Essential oils in the management of the donkey louse, Bovicola ocellatus

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1 Essential oils in the management of the donkey louse, Bovicola ocellatus L. ELLSE*, B. SANDS, F. A. BURDEN and R. WALL Veterinary Parasitology and Ecology Group, School of Biological Sciences, University of Bristol, UK The Donkey Sanctuary, Sidmouth, Devon, UK. Equine Veterinary Journal ISSN DOI: /evj *Correspondence Received: ; Accepted: Summary Reasons for performing study: Chewing lice are widespread and clinically compromising parasites of livestock and equids. Their management is complicated by growing levels of resistance to commonly applied insecticides. Hence, the development of novel approaches to their control is of major clinical interest. Objectives: To assess the effects of incorporating the essential oils of tea tree and lavender into a grooming programme for populations of donkeys with natural infestations of Bovicola ocellatus in the UK and Ireland when louse populations were at their winter seasonal peak. Study design: In vivo field trial. Methods: Suspensions of 5% (v/v) tea tree or lavender oil or an excipient only control were groomed into the coats of winter-housed donkeys (n = 198) on 2 occasions, 2 weeks apart. Louse counts were conducted before each application and 2 weeks later. Results: After 2 applications, the groups groomed with lavender or tea tree oil suspensions had a significant reduction in louse intensity, with a mean decline in louse abundance of 78% (95% confidence interval 76 8%). Louse numbers in the groups groomed with excipient only either did not change or increased significantly. Donkey hair length had no effect on the decline in louse numbers. Conclusions: These results demonstrate that the inclusion of essential oil suspensions during grooming can be used to manage louse populations successfully. The Summary is available in Chinese see Supporting Information. Keywords: donkey; lice; Bovicola; essential oils; ectoparasite Introduction The control of lice has generally centred on herd or flock-level insecticide treatment, often at the point of winter housing. However, routine application of a limited range of insecticides, along with misapplication and the use of inappropriate insecticide formulations [1], has provided a sustained selection pressure for resistance in many louse populations. Within the last 25 years, widespread resistance to pyrethroid insecticides has developed in Australian populations of the sheep chewing louse Bovicola ovis [2 4]. High levels of pyrethroid tolerance have also been found in populations of Bovicola infesting cattle [5] and donkeys [6]. As a result, there has been a growing interest in the use of alternative approaches to ectoparasite control, such as entomopathogenic fungi [7], the toxins of Bacillus thuringiensis [8] and botanical extracts such as essential oils [9]. Essential oils are one of the most promising of these alternatives, since a wide variety of oils from different plants display insecticidal and acaricidal efficacy both in vivo and in vitro [9]. Essential oils are the volatile, hydrophobic fraction of a plant, usually extracted by steam distillation [1]. They consist of up to 8 low molecular weight chemicals such as terpenes and aromatic hydrocarbons [1]. Many of these individual components have cellular or neuronal targets, which accounts for the insecticidal efficacy of the whole essential oil; however, there is evidence that oil constituents may act synergistically to elicit an insecticidal effect [11,12]. Despite promising preliminary findings, most in vivo studies of the efficacy of essential oils against ectoparasites have been relatively limited [9]. Larger scale field trials are now required to demonstrate any clinical value of these compounds. The aims of the study reported here, therefore, were to assess the efficacy of 2 essential oils, tea tree and lavender, used as spray-on grooming aids in a large-scale field trial under realistic farm conditions and to assess the impact of risk factors such as coat length and re-infestation pressure on their efficacy. Materials and methods Study populations Two populations of donkeys were used in this study, one at a farm in Devon, UK and the other in County Cork, Ireland. Both populations had persistent pediculosis. At the Devon farm site these persistent infestations were shown previously to be associated with treatment failure of pyrethroid insecticides [6]. At each site, 3 groups of donkeys, housed in separate barns, were used. These barns were similar in structure, layout and animal management regimen across the 2 sites and, within sites, barns were less than 2 m apart. In Devon, the herd sizes in each barn varied from 45 to 145 animals. In County Cork, herd sizes were smaller, comprising animals. All the barns contained animals that had a history of persistent louse infestation. In Devon, 38 animals from each barn were randomly selected to be included in the trial using Microsoft Excel (213). In County Cork, the whole barn was selected for inclusion. Individual animals were identified by uniquely numbered collars or using a microchip scanner. The veterinarian and grooms responsible for the animals welfare were consulted on the medical history and suitability of each animal; animals that had serious skin complaints or were undergoing treatment for other conditions were not included in the trial. In addition, animals that were likely to be distressed by routine handling were also not included in the study. Therefore, after excluding some animals the group sizes were as follows: Devon, 37, 37 and 35 from barns containing a total of 144, 44 and 141 animals, respectively; and in County Cork, 29, 26 and 32 from barns containing 31, 29 and 35 animals, respectively. Application procedure Tea tree and lavender essential oils were purchased from a commercial supplier a. The tea tree oil complied with ISO (international standard) 473; the lavender oil met a standard that stipulated a 4 42% concentration of the major chemical component, linalyl acetate. At each farm, each barn Equine Veterinary Journal (215) EVJ Ltd 1

2 Essential oils in equine louse control L. Ellse et al. of donkeys was assigned at random, using Microsoft Excel (213), to one of the following grooming regimes: tea tree oil 5% (v/v), lavender oil 5% (v/v) or control (excipient only). The excipient used was water and 1% (v/v) polyvinylpyrrolidone b. From each selected group in each barn, 5 donkeys were selected at random to receive no grooming aid. These animals were housed with animals receiving grooming aid but did not have any liquid applied to their coats: instead, they were simply groomed in the same manner as those groomed with the essential oils or excipient-only control. This was to assess any underlying changes in louse population size within each barn over time as well as to identify any mechanical effect of grooming on louse numbers. Prior to the first application, louse counts were recorded at 1 sites on each animal: supraorbital fossa, behind the ear, neck, axilla and midline on each side. In each area the hair was parted 5 times, in 5 cm lengths. The total number of live adult and nymphal Bovicola ocellatus in all 1 partings was recorded separately. No other species of louse was found to be present. Egg counts were not included in this study. Hair length was also recorded (.5 cm) at each site. All animals were weighed within 6 weeks of the start of the trial. Overall, the mean donkey mass was 18 kg (95% confidence interval [CI] kg) with the largest animal weighing 32 kg and the smallest 12 kg. The mean weight of individuals in each barn was as follows: Devon tea tree barn, 191 kg (95% CI kg); Devon lavender barn, 189 kg (95% CI kg); Devon control barn, 24 kg (95% CI kg); County Cork tea tree barn, 154 kg (95% CI kg); County Cork lavender barn, 153 kg (95% CI kg); County Cork control barn, 197 kg (95% CI kg). Essential oils were applied by Donkey Sanctuary staff using a 5 ml hand spray c at approximately 2 ml/kg bwt to the nearest 5 kg. Prior to application the coat was brushed to remove dirt and loose hair. The suspension was then brushed into the coat with a medium/stiff-bristled brush. Separate brushes were used for each donkey group and within each group were washed between animals in antibacterial solution (Hibiscrub, 4% w/v chlorhexidine gluconate d ). For application to the face, a soft bristled brush or sponge was used, avoiding eyes and nostrils. After application the animals in each group were returned to their barn. The initial louse inspection and oil application was carried out in February 214. Two weeks later each animal had its louse burden recounted and the oil was reapplied in the same manner as before. Finally animals were re-examined 2 weeks after the final application. The animals in the groom-only groups were groomed in the same way and on the same dates as all the other animals, but no spray was applied. Data analysis All statistical analysis was carried out using R (version 2.1.1, 29) e. Except where stated, the pooled louse count of adult and nymphal lice was used as the response variable in statistical analyses. To examine the efficacy of the oil, the pooled total number of lice counted on each animal at each inspection was used as the response variable in a negative binomial, repeated measures, generalised linear mixed model (lme4 package). To investigate any changes in life stage susceptibility to the grooming aids, adult and nymphal louse counts were analysed separately using ANOVA. To examine any changes in distribution of lice throughout thecoat,therelativelouseabundancescorecalculatedbylog 1 [(no. of lice after application +1)/(no. of lice before application +1)] was used in a linear mixed model with donkey ID as a random effect. The effect of hair length on louse numbers was assessed using ANCOVA with the number of lice prior to application as a covariant. In this study treatment was not replicated by barn; however, barn-level variation was determined by assessing the relative changes in louse burden in animals that had been groomed only. The Henderson-Tilton correction (below) was used to calculate the overall reduction in louse numbers. G a = number of lice in the grooming aid group after application; C a = number of lice in the control group after application; C b = number of lice in the control group before application; G b = number of lice in the grooming aid group before application. Corrected mortality ¼ 1 G a C a C b G b Results Essential oil efficacy No adverse reactions were observed following application of either of the essential oils or the excipient-only control. Overall, grooming with 5% (v/v) lavender or 5% (v/v) tea tree oil resulted in a reduction in the total number of lice compared to the control of 78% (95% CI %) and 78% (95% CI %; Henderson-Tilton corrected), respectively, after 2 applications. This difference was highly significant (lavender: P<.1; tea tree: P<.1; Figs 1, 2). There was no significant difference between total louse numbers in the tea tree or lavender groups after 2 applications (P =.8). The reduction in abundance in the 2 treatment groups compared to the controls was similar for both adults and nymphs analysed separately, in both Devon (adults, P<.1; nymphs P<.1) and County Cork (adults P<.1; nymphs P<.1). Over the course of the trial there was no significant effect of animal age (P =.4) or weight (P =.5) on the change in louse numbers. There was no significant change in louse burden in the control groups compared to preapplication louse counts in County Cork (P =.3). In Devon, there was a statistically significant increase in the number of lice in the control group (P<.1). Despite different untreated population sizes, there was no significant difference in the reduction in louse numbers observed between the 2 farms (P =.4). For donkeys that were groomed only, there was no significant change in the louse counts at either the County Cork farm (tea tree barn, P =.3; lavender barn, P =.8) or the Devon farm (tea tree barn: P =.2; lavender barn: P =.1). Mean ± s.e. louse burden Inspection day Fig 1: The mean total louse burden (95% CI) of 3 groups of donkeys in County Cork, Ireland, that were groomed with: 5% (v/v) lavender oil (open circles), 5% (v/ v) tea tree oil (grey circles) or excipient only (1% polyvinylpyrrolidone) control (black circles) on Days 1 and Equine Veterinary Journal (215) EVJ Ltd

3 L. Ellse et al. Essential oils in equine louse control 8 5 Back Supraorbital Ear fossa Neck Axilla Mean ± s.e. louse burden Fig 4: The mean percentage change in louse numbers (95% CI) across 5 infestation sites after 2 applications of 5% (v/v) lavender oil at 2 locations; Devon (black bars) and County Cork (grey bars) Inspection day Fig 2: The mean total louse burden (95% CI) of 3 groups of donkeys in Devon, UK that were groomed with: 5% (v/v) lavender oil (open circles), 5% (v/v) tea tree oil (grey circles) or excipient only (1% polyvinylpyrrolidone) control (black circles) on Days 1 and Back Ear Supraorbital fossa Neck Axilla Fig 3: The mean percentage change in louse numbers (95% CI) across 5 infestation sites after 2 applications of 5% (v/v) tea tree oil at 2 locations; Devon (black bars) and County Cork (grey bars). Effects of hair length and coat distribution on efficacy At the start of the study, donkeys with longer hair were significantly more likely to have lice (P<.1). Hair length varied between sites, with the axilla having the longest mean hair length (P<.1) at 7.19 cm (95% CI, cm); the ear had the shortest mean hair length of 2.52 cm (95% CI, cm). There was no significant difference in mean hair length between barns in Devon (P =.419) or County Cork (P =.475). Adult lice were highly aggregated, with the axillae having the highest abundance of adults (P<.1). Nymphs were also aggregated, although they were more widely dispersed throughout the coat than adult lice. There was no significant effect of hair length on the change in total louse numbers after the second treatment in Devon (P =.6) or County Cork (P =.2). Following treatment, infestation site had a significant effect on therelativechangeinlouseabundanceintheteatreeandlavendergroups in Devon (Fig 3; tea tree: P =.5. Fig 4; lavender: P =.3) and County Cork (Fig 3; tea tree: P =.3. Fig 4; lavender: P<.1), with the ear and supraorbital fossa displaying a less marked reduction in louse numbers (Figs 3, 4). In the control group most infestation sites displayed a proportional increase in louse numbers (Fig 5). Discussion The results reported here demonstrate that grooming with 5% (v/v) tea tree or lavender oil can significantly reduce burdens of B. ocellatus. After2 applications the decline in louse numbers was approximately 78% (95% CI 76 8%): this was consistent between farm sites and no barn level variation was observed. Not all the animals within each barn were treated; in County Cork the majority of the barn was included in the trial, whereas in Devon less than a third of each barn s population was included. Despite this, there was no significant difference between the reductions in lice seen at the 2 farm sites. This suggests that the reinfestation pressure was not sufficiently high to alter the level of decline seen at the Devon farm. In sheep, the spread of Bovicola ovis has been shown to be relatively gradual, with only 63% of a flock of 1 animals being infested 44 weeks after the introduction of 2 infected sheep [13]. Alternatively, the oils may provide a level of louse repellence; indeed, many essential oils have shown repellence characteristics against other ectoparasites [9]. In the presence of the louse attractant ammonium bicarbonate, rosemary, citronella and eucalyptus essential oils repel the human body louse Pediculus humanus, to the same extent as the widely used insect repellent DEET (N, N-diethyl-meta-toluamide) [14]. Equine Veterinary Journal (215) EVJ Ltd 3

4 Essential oils in equine louse control L. Ellse et al Back Ear Supraorbital fossa Neck Axilla Fig 5: The mean percentage change in louse numbers (95% CI) across 5 infestation sites after 2 applications of 1% polyvinylpyrrolidone control at 2 locations; Devon (black bars) and County Cork (grey bars). At the start of this study there was a positive correlation between donkey hair length and louse burden. In addition, whereas adult lice showed a strong tendency to be localised in the axillae, nymphs were relatively more dispersed throughout the coat, highlighting the need for pediculicides to have good coat dispersion and penetration. Hair length did not have a significant effect on louse burdens after application of the essential oils, suggesting that the grooming method was adequate to permeate longer hair coats. However, the decline in louse numbers was not uniform between the infestation sites inspected. Therefore, further investigation into excipient formulations and application methods that provide a consistent coverage and coat penetration maybe beneficial. Essential oils contain a diverse composition of low molecular weight, volatile components. Exposure to the vapour of filter papers impregnated with 5% (v/v) lavender or tea tree oil has been shown to result in 8% mortality of B. ocellatus after 2 h; a similar mortality rate was observed for lice in physical contact with the impregnated filter papers [15]. However, the volatility of many essential oil constituents often means that they have a relatively limited residual activity, resulting in short-lived biocidal efficacy. The acaricidal activity of the cinnamon oil component trans-cinnamic acid against Psoroptes mites decreased significantly 96 h after application [16]. Similarly, exposure to squares of cotton treated with 2% (v/v) tea tree oil thathadbeenlefttodryovernightdidnotresultinsignificantmortalityof B. ovis [17]. The short insecticidal half-life of essential oils may be considered a drawback for ectoparasite control. Indeed, for facultative and temporary parasites, a long residual half-life is desirable to maintain protection from ongoing parasite challenge for a number of weeks or months. However, for the control of permanent ectoparasites, such as lice and mites, persistent residual efficacy is not an essential characteristic provided the whole host population is treated and protected from re-infestation. In addition, a short half-life may be advantageous, resulting in low environmental toxicity and reducing the probability of selection for resistance; slow drug elimination times have often been associated with resistance development owing to the unnecessarily long exposure of the parasite to sublethal concentrations of the treatment, allowing the survival of heterozygous resistant individuals [18]. Any development of resistance to essential oils might be expected to be slow, owing to the chemical diversity of their multiple constituents. Essential oils contain approximately 8 different components, many of which have been isolated and shown to interact with insect neuronal targets. In the case of tea tree oil, terpinen-4-ol, which is typically >3% (v/v) of the oil (ISO 473), has been shown to inhibit the neurotransmitter terminating enzymes acetylcholinesterases [19], whilst a-terpineol (up to 8% of the oil; ISO 473) binds to tyramine receptors leading to changes in intracellular calcium concentrations [2]. Other terpenes from essential oils have also been shown to interact with insect octopaminergic receptors [21 23]. These multiple neuronal targets make resistance development difficult, as the genes coding for the target proteins are distinct and therefore single point mutations seen in the development of resistance to pyrethroids [24] would need to occur at a minimum of 3 different loci. In conclusion, this study has demonstrated that on a large scale 5% (v/v) tea tree or lavender oil minimise equine louse populations. Although this study measured louse populations on a relatively short timescale, as lice are permanent ectoparasites the level of reduction observed is likely to alleviate pediculosis substantially at the peak of infestation. The results suggest that given their anticipated low environmental impact and diverse chemical composition, they are likely to be valuable alternative pediculicides for inclusion in equine louse management programmes. This is particularly the case in populations where chemical alternatives have ceased to be effective or where organic, natural insecticide alternatives are preferred. It is also likely that with further excipient optimisation essential oil-based products could provide greater insecticidal efficacy and persistence. Authors declaration of interests No competing interests have been declared. Ethical animal research The studies described were undertaken as part of the routine grooming regimes implemented by staff at the Donkey Sanctuary. Approval for inclusion of animals was granted by veterinarians responsible for the animals health and welfare. Source of funding This work was funded by The Donkey Sanctuary. Acknowledgements The authors would like to thank Miss Nikki Straddling, Mrs Kristin O Connor and all the Donkey Sanctuary staff members in Devon and County Cork for their assistance. Authorship L. Ellse designed the experiment, supervised and organised field work, carried out field work, analysed data and prepared the manuscript. B. Sands carried out field work and contributed to the analysis of data. F. Burden assisted in the design and organisation of the study and proof read the manuscript. R. Wall assisted in the experimental design and contibuted to the drafting of the manuscript. All authors have approved the publication of this manuscript. Manufacturers addresses a Essential Oils Direct, Oldham, Greater Manchester, UK. b Sigma-Aldrich Co. Ltd., Gillingham, Kent, UK. c Fisher Scientific UK Ltd., Loughborough, Leicestershire, UK. d M olnlycke Health Care Ltd., Oldham, Greater Manchester, UK. e The R Foundation for Statistical computing, ISBN References 1. Levot, G.W. and Hughes, P.B. (199) Laboratory studies on resistance to cypermethrin in Damalinia ovis (Schrank) (Phthiraptera: Trichodectidae). Aust. J. Entomol. 29, Equine Veterinary Journal (215) EVJ Ltd

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