João Ricardo de Souza Martins and co-workers described the first report of fluazuron resistance in R. microplus, and also report the first tick strain that was multi-resistant to six classes of acaricides.
As a result of the indiscriminate use of acaricides to control ticks infesting livestock, tick resistance to acaricides is one of the biggest challenges and a cause of concern for cattle production wherever R. microplus is endemic or in areas prone to invasion by this injurious ectoparasite species. Recently, R. microplus populations with multiple resistances to acaricides have been identified in several ranches, particularly in Brazil and Mexico. Currently, there are six classes of acaricides available on the Brazilian market to control cattle ticks, with fluazuron being the only AI with no previous reports of resistance. Here, we report for the first time an R. microplus tick strain that is resistant to fluazuron. This strain is also the first with documented resistance to the six classes of acaricides used to control cattle tick in Brazil.
In 2010, ticks were collected for the same population from which the Jaguar strain was sourced for the first time, as part of the survey system for cattle tick acaricide resistance in Rio Grande do Sul state, Brazil, performed by IPVDF. At that time, no in vitro test for fluazuron resistance was available, as there was no suspicion of fluazuron resistance. Subsequently, an investigation concerning fluazuron resistance began after ranch employees reported that they could no longer control ticks infesting cattle even using fluazuron. To investigate whether the observation in the field was due to resistance, ticks were sampled and the Jaguar colony was established at IPVDF.
Results from the field trial presented here clearly demonstrated that fluazuron treatment was unable to control ticks of the Jaguar strain, neither by reducing tick count or larvae hatching. This lack of efficacy was more noticeable when comparing fluazuron performance against the SG strain. Field trial studies conducted in Southern Brazil when fluazuron was released on the market achieved more than 99% efficacy at 21 days after treatment, when using 2 mg/kg of this AI. In another field trial, performed in Australia, the adult tick reduction was 100 and 98% at three and six weeks after treatment, respectively. Recently, some authors have shown that fluazuron still gives significant results under field conditions in Brazil. Alves-Branco et al. demonstrated in a study using artificial and natural tick infestations that fluazuron (2.5 mg/kg) efficacy ranged from 96% to 100% between 14 and 49 days after treatment. Lima et al. showed that fluazuron (2.5 mg/kg) treatment reduced more than 99% of natural tick infestation between 14 and 28 days after treatment.
Scientific literature concerning the validation and use of in vitro tests to evaluate fluazuron susceptibility is lacking. This can be attributed to several factors, such as the absence of a resistant reference strain, and the apparent lack of need, since it was assumed that there were no cases of field resistance to fluazuron. Results from the AIT demonstrated a difference in susceptibility to fluazuron between the POA and Jaguar strains. Moreover, the ability of fluazuron to decrease larval hatch was even lower in the Jaguar R selected strain, which indicates there is a genetic basis for the trait that could be increased and selected for in the resistant tick population. Together with the in vivoresults, the AIT has proven to be a useful tool to detect resistance to fluazuron in tick populations. Previously published data showed that a 200 ppm concentration of fluazuron could be used as a discriminating dose (DD) to induce total, or near complete inhibition of larvae hatching in the Parkhurst, Yeerongpilly, and Ulam reference strains from Australia. In our tests, 50 ppm induced more than 99% larvae hatching inhibition in the POA strain, and less than 50% in the Jaguar strain. Larval hatch in the AIT was close to 50% with the Jaguar strain and >80% with the Jaguar R strain using fluazuron at 500 ppm. The results presented here highlight the need for comprehensive studies with the AIT and fluazuron, particularly concerning test reproducibility and the establishment of a discriminating dose to diagnose resistance. Such studies must consider past field exposure of tick populations to fluazuron and, whenever possible, include a comparison between in vitro and in vivo results.
Although the AIT has been questioned as a precise tool for resistance diagnosis in ticks, particularly for systemic acaricides, it is important to note that in contrast to ML, a larval test cannot be employed to investigate fluazuron resistance. This situation is a consequence of the mechanism of action of fluazuron, which inhibits chitin synthase activity and subsequently prevents larval ecdysis. As a consequence, fluazuron is a developmental inhibitor and does not kill adult engorged females or larvae as other acaricides do. Since fluazuron is a systemic acaricide, the artificial feeding technique provided a more similar condition to which the parasitic life stages are exposed to fluazuron than the AIT. Pharmacokinetic studies conducted with fluazuron showed that after the administration of 1.5 mg/kg of live weight, the serum concentration increased to more than 1.3 ppm in cattle. As the fluazuron label rate is 2.5 mg/kg of live weight, the concentration of AI added to blood fed to ticks in the AFA here ranged from 2.5 to 10 ppm. Additionally, the marked differences in mortality after exposure to fluazuron between resistant and susceptible tick strains used in this study lend support to the resistance status of the Jaguar strain. In this sense, the AFA is a technique that is well suited for the characterisation of resistance, especially for systemic drugs with the understanding that, in some cases, biotransformation of the parent drug may be required to produce the pharmacologically active agent.
Investigation of tick resistance and constant survey of population susceptibility status are essential to (i) recognise if acaricide resistance is the cause of tick control failure; (ii) determine the most efficient acaricide for each location/ranch; (iii) understand the epidemiology and spread of resistance; and (iv) develop control strategies that minimise the selection of resistant genotypes. RS state, located in Southern Brazil, has a remarkable history of tick resistance to acaricides as it was the first region in Brazil to report resistance to OP and SP, and the first location in the world to report resistance to ML, mixtures of OP and SP, and in vitro resistance to fipronil. The breeds of cattle, which are predominantly Bos taurus taurus, and high stocking rates are two factors that contribute to the problem of acaricide resistance among populations of R. microplus in RS. These factors lead to heavy tick loads, which, in turn, lead to more treatments per year, and consequently more resistance selection. There may be other factors contributing to the resistance problem in RS requiring further epidemiological studies for its full identification and characterisation. In this sense, information regarding resistance in RS could facilitate the understanding of resistance in other regions of the world where R. microplus impacts livestock production systems.
The resistance mechanism for fluazuron in the Jaguar strain remains to be fully described. A similar situation exists with amitraz, ML, and fipronil. Tick resistance against drugs can be due to target site insensitivity and/or metabolic resistance, including molecular efflux pumps. Studies reported the key role of detoxification mechanisms in resistance to several drugs. It is possible that the intensive use of different acaricides against the Jaguar strain in the field induced several non-specific pathways which may contribute to resistance to fluazuron. In fact, synergist bioassays and enzyme activity quantification studies withMusca domestica , Lucilia cuprina , and Cydia pomonella revealed, at least in part, the role of mixed function oxidase enzymes as a mechanism of resistance to diflubenzuron, a benzoylphenyl urea compound. The occurrence of chitin synthase alteration which could promote insensitivity to fluazuron also remains as a possible resistance mechanism. These mechanisms need to be investigated further. Biological phenomena underlying tick resistance against fluazuron remain to be determined.
Here, the authors documented the first case of fluazuron resistance in R. microplus and the first tick population resistant to six classes of acaricide. Assessing the efficacy of fluazuron against other populations of R. microplus subjected to high selection pressure with acaricides and the characterisation of resistance mechanisms in the Jaguar strain require investigation. This study highlights the spreading and emerging issue of multiple resistance to acaricides in R. microplus, which is a cause of concern worldwide. The inability to control cattle ticks by using only acaricides currently available in an indiscriminate manner demonstrates the need for more research to establish an integrated strategy that includes chemical control, and the development of alternative control tools.
João Ricardo de Souza Martins et al. Veterinary Parasitology (2014), 201, (1-2), 128-136.