Table 1.
Specific primers for the 18s rRNA gene of amoebae.
Fig 1.
Bright field microscopic images demonstrate trophozoite and cyst morphology (A and B) Paravahlkampfia ustiana, (C and D) Acanthamoeba sp., (E and F) Naegleria pagei and (G and H) isolate A-ST39-E1.
P. ustiana, N. pagei and isolate A-ST39-E1 were stained with trypan blue while Acanthamoeba sp. was stained with crystal violet. The letter in images indicate the following: Uf = Uroidal filaments, N = Nucleus, Ac = Acanthopodia, Cv = Contractile vacuole, Fv = Food vacuole, O = Outer-cyst wall, I = Inner-cyst wall, F = Flagella, S = Sub-pseudopodium and U = Uroid.
Fig 2.
Intracellular survival through time of B. pseudomallei in P. ustiana (A), Acanthamoeba sp. (B) and isolate A-ST39-E1 (C).
Time zero represents 3 hours after B. pseudomallei feeding. Bars represent the standard errors of the means of duplicate, three times independent experiments, * p < 0. 0001 using ANOVA.
Fig 3.
B. pseudomallei is internalized into amoebae but could not resist digestion.
CLSM micrographs show the internalized B. pseudomallei in P. ustiana (A-C), Acanthamoeba sp. (D-F) and isolate A-ST39-E1 (G-I) at 0, 3 and 6 h after kanamycin treatment. Orange fluorescence represents CellTracker™ Orange-B. pseudomallei and green fluorescence indicates the amoebae stained with FITC-ConA for visualization.
Fig 4.
Numbers of Acanthamoeba sp. and isolate A-ST39-E1 over time (A-B and C-D respectively) after feeding with B. pseudomallei (▲) or E. coli (positive control) (■) or deprived of bacteria as a negative control (●).
Graphs and figures show no significant differences between amoebae fed on B. pseudomallei and E. coli. However, numbers of amoebae in the negative control group were significantly lower than in the pother groups (p ≤ 0.0001). Data are mean ± SD from duplicates of the three independent experiments.