The Influence of NCTC 109, Serum, and Swine Kidney Cell Cultures on the Morphogenesis of Stephanurus dentatus to Fourth Stage, In vitro

Abstract

Stephanurus dentatus was grown to advanced parasitic stages in a new static in vitro system, free of bacteria and fungi. Criteria for evaluation were based on anatomical characters of the head, stoma, excretory system. and intestine. Descriptions of these characters and observations on morphogenesis not previously reported from in vivo specimens are given in detail. Development to late fourth stage was obtained in cultures consisting of primary monolayers of swine kidney cells overlaid with the following medium (KW-1S): yeast extract (BBL), 112.5 mg; Bacto-peptone (Difco), 140.65 mg; dextrose (BBL), 140.65 mg; swine serum, 50 ml; NCTC 109, 50 ml; and antibiotics to give a final concentration of 1,000 units penicillin G potassium, 1 mg dihydrostreptomycin, and 10 tLg Amphotericin B per ml of medium. When bovine serum was substituted (KW-1) development halted at mid fourth stage. In addition, with both fluid media, the following were observed: only larvae in fourth stage fed on the kidney cell cultures; a definite feeding pattern occurred; larvae fed on rolled cell layers (presumably dead) as well as living cells; digestion and egestion took place, and there was a correlation between feeding behavior and anatomical development of the stoma, excretory system, and intestine. In either medium, without the cell culture, only early fourth stage was attained and yields of this phase of development were reduced to one-fortieth. Media KW-1 and KW-1S evolved from attempts to culture S. dentatus in modifications of Pitts' and Ball's (1953) medium, or in NCTC 109 (McQuilkin et al., 1957) used alone or supplemented with calf serum. The prototype cell-free media, one of which was also supplemented with nonliving tissue, had the following effects on development: (1) the most advanced stage attained was early fourth stage; (2) larvae advanced to late parasitic third stage in media supplemented with either NCTC 109 or serum; (3) development proceeded to early fourth stage when both NCTC 109 and serum were present; (4) a more rapid rate of development to third molt and early fourth stage ensued when NCTC 109 and serum were present at concentrations of 50%, each; and (5) although fourth-stage larvae ingested nonliving tissues when available, development was not enhanced. In swine, Stephanurus dentatus Diesing, 1839 undergoes its entire parasitic development to the fifth stage (young adult) in the liver and to the sexually mature adult in the kidney and perirenal fat (Schwartz and Price, 1929, 1931, 1932; Clunies Ross and Kauzal, 1932). In unpublished observations, one of us (FGT) has noted that young and sexually mature adults invariably have host tissue in their intestine. We assumed, therefore, that host tissues are probably utilized in vivo; and since S. dentatus has been cultured only to early fourth stage in cell-free media (Douvres and Tromba, 1962), we conjectured that host tissue may also be necessary for advanced parasitic development in vitro. Accordingly, attempts were made to cultivate advanced stages of S. dentatus in the following three static systems: (1) eight cell-free media not previously used for S. dentatus; (2) two media (KW and KW-1S) selected from system 1 and Received for publication 8 April 1966. supplemented with nonliving tissues; and (3) two media (KW-1 and KW-1S) selected from system 1 and used as an overlay for primary swine kidney cell cultures. Media KW, KW-1, and KW-1S (Table I) are new formulations that evolved from our attempts to culture S. dentatus in modifications of Pitts' and Ball's (1953) medium or in the chemically defined tissue culture medium NCTC 109 (McQuilkin et al., 1957). The latter was used alone or supplemented with calf serum. Pitts' and Ball's medium, in addition to serum, includes yeast extract, peptone, glucose, and a balanced salt solution (Fenwick, 1939). These investigators were unable to obtain development or growth of Ascaris suum larvae that were hatched from eggs in this medium. Weinstein and Jones (1959), Sawyer and Weinstein (1961, 1962, and 1963), and Weinstein et al. (1963) reported on their attempts to cultivate larval stages of several nematode parasites of vertebrates in NCTC