Elsevier

Experimental Parasitology

Volume 183, December 2017, Pages 143-149
Experimental Parasitology

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

https://doi.org/10.1016/j.exppara.2017.08.008Get rights and content

Highlights

  • A survey on Naegleria spp. occurrence in Central Italy was proposed.

  • A state of the art tool for phylogenetic analysis was applied.

  • High biodiversity was observed albeit the limited study area.

  • Evolutionary relationships among the three detected species were confirmed.

Abstract

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.

Introduction

Naegleria spp. are free-living amoebae included in the Vahlkampfiidae family. These organisms are ubiquitous in natural and man-made aquatic environments, such as lakes, rivers, geothermal waters, discharge from industrial plants, and swimming pools (Marciano-Cabral, 1988). More than 47 species have been classified within the genus using molecular techniques (De Jonckheere, 2014, Khwon and Park, 2017). Based on differences mostly in rDNA sequences, these have been grouped into 8 clusters, two of which contain isolates found only in cold environments (De Jonckheere, 2006, Tyml et al., 2016). However, several of the species remain represented by strains sampled only once, thus limiting an evaluation of the degree of genetic variation within and between species (De Jonckheere, 2004).

Within the genus, Naegleria fowleri is proved to be the only species pathogenic to humans, being the agent of a primary amoebic meningoencephalitis (PAM), i.e. an acute and rapidly fatal infection of the central nervous system (CNS), frequently reported in healthy young persons with a recent history of aquatic activities. However, other species such as Naegleria australiensis, Naegleria italica and Naegleria philippinensis are considered of public health concern, since they have displayed pathogenicity in experimental animals (De Jonckheere, 2002, Schuster, 2002, Visvesvara et al., 2007). In addition, Naegleria lovaniensis has recently attracted attention because it is the only non-pathogenic thermophilic species displaying a close evolutionary relationship with N. fowleri.

Data on Naegleria spp. from Italy are scarce and the distribution of this genus is still poorly investigated. N. australiensis, N. italica, and N. lovaniensis were isolated in different epidemiological surveys carried out in northern Italy, mostly from thermal areas (Scaglia et al., 1987, Scaglia et al., 1983a, Scaglia et al., 1983b). Subsequent molecular studies on these Italian strains, confirmed their identification (De Jonckheere, 2004). In the same geographical area, the presence of N. fowleri was reported in a case of PAM, diagnosed postmortem in a child who probably acquired the infection after swimming in a river (Cogo et al., 2004).

As most of Naegleria spp. are described as tolerant to high temperatures, thermal waters could represent suitable niches for these amoebae. Traditionally, thermal springs in Italy are popular with tourists for recreation and relaxation, and because of their high mineral content, they have also been considered of some therapeutic benefit.

The purpose of this study was to conduct a year-round sampling in two geothermal springs recreational areas in Central Italy in order to: (i) investigate the presence and the distribution of Naegleria spp.; (ii) identify the isolates at the species level using the ribosomal DNA (ITS) as molecular marker; (iii) investigate the contribution of these isolates to the diversity within and between Naegleria species using homologous sequences retrieved from GenBank.

Section snippets

Study area

The two geothermal springs in this study (Site A and Site B) are located in the Latium region, near the city of Viterbo (top of Fig. 1). The Site A is a set of natural pools (Fig. 1), the hot water source being a thermal spring of type sulphureous-sulphate-bicarbonate-alkaline-earthy, with an outlet temperature of about 58 °C. Samples were taken either from the pools themselves or from the origin of the stream. The Site B consists of a natural thermal pool connected to a new concrete swimming

Occurrence and molecular identification of Naegleria

A total of 36 water samples (Site A = 24; Site B = 12) were collected and cultured at 37 °C and 45 °C. Overall, 26 out of 36 water samples (72.2%) provided PCR amplification with the Naegleria genus-specific primer set, from cultures incubated at 37 °C (61%, 22/36) and 45 °C (11%, 4/36). N. fowleri species-specific primer set did not produce any amplicon bands against all isolates.

The prevalence of Naegleria growing at 37 °C was highest in the Site A (70.83%, 17/24) in respect to the Site B

Discussion

The genus Naegleria includes free living amoebae of public health concern. Indeed, among the known species, some have been recognized to be pathogenic to animals; furthermore, Naegleria fowleri is able to infect humans, resulting in fatal central nervous system infections (De Jonckheere, 2014). The interest on Naegleria spp. epidemiology is now growing worldwide, even if data remain mainly focused on N. fowleri so far. These microorganisms tolerate high temperatures and are considered as

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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