Molecular Approach on Classification of Acanthamoeba Korean Isolates Using 18S-srRNA Analysis

Acanthamoeba spp. spread in soil, ponds, air and swimming pool, cause a granulomatous amoebic meningitis and amoebic keratitis in human and experimental animals. Their classification had depended upon morphological characteristics and physiological conditions such as thermophilic status and generation times. However, because amoeba species showed the morphological and physiological diversity in isolates, many attempts have been made on identification of Acanthamoeba spp. Restriction fragments length polymorphism (RFLP) analysis of IBS-small subunit ribosomal RNA (srRNA) was an useful tool for classification among morphologically and genetically closely-related species. In this study, 18S-srDNAs of amoebae were amplified by PCR with primer encoding 18S-srRNA gene and digested with restriction endonudeases, and RFLP analysis was applied on classification of Acanthamoeba Korean isolates (YM-4, YM-5 and YM-7), comparing with reference amoebae, A. culbertsoni, A. polyphaga and A. royreba. Trophozoites of six Acanthamoeba spp. had typical acanthopoda, but did not show any morphological differences. Cyst of Acanthamoeba sp. YM-7 was similar to that of A. polyphaga which was designated as group Ⅱ Acanthamoeba. Morphologically other amoebae belonged to group Ⅲ Acanthamoeba. Digestion of 18S-srDNA with six enzymes, Dde Ⅰ, Hae Ⅲ, Hind Ⅲ, EcoR Ⅰ, Rsa Ⅰ and Sph Ⅰ, resulted in various DNA fragments. Using the method of Nei and U (1979) for RFLP analysis, genetic divergence was not observed between A culbertsoni and Acanthamoeba sp. YM-4. Between A. culbertsoni and Acanthamoeba sp. YM-5, genetic distance was 0.070, 0.364 between Acanthamoeba sp. YM-4 and A. polyphaga, and 0.277 between Acanthamoeba sp. YM-7 and A. polyphaga. In comparison with A. culbertsoni and A. polyphaga, Acanthamoeba sp. YM-7 showed genetic distance of 0.330 and 0.154, respectively. Thus, Acanthamoeba sp. YM-7 is a similar species to A. polyphaga. Acanthamoeba sp. YM-5 is a different species from A. polyphaga, but closely-related to A. culbertsoni. Acanthamoeba sp. YM-4 can be classified as a subspecies or a strain of A. culbertsoni.ope