title: Genotyping of single Cryptosporidium oocysts in sewage by semi-nested PCR and direct sequencing.
abstract: This study describes an approach for genotyping individual Cryptosporidium oocysts obtained from sewage. We isolated single immunofluorescent assay (IFA)-stained Cryptosporidium oocysts from sewage concentrate using glass capillary pipettes and inverted epifluorescence microscopy. Each isolated Cryptosporidium oocyst was analyzed by semi-nested PCR for the 18S rRNA gene and direct sequencing of the PCR products. A total of 74 of 107 oocysts isolated from sewage were genotyped successfully. Of the 74 genotyped isolates, 51% (38 oocysts) were identified as C. parvum genotype 1, 4% (3 oocysts) of C. parvum VF383 human isolates, 20% (15 oocysts) of C. parvum genotype 2, 14% (10 oocysts) of C. meleagridis, 7% (5 oocysts) of C. sp. Pig 1, 3% (2 oocysts) of C. sp PG1-26 pig isolates and 1% (1 oocyst) of C. parvum CPM1 isolated from mouse. The results of this study demonstrate that 18S rRNA-based semi-nested PCR and direct sequencing can be used to characterize individual Cryptosporidium oocysts and also to reveal the distribution of Cryptosporidium genotypes in environmental waters.
authors.affiliation: Department of Civil Engineering, Anan National College of Technology, 265 Aoki, Anan, Tokushima 774-0013, Japan. atsushi@anan-nct.ac.jp
authors.forename: Atsushi
authors.initials: A
authors.lastname: Hashimoto
authors.forename: Hitomi
authors.initials: H
authors.lastname: Sugimoto
authors.forename: Shigemitsu
authors.initials: S
authors.lastname: Morita
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Hirata
mesh_terms: D000818:Animals; D016785:Cryptosporidium parvum / Q000235:genetics*; D005838:Genotype; D033661:Oocysts; D016133:Polymerase Chain Reaction / Q000379:methods*; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D012722:Sewage / Q000469:parasitology*
chemical_list: D012337:RNA, Ribosomal, 18S; D012722:Sewage