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Parasitol Res

DOI 10.1007/s00436-017-5604-x

ORIGINAL PAPER

Evaluation of the acaricidal activity of thymol incorporated


in two formulations for topical use against immature stages
of Rhipicephalus sanguineus sensu lato (Latreille, 1806)
(Acari: Ixodidae)
Camila Delmonte 1 & Paula Barroso Cruz 2 & Viviane Zeringóta 2 & Valéria de Mello 3 &
Felipe Ferreira 3 & Maria da Penha Henriques Amaral 3 & Erik Daemon 1

Received: 10 May 2017 / Accepted: 27 August 2017


# Springer-Verlag GmbH Germany 2017

Abstract The objective of this study was to assess, for the conditions analyzed. The O/W emulsion showed signs of early
first time, the in vitro acaricidal activity of two topical formu- instability at the concentration of 5.0 mg/mL. The results ob-
lations containing thymol, on immature stages of tained indicate that the acaricidal activity of thymol, when
Rhipicephalus sanguineus sensu lato. For this purpose, two included in the proposed formulations, was enhanced against
base formulations were prepared: an oil-in-water (O/W) emul- non-engorged larvae with topical treatment in comparison
sion and a hydroalcoholic solution, containing different thy- with data in the literature. Although there were variations in
mol concentrations (0.5 to 20 mg/mL). We used the larval toxicity between the different stages, these formulations are
packet test for non-engorged larvae and nymphs, and the im- promising for future therapeutic use.
mersion test for engorged larvae and nymphs. For emulsion, a
mortality rate of 94.2% was achieved at 0.75 mg/mL in non-
engorged larvae. For engorged larvae, there was 95.0% mor- Keywords Brown dog tick . Monoterpene . Larvae .
tality at 5.0 mg/mL. Non-engorged nymphs showed 83.3% Nymphs . Emulsion . Hydroalcoholic solution
mortality at 2.5 mg/mL, and for engorged nymphs, 86.0%
mortality was verified at 5.0 mg/mL. For the hydroalcoholic
solution, the mortality found for non-engorged larvae was Introduction
88.1% at 2.5 mg/mL. For engorged larvae, the highest mor-
tality was 25.0% at 20 mg/mL; non-engorged nymphs had Rhipicephalus sanguineus sensu lato (Latreille, 1806), the
91.0% mortality at 1.0 mg/mL and for engorged nymphs; brown dog tick, is widely distributed in the world (Labruna
the maximum value verified was 18.3% mortality at 20 mg/ 2004; Guglielmone et al. 2006). The control of this ectopara-
mL. Preliminary stability tests were carried, and the site should take into account that it is a three-host tick, which
hydroalcoholic solution remained stable under all the means that each stage of its life cycle must feed on a host, and
that oviposition and molts are made in the environment.
Besides the direct damage caused, such as blood spoliation,
* Camila Delmonte
camila_delmonte@hotmail.com discomfort, and allergic reactions, it can also act as a vector for
dissemination of bacteria and protozoa to dogs and humans
(Dantas-Torres 2008). In Brazil, it is considered a potential
1
Programa de Pós Graduação em Ciências Biológicas - vector of human infection by the bacterium Rickettsia
Comportamento e Biologia Animal, Universidade Federal de Juiz de
Fora UFJF, Juiz de Fora, Minas Gerais, Brazil
rickettsii (Serra-Freire et al. 2011).
2
The taxonomic status of this species still lacks consensus.
Programa de Pós Graduação em Ciências Veterinárias, Departamento
de Parasitologia Animal, Universidade Federal Rural do Rio de
In recent years, evidence of genetic, morphological, and epi-
Janeiro, UFRRJ, Seropédica,, Rio de Janeiro, Brazil demiological differences of individuals from different regions
3
Programa de Pós Graduação em Ciências Farmacêuticas,
of the world has been reported, and based on the difficulty of
Universidade Federal de Juiz de Fora, UFJF, Juiz de Fora, Minas determining the neotype R. sanguineus sensu stricto, it has
Gerais, Brazil been established that the most suitable denomination for
Parasitol Res

individuals of this complex is Rhipicephalus sanguineus placed in plastic syringes with the distal end cutoff, sealed
sensu lato (s.l.) (Nava et al. 2015). with hydrophilic cotton, and labeled for identification. The
Control of these parasites is usually by applying synthetic syringes were kept under the same temperature and humidity
chemical substances. However, the indiscriminate use of these conditions, and the larvae were used in the tests 15–25 days
substances causes selection of resistant tick populations, in- after hatching. The unengorged nymphs, derived from
toxication of animals and their handlers, and possible contam- engorged larvae, were tested 15 days after ecdysis and the
ination of the environment with chemical wastes (Chagas engorged larvae and nymphs, obtained by artificial infestation
2004; Coles and Dryden 2014). Since the relationship be- on rabbits, were tested on the day they dropped off the host.
tween humans and domestic animals has been getting increas-
ingly close, with dogs often considered as members of the Formulation development
family, the importance of parasite control and the use of safe
chemicals is emphasized, in order not to compromise the Two base formulations were prepared, one an oil-in-water
health of pets and their guardians. These drawbacks have re- (O/W) emulsion and the other a hydroalcoholic solution.
sulted in growing demand for safer acaricides, in turn, The thymol utilized in the formulations was obtained from
prompting research into substances derived from plants Sigma-Aldrich, with purity ≥ 99%. The O/W emulsion was
(Chagas, 2004; Ellse and Wall 2014; Regnault-Roger and formulated with the following raw materials, identified ac-
Philogène, 2008; Borges et al. 2011). cording to the International Nomenclature of Cosmetic
Among the substances of plant origin already tested for this Ingredients (INCI): cetearyl alcohol, sodium laureth sulfate,
purpose is thymol, an aromatic monoterpene initially isolated metylparaben, propylparaben, glycerin, grape seed oil, and
from plants of the family Lamiaceae. This molecule has water. For preparation, each constituent was separated accord-
shown promising results regarding its potential against vari- ing to its solubility in water or oil. The aqueous phase was
ous species of ticks (Monteiro et al. 2010; Mendes et al. 2011; heated to 80 °C and the oil phase to 75 °C, and then the
Daemon et al. 2012; Matos et al. 2014; Araújo et al. 2015; aqueous phase was added in the oil phase and emulsified with
Novato et al. 2015). a mortar and pestle until completely homogenized. The
For a chemical substance, whether natural or artificial, to be hydroalcoholic solution contained glycerin, methylparaben,
used for therapeutic purposes, it is often necessary to include it and ethanol and was prepared by mixing the components at
in a formulation that makes its application feasible and does room temperature in a graduated glass beaker. Twenty-four
not impair its efficacy (York 2005). Therefore, the aim of this hours after preparation, the samples were evaluated regarding
study was to assess the in vitro acaricidal activity of thymol, homogeneity and organoleptic characteristics, to identify any
incorporated in two formulations for topical use, against im- instability. In both formulations, the thymol, dissolved in ab-
mature stages of R. sanguineus s.l., along with the formula- solute ethanol q.s. (sufficient quantity), was added after prep-
tions’ short-term stability. aration of the base formula.

Bioassays
Material and methods
For the in vitro tests, 18 groups were formed, nine for the emul-
Study location sion and nine for the hydroalcoholic solution: emulsion (or solu-
tion) control, and emulsion (or solution) with thymol at 0.5, 0.75,
The formulations were prepared, and their stability was ana- 1.0, 1.25, 2.5, 5.0, 10.0, and 20.0 mg/mL. These concentrations
lyzed in the Laboratório de Farmacotécnica da Universidade were chosen based on tests previously carried out with thymol on
Federal de Juiz de Fora, Minas Gerais, Brazil. The in vitro R. sanguineus s.l. at concentrations ranging from 2.5 to 20 mg/
tests on ticks were performed in the Laboratório de mL (Daemon et al. 2009; Monteiro et al. 2009; Daemon et al.
Artrópodes Parasitos of the same University. 2012; Senra et al. 2013), and lower thymol concentrations were
also tested to assess possible enhancement of the acaricidal effect
Origin of the ticks caused by the interaction of thymol with the adjuvants in the
formulations. The larval packet test was used for the unengorged
The R. sanguineus s.l. larvae used came from a colony main- phases, as proposed by Stone and Haydock (1962) and adapted
tained by an artificial infestation of rabbits (Oryctolagus by Monteiro et al. (2012). Approximately 100 larvae were placed
cuniculus Linnaeus, 1758—New Zealand x California cross), on sheets of filter paper (6 × 6 cm), which were then folded and
according to the method proposed by Neitz et al. (1971). The sealed with three binder clips. Then each side of the filter paper
engorged female ticks were kept in a climate-controlled cham- envelope was moistened with 90 μL of the formulation to be
ber (27 ± 1 °C and RH 80 ± 10%) for oviposition. After tested, and the envelopes containing the ticks were placed in a
15 days, the eggs were weighed into aliquots of 200 mg, climate-controlled chamber (27 ± 1 °C and RH 80 ± 10%). Each
Parasitol Res

envelope corresponded to a sample unit, and each treatment was of ticks tested. All the tests were performed in clear graduated
repeated ten times. The envelopes were opened after 24 h to count polypropylene tubes, properly sealed. The sensory evaluation
the number of live and dead larvae, using a vacuum pump at- involved judging changes in color, odor, and appearance, after
tached to a hose to collect the live ones. The average mortality subjecting the samples to stress.
was expressed as a percentage, according to the following for- The centrifugation was conducted on 10 mL of each sam-
mula: mortality (%) = (total dead larvae/total of larvae) × 100. ple, at 1509 g for 30 min at room temperature, followed by
The same method was used for the unengorged nymphs, observation of the presence or absence of macroscopic signs
except only five nymphs were placed in each packet. of instability, such as creaming, coalescence, and flocculation.
The engorged larvae were submitted to the immersion test, The pH was measured in with a potentiometer after calibration
as proposed by Drummond et al. (1973). Larvae recovered with buffer solutions at pH 4 and pH 7, on samples diluted
from the artificial infestation of rabbits were separated into 1:10 in recently distilled water, at room temperature
groups of 100 individuals, placed in 10-ml beaker and im- (25 ± 2 °C). Each sample was measured three times, and the
mersed in 5 ml of the formulations for 5 min, with each group results presented correspond to the mean ± standard deviation
corresponding to a treatment. After immersion, the larvae of the three readings. The sensory characteristics were ranked
were dried on paper towels and divided into subgroups of in three categories: normal, without alteration (N); slightly
ten each, which were placed in labeled test tubes and sealed modified (SM); and intensely modified (IM) (Brasil 2004).
with hydrophilic cotton, so that each tube corresponded to a
repetition. Therefore, ten repetitions were performed for each
treatment, with each repetition containing ten engorged larvae.
The mortality rates were assessed after 15 days, during which Results
period the tubes were kept in a climate-controlled chamber at
27 ± 1 °C and RH 80 ± 10%. The average mortality percent- In the tests with the emulsion formulations on unengorged
ages were calculated from the same equation used for the larvae, all the groups showed mortality near 100% starting at
unengorged larvae. the concentration of 0.75 mg/mL, in all cases, significantly
The same technique was used for the tests with engorged different than the control group. For engorged larvae, higher
nymphs, except each experimental unit contained five thymol concentrations were necessary to reach relevant mor-
nymphs, and each treatment was repeated ten times. tality rates, which peaked at 95% for the concentration of
5.0 mg/mL. In turn, in the test with unengorged nymphs,
Data analysis rising mortality rates were observed as the thymol concentra-
tion increased, with rates of 83.3% for the concentration of
The Bioestat version 5.0 software was used for statistical anal- 2.5 mg/mL, rising to 100% with concentration of 20 mg/mL.
ysis. The percent data of mortality were transformed (√arcsin In the test with engorged nymphs, the average mortality was
x) and analyzed by the Kruskal-Wallis and Student-Newman- 86% at the concentration of 5.0 mg/mL (Table 1).
Keuls tests (p < 0.05). For the evaluation of pH averages, In the hydroalcoholic solution tests, the concentration of
Anova and Tukey’s tests were adopted. 2.5 mg/mL caused 88.1% mortality on unengorged larvae
and reached 98.1% at 5.0 mg/mL, and 100% at 10.0 and
Preliminary stability tests 20.0 mg/mL. The activity against engorged larvae was partial,
since the highest concentration tested (20 mg/mL) only caused
The preliminary stability of the formulations was evaluated by 25.0% mortality, a value considered too low for effective tick
accelerated or short-term stability testing (Brasil 2004). In this control (Brasil 1997). In the test with unengorged nymphs,
test routine, the formulations were stored for 15 days under mortality of 91% was achieved at the concentration of
different stress conditions, to accelerate possible instability. 1.0 mg/mL, reaching 100% from 5.0 mg/mL onward, while
The samples were initially evaluated 24 h after preparation, by in the case of the engorged nymphs, the hydroalcoholic solu-
sensory assessment, centrifugation and pH measurement, and tion did not present relevant acaricidal activity, even at the
again after 15 days of stress. The storage conditions were (1) highest concentrations, attaining maximum mortality of only
high temperature (in an oven at 37 ± 2 °C), (2) low temperature 18.3% with the highest concentration (20 mg/mL) (Table 2).
(in a refrigerator at 5 ± 2 °C), (3) room temperature (25 ± 2 °C), When submitting the O/W emulsion and hydroalcoholic
with and without light exposure, and (4) freeze-thaw cycles solution at the concentrations of 2.5 and 5.0 mg/mL to centri-
(24 h at 40 ± 2 °C and 24 h at 4 ± 2 °C, alternately). fugation in the initial stability triage, we noted that the emul-
These tests were only performed on the emulsion and sion at 5 mg/mL was intensely modified, with signs of coa-
hydroalcoholic solution at concentrations of 2.5 and 5.0 mg/ lescence. This sample was, thus, excluded from the subse-
mL, because these were the smallest concentrations that pre- quent stability studies. The O/W emulsion at 2.5 mg/mL,
sented satisfactory acaricidal activity on more than one stage and the hydroalcoholic solution at both concentrations tested
Parasitol Res

Table 1 Mortality rates


(mean ± SD) of immature stages Treatments Unengorged larvae Engorged larvae Unengorged nymphs Engorged nymphs
of Rhipicephalus sanguineus
sensu lato treated with different Control 0.8 ± 1.4a 3.0 ± 4.8ab 4.0 ± 8.4ª 4.0 ± 8.4a
ab
concentrations of thymol 0.5 mg/mL 73.2 ± 11.6 0.0 ± 0.0a 12.5 ± 16.3ab 3.3 ± 10.5a
incorporated in a O/W emulsion 0.75 mg/mL 94.2 ± 9.8bc 5.0 ± 7.1ab 41.7 ± 17.5b 2.0 ± 6.3a
under laboratory conditions
(27 ± 1 °C and RH 80 ± 10%) 1.0 mg/mL 100.0 ± 0.0c 16.0 ± 7.0bc 41.7 ± 15.7b 2.0 ± 6.3a
1.25 mg/mL 99.5 ± 1.3c 29.0 ± 14.5c 40.2 ± 25.2b 0.0 ± 0.0a
2.5 mg/mL 100.0 ± 0.0c 40.0 ± 15.6cd 83.3 ± 22.1c 32.0 ± 23.5b
5.0 mg/mL 100.0 ± 0.0c 95.0 ± 5.3de 86.7 ± 12.4c 86.0 ± 21.2bc
10.0 mg/mL 100.0 ± 0.0c 100.0 ± 0.0e 97.2 ± 8.3c 95.0 ± 9.3bc
20.0 mg/mL 100.0 ± 0.0c 100.0 ± 0.0e 100.0 ± 0.0c 100.0 ± 0.0c

Averages followed by equal letters in the same column do not differ statistically at a significance level of 5%.
Control = emulsion without thymol

did not show alterations after initial centrifugation, so they indiscriminate use and physicochemical characteristics, today
were submitted to the storage challenge tests. the occurrence of selection of resistant populations is becom-
After exposure for 15 days to the different temperature and ing more common. Additionally, these substances cause envi-
lighting regimes, all the samples continued to have stable col- ronmental contamination and intoxication of animals and their
or and characteristic thymol odor. The hydroalcoholic solution owners. Thus, it is necessary to find substances that are effec-
was most stable in all the situations and at both concentrations. tive but have less impact on human and animal health and the
The O/W emulsion at 2.5 mg/mL remained stable at room environment, besides imposing less selective pressure for the
temperature, with and without exposure to light, and when resistance of ticks. Thymol has been studied for several years
kept under continuous refrigeration; but it showed signs of as a bactericide, fungicide, acaricide, and insecticide
instability (creaming) after the final centrifugation in the sam- (Carvalho et al. 2003; Floris et al. 2004; Botelho et al. 2007;
ples maintained in the oven and subjected to the freeze-thaw Vasconcelos et al. 2014), and has been classified as a safe
cycle. The measurement of pH demonstrated a non-significant substance by the US Environmental Protection Agency
variation (p < 0.05) between the initial values (1st day) and (USEPA 1993). One of the reasons for this safety is that it
final ones (15th day) for all the formulations tested, according quickly dissipates in the environment, leaving low residue
to the ANOVA and Tukey test. The results of the sensory levels (Hu and Coats 2008).
analysis, centrifugation, and pH measurement are shown in The results obtained in this study showed a stronger acar-
Table 3. icidal effect of thymol on unengorged larvae in the tests with
the emulsion and hydroalcoholic solution compared to the
results reported by Daemon et al. (2009) and Daemon et al.
Discussion (2012). In the case of the engorged larvae, the emulsion was
more toxic. Indeed, the hydroalcoholic solution did not cause
For many years synthetic substances were used with good relevant mortality rates, reaching levels lower than those ob-
results to control ticks. However, due to their frequent tained when thymol was tested diluted in water + DMSO at

Table 2 Mortality rates


(mean ± SD) of immature stages Treatments Unengorged larvae Engorged larvae Unengorged nymphs Engorged nymphs
of Rhipicephalus sanguineus
sensu lato treated with different Control 0.1 ± 0.3a 7.5 ± 10.4ab 0.0 ± 0.0ª 0.0 ± 0.0a
ab
concentrations of thymol 0.5 mg/mL 3.1 ± 4.7 7.0 ± 8.2ab 4.5 ± 9.6ª 0.0 ± 0.0a
incorporated in a hydroalcoholic 0.75 mg/mL 7.7 ± 4.6bc 12.0 ± 10.3ac 5.0 ± 10.0ª 2.0 ± 6.3a
solution under laboratory
conditions (27 ± 1 °C and RH 1.0 mg/mL 6.5 ± 7.5abc 6.0 ± 8.4ab 91.0 ± 16.6b 0.0 ± 0.0a
80 ± 10%) 1.25 mg/mL 15.2 ± 3.5cd 8.0 ± 7.9a 92.0 ± 10.3b 2.0 ± 6.3a
2.5 mg/mL 88.1 ± 11.9de 0.0 ± 0.0b 96.0 ± 8.4b 0.0 ± 0.0a
5.0 mg/mL 98.1 ± 3.6e 8.0 ± 10.3ab 100.0 ± 0.0b 0.0 ± 0.0a
10.0 mg/mL 100.0 ± 0.0e 11.0 ± 12.9ac 100.0 ± 0.0b 0.0 ± 0.0a
20.0 mg/mL 100.0 ± 0.0e 25.0 ± 17.2c 100.0 ± 0.0b 18.3 ± 24.9ª

Averages followed by equal letters in the same column do not differ statiscally at a significance level of 5%.
Control = hydroalcoholic solution without thymol
Parasitol Res

Table 3 Results of the sensory analysis, centrifugation and pH measurement of the emulsion and hydroalcoholic solution, at concentrations of 2.5 and
5.0 mg/mL, in the accelerated stability test. The pH values are the mean ± SD of three consecutive measurements of each sample. N normal, SM slightly
modified, IM intensely modified

Storage period and conditions Emulsion Hydroalcoholic solution

2.5 mg/mL 5.0 mg/mL 2.5 mg/mL 5.0 mg/mL

Initial (24 h)
Sensory analysis N N N N
pH measurement 6.78 ± 0.05 – 6.12 ± 0.14 6.57 ± 0.03
Centrifugation N IM N N
After 15 days, at room temperature (25 ± 2 °C), with light exposure
Sensory analysis N – N N
pH measurement 6.35 ± 0.15 – 5.75 ± 0.07 6.16 ± 0.10
Centrifugation N – N N
After 15 days, at room temperature (25 ± 2 °C), without light exposure
Sensory analysis N – N N
pH measurement 6.26 ± 0.04 – 5.84 ± 0.08 5.86 ± 0.06
Centrifugation N – N N
After 15 days, in refrigerator (5 ± 2 °C)
Sensory analysis N – N N
pH measurement 6.67 ± 0.21 – 6.04 ± 0.11 5.96 ± 0.10
Centrifugation N – N N
After 15 days, in oven (37 ± 2 °C)
Sensory analysis N – N N
pH measurement 6.65 ± 0.05 – 5.99 ± 0.21 5.91 ± 0.05
Centrifugation SM – N N
After 15 days, under freeze-thaw cycles (40 ± 2 °C/4 ± 2 °C)
Sensory analysis N – N N
pH measurement 6.46 ± 0.10 – 5.80 ± 0.16 6.15 ± 0.03
Centrifugation SM – N N

1% (Daemon et al. 2009). For unengorged nymphs, both the tegument. Although most of this structure defined by
emulsion and solution attained similar mortality rates to those Hackman and Filshie (1982) as a Bheterogeneous, non-
obtained in a previous study using thymol dissolved in ethanol cellular membrane^, is located on the external part of the tick
(Senra et al. 2013). Finally, in the case of the engorged nymphs, body, there is evidence that it extends inside the body at de-
the groups treated with the emulsion suffered higher mortality at termined points of communication with the tracheal system
the concentrations below 5.0 mg/ml, and as of this concentra- and ducts of the dermal glands. Its density is not uniform over
tion the mortality rates were similar to those of the previous the entire surface of the tick, so that there are thinner areas,
study, where water + 1% DMSO was used. The hydroalcoholic mainly found at articulation points, and thicker and harder
solution, in turn, did not reach relevant mortality rates: they areas. The chemical composition of the cuticle is still not
were below those reported by Monteiro et al. (2009). totally resolved, but it is widely assumed to be composed
The variations observed among the different stages tested mainly of lipids, polyphenols, proteins, and chitin (Lees
in this study can be attributed to the anatomic and physiolog- 1947; Hackman and Filshie 1982).
ical difference of Ixodidae (hard ticks) at each stage (larva/ It is believed that the waxes present in the outermost layer
nymph) and phase (engorged/unengorged), and the chemical of the cuticle, called the epicuticle, are secreted by the dermal
properties of the formulation’s components. glands, which are drained by pores to the surface (Sonenshine
Like all arthropods, ticks are covered with a tegument, 1991). This secretion occurs more intensely during and imme-
which besides body coverage and structural strength as the diately after a blood meal or after exposure to light, heat, or
exoskeleton, provide protection against water loss and act as mechanical irritation. This is more evident in nymphs and
a source of reserve energy (Sonenshine 1991; Hackman and females (Lees 1947). Its function has been indicated as pro-
Filshie 1982). The cuticle is the outermost part of the tection against desiccation (Sonenshine 1991).
Parasitol Res

Most water loss of ticks occurs through the cuticle (Lees The results of the tests with engorged larvae showed higher
1947), and the lipid layer present in this structure plays an impor- mortality rates in the groups treated with the emulsion. In the
tant role in regulating this occurrence. This activity appears to case of the hydroalcoholic solution, even at the highest con-
function by means of a physiological control, as demonstrated by centrations tested, the mortality rate was not relevant. In a
the regeneration of the lipid layer of the epicuticle of ticks after previous study investigating thymol dissolved in water +1%
production of artificial injuries by abrasion (Lees 1947) and by DMSO, relevant mortality rates (97%) were only attained
changes in the proportion of lipids during the process of feeding starting at the concentration of 15 mg/mL (Daemon et al.
and ecdysis (Hackman and Filshie 1982). It has been confirmed 2009). The increased toxicity in the treatment with the emul-
that various substances, such as detergents, when applied on the sion on engorged larvae can be related to the use of adjuvants
surface of the cuticle of ticks can increase the transpiration rate, that promote permeation with lipophilic properties, such as
leading to greater water loss (Lees 1947). cetearyl alcohol and grape seed oil. Given the liposolubility
Larvae and nymphs of Ixodidae have been found to be of thymol, we can suggest that the emulsion allowed stronger
more susceptible to water loss than adults (Knüle and interaction of that substance with the cuticle’s fatty layer, fa-
Rudolph 1982). Besides the water loss that occurs through cilitating its entry in the organism, and also keeping it in sur-
the body tegument, additional water is lost by the gas ex- face contact with the tick longer by the possibility of forming a
change process during respiration (Sonenshine 1991). Adult film over the tegument. In the tests of the emulsion on the
hard ticks as well as nymphs have a respiratory system com- engorged nymphs, similar mortality rates were found to those
posed of spiracles and trachea. The larvae, however, do not reported in a previous study using an aqueous solution of 1%
have these structures and perform gas exchange exclusively DMSO as solvent (Monteiro et al. 2009). On the other hand,
through the cuticle (Hackman and Filshie 1982). Nymphs and lower mortality rates were noted in the tests with engorged
adults have a valve mechanism that opens and closes the spi- nymphs and larvae treated with the hydroalcoholic solution.
racles, acting as another physiological control against water To understand this phenomenon, it is important to consider the
loss (Needham and Teel 1991). The control of the opening and higher production of waxes by the dermal glands that happens
closing of the spiracles has been related to the water balance in the engorged phases, as explained previously. It is possible
status of the animal, and according to Sonenshine (1991), that the hydroalcoholic solution generated partial or inade-
engorged ticks are unable to keep the respiratory system quate solubilization of the waxes present on the cuticle’s sur-
closed, causing them to lose water faster. face, making penetration of the thymol less successful. On the
In this study, in both formulations, we used glycerin, a other hand, the interaction of the lipophilic penetration agents
viscous substance with hygroscopic and moisturizing proper- with the thymol and the fatty layer of the cuticle can explain
ties, for the purpose of keeping the formulation from drying the fact the emulsion achieved satisfactory toxicity levels.
out. This component forms a hydrophilic film on the surfaces With respect to the stability studies, the hydroalcoholic
where it is applied (Ribeiro 2006). The mortality rates found solution at concentrations of 2.5 and 5.0 mg/mL remained
in the tests with unengorged larvae suggest a possible delete- stable in all the storage conditions tested. The emulsion at
rious effect caused by glycerin associated with thymol, possi- 2.5 mg/mL, in turn, showed signs of instability (creaming)
bly resulting in increased water loss through the cuticle when subjected to high heat and freeze-thaw cycles.
(Ravindran et al. 2011), or even the formation of a film caus- However, according to Brasil (2004), small alterations in these
ing occlusion of the channels responsible for gas exchange, conditions are acceptable, frequent, and even expected.
since as mentioned above, at this stage, ticks do not have a Besides this, there was no significant variation in the pH
tracheal respiratory system. This effect was not observed in values which are suitable for application in the skin of dogs,
the unengorged nymphs, and the mortality rates were similar considering that the average pH of the skin of a healthy dog
to those recorded in a previous study, where a hydroalcoholic varies between 5.86 and 6.45, with mean of 6.16 (Laurel,
solution of 50% ethanol as the solvent was used (Senra et al. 2005). The emulsion at 5.0 mg/mL showed changes after the
2013). Since nymphs do not depend exclusively on the cuticle initial centrifuging, with signs of coalescence, so it was not
for gas exchange and absorption of water (because they have a tested under other conditions. Because this study is ground-
tracheal system), the occlusive effects of the formulations on breaking regarding the incorporation of thymol as an acaricide
the tegument might have been counterbalanced by its respira- in formulations for topical use, the data presented here have
tory activity, so that the deleterious effect of the adjuvants considerable value as support for new studies.
associated with thymol in the two formulations was attenuat- To better understand the action of the formulations tested
ed. However, since no significant mortality occurred in the here, it will be necessary to conduct further studies to deter-
control groups, the mere obstructive action of glycerin does mine the exact mechanism by which thymol acts, as well as
not explain the enhanced toxic effect of thymol. Thus, other the action of the moisturizing components used.
pharmacological and/or physicochemical hypotheses should Accompanying investigations to adjust proportions of the ad-
be investigated to clarify this enhancement. juvants, and possibly the addition of other excipients, can
Parasitol Res

make the proposed formulations more stable, favoring a study Barros-Battesti DM, Arzua M, Bechara GH (eds) Carrapatos de
importância médico veterinária da região neotropical: um guia
in the near future of their acaricidal effect in vivo, thus, con-
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