Maintenance In vitro of Larval Schistosoma mansoni in Tissues from the Snail, Australorbis glabratus

Abstract

Portions of digestive gland and ovotestis were removed from aquarium-reared Australorbis glabratus about 40 days after infection with Schistosoma mansoni. The tissues were minced and explanted in flasks under sterile conditions. The cultures were maintained at 26 to 28 C either on a complex medium, or, with generally superior results, on a balanced salt solution containing glucose and trehalose (BSS). Medium was changed daily and cultures were evaluated by counting the emerged cercariae and by histologic study of the tissues. Apparently normal cercariae emerged daily in relatively large numbers during the 1st week, and in decreasing numbers during the succeeding 1 to 2 weeks. Omission of the sugars from BSS adversely affected the yields of cercariae; addition of amino acid mixtures to BSS did not increase the amount or duration of cercarial emergence. At least in part, cercarial emergence was found to be influenced by light. A small proportion of culture-derived cercariae was infective for mice by skin penetration or intraperitoneal injection. Tissues harboring infections were also explanted, i.e., from snails 19 days after exposure to S. mansoni. It was demonstrated that cercarial embryos were capable of undergoing maturation in vitro when cercariae appeared in these cultures after a lag of 8 to 13 days. Intramolluscan larval trematodes have received little study in vitro. The few published accounts indicate that immature rediae of Cyclocoelum microstomum from miracidia survived at most for 14 days in complex media (Ingersoll, 1956), and that rediae of Fascioloides magna from snails survived up to 10 days in solutions of individual substrates (Friedl, 1961). The axenic rediae did not grow or produce succeeding larval stages in vitro. Techniques are now described for the study in vitro of larval Schistosoma mansoni in tissues from parasitized Australorbis glabratus. It has been demonstrated, apparently for the first time, that trematode cercariae can develop and emerge in vitro and that under certain circumstances some of them are infective. MATERIALS AND METHODS Maintenance and infection of snails Snails were from a Puerto Rican strain (PR-2) of A. glabratus colonized in our laboratory since 1957 (Chernin and Schork, 1959) and maintained on watercress and romaine lettuce. Groups of 100 snails, 4 to 8 mm in diameter, were mass-exposed in finger bowls to miracidia of a Puerto Rican strain of S. mansoni (Chernin and Dunavan, 1962). Snails were then maintained on lettuce at 25 C, in darkness, in covered, aerated, stainless-steel pans. The snails were isolated in vials and examined for cercarial emergence 38 to 41 days postexposure, except as noted. Snails yielding cercariae at this time were used in experiments within 24 hr; these usually measured 12 to 16 mm in diameter. Composition of solutions Two solutions, previously developed in snail tissue culture studies (Chemrnin, 1963), were compared as media. The first is a complex nutrient medium (TC-NM) consisting mainly of a balanced salt solution and sugars (BSS), plus small amounts of vertebrate serum, amniotic fluid, embryo extract, lactalbumen hydrolysate, and yeast extract. The second solution is the BSS which contains several salts and 100 mg each of glucose and trehalose per 100 ml. The TC-NM was adjusted before use to pH 7.4 to 7.5 with 0.1 N NaOH; the BSS required no adjustment for a similar pH. All media were supplemented with 200 units penicillin G and 200 pg streptomycin sulfate per ml. Securing and processing snail tissues Glassware and instruments were sterile, all procedures were done in a hood, and a stereomicroscope was used in dissection. Snails were blotted dry, wiped with 70% alcohol, and kept temporarily Received for publication 27 April 1964. * These studies were supported in part by research grant AI-00513 and by a Research Career Award from the National Institute of Allergy and Infectious Diseases, U. S. Public Health Service.