Third-stage Larvae to Egg-laying Adults In Vitro

A 2-step roller culture system was designed that supported optimally the development of late-thirdstage larvae (L3) of Ascaris suum, obtained from rabbit lungs, to mature adults in vitro. The culture system consisted of 1) medium API-18 for 7 days, and 2) thereafter, medium API-1 supplemented with bovine hemin. Cultures were gassed with 85% nitrogen/5% oxygen/10% carbon dioxide and incubated at 39°C. Under these conditions, L3 developed to fourth molt, young adults, and mature adults at 14, 20, and 53 days in culture, respectively. Morphogenesis of larval and adult stages in vitro was similar to that of worms of a comparable stage obtained from swine. The rate of development of late L3 to late fourth stage (L4) was similar to that of larvae developing in vivo, but development of late L4 to adults in vitro was delayed. Fertilized eggs were produced by females in cultures containing mature males. The eggs were comparable morphologically to eggs from females isolated from swine, but somewhat smaller. Late L3 developed into mature males and females ovipositing fertilized eggs in 3 other 2-step culture systems and a 1-step culture system. Medium API-1 plus hemin or a modification of API-1 that contained its complex mammalian tissue extracts and peptide digests (medium API-23 plus hemin) was essential. Copulation was observed for the first time between A. suum mature adult males and females in an extant culture of the 1-step culture system. Attempts to grow advanced stages of Ascaris suum in vitro have had varying degrees of success (Taylor and Baker, 1968; Stromberg et al., 1977; Hansen and Hansen, 1978; Urban and Douvres, 1981; Douvres and Urban, 1983; and Urban et al., 1984). The most successful cultivation method was a 3-step roller culture system containing complex, cell-free media KW-2, API-1, and API18 with appropriately added supplements of reducing agents and bovine hemin and precisely applied gas phases that supported the development of second-stage larvae (L2), hatched from eggs, to mature adult males and egg-laying females (Douvres and Urban, 1983). Other investigators have used a variety of culture conditions to obtain high yields of fourthstage larvae (L4) of A. suum in vitro from thirdstage larvae (L3) obtained from the lungs of experimentally infected animals (Sylk et al., 1974; Stromberg etal., 1977; Urban and Douvres, 1981; Urban et al., 1984). The most effective of these was a stationary multi-well system that used RPMI 1640 medium plus serum (Urban et al., 1984). Urban and Douvres (1981) observed that more than 95% of the larvae obtained from A. suum infected rabbits at 7 days after oral inoculation with eggs were in the late phase of L3. 1 Retired, January 1986. This phase predominates as larvae re-enter the intestinal milieu of the natural host and begin development to mature adults in situ (Douvres et al., 1969). These findings suggested that late L3 might require a simpler culture system for in vitro development to mature adults than the L2, and that conditions used to cultivate advanced stages of other intestinal nematodes from infective L3 might effectively support the development of A. suum L3 obtained from rabbit lungs. This concept was tested by inoculating in vivogrown L3 of A. suum into a modified 1 -step roller culture system used to grow Oesophagostomum radiatum from infective larvae to young adults (Douvres, 1983). The system consisted of medium API-1 plus L-glutathione (reduced) for 7 days followed by medium API-1 plus bovine hemin. It supported the development of A. suum L3 to mature adults. However, enhanced development of A. suum L3 to mature adults also was attained with a 2-step culture system that used medium API-18 (Douvres and Urban, 1983) followed by API-1 plus hemin. This report describes the preparation of roller culture systems with combinations of 1 or 2 media (KW-2, API-18, API-1, RPMI 1640) with or without supplements (reducing agents, bovine hemin, or serum) that were used to grow A. suum L3. In addition, 2 new media derived from API-1 (API-22 and API-23) were prepared and