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Abstract：
The objective of this study was to analyze the mRNA transcription levels of ten functional genes of P-glycoproteins (P-gp) in free life stages, eggs and infective larvae (L3) and in endoparasitic stages, fourth larval stage (L4) and adult males of two native isolates of Haemonchus contortus: resistant and susceptible to IVM. The IVM resistant isolate was obtained from sheep naturally infected with H. contortus, and the susceptible isolate (with no pressure to IVM) conserved since 1990. The lethal effect of IVM was evaluated under in vitro conditions, which showed significant differences between susceptible and resistant H. contortus L3 isolates (P < 0.01). The IVM susceptible isolate revealed a lethal effect of 79.22% at 11.42 mM, whereas that resistant isolate showed no lethal effect at any of the four assessed concentrations (1.43, 2.85, 5.71 and 11.42 mM) of IVM. The expression levels of ten Hco-pgp genes (1, 2, 3, 4, 9, 10, 11, 12, 14, and 16) were evaluated in the resistant isolate of H. contortus and compared to the susceptible isolate (as control), using two constitutive genes (GAPDH and β-tubulin). Up-regulation at two statistical significant values (P ≤ 0.05, 0.1) was the criterion to associate IVM resistance with the free life and endoparasitic stages of H. contortus. The expression levels in H. contortus adult nematodes showed 5.64 to 127.56-fold increase for Hco-pgp genes 1, 9, 12, 14, and 16, followed by an increase for Hco-pgp-2 (49.75-fold) and Hco-pgp-10 (106.40-fold) in L4, and for Hco-pgp-16 (2.90-fold) in eggs (P ≤ 0.05). In addition, high expression levels with P < 0.1 were detected in H. contortus L3, L4, and adults for Hco-pgp genes 1, 4, 11, 12, and 16, with changes ranging from 2.17 to 29.72-fold. In conclusion, the highest expression was observed in the adult stage of H. contortus, and the most frequent gene with a significant P-value was Hco-pgp-16, revealing it plays an important role in IVM resistance.
摘要：
本研究的目的是分析10个P-糖蛋白（P-gp）功能基因在自由生命阶段的mRNA转录水平，卵和感染性幼虫（L3）以及内寄生阶段，幼虫的第四期（L4）和成年雄性的两个本地分离株hemonchuscontortus：对IVM具有抗性和易感性。IVM抗性分离株是从自然感染H.contortus的绵羊中获得的，和自1990年以来保存的易感隔离物(对IVM没有压力)。在体外条件下评估了IVM的致死作用，易感和抗性H.contortusL3分离株之间存在显着差异（P<0.01）。IVM敏感分离株在11.42mM时显示出79.22%的致死效应,而该抗性分离株在四个评估浓度（1.43、2.85、5.71和11.42mM）的IVM中的任何一个均无致死作用。在H.contortus的抗性分离株中评估了十个Hco-pgp基因（1、2、3、4、9、10、11、12、14和16）的表达水平，并与易感分离株（作为对照）进行了比较。使用两个组成型基因(GAPDH和β-微管蛋白)。在两个统计学上显着的值（P≤0.05，0.1）上调是将IVM抗性与H.contortus的自由寿命和内寄生阶段相关联的标准。Hco-pgp基因1、9、12、14和16在H.contortus成年线虫中的表达水平增加了5.64至127.56倍，其次是Hco-pgp-2（49.75倍）和L4中的Hco-pgp-10（106.40倍）和Hco-pgp-16（2.90倍）在鸡蛋中（P≤0.05）。此外,在H.contortusL3，L4和成虫中检测到Hco-pgp基因1、4、11、12和16的高表达水平，P<0.1，变化范围为2.17至29.72倍。总之,在扭曲H.的成虫阶段观察到最高的表达，最常见的具有显著P值的基因是Hco-pgp-16，表明它在IVM抗性中起重要作用。
