Samira S. Garboui Æ Thomas G. T. Jaenson Æ K. Pålsson

Transcription

1 Exp Appl Acarol (2006) 40: DOI /s Repellency of MyggA Ò Natural spray (para-menthane- 3,8-diol) and RB86 (neem oil) against the tick Ixodes ricinus (Acari: Ixodidae) in the field in east-central Sweden Samira S. Garboui Æ Thomas G. T. Jaenson Æ K. Pålsson Received: 29 March 2006 / Accepted: 2 October 2006 / Published online: 14 November 2006 Ó Springer Science+Business Media B.V Abstract In the field in south-central Sweden, we tested by randomised, standardised methodology the potential anti-tick repellent activity of two concentrations of MyggA Ò Natural spray (containing PMD) (4.2 and 3.2 g/m 2 ) and one of RB86 (with 70% neem oil containing azadirachtin)(3 g/m 2 ) to host seeking nymphs of Ixodes ricinus. Each substance was applied separately to 1 m 2 cotton flannel cloths. Nymphal ticks on the cloths, pulled over the vegetation, were recorded at 10-m stops. Nymphal numbers recorded differed significantly between treated cloths [4.2 or 3.2 g MyggA Ò Natural spray/m 2 and 3 g RB86/m 2 ] and the untreated control (df =3, v 2 = , P < ). Nymphal numbers also differed significantly among collectors (df =3,v 2 = 15.80, P < 0.001). Repellency of treated cloths, i.e., 4.2 or 3.2 g MyggA Ò Natural spray/m 2 and 3 g RB 86/m 2 declined from day 0 (i.e. the day of impregnation) to day 3 after impregnation from 77 to 24%, 58 to 16% and 47 to 0.5%, respectively. This study suggests that all three treatments have significant repellent activities against I. ricinus nymphs. Keywords Ixodes ricinus ticks Æ Azadirachtin Æ Lemon eucalyptus Æ Para-menthane-3,8-diol Æ Tick repellents Introduction Ticks are important through the direct effects of their feeding and as vectors of various disease agents to man, farm animals and companion mammals. Ixodes ricinus (L.) is the most common and medically most important tick species in Sweden and most other parts of Europe. It is the main vector to humans of the spirochetes (Borrelia burgdorferi s.l.) causing Lyme borreliosis, and the virus causing tick-borne encephalitis (TBE). It is also involved in the transmission from animals to humans of S. S. Garboui Æ T. G. T. Jaenson Æ K. Pålsson (&) Medical Entomology Unit, Department of Systematic Zoology, Evolutionary Biology Center, Uppsala University, Uppsala SE-75236, Sweden Katinka.Palsson@ebc.uu.se

2 272 Exp Appl Acarol (2006) 40: babesiosis (Babesia divergens, B. microti), anaplasmosis/ehrlichiosis (Anaplasma phagocytophilum), Q-fever (Coxiella burnetii) and tularaemia (Francisella tularensis) (Jaenson 1999). Therefore, personal protection against bites of infected I. ricinus is essential. One way of prophylaxis could be to utilise chemical repellents from natural sources, e.g., plant oils, plant extracts or chemicals isolated from plants. Many studies have shown that numerous plant species contain substances, which repel or kill ticks (Ndungu et al. 1995; Malonza et al. 1992; Trigg and Hill 1996; Gardulf et al. 2004; Jaenson et al. 2005). A recently developed product, MyggA Ò Natural is based purely on naturally occurring plant compounds and contains as the main active substance para-menthane-3,8-diol (PMD) from the lemon eucalyptus Corymbia citriodora and small amount of terpene-fractions of essential oils of four different plant species. In a previous study MyggA Ò Natural (roll on) was shown to effectively repel ticks in Sweden (Jaenson et al. 2006). Neem oil from the neem tree Azadirachta indica (family Meliaceae) is known for its insecticidal, antifeedant, and arthropod repellent properties (Mulla and Su 1999). Neem oil extract is toxic to the ticks Amblyomma cajennense Koch and Boophilus microplus Lahille (Williams et al. 1996), and Hyalomma anatolicum excavatum Koch (Abdel-Shafy and Zayed 2002). RB86 is marketed in Sweden as a repellent to be used on horses against ticks and blood-feeding insects and contains about 70% neem oil. The aim of this field study was to investigate if MyggA Natural spray and RB86, containing as the main active compounds g/l PMD and 1 g/l (0.1%) azadirachtin, respectively have significant repellent effects on I. ricinus nymphs, which is the epidemiologically most important stage of the tick. Materials and methods The study area The field trial was carried out at Alsike (59 47 N, E), ~10 km south of Uppsala, east-central Sweden, in a mixed spruce/pine broad-leave woodland with trees, i.e., Picea, Pinus, Populus, Betula, Alnus, Salix, Quercus and Corylus. The ground and field vegetation consisted mainly of lichens, bryophytes, ferns (Dryopteris), grasses (Poaceae), blue-berry and lingon-berry (Vaccinium spp.), meadowsweet (Filipendula) and raspberry (Rubus). Roe deer (Capreolus capreolus), hares (Lepus timidus and L. europaeus), rodents (Clethrionomys glareolus, Apodemus spp.) and shrews (Sorex spp.), and I. ricinus are abundant at the study locality. Substances tested MyggA Natural spray contains g/l of para-menthane-3, 8-diol (PMD) (from the oil of the lemon eucalyptus, Corymbia citriodora) and small amounts of extracts of lavender (Lavandula angustifolia Mill., family: Lamiaceae), geranium (Pelargonium graveolens L Her, fam. Geraniaceae) and roses (Rosa spp., fam. Rosaceae). PMD is the active substance in several mosquito repellent products. According to the manufacturer s suggestions MyggA Natural spray should be applied to the skin in small amounts no more then three times a day, and not be applied on children under three years.

3 Exp Appl Acarol (2006) 40: RB86 contains about 70% neem oil, about 30% rape seed oil, surfactants and perfumes; the active ingredients of RB86 are azadirachtin (0.1%) and citronella (<1%). RB86 is recommended for horses at a dose of 20 ml/day (at a dilution in water) against ticks and bloodsucking arthropods. Field trial with MyggA Natural spray and RB86 neem oil on blankets All substances were tested during 16 days in late July and early August 2005 by randomised, standardised methodology, for potential anti-tick repellent activity. There were two concentrations of MyggA Ò Natural spray (4.2 g/m 2, 3.2 g/m 2 ) and one of RB86 with 70% neem oil (3 g/m 2 ), which were compared with an untreated control for repellency/attractancy to host seeking nymphs of Ixodes ricinus. In the early morning of day 0 three light coloured blankets (1 m 2 each) were impregnated by a paintbrush with 10 ml (MyggA Ò Natural) or 12 ml (RB86) of the test substance to cover the whole downward surface of the test cloth. Substances used were: 4.6 ml (4.2 g) MyggA Ò Natural spray (containing 1000 mg PMD) in 5.4 ml acetone on one blanket; 3.5 ml (3.2 g) MyggA Ò Natural spray (containing 750 mg PMD) in 6.5 ml acetone on another blanket; and 3 ml (3 g) RB86 with 70% neem oil (containing 3 mg azadirachtin) in 9 ml water on a third blanket. In addition, one identical control blanket without any substance was used. The paintbrush was rubbed onto the cloth until the brush was nearly dry. Each white flannel cloth, 1 1 m, was attached to a 1 m long wooden pole to both ends of which a cord was tied (Mejlon and Jaenson 1993). The four cloths, handled by four different persons, were pulled parallel to each other with their treated sides downwards, towards the ground vegetation. By standardised technique each cloth was pulled slowly simultaneously with the other cloths, over an area of 10 m 2 before each cloth was inspected and each tick recorded, removed and put into marked vials. No area was sampled more than once. This procedure was repeated 40 times (400 meters) for each treatment and person each day. Thus, each blanket was pulled for 6,400 meters during the test period. Each person tested one treatment/day. Allocation of treatments and persons were randomised. Four persons needed 16 days to evaluate four cloths, i.e., two concentrations of MyggA Ò Natural, one concentration of neem oil, and one control without any substance. Thus each person tested each substance (and the control) four times. The duration in days of tick repellent activity of MyggA Ò Natural spray and RB86 with neem oil on cloths under natural conditions was estimated and compared with that of the untreated control cloth. When not in use, the cloths were kept in separate, marked plastic bags at room temperature. The cloths were washed and re-impregnated, after 2 4 days, when the test substances had lost most of its activity, i.e., when the number of nymphs recorded on the test blankets were similar to that of the control blanket. Statistical analyses Repellency is calculated using the formula: % repellency = [(no. of nymphs on control blanket no. of nymphs on test blanket)/no. of nymphs on control blanket] 100. A type III generalised linear model with a log link function and a Poisson distribution in the advanced linear/non linear model, using Stat Soft Statistica 7.0 was performed to determine the effect of each treatment and person.

4 274 Exp Appl Acarol (2006) 40: Results Tick repellency of two concentrations of MyggA Ò Natural spray (4.2 g and 3.2 g, i.e., 1000 mg and 750 mg PMD, respectively and one of RB86 with 70% neem oil (3 mg azadirachtin) was tested on blankets during 16 days in Nymphal numbers recorded differed significantly between treated cloths [4.2 g/m 2, 3.2 g/m 2 MyggA Ò Natural spray and RB86 (3 g/m 2 )] and the untreated control (df =3,v 2 = , P < ). Numbers of nymphs on the test blankets impregnated with 4.2 g MyggA Ò Natural/m 2 were significantly different from blankets impregnated with 3.2 g MyggA Ò Natural/m 2 (P < 0.05) and RB86 (P < 0.001)(Fig. 1). Numbers of nymphs on the test blankets impregnated with 3 g/m 2 RB86 were not significantly different (P = 0.5) from blankets impregnated with 3.2 g MyggA Ò Natural spray/m 2 (Fig. 1). Nymphal numbers also differed significantly among collectors [H (df =3,v 2 = 15.80, P < 0.001)] in that one collector s catching efficiency was greater than that of the other collectors (Fig. 2). However, this does not bias the results since all four persons test each substance four times. Repellency of the cloths treated with 4.2 g/m 2 of MyggA Ò Natural spray, 3.2 g m 2 of MyggA Ò Natural or 3 g/m 2 of RB86 declined from day 0, i.e. the day of impregnation, to day 3 after impregnation from 76.8% to 24.3%, 57.6% to 15.8% and 46.6% to 0.5%, respectively (Fig. 3). Discussion Our data suggest that MyggA Natural spray at two concentrations and RB86 have significant repellent activity against nymphs of I. ricinus in this field repellency test. 1,5 1,4 2 M ean number of nymphs/10 m 1,3 1,2 1,1 1,0 0,9 0,8 0,7 0,6 0,5 3.2g MyggA Natural/m2 4.2g MyggA Natural/m2 3g RB 86/m2 Control Fig. 1 Mean number of I. ricinus nymphs/10 m 2 recorded on three cloths impregnated with MyggA Natural spray (4.2 g/m 2, 3.2 g/m 2 ) or RB86 (3 g/m 2 ), and an untreated cloth during 16 days in late July and early August 2005 at Alsike, south-central Sweden. Vertical bars denote 0.95 confidence intervals

5 Exp Appl Acarol (2006) 40: Fig. 2 Mean number of I. ricinus nymphs/person A D recorded on all cloths impregnated with MyggA Natural spray (4.2 g/m 2, 3.2 g/ m 2 ) or RB86 (3 g/m 2 ), and an untreated cloth during 16 days in late July and early August 2005 at Alsike, south-central Sweden. Vertical bars denote 0.95 confidence intervals Mean number of nymphs/person/10 m 2 1,3 1,2 1,1 1,0 0,9 0,8 0,7 A B C D persons Percentage repellncy Percentage tick repellency of test substances MyggA Natural spray (4.2g/m2) MyggA Natural spray (3.2g/m2) RB86 (3g/m2) Day0 Day1 Day2 Day3 Fig. 3 Percentage tick repellency of test substances. Repellencey is based on number of I. ricinus nymphs recorded on treated and untreated cloths dragged over the ground vegetation in a tickinfested locality at Alsike. Day 0 = day of impregnation, % repellency = [(no. of nymphs on control blanket no. of nymphs on test blanket)/no. of nymphs on control blanket] 100 These results conform to those of a previous study in which MyggA Natural (roll-on) was tested in the laboratory and field (Jaenson et al. 2006). In that study we showed that the formulation compound, 1, 2-propanediol, of MyggA Natural does not repel I. ricinus nymphs. This is the reason why we used an untreated cloth as control cloth. MyggA Natural spray and RB86 used on clothing gives significant protection for up to 3 days. In the present study a relatively high concentration of MyggA Natural spray (4.2 g/m 2 ) showed significantly greater repellency than MyggA Natural at a lower concentration (3.2 g/m 2 ) and RB86 (3 g/m 2 ). The latter one was slightly less effective than MyggA Natural spray at a dose of 3.2 g/m 2. It is obvious that a maximum tick repellency of 77% may not be adequate for personal anti-tick protection purposes. However, it might be possible to increase the tick repellency by increasing the concentrations of the main compounds, PMD and azadirachtin and/or by adding other tick-repellent compounds into the formulations. Repellency of MyggA Natural spray at both concentrations and RB86 declined from day 0 to day 3. The initially relatively high repellency (77%) on day 0 for MyggA Natural spray at the high concentration is similar (89%) to that of a blanket treated in a similar way with MyggA containing 19% diethyl-methyl-benzamide (DEET) (Jaenson et al. 2003).

6 276 Exp Appl Acarol (2006) 40: It should be noted that MyggA Natural contains not only PMD but also essential oils of four different plants. Although PMD, most likely is the main tick repellent substance in MyggA Natural at least two of the essential oils also have tickrepellent activity (Jaenson et al. 2006). PMD may have more anti-tick effects than merely to act as a tick repellent. Thus, Trigg and Hill (1996) treated rabbits ears with PMD and showed that PMD was effective in reducing attachment and feeding of I. ricinus nymphs. In several studies, neem oil has been tested for efficacy in repelling insects and Acari. In our study, RB86 with only 0.1% azadirachtin (+<1% citronella) had a significant but not sufficient repellent activity against I. ricinus; the repellency was 47% on the day of impregnation (day 0) and 51% on day 1. The low dosage of RB86 used, with only 3 g azadirachtin/m 2 cloth, is the concentration recommended to be applied on horses according to the manufacturer. It is likely that application of a higher dosage would have increased the tick repellency of RB86. It also contains citronella at <1% concentration, which might contribute to the repellent effect on nymphal I. ricinus (Thorsell et al. 2006). Neem oil was shown to be toxic to Amblyomma cajennense and Boophilus microplus (Williams et al. 1996). The in vitro toxicity of undiluted neem oil against larvae of Amblyomma variegatum (Fabricius) killed all larvae within 48 h (Ndumu et al. 1999). Also, Abdel-Shafy and Zayed (2002) showed that neem oil induced significant mortality rates of newly hatched larvae, unfed larvae, and unfed adults of Hyalomma anatolicum excavatum. Essential oils of a large number of plants have been found to have repellent properties against different hematophagous arthropods, and some have formed the basis of commercial repellent formulations (Curtis et al. 1991). The repellency of these oils appears to be largely associated with the presence of one or more volatile monoterpenoid constituents. Although many formulations were effective when freshly applied, their activity disappeared relatively rapidly (Buescher et al. 1982; Ndungu et al. 1995; Malonza et al. 1992; Gardulf et al. 2004). Extension of the duration of activity of natural tick-repellents has to our knowledge not yet been investigated (Jaenson et al. 2006) but has been explored concerning the synthetic pyrethroids. The activity can be extended and the cost of treatment diminished by employing microencapsulated pyrethroids (Meyer and Hunter III 1991, Sutcliffe 2004). In the so-called long-lasting insecticidal bed net the pyrethroid is either incorporated into the fibres or coated in a wash resistant resin around fibres; the insecticidal activity of the net resists multiple washes and the biological activity lasts as long as the net itself, i.e. 3 5 years (WHO 2005). A product, MK19, contains DEET, which is microencapsulated, the advantage of which is that the dermal absorption of DEET is decreased while its duration of insecticidal and repellent activity is increased (SCI 2004). Similar technology might be used to increase the duration of activity of azadirachtin, PMD and other plantderived substances (Jaenson et al. 2006), which show potential as tick-repellents. Products containing PMD and azadirachtin repel to a high degree I. ricinus nymphs, which are important vectors of Lyme disease spirochetes and other human pathogenic organisms. However, it is desirable to improve these products further so that they will provide a higher degree of and more durable protection against potentially infected ticks. Acknowledgements We are grateful to Emma and David Jaenson, and Mr. Thomas Larsson who assisted in the field work; Mr. Luc Gysen, Jaico Ltd., Belgium and Ms. Elisabeth Hammarberg, Nim Distribution Centre, Stockholm who provided MyggA Natural and RB86, respectively; Bioglan

7 Exp Appl Acarol (2006) 40: Pharma, Lund, Sweden, The Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (Formas/SJFR), and The Swedish International Development Co-operation Agency (Sida/SAREC) for funding this work. References Abdel-Shafy S, Zayed AA (2002) In vitro acaricidal effect of plant extract of neem seed oil (Azadirachta indica) on egg, immature, and adult stages of Hyalomma anatolicum excavatum (Ixodoidea: Ixodidae). Vet Parasitol 106:89 96 Buescher MD, Rutledge LC, Wirtz RA (1982) Tests of commercial repellents on human skin against Aedes aegypti. Mosq News 42: Curtis CF, Lines JD, Lu B, Renz A (1991) Natural and synthetic repellents In: Curtis CF (ed) Control of disease vectors in the community. Wolfe, London, pp Gardulf A, Wohlfart I, Gustafson R (2004) A prospective cross-over field trial shows protection of lemon Eucalyptus extract against tick bites. J Med Entomol 41: Jaenson TGT (1999) Fästingen Ixodes ricinus som sjukdomsöverförare i Skandinavien [The tick Ixodes ricinus as a vector of diseases in Scandinavia]. Växtskyddsnotiser 63:53 59 Jaenson TGT, Lindström A,Pålsson K (2003) Repellency of the mosquito repellent MyggA Ò (N,Ndiethyl-3-methyl-benzamide) to the common tick Ixodes ricinus (L.)(Acari: Ixodidae) in the laboratory and field. Entomol Tidskr 124: Jaenson TGT, Garboui S, Pålsson K (2006) Repellency of oils of lemon eucalyptus (Corymbia citriodora), geranium (Pelargonium graveolens) and lavender (Lavandula angustifolium), and the mosquito repellent MyggA Natural Ò to the common tick Ixodes ricinus (L.) (Acari: Ixodidae) in the laboratory and field. J Med Entomol 43: Jaenson TGT, Pålsson K, Borg-Karlsson A-K (2005) Evaluation of extracts and oils of tick repellent plants from Sweden. Med Vet Entomol 19: Malonza MM, Dipeolu OO, Amoo AO, Hassan SM (1992) Laboratory and field observations on anti-tick properties of the plant Gynandropsis gynandra (L.). Brit Vet Parasitol 42: 136 Mejlon HA, Jaenson TGT (1993) Seasonal prevalence of Borrelia burgdorferi (Acari: Ixodidae) in Ixodes ricinus (Acari: Ixodidae) in different vegetation types in Sweden. Scand J Inf Dis 25: Meyer JA, Hunter JS III (1991) Residual activity of microencapsulated permethrin against stable flies on lactating diary cows. Med Vet Entomol 5: Mulla M, Su T (1999) Activity and biological effects of neem products against arthropods of medical and veterinary importance. J Am Mosq Control Assoc 15: Ndumu PA, George JBD, Choudhury MK (1999) Toxicity of neem seed oil (Azadirachta indica) against the larvae of Amblyomma variegatum a three-host tick in cattle. Phytother Res 13: Ndungu M, Lwande W, Hassanali A, Moreka L, Chabra SC (1995) Cleome monophylla essential oil and its constituents as tick (Rhipicephalus appendiculatus) and maize weevil (Sitophilus zeamais) repellents. Entomol Exp Applic 76: SCI (Swedish Chemicals Inspectorate/Kemikalieinspektionen/KEMI) (2004) MK 19- mikroinkapslat myggmedel. [MK19 - microencapsulated mosquito repellent, In Swedish] Diary number: KEMI F Sutcliffe JS (2004) Prevention and control of arthropod-borne diseases. In: Eldridge BF, Edman JD (eds) Medical entomology. A textbook on public health and veterinary problems caused by arthropods. Kluwer, Dordrecht,The Netherlands & Boston, USA, pp Thorsell W, Mikiver A, Tunon H (2006) Repelling properties of some plant materials on the tick Ixodes ricinus L. Phytomedicine 13: Trigg JK, Hill N (1996) Laboratory evaluation of an eucalyptus-based repellent against four biting arthropods. Phytother Res 10: WHO (2005) Fifth update on long lasting insecticidal nets - current status and programmatic issues, Geneva, 5/01/ [Acessed January 20, 2006] Williams LA, Ajai-Mansingh D, Mansingh A (1996) The insecticidal and acaricidal action of compounds from Azadirachta indica (A. Juss) and their use in tropical pest management. Integr Pest Managem Rev 1: