In vitro Cultivation of Spirometra mansonoides (Cestoda) from the Procercoid to the Early Adult

Abstract

Spirometra mansonoides has been grown in vitro from the procercoid to the young adult using the methods of Mueller (1959) and Berntzen (1961, 1962, 1964). The process involves scolex differentiation, shedding of the larval body, growth from 0.1 mm to 64 mm in length, and segmentation to form the strobila. Successful cultures had a gas phase of 95% N2/5% CO2 or 90% N2/10% CO2 and were preceded by an evaginating solution containing bile salts and trypsin. The medium is complex but does not contain chick embryo extract. Incubation temperature was 39 C. Oxygen is toxic to the cultures. Lack of C02 or omission of the evaginating solution resulted in no growth. Essentially twothirds of the life cycle of this tapeworm has now been accomplished in vitro in the absence of the normal hosts: snake or mouse, and cat. It has been demonstrated that the tapeworms, Hymenolepis diminuta and H. nana, can be grown from the larval to the ovigerous adult stage in vitro using culture apparatus and media described by Berntzen (1961, 1962, 1964). Mueller previously succeeded in culturing the plerocercoid larva of Spirometra mansonoides in vitro from the procercoid stage using methods described in a series of papers (Mueller, 1958, 1959a, 1959b, 1959c). In vitro-reared plerocercoids proved to be infective for cats, and adult worms resulting from them were normal in every detail. Furthermore, the culture system could be made to serve as a surrogate host to the plerocercoid Received for publication 28 May 1964. *Supported by Research Grants Numbers AI04175-01 and AI-01876-06 from the United States Public Health Service, National Institutes of Health, Bethesda, Maryland. t Department of Medical Microbiology and Immunology. t Department of Microbiology. over several successive generations with no indication of impairment to the worm. Since Hymenolepis is a cyclophyllidean genus, the question arose whether the culture methods developed by Berntzen would prove equally successful for rearing a pseudophyllidean cestode from the plerocercoid to the adult stage in vitro. Smyth (1946, 1949) succeeded in bringing about the differentiation to sexual maturity and oviposition of several larval pseudophyllideans (Ligula, Schistocephalus) in vitro, but these large plerocercoids, from the body cavity of fishes, have abundant amounts of stored energy, and little or no increase in mass is involved. In these larvae the primordia of the genitalia are already present, and in Schistocephalus even segmentation is complete. Smyth (1959) also succeeded in inducing differentiation of a small plerocercoid from fishes (Diphyllobothrium dendriticum ?) in vitro. In this case he obtained segmentation and development of genital primordia, but there was no increase in mass, and the resulting