This study identified the optimal age of Trichostrongylus colubriformis larvae (L3) 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 L3 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 % (EC50), 90 % (EC90) and respective 95 % confidence intervals (95 %CI) for exsheathment inhibition were calculated at 60 min after exposure to chlorine solution. Motility of L3 (migration percentages (M%)) was recorded weekly using the larval migration test (LMT) as an indicator of L3 fitness over time. No correlation was found between L3 age or M% and the extract's EC50 or EC90 values. However, the EC50 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 EC50 and EC90 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 L3 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 (r2 = 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 L3 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.

中文翻译：

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在热带条件下进行体外幼虫抑制试验的最适毛滴虫幼虫（L3）年龄。

这项研究确定了在热带条件下的毛滴虫（Trichostrongylus colubriformis）幼虫（L3）的最佳年龄，以便使用幼虫抑制作用试验（LEIT）体外评估植物提取物。本研究使用了两种具有不同驱虫药（AH）敏感性状态的co.colubriformis分离株。两种分离物的L3均保持冷藏（4-5°C）直至使用。对于LEIT，使用不同稀释度（80-1200μg/ mL）的相思丙酮丙酮：水提取物储备液，每周进行分离株测试，持续16周。包括各自的阳性对照（左旋咪唑12.5 mM）和阴性对照（PBS）。在暴露于氯溶液后60分钟，计算出有效的50％（EC50），90％（EC90）和95％置信区间（95％CI）。每周使用幼虫迁移试验（LMT）记录L3的运动能力（迁移百分比（M％））作为L3适应性的指标。L3年龄或M％与提取物的EC50或EC90值之间没有相关性。但是，从第2-10周开始，戊糖提取物的EC50值范围为80至200μg/ mL。在第12周以后，幼虫的排出是不规则的，EC50和EC90值更高，CI范围更广，为95％。对于Paraiso分离株，在第7周的M％降低至85％以下；对于FMVZ分离株，在10周的M％降低。两种T. colubriformis菌株在L3的年龄和M％之间均观察到线性负相关。两个分离株的关系（斜率）相似，因此估计了一个描述所有M％数据的线性方程式（r2 = 0.771，df = 164，P <0.05）。因此，当使用这些T时。在热带条件下使用colubriformis分离株评估LEIT对植物提取物的AH活性时，L3的最佳年龄在2-7周之间，此时M％保持在85％以上。后者可以确保colubriformis的一致和可重现的脱皮结果。每个实验室必须确定最佳条件才能进行LEIT。