title: Novel Parachlamydia acanthamoebae quantification method based on coculture with amoebae.
abstract: Parachlamydia acanthamoebae, belonging to the order Chlamydiales, is an obligately intracellular bacterium that infects free-living amoebae and is a potential human pathogen. However, no method exists to accurately quantify viable bacterial numbers. We present a novel quantification method for P. acanthamoebae based on coculture with amoebae. P. acanthamoebae was cultured either with Acanthamoeba spp. or with mammalian epithelial HEp-2 or Vero cells. The infection rate of P. acanthamoebae (amoeba-infectious dose [AID]) was determined by DAPI (4',6-diamidino-2-phenylindole) staining and was confirmed by fluorescent in situ hybridization. AIDs were plotted as logistic sigmoid dilution curves, and P. acanthamoebae numbers, defined as amoeba-infectious units (AIU), were calculated. During culture, amoeba numbers and viabilities did not change, and amoebae did not change from trophozoites to cysts. Eight amoeba strains showed similar levels of P. acanthamoebae growth, and bacterial numbers reached ca. 1,000-fold (10(9) AIU preculture) after 4 days. In contrast, no increase was observed for P. acanthamoebae in either mammalian cell line. However, aberrant structures in epithelial cells, implying possible persistent infection, were seen by transmission electron microscopy. Thus, our method could monitor numbers of P. acanthamoebae bacteria in host cells and may be useful for understanding chlamydiae present in the natural environment as human pathogens.
authors.affiliation: Department of Medical Laboratory Sciences, Faculty of Health Sciences, Hokkaido University, Sapporo, Hokkaido, Japan.
authors.forename: Junji
authors.initials: J
authors.lastname: Matsuo
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Hayashi
authors.forename: Shinji
authors.initials: S
authors.lastname: Nakamura
authors.forename: Marie
authors.initials: M
authors.lastname: Sato
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Mizutani
authors.forename: Masahiro
authors.initials: M
authors.lastname: Asaka
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Yamaguchi