Amitraz is an acaricide that is widely used in veterinary medicine to control the cattle tick Rhipicephalus microplus. However, controversy exists in the literature regarding the resistance of R. microplus to this product. The present work provides an update on the acaricidal efficacy of amitraz (Triatox®, 12.5 % amitraz) after 15 years without its use on a property. Two in vivo (bovines treated with amitraz and submitted to tick counts, n = 20 animals) and one in vitro (adult immersion test, n = 40 ticks) assays were performed to determine product efficacy. The efficacy of the commercial formulation tested in the first in vivo trial ranged from 14.1 to 47.0%, and in the second from 3.6 to 35.1%, for the 28 days of the experiments. Efficacy for the in vitro trial was 47.38%. The dose recommended by the manufacturer of the product did not cause mortality to most of the ticks of this strain, and efficacy/resistance was not reverted or modified after 15 years (estimated 60 tick generations).
Amitraz belongs to the formamidine class of chemicals, which contains compounds widely used in veterinary medicine and agriculture to control arthropods. Rhipicephalus microplus is the most important livestock tick worldwide and amitraz has been used to control it for more than 40 years. Tick resistance—defined as the ability of ticks to tolerate the indicated dose of an acaricide that would cause mortality to the majority of individuals (FAO 2004)—to amitraz has been reported in a variety of tropical countries, including Australia, South Africa, and countries in Central and South America (i.e., Mexico, Brazil) (Fernández-Salas et al. 2012; Jonsson et al. 2000; Rodriguez-Vivas et al. 2006).
Most acaricides have neurotoxic action, including amitraz, which is an octopamine agonist that causes hyperstimulation of octopaminergic synapses resulting in tremors and seizures (Jonsson et al. 2018). Ticks possess a mixture of resistance mechanisms for each class of acaricide and can acquire resistance metabolically or target site insensitivity. In the former, a tick produces enzymes (esterases, glutathione-S-transferases, and cytochrome P450 monooxygenases) that promote faster detoxification of the acaricide. In the latter, a nucleotide mutation occurs in the coding region of a gene causing a structural change in the protein of the acaricide receptor, thereby reducing or blocking the ability of the acaricide to bind the receptor (Kumar 2019). For R. microplus, however, the mechanism conferring resistance to amitraz has not been definitively identified (Jonsson et al. 2018).
Resistance of R. microplus to the majority of chemical classes available as acaricides has shown to be irreversible; however, some researchers have affirmed the possibility of resistance reversion for the amidine class after 15 to 20 tick generations without contact with products of this group (Foil et al. 2004; Jonsson et al. 2010). On the other hand, Maciel et al. (2015) found that amitraz efficacy/resistance was not reverted or modified in R. microplus after a period of ten years (approximately 40 tick generations). Knowledge of tick susceptibility to acaricides using resistance diagnosis by bioassays and biochemical and molecular tools will help to apply effective tick control programs (Kumar 2019).
In an attempt to update the information provided by Maciel et al. (2015), as mentioned above, in vivo and in vitro acaricidal efficacies of amitraz against the same tick strain of R. microplus were evaluated after 15 years (about 60 tick generations) without contact.
Materials and methods
Site and history of chemical use against R. microplus
This study was performed in January and April 2020 at the same farm of the study of Maciel et al. (2015), who evaluated the acaricidal efficacy of 12.5% amitraz spray against a R. microplus population that had not had any contact with chemical products of this acaricide family for 10 years. The R. microplus populations of Brazil are known to belong clade A (Burger et al. 2014). The ticks of the study region have been estimated to pass through five generations per year due to climatic conditions (Cruz et al. 2020). The individuals found parasitizing bovines at the study farm showed the morphological characteristics described by Nava et al. (Nava et al. 2017), namely, oval body outline, rounded scapulae, and shallow and barely perceptible cervical grooves. Males also presented adanal and accessory adanal shields and a narrow caudal appendage, while females also showed a broad “U”-shaped genital aperture, coxa I with distinct, short internal and external spurs and the remainder of the coxae with indistinct spurs.
A bovine herd (450 animals, Simmental breed) was maintained in nine paddocks with Coast cross grass, each of which was occupied and grazed continuously without any paddocks going animal-free during the entire year. It is important to highlight that no additional animals had been purchased since 1998 when the herd was established, and bovines from neighborhood properties did not have access to the grazing area. The history of chemical use against R. microplus on the farm included treating animals with amitraz (Triatox®, MSD Animal Health) and cypermethrin (Barrage®, Zoetis Animal Health) between January 2002 and December 2004, and different chemical products based on phenylpyrazole, macrocyclic lactones, and benzoylphenyl ureas (fluazuron) between 2005 and 2015. No products of the amidine class were administered in any of the acaricidal treatments performed on the animals between the years 2005 and 2020, except in 2015 when only 10 animals were treated in the study of Maciel et al. (2015).
In vivo evaluation of amitraz efficacy against R. microplus naturally infesting bovines
The experiment was approved by the Ethics Committee on the Use of Animals (#114/17) of the Federal University of Goiás (UFG). Two different trials were conducted in different periods to evaluate the effects of 12.5% amitraz (Triatox®, MSD Animal Health) against R. microplus naturally infesting cattle. The first trial occurred in January 2020 and the second in April 2020. Both trials involved a treatment group and a control group of 10 animals each. The selected bovines (12- to 13-month-old Simmental females) had no contact with any antiparasitic drugs for a minimum of 60 days prior to the beginning of the trials. Animals were randomly designated to treatment or control groups according to a randomized block design. The block formation was based on the arithmetic mean of counts on three consecutive days (–3, –2, and –1) of R. microplus females (between 4.5 and 8.0 mm in length), as described by Wharton and Utech (Wharton and Utech 1970).
The treatment group received the commercial spray formulation, diluted as recommended by the manufacture, once on day 0, while the control group did not receive treatment with acaricide and was only sprayed with water. Each animal received 5 l of the diluted formulation (treatment group) or water (control group). It is important to note that cattle treated with spray formulation did not experience any interference by rain during the first 96 h post-treatment. Animals of the two groups were kept in separate paddocks during the first 3 days post-treatment to prevent any possibility of contaminating the untreated control group, a methodology recommended by Maciel et al. (2016). Both trials (January 2020 and April 2020) were conducted in the same paddocks. Tick counts (Wharton and Utech 1970) were performed on days 3, 7, 14, 21, and 28 post-treatment to determine the therapeutic and residual efficacies obtained by the tested formulation.
The arithmetic mean of the three tick counts was used to calculate efficacy as a percentage, following the formula used by Maciel et al. (2015):
where “Ta” is the number of fully or partially engorged female ticks counted on the treated animals after medication; “Tb” is the number of ticks counted on the treated animals on the three days preceding treatment; “Ca” is the number of fully or partially engorged female ticks counted on untreated control animals after the treatment date; and “Cb” is the number of ticks counted on untreated control animals on the three days preceding treatment.
Tick counts were log10 (count + 1) transformed and analyzed using a mixed linear model. The statistical model included block, animal, and residual as random effects. Treatment, time, and treatment-by-time interaction as fixed effects. All data were analyzed with the aid of SAS V9.4 with a significance level of 5% (P < 0.05).
In vitro evaluation of amitraz efficacy against R. microplus by the adult immersion test
An adult immersion test (AIT) was performed to evaluate in vitro sensitivity of the same R. microplus strain as used in the in vivo trials. One trial was performed in January 2020 using the same commercial formulation used in the in vivo trials and following the methodology of Drummond et al. (Drummond et al. 1973). Approximately 80 fully engorged females of R. microplus were collected from bovines that did not participate in the in vivo trials but lived on the farm. A treatment group and a control group were established with two replicates with 20 ticks for each group. Female reproductive parameters (female weight, egg mass weight), larval hatchability, oviposition reduction, hatchability reduction, and reproductive efficiency were recorded, and efficacy calculated (Drummond et al. 1973; Cruz et al. 2014).
Percentage hatchability was visually estimated using a stereomicroscope with an ocular grid to compare the proportion of larvae in relation to the proportion of whole eggs for each group. The following equations were used to assess the impact of the treatment on oviposition and hatching reduction of females: % oviposition reduction = A − B/A × 100, where A is the average egg mass of the control group and B is the average egg mass of the treated group; % hatching reduction = A − B/A × 100, where A is the average hatchability of the control group and B is the average hatchability of the treated group. The following equations were used to estimate reproductive efficiency and percentage efficacy: reproductive efficiency (ER) = A/B × C, where A is the egg weight (g), B is the female weight (g), and C is the X% hatching × 20,000, with 20,000 being a constant corresponding to an estimate of the number of R. microplus larvae contained in 1 g of eggs (Labruna et al. 1997).
Results and discussion
This work provides an update regarding amitraz efficacy/resistance involving a R. microplus strain on a property located in a tropical region that did not use the chemical compound for over 15 years. The study found that the dose recommended by the manufacturer of the product failed to cause mortality to most of the ticks of this strain, and the efficacy/resistance situation was not reverted or modified.
The results of the first in vivo trial, performed in January 2020, for tick counts and efficacy of 12.5% amitraz are shown in Table 1. Tick counts for the treated group were lower (P ≤ 0.05) than those for the control group on days 3, 7, 14, and 21 post-treatment. The efficacy of the tested commercial formulation ranged from 14.1 to 47.0% on days 14 and 28 post-treatment, respectively. The results of the second in vivo trial, performed in April 2020, for tick counts and efficacy of 12.5% amitraz are shown in Table 2. Tick counts for the treated group were lower (P ≤ 0.05) than those for the control on days 3, 14, and 21 post-treatment. The efficacy of the tested commercial formulation ranged from 3.6 to 35.1% on days 14 and 28 post-treatment, respectively. Results of reproductive parameters, larval hatchability, reduction of oviposition, reduction of hatchability, and reproductive efficiency of engorged R. microplus females from the treatment and control groups are shown in Table 3. The efficacy of 12.5% amitraz in the in vitro assay was 47.4%.
The in vivo (field) and in vitro (laboratory) trials performed in the current study were suitable for evaluating efficacy/resistance, as mentioned by the guidelines of the Food and Agriculture Organization (FAO 2004). The higher efficacy obtained for 12.5% amitraz in the in vivo trials against R. microplus was similar to the efficacy obtained for AIT (in vitro), with both being close to 47%. The efficacy found for AIT for the same compound and the same tick strain was 70% in 2005 and 62% in 2015. The mean efficacy for the in vivo test on day 7 and day 14 post-treatment was 38.8% in the first trial and 25.9% in the second trial. The same in vivo test obtained a mean efficacy of 73.8% in 2005 and 55.2% for day 7 and day 14 post-treatment in 2015 (Maciel et al. 2015). All efficacies against the evaluated tick strain obtained in the present study were considered unsatisfactory, since they were below 90% (emea 2004) and 95% (Brazil 1997; Holdsworth et al. 2006). Thus, these results suggest that there exists resistance by this tick strain to the tested commercial formulation.
It is important to highlight that a decline in efficacy was diagnosed by the in vitro and in vivo trials despite amitraz not being used on infested animals on the property for 15 years. There is divergence in the literature with regard to amitraz resistance in different countries after 15–20 tick generations (Fernández-Salas et al. 2012; Jonsson et al. 2000; Foil et al. 2004; Maciel et al. 2015; Rodríguez-Vivas et al. 2006). Some hypotheses of why amitraz resistance is not reversed and the efficacy of the product is diminished, as found in the present work, include the detoxification capacity of the tick for the acaricide, redefinition of the taxon R. microplus, risk factors for the occurrence of resistance phenomena, and changes in the octopamine receptor of the tick strain.
Due to treatments with other chemicals (pyrethroids, phenylpyrazole, macrocyclic lactones and benzoylphenyl ureas) during the 15-year period, it is likely that the tick strain was exposed to different selection pressures that could alter or otherwise interfere with the proportions of the population of R. microplus that are susceptible and resistant to chemicals of other groups, such as the formamidine amitraz. It may be that this tick strain developed a metabolic resistance to amitraz, making it efficient at detoxifying or eliminating this acaricide. Even if the resistance of amitraz remains without a specific explanation, it is probable that detoxification plays a role in tick resistance to amitraz, as occurs with synthetic pyrethroids (Jonsson et al. 2018).
The mode of application of the formulation of amitraz, as well as cattle movements (purchase) and selection pressure (number of acaricide applications), could be risk factors for amitraz resistance. In addition, the different methods for diagnosing resistance used by various studies are another factor that should be considered with regard to divergent reports of resistance (Foil et al. 2004; Jonsson and Hope 2007). However, for the property of the current study, no animals were purchased since the 1998. Even so, the tick R. microplus was redefined taxonomically such that it now corresponds to a complex with different taxa (Burger et al. 2014; Estrada-Peña et al. 2012; Labruna et al. 2009). Australian ticks differ from American amitraz-resistant strains of R. microplus, given that the ticks from Australia are considered different from those of the Americas (i.e., Texas, Mexico, and Brazil) (Fernández-Salas et al. 2012; Jonsson et al. 2000; Maciel et al. 2015; Rodríguez-Vivas et al. 2006). All these issues could explain the differences found in the literature with regard to amitraz resistance.
Amitraz is known to act on the octopamine receptor, and in arthropods, octopamine has a wide range of functions, such as a neurotransmitter (neuromuscular transmission), as a neurohormone (hemocyte recruitment and phagocytic), and in lipid mobilization during extended motor activity (Farooqui 2012). Amitraz-resistant ticks from the USA, Australia, Latin America, and South Africa possess mutations in the octopamine/tyramine receptor gene (Jonsson et al. 2018; Baron et al. 2015; Baxter and Barker 1999; Chen et al. 2007; Corley et al. 2013), which could explain the different resistance responses in countries that have already reported R. microplus resistance to amitraz. However, by comparing data from 2005 and 2015 for the same R. microplus strain, the present study makes it clear that the efficacy/resistance situation against 12.5% amitraz was not reverted or modified during 15 years without amitraz use. Nonetheless, a molecular evaluation of the ability of ticks to maintain resistance mechanisms against amitraz is required to clearly determine if amitraz resistance was not indeed reverted or modified. Since the process responsible for this amitraz resistance remains unknown, new studies are needed to help in the effective management of resistance and the development of a successful tick control program.
Amitraz did not cause mortality to most of the ticks of the R. microplus strain evaluated in the present study, with its efficacy being below 50% in all trials. Thus, amitraz resistance was not reverted or modified after 15 years without its use.
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Cavalcante, A.S.d., Ferreira, L.L., Couto, L.F.M. et al. An update on amitraz efficacy against Rhipicephalus microplus after 15 years of disuse. Parasitol Res 120, 1103–1108 (2021). https://doi.org/10.1007/s00436-021-07063-5
- Adult immersion test
- Natural infestation
- Tick resistance