Experimental and Applied Acarology (2005) 37:141 146 DOI 10.1007/s10493-005-0359-y Ó Springer 2005 Short Communication In vitro acaricidal activity of four monoterpenes and solvents against Otodectes cynotis (Acari: Psoroptidae) OLIVIER TRAINA 1, CLAUDIA CAFARCHIA 2, GIOIA CAPELLI 3, NICOLA SANTE IACOBELLIS 4 and DOMENICO OTRANTO 2, * 1 Clinique Ve te rinaire du Boulou, Le Boulou, France; 2 Department of Animal Health and Welfare, Faculty of Veterinary Medicine, University of Bari, Str. Prov. per Casamassima Km3, 70010, Valenzano (Bari), Italy; 3 Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro (Padua), Italy; 4 Department of Agri-forest Biology, Defence and Biotechnologies, University of Basilicata, Potenza, Italy; *Author for correspondence: (e-mail: d.otranto@veterinaria.uniba.it; phone: +39080-4679839; fax: +39080-4679839) Received 9 February 2005; accepted in revised form 27 June 2005 Key words: a-pinene, Acaricidal activity, Ear mite, Geraniol, Limonene, Otodectes cynotis, p-cymene, Plant extracts, Psoroptidae Introduction Otodectes cynotis (Psoroptidae), commonly named ear mite, is a frequent nonburrowing mite causing ear infections (otocariasis) in dogs, cats, ferrets, foxes and other wild carnivores (Sweatman 1958). Otocariasis may induce various degrees of pruritus and symptoms, e.g. scratching, rubbing of the ears or violent shaking of the head (Berg and Shomer 1963). Complications are frequent and these may have serious consequences on the host s health (Baker 1999). Treatment of otocariasis includes the mechanical cleaning of the ear canal followed by topical or systemic drug administration (reviewed in Curtis 2004). Aural preparations usually contain acaricides (e.g. thiabendazole, monosulfiram, permethrin), antibiotics and synthetic corticosteroids (Scherk- Nixon et al. 1997). It is well known that the repeated use of acaricides may induce resistance in poultry red mites Dermanyssus gallinae (reviewed in Soon-II Kim et al. 2004), sarcoptid mites parasitizing animals (Coles and Stafford 1999) or humans (Walton et al. 2000), and sheep scab mites Psoroptes ovis (Synge et al. 1995; Clark et al. 1996). Furthermore, insecticides may also cause environmental concerns and become detectable in meats (Halley et al. 1993; O Brien 1999). In the past 10 years the antimicrobial, anti-parasitic and acaricidal effect of plant essential oils has been broadly investigated. In particular, the activity of
142 essential oils has been evaluated for D. gallinae (Soon-II Kim et al. 2004), Psoroptes cuniculi (Perucci et al. 1995; Perucci et al. 1997, 2001) and, within the Sarcoptidae family, for Sarcoptes scabiei (Oladimeji et al. 2000). Preliminary studies have suggested that specific compounds such as linalool and substances with free alcoholic phenolic groups (Perucci et al. 1995) such as the oxygenated terpenoids (terpinen-4-ol) (Walton et al. 2004) show the highest acaricidal activity. Conversely, no studies have been carried out on the acaricidal efficacy of plant essential oils or specific monoterpenes against O. cynotis ear mites. Limonene, a-pinene, p-cymene and geraniol are the most commonly occurring natural compounds of essential oils in plants. Limonene and p-cymene are monocyclic terpenes, a-pinene is a bridged bi-cycle terpene and geraniol is a monoterpene alcohol. For all the above compounds the antifungal and antibacterial activities have been demonstrated (Hammer et al. 2003); in particular the antimicrobial activity has been attributed to their interaction with cellular membranes (Sikkema et al. 1995). The aim of this study is to evaluate a solvent to dilute monoterpenes and test the acaricidal activity in vitro of four purified monoterpenes against O. cynotis. Materials and methods Mites From March to September 2003, ear mites were collected from six cats living outdoors and coming from the same colony (province of Brindisi, southern Italy). The cats were known to be affected and had been monitored for otocariasis for three years before the beginning of this trial (Otranto et al. 2004). No cats were treated for endo- or ecto-parasites throughout the trial period. Mites were collected from all the animals by swabbing both ears. After sampling, the cotton swabs were placed in 60 15 mm Petri dishes and delivered within 12 h to the Parasitology laboratory of the Faculty of Veterinary Medicine (Bari, Italy). The mites were classified as adult male (M), adult female (F), nymph (N) or larva (L), following the morphological description of Sweatman (1958). Solvent selection and monoterpenes In order to solubilize the monoterpenes, three solvents were tested for their acaricidal activity against O. cynotis, i.e. acetone (J.T. Baker-Mallinckrodt Baker BV, Rijsterborgherweg, Holland), paraffin oil (J.T. Baker-Mallinckrodt Baker BV, Phillipsburg, NJ) and 20% glycerol in water (Glycerol 99%, Sigma Aldrich, Milan, Italy). The three solvents were used to take in solution monoterpenes (Perrucci et al. 1995; Morsy et al. 2002, 2003) and saline
143 solution (NaCl 0.9%) was used as a control since it displayed no acaricidal activity in a previous trial (Otranto et al. 2004). Limonene, a-pinene, p-cymene and geraniol were commercially available as samples purified to 97% minimum (Sigma Aldrich, Milan, Italy). The four monoterpenes were first used diluted to 10% in paraffin oil, which was found to be the most effective solvent among those tested and when necessary, further dilutions of monoterpenes to 5% and 1% were used. The trial was repeated three times for each monoterpene and dilution. Experimental procedures A total of 1080 O. cynotis mites were used (438 L, 298 N, and 344 adults 233 M and 111 F) to test monoterpenes. After identification under the stereomicroscope, the various life-cycle stages were placed in different Petri dishes and weak or dead mites were immediately removed from the dishes. The remaining mites were evenly divided into groups (30 mites per group) and placed in Petri dishes. All the dishes were examined hourly by stereomicroscopy till the fourth hour. The observation period began when the 15th mite was placed in the test plate (the beginning of the trial). After the 4th hour, the Petri dishes were examined every twelve hours till the 60th hour and every 24 h until the death of the last mite (the end of the trial). Lack of natural movements, even when stimulated with a needle, was considered indicative of death. Dead mites were identified and removed from the test plate. Each trial was carried out using 30 mites (different stages in the same or similar percentages) and repeated three times. The first part of the trial evaluated the activity of the solvents used to dilute the monoterpenes. Thereafter, four purified monoterpenes were tested for their acaricidal activity against O. cynotis. The experiments employed Petri dishes (4 cm diameter) lined with blotting paper discs (5 cm diameter) fitted on their lids. After several attempts with different amounts of liquids, 100 ll was considered most suitable to add to the blotting paper. After 5 min the fluid had spread completely and uniformly over the paper and the mites were transferred for the various tests. The Petri dishes were then placed in a 25 15 5cm plastic box with waterlogged absorbent tissue and covered with aluminium foil to guarantee saturated relative humidity and to reduce evaporation of the test products. The plastic box containing the Petri dishes was then placed in a constant temperature cabinet at 35 C. Statistical analysis The Mean Survival Time (MST) was calculated for each group by measuring the mean survival in hours of all the mites within each experiment block. The MSTs obtained from testing each solvent against the saline solution, and from
144 testing the monoterpenes against paraffin oil, were statistically compared using the non parametric Mann Whitney U-test. Results After initial tests with glycerol, acetone and paraffin oil, the latter was chosen as a solvent to take in solution the four monoterpenes because it induced a longer MST (66 h) on O. cynotis than the others (i.e. glycerol = 28 h 34 min and acetone = 38 h 48 min). Geraniol 10% displayed the best results with all the mites dead within 1 h of placing in the test Petri dishes (Figure 1). The acaricidal effect of the others monoterpenes was less, with limonene displaying the lowest MST, followed by p-cymene and a-pinene. In addition, geraniol was further diluted causing the death of all the mites within one hour when diluted to 5%, and after 14 h when diluted to 1% (Figure 1). Discussion None of the three test solvents (i.e. acetone, paraffin oil and glycerol 20%) displayed significant acaricidal activity compared to saline solution. Nevertheless, the choice of paraffin oil was also determined by the physical and Figure 1. Mean survival time (MST) and standard deviation (in brackets) for each monoterpene and paraffin oil of Otodectes cynotis (in hours). Sample sizes: n = 3 for each monoterpene, n =18 for paraffin oil. * Significant difference between a monoterpene and the control (paraffin oil), based on Mann Whitney U-test (p < 0.05).
145 chemical properties of the test substance. In fact, despite the satisfactory solubilization of monoterpenes in acetone and its low viscosity, this solvent is excessively volatile, which may cause the monoterpene concentration to increase during the trial. Conversely, glycerol 20% displayed the lowest monoterpene solubilization capability and highest viscosity. These properties may interfere with mite movements and monoterpene distribution on the surface of the blotting paper. Finally, the viscosity and volatility properties of paraffin oil and its ability to effectively solubilize monoterpenes makes paraffin oil a satisfactory solvent for the above substances. Although monoterpenes are highly volatile, this trial aimed to evaluate their acaricidal activity by direct contact with the mites. Of the four monoterpenes tested, geraniol and limonene displayed the strongest acaricidal activity against O. cynotis. Geraniol is also very effective in a 5% dilution. The promising results obtained for gerianol and limonene reported in this study suggest the need for further investigation to assess the mechanisms of action of the substances, and to evaluate possible synergistic effects of different monoterpenes against O. cynotis and other parasites. The non-toxicity of monoterpenes for animal skin should also be tested in vivo. This study goes some way toward demonstrating that monoterpenes, the principal components of essential oils and other plant extracts, may be used effectively as acaricides, with a presumably low environmental impact. References Baker A.S. 1999. Mites, Ticks of Domestic Animals An Identification Guide and Information Source. The Natural History Museum, London, UK, pp. 89 91. Berg P. and Shomer R.R. 1963. Otocariasis in the dog and cat. J. Am. Vet. Med. Assoc. 1430: 1224 1226. Clark A.M., Stephen F.B., Cawley G.D., Bellworthy S.J. and Groves B.A. 1996. Resistance of the sheep scab mite Psoroptes ovis to propetamphos. Vet. Rec. 139: 451. Coles G.C. and Stafford K.A. 1999. The in vitro response of sheep scab mites to pyrethroid insecticides. Vet. Parasitol. 83(3 4): 327 330. Curtis C.F. 2004. Current trends in the treatment of Sarcoptes, Cheyletiella and Otodectes mite infestations in dogs and cats. Vet. Dermatol. 15(2): 108 114. Halley B.A., Vandenheuvel W.J.A., Wislock P.G., Herd R., Strong L. and Wardhaug K. 1993. Environmental effects of the usage of avermectines in livestock. Vet. Parasitol. 48: 109 125. Hammer K.A., Carson C.F. and Riley T.V. 2003. Antifungal activity of the components of Melaleuca alternifolia (tea tree) oil. J. Appl. Microbiol. 95(4): 853 860. Morsy T.A., Morsy G.H. and Sanad E.M. 2002. Eucalyptus globulus (camphor oil) in the treatment of human demodicidosis. J. Egypt. Soc. Parasitol. 32(3): 797 803. Morsy T.A., Rahem M.A., el-sharkawy E.M. and Shatat M.A. 2003. Eucalyptus globulus (camphor oil) against the zoonotic scabies, Sarcoptes scabiei. J. Egypt. Soc. Parasitol. 33(1): 47 53. O Brien D. 1999. Treatment of psoroptic mange with reference to epidemiology and history. Vet. Parasitol. 83: 177 185. Oladimeji F.A., Orafidya O.O., Ogunniyi T.A. and Adewunmi T.A. 2000. Pediculocidal and scabicidal properties of Lippa multiflora essential oil. J. Ethnofarmacol. 72(1 2): 305 311.
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