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Fig 1.

Morphological observation of Naegleria -like cyst at 100X magnification.

Zone of Escherichia coli clearing progressed and the number of cysts increased markedly.

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Fig 2.

Amplicon bands revealed by genus and species specific primers against Naegleria isolates for representative samples.

Genus Naegleria specific amplification (Lane 1–13), Lane 1 Ladder 100bp, Lane 2 Negative control, Lane 3 Positive control, Lane 4–13 Positive samples. N. fowleri specific amplification (14–17), Lane 14. Ladder 100bp, Lane 15 Negative control, Lane 16 Positive control, Lane 17 Positive sample.

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Table 1.

Overview of geographic sites sampled for Naegleria spp.

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Table 1 Expand

Fig 3.

Multiple sequence alignment for amplicons NGES1-NGES10 using ClustalW program.

Black colour dash (—-) indicates gap, red colour fonts base substitution or base insertion, forward and reverse primers are underlined.

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Table 2.

Result of DNA sequencing of the selected amplicons (NGES1-NGES10).

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Table 2 Expand

Table 3.

Length and position of base in the ITS-5.8S-ITS2-28S sequence of the selected isolates (NGES1- NGES10).

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Fig 4.

Neighbour-joining tree depicting the relationships between test isolates with amplicons (NGES1-NGES10) and reference strains of Naegleria.

Numbers at the notes are percentage-bootstrapping values on 1000 replicates, and only values of > 50% are shown. GenBank accession numbers for reference sequences are indicated at the ends of the species designations.

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Fig 5.

Graph depicting the association between temperature of the sampling sites and isolation frequency of Naegleria spp.

Y axis denotes the number of isolation and X axis denotes minimum and maximum temperature range of the sampling sites during the entire season (March to August).

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