A comparison of two in vitro bioassays to detect resistance of the cattle tick Rhipicephalus microplus to fipronil.

The aim of this work is to compare the sensitivity of two in vitro bioassays to detect resistant to fipronil in Argentinean populations of the cattle tick Rhipicephalus microplus. Two different larval bioassays prepared with technical grade (97%) fipronil were compared: larval immersion test (LIT) and larval packet test (LPT). Seven strains from different provinces were treated with both assays. Colonia Tabay, Colonia Benítez, Intiyaco and Quimili strains were considered resistant in both LIT and LPT bioassays. The 95% confidence intervals (IC95) for lethal concentration 50% (LC50) did not overlap with the susceptible reference strain (SRS) and all the values of RR50 obtained were higher than 2. Garabato and Federal strains were considered as susceptible for both techniques because the IC95 for the LC50 overlapped with those of the SRS and the RR50 values were lesser than 2. An ambiguous situation occurs with Reconquista strain. This strain was considered as susceptible with LPT and with incipient resistant after LIT trial. The analysis of the results indicates that both LIT and LPT trials have enough sensibility to differentiate resistant and susceptible strains, but LIT was more sensitive than LPT when the resistance is incipient.

In vitro diagnosis of resistance of the cattle tick Rhipicephalus (Boophilus) microplus to fipronil in Argentina.

The aim of this work was to adapt and apply the in vitro larval bioassay packet test (LPT) to diagnose resistance of Rhipicephalus microplus ticks to fipronil in Argentina. Ten serial dilutions of fipronil were included in the LPT bioassay. Seven populations of R. microplus from six Argentinean Provinces were evaluated. Samples from Colonia Benítez, Quimilí and Intiyaco were unambiguously classified in the category 'resistant'. In all these cases the 95% confidence intervals did not overlap with that of the susceptible reference strain and the slope values were lower than the slope value obtained for the susceptible reference strain. The resistance ratio (RR50) values of the populations from Colonia Benítez I and II, Quimilí and Intiyaco were 2.23, 3.49, 6.84 and 3.39, respectively. Populations from El Colorado I and II, Acambuco, Vera and Feliciano were classified as 'susceptible' because the 95% confidence intervals overlapped with that of the susceptible reference strain and the RR50 values never exceeded 1.32. The results of these LPT bioassays indicate the presence of fipronil resistant R. microplus populations in Argentina.

Resistance of the cattle tick Rhipicephalus (Boophilus) microplus to ivermectin in Argentina.

Resistance to ivermectin in populations of the cattle tick Rhipicephalus microplus in Argentina was diagnosed in this work. The in vitro larval immersion test (LIT) was used to determine quantitatively the levels of resistance to ivermectin in different populations of R. microplus. Additionally, field trials to control natural infestations of R. microplus on cattle with a commercial formulation of ivermectin 3.15% were carried and jointly analyzed with the in vitro assays. The phenotypic response of the populations analyzed was not uniform. Five of them were classified as susceptible, four populations as resistant, and one in the category "incipient resistance". Regarding the field trials, the therapeutic efficacy in a population classified with LIT as susceptible achieved values higher than 94% two weeks after treatment, and no reproductively viable females were observed after the second day post-treatment. Conversely, the values of efficacy percentage in a population (named as "San Martín") classified with LIT in the category "incipient resistance" never exceeded the 70.8%, and engorged females were collected in practically all counts. The population "San Martín" was classified in the category "incipient resistant" with LIT analysis, but the field trial unambiguously shows that this tick population is resistant. The comparison of the results obtained with LIT in vitro assays and through field trials shows that biased estimations of resistance levels may occur when resistance ratios (RR) values are ≤2, and additional field efficacy trials could be needed to know with precision the status of the tick populations evaluated.