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. 2020 Jan 31;9(2):90.
doi: 10.3390/pathogens9020090.

Isolation of Naegleria Spp . From a Brazilian Water Source

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Free PMC article

Isolation of Naegleria Spp . From a Brazilian Water Source

Natália Karla Bellini et al. Pathogens. .
Free PMC article

Abstract

The genus Naegleria, of the free-living amoeba (FLA) group, has been investigated mainly due to its human health impact, resulting in deadly infections and their worldwide distribution on freshwater systems. Naegleria fowleri, colloquially known as the "brain-eating amoeba," is the most studied Naegleria species because it causes primary amoebic meningoencephalitis (PAM) of high lethality. The assessment of FLA biodiversity is fundamental to evaluate the presence of pathogenic species and the possibility of human contamination. However, the knowledge of FLA distribution in Brazil is unknown, and to rectify this situation, we present research on identifying Naegleria spp. in the Monjolinho River as a model study. The river is a public Brazilian freshwater source that crosses the city of São Carlos, in São Paulo state, Brazil. Five distinct sampling sites were examined through limnological features, trophozoites culturing, and PCR against internal transcribed spacer (ITS) regions and 5.8S rRNA sequences. The results identified N. philippinensis, N. canariensisi, N. australiensis, N. gruberi, N. dobsoni sequences, as well as a Hartmannella sequence. The methodology delineated here represents the first Brazilian Naegleria spp. study on a freshwater system. Our results stress the urgency of a large scale evaluation of the presence of free-living amoebas in Brazil.

Keywords: Brazil; Monjolinho River; Naegleria spp.; PCR; free-living amoeba.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Limnological profile throughout the sampling sites in the Monjolinho River. (A) DO (dissolved oxygen) and conductivity values plotted along the river samples. (B) Temperature and pH values plotted along the river samples.
Figure 2
Figure 2
Light microscopy images of amoeba cells on non-nutrient agar (NNA) plates from sampling site D that were cultivated at 26 °C (A), 37 °C (B), and 44 °C (C). Arrows indicate trophozoite (T) and cyst (C) cells that were observed with an inverted microscope (Nikon TS 100) at ×400 magnification.
Figure 3
Figure 3
PCR amplification of Naegleria internal transcribed spacer (ITS) rDNA region. In (A), the lanes L1 and L2 correspond to the isolates of sampling site C, and L3 corresponds to the isolates of sampling site E. In (B), the lanes L4 and L5 correspond to sampling site D cultivated at 37 and 44 °C, respectively. The arrows indicate the amplified products that correspond to Naegleria ITS genes (N1–N5) and the Hartmannella (H1) gene. The molecular marker (M, Gene Ruler 1 kb Plus DNA Ladder, Thermo Fisher Scientific), positive (+) control, and negative (-) control are also displayed.
Figure 4
Figure 4
Evolutionary relationship among Naegleria spp., Hartmannella spp. and Vahlkampfia spp. based on internal transcribed sequences (ITS1 and ITS2 regions) and 5.8S sequence. The bootstrap consensus tree was inferred from 1000 replicates. Branches corresponding to partitions reproduced in less than 50% of the bootstrap replicates are collapsed. Hartmannella vermiformis and Acanthamoeba pearcei were used as outgroups for the Vahlkampfia and Naegleria clades.
Figure 5
Figure 5
Cadastral map of the Monjolinho River Basin, São Carlos, São Paulo (Brazil) that shows the Monjolinho river (blue line) and sampling sites A–E (in red letters and circles) that were investigated in this study.

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