[Use of the presence of cellulose in cellular wall of Acanthamoeba cysts for diagnostic purposes].

Abstract

Species identification within the genus Acanthamoeba is based predominantly on morphological and biochemical features. It is labor-intensive and requires cloning and axenization. We described a novel immunocytochemical method for the identification of Acanthamoeba spp. based on selective binding of Clostridium cellulovorans cellulase to protozoan cyst wall cellulose. Free-living amoebae isolated from different water sources by filtration and subsequent cultivation on non-nutrient agar were assigned to genera Acanthamoeba, Naegleria or Hartmannella using morphological taxonomic criteria. Tissues samples from experimentally infected mice were fixed in formalin and for sectioning embedded in paraffin or snap frozen. The Cellulose-Binding Domain of C. cellulovorans cellulase (CBD) obtained as a recombinant protein, were coupled to the fluorescent dye using Alexa Fluor350, 488, 568 - Protein Labelling Kit or labelled with the biotin using EZ-Link Sulfo-NHS-Biotin. All coupling procedures were performed according to the methods provided by manufacturers. For staining with CBD conjugate, slides containing cysts collected from the agar plates or tissue sections were immersed with PBS and incubated with CBD for 30 min at room temperature, washed 3 times with PBS. For staining with CBD-biotin slides containing cysts were incubated with biotinylated CBD for 30 min at room temperature. Subsequent washings in changes of PBS were followed by the incubation with Strept ABComplex/HRP, for 30 min at room temperature, than 3,3 diaminobenzidine tetrahydrochloride was added for 15 min. Slides were rinsed with water, dried and examined in the light microscope. We showed that cellulose could be easily detected by immunofluorescence using conjugated CBD in the inner cyst wall of Acanthamoeba spp. The reference strains of Acanthamoeba spp. and all Acanthamoeba strains isolated from water and from tissues of infected animals gave positive reaction. CBD prepared as a biotynylated protein can be also used for the demonstration of Acanthamoeba cyst in infected tissues and environmental samples.