Determination of the susceptibility of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) to ivermectin and fipronil by Larval Immersion Test (LIT) in Uruguay.

Rhipicephalus (Boophilus) microplus is an important cattle pest in Uruguay, and the law regulates its control. It is resistant to organophosphates, synthetic pyrethroids and, as recently discovered, to fipronil. Resistance to macrocyclic lactones (MLs) and amitraz have not been documented; however, veterinarians and farmers have reported treatment failures. The objective of the present work was to study the susceptibility of cattle tick strains from different Uruguayan counties to ivermectin (IVM) and fipronil by using the Larval Immersion Test (LIT). The Mozo strain was used as the susceptible reference strain. From 2007 to 2009, twenty-eight tick populations were collected from different cattle farms with and without history of IVM or fipronil use. A probit analysis estimated dose-mortality regressions, lethal concentrations (LC), and confidence intervals. The resistance ratio (RR) was determined at the LC(50) and LC(90) estimates. To classify a tick population in relation to resistance, three categories based on a statistical analysis of LC and RR between field populations and Mozo strains were defined: susceptible (no differences), incipient resistance (differences and RR(50)<2) and resistant (differences and RR(50)≥2). Eighteen field populations were tested with IVM and five of them presented a RR(50) range between 1.35 and 1.98 and the LC(50/90), which is statistically different from the Mozo strain (incipient resistance). However, the RR(90) increases ≥2 in four of the populations, confirming that tick resistance to IVM is emergent. The low RR values obtained could be a result of a low frequency of treatments with IVM. Twenty-seven tick populations were tested with fipronil and six were diagnosed as resistant according to the LIT. Cross-resistance was not observed between fipronil and IVM on these tick populations. The current study presents different R. (B.) microplus populations with an incipient resistance to IVM, and indicates that the fipronil tick resistance is restricted to certain areas in Uruguay.

Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) resistance to fipronil in Uruguay evaluated by in vitro bioassays.

Rhipicephalus (Boophilus) microplus obtained from four local populations in Uruguay (2007-2008) were subjected to various bioassay techniques to determine the presence of fipronil resistance within the country. Resistance ratios (RRs) obtained by larval immersion test varied between 3.3 and 3635 for tick populations subjected to treatment with fipronil for the last 3-7 years. The highest RR was observed in the population which received fewer treatments. Using discriminating concentration (8ppm) for larval immersion test, all field strains were correctly diagnosed as fipronil-resistant. This study presents the first diagnoses of cattle tick resistance to fipronil in Uruguay's field populations. It also highlights the importance of the possible conflict between programs to control agricultural pests and cattle ticks. The findings provide valuable information for selection and adoption of new control alternatives to manage drug resistance exhibited by cattle ticks.

In vitro tests to establish LC50 and discriminating concentrations for fipronil against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) and their standardization.

Laboratory test was carried out on larvae and adults of the cattle tick, Rhipicephalus (Boophilus) microplus, to determine fipronil toxicity. Adult immersion test (AIT, N=26), larval immersion test (LIT, N=71) and larval packet test (LPT, N=41) were standardized using susceptible strain (Mozo). Dose-response curves were compared with a fipronil resistant strain. Four variables were analyzed from AIT results: mortality, weight of eggs on day 7 and on day 14, index of fertility, and index of fecundity. For larval test, dose mortality curves were analyzed. In spite of the high LC(50) variability, all variables determined for AIT were appropriate to discriminate both strains. AIT and LIT had more sensitivity than LPT, with larger resistance factors. It was used two times LC(99.9) as discriminating doses (DCs) following FAO suggestion. For mortality by AIT, LIT and LPT the DCs were estimated: 4.98ppm, 7.64ppm and 2365.8ppm, respectively, for Mozo strain. DCs mortality values estimated for resistant strain by AIT, LIT and LPT were: 6.96x10(5)ppm, 343.26ppm and 5.7x10(3)ppm, respectively and their respective resistant factors were: 202.4, 5.36 and 1.52. Protocols for AIT, LIT and LPT have been presented in this paper.