Gray M A, Cunningham I, Gardiner P R, Taylor A M, Luckins A G
Parasitology. 1981 Feb;82(1):81-95. doi: 10.1017/s0031182000041883.
Two stocks of Trypanosoma congolense were established in culture at 28 degrees C using trypanosomes from the proboscides of infective Glossina morsitans. Successful primary cultures were initiated by placing an infected tsetse proboscis beside a bovine dermal collagen explant in Eagle's minimum essential medium supplemented with foetal calf serum. The trypanosomes multiplied rapidly in the medium and also gradually formed an adherent layer o the plastic surface of the culture vessel. Three primary cultures produced organisms infective for mice from 14, 20 and 35 days after initiation and thereafter continuously until days 76, 76 and 52 when they were discarded. Four attempts to initiate infective cultures using infected tsetse proboscides in medium without dermal explants were unsuccessful. When trypanosomes from primary cultures were placed in culture medium with proboscides from uninfected tsetse flies, the parasites multiplied, formed an adherent layer in the culture flasks and were seen in the proboscides within 24 h. A line of 1 stock was serially sub-passaged in this way 4 times during a period of 215 days. Infectivity titrations in mice indicated that primary and sub-passaged cultures each contained similar numbers of infective organisms. Another line of the same stock was also sub-passaged 4 times in medium alone over a period of 186 days. These sub-cultures again retained infectivity for mice, but titrations showed a decrease in infective organism production in the 4th sub-culture. Primary and sub-passaged cultures all included a variety of morphologically different developmental forms of T. congolense, closely resembling those described in the labrum and hypopharynx of Glossina by previous workers. Short metacyclic-like trypanosomes and organisms with proteinaceous surface coats were present in infective cultures. Cultures were successfully re-established after cryopreservation at -196 degrees C and retained the ability to produce infective organisms.
使用来自感染性采采蝇(Glossina morsitans)喙部的锥虫,在28摄氏度下建立了两株刚果锥虫(Trypanosoma congolense)培养物。通过将感染的采采蝇喙部放置在补充有胎牛血清的伊格尔最低限度基本培养基中的牛真皮胶原外植体旁边,成功启动了原代培养。锥虫在培养基中迅速繁殖,并逐渐在培养容器的塑料表面形成附着层。三次原代培养在启动后14、20和35天产生了对小鼠有感染性的生物体,此后一直持续到第76、76和52天被丢弃。四次尝试在没有真皮外植体的培养基中使用感染的采采蝇喙部启动感染性培养均未成功。当将原代培养物中的锥虫置于含有未感染采采蝇喙部的培养基中时,寄生虫繁殖,在培养瓶中形成附着层,并在24小时内在喙部中可见。在215天的时间里,一株培养物以这种方式连续传代4次。小鼠感染性滴定表明,原代培养物和传代培养物中每种都含有相似数量的感染性生物体。同一株的另一系也在仅培养基中在186天的时间里传代4次。这些传代培养物再次对小鼠保持感染性,但滴定显示在第4次传代培养中感染性生物体的产生有所减少。原代培养物和传代培养物均包括刚果锥虫各种形态不同的发育形式,与先前研究人员在采采蝇唇和下咽中描述的非常相似。感染性培养物中存在短的类循环后期锥虫和具有蛋白质表面被膜的生物体。在-196摄氏度冷冻保存后,培养物成功重建,并保留了产生感染性生物体的能力。