Continuous in vitro propagation and differentiation of cultures of the intramolluscan stages of the human parasite Schistosoma mansoni

Abstract

The metazoan parasitic blood flukes, Schistosoma spp., infect over 200 million people worldwide and cause extensive human morbidity and mortality. Research strategies for development of anti-schistosomal agents are impeded by the organism's complex molluscan-mammalian life cycle, which limits experimental approaches and availability of material. We derived long-term continuously proliferative cultures of Schistosoma mansoni sporocysts capable of generating cercariae in vitro. Cultured organisms retained the ability to parasitize the host, and they exhibited developmental regulation of candidate stage-specific genes in the host-free culture system. Evidence for expression of a reverse transcriptase also was found in the cultured organisms, pointing to this activity as a possible mechanistic contributor to the dynamic relationship between the parasite and its hosts. Continuous in vitro propagation of the asexual sporocyst stage allows isolation of clonally derived parasite populations and provides a means to study schistosomal molecular genetics, metabolism, and evasion of host defenses.

Figure 1

Proliferating sporocysts and cercaria produced in vitro. (Top) Daughter sporocyst (DS) and developing daughter sporocyst embryos (DSE) inside a mother sporocyst (MS), cocultured with Bge cells. (Middle) Cercaria produced from proliferating daughter sporocyst culture maintained for 8 months in vitro. (Bottom) Sequestered sporocysts separated from Bge cells by a permeable membrane. Cultures were initiated by transformation of parasitized rodent-derived miracidia and maintained as described in the text. (×150.)

Figure 2

RT-PCR assay of mRNA expression of cercarial-marker serine protease and the calcium-binding protein gene. (A) MR, miracidia; CR, cercariae; NDG, non-infected digestive gland of snail; 28DG, digestive gland 28 days after infection, containing sporocysts; 56DG, digestive gland 56 days after infection, containing cercariae. For each sample, the left lane shows RT-PCR for protease, and the right lane shows RT-PCR for the calcium-binding protein gene. (B) Bge, established embryonic snail cell culture; MS, mother sporocysts after 2 days of culture (freshly transformed); MS+DSE, in vitro culture of mother sporocysts containing developing sporocyst embryos; MS+DS, culture containing mother sporocysts and emergent daughter sporocysts; MC, coculture of Bge and sporocysts in which cercarial differentiation had been detected.

Figure 3

Localization of alkaline phosphatase in developing daughter sporocysts. Sporocyst samples were washed with Chernin’s balanced salt solution and fixed with 3% glutaraldehyde in the salt solution for 30 min at room temperature. Samples were then stained for endogenous alkaline phosphatase by using a kit from Sigma. (×400.)