Expression of virulence factors in Acanthamoeba pretreated with different water conditions. The relative mRNA expression levels of SEP, MBP, AhLBP, and CP3 in Acanthamoeba pretreated with PYG medium, PAS, domestic tap water, CaCl₂, or DDW for one hour were quantified using ImageJ software. The expression levels of the ARP2 gene served as an internal control. Bar graphs represent the mean ± SD (p ≤ 0.05).