Cross-resistance between macrocyclic lactones in populations of Rhipicephalus microplus in Brazil’s semiarid region

doi:10.21203/rs.3.rs-1506887/v1

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Cross-resistance between macrocyclic lactones in populations of Rhipicephalus microplus in Brazil’s semiarid region

https://doi.org/10.21203/rs.3.rs-1506887/v1

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Rhipicephalus microplus, also known as the cattle tick, is the parasite with the greatest impact on cattle in Brazil. The most common method for controlling this tick is the application of synthetic chemical acaricides, especially ivermectin, which belongs to the group of macrocyclic lactones (MLs). However, because ivermectin is widely used, there is concern about the development of cross-resistance within this chemical class. Thus, engorged females were collected from farms with a history of resistance to ivermectin, which was the only one among the MLs that was used as an anthelmintic drug. Using the larval immersion test (LIT) technique, bioassays were performed with ivermectin, moxidectin and eprinomectin on populations of R. microplus from the semiarid region of the states of Paraíba and Ceará. Epidemiological questionnaires were applied to collect information about tick control management. All the evaluated populations showed cross-resistance between ivermectin and moxidectin, but only one population showed cross-resistance between ivermectin and eprinomectin. Weekly or monthly administration of injectable 1% ivermectin on farms was reported. It was concluded that the frequent use of ivermectin may lead to the development of cross-resistance to moxidectin. For eprinomectin, despite the structural similarity, cross-resistance was not observed in three tick populations.

acaricides

cattle tick

eprinomectin

ivermectin

moxidectin

Rhipicephalus microplus infests cattle in tropical and subtropical regions. Larva, nymphs and adults attach to and develop in a single host, characterizing its life cycle as monoxenous (Taylor, 2019). Although this tick is present throughout the year in Brazil, it reaches its peak in the semiarid region of the country’s northeast during the rainy season, which tends to occur from January to May or June (Barros et al., 2017). R. microplus is the parasite that has the greatest impact on cattle, causing the transmission of several pathogens, including Babesia spp. and Anaplasma sp. (Costa et al., 2013). That is why the effective control of this ectoparasite is of utmost concern.

R. microplus populations have developed resistance to all acaricides available on the market (Rodriguez-Vivas et al., 2014; Reck et al., 2014; Villar et al., 2020; Vilela et al., 2020; Valsoni et al., 2020; 2021). The excessive use of acaricides in the absence of knowledge about the life cycle of the arthropod, allied to failures in its detection and mismanagement in animal husbandry, leads to the development of resistance to almost all classes of veterinary drugs on the market, and the more widely these acaricides are applied, the higher the percentage of drug resistance (Kumar et al., 2020; Villareal et al., 2021).

Since its discovery in 1980 (Chabala et al., 1980), ivermectin has been one of the most commonly used drugs in the treatment of R. microplus infestations. Ivermectin belongs to the group of macrocyclic lactones (MLs), which is composed of avermectins (ivermectin, eprinomectin and doramectin) and milbemycins (moxidectin). The substances naturally produced by Streptomyces avermitilis have been identified as A1a, A1b, A2a, A2b, B1a, B1b, B2a and B2b, with A2a, B1a and B2a being the main products of its fermentation (Brossi, 2018). Avermectin B1 (abamectin) is important because other avermectins are produced from it. Ivermectin (22,23-Dihydroavermectin B1a) is obtained via the hydrogenation of carbon atoms C-22 and C-23 of abamectin, while eprinomectin (400-epi-acetylamino-400-deoxyavermectin B1) is obtained through the addition of the acetylamino group at C-4 (Danaher et al., 2006). Hibbs and Gouaux (2011), as well as Prichard et al. (2012), stated that the composition of ivermectin and moxidectin differs because fermentation occurs through different organisms and they have dissimilar chemical structures. The main structural difference is that avermectins have sugar groups at carbon 13(C13) of the macrocyclic ring, whereas moxidectin has protons at C13. In addition, moxidectin has other differences, including a methoxime at C23 that can prevent H binding to an M3 loop in the act of drug binding to the receptor, which can cause some molecular shift of this loop. Moreover, the absence of the disaccharide substituent should result in moxidectin lacking two van der Waals binding sites (for M2-M3 loops) for the glutamate (GluCl)-controlled Chlorine Channel of the ectoparasite, thereby possibly causing interactive changes through the difference in methyl to ethyl binding in this position, affecting its power.

MLs act as an analogue of the neurotransmitter γ-aminobutyric acid (GABA) and in GluCl. By binding to chlorine channels, they cause the uninterrupted influx of Cl- ions into neurons at the neuromuscular junction, blocking neurotransmission. Thus, they paralyze the tick’s somatic and pharyngeal muscles (Omura, 2008; Klafke, 2011).

The first report of resistance to ivermectin in the tick R. microplus (São Gabriel strain) in Brazil was described by Martins and Furlong (2001). Since then, several studies have shown the occurrence of this resistance in different locations (Klafke et al., 2006; 2012; Andreotti, 2010; Lopes et al., 2013; Vilela et al., 2020). In addition, Martins and Furlong (2001) reported cross-resistance between doramectin, moxidectin and ivermectin in studies carried out with the São Gabriel strain. However, the existence of cross-resistance to eprinomectin and moxidectin has not yet been reported in field populations, although ticks showed susceptibility in laboratory experiments (Nazir et al., 2013; Nascimento et al., 2020). However, in studies with moxidectin conducted by Lovis et al. (2013), the population of R. microplus on a farm in Argentina proved to be resistant. Nevertheless, the author points out the weakness of her results, as there were no repetitions in the trials. In Brazil, these two compounds are used only occasionally, and the use of ivermectin is more widespread.

Due to the drug resistance of R. microplus, particularly against ivermectin, there is a concern about the possibility of cross-resistance between MLs, given their similar molecular structures and mechanisms of action. Therefore, this work aimed to evaluate the existence of cross-resistance between MLs in R. microplus populations in the semiarid region of Northeast Brazil, based on in vitro larval bioassays.

Collection of samples and location of experiments

Samples were collected at four cattle farms in Brazil’s northeast semiarid region, covering the municipalities of São João do Rio do Peixe – PB (Farm 1), Sousa – PB (Farm 2), Barro – CE (Farm 3) and Várzea Alegre – CE (Farm 4) (Fig. 1). The farms were selected based on their history of use and anthelmintic resistance to ivermectin.

The experimental tests were carried out at the Veterinary Parasitology Laboratory of the Federal Institute of Paraíba – IFPB at the city of Sousa.

Characterization of the farms

In order to build a profile of the farms, a questionnaire was applied containing the following items: bovine population, breed of animals, production system (intensive, extensive or semi-intensive), main purpose (dairy or beef cattle), and size of grazing area. For the management profile for tick control, the following were considered: acaricides applied in the last two years, acaricide applied in the last treatment before ticks were collected, interval between treatments, performance of treatments according to the manufacturers’ recommendations, application method (spray, injectable or pour-on formulations), and influence for the choice of a specific acaricide: recommended by a veterinary doctor, veterinary pharmaceutical sales representatives, by neighbors/cooperatives, or determined by product price.

Ticks

The isolate Porto Alegre (POA) was used as a reference susceptible strain. Since it was first isolated, the POA strain has been widely used as a susceptible reference strain (Reck et al., 2014; Vilela et al., 2020).

Approximately 100 engorged females were collected at each farm from cattle that had been treated with topical acaricide at least 30 days earlier, or 45 days after treatment with injectable acaricide, to ensure the absence of interference in the results. Ticks were removed directly from infested cattle and placed in plastic containers (105 mm high, 80 mm in diameter) with holes in the lid to allow the passage of air. The containers were kept in a polystyrene box until their arrival at the Veterinary Parasitology Laboratory (LPV) of the Federal Institute of Paraíba (IFPB) at Sousa.

Ticks were processed at the aforementioned laboratory of IFPB, as specified by FAO (2004). The ticks were washed in distilled water, dried on paper towels, placed on plastic Petri dishes (90 mm in diameter × 22 mm in height) and incubated in a BOD (Bio-Oxygen Demand) chamber in the dark, at temperatures between 27 and 28°C, with relative humidity between 85 and 90%. The female ticks were left in the BOD chamber for two weeks, giving them time to lay eggs. Egg masses (250 mg aliquots) were then placed in glass vials (10 mL), which were closed with a cotton lid, allowing air and moisture to pass through. To enable the larvae to hatch, the eggs were incubated under the same conditions as the engorged females.

Acaricides

Technical grade ivermectin, eprinomectin and moxidectin (Sigma Chemical Co., St. Louis, MO, USA) were used.

Larval immersion test (LIT)

Acaricides were diluted in 2% Triton X-100 solution in absolute ethanol (ETH-TX2%) in the case of eprinomectin and 2% Triton X-100 with pure acetone (ACTX-2%) in that of ivermectin and moxidectin dilution. The solutions for dilution were different to ensure a more homogeneous dilution of the acaricides.

Ivermectin, eprinomectin and moxidectin were evaluated with the larval immersion test (LIT), as described by Klafke et al. (2012). A diluent without acaricide was used as a control for all acaricides.

The bioassay was performed using 500 microliters of each immersion solution distributed in three 1.5 ml microcentrifuge tubes. Using a brush, approximately 100 larvae were transferred to each tube, which was then closed and shaken vigorously to ensure the larvae were completely submerged. After 10 min of immersion, the larvae were removed from the tube with a clean brush, allowed to dry on a paper towel, then transferred to a filter paper package folded in half and closed on the sides with metal clips. After adding the larvae, the package was sealed with a third clip and incubated in the dark in an environmental chamber at 27–28°C and 85–90% relative humidity. After 24 h, larval mortality was determined by counting the total number of live and dead individuals. Larvae that were paralyzed or moving only their appendages, but unable to walk, were considered dead.

Statistical analysis

Probit analysis was performed on the bioassay results, using Polo-Plus software (LeOra Software, 2003). The following parameters were estimated for each test: LC50 (50% lethal concentration) with its 95% confidence interval (95%CI) and the slope of the regression line. Resistance was calculated based on a comparison of the susceptibility of the field population and the susceptible reference strain, determining the LC50 for each field population and the LC50 for the POA strain, in the LIT.

The four visited farms had an average of 272.9 (16.5–600) hectares (ha), with areas of native Caatinga pasture and areas with cultivated grasses (Pennisetum purpureum and Cenchrus ciliaris). The average population of the herds was 123 (52–200) cattle. Two farms (1 and 3) are exclusively dairy, while the others have mixed production (beef and dairy). Farms 1 and 3 had Holstein cattle, farm 2 had Holstein and Gyr breeds, and farm 4 had crossbred cattle.

The main health problems reported on the farms were tick infestations (farms 1, 3 and 4), while the main problem reported on farm 2 was the occurrence of bovine parasite sadness. The farms have a history of using sprays and pour-on with synthetic and organophosphate pyrethroids to control ticks. However, the most frequent administration is by injection, with weekly or monthly application of 1% ivermectin to control R. microplus and/or Haematobia irritans. Producers stated they had never administered eprinomectin and moxidectin against endo- or ectoparasites to their animals. At all the farms, the control of R. microplus started when the producers noticed the effect of telegony in the animals.

All the R. microplus populations showed resistance to ivermectin when compared to the POA strain. As can be seen in Table 1, ivermectin presented a resistance factor (RF) ranging from 6.3 to 38.9. Only the Barro-CE population demonstrated resistance to eprinomectin (RF = 31.4). Moxidectin presented RF values ranging from 5.6 to 339.2.

Table 1

Cross-resistance status of *Rhipicephalus microplus* populations in the states of Paraíba and Ceará, Brazil, using the Larval Immersion Test (LIT).
Farm	Population	Treatment	No.	Slope (S.E.)	LC₅₀ (CI 95%)	RF
-	POA	IVM	2.087	2.865 (0.154)	26.228 (15.368–39.439)	-
		EPM	2.006	3.541 (0.178)	22.439 (15.998–31.032)	-
		MOX	2.788	10.454	0.297 (0.281–0.313)	-
1	S. J. Rio do Peixe - PB	IVM	1473	1.447 (0.094)	167.214 (146.189-263.727)	6.375
		EPM	2.306	3.445 (0.177)	5.277 (4.814–5.754)	0.235
		MOX	2.700	1.338 (0.060)	100.748 (65.928–143.057)	339.281
2	Sousa - PB	IVM	3.383	1.305(0.052)	1021.235 (725.243-1525.858)	38.936
		EPM	1.855	0.911 (0.047)	12.434 (7.267–18.950)	0.554
		MOX	3.297	4.985 (0.312)	2.957 (2.712–3.201)	9.956
3	Barro - CE	IVM	2478	1.105 (0.039)	221.170 (153.254-329.165)	8.432
		EPM	1.713	0.958 (0.047)	705.417 (473.761–1141.840)	31.437
		MOX	2.400	7.870 (0.866)	6.789 (5.584–7.731)	22.888
4	Várzea Alegre - CE	IVM	2.407	3.105(0.212)	527.093 (234.052-782.235)	20.096
		EPM	1.582	0.620 (0.052)	1.308 (0.201–3.711)	0.058
		MOX	2.288	5.057 (0.272)	1.688 (1.593–1.784)	5.683
S.E.: Slope; LC50: lethal concentration for 50% of the population; CI 95%: 95% confidence interval; IVM: ivermectin; EPM: eprinomectin; MOX: moxidectin.
RF: resistance factor = LC50 test population / LC50 susceptible reference strain POA.

All the populations showed cross-resistance between ivermectin and moxidectin. However, only in the Barro-CE population showed collateral resistance between ivermectin, eprinomectin and moxidectin.

The farms visited in this study engage in practices that are known to predispose to the development of acaricide resistance. The profiles of the farms indicated that factors such as high animal stocking rate, dairy production and predominance of taurine breeds, as described in the literature (Utech et al., 1978; Veríssimo et al., 2002; Vilela et al., 2002; Vilela et al. al., 2020), were factors that favored the development of parasite drug resistance.

Pereira et al. (2010) reported that inadequate management of acaricides, including incorrect application, continuous use of spray formulations, high frequency of treatments, initiation of treatments only after the discovery of engorged female ticks, and incorrect interval between treatments, facilitate the development of drug resistance. These practices were observed on all the farms that participated in this study.

It was found that ivermectin was the only macrocyclic lactone used on the farms, and that the animals had no contact with eprinomectin and moxidectin. However, resistance to the acaricides ivermectin, eprinomectin and moxidectin was observed in one tick population, and to ivermectin and moxidectin in the other three populations. This is evidence of cross-resistance and underscores the importance of adopting strategies to control the bovine tick in Brazil’s semiarid region, mainly due to overuse of macrocyclic lactones.

given their similar structural formulations and mechanisms of action, high cross-resistance between ivermectin and eprinomectin (avermectins) was expected, since they are amino-avermectins derived from Avermectin B1, which have a modified terminal oleandrose moiety called 400-Deoxy-400-epi-acetylamino-avermectin B1. However, cross-resistance between these drugs was only observed in the Barro-CE population. In studies with nematodes, Ménez et al. (2016) demonstrated that with the multidrug-resistant phenotype selected by ivermectin and moxidectin, the nematodes were also more resistant to eprinomectin than the wild type strain, with eprinomectin being the less potent drug after ivermectin and moxidectin selection pressure. Thus, further studies are needed to better characterize the resources the tick R. microplus uses to build up its eprinomectin resistance.

Acaricide resistance may be metabolic, via an increase in the detoxification ability of the acaricide; by structural alterations in the exoskeleton, in which the penetration of acaricides is reduced; or molecular, involving target-site mutation, the latter being described in ivermectin-resistant populations (Pohl et al., 2012; Guerrero et al., 2013).

Despite the similarities between macrocyclic lactones, there are important differences between ivermectin and moxidectin, such as plasma and tissue kinetics. Moxidectin can maintain activity for a long period of time (150 days) in different long-acting formulations, such as the drug Cydectin 10% LA (Zoetis, S.A, Belgium). According to Prichard et al. (2012), when resistance to avermectins first develops, it usually influences the effectiveness of all avermectins. However, as a milbemycin, moxidectin is generally still highly effective against avermectin-resistant parasites at its recommended dose rate. According to Ranjan et al. (2002), parasite resistance to moxidectin develops more slowly than to ivermectin. However, the findings of this study suggest that resistance to moxidectin may occur as a result of continuous selection pressure through the use of ivermectin.

Observing and comparing the RF results of the tick populations, it can be stated that there are different types of receptors and/or existing connections. These differences in the responses to ivermectin and moxidectin suggest that there are differences in the level of interaction of GluCl with these molecules, or that the involvement of other amino-restricted chlorine channels with the actions of avermectins and moxidectin differ, as suggested by Lespine et al. (2012) in their studies of nematodes.

In view of the findings of this study, it can be stated that R. microplus populations from Brazil’s northeastern semiarid region exhibit collateral resistance between the macrocyclic lactones ivermectin and moxidectin, which may be caused by selection pressure, favored by the long-term use of ivermectin. However, although eprinomectin is structurally similar to ivermectin, this type of resistance was not observed.

Funding

This work was supported by CNPq – Brazil’s National Council for Scientific and Technological Development and CAPES – Brazil’s Federal Agency for the Support and Improvement of Higher Education.

Conflict of interest statement

The authors declare that they have no conflict of interest relevant to the content of this article.

Ethical approval

The activities involved in this research were approved by the Ethics Committee for Animal Use of our institution (CEUA/IFPB), under protocol number 23000.000558.2021-23.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

Datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Code availability

Not applicable.

Author contributions

All the authors contributed to the conception and design of the study. Material preparation, data collection and analysis were carried out by Larissa C. Ferreira, Estefany F. Lima, Ana Luzia P. Silva, Clarisse Silva M. Oliveira, Geraldo M. Silva Filho, Luana C. Sousa, Guilherme M. Klafke, Thais F. Feitosa and Vinícius Longo R. Vilela. The first version of the manuscript was written by Larissa C. Ferreira, Guilherme M. Klafke and Vinícius Longo R. Vilela; and all the authors made comments in previous versions of the manuscript. All the authors read and approved the final manuscript.

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Cross-resistance between macrocyclic lactones in populations of Rhipicephalus microplus in Brazil’s semiarid region

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Abstract

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Introduction

Material And Methods

Collection of samples and location of experiments

Characterization of the farms

Ticks

Acaricides

Larval immersion test (LIT)

Statistical analysis

Results

Discussion

Conclusions

Declarations

References

Additional Declarations

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