Optimal age of Trichostrongylus colubriformis larvae (L) for the in vitro larval exsheathment inhibition test under tropical conditions.

This study identified the optimal age of Trichostrongylus colubriformis larvae (L) under tropical conditions for the in vitro evaluation of plant extracts using the larval exsheathment inhibition test (LEIT). Two T. colubriformis isolates with different anthelmintic (AH) susceptibility status were used for this study. The L of both isolates were maintained on refrigeration (4-5 °C) until use. For the LEIT, the isolates were tested every week during 16 weeks, using a stock solution of Acacia pennatula acetone:water extract at different dilutions (80-1200 μg/mL). Respective positive controls (levamisole 12.5 mM) and negative controls (PBS) were included. Effective concentrations 50 % (EC), 90 % (EC) and respective 95 % confidence intervals (95 %CI) for exsheathment inhibition were calculated at 60 min after exposure to chlorine solution. Motility of L (migration percentages (M%)) was recorded weekly using the larval migration test (LMT) as an indicator of L fitness over time. No correlation was found between L age or M% and the extract's EC or EC values. However, the EC values for the A. pennatula extract ranged from 80 to 200 μg/mL from weeks 2-10. Beyond week 12, larval exsheathment was irregular, with higher EC and EC values and wider 95 %CI. The M% decreased below 85 % on week 7 for Paraiso isolate, and on week 10 for FMVZ isolate. A linear negative relationship was observed between the age of L and M% for both T. colubriformis isolates. The relationship (slope) for both isolates was similar therefore a single linear equation was estimated describing all M% data (r = 0.771, df = 164, P < 0.05). Thus, when using these T. colubriformis isolates under our tropical conditions for the evaluation of AH activity of plant extracts with LEIT, the optimal age of L is between weeks 2-7, when M% remained above 85 %. The latter may ensure consistent and reproducible exsheathment results for T. colubriformis. Each laboratory must identify optimal conditions to perform the LEIT.