Proc. Helminthol. Soc. Wash. 53(2), 1986, pp. 256-262 Development of Ascaris suum from In Vivo-derived Third-stage Larvae to Egg-laying Adults In Vitro F. W. DOUVRES1 AND J. F. URBAN, JR. Helminthic Diseases Laboratory, Animal Parasitology Institute, Agricultural Research Service, USDA, Beltsville, Maryland 20705 ABSTRACT: A 2-step roller culture system was designed that supported optimally the development of late-third- stage 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 This phase predominates as larvae re-enter the suum in vitro have had varying degrees of success intestinal milieu of the natural host and begin (Taylor and Baker, 1968; Stromberg et al., 1977; development to mature adults in situ (Douvres Hansen and Hansen, 1978; Urban and Douvres, et al., 1969). These findings suggested that late 1981; Douvres and Urban, 1983; and Urban et L3 might require a simpler culture system for in al., 1984). The most successful cultivation meth- vitro development to mature adults than the L2, od was a 3-step roller culture system containing and that conditions used to cultivate advanced complex, cell-free media KW-2, API-1, and API- stages of other intestinal nematodes from infec- 18 with appropriately added supplements of re- tive L3 might effectively support the develop- ducing agents and bovine hemin and precisely ment of A. suum L3 obtained from rabbit lungs. applied gas phases that supported the develop- This concept was tested by inoculating in vivo- ment of second-stage larvae (L2), hatched from grown L3 of A. suum into a modified 1 -step eggs, to mature adult males and egg-laying fe- roller culture system used to grow Oesophagos- males (Douvres and Urban, 1983). tomum radiatum from infective larvae to young Other investigators have used a variety of cul- adults (Douvres, 1983). The system consisted of ture conditions to obtain high yields of fourth- medium API-1 plus L-glutathione (reduced) for stage larvae (L4) of A. suum in vitro from third- 7 days followed by medium API-1 plus bovine stage larvae (L3) obtained from the lungs of ex- hemin. It supported the development of A. suum perimentally infected animals (Sylk et al., 1974; L3 to mature adults. However, enhanced devel- Stromberg etal., 1977; Urban and Douvres, 1981; opment of A. suum L3 to mature adults also was Urban et al., 1984). The most effective of these attained with a 2-step culture system that used was a stationary multi-well system that used medium API-18 (Douvres and Urban, 1983) fol- RPMI 1640 medium plus serum (Urban et al., lowed by API-1 plus hemin. 1984). Urban and Douvres (1981) observed that This report describes the preparation of roller more than 95% of the larvae obtained from A. culture systems with combinations of 1 or 2 me- suum infected rabbits at 7 days after oral inoc- dia (KW-2, API-18, API-1, RPMI 1640) with or ulation with eggs were in the late phase of L3. without supplements (reducing agents, bovine hemin, or serum) that were used to grow A. suum L3. In addition, 2 new media derived from 1 Retired, January 1986. API-1 (API-22 and API-23) were prepared and 256 Copyright © 2011, The Helminthological Society of Washington 257 tested to determine the active ingredients in API-1 Table 1. Comparison of media composition of API-1, necessary to promote development of A. suum API-22, and API-23. to mature adults. API-1 stock solutions* API-22 API-23 Materials and Methods Chemically undefined PREPARATION OF MEDIA: Medium API-1 (Douvres Bovine calf serum and Malakatis, 1977) was used alone or supplemented Rabbit embryo extract with 10 mM glutathione (reduced). Media KW-2 and Calf liver extract API-18 (Douvres and Urban, 1983) and API-1 were Bactopeptone used with and without bovine hemin (Hemin Type I: Trypticase Bovine, Sigma Chemical Company, St. Louis, Mis- Yeast extract souri) at a final concentration of 24 /ug/ml. Casein Medium RPMI 1640+S was prepared with RPMI 1640 (M. A. Bioproducts, Walkersville, Maryland) plus Chemically denned a supplement of 25% (v/v) bovine calf serum (obtained MEM essenital amino acids from 4-mo-old calves maintained helminth-free at the MEM nonessential amino acids Animal Parasitology Institute) that had been heat-in- L-cysteine-HCl, ascorbic acid activated for 30 min at 56°C. Media API-22 and API- L-glutamine 23 were new formulations (Table 1) that evolved from N-acetyl-D-glucosamine modifications of medium API-1; they were used freshly Vitamin mixture prepared or after storage at - 20°C for up to 6 months. Nucleic acids These media were used alone or with a supplement of Sugar mixture 24 /ug/ml bovine hemin. Carboxylic acids All media contained 1,000 units/ml of penicillin G Salt mixture potassium, 1 mg/ml of streptomycin sulfate and 10 ng/ ml Fungizone, and were adjusted to pH 6.8 with the * Groups of stock solutions used to prepare medium API-1 addition of either 1 N HC1 or 1 N NaOH as needed. (Douvres and Malakatis, 1977). ANIMAL INFECTIONS AND PREPARATION OF LARVAL INOCULUM: New Zealand white rabbits (males and fe- and 35 (Douvres et al., 1966). Large larval stages or males) of approximately 2-3 kg were obtained locally adults were transferred with a pick to avoid damage and maintained in wire-based cages. Rabbits were in- (Douvres and Urban, 1983). oculated orally with 75,000 decoated, embryonated eggs Checks for sterility of all stocks of media and eval- of A. suum (Urban and Douvres, 1981), and lungs were uation of development of the cultured nematodes were removed 7 days after infection. Larvae were recovered performed (Douvres et al., 1966). Cultures were judged from the lungs of infected rabbits, freed of host tissue, free of contamination if there was no visible evidence and prepared for inoculation of cultures (Urban and of fungi or bacteria. Adults were killed and fixed in hot Douvres, 1981). The stage of development of larvae buffered 5% formalin, cleared in phenol-alcohol, and was determined by the morphological characters de- were studied in temporary wet mounts. The larval stages scribed by Douvres et al. (1969). The recovered larval and adults were classified according to Douvres et al. population was composed of greater than 95% mid to (1969), Pilitt et al. (1981), Tromba (1978), and from late L3; the remainder were early L3. original observation. Fertilized and unfertilized eggs PREPARATION AND HANDLING OF CULTURES: Proto- oviposited by adult females were identified according cols for preparation and handling of 10 different roller to Alicata (1936) and Poor (1967). culture systems (A through J) and data on the number of trials and cultures involving each system are given in Table 2. Systems A, B, and C each used a distinct Results medium for the first 7 days of culture that was then Advanced development from L3 was obtained altered only by the addition of hemin from day 7 to in all trials of the 10 culture systems (Table 2). termination. Systems D and E contained the same me- dium from day 0 to 7 in culture, but it was altered Development from L3 to mature adult males and subsequently by the removal of glutathione (reduced) ovipositing females was obtained in 5 systems (system D) or the removal of glutathione (reduced) and (E, G, H, I, J), but was best in systems E and J. the addition of hemin (system E). These systems were Only results from systems E and J will be de- considered as involving a single basic medium com- scribed in detail. position and, for the purposes of discussion, were de- scribed as 1-step systems. Systems F through J each MORPHOGENESIS, HEALTH, AND GROWTH: Third- involved marked changes in medium composition af- stage larvae obtained after 7 days in rabbits de- ter the first 7 days of culture and were, therefore, de- veloped to mature adult males and egg-laying scribed as 2-step systems. females by day 53 in culture (systems E and J, The roller bottle cultures were prepared and gassed Table 3). In system E, L3 advanced to early L4 with a mixture of 85% N2/5% O2/10% CO2 (Douvres and Malakatis, 1977).