Strigeid trematodes (Cotylurus lutzi) cultured in vitro: production of normal eggs with continuance of life cycle.

Tetracotyles of Cotylurus lutzi were incubated to adults in a basic medium of 40% NCTC135 and 40% chicken serum, supplemented with 20% extract of chicken heart, liver, muscle, intestine wall, or mucosa. Ten per cent or more of eggs produced in media containing intestine extracts appeared normal, but miracidial development occurred only when upper small intestine mucosal extract was used. Five Biomphalaria glabrata snails became infected by such miracidia, shedding cercariae which encysted as tetracotyles and developed into normal adults in zebra finches. Dialysates of the effective mucosal extract failed to stimulate production of normal eggs when added to the basic medium. The strigeid trematode Cotylurus lutzi Basch, 1969, can be grown in vitro from tetracotyle to adult by the method of Voge and Jeong (1971). Their medium contains equal parts of NCTC-135 and chicken serum (CS), plus 100 units of penicillin and 100 tug of streptomycin per ml. Worms develop well in this but produce only fragile, shell-less eggs incapable of embryonation. We have modified this medium so that normal eggs, yielding infective miracidia, are produced by worms grown entirely in vitro. MATERIALS AND METHODS Young adult snails, Biomphalaria glabrata (Say), were exposed to large numbers of cercariae of C. lutzi. After 1 to 2 months the snails were washed and their shells gently crushed in 0.34% NaCl in distilled water. The ovotestes, containing encysted tetracotyles, were removed and teased apart in sterile 0.34% saline containing 10 ,ug gentamicin, 50'0 units of penicillin, and 500a ,g streptomycin per ml. Freed organisms were rinsed 4 times in this solution. Cultures were set up in plain-lipped 25by 150-mm glass test tubes containing 5 ml of medium. About 15 well-encysted tetracotyles were placed into each tube, which was closed by a silicone rubber stopper and then sealed by a tightly wound strip of parafilm. No special gas phase was used. Basic culture medium contained 40% NCTC-135 and 40% CS (Grand Island Biological Co.), plus 100 units of penicillin, 100 jug streptomycin, and 10 jug gentamicin per ml. This was supplemented Received for publication 29 August 1972. * Supported by Grant AI-10271 from the NIAID, NIH, Bethesda, Maryland. t At present student, School of Medicine, UCLA, Los Angeles, California. with various extracts of chicken origin. From 4 freshly killed 2to 3-kg adult white leghorn chickens, separate extracts of liver, breast muscle, heart, and intestinal wall muscle were prepared. About 15 g of tissue was finely minced, then ground with a mortar and pestle, and finally squeezed through silk bolting cloth. The resulting liquid was made up to 20 ml with normal saline and centrifuged for 60 min at 3,000 rpm at 5 C. The supernatant fluid was filtered twice through paper, then through 0.65and 0.45-Am Millipore filters for sterilization. For extracts of chicken intestinal mucosa, the small intestine was cut at the gizzard and at the beginning of the large intestine (as determined by texture changes and the appearance of fecal masses). This portion, approximately 70 cm, was divided into 2 equal lengths, referred to as "upper" and "lower" small intestine. Each half was cut open longitudinally and washed in several changes of cold (5 C) normal saline. A glass microscope slide was used to scrape off the mucosal cell layer, which was then collected and homogenized in a glass tissue grinder with teflon plunger. Mucosal homogenates from the upper and lower small intestine were then treated as described above. Dialysis of the upper small intestine mucosal extract was done through cellulose tubing (pore size 2.4 nm) overnight at 5 C against 1,000 ml of normal saline or of distilled water. In the former case, 6 ml of extract was used; in the latter, 7 ml. Following dialysis against distilled water, NaCl was added to restore the saline concentration of the dialysate, which in both cases was sterilized using 0.65and 0.45-utm Millipore filters. After filter sterilization, all extracts were stored at -10 C until used. Egg yolk medium was prepared as described by Berntzen and Macy (1969). Extracts were added to the basic medium as shown in Table I. Media were changed on alternate days. In any one tube, organisms were maintained in the same formulation throughout; when extract prepared from one chicken was used up, the homologous material from the next chicken