猪蛔虫不同发育期幼虫的收集方法研究

Collection of Larvae of Different Developmental Stages of Ascaris suum

目的研究猪蛔虫不同发育期幼虫的收集方法。方法采用7.5%次氯酸钠氧化感染期蛔虫卵,37℃过夜后,用灭菌生理盐水离心数次,充分洗去次氯酸钠,将沉淀用玻璃珠在振荡器上振荡至全部卵壳破裂,幼虫孵出。用淋巴细胞分离液通过密度梯度离心法将幼虫与卵壳分离,最后收集底层的脱鞘第三期(L3)幼虫。在贝尔曼原理的基础上,在漏斗中加入低浓度(0.4%)琼脂凝胶,分别将绞碎的含幼虫的猪肝、肺和小肠内容物及其肠粘膜平均分装在数个漏斗中,放入40℃温箱中孵育3h,待大部分幼虫游离到管底,最后用400目分样筛过滤分离肝中的L3,用300目分样筛过滤分离肺中的L3,用200目分样筛过滤分离肠内容物及其粘膜上的第四期幼虫(L4)。结果次氯酸钠氧化加玻璃珠震荡法能够使感染期蛔虫卵几乎完全脱壳,配合淋巴细胞分离液的密度梯度离心法能收集到纯度较高且活力较强的体外脱鞘幼虫。与传统贝尔曼氏分离法相比,采用低浓度琼脂糖凝胶液能获得杂质少、高纯度的猪内脏中的不同发育期幼虫。结论次氯酸钠氧化加玻璃珠震荡法是体外脱鞘分离感染期幼虫的一种较为理想的方法,通过在漏斗内加低浓度的琼脂凝胶的方法是从猪内脏中获得高纯度的不同发育期幼虫的一种有效的方法。本方法不仅适用于猪蛔虫,而且对其它动物和人的寄生线虫幼虫的收集也有参考价值。

Objective To develop a more effective method for the collection of larvae of different developmental stages of A scaris s uurrt. Methods To isolate the infective third-stage larvae （L3）, infective eggs were treated with 7.5% NaClO at 37℃ for overnight. The egg shell layer was removed by mixing the eggs with glass-beads. The infective L3 were separated from shells by density gradient centrifugation with lymphocyte separating medium. L3 in livers and lungs, and L4 in intestines were isolated with a modified conventional Baerman＇s technique by using 0.4% agar-gel. Briefly, the minced liver and lung tissues and the content of intestine and intestinal mucosa were put into the funnels. The minced tissues were incubated at 40℃ for 4 hours, and larvae of different developmental stages were collected by passing through meshes of different sizes. Results Pure and viable larvae of different developmental stages were collected by this method. Egg shells were removed from infective eggs by the combination of NaClO treatment and shaking with glass-beads. The decorticated infective L3 could be isolated by density gradient centrifugation with lymphocyte separating medium. A clean suspension of L3 and L4 larvae could be obtained using the modified conventional Baerman＇s technique. Conclusion Combining both NaClO treatment and shaking with glass-beads is an effective method for isolating the infective larvae from the eggs. The modified Baerman＇s technique （with 0.4% agargel） is suitable for collecting L3 from liver and lung tissues and L4 from intestines. The establishment of this method would enable us to collect a large number of larvae for molecular and parasitological studies.