was shown by O. vulgare, F. vulgare, S. montana, two samples of T. vulgaris and S. hortensis, EOs containing compounds such as carvacrol, thymol, anethol, p-cymene and y-terpinene in high percentages.. However, an additional in vivo trial with modified conditions (higher doses, a different means of application or the use of encapsulation methods) for samples tested in vivo (T. vulgaris EO and linalool:estragole combination), or a new trial with other mentioned EOs should be conducted to reach higher efficacy in field conditions. Nevertheless, this study is another confirmation of the possible role of botanical anthelmintics in the control of sheep GINs. Thus, and looking from a future perspective, the results of the present study may be of practical importance in combating anthelmintic resistance.

Funding:
The financial support for the present study was obtained from the COST network (Networking tool reference ECOST-STSM-CA16230-201019-113354). Within that context, many thanks to Smaragda Sotiraki, a Grant Holder Manager, for approving funding for this STSM from the COST network.

Institutional Review Board Statement:
The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of the University of Naples (PG/2021/0130480 del 16/12/2021).

Informed Consent Statement:
Informed consent was obtained from farm owner involved in the study.
Data Availability Statement: All data generated or analysed during this study are included in this published article. The datasets used and/or analysed during the present study available from the corresponding author upon reasonable request.