title: Validation of cell-free culture using scanning electron microscopy (SEM) and gene expression studies.
abstract: A cell-free culture system for Cryptosporidium parvum was analysed using scanning electron microscopy (SEM) to characterise life cycle stages and compare gene expression in cell-free culture and cell culture using HCT-8 cells. Cryptosporidium parvum samples were harvested at 2 h, 8 h, 14 h, 26 h, 50 h, 74 h, 98 h, 122 h and 170 h, chemically fixed and specimens were observed using a Zeiss 1555 scanning electron microscope. The presence of sporozoites, trophozoites and type I merozoites were identified by SEM. Gene expression in cell culture and cell-free culture was studied using reverse transcriptase quantitative PCR (RT-qPCR) of the sporozoite surface antigen protein (cp15), the glycoprotein 900 (gp900), the Cryptosporidium oocyst wall protein (COWP) and 18S ribosomal RNA (rRNA) genes in both cell free and conventional cell culture. In cell culture, cp15 expression peaked at 74 h, gp900 expression peaked at 74 h and 98 h and COWP expression peaked at 50 h. In cell-free culture, CP15 expression peaked at 98 h, gp900 expression peaked at 74 h and COWP expression peaked at 122 h. The present study is the first to compare gene expression of C. parvum in cell culture and cell-free culture and to characterise life cycle stages of C. parvum in cell-free culture using SEM. Findings from this study showed that gene expression patterns in cell culture and cell-free culture were similar but in cell-free culture, gene expression was delayed for CP15 and COWP in cell free culture compared with the cell culture system and was lower. Although three life cycle stageswere conclusively identified, improvements in SEM methodology should lead to the detection of more life cycle stages.
authors.affiliation: School of Veterinary and Life Sciences, Vector- and Water-Borne Pathogen Research Group, Murdoch University, Murdoch, Western Australia 6150, Australia.
authors.forename: R
authors.initials: R
authors.lastname: Yang
authors.affiliation: School of Veterinary and Life Sciences, Vector- and Water-Borne Pathogen Research Group, Murdoch University, Murdoch, Western Australia 6150, Australia.
authors.forename: Y
authors.initials: Y
authors.lastname: Elankumaran
authors.affiliation: Department of Medical Laboratory Sciences, Faculty of Allied Health Sciences, The Hashemite University, PO Box 150459, Zarqa 13115, Jordan.
authors.forename: N
authors.initials: N
authors.lastname: Hijjawi
authors.affiliation: School of Veterinary and Life Sciences, Vector- and Water-Borne Pathogen Research Group, Murdoch University, Murdoch, Western Australia 6150, Australia. Electronic address: Una.Ryan@murdoch.edu.au.
authors.forename: U
authors.initials: U
authors.lastname: Ryan
mesh_terms: D002460:Cell Line; D003457:Cryptosporidiosis; D016785:Cryptosporidium parvum / Q000235:genetics / Q000254:growth & development / Q000378:metabolism / Q000648:ultrastructure*; D006801:Humans; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D034101:Sporozoites / Q000254:growth & development / Q000378:metabolism / Q000648:ultrastructure; D053058:Trophozoites / Q000254:growth & development / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D015800:Protozoan Proteins
keywords: 18S rRNA; COWP; Cryptosporidium; RT-qPCR; SEM; cp15; gp900; mRNA expression