Detection and Molecular Characterization of Acanthamoeba from Malaysian Environment

Abstract

This study was carried out in Malaysian environment to investigate the presence and the molecular characterization of Acanthamoeba. A total of 80 samples were collected from University Malaya environment sources, lakes, dust, sewages and air condition. All samples were cultured on non nutrient agar spread with E. coli lawn, then tested microscopically, only 14 samples were positive for Acanthamoeba while another 66 were negative. The morphology of cysts and trophozoites of these 14 Acanthamoeba isolates were detected by Trichrome and Modified Field stain respectively. The cysts were stained red with spherical or wrinkled outer wall, while the trophozoites appeared blue with or without acanthopodia. Furthermore, these 14 positive samples were used for DNA extraction and purification by Advanced silica-gel-membrane technology. The purified DNA was used in Polymerase Chain Reaction (PCR) with primers, forward primer ACARNA.for1383 (TCCCCTAGCAGCTTGTG) and reverse primer ACARNA.rev1655 (GTTAAGGTCTCGTTCGTTA), ACARNA.for1345 ( CGC GAG GGC GGT TTA ) and ACARNA.rev1830 ( GCT GGC TAG GCG CGC AG ) to detect the genus and pathogenicity respectively. Out of these 80 isolates tested, 14 produced 272 bp.for the genus and many different sizes for the pathogenicity. These PCR product seemed to be similar with the result reported by Michael H. et al., (1992) which showed Acanthamoeba genus and it’s pathogenicity. The third primers were forward primer ACA18.for 2209 (CGGGCTTGTGAGGTCTC) and reverse primer ACA58.rev 92 (GATGATTCACTGATCCCTG) were used to detect the species of Acanthamoeba. The 14 positive isolates for Acanthamoeba were divided into three species, Eight isolates were categorised as A. Castellani, Three isolates were categorised as Acanthamoeba spp Lb. and three isolates were categorised as Acanthamoeba spp. Gc.