Age of Haemonchus contortus third stage infective larvae is a factor influencing the in vitro assessment of anthelmintic properties of tannin containing plant extracts

The larval exsheathment inhibition assay (LEIA) of infective larvae (L-3) is an in vitro method used to evaluate the anthelmintic (AH) activity of tannin-containing plant extracts against different species of gastrointestinal nematodes, including Haemonchus contortus. Some conditions remain to be defined in order to standardize the LEIA, i.e. the optimal age of larvae produced from donor animals to use in the assays. Therefore, this study aimed at identifying the effect of age and age-related vitality of H. contortus infective larvae produced under tropical conditions, on the in vitro AH activity measured with the LEIA. The same acetone:water (70:30) extract from Acacia petutatula leaves was used to perform respective LEIA tests with H. contortus L-3 of different ages (1-7 weeks). Each week, the L3 were tested against different concentrations of extract (1200, 600, 400, 200, 100, 40 mu g/mL of extract) plus a PBS control. Bioassays were performed with a benzimidazole (Bz) resistant H. contortus (Paraiso) strain. In order to identify changes in L3 vitality on different weeks (1-7), two assays testing larval motility were included only with PBS: the larval migration assay (LMA) and the larval motility observation assay (LMOA). Mean effective concentrations causing 50% and 90% exsheathment inhibition (EC30, EC90) were obtained for every week using respective Probit analyses. On the first week, the larvae had lowest EC50 and EC90 (39.4 and 65.6 mu g/mL) compared to older larvae (P < 0.05). The EC50 and EC90 for weeks 2-5 were similar (P > 0.05), while older larvae tended to show higher EC30 and EC90 (P < 0.05). Motility showed strong negative correlations with age of larvae (r >= -0.83; P < 0.05) and EC50 (r >= -0.80; P < 0.05), suggesting that the lower extract efficacy could be associated with decaying vitality of larvae associated with age. More stable efficacy results were found between two to five weeks of age.