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

Abstract

The metazoan parasitic blood f lukes, 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.Schistosoma mansoni, Schistosoma haematobium, and Schistosoma japonicum create widespread disease in tropical developing countries (1). These parasites thwart attempts to produce practical and effective vaccines, and pharmacological approaches are problematic (2). Schistosome eggs are laid by females paired with males and residing in the vasculature of parasitized vertebrate hosts. To be successfully infective, eggs must penetrate the intestinal wall and pass in feces to fresh water. From these released eggs hatch free-swimming miracidia, the first larval form. Successful miracidia encounter and penetrate the appropriate species of snail and develop into sporocysts, the second larval from. In the following weeks, these primary sporocysts asexually generate first-generation and second-generation daughter sporocysts in the snail. From the later-generation sporocysts develop the third larval form, free-swimming cercaria. To be successfully infective in the human or other vertebrate host, cercaria must encounter and penetrate the skin of the host and undergo further changes to form schistosomula. These move through several further developmental stages, ultimately leading to sexual pairing in the veins of the parasitized host and egg production.Approaches to elucidate the mechanisms schistosomes employ to escape host immunity, as well as molecular genetic and pharmacological investigations, have been limited by the lack of an in vitro system for the continuous proliferative culture of the parasite in the absence of the host. Recently, Yoshino and colleagues (3, 4) have reported significant success in limited culture of the intramolluscan stages of S. mansoni and S.japonicum, but these techniques do not allow extended culture of proliferating sporocysts.Here we describe long-term cultures of S. mansoni in vitro and the development from the snail-infective miracidial stage, through generations of mother and daughter sporocysts, t...

Saci-1, -2, and -3 and Perere, Four Novel Retrotransposons with High Transcriptional Activities from the Human Parasite Schistosoma mansoni

“…Four retroelements belonging to LTR and non-LTR classes have been previously characterized for S. mansoni (10,14,15). The presence of RT activity in Schistosoma extracts suggests that some of these elements are active (23).…”

Boudicca , a Retrovirus-Like Long Terminal Repeat Retrotransposon from the Genome of the Human Blood Fluke Schistosoma mansoni

“…This suggests that Boudicca may be transposing within the genome of S. mansoni at this point in evolutionary time. Other recent reports have also suggested the activity of reverse transcriptase enzymes and/or mobilization of retrotransposable elements in schistosomes (29,36). Given the phylogenetic proximity of Boudicca to the errantiviruses and the possible presence of an envelope protein, Boudicca may be capable of both vertical and horizontal transmission.…”

“…Schistosome eggs were isolated from the livers of infected mice at 8 weeks postinfection. For the initiation of in vitro cultures, miracidia were transformed into primary (mother) sporocysts by overnight culture in MEMSE-J with 10% fetal calf serum (31) or medium F with 10% bovine serum albumin (29) at 26°C and 5% CO 2 . Shed ciliated plates were washed away.…”

“…Although active replication of these elements has not been definitively proven, mRNA transcripts encoding reverse transcriptase and endonuclease have been detected (34,36), as has reverse transcriptase activity in schistosome extracts (29), suggesting that at least some of these elements are actively mobile within the genome. Indeed, actively replicating mobile genetic elements in other platyhelminths have been described as RNA intermediates (4) and DNA transposons (3,53).…”

Transplantation of in vitro-generated Schistosoma mansoni mother sporocysts into Biomphalaria glabrata

“…They obtained the continuous development from miracidia to mother sporocysts and from mother sporocysts to daughter sporocysts using co-cultivation with Bge cells. Finally, Ivanchenko et al (1999) succeeded in completing the entire intramolluscan cycle of S. mansoni (miracidium to cercariae) within 8 months in the presence of Bge cells, and improved media formulations.…”