The sensitivity of Demodex canis (Acari: Demodicidae) to the essential oil of Melaleuca alternifolia – an in vitro study estudo in vitro

nevesrita@id.uff.br Abstract The essential oil of the Melaleuca alternifolia (Maiden & Betche) (tea tree oil) has been effective in previous studies, in the treatment of infestation by Demodex mites in humans. The present study aimed at evaluating the in vitro acaricidal effect of this herbal medicine on Demodex canis . For the parasitological examination, samples were collected from a dog’s skin and examined using optical microscopy. Only samples with intact mites and with evident movement of chelicerae and tarsi were selected. Twenty-one samples were tested with the oil, in seven different concentrations: 100%; 50%; 25%; 12.5%; 6.25%; 5.0% and 3.13%. Three samples were tested with the positive control amitraz, and three with the negative control neutral shampoo. The interval between the time the solution was added and the moment the movement of the last mite ceased defined the survival time in the sample. By comparing the times at different concentrations and controls, the results showed that the higher the concentration of the oil, the more quickly its lethal effect occurred, and that the survival times in the controls were longer than in the different concentrations of melaleuca chloroform injected chromatograph. components were identified comparing their mass spectra with the Kovats retention


Introduction
Mites of the genus Demodex are commensal on the skin of mammals (Patra et al., 2019) causing severe inflammatory skin disease when a population of these parasites exceeds the point of equilibrium. The pathogenic role of these mites is well documented in dogs, in which Demodex canis Leydig 1859 causes demodicosis (Jarmuda et al., 2012), which usually requires several months of treatment to achieve clinical resolution (Mueller et al., 2012).
For decades, in many countries, the gold-standard treatment for demodicidosis has consisted of the application of an aqueous dilution of amitraz, a product of the formamidine group (Adams, 2003). Despite its variable efficacy (Arsenović et al., 2015), it has been responsible for numerous cases of intoxication (Gortel, 2006;Barretto et al., 2017). The acaricidal activity of amitraz is based on disrupting the transmission of nerve impulses, because it is an antagonist at the octopamine receptor level (Hugnet et al., 1996;Rhodes, 2004). Systemic macrocyclic lactones, some orthodox and some off-label, such as ivermectin, doramectin, and moxidectin, subsequently became available for use as more convenient forms of treatment in veterinary medicine (Delayte et al., 2006) and continues to be used in small animal clinics, although there have been some reports of adverse reactions (Singh et al., 2011;Perego et al., 2019).
In the search for more effective and less toxic drugs, herbal medicines have arisen as potential alternatives, with few residual effects and a low incidence of resistance (Castro et al., 2018). Among these, essential oils constitute an appealing option because they contain a variety of volatile secondary metabolites known to play a role in plant defense mechanisms (Nazzaro et al., 2013). Tea tree oil, derived from the leaves of the Australian tea tree Melaleuca alternifolia (Maiden & Betche, 1905), has been reported to have antibacterial properties (Neves et al., 2018;Puvača et al., 2019), anti-inflammatory properties (Caldefie-Chézet et al., 2004;Neves et al., 2018), and antiparasitic properties (Neves et al., 2013;Puvača et al., 2019;Savla et al., 2019;Zulkarnain et al., 2019).
Despite the tea tree oil having been tested in terms of its efficacy against mites of the Demodex genus in humans (Gao et al., 2005;Savla et al., 2019), its efficacy against the etiological agent of canine mange has never been compared. Therefore, the aim of this study was to evaluate the in vitro acaricidal effect of tea tree oil on Demodex canis mites.

Ethical considerations
All procedures were performed in compliance with the Ethical Principles on Animal Experimentation established by the Brazilian Society for the Ethical Use of Laboratory Animals in Scientific Research. The study was approved by the Animal Research Ethics Committee of the Universidade Federal Fluminense (Protocol numbers 853 and 4235270918, ID 000562).

Sample collection
For the in vitro study, mites from an approximately six-month-old female pit bull dog from the city of Niterói, in the Brazilian state of Rio de Janeiro were collected. It had been diagnosed for the first time with Demodex sp., presented a high parasite load, and had not previously been treated with any ectoparasiticide. Only one patient was used in order to minimize individual differences between mites, a procedure followed in other studies with similar objectives to this (Walton et al., 2004;Tighe et al., 2013).
Mites were collected from the lesions by using adhesive tapes (Zhao et al., 2006(Zhao et al., , 2011Pereira et al., 2012). The tapes, measuring 6 × 2.5 cm, were then adhered to microscopy slides and observed under light microscopy at magnifications of 100× and 400×. As the collection of equivalent samples is difficult to be performed, given the uneven distribution of the mite population on the skin, it was not possible to standardize for a single number of mites per slide. Thus, slides that contained between 5 and 7 adult mites, evidently mobile, were chosen for the test. As in previous studies, the evident movement of chelicerae and tarsi was the criterion of choice for selecting viable samples (Gao et al., 2005;Tighe et al., 2013). Twenty seven samples were selected and distributed in triplicates (seven concentrations of the oil and two controls, positive and negative). In order to make the total of mites equal in each triplicate, this distribution rendered each replica with a difference of one or two mites in relation to the other two.
Some of the collected mites were fixed in 70% alcohol and sent to the Fundação Oswaldo Cruz for taxonomic identification. The mites were identified according to the key proposed by Domrow (1991).

Analysis of the test product
The chemical composition of the tea tree oil employed (Laszlo Aromaterapia, Belo Horizonte, Brazil) was analyzed by gas chromatography. The qualitative and quantitative analyses were both performed in a high-resolution gas chromatograph (7820A; Agilent, Santa Clara, CA, USA), equipped with a flame ionization detector and a 30 m × 0.32 mm × 0.25 mm column (HP-5; Agilent), at an initial temperature of 70°C, the temperature being increased at a rate of 3°C/min until reaching 240°C. The injector and flame ionization detector were operated at 250°C and 260°C, respectively. Hydrogen was used as a carrier gas, at a flow rate of 3 mL/min, and the split ratio was 1:30. The oil was diluted with chloroform (1%), and 1 µL was injected into the chromatograph. The components of the oil were identified by comparing their mass spectra with the Kovats retention index. Data were acquired and processed with EZChrom Elite Compact software, version A.04.05 (Agilent).

Experimental procedures
The 27 samples selected were divided into three groups: TTO (n = 21), to be treated with tea tree oil; AMZ (n = 3) to be treated with the amitraz solution (Triatox; MSD Saúde Animal, São Paulo, Brazil); and JBS (n = 3), to be treated with Johnson's Baby Shampoo (Johnson & Johnson, Sao Paulo, Brazil), in its original factory concentration. The tapes were carefully detached from the microscopy slides, and 200 µL of the respective substances were applied, covering the entire face of the glass slides. After the substances had been applied, the adhesive tapes with the samples were again placed onto the slides.
For the TTO group, the samples were divided into seven subgroups (n = 3 samples/subgroup), in which the test product was applied in pure form and in six different dilutions: 50.0%, 25.0%, 12.5%, 6.25%, 5.0% and 3.13%. The shampoo used in the tea tree oil dilutions was the same as that used in the JBS group. In the AMZ (positive control) group, all three samples were treated with a solution of 4 ml of 12.5% amitraz diluted in 1 L of water (a final solution of 0.05% amitraz, or 50 ppm). This ratio is recommended by the manufacturer when used in therapeutic baths of animals suffering from mange (Viana, 2014). In the JBS (negative control) group, all three samples were treated with the neutral shampoo in its original concentration, as used in the dilution of the tea tree oil. All dilutions were prepared at the time of use.

Microscopy
The samples were observed under microscopy, the parameters of interest being mite mobility and changes in aspects of mite bodies. The initial observations were made at magnifications of 100×, following a zigzag line, for 5 min and at 5-min intervals thereafter, until the death of each mite was suspected, after which a magnification of 400× was employed, and the chelicerae and tarsi of the mites were observed continuously for 60s. The mobility (or lack thereof) of those structures over time attested to whether a mite was alive or dead (Gao et al., 2005;Tighe et al., 2013). The survival time was defined by the lapse of time between the application of the solution and the moment when the movement of the last mite in the sample ceased. Therefore, the time at which the last mite in each sample died was recorded was considered the total time required for the extermination of the mites in the sample and concentration in question. The same procedure was followed for all mites in each sample. At 30 min after confirmation of the last death, another confirmatory reading was taken.
The temperature and relative humidity in the laboratory were measured at 60-min intervals with a thermo-hygrometer (Incoterm, Porto Alegre, Brazil).

Results
The mites were identified as D. canis and were deposited in the Entomological Collection of the Fundação Oswaldo Cruz, Registration numbers ACA -4242, 4243 and 4244.
The compounds identified and quantified in the tea tree oil are shown in Table 1. Gas chromatography revealed that the tea tree oil used in this study had concentrations of 45.9% for terpinen-4-ol and 1.6% for 1,8-cineol.
In the TTO group samples, after application, the deaths occurred between 4 and 214 min. (Table 2). Mite agitation and opisthosomal wrinkling (Figure 1) were observed when the samples came into contact with the tea tree oil, especially at higher concentrations. Immediately before the death of each mite, there was progressive slowing of the movements of the chelicerae and tarsi.
In the AMZ group samples, the deaths, after application of the product, occurred between 268 and 434 min. (Table 2), and in the JBS group samples, the survival time was between 436 and 540 minutes ( Table 2).  The lowest mortality time observed in the AMZ group (268 minutes) was higher than the maximum time detected in the TTO group with the lowest concentration tested (214 minutes) ( Table 2). Although it was observed that the higher the concentration in the TTO group, the shorter the mite survival time was, in the concentration of 25% the death time of the last parasite was 14 minutes and in the 50% concentration it was 18 minutes ( Table 2).With the exception of the 25% and 12,5% treatment, all others had greater variability than that observed in the AMZ group. A high variation between replicates (Table 2) occurred mainly between treatments with lower oil concentrations (5% and 3.13%).
There was no high variation in the temperature and humidity of the laboratory during the experiment. The average temperature was 26.4°C (range: 23.0-27.1°C), and the average relative humidity was approximately 70% (range: 67-73%).

Discussion
Gas chromatography showed that the commercial quality of the tea tree oil used in the present study was within the standards established by the ISO 4730:2004and AS 2782-2009(Silva et al., 2002Simões et al., 2002;Walton et al., 2004;Mondello et al., 2006;Malhi et al., 2017).
In vitro, tea tree oil was effective against Demodex canis mites, being most efficient, in terms of its speed, at higher concentrations. As the evidence showed, a higher concentration was efficient in a shorter time and, conversely, a lower concentration required a longer time for an acaricidal effect to be achieved in the entire sample. Our data are consistent with the reports of Gao et al. (2005) concerning the effects of melaleuca oil on Demodex folliculorum Simon (1842), wherein the authors found concentration-and-time-dependent increases in mortality. Although the results of time of death between the 25% and 50% concentration groups were inverse to what was expected, the values are very close. This suggests that the time of death is dose-dependent, but the in interval between 25% and 50% not much difference should occur. This hypothesis deserves to be better analyzed in future investigations. In another study, Zhao et al. (2006) found that eucalyptus oil, from the same family as the Melaleuca alternifolia, was a potent acaricide with concentration-and-time-dependent increases in mortality of Demodex folliculorum and Demodex brevis Akbulatova (1963). These authors found that the eucalyptus oil was least effective in killing the mites with a concentration of 6.25%, whereas the most suitable, effective concentration for the in vitro killing of these mites was 12.5%. In the present study, even in the low dilutions tested (3.13%, 5.0% and 6.25%), the velocity of killing mites was considerably faster than the AMZ group, and the concentration 25% showed the lowest variability (CV = 16.7%) in relation to the other concentrations of the TTO group, suggesting a more homogeneous action in the elimination of the mite. The agitation of mites soon after contact with tea tree oil, as well as the reduction in their mobility thereafter, is also similar to what was found by Zhao et al. (2006). It is possible that the action of these two oils may be similar, since Eucalyptus and Melaleuca, although with different chemical concentrations, have some similar chemical components (Noumi et al., 2011).
Although it is believed that the antiparasitic action of tea tree oil is attributable to terpinen-4-ol, the mechanism by which these constituents promote the death of Demodex mites has yet to be elucidated (Tighe et al., 2013). However, tea tree oil could disrupt various membrane structures of bacteria, fungi, protozoa, and enveloped viruses (Low et al., 2017;Lam et al., 2018). Mills et al. (2004) suggested that tea tree oil competitively blocks the neurotransmitter terminating enzyme acetylcholinesterase in parasites and may contribute to the arthropodicidal effect. However, it has been suggested that the inhibition of acetylcholinesterase by tea tree oil may be more potent than that of the primary monoterpenoid constituents, because of the synergistic or additive effect of a combination of components instead of a one single strong inhibitor (Miyazawa & Yamafuji, 2006). Furthermore, another in vitro study showed that terpinen-4-ol can decrease the production of pro-inflammatory cytokines such as interferon gamma, interleukin IL-1β, IL-6, and IL-17, as well as that of anti-inflammatory cytokines such as IL-4 and IL-10 (Nogueira, 2013).
The efficacy in vitro acaricidal action of tea tree oil was also reported by Walton et al. (2004) and Neves et al. (2013), both of whom used it at a concentration of 5% against mites of the species Sarcoptes scabiei De Geer, 1778 and Otodectes cynotis (Hering, 1838), respectively. Walton et al. (2004), Gao et al. (2005) and Yang et al. (2013) also conducted in vivo studies of tea tree oil, all showing that it had efficacious acaricidal action against various species of mites in a variety of hosts. According to those authors, the cumulative acaricidal, antibacterial, antipruritic, anti-inflammatory, and wound-healing effects of tea tree oil may have the potential to successfully reduce the burden of scabies infection and associated bacterial complications. The same mechanisms are believed to be involved in the effects of tea tree oil as a treatment for human ocular demodicosis (Gao et al., 2005;Tighe et al., 2013). Therefore, it is likely that these mechanisms would act on canine demodicosis in its natural environment.
In this study, amitraz was chosen as the positive control because it is a topical agent and could therefore be compared directly to the tea tree oil, which is also applied topically. It is noteworthy that the maximum time for mite mortality was longer in the AMZ group samples than in the TTO group samples. However, AMZ showed less variation between replicates.
Although neutral shampoo has no proven acaricidal action, the mites in the JBS group samples also died over time. That was likely due to desiccation and lack of nutrition, as remaining between the slide and the coverslip eventually led to dehydration and a natural difficulty in surviving away from the skin of the host. Gao et al. (2005) and Tighe et al. (2013) also reported that the controls had no acaricidal action, when observed for up to 150 minutes.
In the present study, the mites were observed in vitro, arranged on microscopy slides, and submitted to average temperatures of 26.4°C at 70% humidity. The suitable development temperature range, previously described for Demodex folliculorum, was 20 -30°C, of which the most suitable temperature range was 25-26°C (Zhao et al., 2005(Zhao et al., , 2009). The authors did not evaluated the extreme temperatures, because it was not the aim of this study, but the average temperature of 26.4 seems to be suitable for this species, considering the maintenance of structural integrity and movements on the neutral shampoo group. It was not possible to report the acaricidal effects of melaleuca oil on Demodex canis mite directly on the skin of the dogs, especially considering that the mite insertion environment would be substantially different.

Conclusions
Tea tree oil, at any concentration, appears to have satisfactory acaricidal effects on mites of the species Demodex canis in vitro. In addition, the acaricidal effects of tea tree oil were faster than were those of amitraz.