, 2001). The authors tested a susceptible strain, Yeerongpilly, against commercial and technical

formulations of MLs, established their lethal concentrations and determined the discriminating dosages for the detection of resistance to MLs in Australia. In Brazil ( Klafke et al., 2006) and Mexico ( Perez-Cogollo et al., 2010a and Perez-Cogollo et al., 2010b), the existence of IVM-resistant populations was confirmed using the LIT technique. Currently, GDC-0449 price the LIT is been used to monitor IVM resistance in cattle tick outbreaks occurring in the USA (Miller, R.J., 2010 – personal communication). In Uruguay, the LIT was demonstrated to be a very sensitive assay, with which it was possible to diagnose IVM resistance in some populations of cattle ticks before this resistance could be observed through efficacy failures or complains from ranchers ( Castro-Janer et al., 2011). In this article, we present a critical analysis of the performance of classical tests to detect acaricide resistance in the diagnosis of resistance to Raf targets IVM in R. microplus. The following strains of R. microplus were used: Mozo, originating in Uruguay, is the FAO reference strain to diagnose acaricide resistance in Latin America; ZOR, originating in the municipality of Ipiguá (state of São Paulo, Brazil), was isolated from an IVM-resistant field population in February 2008 and maintained under selection

for resistance to IVM. Both strains were maintained at the Instituto Biológico de São Paulo, Brazil. The field populations were collected in ranches located in the states of São Paulo (populations APO, TPA, FIG, JS, AR, PIQ, STO and VIS) and Mato ADP ribosylation factor Grosso do Sul (population StaP). Three populations (JS, AR and StaP) have never been exposed to ivermectin. The populations APO, TPA, FIG, PIQ, STO and VIS had been exposed to ivermectin for three consecutive years prior to the collection of ticks. Six-month-old calves (Holstein-Friesian), free of ticks, were housed in individual stalls (measurements: 2.30 m × 3.00 m) located in an experimental barn, in which they remained isolated. During the experiment, the animals had free access to hay, rations, mineral salt, vitamins and

water. The handling procedures of the animals followed the rules of the ethics committee of the Institute of Biomedical Sciences of the University of São Paulo (protocol number 44/05-CEEB/ICB). The IVM-resistant strain (ZOR) was kept under selective pressure in calves treated with subcutaneous injections of 1% ivermectin at the label rate (200 μg/kg) (IVOMEC® – Merial Saúde Animal, Campinas, Brazil) at the time as the artificial infestation with 200 mg of larvae (approximately 4000 individuals). In the present study, the fourth generation of the ZOR strain was used (ZORF4). This generation of larvae was obtained from 161 engorged females that had been recovered from a calf treated with IVM. The susceptible strain (Mozo) was maintained in cattle as described above, without acaricide treatment.