Residual and Ovicidal Efficacy of Essential Oil-Based Formulations in Vitro Against the Donkey Chewing Louse Bovicola Ocellatus

Medical and Veterinary Entomology (2016) 30,78–84 doi:10.1111/mve.12148

Residual and ovicidal efﬁcacy of essential oil-based formulations in vitro against the donkey chewing louse Bovicola ocellatus

B. S A N D S, L. E L L S E and R. W A L L Veterinary Parasitology and Ecology Group, University of Bristol, Bristol, U.K.

Abstract. Essential oils have shown good experimental potential as novel veterinary ectoparasiticides. However, if they are to be used as veterinary products, they must be available in formulations that are suitable for practical application against specifc ectoparasites. Here, the effcacies of formulations containing 5% (v/v) lavender or tea tree oil, in combination with two emulsifers [a surfactant, 5% (w/v) N-lauroylsarcosine sodium salt (SLS), and a soluble polymer, 5% (w/v) polyvinylpyrrolidone (PVP)], with or without 10% coconut oil, were tested in contact bioassays against the donkey chewing louse Bovicola ocellatus (Piaget) (Phthiraptera: Trichodectidae). Residual activity was quantifed in open and closed containers; ovicidal effcacy was also examined. Exposure to either of 5% (v/v) lavender or tea tree oils with SLS or PVP resulted in louse mortality of 100%, but when coconut oil was included as an excipient, signifcantly lower effcacy was recorded. However, the formulations became signifcantly less effective after 2h in open containers and 40 h in closed containers. The results confrm that the residual activity of essential oils is relatively transitory and the addition of 10% coconut oil does not prolong the period of insecticidal activity by slowing essential oil evaporation. Too short a period of residual activity is likely to be a signifcant impediment to the effective practical use of essential oils. However, unlike many synthetic pediculicides, the essential oils tested here were highly ovicidal, which suggests that prolonged residual activity may not be essential to kill newly hatched nymphs after treatment. Key words. Control, ectoparasite, livestock, parasiticide, pest, phytotherapy.

Introduction to allow this a number of issues must be resolved. Firstly, essential oils must be combined with excipients to create topical The need to identify novel, cost-effective treatments for the con- formulations that can be applied to and will disperse over trol of ectoparasites has stimulated considerable interest in the animals; essential oils are hydrophobic and therefore emulsifers use of natural botanical insecticides such as essential oils. In pre- must be included in aqueous formulations to facilitate their vious work, essential oils have been shown to cause signifcant dispersal. Secondly, the period of residual activity of these mortality in vitro against a wide range of ectoparasites, including volatile oils must be determined and if necessary extended so mites (Fichi et al.,2007;Georgeet al., 2009), ticks (Iori et al., that it is appropriate to the generation time, off-host survival 2005), fies (Kumar et al.,2011;Callander&James,2012),and and specifc parasite–host interaction pattern; excipients that lice (James & Callander, 2012; Ellse et al.,2013),afterbothcon- reduce volatility may be able to prolong the period of residual tact and exposure to vapour. activity. Finally, the effects of essential oils on eggs must The experimental evidence gives good reason to suggest that be established because signifcant levels of ovicidal activity essential oils may well form the basis of effective novel parasite will infuence the frequency of application required (Ellse & management products for the future (Ellse & Wall, 2013), but Wall, 2013).

Correspondence: Bryony Sands, Veterinary Parasitology and Ecology Group, University of Bristol, Life Sciences Building, 24 Tyndall Avenue, Bristol BS8 1TQ, U.K. Tel.: + 44 117 928 9000; Fax: + 44 117 929 8383; E-mail: [email protected]

Excipients, which make up the bulk of the formulation, are exudate and debris from the upper layers of the host epidermis, usually inert and can have a number of functions, including and infestation may result in irritation, unthriftiness, sleepless- those of stabilizing, synergizing and preserving the active ingre- ness and anorexia in the host. In the U.K., these symptoms usu- dient, as well as aiding the application and dispersal of the prod- ally manifest in winter, when animals have long coats and are uct (Ellse et al.,2012;Premjeetet al.,2012).Somecommonly housed indoors (Ellse et al., 2014). used pyrethroid formulations contain more than 90% hydropho- The aims of the present study were, frst, to examine the bic excipients such as butyl dioxitol and caprylic triglyceride. effcacy and ovicidal activity of different excipient formulations In some cases these oily excipients may contribute as much to against B. ocellatus, in combination with two essential oils of the effcacy of the formulation as the active ingredient itself: known pediculicidal activity, lavender oil and tea tree oil (Ellse butyl dioxitol was found to account for a signifcant level of et al.,2013),asasteptowardsdevelopingformulationsthatcan concentration-dependent mortality in pyrethroid-tolerant lice be applied to animals. Secondly, the study aimed to assess the and was as effective as 4% permethrin (Ellse et al., 2012). residual activity of the oils in the context of free or restricted Immersion of Bovicola ocellatus in grapeseed or silicone oils levels of evaporation. resulted in 86% and 100% mortality, respectively (Talbert & Wall, 2012). A pesticide-free, coconut oil-based shampoo for head lice Pediculus humanus capitis De Geer (Phthiraptera: Materials and methods Pediculidae) (Schooltime Shampoo®;BiosafeTechnologies Ltd) gave a 61% cure rate in children after two applications, Lice compared with a 14% cure rate achieved by treatment with a permethrin-based shampoo (Connolly et al., 2009). An appro- Bovicola ocellatus adults, nymphs and eggs were collected priate excipient may therefore enhance the effcacy of an insec- from naturally infested donkeys at a farm in Devon, southwest ticidal formulation. U.K. Lice were collected either by grooming or from the Essential oils consist of low molecular weight, highly volatile hair of animals that had been clipped as part of their routine terpenes and terpenoids. The high volatility leads to low per- management. Lice were placed in 60-mL plastic specimen pots sistence and, although this may be considered environmentally (Scientifc Laboratory Supplies Ltd, Nottingham, U.K.) and advantageous, provides a short period of protection from par- taken to the laboratory within 24 h of collection. They were asite re-infestation if hosts are under repeated challenge. For held in darkness at 35 ∘Cand75%relativehumidity(RH)in ectoparasites that spend the majority of their lives on the host, an incubator (MLR-351H; Sanyo, Panasonic Biomedical Sales residual activity should ideally at least match the likely survival Europe BV, Loughborough, U.K.). Donkey hair and scurf, time in the environment. However, most studies report low resid- collected from the same group of animals, were provided for ual activity. For example, the insecticidal effcacy of wool treated food. Preliminary observations had indicated good survival over with 1% tea tree oil against Bovicola ovis Schrank declined a period of several days under these conditions (Ellse, 2013) from 100% to 15% over a period of 1.5 h (James & Callander, and lice were used for assays within 3 days of collection. 2012). Similarly, the acaricidal effcacy of lavender essential oil All the nymphal and adult lice used for assays were female; dropped from > 80% to 8–11% over a 24-h period when poultry observations indicated that fewer than 1% of the population mites, Dermanyssus gallinae (De Geer) (Mesostigmata: Der- were male. manyssidae), were applied to pretreated surfaces (George et al., 2008). Not only must residual activity last long enough to protect Excipients against re-infestation from the environment or from other animals, but if compounds are not ovicidal, they must remain Suspensions of 5% (v/v) tea tree and 5% (v/v) lavender in situ long enough to kill newly hatched juvenile stages, or essential oils (Essential Oils Direct, Oldham, U.K.) were made they must be reapplied. The ovicidal effcacy of essential oils in distilled water with one of two emulsifers, the soluble has been widely reported against P. h . c a p i t i s (Priestley et al., polymer, polyvinylpyrrolidone (PVP) (Sigma-Aldrich Co. Ltd, 2006; Toloza et al.,2008;Yanget al.,2009).EggsofB. ovis that Gillingham, U.K.) and the surfactant N-lauroylsarcosine sodium were applied to wool freshly dipped in 1% tea tree oil showed salt (SLS) (Sigma-Aldrich Co. Ltd). The chemical composition 100% reductions in hatchability and embryonic development of tea tree oil (terpinen-4-ol type) complied with international (James & Callander, 2012). Immersion for 10 min in the essen- standards (ISO 4730). The lavender oil contained 40–42% of its tial oils of camphor (22%), onion (47%), peppermint (57%) major constituent, linalyl acetate. Formulations of the lavender and chamomile (54%) resulted in reductions of 78–95% in the or tea tree oils were made with either 5% (w/v) SLS or 5% (w/v) hatchability of eggs of the buffalo louse Haematopinus tuber- PVP. Formulations of the essential oils and emulsifers were also culatus (Burmeister) (Phthiraptera: Haematopinidae), compared examined with and without the addition of 10% coconut (Cocos with 62% for the pyrethroid d-phenothrin (Khater et al., 2009). nucifera) oil (Sigma-Aldrich Co. Ltd). Excipient-only controls By contrast, the ovicidal effcacy of many of the common syn- were used in all cases. thetic pediculicides, such as lindane, pyrethrins with piperonyl The effcacy of each formulation was tested against butoxide and permethrin, is poor (Meinking et al., 2001). B. ocellatus using contact assays. Filter papers (90 mm in Chewing lice of domestic animals, such as the donkey louse diameter; Whatman No. 1) were placed in 90-mm diameter B. ocellatus,areofclinicalsignifcanceworldwide(Kaufmann, polystyrene Petri dishes and quantities of 800 !Lofeachsus- 1996; Svendsen, 2008). Feeding occurs by scraping epidermal pension were pipetted evenly onto separate flter papers. This

© 2015 The Royal Entomological Society, Medical and Veterinary Entomology, 30,78–84 80 B. Sands et al. volume was enough to fully saturate the flter paper. The Petri Statistical analysis dishes were then placed in a fume cupboard for 5 min to allow the flter papers to absorb the liquid. Ten adult female lice were All statistical analyses were carried out using R Version 3.0.3 placed onto each flter paper, lids were ftted, and the Petri RCoreTeam,2013).Thetoxicityoftheessentialoiland dishes were maintained in darkness in an incubator at 35 ∘C excipient formulations to B. ocellatus were examined at 2 h and 75% RH. Lice were checked for mortality at 15 min, and post-exposure. Louse mortality was the dependent variable in at 1 h, 2 h, 3 h, 4 h and 24 h after exposure to the flter paper. a three-way analysis of variance (anova) with life stage (adult During these checks the Petri dishes were removed from the or nymph), essential oil and excipient as factors. Tukey multiple incubator and mortality was assessed by observing the lice range tests were used for post hoc analysis. The median time (h) under a dissecting microscope (×30); absence of movement of required to kill 50% of lice after exposure to the formulations the legs, mouthparts, antennae or abdomen even when stroked (LT50)wascalculatedforeachtimeperiodforwhichflterpapers with a dissecting needle was taken as indicating death. The Petri were left before lice were introduced, for adults and nymphs in dishes were immediately returned to the incubator. The assays open and closed containers. The LT50 was used as the response were repeated three times with adult lice and also three times variable in a general linear model (GLM) with pre-exposure time with small nymphs that were considered to be frst or second as a continuous variable and excipient type as a factor. Egg hatch instars. data were analysed using chi-squared analysis.

Results Residual activity Excipients The residual activity of the lavender and tea tree essential The results of exposure of lice to formulations containing oils was examined using formulations in PVP, with and without lavender essential oil for 2 h showed a signifcant difference in 10% coconut oil. Residual activity was observed in open (free evaporation) and closed (reduced evaporation) Petri dishes. Contact assays were carried out as described previously, except that dosed flter papers were left for 0 h, 1 h, 2 h and 5 h with the Petri dish lids off, or for 0 h, 20 h or 40 h with the Petri dish lids on, before lice were exposed. Lice were examined for mortality after 1 h, 4 h and 24 h. The assays were repeated three times with adults and three times with small nymphs that were considered to be frst or second instars. Excipient-only controls were used.

Ovicidal effcacy

The ovicidal activities of the essential oils of tea tree and lavender on B. ocellatus eggs were investigated using flter paper contact assays. Suspensions of 5% (v/v) tea tree oil and lavender oil were made in distilled water with a 5% (w/v) PVP excipient. As a hydrophobic control, 5% (w/v) coconut oil in distilled water with a 5% (w/v) PVP excipient was used (5% coconut oil was used here for comparison with the essential oils). There was also an untreated (dry flter paper) control. Filter papers were treated in the same way as described above and left in a fume cupboard for 5 min before 20 B. ocellatus eggs were removed from the hair to which they were attached and placed onto each flter paper using a fne paint brush and forceps. Lids were placed on the Petri dishes, which were then maintained at 35 ∘C, 75% RH. The numbers of eggs that hatched were recorded on days 1, 4, 8 and 12 after their introduction, after which no further eggs hatched Fig. 1. Mean ± standard error mortality in three replicates of 10 Bovi- in any treatment. For observations of hatch, Petri dishes were cola ocellatus (A) adult females or (B) nymphs after exposure to 5% removed from the incubator and the eggs were inspected under (v/v) lavender essential oil formulations containing excipients of either 10× magnifcation; lice were considered to have hatched if the 5% (w/v) polyvinylpyrrolidone (PVP) ( ), 5% (w/v) N-lauroylsarcosine head and thorax had emerged from the chorion. This trial was sodium salt (SLS) ( ), 5% PVP and 10% (w/v) coconut oil ( ), or 5% carried out in triplicate. SLS and 10% (w/v) coconut oil ( ). Points are joined for clarity.

not signifcantly reduce mortality compared with formulations

containing SLS or PVP alone in either adults (F3,11 = 2.77, P = 0.092) (Fig. 2A) or nymphs (F3,8 = 3.35, P = 0.076) (Fig. 2B), although the differences did approach signifcance. After exposure to control (excipient-only) formulations for 2 h, there were no signifcant differences in mortality between

excipient types in either adult lice (F3,11 = 0.64, P = 0.60) or nymphs (F3,8 = 1.07, P < 0.42); mortality amounted to ≤ 3% in adult lice and ≤ 2% in nymphs. Mortality after 24 h was 12–50% in adult lice exposed to control formulations, whereas it was 100% for both excipient types with lavender and tea tree oils (Table 1). Nymph mortality after 24 h of exposure was 10–33% in controls, 90–100% in nymphs exposed to lavender oil, and 73–97% in nymphs exposed to tea tree oil.

Residual activity

In open Petri dishes in which flter papers had been treated with essential oils less than 5 min previously, 50% of adult lice died within an average of 96 min. However, the time taken

for 50% of lice to die (LT50) was greater than 24 h when they were exposed to flter papers that had been treated more than

2h previously (Fig.3). The LT50 increased signifcantly with increasing flter paper drying time for formulations containing tea tree (F3,20 = 17.47, P < 0.001) and lavender (F3,20 = 16.42, P < 0.001) oil. There was no effect of the presence of coconut oil with the PVP excipient. The mortality of adult lice in Fig. 2. Mean ± standard error mortality in three replicates of 10 Bovi- contact with excipient-only controls was low and showed lit- cola ocellatus (A) adult females or (B) nymphs after exposure to 5% tle change with increasing pre-exposure period (F1,22 = 0.078, (v/v) tea tree essential oil formulations containing excipients of either P = 0.78). Similar results were obtained with nymphs for both 5% (w/v) polyvinylpyrrolidone (PVP) ( ), 5% (w/v) N-lauroylsarcosine tea tree (F2,21 = 8.56, P < 0.05) and lavender (F3,20 = 22.91, sodium salt (SLS) ( ), 5% PVP and 10% (w/v) coconut oil ( ), or 5% P < 0.001) oils. SLS and 10% (w/v) coconut oil ( ). Points are joined for clarity. For essential oil formulations placed in closed containers,

average LT50 was 102 min in adult lice exposed to flter papers mortality between the excipient types for adults (F3,11 = 15.26, treated < 5 min prior to louse introduction. When flter papers P < 0.001) and nymphs (F3,8 = 27.31, P < 0.001). There was no were treated 20 h before testing, adult LT50 was < 16 h (Fig. 4). signifcant difference in mortality among control lice exposed to When flter papers had been treated 40 h previously, adult LT50 SLS or PVP alone (P = 0.39), or SLS and PVP with coconut oil was > 24 h (Fig. 4). This change was highly signifcant for both (P = 0.92). Formulations containing essential oil and coconut tea tree (F1,12 = 229.42, P < 0.001) and lavender (F1,12 = 87.44, oil caused lower adult and nymphal mortality than formulations P < 0.001) essential oils. The presence of coconut oil signif- that did not contain coconut oil (P < 0.05) (Fig. 1). In tea tree cantly reduced the effcacy of both lavender (t = 2.37, P < 0.05) oil formulations exposed for 2 h, the presence of coconut oil did and tea tree (t = 2.38, P < 0.05) essential oils. For nymphs, there

Table 1. Mean ± standard error percentage mortality in three replicates of 10 Bovicola ocellatus after 24 h of exposure to flter papers treated with tea tree or lavender essential oils in combination with excipients of polyvinylpyrrolidone (PVP) and N-lauroylsarcosine sodium salt (SLS), with or without coconut (Cocos nucifera)oil.

Mortality, % PVP (5% w/v) and SLS (5% w/v) and Stage Essential oil PVP (5% w/v) SLS (5% w/v) coconut oil (10% w/v) coconut oil (10% w/v)

Adults Tea tree (5% v/v) 100 ± 0% 100 ± 0% 100 ± 0% 100 ± 0% Lavender (5% v/v) 100 ± 0% 100 ± 0% 100 ± 0% 100 ± 0% Control 50.0 ± 18.0% 30.0 ± 11.0% 20.0 ± 13.0% 12.5 ± 7.0% Nymphs Tea tree (5% v/v) 73.3 ± 27.0% 96.7 ± 3.0% 96.7 ± 3.0% 90.0 ± 0.0% Lavender (5% v/v) 90.0 ± 10.0% 100 ± 0% 96.7 ± 3.0% 93.3 ± 7.0% Control 26.7 ± 9.0% 20.0 ± 0.0% 10.0 ± 6.0% 33.3 ± 9.0%

Fig. 3. Time (h) required for 50% of 10 adult female Bovicola ocellatus Fig. 4. Time (h) required for 50% of 10 adult female Bovicola ocellatus to die (LT50)afterbeingplacedontreatedflterpapersthatwereleftin to die (LT50)afterbeingplacedontreatedflterpapersthatwereleftin open containers for 0 h, 1 h, 2 h or 5 h pre-exposure of lice. Filter papers closed containers for 0 h, 20 h or 40 h pre-exposure of lice. Filter papers were treated with (A) 5% (v/v) tea tree or (B) 5% (v/v) lavender essential were treated with (A) 5% (v/v) tea tree or (B) 5% (v/v) lavender essential oils formulated with excipients of 5% (w/v) polyvinylpyrrolidone (PVP) oils formulated with excipients of 5% (w/v) polyvinylpyrrolidone (PVP) ( ) or 5% PVP and 10% (w/v) coconut oil ( ). ( ) or 5% PVP and 10% (w/v) coconut oil ( ). was a signifcant increase in LT50 with flter paper pre-exposure Table 2. Mean ± standard deviation (SD) numbers of hatched Bovicola period for both lavender (F1,12 = 560.86, P < 0.001) and tea ocellatus eggs in three replicates of 20 eggs exposed to flter papers tree (F1,12 = 25.47, P < 0.001) essential oils, but the presence of treated with essential oils. coconut oil did not affect the change in LT50.Liceexposedto flter papers treated with only the excipient showed no change Hatched eggs, mean ± SD in LT50 as the flter paper pre-exposure period increased in 1day 4days 8days 12days either adults (F1,16 = 2.81, P = 0.11) or nymphs (F1,16 = 0.22, P = 0.64). Tea tree oil 5% 0 0 0 0 Lavender oil 5% 0 0 0 0 Coconut oil 5% 0.3 ± 0.6 1.7 ± 2.1 9.7 ± 2.9 14.3 ± 1.5 Filter paper only 0 6.3 ± 1.7 14.3 ±2.9 14.3 ± 2.9 Ovicidal effcacy

No louse eggs in contact with flter papers treated with 5% lavender or tea tree oil hatched, whereas a mean ± standard mortality after contact (Ellse et al.,2013).Ithasbeendemon- deviation of 72 ± 10% of eggs exposed to 5% coconut oil strated that the insecticidal effect is the result of a combination hatched (Table 2). The difference was signifcant for both tea of exposure to volatile insecticidal components of the oil and 2 2 tree (" = 67.01, d.f. = 1, P < 0.01) and lavender (" = 67.01, direct mechanical effects (Ellse et al., 2012, 2013). The frst pur- d.f. = 1, P < 0.01) oil. The hatch rate of eggs exposed to 5% pose of the present work was to identify excipients suitable for coconut oil alone did not differ signifcantly from that in eggs use in formulations of these oils that would maximize their eff- 2 exposed to the dry flter paper control (" = 0, d.f. = 1, P > 0.9). cacy, aid their suspension in water, and give favourable qualities for in vivo application. The two emulsifers tested, PVP (a soluble polymer) and SLS (a surfactant), are both already used in Discussion human cosmetic products and neither adversely affected the high louse mortality achieved with both essential oils. Therefore both Lavender and tea tree oils in a 0.2% polysorbate-80 excipient excipients are potential candidates for on-animal application. have previously been shown to facilitate high levels of louse However, the residual activity, measured as LT50,wasrelatively

© 2015 The Royal Entomological Society, Medical and Veterinary Entomology, 30,78–84 Essential oils for louse control 83 short with both excipients, extending to 2 h in open containers In the present work, lavender and tea tree oils were shown to and 40 h in closed containers. Previous work has also highlighted be effective against B. ocellatus adults, nymphs and eggs; hence the short residual activity of most essentials oils; for example, both oils represent potential pediculicides that could eliminate the effects of lavender oil against the poultry mite D. gallinae all louse life stages at once, but are subject to limitations in were shown to fall to < 11% when flter papers were left in a their residual activity. Therefore, simultaneous treatment of a fume cupboard for 24 h pre-exposure of mites (George et al., closed population of animals with essential oils may provide 2008). effective control of permanent ectoparasites. In this context It was hypothesized that the coconut oil might increase louse prolonged residual activity is not essential because there is little mortality, given the recorded effcacy of a coconut oil-derived or no immigration from untreated animals, particularly with high shampoo against head lice (Connolly et al.,2009).Inaddition, levels of ovicidal activity as seen here. Indeed, non-persistence it was considered that coconut oil might reduce the evapo- may help to minimize the risk for non-target effects and the ration of the essential oils, thereby increasing their residual development of resistance. activity. However, when 10% coconut oil was included into the formulation, the effcacy of the essential oils was decreased signifcantly, but the residual activity of the essential oils, when Acknowledgements combined with PVP, was not signifcantly increased. Hence, if coconut oil did reduce evaporation, it was not suffcient to The authors would like to thank The Donkey Sanctuary for their prevent the rapid loss of insecticidal effcacy. The reduction assistance in collecting lice and eggs. We would also like to in effcacy attributable to the presence of coconut oil was thank the anonymous reviewer for their helpful input. greater in formulations containing lavender essential oil than in those containing tea tree oil: after 4 h of exposure, lavender formulations containing coconut oil achieved louse mortality of approximately 60%, whereas those without coconut oil References achieved louse mortality of 100%. Similarly, formulations of Callander, J.T. & James, P.J. (2012) Insecticidal and repellent effects tea tree oil with grapeseed oil have been shown to require higher of tea tree (Melaleuca alternifolia)oilagainstLucilia cuprina. concentrations of tea tree oil to give signifcant mortality in Veterinary Parasitology, 184,271–278. in vitro assays simulating fumigation with B. ovis (James & Connolly, M., Stafford, K.A., Coles, G.C., Kennedy, C.T.C. & Downs, Callander, 2012). Here, although the coconut oil reduced the A.M.R. (2009) Control of head lice with a coconut-derived emulsion rate of kill, it did not prevent it overall. There may therefore shampoo. Journal of the European Academy of Dermatology and be a compromise between prolonging residual activity and Venereology, 23,67–69. maintaining insecticidal effcacy by ensuring vapour pressure is Ellse, L. (2013) The epidemiology and control of the louse Bovicola not overly reduced. This would require further investigation. ocellatus (Piaget) in donkeys. PhD Thesis. University of Bristol, The data presented in this and previous studies suggest that Bristol. when an essential oil-based formulation is applied topically Ellse, L. & Wall, R. (2013) The use of essential oils in veterinary to an animal, its residual activity is likely to be poor. Low ectoparasite control: a review. Medical and Veterinary Entomology, levels of residual activity have previously been demonstrated 28,233–243. on hair fbres in contact assays: when 5% tea tree oil was Ellse, L., Burden, F. & Wall, R. (2012) Pyrethroid tolerance in the chew- applied to sheep wool and allowed to dry for 1.5 h before ing louse Bovicola (Werneckiella) ocellatus. Veterinary Parasitology, applying B. ovis, louse mortality was not signifcantly different 188,134–139. from that in untreated controls (James & Callander, 2012). A Ellse, L., Burden, F.A. & Wall, R. (2013) Control of the chewing louse short drug half-life would lead to a high chance of re-infestation Bovicola (Werneckiella) ocellatus in donkeys, using essential oils. if there was subsequent exposure to untreated animals. This Medical and Veterinary Entomology, 27,408–413. Ellse, L., Burden, F.A. & Wall, R. (2014) Seasonal infestation of has consequences for the future application of essential oils as donkeys by lice: phenology, risk factors and management. Veterinary ectoparasiticides for animals of agricultural importance, such Parasitology, 203,303–309. as cattle and sheep, in contexts in which treated and untreated Fichi, G., Flamini, G., Giovanelli, F., Otranto, D. & Perrucci, S. animals may mix and a time-consuming and repetitive treatment (2007) Effcacy of an essential oil of Eugenia caryophyllata against regime is impractical. Psoroptes cuniculi. Experimental Parasitology, 115,168–172. Prolonged residual activity is usually considered to be essen- George, D.R., Callaghan, K., Guy, J.H. & Sparagano, O.A.E. (2008) tial with synthetic chemical insecticides, such as pyrethroids, Lack of prolonged activity of lavender essential oils as aracicides because these compounds have no reported ovicidal activity. against the poultry red mite (Dermanyssus gallinae) under laboratory Hence, they must either persist on the animal long enough to conditions. Research in Veterinary Science, 85,540–542. destroy newly hatched nymphs (at least 2 weeks), or be reap- George, D.R., Smith, T.J., Shiel, R.S., Sparagano, O.A.E. & Guy, J.H. plied. Here, contact assays showed that lavender and tea tree oils, (2009) Mode of action and variability in effcacy of plant essential oils at concentrations of 5%, are both highly ovicidal. This has also showing toxicity against the poultry red mite, Dermanyssus gallinae. been demonstrated for the essential oil constituents nerolidol, Veterinary Parasitology, 161,276–282. geraniol and thymol (Priestley et al.,2006;James&Callander, Iori, A., Grazioli, D., Gentile, E., Marano, G. & Salvatore, G. (2005) 2012). In the present study, the coconut oil control did not inhibit Acaricidal properties of the essential oil of Melaleuca alternifolia egg development or hatching, suggesting that mortality was not Cheel (tea tree oil) against nymphs of Ixodes ricinus. Veterinary aresultofasphyxiationoranymechanicaleffect. Parasitology, 129,173–176.

James, P.J. & Callander, J.T. (2012) Bioactivity of tea tree oil from Priestley, C.M., Burgess, I.F. & Williamson, E.M. (2006) Lethality Melaleuca alternifolia against sheep lice (Bovicola ovis Schrank) in of essential oil constituents towards the human louse, Pediculus vitro. Veterinary Parasitology, 187,498–504. humanus, and its eggs. Fitoterapia, 77,303–309. Kaufmann, J. (1996) Stages on the body surface. Parasitic Infections of RCoreTeam(2013)R: A Language and Environment for Statistical Domestic Animals: A Diagnostic Manual, p. 195. Birkhäuser Verlag, Computing. R Foundation for Statistical Computing, Vienna. Basel. Svendsen, E. (2008) The Professional Handbook of the Donkey, 4th edn. Khater, H.F., Ramadan, M.Y. & El-Madawy, R.S. (2009) Lousicidal, Whittet Books, Yatesbury. ovicidal and repellent effcacy of some essential oils against lice and Talbert, R. & Wall, R. (2012) Toxicity of essential and non-essential fies infesting water buffaloes in Egypt. Veterinary Parasitology, 164, oils against the chewing louse, Bovicola (Werneckiella) ocellatus. 257–266. Research in Veterinary Science, 93,831–835. Kumar, P., Mishra, S., Malik, A. & Satya, S. (2011) Repellent, larvicidal Toloza, A.C., Vassena, C. & Picollo, M.I. (2008) Ovicidal and adulti- and pupicidal properties of essential oils and their formulations cidal effects of monoterpenoids against permethrin-resistant human against the housefy, Musca domestica. Medical and Veterinary head lice, Pediculus humanus capitis. Medical and Veterinary Ento- Entomology, 25,302–310. mology, 22,335–339. Meinking, T.L., Entzel, P., Villar, M.E., Vicaria, M., Lemard, G.A. & Yang, Y.C., Lee, S.H., Clark, J.M. & Ahn, Y.J. (2009) Ovicidal and Porcelain, S.L. (2001) Comparative effcacy of treatments for pedicu- adulticidal activities of Origanum majorana essential oil constituents losis capitis infestations: update 2000. Archives of Dermatology, 137, against insecticide-susceptible and pyrethroid/malathion-resistant 287–292. Pediculus humanus capitis (Anoplura: Pediculidae). Journal of Premjeet, S., Ajay, B., Sunil, K., Bhawana, K., Sahil, K., Divashish, R. Agricultural and Food Chemistry, 57,2282–2287. & Sudeep, B. (2012) Additives in topical dosage forms. International Journal of Pharmaceutical, Chemical, and Biological Sciences, 2, Accepted 7 August 2015 78–96. First published online 2 November 2015