Identification and phylogenetic position of Naegleria spp. from geothermal springs in Italy

Naegleria spp. are free-living amoebae belonging to the family Vahlkampfiidae, in the class Heterolobosea. Among the recognized species, Naegleria fowleri causes primary amoebic meningoencephalitis (PAM), while two other species, Naegleria australiensis and Naegleria italica, have been reported as pathogenic in experimental animals. Due to the thermotolerance properties of some species, geothermal water sources including hot springs represent suitable habitats for their proliferation. The main aim of this study was a year-round sampling in two geothermal springs in Central Italy, to investigate the presence of Naegleria spp. using PCR/DNA sequencing based methods. The affinities between the sequences generated here and others reported in the literature were explored by using POY, which implements the concept of dynamic homology. Naegleria australiensis, Naegleria italica, and Naegleria lovaniensis, plus an unassigned Naegleria spp. were detected. Indels in the rDNA ITS1 and ITS2 turned out to be critical to distinguish the three species and confirmed their phylogenetic relationships. This is the first molecular report on the Naegleria spp. occurrence in geothermal waters in Central Italy, coupled with a fine genetic characterization