Ticks are obligate hematophagous ectoparasites [1] and are the most important vectors of pathogens within the phylum Arthropoda [2] . Worldwide, there are 84 species [3] , with Rhipicephalus sanguineus, also known as the brown dog tick, being the one with the greatest distribution [2] . In Mexico, R. sanguineus is one of the most important species and has been identified in Cuernavaca, Morelos, (central part of Mexico), Mexicali, Baja California and Culiacán, Sinaloa (northwest part of Mexico), among others [1] . In addition, Rhipicephalus sanguineus has been often found in other hosts, including humans [1] [2] . It is a vector of different pathogens, such as Ehrlichia canis, E. chaffensis, Babesia canis, B. vogeli, Rickettsia rickettsii, R. conorii, Coxiella burnetti and Hepatozoon canis, some of them of zoonotic importance [1] - [3] .

The control of ticks is based mainly on chemical treatment, through synthetic acaricides applied by spraying, in baths or spray, and in subcutaneous injections.

Some of these chemicals also have a persistent effect lasting for several weeks after their initial application [4] , while others are effective for several months [5] .

On the other hand, permethrin is an insecticide belonging to the group of pyrethroids, which act at the level of the insect’s nervous system, producing a series of disordered nervous impulses, accelerating tissue degeneration, and inhibiting their feeding process [6] . It is of low toxicity for mammals since it is excreted quickly after oral or topical exposure [7] .

Various studies have verified the effectiveness of permethrin against the brown dog tick in different presentations and concentrations, reaching from 60.8% to 100% efficacy, as a single treatment or in combination with other acaricides [3] [4] [8] [9] .

Another control strategy has been the use of ixodicides with synergists, which are not considered toxic by themselves [10] ; intervene on different enzymes in order to potentiate the effect of ixodicides, such as piperonyl butoxide [10] [11] , which acts at the microsomal level as inhibitor of cytochrome P450 monooxygenases [11] , causing the inhibition of the oxidative functions present in the metabolism of pyrethroids [12] [13] .

The aim of the present study was to compare the efficacy of a single treatment of 1% permethrin applied as a shampoo-bath, versus a 2% permethrin combined formulation of 1% piperonyl butoxide applied as powder, against Rhipicephalus sanguineus ticks in naturally infested dogs.

A summary of the tick counts and efficacy results are shown in Table 1 and Table 2 .

Note: *Day of treatment.

Note: ^a,bDifferent letters in the same column represent statistically significant differences (p < 0.05) between average effectiveness.

Group 1 (permethrin applied as shampoo-bath) showed a remarkable reduction of ticks, observing that of 320 specimens on the group counted at day 0, this number decreased to 103 on day 1 and to 37 ticks on day 7 after treatment. By day 14 after initial of the study, the number of ticks observed increased to 57, indicating that the initial effect of the compound has worn of Table 1 .

In terms of efficacy, the percentage obtained for this group was 81.0% for day 1 and 93.1% for day 7. For day 14 after treatment, the efficacy conferred was reduced to 89.6%, suggesting that tick-killing activity of shampoo had decreased ( Table 2 ).

Group 2 (2% permethrin + 1% piperonyl butoxide applied as powder) showed a drastic decrease in ticks, where of 164 specimens observed at day 0, this count was reduced to only 6 ticks. On day 7 after treatment, the specimens observed were 12. On day 14 after treatment, the number of ticks increased to 16, indicating that in the same way, the effect of the combined application of permethrin + piperonyl butoxide was already declining ( Table 1 ).

The efficacy conferred for this group was 98.8%, 97.7% and 97.0%, for days 1, 7 and 14, respectively ( Table 2 ).

In relation to Group 3 (non-treated control), the tick count was always high, recording a total number of 543, 154, 139 and 147, for days 0, 1, 7 and 14 after treatment, respectively. Here, the tick counts recorded throughout the study on the untreated dogs, confirms that the study complies with the guideline recommendations suggested by [14] .

Forty-five days after the treatment, all dogs were showing increasing numbers of ticks, since the study area is highly prevalent in these arthropods.

It is important to note that no adverse health effects related to the treatment occurred during the study.

The statistical analysis indicated that significant differences were found between the experimental groups (G1 - G2) in the count of ticks retrieved for days 1 and 14, but not for day 7 (p < 0.05); regarding control group (G3), significant differences were found between it and G2 for days 1, 7 and 14, and between G3 and G1 only on day 7 (p < 0.05).

Several chemicals, or combinations of chemicals, with acaricidal or insecticidal properties and which are appropriate and safe for treatment of domestic dogs and cats, have been formulated for application either orally, parentally, topically, or as medicated collars [16] [17] .

Permethrin has been a widely used acaricide for control and prevention of parasites in both dogs and large animals [6] [10] [18] . In dogs, it is most administrated as topical treatment via spot-on, although it has also been formulated as collars. The active ingredient is distributed through the coat from the application site and along stratum corneum, however, studies suggest that the most distal parts may not receive the same concentration of the product as those closest application site [19] .

In the present study, two products applied topically were compared to review the tick-killing efficacy of permethrin alone at 1%, applied as shampoo-bath and the combination of 2% permethrin with 1% piperonyl butoxide powder. In the case of these two presentations, they allow a more uniform distribution since they are applied throughout the animal’s body and not in a single place, which could ensure adequate drug concentrations in the sites where ticks commonly lodge, such as interdigital spaces, armpits, groin and ears.

The results obtained demonstrated a greater efficacy with the combination of permethrin and piperonyl butoxide at day 1 (98.8%), although for days 7 and 14, the effect was in slight decline (97.7% and 97%, respectively), the efficacy remained above 90%, unlike 1% permethrin applied alone, which on day 7 obtained an efficacy of 99.8%, however by day 14 it decreased to 89.66%.

These percentages coincide with diverse studies in which the effectiveness of permethrin in different combinations has been analyzed, with the efficacy and residual effect of imidacloprid + permethrin being one of the most studied to measure. Epe et al. (2003) [20] , found that this combination offered a preventive efficacy of 94% at day 9 post-application as spot-on and remained the efficacy above 90% against R. sanguineus for a period of five weeks. Otranto et al. (2005) [21] , also recorded that this combination got an efficacy above 95% against ticks on naturally infested dogs from day 14 to final day of observation period (56 days). In another study of [22] , compared three commercial products applied as spot-on, two of them including permethrin as an active ingredient (imidacloprid/permethrin, IP and dinotefuran/pyriproxyfen/permethrin, DPP) which achieved an efficacy of 99.6% on day 9 post-infestation, reducing this percentage till day 29 to 96.7% for the DPP treated group and to 91.7% for the IP treated group.

Following the same line, the combination dinotefuran/pyriproxyfen/permethrin was challenged by [23] , where the immediate efficacy was 79.9% and the persistence of efficacy above 90% sustained for three weeks.

Other combinations have been examined, such as methoprene/permethrin, which achieved an efficacy greater than 90% at four weeks and with a residual effect that remained for 28 days [8] , and fipronil/permethrin [24] , where the acaricidal effect started at 4 h after treatment with a percentage of ±94.7% from day 2 to day 21 postexposure.

On the other hand, the results of the present study suggest the synergistic action of piperonyl butoxide, coinciding with previous studies [25] , where it was demonstrated that the combination of amitraz and piperonyl butoxide acts additively against Boophilus microplus ticks. In another more recent study [26] , it was shown that the enzymatic activity of oxidases as a mechanism of resistance to ixodicides when verifying the synergistic effect of piperonyl butoxide with cypermethrin, generating the inhibition of this activity in R. sanguineus larvae.

It is worth highlighting the importance of maintaining high percentages of efficacy for a long period; however, it is also necessary to consider a short latency period, to reduce the risk of disease transmission by vectors. Horak et al. (2012) [23] , mentioned that tick burdens exceeding 10 individuals after treatment are quite adequate for the transmission or acquisition of organisms responsible for tick-borne diseases in the field.

A reduction in parasite numbers will inevitably influence the prevalence of the diseases that they transmit. It is also possible that chemicals that have a repellent, or a particularly rapid killing effect, could eliminate ticks before they can transmit vector-borne organisms with which they may be infected. For instance, R. sanguineus is a vector of Babesia canis, Babesia vogeli, Babesia gibsoni, Hepatozoon canis and Ehrlichia canis, the causative organisms of tick-borne diseases that affect dogs in different regions of the world [27] .

Permethrin has been compared with other ixodicides such as fipronil, fluralaner, afoxolaner or sarolaner to determine the time of onset of action; here [28] found this beginning of activity two hours after spot-on application with an effectiveness of 36.9%, which slowly increased in the following 48 hours to 80.1%, in this study, permethrin in combination with imidacloprid failed to reach the minimum percentage to be considered the first option, since it is also mentioned, in general terms, that the time necessary for the transmission of pathogens through an infected tick can be 24 - 48 hours after feeding. The same product was analyzed by [29] , in a study to verify its effectiveness against the transmission of Ehrlichia canis by R. sanguineus in dogs, resulting in 100% blocking of transmission.

Although the latency period or effectiveness against disease transmission was not analyzed in the present study, the results obtained on day 1 post-treatment in Group 2 suggest a high probability of disease prevention, since a total of 6 live ticks were counted (98.8% of efficacy), which means a lower risk of infection.

Several chemicals, or combinations of chemicals, have been formulated for application either orally, parentally, topically, or as medicated collars.

Since pet owners do not comply with the recommended time periods between the administrations of these tick remedies, serious gaps in efficacy may occur. However, in some populations, the indiscriminate use of this type of control has generated resistance to certain active ingredients, as well as it can cause a negative environmental impact, potentially affecting the natural insect fauna [26] [30] .

Besides, it is expected that dogs are inevitably going to be washed or shampooed, or swim or go out into the rain while being walked by their owners or working in the field. Therefore, the activity of any treatment applied may lead to a reduction of activity of the drug used.

Under the conditions in which the present study was carried out, it was observed that the combination of 2% permethrin + 1% piperonyl butoxide produces additional efficacy that benefits the treatment of R. sanguineus infections in naturally infested dogs.

It is worth highlighting the importance of always maintaining assertive and effective communication between veterinarians and pet guardians, as well as considering the rational use of chemical products to ensure the correct use of the ectoparasiticide, reducing the gaps in efficacy and the negative effects that could be caused by mishandling.

A combination of 2% permethrin + 1% piperonyl butoxide applied as a single powder treatment was highly effective at rapidly repelling and killing Riphicephalus sanguineus ticks on naturally infected dogs, showing a significant effect on days 1, 7 and 14 after tick exposure.

The sustained high and quick level of efficacy of this combination may well interfere with the transmission of Riphicephalus sanguineus ticks.

The authors are indebted to Laboratorios Salud Animal, S.A de C.V. for kind financial support.

All datasets are included in this manuscript.

The study was funded by Laboratorios Salud Animal, S.A de C.V., Mexico.

AMA contributed to the study design, carried out the field study, ticks counting and results interpretation. FIV contributed to the study design, carried out the field study, ticks counting, results interpretation, manuscript revision and discussion. JAM collaborated with the field study, ticks counting and results interpretation and provided financial support. RA-G carried out the statistical analysis. All authors read and approved the final version of the manuscript.

The work was carried out adhering to the guidelines of the Institutional Committee for Use and Care of Experimental Animals of the institution, according to the Mexican Official Regulation NOM-062-ZOO-1999 and Animal Research: Reporting of in Vivo Experiments guidelines followed.