Development of 18S rRNA-targeted oligonucleotide probes for specific detection of Hartmannella and Naegleria in Legionella - positive environmental samples

Aquatic protozoa are natural hosts of the human pathogen Legionella pneumop hila. The fluorescence labeled 16S rRNA-targeted oligonucleotide probe LEGP NE1 has recently been shown to specifically detect extracellular legionella e as well as intracellular legionellae parasitizing protozoa. In this study we designed oligonucleotide probes which are complementary to distinct reg ions of the 18S rRNA of the Legionella host organisms of the genera Hartman nella and Naegleria. The specificity of the probes, HART498 and NAEG1088, w as rested by in situ hybridization of various laboratory reference strains. In order to evaluate the fluorescent probes for environmental studies thre e selected Legionella-positive cold water habitats were examined for the pr esence of these protozoa. Traditional culture methods followed by morpholog ical identification revealed an almost consistent presence or Naegleria spp . in cold water habitats. Other protozoa species including Acanthamoeba spp ., Echinamoeba spp., Hartmanella spp., Platyamoeba placida, Saccamoeba spp. , Thecamoeba quadrilineata, and Vexillifera spp. were found sporadically. C oncomitant analysis of the pH, conductivity and temperature of the water sa mples revealed no preference of Legionella or the respective protozoa for c ertain environmental conditions. The specificity of the newly designed 18S rRNA probes demonstrates that they are valuable and rapid tools for the ide ntification of culturable environmental protozoa.