Fig 1.
Flow-chart showing the steps of the current study.
Table 1.
The primers ’sequences used in PCR reaction in the present study.
Fig 2.
Prevalence of FLA, Acanthamoeba, Vahlkampfiidae (Naegleria-like), and mixed genera in tap and tank water samples.
Fig 3.
Correlation between the frequency of FLA and water temperature at the sampling time.
Fig 4.
A. Acanthamoeba trophozoite showing characteristic acanthopodia (black arrows) with a single nucleus (red arrowhead). B. Acanthamoeba cyst showing an outer wall “ectocyst” (black arrow), inner wall "endocyst" (red arrowhead). C. Naegleria trophozoite showing blunt hemispherical bulge (lobopodium) at the anterior end (black arrow) and a single nucleus (red arrowhead). D. Naegleria flagellate stage, temporary pear-shaped with a pair of flagella (black arrows). E. Naegleria cyst with a single nucleus (red arrowhead). F. Vahlkampfiidae trophozoite showing blunt lobopodium (black arrow) and a single nucleus (red arrowhead). G. Vahlkampfiidae cyst with a single nucleus (red arrowhead). (D, E) unstained (A, B, C, F, G) stained with Lugol’s iodine stain. All images are at x1000.
Fig 5.
A. astronyxis, showing an average cyst diameter of 19–22 μm. Ectocyst was smoothly circular, while the endocyst was stellate with mainly five rays. B. A. comandoni, showing an average cyst diameter of 20–25 μm, stellate endocyst with mainly 6–9 rays, thin and little wrinkled ectocyst encircling the endocyst. C. A. echinulata, showing an average diameter of 25 μm, stellate endocyst with a higher number of rays (up to 12–14) and thin, little wrinkled ectocyst. D. A. triangularis, showing an average diameter of 13 μm. The endocyst was triangular with broad rays. The ectocyst was thick, wrinkled, and corrugated but not spherical. (E, F) A. polyphaga, showing an average cyst diameter of 14 μm. The endocyst was irregularly polyhedral, usually quadrangular or pentagonal. The ectocyst was folded and loosely applied to the endocyst. G. A. lenticulata, showing a cyst diameter of 11–13 μm. The endocyst was nearly rounded. The ectocyst closely followed the endocyst contour and pleated forming saw-teeth appearance all-around the endocyst. H. A. culbertsoni, showing a cyst diameter of 15–18 μm. The ectocyst was more wrinkled and thinner than the endocyst. The endocyst was nearly rounded or with only slight angles. [Cysts of A, B, C belonged to the group (I); D, E, F belonged to the group (II); G and H belonged to the group (III)]. (C, H) unstained (A, B, D, F, G) stained with lactophenol cotton blue stain.
Fig 6.
Agarose gel 1.5% stained with ethidium bromide showing the PCR products of 9 positive samples amplifying ASA.S1 region of Acanthamoeba using JDP1-2 primers showing a single band of 550 bp. in lanes (2–10), lanes (12–15) are negative samples, lane 11 = negative control, and lane 1 (M) marker = 100 bp DNA ladder.
Fig 7.
Agarose gel 1.5% stained with ethidium bromide showing the PCR products of 8 positive samples amplifying ITS region of Vahlkampfiidae using ITS1-2 primers showing a single band of 600 bp. (Vahlkampfia) in lanes (2,3,4), a single band of 500 bp. (Allovahlkampfia) in lane (1) and a single band of 400 bp. (Naegleria) lanes (6, 7, 8, 9).
Lane 5; (M) marker = 100 bp DNA ladder.
Fig 8.
Phylogenetic tree showing the evolutionary relationship among the investigated isolates.
Sequences with black circles are the ones analyzed in the current study. The scale below shows the number of substitutions in 100 nucleotides and corresponds to the branch length. Dashed branches indicate negative branches. The values at bifurcations show the node support value in the 1000 bootstrap trials. NA indicates the absence of bootstrapping for the respective node.
Fig 9.
Phylogenetic tree showing the evolutionary relationship among Allovahlkampfia spp.
The scale below shows the number of substitutions in 100 nucleotides and corresponds to the branch length. Dashed branches indicate negative branches. The values at bifurcations show the node support value in the 1000 bootstrap trials. NA indicates the absence of bootstrapping for the respective node.
Table 2.
Genotyping results and water source of FLA mono-isolates in the present study (n = 6).