Influence of pH, Serum, and Cell Cultures on Development of Ascaris suum to Fourth Stage In vitro

Abstract

Ascaris suum larvae hatched from decoated eggs developed to parasitic stages in media KW-1, KW-1A, KW-1S, and KW-1SA, when each was used alone or as the culture fluid with swine kidney cell cultures. Media KW-1 and KW-1S were formulated (Douvres et al., 1966) with 50% bovine or swine serum, respectively, and medium NCTC 109, yeast extract, peptone, and dextrose; media KW-1A and KW-1SA are variations that were made by adjusting the pH to 6.9. Based on a culture period of 30 days, larvae developed to early or late phases of third stage in all 8 combinations, but to fourth stage only in systems with medium KW-1A. Despite yields of up to 90% late thirdstage larvae, only a few fourth stage were produced. The most rapid development, highest rate of survival, and largest yields of advanced stages were obtained in cultures of medium KW-1A with cells. The primary factors influencing development in the 8 culture combinations were pH, serum source, and the presence of cultivated cells. Previous attempts to cultivate Ascaris suum in complex cell-free media have had limited success (Pitts and Ball, 1953, 1955; Pitts, 1960, 1962, 1963; Cleeland and Laurence, 1962; Cleeland, 1963; Levine and Silverman, 1969). Since ascaris larvae traverse host tissues, it occurred to us that the cell culture system developed for in vitro cultivation of Stephanurus dentatus (Douvres et al., 1966) might provide a more favorable environment. Morphological criteria used to identify larval stages grown in vitro were developed chiefly from observations made during the present study and, to a lesser extent, on reevaluations of previous in vivo work (Roberts, 1934; Nichols, 1956). These criteria were subsequently used as the basis for a study of morphogenesis in vivo. Since the detailed descriptions arising from this work (Douvres et al., 1969) pertain equally to in vitro-grown larvae, we have limited descriptions in this report to certain features (stunting, delayed organogenesis, abnormalities) which are peculiar to larvae grown in vitro. MATERIALS AND METHODS Host tissues and sera used in the present study were obtained from animals raised and maintained as previously described (Douvres et al., 1966). Decoated embryonated ascaris eggs were from Stock I, prepared as previously described (Douvres et al., 1969). Media and cell cultures Eight combinations of media and cell cultures were used (Table I). Media KW-1 and KW-1S Received for publication 18 July 1969. were prepared as previously described (Douvres et al., 1966). After preliminary work with these media indicated that a lower initial pH was more favorable for ascaris, media KW-1A and KW-1SA were made by adjusting the pH from 7.9 to 6.9 with HC1. All pH measurements of stock media and of transferred and terminated cultures were made with a glass electrode in an open vessel. Primary cell cultures in 4-oz prescription bottles and in milk dilution bottles were prepared essentially as described by Douvres et al. (1966). However, they were grown and maintained in medium 199 (Morgan et al., 1950) supplemented with 20% calf serum. A cell line, subcultured from primary cells, was also grown and maintained in this medium in 32-oz prescription bottles. All vessels were provided with teflon-lined screw caps. Cell cultures were used when they showed a confluent sheet of cells, usually after 4 to 6 days. Adherence of the cells to the vessel was the criterion of viability. Hatching and preparation of the larval inoculum These procedures, with some exceptions, were essentially identical to those described by Cleeland and Laurence (1962). Eggs stored in 2% formalin at 5 C for 5 to 8 months were transferred to centrifuge tubes and placed in a water bath at 37 to 39 C for 2 hr. After 6 to 8 washes in sterile balanced salt solution (Fenwick, 1939), they were washed twice with medium NCTC 109 containing 1,000 units penicillin G potassium, 1 mg dihydrostreptomycin sulfate, and 10 jug Amphotericin B per ml. This preparation was also used as the hatching fluid. About 2 to 4 million eggs were transferred to 125-ml screw-capped flasks containing a spinner bar and the volume brought to 20 ml. The flasks were then held in a water bath (37 to 39 C) over a magnetic stirrer. After about 3 hr, 40 to 50% of the eggs had hatched. The contents of the flasks were transferred to tubes, centrifuged at 700 g for 5 min, and the supernatant fluid dis-