Introduction

The integrated control of cattle ticks involves the simultaneous combination of two or more tools, one of which should not be chemical. The application of synthetic chemical acaricides, the pasture spelling and the use of cattle breeds with natural resistance to tick infestation are the three main tools with proven efficacy that, in practice, can be used in integrated control of Rhipicephalus microplus (Canestrini, 1888), the principal tick species affecting cattle in the world. There are other tools such as anti-tick vaccines, biological control and non-synthetic acaricides (e.g. essential oils) which have been evaluated to enhance cattle ticks control (Samish and Rehacek 1999; Samish and Alekseev 2001; Jonsson and Piper 2007; Webster et al. 2015; Lew-Tabor and Rodríguez Valle 2016; De la Fuente et al. 2023; Bishop et al. 2023; Teixeira et al. 2023), but in practice none of them are currently available to farmers as an operational option to control high tick infestation on cattle.

The application of synthetic chemical acaricides is the principal method used against R. microplus in America, but its efficacy is significantly diminished by the emergence of tick populations resistant to one or more chemical groups. In fact, there are reports of resistance of R. microplus to almost all available classes of chemical groups used as acaricides in America, including tick populations with multiple-resistance (Guerrero et al. 2012; Castro-Janer et al. 2010, 2011; Fernandez-Salas et al. 2012; Cutulle et al. 2013; Reck et al. 2014; Rodriguez-Vivas et al. 2014; Klafke et al. 2017; Torrents et al. 2020). Pasture spelling constitutes an efficient tool to be used as alternative or complement to the chemical control (Wharton 1972; Sutherst et al. 1979; Johnston et al. 1981; Norton et al. 1983; Rossner et al. 2022), but the application of this method is constrained because it requires specific management conditions in cattle properties and an accurate knowledge of tick ecology, and because sometimes led to underutilization of forage resources.

In a context where R. microplus resistance to at least one chemical group is practically ubiquitous, the incorporation of cattle breeds with high levels of natural resistant to tick infestation could be a feasible tool in a plan of integrated control, not only to reduce the annual frequency of acaricide treatments, but also to improve the performance of chemical acaricides when their efficacy levels are diminished by the presence of resistant specimens in a tick population. Cattle breeds present different levels of natural resistance to tick infestation because of the co-evolution of cattle with ticks and due to selective breeding over long periods of time (Biegelmeyer et al. 2015; Burrow et al. 2019). Bos indicus breeds have higher resistance levels to infestation with R. microplus than Bos taurus breeds, while different resistance degrees have been reported in synthetic breeds (e.g. Braford and Brangus, among others), always higher than in pure B. taurus breeds, essentially related to the proportion of the zebuin component (Utech et al. 1978; Sutherst and Utech 1981; Guglielmone et al. 1990, 1992a,b; Jonsson et al. 2014). Also, the natural resistance to tick infestation in Creole breeds is higher than in other non-Iberian B. taurus breeds (Ortega et al. 2023). The aim of this work was to comparatively analyze the evolution of R. microplus infestation in two bovine herds with different degrees of natural resistance (i.e., Hereford and Braford) to ticks subjected to an identical chemical treatment scheme at the same farm, to demonstrate how the incorporation of a more resistant bovine breed improves the tick control in a context where resistance of cattle ticks to chemical acaricides is almost ubiquitous.

Materials and methods

The study was performed in a cattle farm located in the vicinity of Mariano Loza (29°22ʹS 58°11′W), Corrientes Province, northeastern Argentina. The analysis included B. taurus (Hereford) and B. taurus x B. indicus (Braford) cattle raised under an extensive rearing system grazing in native grassland.

Two groups of ten Hereford and Braford cows each, naturally infested with R. microplus, were subjected to the following scheme of treatments with chemical acaricides: (i) 1 mL/10 kg of a pour-on formulation of fluazuron (ACATAK®, Elanco Animal Health) on 10th September 2022; (ii) 1 mL/10 kg of a commercial pour-on formulation of fipronil 1% (ECTOLINE®, Boehringer Ingelheim Animal Health) on 27th October 2022; (iii) 1 mL/10 kg of ECTOLINE® on 17th November 2022; (iv) 1 mL/50 kg of a commercial injectable (subcutaneous) formulation of ivermectin 3.15% (IVERVET®, Vetanco) on 8th December 2022; (v) 1 mL/10 kg of ACATAK® on 18th January 2023; (vi) immersion in a dipping vat with a combination of cypermethrin 10% and ethion 40% (DERRIBANTE SM®, ACA) on 6th April 2023; (vii) immersion in a dipping vat with DERRIBANTE SM® on 12th April 2023; (viii) immersion in a dipping vat with DERRIBANTE SM® on 27th May 2023; (ix) immersion in a dipping vat with DERRIBANTE SM® on 10th July 2023; (x) 1 mL/10 kg of ACATAK® on 20th September 2023; (xi) 1 mL/10 kg of ACATAK® on 20th October 2023. Cows of both groups were kept in two different paddocks during the entire study period with a stocking rate of 0.7 animal per hectare.

Counts of female ticks (4.5–8.0 mm long) were monthly performed simultaneously on one side of ten cows of each group from August 2022 to November 2023. Counts were made during the 2 week of each month. The number of ticks collected on cows was multiplied by two for statistical analyses. Prevalence and mean abundance as defined in Bush et al. (1997) were calculated. Distribution-free 2-sample bootstrap t-tests were used to compare mean abundances (each with 2,000 replicates) (Reiczigel et al. 2019). The tick distribution within each group of bovines was examined by calculating the index of discrepancy D (Poulin 2007). This index quantifies aggregation as the departure between the observed parasite distribution and a perfectly uniform distribution, In D, 0 means null aggregation (all hosts with equal level of infestation) and 1 means complete aggregation (all members of a parasite population on one individual host) (Poulin 2007). Comparison of D between groups was performed by the comparison of 95% confidence intervals. All these calculations were done with the program Quantitative Parasitology 3.0 (Reiczigel et al. 2019).

The status of susceptibility/resistance to different chemical groups used as acaricides of the tick population exposed to the treatments was determined by in vitro bioassays. The larval immersion test (LIT) bioassay was performed to evaluate the level of ivermectin resistance in tick samples following the methods described in Torrents et al. (2020). The technique was performed by using technical grade ivermectin (22,23-dihydroavermectin B1, batch number MKCK0618, Sigma-Aldrich, USA). The technical ivermectin was dissolved at 1% in acetone to prepare a stock solution, which was diluted 1:100 with 1% acetone and 0.02% Triton X-100 in distilled water to obtain an initial solution of ivermectin 100 ppm. This initial solution was employed to prepare the following solutions (in ppm): 4, 5.8, 8.2, 11, 16.8, 32.4, 43.96, 51.4, and 100. The LIT bioassay as described in Torrents et al. (2023) was also performed to evaluate the level of fipronil resistance in tick samples. The stock solution was made with technical grade (97%) fipronil (Jiangsu Tuoqiu Agrochemicals Co. Ltd., China, batch number 18061201) at 0.01% in diluent, which was prepared with 0.02% Triton X-100, 1% acetone and distilled water. The gradient of dilutions in ppm was 0.1, 0.3, 0.6, 1, 1.5, 2, 2.5 and 3. The mortality values obtained were processed through probit analysis with POLO PLUS software (LeOra Software, 2003). Values of lethal concentrations (LC) for 50% with their confidence intervals (CI) and the slope of the regressions obtained for each sample were compared with the results obtained for ticks of the susceptible reference strain (SRS). Differences between LC50 values of each tick population were considered significant when the 95% CIs did not overlap with those of the SRS. The adult immersion test (AIT) as described by Klafke et al. (2017) was employed to determine the status of susceptibility/resistance to the combination of pyrethroids and organophosphates. Fifty microlites of a commercial acaricide mixture of cypermethrin 10% and ethion 40% (100 ppm y 400 ppm respectively) was diluted in 50 mL of diluent (1% acetone, 0.02 ul TRITON-X and distilled water). Homogeneous groups of ten engorged females were immersed in the solution containing the acaricide and in diluent only as control group. Tick mortality and egg-mass weight was recorded. The percentage of efficacy for AIT was calculated as: ((eggs weight of control group-eggs weight of treated group)/eggs weight of control group) × 100. Finally, the adult immersion test (AIT) based on technical grade fluazuron (batch number BCBT8190; Sigma-Aldrich, St. Louis, MO, USA) was performed to test the susceptibility of the tick population in an in vitro bioassay. The AIT bioassay with fluazuron was carried out following the methods described in Reck et al. (2014) and Torrents et al. (2022). Briefly, technical grade fluazuron was diluted in technical grade acetone to obtain a 1% fluazuron solution, which was diluted 1:200 in diluent to give a dipping solution of 50 ppm. The diluent is formed by 0.02% Triton X-100 and 1% of technical grade acetone in distilled water. Ten engorged females were immersed for 1 min. Because the absence of both susceptible and resistant reference strains to fluazuron in Argentina, the comparison was performed with ticks from Goya (Corrientes, Argentina) and Monte Caseros (Corrientes, Argentina) populations, whose phenotypic response to fluazuron in a file trial was classified as resistant and susceptible, respectively. Engorged females were drier after immersion with a paper towel and incubated at 25 ± 1 °C and 83% R.H, and the egg hatching rate was calculated as the total number of hatched larvae divided by the number of total eggs produced by each female. Larvae and unhatched eggs were counted as described in Guglielmone et al. (1989). In vitro bioassays were performed at the Laboratorio de Inmunologìa y Parasitologìa of INTA E.E.A. Rafaela.

Handling of animals was made in accordance with the institutional guide for the care and use of experimental animals, with the approval of the Institutional Committee for Care and Use of Experimental Animals, CICUAE-INTA, Argentina (council resolution number P23–043).

Results and discussion

The two groups of cows were simultaneously exposed to the same 11 acaricidal treatments between August 2022 and October 2023. The R. microplus population of the study was classified as susceptible to ivermectin and resistant to fipronil according to the LIT tests (Table 1). The percentage of efficacy obtained in the AIT for the combination of pyrethroids and organophosphates was 95%. The reduction in larval hatch of the eggs produced by the engorged females from Goya (resistant), Monte Caseros (susceptible) and Mariano Loza (tested in this work) strains exposed to technical grade fluazuron (50 ppm) was 48%, 89% and 99%, respectively. These results indicate that, at the beginning of the trial, the tick population was susceptible to ivermectin, fluazuron and to the combination of pyrethroids and organophosphates but resistant to fipronil.

Table 1 Results of the larval immersion test (LIT) with ivermectin applied to larvae of Rhipicephalus microplus from the study site. SRS: Susceptible reference strain
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The values of prevalence, mean abundance, and aggregation (expressed as D) of the tick infestation in both Hereford and Braford groups of cows are show in Table 2, with the statistical comparison. The tick infestation was significantly greater in the Hereford cows than in the Braford cows in the counts from August 2022 to March 2023, but no significant differences were found between both groups from April 2023 to November 2023 (Table 2). The evolution of tick infestation in each group is graphed in Fig. 1. The levels of aggregation were low in both Hereford and Braford groups when prevalence was 100% (values ranged from 0.20 to 0.33), and there were no significant differences between the two groups when D values were compared.

Table 2 Values of prevalence (P), mean abundance (MA), and index of discrepancy (D) obtained from the counts of Rhipicephalus microplus females 4.5–8.0 mm long in the groups of Hereford and Braford cows. Counts of female ticks were performed on one side of each host. The number of ticks counted on cows was multiplied by two for statistical analyses. Values in parentheses indicate 95% bootstrap confidence limits around the mean abundance (2000 replications)
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Fig. 1
figure 1

Mean abundance of Rhipicephalus microplus ticks in groups of Hereford and Braford cows, based on counts of female ticks (4.5–8.0 mm long) on one side of each host. The number of ticks collected on cows was multiplied by two for statistical analyses

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Undoubtedly the R. microplus infestation in the Hereford cows was greater than in the Braford cows. This result is not unexpected because it is widely recognized that synthetic breeds (B. indicus x B. taurus crosses) are more resistant to tick infestation than pure B. taurus breeds (Utech et al. 1978; Sutherst and Utech 1981; Guglielmone et al. 1990, 1992a, b; Jonsson et al. 2014). However, the difference in the infestation level between groups became manifest between November 2022 and March 2023, but not from April 2023 to November 2023. The series of chemical treatments applied to the cows from March 2023 to November 2023 include four dips with a combination of cypermethrin 10% and ethion 40%, and two pour-on applications of fluazuron. Ticks were shown to be susceptible to both fluazuron and the combination of cypermethrin 10% and ethion 40% in the in vitro trials. Five treatments were applied between August 2022 and March 2023, but a different combination of chemical groups was used. This series consisted of one treatment with fluazuron, two with fipronil, one with ivermectin, and a last treatment with fluazuron. That is, two of the five treatments applied in this series were made with drugs against which the treated tick population showed to be highly resistant (RR50 of LIT: 6.5; see Table 2). The interpretation that emerges from these results is that tick infestation in both Hereford and Braford breeds was similar when treatment with functional drugs was applied, but when a block of the treatments was done with drugs with decreased functionality due to resistance, treatment failure was manifested more strongly in the most susceptible breed (i.e., Hereford). The biological explanation of this phenomenon is given by the fact that resistance of cattle to ticks is expressed as the percentage of R. microplus larvae that failed to mature into engorged females (Utech et al. 1978). If the initial infestation consists of 10,000 larvae, cattle with 100 and 1000 engorged females would have 99% and 90% resistance, respectively. Utech et al. (1978) obtained resistance values of 96% in Braford (classified as moderate resistance), and between 82 and 89% in Hereford (classified as very low resistance). The number of adult ticks generated in a susceptible breed will be greater than that produced in a breed with more natural resistance when they are exposed to a similar tick challenge and in the absence of a disruptive factor as an effective acaricidal treatment. This differential production of adult ticks will become more evident when a treatment scheme is applied to a tick population with resistance to some of the drugs used.

The genetic resistance of the host, besides reducing the proportion of immature ticks that reach the adult stage, also has a differential effect on the duration of the tick feeding phase and therefore on the weight of the engorged females, which will result in less offspring and infestation of pastures with larvae. The incorporation of cattle breeds with moderate or high resistance to R. microplus is instrumental to optimize the efficacy and sustainability of chemical control of ticks in a scenario where resistance to one or more chemical groups is almost ubiquitous, because it favors the biological control of this parasite. Thus, bovine genetics could be a decisive component for the success or failure of a plan of integrated tick control in cattle farms heavily infested with R. microplus. Because there is also a difference in natural tick resistance among bovines within the same breed of cattle (Burrow et al. 2019; Morè et al. 2019; Mantilla-Valdivieso et al. 2002), further research on the genetic traits involved in the development of resistant to tick infestation in cattle are necessary to also incorporate genomic selection as an additional item in the tick control strategies.