Discovering Acaricide Resistance in Rhipicephalus microplus: The Innovative Resistance Intensity Test (RIT) Explained

Ticks

In November and December 2023, researchers collected samples of R. microplus ticks from six ranches in Rio Grande do Sul, Brazil. Farmers and veterinarians submitted engorged female ticks to the Laboratory of Parasitology at the Instituto de Pesquisas Veterinárias Desidério Finamor. These samples were part of a program to check acaricide resistance. Farmers received guidance from the FAO on how to collect and submit ticks. Each sample contained around 200 ticks, sourced from 5 to 10 cattle, and was submitted within 48 hours of collection.

Preparation of Ticks

Researchers followed FAO guidelines for handling ticks. Upon arrival, ticks were washed, dried, and selected based on size (greater than 8.0 mm), condition, and vitality. Ninety ticks from each sample were used for the Adult Immersion Test (AIT). Additionally, 30 engorged female ticks were placed in a Petri dish and incubated at 27 ± 1 °C with 80-90% humidity for 14 days to allow egg laying. The eggs were later pooled and incubated in sealed serum vials to promote larval hatching. Larvae from 14-28 days old were then used in the Resistance Intensity Test (RIT).

Adult Immersion Test (AIT)

Researchers employed the AIT protocol by Drummond et al. to test the effectiveness of acaricides on adult ticks. They diluted acaricide solutions according to product labels: deltamethrin at 1:1000 and a combination product (cypermethrin, chlorpyrifos, and piperonyl butoxide) at 1:800. Groups of ten engorged female ticks, categorized by weight, were immersed in 100 mL of solution for 5 minutes. Control groups received distilled water. After immersion, ticks were dried and incubated at the same temperature and humidity conditions for 14 days. Egg production was assessed by weighing the egg mass and estimating larval hatching under a stereomicroscope. Each treatment was replicated three times.

Resistance Intensity Test (RIT)

For the RIT, researchers used commercial deltamethrin and Vectoclor, along with higher concentrations of each. Deltamethrin concentrations were 0.25 mg/mL, 1.25 mg/mL, and 2.5 mg/mL; cypermethrin concentrations were 0.56 mg/mL, 2.8 mg/mL, and 5.5 mg/mL; chlorpyrifos concentrations were 0.94 mg/mL, 4.69 mg/mL, and 9.94 mg/mL. The acaricide was diluted in a mixture of acetone and olive oil, then applied to filter papers. Each filter paper was folded and sealed for testing. Larvae from the engorged females were used in the RIT, and researchers recorded the number of live and dead larvae after 24 hours.

Test Read-Out and Statistical Analysis

In the AIT, researchers measured engorged female weights, egg mass weights, and larval hatch percentages. They calculated the index of fertility (IF) and in vitro efficacy using standard formulas. Statistical analysis included ANOVA and Tukey tests, with a decision table assessing tick resistance levels. Researchers also performed a Pearson correlation analysis on mortality percentages of larvae from RIT and AIT results.