The Use of Essential Oils in Veterinary Ectoparasite Control: a Review

Medical and Veterinary Entomology (2014) 28, 233–243 doi: 10.1111/mve.12033

REVIEW ARTICLE

The use of essential oils in veterinary ectoparasite control: a review L.ELLSE andR.WALL Veterinary Parasitology and Ecology Group, School of Biological Sciences, University of Bristol, Bristol, UK

Abstract. There is a growing body of evidence indicating the potential value of essential oils as control agents against a range of arthropod ectoparasites, particularly lice, mites and ticks. Toxicity has been demonstrated following immersion and physical contact with treated surfaces, as well as after exposure to the vapour of these oils; the last of these factors implies that there is a neurotoxic, rather than simply a mechanical, pathway in their mode of action. However, the volatile nature of essential oils suggests that their residual activity is likely to be short-lived. A possible advantage of essential oils over conventional ectoparasite treatments may refer to their reported ovicidal efficacy, although it is unclear whether this results from neurotoxicity or mechanical suffocation. There are many difficulties in comparing the findings of existing studies of essential oil toxicity. One major issue is the wide variation among batches in the relative concentrations of oil constituents. A second issue concerns the fact that many experimental designs make it difficult to confirm that the effect seen is attributable to the oil; in many cases inappropriate controls mean that the effects of the excipient on mortality cannot be distinguished. Hence, it is important that an excipient-only control is always included in these bioassays. Furthermore, in direct contact assays, when attempting to identify the toxicity pathway of the essential oil tested, it is important to include a hydrophobic control. Without this, it is impossible to distinguish simple mechanical effects from neurological or other cellular toxicity. The use of essential oils in the control of veterinary ectoparasites is an area which holds considerable potential for the future and research into their use is still at an early stage. More extensive field trials, the standardization of components, the standardization of extraction, the standardization of good experimental design, mammalian toxicology profiling and excipient development, as well as further investigation into the residual activities and shelf-lives of these oils are all required to allow the full realization of their potential. Key words. Botanical products, ectoparasites, essential oils, flies, lice, mange, mites, myiasis, pediculosis, ticks.

Introduction their effects on human health (Kolaczinski & Curtis, 2004) and the environment (Ramwell et al., 2009), combined with The control of ectoparasites of veterinary importance using a growing interest in organic farming practices, have led to synthetic neurotoxic insecticides has been progressively under- an increase in research into the development of alternative mined by the development of insecticide resistance. This is approaches to ectoparasite management, amongst which botan- seen particularly clearly in the control of lice (Johnson et al., ical alternatives, such as essential oils, are currently receiving 1992; James et al., 1993; Levot, 1995; Ellse et al., 2012), particular attention. mites (Beugnet et al., 1997) and ticks (Foil et al., 2004). In Essential oils are blends of approximately 20–80 different addition, restrictions on the use of some insecticides, such as plant metabolites which are usually extracted from plants organochlorines, organophosphates and pyrethroids, because of through steam distillation (Bakkalai et al., 2008). These

Correspondence: Lauren Ellse, Veterinary Parasitology and Ecology Group, School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG, U.K. Tel.: + 1179289182; Fax: + 44 1173317985; E-mail: [email protected]

© 2013 The Royal Entomological Society 233 234 L. Ellse and R. Wall metabolites are volatile molecules of low molecular weight. Table 1. Common and systematic names of plant essential oils Essential oils usually contain two or three major terpene or investigated in the papers included in this review. terpenoid components, which constitute up to 30% of the oil (Bakkalai et al., 2008). The insecticidal or acaricidal efficacy Common name Systematic name of many essential oils has been well documented in a variety Allspice Pimenta dioica of pests. This efficacy is often attributed to the oil’s major Basil Ocimum basilicum component(s); however, there is also evidence that the various Bay Laurus nobilis oil components may work in synergy (Yang et al., 2003). This Bog myrtle Myrica gale may occur because some oil components aid cellular accumu- Cade Juniperus oxycedrus lation and absorption of other toxic components (Cal, 2006). Camphor Cinnamomum camphora Carnation Dianthus caryophyllus Nevertheless, the mode of action of many essential oils or their Catnip Nepeta cataria components is largely unknown, although there is evidence Chamomile Matricaria chamomilla of a toxic effect on the insect nervous system. For example, Cinnamon Cinnamomum zeylanicum terpinen-4-ol, a monoterpenoid found at high concentrations Citronella Cymbopogon nardus in tea tree oil, inhibits arthropod acetylcholinesterase, an Clove Eugenia caryophyllata enzyme essential for transmission of action potentials (Mills Coriander Coriandrum sativum et al., 2004; Lopez & Pascual-Villalobos, 2010). Alternatively, Cumin Cuminum cyminum the hydrophobic nature of the oils may simultaneously exert Eucalyptus (blue gum) Eucalyptus globulus mechanical effects on the parasite such as by disrupting the Garlic Allium sativum cuticular waxes and blocking the spiracles, which leads to Geranium Pelargonium roseum Horsemint Mentha longifolia death by water stress (Burgess, 2009) or suffocation. Horseradish Armoracia rusticana A great deal of the recent research into the efficacy of essen- Lavender Lavandula angustifolia tial oils as agents for the control of arthropod ectoparasites of Lemon eucalyptus Corymbia citriodora veterinary importance has been carried out using such widely Manuka Leptospermum scoparium different extraction procedures, formulations, application meth- Mustard Brassica juncea ods and approaches to toxicity measurement that it can be dif- Nutmeg Myristica fragrans ficult to replicate or compare studies. The experimental designs Onion Allium cepa used in a large number of cases are not ideal, particularly in Oregano Origanum bilgeri relation to the inclusion of appropriate control groups, and Origanum onites Origanum minutiflorum undermine the conclusions drawn. This review therefore aims Pennyroyal Mentha pulegium to present a comparative synopsis of current research, against Peppermint Mentha piperita which the relative efficacies of these compounds are assessed Savoury Satureja thymbra and good experimental practice is highlighted. For ease of ref- Tea tree Melaleuca alternifolia erence, the common and systematic names of the plants from Thyme Thymus vulgaris which the essential oils are derived are shown in Table 1. Wormwood Artemisia absinthium Ylang ylang Cananga odorata Unknown Ageratum houstonianum Unknown Alpinia officinarum Mites Unknown Calea serrata Unknown Cupressus macrocarpa Poultry red mite. The poultry red mite, Dermanyssus Unknown Dorystoechas hasata gallinae (De Geer) (Mesostigmata: Dermanyssidae), has been Unknown Gynandropsis gynandra the subject of extensive acaricide assays using a wide range of Unknown Hesperozygis ringens essential oils (Kim et al., 2004; George et al., 2009a, 2010a, Unknown Hypericum polyanthemum 2010b). At a concentration of 0.35 mg/cm2 of filter paper, Unknown Laurus novocanariensis 50 of 56 essential oils tested resulted in 100% mortality Unknown Lippia sidoides after 24 h of contact with impregnated filter papers in a Unknown Lippia triplinervis Unknown Rhododendron tomentosum closed chamber and 12 of the tested oils produced 100% mite mortality at concentrations as low as 0.07 mg/cm2 (Kim et al., 2004). A second study by the same authors suggested that the contact activity of some essential oils, when exposure 100% mortality of mites after 24 h of exposure to the was measured in a closed chamber, was comparable with essential oils of cade (Juniperus oxycedrus), clove (Eugenia that of commercially available acaricides (Kim et al., 2004). caryophyllata), coriander (Coriandrum sativum), horseradish These effects of exposure to the vapour from essential oils (Armoracia rusticana) and mustard (Brassica juncea)(Kim suggested that the insecticidal efficacy could be attributed et al., 2004). Subsequent similar experiments with D. gallinae to the volatile components of the oils (Kim et al., 2004, supported these findings (Kim et al., 2007). 2007; George et al., 2009a). Placing mesh-ended cylinders The acaricidal efficacy of essential oils appears to be linked containing D. gallinae into sealed vials containing filter papers to the vapour pressure to which the mites are exposed, impregnated with 0.28 mg/cm2 of essential oil resulted in presumably because this affects the concentration of volatile

© 2013 The Royal Entomological Society, Medical and Veterinary Entomology, 28, 233–243 Essential oils in veterinary ectoparasite control 235 components. For example, a study in which mites were allowed D. gallinae (George et al., 2010b). Whereas the essential oils contact with oils in open or closed chambers showed that of cinnamon, garlic (Allium sativum) and pennyroyal (Mentha even the most effective of the three oils tested, essential oil pulegium) were found to have significant ovicidal activity at of manuka (Leptospermum scoparium), resulted in mortality concentrations equal to the 50% lethal concentration (LC50) of just 30% in contact assays carried out in open chambers for adult D. gallinae, essential oils of clove, thyme, cade and compared with > 80% in contact assays conducted in closed manuka had no effect on egg hatch at their corresponding adult chambers at the same concentration (George et al., 2009a). LC50 levels (George et al., 2010b). This result may reflect Similarly, > 90% mortality was observed after 24 h of exposure differences in the modes of action of the oils. However, an to the vapour of essential oil of thyme (Thymus vulgaris) alternative explanation may lie in the experimental design, as in closed chambers, whereas vapour assays conducted in the study used LC50 concentrations for adults which had been open chambers using the same concentration resulted in only determined previously, and this meant that some of the oils approximately 10% mortality (George et al., 2009a). which appeared to be non-ovicidal (e.g. essential oil of thyme) The highly volatile nature of essential oils may be respon- were used at concentrations at least five times lower than those sible for the relatively short persistence of the acaricidal effi- of the effective oils (George et al., 2010a). cacy of some oils. Indeed, contact exposure to 0.21 mg/cm2 of As yet, no major in vivo tests of the acaricidal efficacy of essential oil of lavender (Lavandula angustifolia) in a closed essential oils against D. gallinae appear to have been published. chamber resulted in > 70% mortality if the oil solution was A repellence experiment, in which red mite traps which had applied to the filter paper 3 min prior to exposure, whereas been treated with two different strains of catnip essential oil mortality dropped significantly to approximately 11% if the were placed into hen houses, found a significant reduction filter paper had been left in a fume cupboard for 24 h prior to in the number of mites caught in the essential oil-treated the introduction of the mites (George et al., 2008). However, trap compared with the control trap (Birkett et al., 2011). despite the decrease in acaricidal efficacy, olfactory experi- An in vitro study looking at the impact of environmental ments indicate that the volatile nature of essential oil of thyme factors in hen houses, such as temperature, humidity and dust may be sufficient to repel D. gallinae forupto13days(George levels, suggested that some essential oils may be particularly et al., 2009b). appropriate for further investigation in the field (George et al., Another limitation of essential oils identified in bioas- 2010a). However, a study of the impact of spraying pennyroyal says against D. gallinae is the variance between experiments and thyme essential oils in hen houses suggested there were a in the toxicity of the same essential oil (Kim et al., 2004; range of negative impacts associated with the presence of high George et al., 2010a), even when the oils are supplied by the concentrations of pennyroyal essential oil: two chickens died same source (George et al., 2010a). In addition, essential oils after chronic exposure to treated hen houses, and reductions in produced from different varieties of the same plant species both chicken weight gain and egg production were observed showed marked differences in toxicity against D. gallinae in (George et al., 2010c). By contrast, thyme essential oil did not experiments using essential oils of lavender (George et al., have any negative effects on the birds used (George et al., 2010a) and cinnamon (Cinnamomum zeylanicum)(Naet al., 2010c). 2011). Indeed, gas chromatography–mass spectrometry (GC- MS) found that the major constituents of two different strains of essential oil of catnip (Nepeta cataria) differed greatly Psoroptes spp. Early in vitro studies found that lavender from one another in that one strain was comprised of 92% essential oil and many of its constituents were effective against R-enantomer nepetalactone and 8% caryophyllene, whereas Psoroptes cuniculi (Hering) (Sarcoptiformes: Psoroptidae) the other was comprised of 17% R-enantomer nepetalactone, (Perrucci et al., 1994, 1996). These studies used contact and 70% S-enantomer nepetalactone and 13% caryophyllene (Bir- vapour exposure assays to show that there was a structural basis kett et al., 2011). This inconsistency in oil composition among to the acaricidal properties of essential oil derivatives (Perrucci batches is an inherent problem in experimental work and com- et al., 1995). In the latter study, monoterpene hydrocarbons, mercial applications. Such differences are important because limonene and γ -terpinene were found to be ineffective against the precise composition of an essential oil may determine its mites in open-chamber contact assays and closed-chamber acaricidal efficacy. Individual constituents of cinnamon and vapour assays at concentrations up to 1% (v/v) (Perrucci cassia oils have been shown to have acaricidal efficacy against et al., 1995), whereas the phenols, thymol and eugenol were D. gallinae (Na et al., 2011). Closed-chamber vapour assays extremely effective at killing mites, even at concentrations of of 34 compounds derived from Cassia spp. and Cinnamo- 0.125%, in open-chamber contact assays and closed-chamber mum spp. showed that α-methyl-E-cinnamaldehyde and E- vapour assays. Both linear (linalool, geraniol and nerol) and cinnamaldehyde had acaricidal efficacy comparable with that cyclic (terpinen-4-ol and menthol) terpene alcohols were highly of the acaricide dichlorvos with 50% lethal doses (LD50) effective at concentrations of 0.125%, with mortality rates of of 0.45 μg/cm3, 0.66 μg/cm3 and 0.3 μg/cm3, respectively, > 97% in open-chamber contact assays; however, mortality in 3 which were significantly better than the LD50 of 11.79 μg/cm closed-chamber vapour assays varied widely (Perrucci et al., attributed to the most effective whole cinnamon essential oil 1995). The structure of the components of essential oils (technical grade) (Na et al., 2011). has also been used to explain marked differences in the The chemical compositions of the various essential oils efficacy of the oils of two Laurus spp. against P. cuniculi may affect their mechanisms of action, which may explain in vitro (Macchioni et al., 2006). Experiments using GC- differences in the ovicidal efficacy of some oils against MS found that essential oil of bay (Laurus nobilis), which

© 2013 The Royal Entomological Society, Medical and Veterinary Entomology, 28, 233–243 236 L. Ellse and R. Wall provided relatively poor acaricidal effects with a 10% (v/v) 2010). In vivo trials in which 1% emulsions of seven differ- concentration resulting in mortality of only 73% after 24 h of ent essential oils were applied to groups of six S. scabiei var. contact exposure, is comprised mainly of 1,8-cineole (39.2%), suis-infested pigs showed that the essential oils of tea tree a monterpeniod. By contrast, Laurus novocanariensis had (Melaleuca alternifolia), pennyroyal and citronella (Cymbo- greater acaricidal effects, with a > 5% (v/v) concentration pogon nardus) all caused a reduction in mite numbers to < 7% resulting in 100% mite mortality; its major constituents were of the original infection 4 weeks post-treatment; essential oil α-pinene (10.4%), 1,8-cineole (9.6%) and β-selinene (7.2%), of tea tree was the most effective, with only 1.45% of original which are sesquiterpenes. However, neither oil was effective infection present by the end of the trial (Magi et al., 2006). at the lower concentration of 3% (Macchioni et al., 2006). In vivo treatment of Psoroptes sp. infections with the essential oil components linalool (Perrucci et al., 1997) and Otodectes cynotis. In vitro contact assays using the dog trans-cinnamic acid (Wall & Bates, 2011) resulted in high ear mite, Otodectes cynotis (Hering) (Astigmata: Psoroptidae), mite mortality. Rabbits that had been experimentally infected found that the essential oil constituent, geraniol, killed all mites with ear mites were cleared of infestation when treated twice within 1 h at concentrations of ≥ 5% (v/v), whereas limonene, weekly for 3 weeks with a 5% linalool, saline and petroleum p-cymene and α-pinene were less effective, taking in excess oil solution (Perrucci et al., 1997). The decrease in mite of 19 h to achieve 100% mortality at concentrations of 10% abundance was comparable with that provided by conventional (Traina et al., 2005). insecticide treatments (Perrucci et al., 1997). These positive results suggest that a similar solution may serve as the basis of future scab control programmes. However, some of the animals Ticks treated with higher concentrations of linalool (10%) had a short-lived reddening of the ear (Perrucci et al., 1997). Seven There has been extensive research in the last decade into the of eight experimentally infected sheep treated with 10% trans- repellent and acaricidal effects of many essential oils against cinnamic acid were completely cured of scab and the eighth ticks. The majority of these studies have focused on species sheep exhibited a significant reduction in mite abundance of Rhipicephalus and Ixodes (both: Ixodida: Ixodidae) ticks, and scab size; this protection was retained for 56 days post- largely in vitro. However, data on the effects of essential oils application and no adverse effects of treatment were reported as tick treatments or repellents in vivo are very limited. One (Wall & Bates, 2011). of the few studies in the latter category used a soap containing μ Ageratum houstonianum Whole essential oils of cinnamon leaf and clove have 0.03 L/g of essential oil of against ticks biting goats. However, the concentration actually applied been shown to have high levels of acaricidal efficacy to the goat is unclear from the methodology presented. Eight against P. cuniculi in rabbits at concentrations of 2.5% (Fichi days after goats were treated there was a 95% reduction in et al., 2007a, 2007b). After application, levels of excoriation, the numbers of biting ticks compared with a 23% reduction in scabbing and mite abundance decreased to show no significant the soap-only control (Pamo et al., 2005). Whether this was differences with those achieved by the positive control, ® the result of mortality, irritation leading to detachment or the Acacerulen (Teknofarma SpA, Turin, Italy) (25% pyrethrins) action of the soap/oil as a repellent that deterred the attachment in either essential oil treatment group (Fichi et al., 2007a, of further questing ticks is unclear. 2007b). An irritant effect of the essential oil of cinnamon leaf was evident, with reddening of the pinna and more regular ear scratching (Fichi et al., 2007b). No irritant effect was Immersion and contact assays. Mortality in Rhipicephalus observed in the clove oil-treated group, which the authors microplus (Canestrini) after exposure to essential oils has attributed to the second most abundant component of clove been particularly widely examined in vitro. Typically, in vitro essential oil, β-caryophyllene (24.9%) (Fichi et al., 2007a). trials involve the temporary immersion of adults or mesh This sesquiterpene, which itself showed no acaricidal efficacy bags containing newly hatched larvae into various solutions in vitro, may have a local anaesthetic effect (Ghelardini et al., of essential oil before mortality is assessed over subsequent 2001; Fichi et al., 2007a). days. The potency of essential oils against tick larvae varied con- siderably among plant species. The results of a larval immer- Sarcoptes scabiei. Contact assays against Sarcoptes sca- sion test using three essential oils derived from plants of the biei (De Geer) (Sarcoptiformes: Sarcoptidae) found that the Lamiaceae family [thyme, horsemint (Mentha longifolia)and essential oil of clove and derivatives of its major constituent, Dorystoechas hasata] showed 100% mortality in R. microplus eugenol, caused significant mortality at concentrations as low larvae 24 h after a 5-min submersion in 0.1% solutions of each as 1.5%, whereas essential oils of ylang ylang (Cananga oil (Koc et al., 2012). Cumin (Cuminum cyminum) and allspice odorata) and nutmeg (Myristica fragrans) had limited effects (Pimenta dioica) essential oils were also effective against the (Pasay et al., 2010). Notably, mortality appeared to differ same tick species in larval immersion tests at concentrations between permethrin-resistant and -susceptible populations of of 1.25% and 2.5%, respectively. However, basil (Ocimum S. scabiei, with resistant mites having a higher 50% lethal tox- basilicum) essential oil had no larvicidal properties, even at icity (LT50); however, the sample size was not sufficient to concentrations of 20% (Martinez-Velazquez et al., 2011). Mor- support a statistical confirmation of this trend (Pasay et al., tality was also high in Rhipicephalus sanguineus (Latreille)

© 2013 The Royal Entomological Society, Medical and Veterinary Entomology, 28, 233–243 Essential oils in veterinary ectoparasite control 237 and R. microplus larvae immersed in hexane-extracted Calea to have mortalities of 79.2% after 24 h and 83.3% after serrata essential oil diluted in ethanol. However, mortality of 48 h, respectively. Neither of the oils gave 100% adult 100% was not achieved until a concentration of 3.25% was mortality at the concentrations tested and many had very poor used (Ribeiro et al., 2008). In many cases it is difficult to acaricidal efficacy. Essential oil of eucalyptus (Eucalyptus compare the efficacies of essential oils between studies because globulus) killed just 37.5% of adult ticks at 6 days after inappropriate controls mean that the effects of the excipients immersion in a 5% solution, and some oils, including those used in each test cannot be distinguished. In addition, oil of H. polyanthemum (Ribeiro et al., 2007), C. serrata (Ribeiro extraction techniques, which may also affect subsequent effi- et al., 2008) and Lippia triplinervis (Lage et al., 2013), have cacy, vary widely. For example, methanol and hexane extrac- been shown to have no adult acaricidal effects at all. tions from the same plant resulted in differences in R. microplus A significant decrease in the egg mass laid by female larval mortality (Ribeiro et al., 2007). Steam distillation may Rhipicephalus was observed after immersion in the essential yield an essential oil with different properties again. These oils of L. triplinervis (Lage et al., 2013), geranium (Pirali- differences in efficacy may be attributed to the concentration Kheirabadi et al., 2009) and H. ringens (Ribeiro et al., 2010), of the oil’s major component or the relative concentrations of suggesting that sub-lethal doses of essential oils may have oil constituents and their interactions. Analysis of the larvici- an effect on tick fecundity. The viability of eggs from adults dal efficacy of the oil of Hesperozygis ringens and its major exposed to essential oils may also be affected. Immersion in component, pulegone, indicate that the oil as a whole has a oil of L. triplinervis caused a significant decrease in tick egg superior efficacy (Ribeiro et al., 2010), which may reflect the hatch (Lage et al., 2013). synergism of the oil’s components. The effect of prolonged contact with essential oils on adult Essential oil toxicity is also dependent upon arthropod tick mortality is not well studied. In filter paper contact assays susceptibility. Whereas two species of tick, R. sanguineus and in an open-chamber environment, oil of oregano (Origanum R. microplus, displayed no significant difference in mortality in onites)hadanLC50 of 2.34% (v/v) after 24 h against engorged response to the oil of C. serrata (Ribeiro et al., 2008), different Rhipicephalus turanicus (Pomerantsev) (Coskun et al., 2008). genera of ticks have shown marked differences in mortality Further studies in this area may be of considerable importance in response to the same essential oil (Gomes et al., 2012). to allow the range of potential impacts of essential oils on ticks Rates of mortality in larvae of Dermacentor nitens (Neumann) to be assessed. (Ixodida: Ixodidae) and R. microplus indicated LC50 values of 11.13 μL/mL and 5.59 μL/mL, respectively, after a 5-min immersion in essential oil of Lippia sidoides diluted in water ® Acaricidal vapour assays. As with mites, there is evidence and 2% Tween 80 (Gomes et al., 2012). that the acaricidal efficacy of essential oils against ticks Comparisons of the efficacy of these oils are hampered by may in part be attributable to their volatile components differences in the excipients used. For example, although the (Iori et al., 2005; Cetin et al., 2009, 2010). Exposure to control mortality of excipients such as ethanol (Ribeiro et al., closed vapour chambers containing the essential oils of tea 2008, 2010) or a trichloroethylene/olive oil mixture (Martinez- tree (Iori et al., 2005), oregano (Origanum minutiflorum) Velazquez et al., 2011) may be acceptably low, it cannot be (Cetin et al., 2009) and savoury (Satureja thymbra) (Cetin assumed that essential oils will interact in the same manner in et al., 2010) demonstrated acaricidal efficacy against Ixodes such disparate media. In addition, in some cases the units used ricinus (Linnaeus), R. turanicus and Hyalomma marginatum to record the concentrations are not intuitive and therefore it is (Koch) (Ixodida: Ixodidae), respectively. The toxicity of extremely difficult to compare the efficacy of oils tested in one O. minutiflorum study with that of oils tested in another. For example, the use the vapour phase of oregano ( ) essential oil of mg/mL as a unit of measurement is surprising in a context in against engorged R. turanicus was particularly high, with 95% μ which the diluted product is a liquid and the specific weight is mortality after 120 min of exposure to 5 L/L oil (Cetin et al., not stated (Ribeiro et al., 2007; Lage et al., 2013). Using this 2009). This effect was dependent on exposure time, with unit of measurement because it refers to the weight of the plant shorter exposure times resulting in lower mortality, even at prior to oil extraction is also problematic as different plants and higher concentrations (Cetin et al., 2009). High mortality was even sections of plants contain vastly different concentrations also seen in unfed adult H. marginatum (Cetin et al., 2010) and of essential oil. nymphal I. ricinus (Iori et al., 2005) exposed to essential oil of The susceptibility of adult Rhipicephalus spp. to essential savoury. The standardization of exposure time is undoubtedly oils appears generally to be substantially lower than that of an important factor in any attempt to compare levels of the larvae of this genus (Ribeiro et al., 2007, 2008, 2010). No essential oil toxicity against ticks. For example, a contact study significant mortality was observed after the immersion of adult on the effect of exposure time on mortality in I. ricinus nymphs Rhipicephalus in essential oils of Hypericum polyanthemum found that > 4.5 h of contact time was required to achieve (Ribeiro et al., 2007), C. serrata (Ribeiro et al., 2008) or 100% mortality in nymphs placed on filter papers impregnated 2 H. ringens (Ribeiro et al., 2010), although larvae immersed with 0.18 mL/cm of the oil of lemon eucalyptus (Corymbia in the same oils experienced high levels of mortality. citriodora) (Elmhalli et al., 2009). However, engorged adults of Rhipacephalus spp. immersed in 5% essential oil of geranium (Pelargonium roseum) (Pirali-Kheirabadi et al., 2009) and 0.8% essential oil of Tick repellence assays. Studies have shown that essential oregano (Origanum bilgeri)(Kocet al., 2013) were found oil of Gynandropsis gynandra (Lwande et al., 1999) and two

© 2013 The Royal Entomological Society, Medical and Veterinary Entomology, 28, 233–243 238 L. Ellse and R. Wall varieties of essential oil of catnip (Birkett et al., 2011) deter et al., 2011). These oils also demonstrated high larvicidal 2 unfed ticks from questing. A concentration of 0.1 μLofthe efficacy: LC50 values were 5 μg/cm for peppermint essential oil of G. gynandra was shown to repel 98.9% of nymphs of oil and 7 μg/cm2 for eucalyptus essential oil. Mortality of Rhipicephalus appendiculatus (Neumann) from questing; the 100% was achieved when pupae were exposed to 10% deterring effects of the oil’s major constituents were not as formulations of both oils (Kumar et al., 2011). However, effective as those of the oil as a whole, suggesting a synergistic the same authors found that essential oil of peppermint was effect (Lwande et al., 1999). However, the oil’s repellent effective at repelling M. domestica on cattle in vivo only at effect may have been attributable to the presence of the trace concentrations of 100%; 10% formulations applied to cattle element (0.1%), nerolidol, which was extremely effective, with had no effect (Kumar et al., 2011). On barn surfaces, a 0.001 μL causing 98.3% repellency (Lwande et al., 1999). Two 10% peppermint oil formulation was effective at deterring different strains of essential oil of catnip were shown, through M. domestica from landing (Kumar et al., 2011). However, GC-MS analysis, to have marked differences in chemical the control used in the in vivo studies was water, whereas composition which were associated with significant differences the excipient for the peppermint formulation contained 45% in effect: 50% repellency dosages (RD50) were 0.05 mg and xylene, 3% butane, castor oil ethoxylate and nonylphenol. No 0.0012 mg, respectively (Birkett et al., 2011). excipient-only control was used and hence the contribution Repellency of I. ricinus nymphs has also been shown of the excipient to any repellent effect cannot be determined. in response to essential oils of Rhododendron tomentosum This finding of relatively poor efficacy of peppermint oil (Jaenson et al., 2005) and carnation (Dianthus caryophyllus) as a deterrent in the field does not agree with earlier (Tunon´ et al., 2006). A suspension of 10% carnation essential observations made in water buffalo (Khater et al., 2009). oil repelled 100% of ticks at 4 h after treatment, which was Numbers of three species of nuisance fly, Stomoxys calcitrans comparable with the effects exerted in positive control groups (Linnaeus) (Diptera: Muscidae), M. domestica and Hippobosca using N,N-diethyl-meta-toluamide (DEET) and mandelic equina (Linnaeus) (Diptera: Hippoboscidae), were found to acid diethylamide (DEM) (Tunon´ et al., 2006). This efficacy decrease significantly on cattle treated for lice infestations dropped over time, but the suspension still gave a repellence with essential oils of camphor (Cinnamomum camphora), of 91% at 8 h after treatment. In this study, variation in the peppermint and chamomile (Matricaria chamomilla) until effects of various oil constituents was evident; suggesting that 6 days post-treatment; however, this experiment used only an some oil components may have better longevity than others untreated control and thus did not account for any possible (Tunon´ et al., 2006). repellent effects of a hydrophobic solution (Khater et al., In a feeding repellence assay, 95% of I. ricinus were 2009). deterred from feeding when a concentration of 10% of essential oil of R. tomentosum was introduced, but essential oil of bog myrtle (Myrica gale) showed a less marked degree of Haematophagous flies. A study of the repellent effects of repellency of 59% at the same concentration and essential oil essential oils of M. gale against the biting midge Culicoides of wormwood (Artemisia absinthium) had no effect on tick impunctatus (Goetghebuer) (Diptera: Ceratopogonidae) used a feeding (Jaenson et al., 2005). From a practical perspective, behavioural assay in which solutions of bog myrtle essential oil the use of wormwood as an acaricide would be limited by its and its constituents were diluted until the ratio of control : test high mammalian toxicity. landings equalled 1 (Stuart & Stuart, 1998). The essential oil of bog myrtle was diluted 512 times to reach this point. Notably, the various oil components had a wide range of efficacies: Flies terpinen-4-ol was the most repellent and required to be diluted 2048 times for repellence to cease (Stuart & Stuart, 1998). Musca domestica. In larvicidal tests in vitro against the High mortality was also observed and was attributed to the housefly, Musca domestica L. (Diptera: Muscidae), in which highly volatile fraction. early-stage larvae were submerged for 1 min in acetone and Three essential oils and their major constituents were tested essential oil extracts at concentrations of 100–300 p.p.m. in vitro and in vivo against S. calcitrans (Zhu et al., 2012). In a (0.01–0.03%), the most effective essential oil, peppermint repellency test, adult flies were starved for 48 h prior to being (Mentha piperita), was found to have an LC50 of 104 p.p.m. introduced into cages containing citrated bovine blood-soaked (Morey & Khandagle, 2012). Peppermint essential oil also sanitary towels, the membranes of which had been impregnated demonstrated a repellent effect when newly emerged adults with diluted essential oil. After 4 h flies were squashed to were placed into cages containing two conical flask traps, ascertain whether or not they had taken a bloodmeal. Essential of which one contained 1% essential oil in milk and oil of catnip was found to be a highly effective feeding and the other contained only milk; 96.8% of the flies were oviposition deterrent at 6.7%; the component nepetalactones found in the milk-only trap (Morey & Khandagle, 2012). were also 100% effective as deterrents at this concentration In the same study, peppermint essential oil also deterred (Zhu et al., 2012). In vivo, 250 mL of 15% and 30% catnip oviposition (Morey & Khandagle, 2012). Similarly, essential essential oil in TrintonX (3%) and water caused a significant oils of both peppermint and eucalyptus were shown to be decrease in the number of flies landing on cattle legs for effective repellents at doses of approximately 70 μg/cm2, up to 6 h (> 95% reduction). However, the repellence of the with repellencies of 86% and 76%, respectively (Kumar 15% solution was fairly short-lived; no significant difference

© 2013 The Royal Entomological Society, Medical and Veterinary Entomology, 28, 233–243 Essential oils in veterinary ectoparasite control 239 between this solution and the control were found at 8 h 2012b) and donkeys (Ellse et al., 2013) and only one study (Zhu et al., 2012). Interestingly, a mineral oil control also has investigated the effect of essential oils on the sucking demonstrated a significant repellent effect in the field and in louse, Haematopinus tuberculatus (Burmeister) (Phthiraptera: the feeding repellence assay (Zhu et al., 2012). This highlights Haematopinidae) (Khater et al., 2009). the need to include non-essential (e.g. mineral) oil controls in In vitro investigation into the efficacy of essential oil of toxicity and repellence assays in order to account for the effect tea tree against Bovicola ovis (Schrank) suggested that it has of the hydrophobic nature of essential oils on ectoparasite high insecticidal efficacy in closed-chamber contact and vapour survival and behaviour. assays; however, its residual activity may be short-lived (James & Callander, 2012a). Assays showed that exposure to a 1% concentration of essential oil of tea tree caused 100% mortality Myiasis and carrion flies. Studies have suggested that of B. ovis adults and eggs in contact with dipped wool, and essential oils may provide effective prevention and control mortality was also high in adults exposed to tea tree essential of the myiasis-causing fly Lucilia cuprina (Weidemann) oil vapour (James & Callander, 2012a). The efficacy of a 1% (Diptera: Calliphoridae) (Callander & James, 2012) and the concentration of essential oil of tea tree as a sheep dip or jet necrophagous Synthesiomyia nudiseta (Van De Wulp) (Diptera: product against B. ovis was confirmed in the field (James & Muscidae) (Khalaf et al., 2009). An extensive in vitro study Callander, 2012b). Sheep dipped in a 1% solution of essential found essential oil of tea tree to have significant repellent oil of tea tree had no clinical pediculosis for 20 weeks after effects against adults and larvae, as well as larvicidal and treatment. Jetting fully fleeced sheep with the same formulation ovicidal activity against L. cuprina (Callander & James, 2012). resulted in a 94% reduction in louse numbers, which did not No oviposition was observed when gravid female L. cuprina increase again for 12 weeks. However, both of these in vivo were given oviposition media treated with 3% (v/v) tea trials used untreated animals as controls and therefore some tree essential oil. The mean ± standard deviation number of mortality may be attributed to the emulsifying agent used in egg masses in the excipient-only control, which consisted the suspensions. In addition, the field efficacy of essential oil of a 3% aqueous emulsion of ethoxylated castor oil and of tea tree may not be as great if the animals were subject to ethoxylated oleic acid, was 20 ± 1.56 (Callander & James, repeated louse challenge. Indeed, only low levels of mortality 2012). Furthermore, in choice experiments, female L. cuprina were seen when lice were introduced to cotton fabric that had placed in cages with two oviposition sites, of which one been treated with essential oil of tea tree 1 day previously was treated with 3% tea tree essential oil and the other (James & Callander, 2012a). with excipient, did not lay any eggs on the tea tree oil- The essential oil of tea tree used in the experiments treated media for 44 days, indicating that essential oil of tea described by James & Callander (2012a, 2012b) complied tree has a substantial period of residual activity in treated with international standard ISO 4730 with respect to its wool (Callander & James, 2012). Second- and third-stage major constituents. The in vitro comparison of the toxicity larvae were also repelled from feeding media treated with of its major component, terpinen-4-ol, against B. ovis found tea tree essential oil (Callander & James, 2012). However, that it also resulted in similarly high rates of mortality in there was marked variance in life stage susceptibility; the oil vapour assays at the range of concentrations specified by its was highly toxic to L. cuprina eggs and first-stage larvae at ISO number (30–48%). The authors therefore attributed the 1% (v/v) concentrations, but failed to prevent the emergence mortality caused by essential oil of tea tree primarily to the of adults from third-stage larvae immersed even in very high presence of terpinen-4-ol. Contact assays using terpinen-4-ol concentrations of oil (Callander & James, 2012). against the donkey chewing louse, Bovicola ocellatus (Piaget), In S. nudiseta, the introduction of the essential oils of agreed with these findings; in this study, the 50% lethal time Cupressus macrocarpa and Alpinia officinarum into larval (LT50) of 3% terpinen-4-ol was much lower than that of 3% feeding media showed the oils to have larval LC50 values of tea tree essential oil, at 16.3 min compared with 30.2 min, 1.11% and 2.37%, respectively, both of which were statistically respectively (Talbert & Wall, 2012). Closed contact assays of lower than that of the excipient, acetone (Khalaf et al., 2009). six oils against B. ocellatus found the essential oils of lavender In addition, at the larval LC50 concentrations, 85% of surviving and tea tree to be the most toxic, with LC50 values of 0.76% larvae failed to pupate and 91% of those pupae failed to and 0.98%, respectively (Talbert & Wall, 2012). The efficacy emerge, whereas in the excipient-only control, 90% of larvae of the vapour phases of these oils was confirmed against pupated and 87% of pupae emerged (Khalaf et al., 2009). The B. ocellatus, which demonstrated approximately 80% mortality oil’s toxicity may be attributed to histopathology observed in after 2 h of exposure to 5% solutions of tea tree or lavender the fat bodies and salivary glands of the exposed larvae. essential oil (Ellse et al., 2013). In a limited in vivo study, spray application of 2 mL/kg of a 5% solution of lavender or ® 5% tea tree oil in water and emulsifier (2% Tween 80) to Lice groups of 10 donkeys with natural infestations of B. ocellatus resulted in a significant decline in louse numbers for 2 weeks A wide range of in vitro and in vivo studies have exam- after application (Ellse et al., 2013). ined the effects of various essential oils against lice of In the cattle sucking louse, H. tuberculatus, in vitro contact veterinary importance. The majority of research has con- assays comparing a range of oils found essential oil of centrated on chewing lice of the genus Bovicola (Phthi- camphor to be the most toxic with an LC50 of 2.74% and raptera: Trichodectidae) in sheep (James & Callander, 2012a, a significant ovicidal effect on eggs immersed for 10 min

© 2013 The Royal Entomological Society, Medical and Veterinary Entomology, 28, 233–243 240 L. Ellse and R. Wall in 22% camphor solution (Khater et al., 2009). However, residual repellence of facultative parasites by essential oils the pyrethroid positive control, D-phenothrin, also caused a is longer-lived than their insecticidal or acaricidal efficacy, 62% reduction in egg hatch, which was unexpected because presumably because lower doses are required to achieve pyrethroids are not usually considered to be ovicidal (Khater deterrence (Callander & James, 2012). A possible advantage of et al., 2009). Notably, essential oil of camphor was the least essential oils over conventional ectoparasite treatments, such as effective (LC50 of 8.6%) of the oils tested in vitro against pyrethroids, may lie in their ovicidal efficacy. As highlighted B. ocellatus by Talbert & Wall (2012). In vivo application here, significant egg mortality has been reported in mites of the oils of camphor, cinnamon, onion (Allium cepa)and (George et al., 2010b) and flies (Callander & James, 2012) peppermint to water buffalo, at the same concentrations treated with essential oils. It is unclear, however, whether used in the laboratory study, all resulted in a large initial this also results from neurotoxic rather than mechanical decline in H. tuberculatus numbers; however, just 6 days after suffocation and the ovicidal mode of action warrants further application, louse numbers had recovered (Khater et al., 2009). more detailed investigation, particularly into the importance Interestingly, the treated animals in the positive control (D- of oil components on ovicidal activity. Ovicidal effects may phenothrin) group also recovered their infection 10 days post- be particularly beneficial in the control of permanent parasites treatment (Khater et al., 2009). The difference in efficacy seen as they would reduce the need for multiple treatments to kill in the laboratory and field and between Bovicola sp. and newly hatched nymphs and thereby decrease treatment costs. H. tuberculatus may reflect climatic differences or species- There are many difficulties in comparing the studies of specific differences in louse susceptibility. essential oil toxicity that have been undertaken to date. One major issue refers to the variation in the relative concentrations of oil constituents between batches. This can lead to dramatic differences in the oils’ efficacy against ectoparasites (George Fleas et al., 2009a; Birkett et al., 2011). This also makes the comparison of oils between studies difficult without GC-MS To the authors’ knowledge, no specific studies on the effi- profiles of the specific batch of oil used. The widely used and cacy of whole essential oils against fleas have been reported commercially important oil, essential oil of tea tree, is the only in the primary scientific literature. However, it has been sug- oil for which set guidelines for constituent concentrations are gested that the citrus oil component limonene is toxic to the cat outlined in an international standard. This ensures that essen- flea Ctenocephalides felis (Bouche)´ (Siphonaptera: Pulicidae) tial oil of tea tree, complying with this standard, can be used (Collart & Hink, 1986; Hink & Fee, 1986) and the essential to provide consistency in ectoparasite control studies (James oil constituents carvacrol and nootkatone have been shown & Callander, 2012b). For large-scale control of ectoparasites, to cause high flea mortality in vitro (Panella et al., 2005). the products that make up an essential oil would need to be of However, essential oils may be of limited use against fleas as similar quantification in order to support the making of replica- they may not have a residual on-host phase that is sufficiently ble comparisons. A second issue is that in many experimental sustained to prevent re-infestation from the environment. designs it is difficult to confirm whether or not the effect seen is attributable to the oil at all; in many cases inappropriate controls mean that the effects of the excipient used in each Discussion test cannot be distinguished. It is important to always include an excipient-only control in the study design. Furthermore, There is a growing body of evidence supporting the efficacy of when attempting to identify the mechanism of toxicity of the essential oils as control agents against arthropod ectoparasites; essential oil tested in a direct contact assay, it is important their efficacy has not only been apparent following immersion to include a non-essential oil (e.g. a mineral oil) as a control. and physical contact with treated surfaces, but also after Without this, it is impossible to distinguish simple mechanical exposure to the vapour of the oils. The latter implies that there effects, such as blockage of the spiracles and suffocation, from is a neurotoxic rather than simply a mechanical pathway in essential oil-specific neurotoxicity or other cellular toxicity. their mode of action. This may be beneficial in ectoparasite The circumstances in which water alone can serve as an control. However, the volatile nature of essential oils may be informative control in any study design are likely to be rare. an obstacle to their use as ectoparasite treatments because their In order to maximize essential oil efficacy, further study insecticidal or acaricidal efficacy is likely to be relatively short- of the development of appropriate excipients for on-host lived. This has been demonstrated in studies in which essential application is required. Indeed, most of the in vivo trials oil preparations have been allowed to dry in open environments reported here have simply used water and a small amount of for hours prior to the introduction of the test arthropods. These emulsifier as the excipient. By contrast, James & Callander studies in mites (George et al., 2008; Wall & Bates, 2011) (2012b) used an ethoxylated castor oil, oleic acid and water and lice (James & Callander, 2012a) all reported extremely mix for the application of tea tree oil solution to sheep. Altering poor mortality rates in these treatment groups. These findings the hydrophobicity of the excipient is clearly likely to be of suggest that the application of essential oils may be more value because it may facilitate the better penetration of the appropriate for the control of permanent parasites because the oil into the animal’s coat and may also aid retention of the oils’ low levels of residual activity may not afford protection essential oil and hence prolong its activity. from continual environmental challenges by parasites with Given the poor public opinion of traditional chemical free-living stages. By contrast, studies have suggested that the treatments, essential oil-based insecticides are likely to appeal

© 2013 The Royal Entomological Society, Medical and Veterinary Entomology, 28, 233–243 Essential oils in veterinary ectoparasite control 241 to many pet owners. In addition, the shortfalls of essential oils, onites L. essential oil against Rhipicephalus turanicus (Ixodidae). such as a limited half-life, are likely to be less problematic in Parasitology Research, 103, 259–261. a pet care context than in livestock management. Companion Ellse, L., Burden, F. & Wall, R. (2012) Pyrethroid tolerance in animals are usually kept singly or in very small groups and the chewing louse Bovicola (Werneckiella) ocellatus. Veterinary thus re-infestation rates should be minimal and owners are Parasitology, 188, 134–139. often willing to reapply products. However, caution should Ellse, L., Burden, F. & Wall, R. (2013) Control of the chewing louse always be taken in applying essential oils to animals, especially Bovicola (Werneckiella) ocellatus in donkeys, using essential oils. those oils which are less widely available; toxicity data are Medical and Veterinary Entomology. doi: 10.1111/mve.12004. often missing and prolonged exposure to high concentrations Elmhalli, F.H., Palsson, K., Orberg, J. & Jaenson, T.G.T. (2009) Aca- of certain oils can have deleterious effects on the behaviour, ricidal effects of Corymbia citriodora oil containing para-menthane- health and welfare of the host (George et al., 2008). The human 3,8-diol against nymphs of Ixodes ricinus (Acari: Ixodidae). Exper- health implications of many more commonly used essential oils imental and Applied Acarology, 48, 251–256. have been documented and some oil components have been Fichi, G., Flamini, G., Giovanelli, F., Otranto, D. & Perrucci, S. Eugenia caryophyllata found to demonstrate irritant and potentially mutagenic effects (2007a) Efficacy of an essential oil of against Psoroptes cuniculi. Experimental Parasitology, 115, 168–172. (Tunon´ et al., 2006). Fichi, G., Flamini, G., Zaralli, L.J. & Perrucci, S. (2007b) Efficacy In conclusion, the use of essential oils in the control of an essential oil of Cinnamomum zeylanicum against Psoroptes of veterinary ectoparasites is an exciting area which holds cuniculi. Phytomedicine, 14, 227–231. huge potential for the future. However, research into the use Foil, L.D., Coleman, P., Eisler, M. et al. (2004) Factors that influence of essential oils as control agents is still at a preliminary the prevalence of acaricide resistance and tick-borne diseases. stage. 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