# Functional and sequence-based comparison of Ctenocephalides felis and Rhipicephalus sanguineus sensu lato isolates from different geographic regions

## TL;DR

This study compares fleas and ticks from different regions to see if they respond similarly to a pesticide, suggesting fewer tests may be needed for approval in Europe.

## Contribution

The study demonstrates that ectoparasite isolates from different countries are interchangeable for regulatory testing purposes.

## Key findings

- No mutations affecting pesticide sensitivity were found in Ctenocephalides felis or Rhipicephalus sanguineus isolates.
- High intraclass correlation coefficients confirmed similar in vitro pesticide sensitivity across isolates from different regions.
- Results support reducing in vivo studies for European marketing authorization of ectoparasiticides.

## Abstract

Current regulatory requirements for European marketing authorization of canine and feline ectoparasiticides include dose confirmation studies conducted with European isolates of each ectoparasite species indicated in the proposed labeling, in addition to any studies conducted against non-European ectoparasite isolates. This regulatory requirement may be deemed unnecessary if no significant differences exist among laboratory ectoparasite isolates obtained from various countries. Between-country interchangeability was examined through various comparative studies by using Ctenocephalides felis fleas and Rhipicephalus sanguineus sensu lato ticks sourced from multiple countries. To detect potential alterations that may influence fluralaner binding efficacy, comparative complementary DNA (cDNA) and genomic DNA sequence analyses were performed. These analyses focused on the regions coding for the amino acid residues involved in fluralaner binding in the γ-aminobutyric acid receptor (GABAR, subunits encoded by Rdl) and the glutamate-gated chloride channel (GluCl) of the mentioned parasite isolates. Additionally, their in vitro fluralaner sensitivities were compared.

Laboratory in vivo-reared C. felis and R. sanguineus isolates were sourced from Australia (fleas only), Europe, and USA (both fleas and ticks). Genomic DNA and cDNA sequences coding for GABAR and GluCl were analyzed for variations that could result in alterations of fluralaner and dieldrin binding. For in vitro testing, three replicates of 20 fleas per isolate were exposed to fluralaner-impregnated filter paper at increasing concentrations. In total, three replicates of ten ticks per concentration were immersed for approximately 5 min in fluralaner dilutions. Untreated control replicates were included for all comparisons. Intraclass correlation coefficients were calculated to assess the overall similarity of tick/flea mortality and inhibition rates between different isolates.

No mutation or alteration that could affect the GABAR or GluCl isoxazoline binding efficacy was found in any of three C. felis or two R. sanguineus isolates. The predicted lethal and effective concentrations of all tested isolates fell within a narrow range. High intraclass correlation coefficients confirmed an overall similarity between tick (Europe and USA) and flea isolates (Europe, USA, Australia) in the in vitro fluralaner sensitivity assay, aligning with the expected susceptibility based on the DNA sequence analysis of each isolate.

There was no evidence of target-related or functional differences in fluralaner sensitivity between either C. felis or R. sanguineus laboratory isolates from different countries. These findings indicate the between-country interchangeability of results of in vivo dose confirmation studies and justify reducing the number of in vivo studies required for European marketing authorization of canine and feline ectoparasiticides.

The online version contains supplementary material available at 10.1186/s13071-025-06806-y.