Exp Appl Acarol (2014) 63:77–83 DOI 10.1007/s10493-013-9765-8
Influence of tea tree oil (Melaleuca alternifolia) on the cattle tick Rhipicephalus microplus Rafael Pazinato • Vanderlei Klauck • Andreia Volpato • Alexandre A. Tonin • Roberto C. Santos • Ma´rcia E. de Souza • Rodrigo A. Vaucher • Renata Raffin • Patrı´cia Gomes • Candice C. Felippi • Lenita M. Stefani • Aleksandro S. Da Silva
Received: 29 October 2013 / Accepted: 14 December 2013 / Published online: 25 December 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract The aim of this study was to verify the influence of tea tree oil (TTO) (Melaleuca alternifolia) tested in its pure and nanostructured (TTO nanoparticles) forms on the reproduction of female Rhipicephalus microplus. For our purpose, female ticks were collected from naturally infected animals and treated in vitro with TTO (1, 5, and 10 %) and TTO nanoparticles (0.075, 0.375, and 0.75 %). In order to validate the tests, they were performed in triplicate using positive (amitraz) and negative (untreated) controls. It was possible to observe that pure TTO (5 and 10 %) and TTO nanoparticles (0.375 and 0.75 %) showed 100 % reproductive inhibition on female ticks. Additionally, pure TTO (1 %) also showed an acaricide effect (70 %), similarly to the positive control (78.3 %). This is the first study demonstrating the activity of pure TTO and TTO nanoparticles on female ticks. Therefore, based on these results, we were able to show that both forms and all concentrations of M. alternifolia affected tick reproduction by inhibiting egg laying and hatching.
R. Pazinato V. Klauck A. Volpato A. S. Da Silva (&) Department of Animal Science, Universidade do Estado de Santa Catarina, Chapeco´, SC, Brazil e-mail: [email protected] A. A. Tonin Department of Microbiology and Parasitology, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil R. C. Santos M. E. de Souza R. A. Vaucher Laboratory of Microbiology, Center of Health Science, Centro Universita´rio Franciscano-UNIFRA, Santa Maria, RS, Brazil R. C. Santos M. E. de Souza R. Raffin P. Gomes Laboratory of Nanotechnology, Programa de Po´s-Graduac¸a˜o em Nanocieˆncias, UNIFRA, Santa Maria, RS, Brazil C. C. Felippi Inventiva, Porto Alegre, RS, Brazil L. M. Stefani Graduate School of Animal Science, Universidade do Estado de Santa Catarina, Lages, SC, Brazil
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We were also able to show that TTO nanoparticles potentiated the inhibitor effect of pure TTO on the reproduction of R. microplus. Keywords
Ticks Natural products Tea tree oil Nanotechnology
Introduction Rhipicephalus (Booophilus) microplus is the tick that causes the most damage to livestock in Brazil and also affects the majority of all cattle herds in the world (Evans 1992). These parasites are favored due to the tropical and subtropical Brazilian climate, representing the main ectoparasite of cattle. Besides the damage caused directly on the animals, R. microplus can also act as a transmitter of other diseases, usually leading to economic losses (Constantinoiu et al. 2010). Tick control is usually done by chemicals, however, the indiscriminate use of them triggered some problems, specially natural selection of this parasites, leading to resistance to many drugs employed in disease treatment (Monteiro 2010). Furthermore, drug residue can be toxic, causing damage to the animal, or even to men through the consumption of contaminated animal products (Chagas et al. 2003). An alternative parasite control is the employment of natural products, which in many cases can help to reduce the resistance process, without harming the environment, since they are biodegradable (Chagas 2004). Among them, the most employed product is a plant from the Meliaceae family (Mulla and Su 1999), represented in this study by Melaleuca alternifolia. It is also called tea tree, an Australian plant, commonly found around swamps and rivers. The M. alternifolia oil (tea tree oil—TTO) is extracted from leaves, and it is chemically rich in terpinen-4-ol, which acts as an efficient antifungal and antibacterial agent. The oil extracted from the bark and trunk of adult trees has a methylated derivative, along with five cyclic penta-triterpenes (Vieira et al. 2004). The nanotechnology has been used in order to improve its efficiency using reduced volumes. This is possible by molecule manipulation in order to form new structural organization of their atoms (Martinez et al. 2011). Nanotechnology is one of the most promising areas of research in modern science. Nanoparticles show completely new or improved properties based on specific characteristics such as size, distribution, and morphology. The emergence of this technology in the last decade opens new opportunities for exploring the effects of nanoparticles in bacteria, fungi, and parasites (Kora and Arunachalam 2011). Faced to the inappropriate use of conventional chemicals, added to the increased resistance problems in the majority of rural properties, it is clear the need for new alternatives to fight ectoparasites. Natural products might be an option taking into account their low cost, good efficiency, less environmental damage, and low toxicity. Therefore, the aim of this study was to investigate, for the first time, the influence of M. alternifolia oil in its pure (TTO) and nanostructured (TTO nanoparticles) forms on the reproduction of female R. microplus.
Materials and methods TTO and TTO nanoparticles TTO was purchased from Importadora Quı´mica Delaware, Brazil. TTO nanoparticles were obtained from InventivaÒ (Porto Alegre, Brazil). Briefly, solid lipid nanoparticles were
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prepared with 7.5 % of tea tree oil using a patented method (InventivaÒ), based on high pressure homogenization. Cetyl palmitate was used as solid lipid and polisorbate 80 as surfactant. Total solid content was 18.6 %. Particle size and zeta potential were evaluated in diluted samples (5009) using Zeta Sizer Nanoseries, Malvern. The pH was assessed by direct use of Digimed potentiometer. Ticks More than 330 ticks were obtained from naturally infected cows from a dairy farm located in Quilombo, Santa Catarina State, Brazil. Engorged females were stored in plastic bottles and kept at 13 °C in styrofoam boxes, and immediately transported to the laboratory. Bioassays Engorged females were divided according to their weight into Petri dishes (10 each). Tests were performed simultaneously and in triplicate for all treatments. The tests were carried out at different concentrations of pure TTO (1, 5, and 10 %) and TTO nanoparticles (0.075, 0.375, and 0.75 %). Five control groups were used to validate the tests: one group did not receive any treatment; two groups had Triton (5 and 10 %) used as a TTO diluent according to the methodology previously described (Chagas et al. 2003; Farias et al. 2007); a fourth group with only nanocapsules with cetyl palmitate and polisorbate 80 without Triton (control 0.75 %); and the last group was a positive control based on amitraz 10 %). In order to decide which dilutions to use, a pilot study was performed previously. Once distributed in Petri dish, the engorged ticks were immerged during 30 s into the test solutions, subsequently dried with paper towel, and placed under controlled conditions (27 °C, 75 % RH). The same procedure was performed for all groups (Tables 1, 2). Seven to fourteen days after the beginning of the experiment, the number of ticks that were able to lay eggs was recorded, and their eggs were weighed. The eggs collected from each Petri dish were placed in test tubes, capped with cotton, and stored in BOD incubators up to 35 days. Elapsed this time, egg hatchability was recorded. Therefore, in order to assess treatment efficacy the following variables were considered: posture, egg’s weight, and the rate of hatched larvae (Drummond et al. 1973; Camillo et al. 2009). Reproductive efficiency and treatment effectiveness were calculated based on mathematical model suggested by Drummond et al. (1973). Statistical analysis Normality test was applied, which showed normal data distribution. Then the data were statistically analyzed by ANOVA and Duncan test (a = 0.05).
Results TTO nanoparticles The TTO nanocapsules were evaluated regarding their physical and chemical properties. The particle size was 287 (±2 nm) and the polydispersion index was 0.203 (±0.022) with a zeta potential of -14.2 ± 1.7 mV.
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Table 1 Mean (± SD) of the number of ticks (Rhipicephalus (Boophilus) microplus) that posture, egg weight and hatching eggs from each treatment with Melaleuca alternifolia oil in pure (TTO oil) and nanostructured (TTO nanoparticles) form Treatment
Number posture by treatment (n = 10)
Control (untreated)
10.0 ± 0.0a
Weighing eggs per treatment (g)
Eggs hatch (yes or not)
0.46 ± 0.035a
Yes
Control (5 % triton*)
ab
9.75 ± 0.50
0.44 ± 0.036a
Yes
Control (10 % triton*)
9.25 ± 0.95ab
0.42 ± 0.057a
Yes
Control (0.75 % nanocapsules?)
9.75 ± 0.50ab
0.41 ± 0.068a
Yes
TTO oil (1 %)
6.0 ± 0.81c
0.17 ± 0.028c
Yes
TTO oil (5 %)
1.5 ± 0.57e
0.076 ± 0.005d
Not
TTO oil (10 %)
1.0 ± 0.81e
0.012 ± 0.001e
Not
TTO nanoparticles (0.075 %)
8.5 ± 1.29b
0.30 ± 0.043b
Yes
TTO nanoparticles (0.375 %)
4.0 ± 0.81d
0.175 ± 0.054c
Not
TTO nanoparticles (0.75 %)
1.25 ± 0.95e
0.181 ± 0.025c
Not
4.0 ± 0.50d
0.20 ± 0.034c
Yes
Amitraz (10 %)—positive control
Means within a column followed by the same letter do not differ significantly (Duncan test, P [ 0.05) * Triton diluent was used for the M. alternifolia oil (1v/v) ?
Nanocapsules formed by cetyl palmitate and polisorbate 80
In vitro tests The results of posture, egg weight, and hatched larvae are shown in Table 1. The negative control groups did not differ statistically in posture, since they were not treated, i.e. ticks immerged in pure water and water plus 5 and 10 % of Triton showed 100, 97, and 92 % of egg laying rate, respectively. The control group containing only TTO nanocapsules showed 97 % of egg laying rate. On the other hand, TTO when used pure at concentrations of 1, 5, and 10 % caused a reduction in the number of females able to perform posture (60, 15, and 10 %, respectively). However, when TTO nanoparticle was tested at 0.075 %, no significant difference was observed when compared with the negative control groups. However, groups subjected to the concentrations of 0.375 and 0.75 showed reductions of 40 and 12 % in their number of ticks that performed posture, respectively. Between 0 and 20 % of engorged females did not die after the immersion bath, while the remainder were alive 15 days after the beginning of the test. There was a great difference in egg weight between the untreated groups and the groups subjected to pure TTO and TTO nanoparticles at all concentrations (P \ 0.05). Although some female ticks were able to perform posture (even at under lower rates), the eggs apparently were infertile when higher concentrations of pure TTO (5 and 10 %) and TTO nanoparticles (0.375 and 0.75 %) were used. We were able to support this finding, since after 35 days of observation larvae hatching was not observed, differently from the other groups (Table 1). In general, ticks immerged in pure and nanocapsules of TTO had reproduction rates much lower than negative control groups (Table 2). Therefore, treatment with oil, in its higher concentrations, showed an efficacy of 100 %, since at concentrations of 1 and 0.075 % the efficacy reached 70.4 (TTO) and 47.8 % (TTO nanoparticles), respectively. Amitraz used as positive control in this study showed lower efficacy in comparison to groups treated with higher concentrations of the tested oil (both in pure and nanostructure
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forms). The in vitro efficacy of amitraz was 78.2 % against R. (B.) microplus, demonstrating tick resistance to this drug (Tables 1, 2).
Discussion Tick high resistance to chemical products used for control, such as amitraz used in this study, has generated great farm concern, a fact that asks for alternative options in controlling these parasites. One that shows less resistance, safety in applying, lower cost, and less environmental damage. In addition, there is a high commercial demand for this type of natural product with acaricide properties and a high interest from researchers and pharmaceutical companies. Therefore, in this study, we are showing a novel acaricide effect of tea tree in its nanocapsuled presentation. To date, studies with acaricide TTO effect on ticks are unknown. Thus, this study represents a pioneering research in this area, since it was found that TTO in its pure formula was able to inhibit and reduce posture, according to the concentration of TTO used, and therefore, influencing directly the reproduction of this parasite, leading to high indexes of parasite control. In this study, probably posture did not occur by the R. (B.) microplus at concentrations of 5 and 10 %, due to death or damage in the reproductive system of the tick. The acaricidal activity of TTO on Otodectes cynotis and Sarcoptes scabiei were already described (Walton et al. 2004; Neves et al. 2012). There are several papers showing the toxic effects of TTO on some insect’s life cycle. Callander and James (2012) showed that this oil was capable of eradicating the Australian sheep blowfly (Lucilia cuprina) and sheep body lice (Bovicola ovis Schrank). In this same study, Sarcoptes scabiei var. hominis and Pediculus capitis (head lice) are also sensitive to TTO. The acaricide mechanism of action of this oil is unknown, however suffocation by breathing spiracles blockage has been suggested (Barker and Altman 2011). Table 2 Degree of reproductive efficiency and efficacy of treatment with Melaleuca alternifolia oil in pure (TTO Oil) and nanostructured (TTO nanoparticles) form against a strain of tick (Rhipicephalus (Boophilus) microplus) Treatment
Reproductive efficiency (%)
Efficacy of treatment (%)
Hatchability (%)
Control (untreated)
92a
0.0e
100a
Control (5 % triton*)
88a
4.3e
100a
Control (10 % triton*)
84a
8.7e
100a
Control (0.75 % nanocapsules?)
85a
5.2e
100a
TTO oil (1 %)
27.2c
70.4c
80.2b
TTO oil (5 %)
0.0d
100a
0.0d
TTO oil (10 %)
0.0d
100a
0.0d
TTO nanoparticles (0.075 %)
48b
47.8d
78.5b
TTO nanoparticles (0.375 %)
0.0d
100a
0.0d
TTO nanoparticles (0.75 %)
0.0d
100a
0.0d
Amitraz (10 %)—positive control
20c
78.3b
50.0c
Means within a column followed by the same letter do not differ significantly (Duncan test, P [ 0.05). The calculations of reproductive efficiency and efficacy of the treatment is detailed in paper of Drummond et al. (1973) and Camillo et al. (2009) * Triton diluent was used for the M. alternifolia oil (v/v) ?
Nanocapsules formed by cetyl palmitate and polisorbate 80
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Other previous studies showed that the effect of basil essential oil (Ocimum basilicum L.) (Santos et al. 2012), and neem oil (Azadirachta indica A. Juss) (Broglio-Micheletti et al. 2010) are able to interfere on tick reproduction. Additionally, andiroba oil (C. guianensis) has shown natural acaricide properties on tick control (Farias et al. 2009), mainly due to its cytotoxic effects on oocytes of engorged female Rhipicephalus sanguineus (Vendramini et al. 2012). The same damage may have occurred in our study, and therefore, TTO probably leads to damage on the reproductive system of the ticks. The essential oils act on insects, ticks, and in many other pathogens, mainly due to their toxic effect, interfering with the biochemical and physiological functions of them (Salisbury and Ross 1992). The main active components of these oils are terpenoids oxygenated (Walton et al. 2004), with the most significant the 1,8 cineole and the terpinen-4-ol. The cineol is known as skin irritant, while the terpinen-4-ol has antimicrobial activity (Simo˜es et al. 2002). Additionally, terpinen-4-ol from TTO was accounted for the acaricide effect against S. scabiei (Walton et al. 2004). Thus, this component might be involved in the inhibition of tick posture, when immerged into TTO during our experiment. TTO nanoparticles used in a concentration lower than the pure oil was more effective in tick control, primarily related to posture inhibition. When at concentrations of 0.375 and 0.75 % the nanocapsuled form showed superior efficacy than the pure TTO at 1 %, demonstrating potential acaricide effect of TTO in nanoemulsion. While various hypotheses have been proposed to explain the enhancement or maintenance of nanoparticles activity, it is widely believed that these nanoparticles are incorporated in the cell membrane causing leakage of intracellular substances and eventually causing cell death (Salopek-Sondi 2004; Cho et al. 2005). Some of the nanoparticles also penetrate into the cells. For example, it is reported that the nanoparticle bactericidal ability decreases as its size increases and the effect is also influenced by its shape. Although most studies have used spherical particles, truncated triangular shaped are reported to have greater effect compared to spherical and rod-shaped particles (Panacek et al. 2006; Pal et al. 2007). The emergence of nanoscience and nanotechnology in the last decade opens many opportunities for exploring the bioactive effects of nanoparticles, however there are no studies using nanoparticles to fight R. microplus infestation and to our knowledge this is the first study demonstrating the use of nanotechnology to control such important parasite. Based on the results presented above, it is possible to conclude that the oil of M. alternifolia in its pure (TTO) and nanostructured (TTO nanoparticles) form has a potential acaricide effect on controlling R. (B.) microplus. In addition, the TTO inhibits or reduces posture, besides its action of derail the remaining eggs. In doses of 5 and 10 % (TTO pure) and 0.375 and 0.75 % (TTO nanoparticles) 100 % of effectiveness was reached in tick control, differently of amitraz that showed poor effect due to tick resistant. The use of perspective non-toxic compounds could represent a natural alternative to synthetic drugs in the control of R. microplus infestation. In this way, TTO nanoparticles could be potentially an useful alternative against this parasite, a major cause of problems for cattle livestocks.
References Barker SC, Altman PM (2011) An ex vivo, assessor blind, randomized, parallel group, comparative efficacy trial of the ovicidal activity of three pediculicides after a single application-melaleuca oil and lavender oil, eucalyptus oil and lemon tea tree oil, and a ‘‘suffocation’’ pediculicide. BMC Dermatol 11:2–7 Broglio-Micheletti SMF, Dias NS, Valente ECN, Souza LA, Lopes DOP, Sanros JM (2010) Ac¸a˜o de extrato e o´leo de nim no controle de Rhipicephalus (Boophilus) microplus em laborato´rio. Rev Bras Parasitol Vet 19:44–48
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Callander JT, James PJ (2012) Insecticidal and repellent effects of tea tree (Melaleuca alternifolia) oil against Lucilia cuprina. Vet Parasitol 184:271–278 Camillo G, Vogel FF, Sangioni LA, Cadore GC, Ferrari R (2009) Eficieˆncia in vitro de acaricidas sobre carrapatos de bovinos no Estado do Rio Grande do Sul, Brasil. Cieˆncia Rural 39:490–495 Chagas ACS (2004) Controle de parasitas utilizando extratos vegetais. Rev Bras Parasitol Vet 13:156–160 Chagas ACS, Leite RC, Furlong J, Prates HT, Passos WM (2003) Sensibilidade do carrapato Boophilus microplus a solventes. Cieˆncia Rural 33:109–114 Cho K, Park J, Osaka T, Park S (2005) The study of antimicrobial activity and preservative effects of nanosilver ingredient. Electrochi Acta 51:956–960 Constantinoiu CC, Jackson LA, Jorgensen WK, Lew-Tabor AE, Piper EK, Mayer DG, Venus B, Jonsson NN (2010) Local immune response against larvae of Rhipicephalus (Boophilus) microplus in cattle. Int J Parasitol 40:865–875 Drummond RO, Ernst SE, Trevino JL, Gladney WJ, Graham OH (1973) Boophilus annulatus and B. microplus: laboratory tests of insecticides. J Econ Entomol 66:130–133 Evans DE (1992) Tick infestation of livestock and control methods in Brazil: a situation reports. Insect Sci Appl 13:629–643 Farias MPO, Sousa DP, Arruda AC, Arruda MSP, Wanderley AG, Alves LC, Faustino MAG (2007) Efica´cia in vitro do o´leo da Carapa guianensis Aubl. (andiroba) no controle de Boophilus microplus (Acari: ixodidae). Revista Brasileira de Plantas Medicinais 9:68–71 Farias MPO, Sousa DP, Arruda AC, Wanderley AG, Teixeira WC, Alves LC, Faustino MAG (2009) Potencial acaricida do o´leo de andiroba Carapa guianensis Aubl. Sobre feˆmeas adultas ingurgitadas de Anocentor nitens Neumann, 1897 e Rhipicephalus sanguineus Latreille, 1806. Arq Bras Med Vet Zootec 61:877–882 Kora AJ, Arunachalam J (2011) Assessment of antibacterial activity of silver nanoparticles on Pseudomonas aeruginosa and its mechanism of action. World J Microbiol Biotechnol 27:1209–1210 Martinez HR, Abdala HM, Trevin˜o E, Garza G, Pozas A, Rivera G (2011) Aplicacio´n de la alternifolia a em alternifolia: nano-odontologı´a. CES Odontol 24:87–91 Monteiro SG (2010) Parasitologia Veterina´ria. Roca, Sa˜o Paulo, p 352 Mulla MS, Su T (1999) Activity and biological effects of NET products against arthropods of medical and veterinary importance. J Am Mosq Control Assoc 15:133–152 Neves RCSM, Ferraz RHS, Mendonc¸a AJ, Lima SR, Barros LA (2012) Acaricide effect of the Melaleuca alternifolia essential oil on Otodectes cynotis. Braz J Vet Res Anim Sci 19:144–148 Pal S, Tak YK, Song JM (2007) Does the antimicrobial activity of silver nanoparticles depend on the shape of the nanoparticle? A study of the Gram-negative bacterium Escherichia coli. Appl Environ Microbiol 73:1712–1720 Panacek A, Kvı´tek L, Prucer R, Kolar M, Vecerova R, Pizu´rova N, Sharma VK, Nevecna T, Zboril R (2006) Silver colloid nanoparticles: synthesis, characterization, and their antibacterial activity. J Phys Chem B 110:16248–16253 Salisbury FB, Ross CW (1992) Plant physiology. Wadsworth, Belmont, pp 357–407 Salopek-Sondi B (2004) Silver nanoparticles as antimicrobial agent: a case study on E. coli as a model for Gram-negative alternif. J Colloid Interface Sci 275:177–182 Santos FCC, Vogel FSF, Monteiro SG (2012) Essential basil oil effect (Ocimum basilicum L.) on cattle tick Rhipicephalus (Boophilus) microplus in vitro experiments. Semina Cieˆncias Agra´rias 33:1133–1140 Simo˜es RP, Groppo FC, Sartorato A, Del Fiol FS, Filho TRM, Ramacciato JC, Rodrigues MVN (2002) ´ leo de Melaleuca alternifolia sobre a infecc¸a˜o estafilo´cica. Revista Lecta 2:143–152 Efeito do O Vendramini MCR, Camargo-Mathias MI, Faria AU, Bechara GH, Oliveira PR, Roma GC (2012) Cytotoxic effects of andiroba oil (Carapa guianensis) in reproductive system of Rhipicephalus sanguineus (Latreille, 1806) (Acari: ixodidae) semi-engorged females. Parasitol Res 111:1885–1894 Vieira TR, Barbosa LCA, Maltha CRA, Paula VF, Nascimento EA (2004) Constituintes quı´micos de Melaleuca alternifo´lia (Myrtaceae). Quim Nova 27:536–539 Walton S, Mckinnon M, Pizzutto S, Dougall A, Williams E, Currie BJ (2004) Acaricidal activity of Melaleuca alternifolia (tea tree) oil: in vitro sensitivity of Sarcoptes scabiei var hominis to terpinen-4ol. Arch Dermatol 140:563–566
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Influence of tea tree oil (Melaleuca alternifolia) on the cattle tick Rhipicephalus microplus Rafael Pazinato • Vanderlei Klauck • Andreia Volpato • Alexandre A. Tonin • Roberto C. Santos • Ma´rcia E. de Souza • Rodrigo A. Vaucher • Renata Raffin • Patrı´cia Gomes • Candice C. Felippi • Lenita M. Stefani • Aleksandro S. Da Silva
Received: 29 October 2013 / Accepted: 14 December 2013 / Published online: 25 December 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract The aim of this study was to verify the influence of tea tree oil (TTO) (Melaleuca alternifolia) tested in its pure and nanostructured (TTO nanoparticles) forms on the reproduction of female Rhipicephalus microplus. For our purpose, female ticks were collected from naturally infected animals and treated in vitro with TTO (1, 5, and 10 %) and TTO nanoparticles (0.075, 0.375, and 0.75 %). In order to validate the tests, they were performed in triplicate using positive (amitraz) and negative (untreated) controls. It was possible to observe that pure TTO (5 and 10 %) and TTO nanoparticles (0.375 and 0.75 %) showed 100 % reproductive inhibition on female ticks. Additionally, pure TTO (1 %) also showed an acaricide effect (70 %), similarly to the positive control (78.3 %). This is the first study demonstrating the activity of pure TTO and TTO nanoparticles on female ticks. Therefore, based on these results, we were able to show that both forms and all concentrations of M. alternifolia affected tick reproduction by inhibiting egg laying and hatching.
R. Pazinato V. Klauck A. Volpato A. S. Da Silva (&) Department of Animal Science, Universidade do Estado de Santa Catarina, Chapeco´, SC, Brazil e-mail: [email protected] A. A. Tonin Department of Microbiology and Parasitology, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil R. C. Santos M. E. de Souza R. A. Vaucher Laboratory of Microbiology, Center of Health Science, Centro Universita´rio Franciscano-UNIFRA, Santa Maria, RS, Brazil R. C. Santos M. E. de Souza R. Raffin P. Gomes Laboratory of Nanotechnology, Programa de Po´s-Graduac¸a˜o em Nanocieˆncias, UNIFRA, Santa Maria, RS, Brazil C. C. Felippi Inventiva, Porto Alegre, RS, Brazil L. M. Stefani Graduate School of Animal Science, Universidade do Estado de Santa Catarina, Lages, SC, Brazil
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We were also able to show that TTO nanoparticles potentiated the inhibitor effect of pure TTO on the reproduction of R. microplus. Keywords
Ticks Natural products Tea tree oil Nanotechnology
Introduction Rhipicephalus (Booophilus) microplus is the tick that causes the most damage to livestock in Brazil and also affects the majority of all cattle herds in the world (Evans 1992). These parasites are favored due to the tropical and subtropical Brazilian climate, representing the main ectoparasite of cattle. Besides the damage caused directly on the animals, R. microplus can also act as a transmitter of other diseases, usually leading to economic losses (Constantinoiu et al. 2010). Tick control is usually done by chemicals, however, the indiscriminate use of them triggered some problems, specially natural selection of this parasites, leading to resistance to many drugs employed in disease treatment (Monteiro 2010). Furthermore, drug residue can be toxic, causing damage to the animal, or even to men through the consumption of contaminated animal products (Chagas et al. 2003). An alternative parasite control is the employment of natural products, which in many cases can help to reduce the resistance process, without harming the environment, since they are biodegradable (Chagas 2004). Among them, the most employed product is a plant from the Meliaceae family (Mulla and Su 1999), represented in this study by Melaleuca alternifolia. It is also called tea tree, an Australian plant, commonly found around swamps and rivers. The M. alternifolia oil (tea tree oil—TTO) is extracted from leaves, and it is chemically rich in terpinen-4-ol, which acts as an efficient antifungal and antibacterial agent. The oil extracted from the bark and trunk of adult trees has a methylated derivative, along with five cyclic penta-triterpenes (Vieira et al. 2004). The nanotechnology has been used in order to improve its efficiency using reduced volumes. This is possible by molecule manipulation in order to form new structural organization of their atoms (Martinez et al. 2011). Nanotechnology is one of the most promising areas of research in modern science. Nanoparticles show completely new or improved properties based on specific characteristics such as size, distribution, and morphology. The emergence of this technology in the last decade opens new opportunities for exploring the effects of nanoparticles in bacteria, fungi, and parasites (Kora and Arunachalam 2011). Faced to the inappropriate use of conventional chemicals, added to the increased resistance problems in the majority of rural properties, it is clear the need for new alternatives to fight ectoparasites. Natural products might be an option taking into account their low cost, good efficiency, less environmental damage, and low toxicity. Therefore, the aim of this study was to investigate, for the first time, the influence of M. alternifolia oil in its pure (TTO) and nanostructured (TTO nanoparticles) forms on the reproduction of female R. microplus.
Materials and methods TTO and TTO nanoparticles TTO was purchased from Importadora Quı´mica Delaware, Brazil. TTO nanoparticles were obtained from InventivaÒ (Porto Alegre, Brazil). Briefly, solid lipid nanoparticles were
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prepared with 7.5 % of tea tree oil using a patented method (InventivaÒ), based on high pressure homogenization. Cetyl palmitate was used as solid lipid and polisorbate 80 as surfactant. Total solid content was 18.6 %. Particle size and zeta potential were evaluated in diluted samples (5009) using Zeta Sizer Nanoseries, Malvern. The pH was assessed by direct use of Digimed potentiometer. Ticks More than 330 ticks were obtained from naturally infected cows from a dairy farm located in Quilombo, Santa Catarina State, Brazil. Engorged females were stored in plastic bottles and kept at 13 °C in styrofoam boxes, and immediately transported to the laboratory. Bioassays Engorged females were divided according to their weight into Petri dishes (10 each). Tests were performed simultaneously and in triplicate for all treatments. The tests were carried out at different concentrations of pure TTO (1, 5, and 10 %) and TTO nanoparticles (0.075, 0.375, and 0.75 %). Five control groups were used to validate the tests: one group did not receive any treatment; two groups had Triton (5 and 10 %) used as a TTO diluent according to the methodology previously described (Chagas et al. 2003; Farias et al. 2007); a fourth group with only nanocapsules with cetyl palmitate and polisorbate 80 without Triton (control 0.75 %); and the last group was a positive control based on amitraz 10 %). In order to decide which dilutions to use, a pilot study was performed previously. Once distributed in Petri dish, the engorged ticks were immerged during 30 s into the test solutions, subsequently dried with paper towel, and placed under controlled conditions (27 °C, 75 % RH). The same procedure was performed for all groups (Tables 1, 2). Seven to fourteen days after the beginning of the experiment, the number of ticks that were able to lay eggs was recorded, and their eggs were weighed. The eggs collected from each Petri dish were placed in test tubes, capped with cotton, and stored in BOD incubators up to 35 days. Elapsed this time, egg hatchability was recorded. Therefore, in order to assess treatment efficacy the following variables were considered: posture, egg’s weight, and the rate of hatched larvae (Drummond et al. 1973; Camillo et al. 2009). Reproductive efficiency and treatment effectiveness were calculated based on mathematical model suggested by Drummond et al. (1973). Statistical analysis Normality test was applied, which showed normal data distribution. Then the data were statistically analyzed by ANOVA and Duncan test (a = 0.05).
Results TTO nanoparticles The TTO nanocapsules were evaluated regarding their physical and chemical properties. The particle size was 287 (±2 nm) and the polydispersion index was 0.203 (±0.022) with a zeta potential of -14.2 ± 1.7 mV.
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Table 1 Mean (± SD) of the number of ticks (Rhipicephalus (Boophilus) microplus) that posture, egg weight and hatching eggs from each treatment with Melaleuca alternifolia oil in pure (TTO oil) and nanostructured (TTO nanoparticles) form Treatment
Number posture by treatment (n = 10)
Control (untreated)
10.0 ± 0.0a
Weighing eggs per treatment (g)
Eggs hatch (yes or not)
0.46 ± 0.035a
Yes
Control (5 % triton*)
ab
9.75 ± 0.50
0.44 ± 0.036a
Yes
Control (10 % triton*)
9.25 ± 0.95ab
0.42 ± 0.057a
Yes
Control (0.75 % nanocapsules?)
9.75 ± 0.50ab
0.41 ± 0.068a
Yes
TTO oil (1 %)
6.0 ± 0.81c
0.17 ± 0.028c
Yes
TTO oil (5 %)
1.5 ± 0.57e
0.076 ± 0.005d
Not
TTO oil (10 %)
1.0 ± 0.81e
0.012 ± 0.001e
Not
TTO nanoparticles (0.075 %)
8.5 ± 1.29b
0.30 ± 0.043b
Yes
TTO nanoparticles (0.375 %)
4.0 ± 0.81d
0.175 ± 0.054c
Not
TTO nanoparticles (0.75 %)
1.25 ± 0.95e
0.181 ± 0.025c
Not
4.0 ± 0.50d
0.20 ± 0.034c
Yes
Amitraz (10 %)—positive control
Means within a column followed by the same letter do not differ significantly (Duncan test, P [ 0.05) * Triton diluent was used for the M. alternifolia oil (1v/v) ?
Nanocapsules formed by cetyl palmitate and polisorbate 80
In vitro tests The results of posture, egg weight, and hatched larvae are shown in Table 1. The negative control groups did not differ statistically in posture, since they were not treated, i.e. ticks immerged in pure water and water plus 5 and 10 % of Triton showed 100, 97, and 92 % of egg laying rate, respectively. The control group containing only TTO nanocapsules showed 97 % of egg laying rate. On the other hand, TTO when used pure at concentrations of 1, 5, and 10 % caused a reduction in the number of females able to perform posture (60, 15, and 10 %, respectively). However, when TTO nanoparticle was tested at 0.075 %, no significant difference was observed when compared with the negative control groups. However, groups subjected to the concentrations of 0.375 and 0.75 showed reductions of 40 and 12 % in their number of ticks that performed posture, respectively. Between 0 and 20 % of engorged females did not die after the immersion bath, while the remainder were alive 15 days after the beginning of the test. There was a great difference in egg weight between the untreated groups and the groups subjected to pure TTO and TTO nanoparticles at all concentrations (P \ 0.05). Although some female ticks were able to perform posture (even at under lower rates), the eggs apparently were infertile when higher concentrations of pure TTO (5 and 10 %) and TTO nanoparticles (0.375 and 0.75 %) were used. We were able to support this finding, since after 35 days of observation larvae hatching was not observed, differently from the other groups (Table 1). In general, ticks immerged in pure and nanocapsules of TTO had reproduction rates much lower than negative control groups (Table 2). Therefore, treatment with oil, in its higher concentrations, showed an efficacy of 100 %, since at concentrations of 1 and 0.075 % the efficacy reached 70.4 (TTO) and 47.8 % (TTO nanoparticles), respectively. Amitraz used as positive control in this study showed lower efficacy in comparison to groups treated with higher concentrations of the tested oil (both in pure and nanostructure
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forms). The in vitro efficacy of amitraz was 78.2 % against R. (B.) microplus, demonstrating tick resistance to this drug (Tables 1, 2).
Discussion Tick high resistance to chemical products used for control, such as amitraz used in this study, has generated great farm concern, a fact that asks for alternative options in controlling these parasites. One that shows less resistance, safety in applying, lower cost, and less environmental damage. In addition, there is a high commercial demand for this type of natural product with acaricide properties and a high interest from researchers and pharmaceutical companies. Therefore, in this study, we are showing a novel acaricide effect of tea tree in its nanocapsuled presentation. To date, studies with acaricide TTO effect on ticks are unknown. Thus, this study represents a pioneering research in this area, since it was found that TTO in its pure formula was able to inhibit and reduce posture, according to the concentration of TTO used, and therefore, influencing directly the reproduction of this parasite, leading to high indexes of parasite control. In this study, probably posture did not occur by the R. (B.) microplus at concentrations of 5 and 10 %, due to death or damage in the reproductive system of the tick. The acaricidal activity of TTO on Otodectes cynotis and Sarcoptes scabiei were already described (Walton et al. 2004; Neves et al. 2012). There are several papers showing the toxic effects of TTO on some insect’s life cycle. Callander and James (2012) showed that this oil was capable of eradicating the Australian sheep blowfly (Lucilia cuprina) and sheep body lice (Bovicola ovis Schrank). In this same study, Sarcoptes scabiei var. hominis and Pediculus capitis (head lice) are also sensitive to TTO. The acaricide mechanism of action of this oil is unknown, however suffocation by breathing spiracles blockage has been suggested (Barker and Altman 2011). Table 2 Degree of reproductive efficiency and efficacy of treatment with Melaleuca alternifolia oil in pure (TTO Oil) and nanostructured (TTO nanoparticles) form against a strain of tick (Rhipicephalus (Boophilus) microplus) Treatment
Reproductive efficiency (%)
Efficacy of treatment (%)
Hatchability (%)
Control (untreated)
92a
0.0e
100a
Control (5 % triton*)
88a
4.3e
100a
Control (10 % triton*)
84a
8.7e
100a
Control (0.75 % nanocapsules?)
85a
5.2e
100a
TTO oil (1 %)
27.2c
70.4c
80.2b
TTO oil (5 %)
0.0d
100a
0.0d
TTO oil (10 %)
0.0d
100a
0.0d
TTO nanoparticles (0.075 %)
48b
47.8d
78.5b
TTO nanoparticles (0.375 %)
0.0d
100a
0.0d
TTO nanoparticles (0.75 %)
0.0d
100a
0.0d
Amitraz (10 %)—positive control
20c
78.3b
50.0c
Means within a column followed by the same letter do not differ significantly (Duncan test, P [ 0.05). The calculations of reproductive efficiency and efficacy of the treatment is detailed in paper of Drummond et al. (1973) and Camillo et al. (2009) * Triton diluent was used for the M. alternifolia oil (v/v) ?
Nanocapsules formed by cetyl palmitate and polisorbate 80
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Other previous studies showed that the effect of basil essential oil (Ocimum basilicum L.) (Santos et al. 2012), and neem oil (Azadirachta indica A. Juss) (Broglio-Micheletti et al. 2010) are able to interfere on tick reproduction. Additionally, andiroba oil (C. guianensis) has shown natural acaricide properties on tick control (Farias et al. 2009), mainly due to its cytotoxic effects on oocytes of engorged female Rhipicephalus sanguineus (Vendramini et al. 2012). The same damage may have occurred in our study, and therefore, TTO probably leads to damage on the reproductive system of the ticks. The essential oils act on insects, ticks, and in many other pathogens, mainly due to their toxic effect, interfering with the biochemical and physiological functions of them (Salisbury and Ross 1992). The main active components of these oils are terpenoids oxygenated (Walton et al. 2004), with the most significant the 1,8 cineole and the terpinen-4-ol. The cineol is known as skin irritant, while the terpinen-4-ol has antimicrobial activity (Simo˜es et al. 2002). Additionally, terpinen-4-ol from TTO was accounted for the acaricide effect against S. scabiei (Walton et al. 2004). Thus, this component might be involved in the inhibition of tick posture, when immerged into TTO during our experiment. TTO nanoparticles used in a concentration lower than the pure oil was more effective in tick control, primarily related to posture inhibition. When at concentrations of 0.375 and 0.75 % the nanocapsuled form showed superior efficacy than the pure TTO at 1 %, demonstrating potential acaricide effect of TTO in nanoemulsion. While various hypotheses have been proposed to explain the enhancement or maintenance of nanoparticles activity, it is widely believed that these nanoparticles are incorporated in the cell membrane causing leakage of intracellular substances and eventually causing cell death (Salopek-Sondi 2004; Cho et al. 2005). Some of the nanoparticles also penetrate into the cells. For example, it is reported that the nanoparticle bactericidal ability decreases as its size increases and the effect is also influenced by its shape. Although most studies have used spherical particles, truncated triangular shaped are reported to have greater effect compared to spherical and rod-shaped particles (Panacek et al. 2006; Pal et al. 2007). The emergence of nanoscience and nanotechnology in the last decade opens many opportunities for exploring the bioactive effects of nanoparticles, however there are no studies using nanoparticles to fight R. microplus infestation and to our knowledge this is the first study demonstrating the use of nanotechnology to control such important parasite. Based on the results presented above, it is possible to conclude that the oil of M. alternifolia in its pure (TTO) and nanostructured (TTO nanoparticles) form has a potential acaricide effect on controlling R. (B.) microplus. In addition, the TTO inhibits or reduces posture, besides its action of derail the remaining eggs. In doses of 5 and 10 % (TTO pure) and 0.375 and 0.75 % (TTO nanoparticles) 100 % of effectiveness was reached in tick control, differently of amitraz that showed poor effect due to tick resistant. The use of perspective non-toxic compounds could represent a natural alternative to synthetic drugs in the control of R. microplus infestation. In this way, TTO nanoparticles could be potentially an useful alternative against this parasite, a major cause of problems for cattle livestocks.
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