Syringe immersion test as in vitro bioassay against Rhipicephalus microplus: Macrocyclic lactones dose-response relationship

Abstract

Background: For the diagnosis of tick sensitivity against different acaricides, there are in vitro and in vivo methods. The
main in vivo method, the stable test, is considered a defining methodology. In Uruguay, the Rhipicephalus microplus
(R. microplus) strain Mozo is used as the standard susceptible strain by the regulatory authorities. In vitro techniques
applied both on adult and larvae stages are validated by FAO and can serve as an orientation diagnosis of the resistance
profile developed in field conditions. An alternative was proposed as a modification of the larval immersion test (LIT),
where syringes were used seeking to reduce the work necessary to perform the original technique, resulting in the syringe immersion test (SIT).
Aim: The aim of this study was to expand the SIT for the characterization of sensitivity to Macrocyclic Lactones (MLs) in  R. microplus and provide information on field strain sensitivity of R. microplus larvae.
Methods: Log-logistic dose-response model for Ivermectin (IVM), Doramectin (DRM), and Moxidectin (MOX) were
performed using concentrations ranging from 0.01 to 20.0 ppm (n = 6, 3 replicates per level on each drug). Larvae
sensitivity results were determined after 24 hours of incubation at 27°C/90% RH, counting live/dead larvae. The final
model will be decided as the best fit according to the model selection AIC criteria for each drug. Pharmacodynamic
parameters [lower limit, slope, and effective dose at different levels (ED20, ED50, ED80, and ED95)] and its 95% confidence interval were considered for drug comparison.
Results: Dose-response models were fitted for IVM, DRM, and MOX. MOX had the lowest ED50 of the three drugs, implying that MOX is of higher potency (two folds) when compared to IVM and DRM on R. microplus larvae using SIT.  DRM had a different slope compared to IVM and MOX (p < 0.05), while IVM and MOX showed a similar slope (p > 0.05).
Conclusion: This study allowed us to standardize the technique for larvae immersion for each ML, granting a new tool
for in vitro test as a screening technique for tick sensitivity.