Introduction

Ticks are hematophagous ectoparasites that are important in human and animal health due to the damage they cause by transmitting pathogens and feeding¹. Rhipicephalus microplus is the most important species in cattle farming because it is the main vector of hemoparasites such as Babesia spp. and Anaplasma spp., in addition to this, the economic losses it causes at the productive level in Mexico amount to more than 573.6 million dollars per year². This species is dispersed in the tropical, subtropical, and semiarid regions of all continents, except Europe³. The geographical distribution of R. microplus in the country is recorded by SENASICA, which states that 30.60 % of the country is free of ticks, 3.44 % is under eradication, and 65.96 % are natural free zones and control zones⁴.

Ixodicides have been used for years for the control of R. microplus, such as: organophosphates, amidines, synthetic pyrethroids, growth regulators, phenylpyrazolones, and macrocyclic lactones (MLs); the latter are a broad-spectrum family (endectocide) and act by binding to the transmembrane (TM) domains of Cys-loop receptors, such as the glutamate-gated chloride channel (GluCl), which are expressed in the motor and sensory systems of arthropods and nematodes, causing hyperpolarization and ultimately death^5,6,7.

What all these drugs have in common is that they have generated resistance due to operational factors such as inappropriate and continuous use⁸. In Mexico, in 2010, resistance to ivermectin was reported for the first time in R. microplus populations⁹, and it has been used since the beginning of the 21st century and currently there are few studies on resistance to MLs in Mexico, which are scarce in the northeast of the country. Therefore, the objective of this research was to determine the status of ivermectin resistance in R. microplus in cattle ranches in northeastern Mexico, as well as the possible risk factors associated with such resistance.

Material and methods

Results

Discussion

Chemical control of ticks in Mexico and the world has become ineffective, given the emergence of populations resistant and multi-resistant to ixodicides^16,17,18. Since its introduction in the 1980s, IVM has been the most important animal health product worldwide¹⁹. There have been few studies on the status of resistance to IVM in R. microplus in Mexico^8,9,15. This highlights the importance of conducting studies on the evaluation and diagnosis of resistance to this drug in the northeast of the country.

Applying the LIT and following the Probit methodology, the LC50 and LC99 of the study populations were determined. In the results obtained, a significant difference was found with the reference strain Deutch, with a susceptible population (5 %) (RR50= 0.73), three populations with incipient resistance (15 %) (RR50= 1.20-1.61), and the rest (80 %) with resistance (RR50= 2.07-11.14). These results coincide with those reported for the first time in Mexico⁹, where 100 % of the populations analyzed showed resistance to IVM, with RR50= 2.04-8.59 and RR99= 2.67-87.86, in addition to exponential growth in different sampling periods. The importance of using a susceptible reference strain lies in the fact that it is a reference parameter for biochemical and molecular resistance studies²⁰. In addition, they are regulated by international organizations. In the study carried out in 2006⁹, a comparison was made between the results obtained in their research using the Deutch strain and another study¹⁵, which used the Porto Alegre strain. This study⁹ highlights that the result obtained by this team is superior to those of the second, even so, slightly higher or equal RR50 values were obtained. In the present research, similar results were found when analyzing the Porto alegre, Mozo and Deutch strains^9,13,21 as possible candidates for the reference strain, so it was decided to select the Deutch strain because, when analyzing the results of the three, there was no significance at the time of determining the already stipulated classification, and it was more in line with what was desired. On the other hand, the Mexican strain Media Joya is only susceptible to organophosphates, synthetic pyrethroids and amidines, and there is no toxicological characterization of susceptibility to ivermectin²².

Authors²³ mention that resistance is given by biochemical/genetic factors, operational factors, and ecological factors; the latter include intrinsic traits and interactions of populations with their surroundings and environment. In addition, the development of resistant individuals is dependent on the frequency of occurrence and the selection pressure^9,24,25. In addition, in different studies of Latin American countries, resistant populations of between 40 and 100 % of the populations analyzed were obtained^26,27,28.

The response of populations to dose increase (slope) is an important indicator of resistance. A low slope ≤ 2 and a high LC (higher than the reference strain) are common in resistant populations, while a high slope ≥ 2 and low LC are common in susceptible populations with heterogeneous response^13,29. In the present study, populations that respect this statement were found: JCG4 (S.L.P.), JAM5 (Ver), JNSE (Tamps), VMA1 (N.L.), and the JPN1 population (Ver), while, surprisingly, three populations from Tamaulipas (ETHM, RAMT, and MRNA), four from Nuevo León (JVML, SNTM, PRVA, and DALC), five from Veracruz (ANGS, LEX15, ESHP, VIHM, and JHE2) and three from San Luis Potosí (KML1, EBEV, and ISALI) showed high LCs and slopes. To date, there are no reports that determine a strain of R. microplus that is highly resistant to IVM²⁸; according to these statements, the populations described have suffered a loss of heterogeneity and susceptible genes, demonstrating for the first time in the present research that resistant alleles are fixed in the population and they present a homogeneous resistance response. Other studies mention that the heterogeneity of resistant alleles would lead to the loss of susceptible populations and the emergence of resistant populations with homogeneous alleles^9,30,31.

Of the resistant populations obtained in this study, two were classified as highly resistant (RR50= 6.98 and RR50= 11.14), results that are similar to those that showed the highest values of resistance (RR50= 6.84, 7.37 and 10.23) and RR50= 5.89, 6.25 and 8.21^8,9,15. Even so, molecular studies are needed to analyze all frequencies of resistant alleles in populations.

On the other hand, frequencies were analyzed based on the responses obtained in the epidemiological questionnaire (Table 3). The municipalities included in this study are located between parallels 26° N to 21° N, relative humidity between 65 and 79 %, average temperatures of 21° C, and an average water evaporation between 1,200-1,400 mm, optimal conditions for the development, distribution, and survival of the tick, as well as for the increase of generations per year^32,33,34. Some authors mention that geographic location and abiotic niche are factors that promote the greater development of ticks^3,35.

Of the 14 variables studied, two showed significances of P≤0.20: frequency of treatments (P=0.026) and formulation administered (P=0.1531), which were included in the binomial logistic regression model.

Animal management systems, as well as the number of annual treatments, are considered factors that influence the efficacy of drugs, playing an important role in the development of resistance⁴. In 55 % of the ranches, IVM treatment is applied 4 to more than 5 times per year, similar to that obtained by Fernández-Salas et al³⁶, where cattle ranches that apply MLs 4 or more than 5 times a year are up to 13 times more likely to develop resistance⁸. IVM has a period of decrease in concentration after application, but due to its high affinity to fat and its persistence in tissues, it is not completely eliminated, so prolonged exposure to therapeutic doses favors the emergence of resistant organisms^9,15,36. This assumption is known as the “tail effect”; if organisms are present during this period, the selection of IVM-resistant organisms is possible^37,38. R. microplus reacts quickly to selection pressure and higher concentrations of ixodicides³⁹, therefore, the application of the chemical should be carried out less frequently at 30-d intervals with the intention of reducing this pressure, not only for the tick, but also for non-target organisms such as helminths^40,41.

By applying the binomial logistic regression, it was observed that a P≤0.0134 was obtained for the variable of frequency of treatments, a result that proved to be significant, but with an undefined OR due to the fact that in one of the groups of the 2 x 2 contingency table, there was a box in which there was no susceptible population and that the IVM was applied 4 or more than 5 times a year, which had to be computed as a zero; since the OR is the quotient of two ratios⁴². Including a zero in the division generates an incalculable result. It was determined that the administration of the treatment 4 or more than 5 times a year may be a risk factor since, on the one hand, the calculated frequency measures resulted in values greater than 1; the relative risk obtained was 1.8 and the ORs are in a range from 1.27 to infinity. Therefore, the increase in frequency in the exposed group can be considered to be due to the effect of the independent variable. One way to solve the fact that the OR is incalculable is to proportionally increase the values of each box⁴³, so when doing so, a value of OR= 11.14 and P=0.032 was obtained; although this result cannot be taken as reliable, it leaves open the possibility that, in future studies, including a larger number of farms studied, the increase in ORs for farms that apply treatments 4 or more times a year can be verified.

Regarding the independent variable of formulation administered, it was observed that more than half of the farmers use IVM-LA formulations of 3.15 % to 4 % due to the lack of efficacy of the 1 % formulation. IVM-LA formulations have a higher risk of generating resistant populations when applied with high frequency compared to 1 % short-acting formulations¹. This is due to several factors, such as a higher concentration of the active ingredient in IVM-LA formulations, an applied dose that is three times higher (630 μg/kg), a prolonged withdrawal period, a decrease in natural immunity and a faster selection of resistant parasites^41,44,45. The binomial logistic regression analysis showed that for the variable of formulation administered, a P≤0.1101 (OR= 6.59, 95 % CI= 0.5428 and SE= 1.27) was obtained, which was not significant as a possible risk factor, but with a positive association. With these data, the only susceptible population (VMA1) was related to the possible associated risk factors due to the fact that, in this population, a lower frequency of treatments was found: 1-3 per year and a lower formulation administered: IVM at 1 %.

Conclusions and implications

Based on the results obtained, it was shown that, in the states of Veracruz, San Luis Potosí and Tamaulipas, there are no populations susceptible to IVM and 14 to 25 % of these have incipient resistance. On the other hand, in the state of Nuevo León, only one susceptible population was found. R. microplus is resistant to IVM in northeastern Mexico (80 %). Currently, the frequency of applications of 4 or more than 5 times a year is the only risk factor that could be associated with the presence of resistant populations. Therefore, it is necessary to migrate to new control methods, such as including several families of ixodicides, carrying out integrated control, responsible management, and a culture of diagnosis in order to reduce the selection pressure to which populations are exposed.