Introduction
Gastrointestinal nematode (GIN) infection hampers animal welfare and productivity, resulting in reduced weight gain, carcass quality, milk production and fertility (Charlier et al., Reference Charlier, Hoglund, von Samson-Himmelstjerna, Dorny and Vercruysse2009). Gastrointestinal parasitism is often characterized by a combination of several species infecting the host (Kaplan & Vidyashankar, Reference Kaplan and Vidyashankar2012). The most common cattle GINs recovered from pastures in Ontario, Canada, are Cooperia spp. and Ostertagia spp. (Nodtvedt et al., Reference Nodtvedt, Dohoo, Sanchez, Conboy, DesCoteaux, Keefe, Leslie and Campbell2002; Scott, Reference Scott2017). Recently, following an epidemiological study on 306 farms in Canada, the prevalent GIN species present were found to be Cooperia oncophora and Ostertagia ostertagi, with heifers having access to pasture bearing higher faecal egg counts (FECs) than animals in non-pastured herds (Scott, Reference Scott2017). Owen et al. (Reference Owen, Slocombe and Curtis1989) recovered Cooperia sp. (46%), Ostertagia sp. (39%) and Haemonchus sp. (15%) larvae in a herd of cow/calf pastured in September in Ontario, Canada. The trichostrongyles group of nematodes, including all of the aforementioned species, have a general life cycle in which cattle become infected by the ingestion of L3 (Verschave et al., Reference Verschave, Levecke, Duchateau, Vercruysse and Charlier2015). The control of parasites in dairy cattle relies most commonly on commercially available anthelmintic drugs; however, resistance to anthelmintic drugs by GINs has increased over the last few years (Gasbarre, Reference Gasbarre2014).
The use of plants or plant-derived products, such as condensed tannins or saponins, for the treatment of GIN infections has increased significantly worldwide (Sandoval-Castro et al., Reference Sandoval-Castro, Torres-Acosta, Hoste, Salem and Chan-Pérez2012). Most of the work performed with plant-based phytochemicals has focused on the control of parasites in small ruminants (Sandoval-Castro et al., Reference Sandoval-Castro, Torres-Acosta, Hoste, Salem and Chan-Pérez2012), while few studies have tested the antiparasitic effects of different plants and plant-derived products to control parasite infections in large ruminants (Shepley et al., Reference Shepley, Vasseur, Bergeron, Villeneuve and Lachance2015).
Several of the common major components of essential oils (EOs), such as thymol, menthol, limonene and geraniol, have shown significant antiparasitic effects for the control of animal diseases (Hrckova & Velebny, Reference Hrckova and Velebny2013). EOs are volatile, natural, complex compounds derived from aromatic plants, and have been found to possess antimicrobial, antifungal, antiparasitic, anti-oxidant and anti-inflammatory activities (Bakkali et al., Reference Bakkali, Averbeck, Averbeck and Idaomar2008), as well as being repellent for cattle pest flies (Lachance & Grange, Reference Lachance and Grange2014). In ruminant nutrition, EOs have been used as feed additives to improve rumen fermentation efficiency (Cobellis et al., Reference Cobellis, Trabalza-Marinucci and Zhontang2016). The potential use of EOs from plants is a promising line of research that may give rise to the improved treatment of helminth infections (Grando et al., Reference Grando, de Sa and Baldissera2016), in addition to the other benefits. To the best of our knowledge, no studies have been carried out to test cattle nematode control using EOs.
In vitro techniques have been developed to investigate and validate the efficacy of plant extracts or plant EOs against GINs of ruminants, such as the egg hatch, larval development, larval migration inhibition (LMI), larval feeding inhibition and larval exsheathment assays. In vitro assays seldom reflect in vivo bioavailability (Hrckova & Velebny, Reference Hrckova and Velebny2013), but they are highly reproducible, convenient, rapid to perform and constitute low-cost pre-screening tests to evaluate the anthelmintic activity of secondary metabolites of plants (Novobilsky et al., Reference Novobilsky, Mueller-Harvey and Thamsborg2011).
Before testing therapeutic plant extracts in effort-intensive in vivo trials with dairy cows, it is important to select extracts more likely to have an anthelmintic effect. The present study was, therefore, conducted to evaluate the in vitro anthelmintic efficacy of selected EOs on egg hatch, mobility and mortality of GINs collected and cultured from naturally infected cattle.
Materials and methods
EOs
EOs were purchased from Aliksir Inc. (Grondines, QC, Canada). The eight experimental EOs were: Solidago canadensis L. (Asteraceae), Eucalyptus globulus Labill. (Myrtaceae), Pelargonium asperum Willd. cv. Bourbon (Geraniaceae), Ocimum basilicum L., Thymus vulgaris L., Mentha x piperita L. (Lamniaceae), Cymbopogon citratus (DC.) Stapf and Cymbopogon martinii (Roxb.) Wats. (Poaceae). A chemical high-performance gas chromatography analysis was provided by the supplier for each EO tested (supplementary table S1). The EOs were kept at 4°C until use. The EOs were chosen based on the lack of potential adverse effects on animals and efficacy (Bakkali et al., Reference Bakkali, Averbeck, Averbeck and Idaomar2008; Sandoval-Castro et al., Reference Sandoval-Castro, Torres-Acosta, Hoste, Salem and Chan-Pérez2012), cost and availability. The dose of 8.75 mg/ml used for the first assays was selected based on doses used in other studies (Camurca-Vasconcelos et al., Reference Camurca-Vasconcelos, Bevilaqua and Morais2007; Macedo et al., Reference Macedo, Bevilaqua, de Oliveira, Camurca-Vasconcelos, Vieira and Amora2011).
Collection and culture of faecal samples
Faecal samples were collected weekly from mid-September to October 2014 from a 27-head milking animals organic dairy farm in eastern Ontario, Canada, from three highly parasitized dairy cattle, directly from the rectum. The samples were placed in plastic bags, labelled and brought to the Organic Dairy Research Centre, University of Guelph – Campus d'Alfred for processing. Procedures involving animals in this study were approved (Protocol #1641) by the animal care committee at the University of Guelph, which adheres to the Canadian Council on Animal Care guidelines (CCAC, 2009).
Several fresh faecal samples from the three animals containing the highest number of eggs (average 17 eggs per gram; modified Wisconsin technique), over several collection days, were cultured to obtain the infective nematode stages (L3) for the experiments. About 100 g of faeces were transferred into a 500 ml glass container and mixed with 20 g of vermiculite for culture. Vermiculite was mixed with the manure to provide aeration and to absorb excessive moisture from faeces. A plastic lid with a 2 cm diameter hole covered with mesh was screwed to the glass container. The rearing conditions were 22 ± 2°C and 16:8 L:D. The mixture was stirred twice every day until extractions of L3 larvae, 8–10 days after the start of the culture.
The third-stage larvae (L3) were recovered using a modified Baermann technique. Fifteen grams of faecal samples were placed on the Baermann apparatus and allowed to stand in tap water for 24 h. During this time, larvae settled down the tube of the funnel. Five millilitres of the fluid in the tube were then drawn into a 15 ml conical tube and centrifuged at 123 g for 2 min. The supernatant was siphoned out and the 2 ml sediment of several funnels transferred and mixed in a conical flask and kept in the fridge until used for the experiments, up to a maximum of two months.
For genus identification, the third-stage larvae from culture were killed by adding a drop of diluted Lugol's iodine solution to a drop of the larval suspension on a microscope slide and examined under 400× magnification. The larvae were identified to the genus level on the basis of the characteristic tail length according to Van Wyk & Mayhew (Reference Van Wyk and Mayhew2013). The percentage of each genus was calculated on a mixture of 200 larvae cultured from the three experimental cows.
Egg hatch assay (EHA)
The EHA was conducted using a modified method described by Coles et al. (Reference Coles, Bauer, Borgsteede, Geerts, Taylor and Waller1992). Briefly, 20 g of faecal samples were mixed with 30 ml tap water and filtered through 250, 212, 150 and 28 µm sieve sizes, the latter retaining the eggs. The material retained by the 28 µm sieve was processed (Coles et al., Reference Coles, Bauer, Borgsteede, Geerts, Taylor and Waller1992) to extract the eggs. The concentration of eggs was estimated by counting the eggs in aliquots of 50 µl at 25× magnification, and concentrations of 50–100 eggs in 100 µl of solution were used for the experiment.
The eight EOs were prepared by dissolving them in water and 2% Tween 20, to improve solubility. The solutions (water, EO and Tween 20) were mixed in a vortex shaker (Mini Vortexer, Fisher Scientific, USA) for 10 min. A solution of 8.75 mg/ml was prepared for each EO. One hundred microlitres of the egg solution was pipetted in each tested well of a 24-well polystyrene tissue culture plate (Corning Incorporated, NY, USA). Then, 1600 µl of EO solution were added in each well containing the eggs. The control consisted of 1600 µl of a solution of 2% Tween 20 and distilled water, and an additional treatment of only distilled water was added. The tissue culture plates were then placed by groups of ten on top of wet paper towels in a sealed polyethylene container, to ensure high relative humidity, and incubated at 26°C. After 48 h, a drop of Lugol's iodine solution was added to each well to stop the egg hatching and to kill hatched larvae. All the larvae (L1) and unhatched eggs were then counted. Five replicates of each treatment were performed. The percentage of hatched eggs for each treatment was calculated using the following equation:
where L = number of larvae in well and E = number of unhatched eggs in well.
LMI assay
The technique from Rabel et al. (Reference Rabel, Mcgregor and Douch1994) was used. Briefly, a solution containing 100–150 larvae per 100 µl was prepared from reared L3 larvae. Then, 100 µl of this solution was added into each tested well of a 24-well tissue culture plate and 1600 µl of the 8.75 mg/ml EO solutions were then added to tested wells. The plates were incubated for 3 h at room temperature. After incubation for 3 h at 21 ± 2°C, the content of each well was transferred into a sieve placed in the next corresponding well and incubated for 24 h. The sieves were constructed from translucent acrylic tubing (2 mm in length, 1 mm internal diameter and 1.3 mm external diameter). One end of the sieve was covered with a 28 µm mesh (Sefar Nitex 03-28/17 102 cm, Sefar Inc, Depew, NY, USA) glued with cyanoacrylate adhesive. The 28 µm mesh size was selected to permit active migration of cattle live larvae (Demeler et al., Reference Demeler, Küttler and von Samson-Himmelstjerna2010) through the mesh. Each sieve was held in place 3 mm above the bottom of the cell with an acrylic plate holder consisting of holes where the sieves were inserted.
After the 24-h incubation period, the sieves were raised and the content allowed to drain in each well. The outside of the sieves was washed gently with distilled water. The number of dead (not moving when prodded) and live larvae retained in sieve or having migrated and present in the well was counted by using a stereo microscope at 25× magnification. There were five replicates for each treatment. The percent migration of larvae was calculated with the formula:
where R = number of larvae retained in sieve (dead and alive) and M = number of larvae migrated through sieve (dead and alive).
The percentage of dead larvae was also calculated using the formula:
where T = total number of larvae deposited in sieve and D = number of dead larvae after 24 h (in sieve or having migrated).
Dose–response of three EOs on larvae
Based on the results of migrating larvae of the previous experiment, three EOs were selected for dose–response testing. The EOs were C. citratus, C. martinii and O. basilicum, and doses of 35.00, 17.50, 8.75, 4.38, 1.75, 0.88, 0.09 and 0.04 mg/ml of each EO were chosen for the assays. The LMI assay was performed and we used the same methods as described above. Three replicates were performed for each dose. The percent migration of larvae and the percentage of dead larvae were also calculated using the same methods as described above.
Statistical analysis
Percentage egg hatch, migrating larvae and mortality were analysed in generalized linear mixed models using the GLIMMIX procedure of SAS Software, version 9.4 (SAS Institute Inc., 2012). The residuals were tested for normality, and the normal distribution was used as it was the best fit for the data. The PDIFF option was used in the LSMEANS statement with the Tukey multiple comparison test for analysing differences between treatments (SAS Institute Inc., 2012). Data are reported as LSMEANS ± standard error of mean, and differences among treatments were considered at a significance level of α = 0.05.
Mortality and migrating larvae responses vs. concentration were analysed using a chi-square test to measure goodness-of-fit, describing the relationship between dosage levels and observed and expected data. Lethal concentration 50 and 90% (LC50 and LC90) for larval mortality, and inhibition concentration (IC) 50 and 90% (IC50 and IC90) for larval migration, were calculated using PROC PROBIT. The predicted values were corrected if necessary using the procedure LACKFIT, when the goodness-of-fit statistic test P-value was <0.1, where variances and covariances are adjusted by a heterogeneity factor (the goodness-of-fit chi-square divided by its degrees of freedom) and a critical value from the t distribution is used to compute the fiducial limits (SAS Institute Inc., 2012). The LC50 and IC50 values of the three EOs were compared to one another using a ratio test (Robertson et al., Reference Robertson, Russell, Preisler and Savin2007) to determine differences in GIN susceptibility to the EOs.
Results and discussion
No significant differences were observed between treatments with distilled water only and distilled water and 2% Tween 20, with hatching rates of 94.5 and 92.0%, respectively (fig. 1). All EOs had a significant direct impact on the hatching rates of nematode eggs (fig. 1). The treatment with C. martinii had the lowest percentage of hatched eggs (8.1%), while S. canadensis had the highest percentage of hatched eggs (19.0%) of all the EOs tested (fig. 1).
Percentages of nematode egg hatch, larval migration and dead larvae at 8.75 mg/ml concentration for eight EOs. Data with the same letter for the same test are not significantly different based on Tukey's Honest Significant Difference test at P < 0.05 (n = 5).
In the control groups (distilled water; distilled water and 2% Tween 20), the percentage of migrating L3 larvae was higher than 92.6% (fig. 1). All EOs significantly inhibited larval migration, to various degrees, compared to the distilled water control (fig. 1). The EOs from the genus Cymbopogon had the most significant impacts, contributing to the lowest migration percentage of larvae (54.9% for C. citratus and 60.5% for C. martinii) (fig. 1). Although significantly different than the water-only control, larval migration still occurred for 85.9% of larvae with S. canadensis EO, and was no different than with P. asperum and T. vulgaris (fig. 1).
In the larval migration assay, the number of dead larvae were also counted. The percentage of dead larvae was highest for C. citratus (63.3%) and C. martinii (56.3%), and lowest in S. canadensis (13.9%) (fig. 1). All EOs had an effect on the mortality of larvae compared to the distilled water and Tween control treatments (fig. 1).
The mortality rate of larvae (L3) was dose dependent and the results of the Probit analysis are shown in table 1. Cymbopogon citratus showed the lowest estimated LC50 at 3.89 mg/ml (table 1) when compared to C. martinii and O. basilicum. At our higher dosage level of 35 mg/ml, C. martinii yielded a 87.1% mortality rate, while C. citratus and O. basilicum yielded mortality rates of 78.6 and 84.7%, respectively (not shown). In the control groups (distilled water and 2% Tween 20), the percentage of dead larvae was below 5%.
Larval lethal concentration (LC) and migration inhibitory concentration (IC) values (mg/ml) with fiducial limits for C. citratus, C. martinii and O. basilicum EOs.
Values calculated from Probit analysis.
N, total number of nematodes used in generating the probit regression estimates; FL, 95% fiducial limits; SE, standard error of the estimate; χ2 values for goodness-of-fit model.
The migration inhibition (IC) increased with dose (table 1). Cymbopogon citratus had the lowest estimated IC50 (7.19 mg/ml) (table 1). Ocimum basilicum, on the other hand, showed the lowest estimated IC90 (158.42 mg/ml) of the three EOs (table 1). At our highest dose of 35 mg/ml, the EO from C. citratus showed 72.8% inhibition of larval migration, whereas O. basilicum and C. martinii showed a 60.6 and 56.3% inhibition, respectively (not shown). In the control groups (distilled water and 2% Tween 20), the migration inhibition was less than 9% (not shown).
Ratio tests showed that C. martinii had a significantly higher LC50 and IC50 than C. citratus, therefore indicating that GIN larvae were more susceptible to the latter (table 2). Further, when compared with O. basilicum, C. citratus had a significantly lower LC50 and IC50 (table 2). No significant differences in values between C. martinii and O. basilicum were found, since the 95% confidence intervals for the ratios included 1 (table 2).
Comparisons of LC50 (lethal concentration) and IC50 (migration inhibitory concentration) values between C. citratus, C. martinii and O. basilicum EOs, using a ratio test.
*Significant (P < 0.05).
a The essential oil mentioned first has a higher LC50 and IC50 value.
b When the 95% confidence interval (CI) includes 1, the LC50 or IC50 is not significantly different.
Using both EHA and LMI, the current study showed a significant impact of EOs on egg hatching rates and on the migration and mortality rates of larvae. The relative effect of each EO was somewhat similar for the eggs and the L3 larvae, with the two plant extracts of Cymbopogon being the most efficient as an anthelmintic for all performed bioassays. All of the EOs tested showed an important reduction in egg hatch, as a concentration of 8.75 mg/ml decreased egg hatch to rates lower than 20% for all EOs, compared to controls that had hatching rates above 90%.
Macedo et al. (Reference Macedo, Bevilaqua and de Oliveira2009) demonstrated that EOs from E. globulus at 17.4 mg/ml inhibited Haemonchus contortus egg hatching by 87.3%, while, in our study, half of that dose inhibited egg hatching by 81.7%. In our study, E. globulus was efficient at decreasing egg hatch, but not to the level reported for Eucalyptus citriodora by Macedo et al. (Reference Macedo, Bevilaqua, de Oliveira, Camurca-Vasconcelos, Vieira and Amora2011), which inhibited egg hatching by 99.8% in goat GINs at a dose of 5.3 mg/ml. Different proportions of active ingredients, such as cineole, α-pinene and limonene, in the two plant species (E. globulus and E. citriodora) may, in part, explain the differences. Katiki et al. (Reference Katiki, Chagas, Bizzo, Ferreira and Amarante2011) reported 99% egg hatch inhibition (LC99) values on sheep nematodes (95% H. contortus and 5% Trichostrongylus spp.) using 0.27, 0.61 and 1 mg/ml for Cymbopogon schoenanthus, C. martinii and Mentha piperita, respectively, showing lower LC99 values for the two Cymbopogon spp. than for M. piperita. The lower efficiency rates of egg hatch inhibition obtained in the present study (91.9 and 88.8% for C. martinii and M. piperita, respectively), using 8.75 mg/ml, may be due, in part, to the nematodes tested being a mixture from different genera following isolation from cattle faeces. In fact, the proportion of L3 larvae identified from cultured eggs collected in September–October were highest for Haemonchus spp. (55.5%). Other genera found were Trichostrongylus spp. (28.0%), Cooperia spp. (15.0%) and Oesophagostomum spp. (1.5%). Rossanigo & Gruner (Reference Rossanigo and Gruner1994) reported that Haemonchus spp. egg deposition predominates in the fall season for cattle. It was, however, surprising to find Haemonchus as the dominant genus in cattle, as previous studies have mainly found Ostertagia and Cooperia as the two main genus present in Canada, and specifically Ontario (Nodtvedt et al., Reference Nodtvedt, Dohoo, Sanchez, Conboy, DesCoteaux, Keefe, Leslie and Campbell2002; Scott, Reference Scott2017). The Haemonchus genus, and specifically the species H. contortus, is generally considered to prefer warmer climates typical of more southern areas (Emery et al., Reference Emery, Hunt and Le Jambre2016), although H. contortus in sheep has been reported to expand to northern cooler climates (Domke et al., Reference Domke, Chartier, Gjerde, Leine, Vatn and Stuen2013).
Although not identified to the species level, it is likely that the species found to be more common in our samples was Haemonchus placei, the bovine Haemonchus sp., even though calves may be susceptible to H. contortus infections (Zajac, Reference Zajac2006). The bioassays performed were not designed to discriminate between the major genera of nematodes found in the samples, as the tests were performed on live reared parasites of naturally infected cattle. In vitro tests with a single species would be necessary to determine species-specific sensitivity to the various EOs, as resistance to anthelmintic is a species-specific character (Kaplan & Vidyashankar, Reference Kaplan and Vidyashankar2012).
Significantly fewer larvae were found migrating with the genus Cymbopogon compared with the other EOs tested. In addition, the number of dead larvae was the highest with Cymbopogon species. Several of the L3 larvae able to move through the sieve subsequently died during the 24-h period. This suggests that EOs action on nematodes is gradual. Two major constituents of C. martinii and C. citratus are geraniol and geranial. Geraniol and citronellol are the main components of Pelargonium, and both constituents have shown nematicidal properties in Caenorhabditis elegans (Abdel-Rahman et al., Reference Abdel-Rahman, Alaniz and Saleh2013). In our study, P. asperum showed some anthelmintic activity in regard to egg hatching, migration and mortality of larvae, but not to the extent of the EO of the Cymbopogon spp. As single constituents were not tested in the present study, it is difficult to pinpoint the EO constituent(s) with the most active principles against GINs. However, Macedo et al. (Reference Macedo, de Oliveira, Ribeiro, dos Santos, das Chagas Silva, de Araujo Filho, Camurca-Vasconcelos and Bevilaqua2015) concluded that citral (geranial and neral) was responsible for the anthelmintic activity of C. citratus on H. contortus in vitro and showed 38.5% reduction of H. contortus when the EO was fed to gerbils using an oral dose of 800 mg/kg. Variations in extraction methods, plant parts and varieties used, as well as geographic location and harvest time, can affect the chemical content of bioactive compounds in plants (Sandoval-Castro et al., Reference Sandoval-Castro, Torres-Acosta, Hoste, Salem and Chan-Pérez2012).
The L3 stage is usually more resilient to adverse substances than the first-stage larvae due to the double-sheath exoskeleton, and is also less sensitive to paralysis of its pharynx muscles (Molan et al., Reference Molan, Waghorn and McNabb2002). In a development bioassay, Katiki et al. (Reference Katiki, Chagas, Bizzo, Ferreira and Amarante2011) demonstrated that C. martinii and M. piperita had low LC50 values of 0.15 and 0.26 mg/ml, respectively, for H. contortus L1 larvae. Bioassays with L3, such as in the present study, will likely result in higher lethal concentrations than in those using L1. Using L3 for in vitro tests may be more biologically relevant if the objective is to use the EO directly as a feed supplement with anthelmintic properties, although results of assays in vitro would not be sufficient to suggest direct anthelmintic efficiency in field situations (Sandoval-Castro et al., Reference Sandoval-Castro, Torres-Acosta, Hoste, Salem and Chan-Pérez2012).
Our test showed a LC50 of 3.89 mg/ml using C. citratus, significantly better than the two other EOs tested (C. martinii and O. basilicum) for dose–response. Katiki et al. (Reference Katiki, Chagas, Bizzo, Ferreira and Amarante2011) also had consistently better efficacy using another Cymbopogon species, C. schoenanthus, than with C. martinii. The results seem to confirm that C. martinii should not be the preferred Cymbopogon species to use for in vivo tests. Cymbopogon citratus and C. martinii contain approximately 20 constituents, being rich in geraniol, geranial, neral, geranyl and myrcene. These are terpenoid compounds, which can also cause insect death (Bakkali et al., Reference Bakkali, Averbeck, Averbeck and Idaomar2008) and may likely be involved in the inhibition, retarded growth, reduced reproduction capacity and damage to the mature larvae.
EOs administered alone or in combination to the animals can have a variety of effects, such as killing GINs, reducing the establishment or development of nematodes in the host, decreasing contamination by nematode eggs or reducing egg hatching (Athanasiadou & Kyriazakis, Reference Athanasiadou and Kyriazakis2004). Andre et al. (Reference Andre, Ribeiro and Cavalcante2016) showed that carvacryl acetate, when fed to sheep at 250 mg/kg fresh weight, reduced FEC 16 days post-treatment, but nonetheless concluded that its effectiveness could be increased, possibly by encapsulation. Sandoval-Castro et al. (Reference Sandoval-Castro, Torres-Acosta, Hoste, Salem and Chan-Pérez2012), however, identified several challenges that need to be overcome to confirm in vivo efficacy of EOs. For instance, the sole use of EOs as anthelmintic might be problematic, given the high concentration that would be necessary in the gastrointestinal tract to directly affect the nematodes, without causing any adverse effect to the animals. The rumen of 12.5-month-old heifers can contain a volume of digesta ranging from 50.1 to 63.6 l, 5 h post-feeding, depending on the diet (Suarez-Mena et al., Reference Suarez-Mena, Lascano and Heinrichs2013). Digesta volumes, which can be fairly large, can give indications to the quantity of EOs needed to be delivered to the digestive tract of heifers, if the goal is to reach concentrations directly affecting GINs in the gastrointestinal system.
The effect of the EOs on maturation of infective larvae to adult GINs, establishment in the gastrointestinal tract, and reduction in adult fecundity, has not been evaluated in the present study. Relying solely on assessing the direct impact of EO on mobility and mortality of L3 is not sufficient to extrapolate to the in vivo effect. However, when the findings of in vitro assay are conclusive, in vivo assays can be used in subsequent investigation in field conditions, to confirm in vitro results (Sandoval-Castro et al., Reference Sandoval-Castro, Torres-Acosta, Hoste, Salem and Chan-Pérez2012). Anthelmintic plant extracts such as EOs can also provide a lower risk of GIN resistance development than synthetic anthelmintic drugs, due to the potential synergistic effect of the combination of secondary metabolites present in each EO. Further research on the practical use of EOs needs to be carried out to standardize the doses needed and to develop practical delivery methods.
Supplementary material
To view supplementary material for this article, please visit https://doi.org/10.1017/S0022149X19001081
Acknowledgements
We thank the late Grant Hepburn for his generous assistance with the collection of animal faeces. We would also like to thank Elyna Pierre-Gilles and Elise Shepley for their assistance with data collection throughout the study, and Fannie D'amour, from the Diagnostic Services at Université de Montréal, for help in the identification of nematodes. We would also like to thank Dr Renée Bergeron for reviewing an earlier version of the manuscript.
Financial support
This work was supported by the AgriInnovation Program of Agriculture and Agri-Food Canada's Growing Forward 2 Policy Framework (a federal, provincial, territorial initiative) and Dairy Farmers of Canada (Ottawa, Ontario, Canada) through the Organic Science Cluster II (Grant no. D.45.71), an industry-supported research and development endeavour initiated by the Organic Agriculture Centre of Canada at Dalhousie University (Truro, Nova Scotia, Canada) in collaboration with the Organic Federation of Canada (Montreal, Quebec, Canada).
Conflicts of interest
None.
Ethical standards
The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional guides on the care and use of laboratory animals.
