title: Quantitative analysis of Cryptosporidium growth in in vitro culture--the impact of parasite density on the success of infection.
abstract: Cryptosporidium is an important waterborne pathogen for which no treatment or vaccination is available. This study set out to quantify DNA replication of Cryptosporidium parvum in vitro. Cryptosporidium DNA could be detected at up to 60 % of input level in both host-cell-free and host cell containing cultures 6 days after infection with living sporozoites, but was lost within 2 days in cultures inoculated with UV-inactivated sporozoites. Total DNA increased between days 2 and 6, evidence of successful DNA replication in both cell-free and host-cell-containing cultures. Overall however, only a small fraction (up to 5 %) of parasite DNA could be found associated with host cells or bound to plastic of the cell-free cultures, and the majority of parasite DNA was present in the cell culture medium, separable by simple decantation. After 2 days, in host-cell-containing cultures, the parasite DNA could be concentrated by slow centrifugation, suggesting that it was associated with intact parasite cells, but at 6 days, the majority could not be centrifuged and is therefore thought to have represented copies associated with dead and degraded parasites. In cell-free cultures and in larger plates, the majority of DNA was in this form. Performance of the parasite was best in small culture plates, and least in the largest plate sizes. We interpret these results as suggesting that Cryptosporidium sporozoites first bind to the host cell monolayer or to the plasticware, but then by 2 days, there has been a substantial release of parasites back into the medium. Host-cell-free cultures also supported modest replication and may have represented DNA synthesis in cells beginning merogony. The role of the host cells is unclear, as so much of the parasite DNA is released into the medium. Host cells may provide a feeder role, conditioning the medium for Cryptosporidium development.
authors.affiliation: KIT Biomedical Research, Royal Tropical Institute, Meibergdreef 39, 1105 AZ, Amsterdam, The Netherlands. a.paziewska@kit.nl.
authors.forename: Anna
authors.initials: A
authors.lastname: Paziewska-Harris
authors.affiliation: Current address: Laboratory of Immunogenetics, Department of Medical Microbiology and Infection Control, VU University Medical Center, Van der Boechorststraat 7, 1081 BT, Amsterdam, The Netherlands.
authors.forename: Martin
authors.initials: M
authors.lastname: Singer
authors.affiliation: KIT Biomedical Research, Royal Tropical Institute, Meibergdreef 39, 1105 AZ, Amsterdam, The Netherlands.
authors.forename: Gerard
authors.initials: G
authors.lastname: Schoone
authors.affiliation: KIT Biomedical Research, Royal Tropical Institute, Meibergdreef 39, 1105 AZ, Amsterdam, The Netherlands.
authors.forename: Henk
authors.initials: H
authors.lastname: Schallig
mesh_terms: D003457:Cryptosporidiosis / Q000469:parasitology; D016785:Cryptosporidium parvum / Q000235:genetics / Q000254:growth & development* / Q000528:radiation effects; D003470:Culture Media; D004261:DNA Replication; D016054:DNA, Protozoan / Q000032:analysis* / Q000302:isolation & purification; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction; D034101:Sporozoites / Q000254:growth & development / Q000528:radiation effects; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
chemical_list: D003470:Culture Media; D016054:DNA, Protozoan
keywords: Cryptosporidium parvum; HCT-8 human adenocarcinoma cells; Host-cell-free culture; qPCR