Abstract
Here, we carried out a survey to determine the prevalence of free-living amoebae (FLA) in tap-water sources from rivers and water treatment plants located in Osaka Prefecture, Japan. A total of 374 raw water samples were collected from 113 sampling points. The samples were filtrated and transferred to non-nutrient agar plates seeded with a heat-killed suspension of Escherichia coli and incubated for 2 to 7 days at 30°C or 42°C. The plates were examined by microscopy to morphologically identify FLA families, and polymerase chain reaction and sequence analysis were then performed to define the species of the detected Naegleria and Acanthamoeba isolates. A total of 257 of 374 samples (68.7%) were positive for FLA by microscopy, and among these there were 800 FLA isolates, including Acanthamoeba and Naegleria species. Sequence analysis identified five Acanthamoeba spp. isolates of the known pathogenic T4 genotype and 43 Naegleria australiensis isolates, a reported pathogen to mice and also of concern as a potential pathogen to humans. Our results suggest a wide distribution of FLA, including potential pathogenic species, in tap-water sources of western Japan.
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Behets J, Declerck P, Delaedt Y, Verelst L, Ollevier F (2007) Survey for the presence of specific free-living amoebae in cooling waters from Belgian power plants. Parasitol Res 100:1249–1256
Chong EM, Dana MR (2007) Acanthamoeba keratitis. Int Ophthalmol Clin 47:33–46
De Jonckheere JF (2004) Molecular definition and the ubiquity of species in the genus Naegleria. Protist 155:89–103
De Jonckheere JF (2006) Isolation and molecular identification of free-living amoebae of the genus Naegleria from Arctic and sub-Antarctic regions. Europ J Protistol 42:115–123
De Jonckheere JF (2007) Molecular identification of free-living amoebae of the Vahlkampfiidae and Acanthamoebidae isolated in Arizona (USA). Europ J Protistol 43:9–15
De Jonckheere JF, Yagita K, Kuroki T, Endo T (1991) Furst isolation of pathogenic Naegrelia fowleri in Japan. Jpn J Parasitol 40:352–357
Ettinger MR, Webb SR, Harris SA, McIninch SP, Garman G, Brown BL (2003) Distribution of free-living amoebae in James River, Virginia, USA. Parasitol Res 89:6–15
Gornik K, Kuzna-Grygiel W (2004) Presence of virulent strains of amphizoic amoebae in swimming pools of the city of Szczecin. Ann Agric Environ Med 11:233–236
Greub G, Raoult D (2004) Microorganisms resistant to free-living amoebae. Clin Microbiol Rev 17:413–433
Hoffmann R, Michel R (2001) Distribution of free-living amoebae (FLA) during preparation and supply of drinking water. Int J Hyg Environ Health 203:215–219
Horn M, Wagner M (2004) Bacterial endosymbionts of free-living amoebae. J Eukaryot Microbiol 51:509–514
Huizinga HW, McLaughlin GL (1990) Thermal ecology of Naegleria fowleri from a power plant cooling reservoir. Appl Environ Microbiol 56:2200–2205
Izumiyama S, Yagita K, Furushima-Shimogawara R, Asakura T, Karasudani T, Endo T (2003) Occurrence and distribution of Naegleria species in thermal waters in Japan. J Eukaryot Microbio 50:514–515
John DT, De Jonckheere JF (1985) Isolation of Naegleria australiensis from an Oklahoma Lake. J Protozool 32:571–575
John DT, Howard MJ (1995) Seasonal distribution of pathogenic free-living amebae in Oklahoma waters. Parasitol Res 81:193–201
Kandori M, Inoue T, Hori Y, Maeda N, Tano Y (2007) Clinical management and prognosis of Acanthamoeba keratitis: a retrospective study of 11 cases at the department of ophthalmology of Osaka university medical school. Folia Ophthalomol Jpn 58:691–636
Karanis P, Kourenti C, Smith H (2007) Waterborne transmission of protozoan parasites: a worldwide review of outbreaks and lessons learnt. J Water Health 5:1–38
Kilvington S, Gray T, Dart J, Morlet N, Beeching JR, Frazer DG, Matheson M (2004) Acanthamoeba keratitis: the role of domestic tap water contamination in the United Kingdom. Invest Ophthalomol Visual Sci 45:165–169
Ledee DR, Hay J, Byers TJ, Seal DV, Kirkness CM (1996) Acanthamoeba griffini. Molecular characterization of a new corneal pathogen. Invest Ophthalmol Visual Sci 37:544–550
Ledee DR, Iovieno A, Miller D, Mandal N, Diaz M, Fell J, Fini ME, Alfonso EC (2009) Molecular identification of T4 and T5 genotypes in Acanthamoeba keratitis patients. J Clin Microbiol 47:1458–1462
Lorenzo-Morales J, Lindo JF, Martinez E, Calder D, Figueruelo E, Valladares B, Ortega-Rivas A (2005a) Pathogenic Acanthamoeba strains from water sources in Jamaica, West Indies. Ann Trop Med Parasitol 99:751–758
Lorenzo-Morales J, Ortega-Rivas A, Foronda P, Martinez E, Valladares B (2005b) Isolation and identification of pathogenic Acanthamoeba strains in Tenerife, Canary Islands, Spain from water sources. Parasitol Res 95:273–277
Lorenzo-Morales J, Ortega-Rivas A, Martinez E, Khoubbane M, Artigas P, Periago MV, Foronda P, Abreu-Acosta N, Valladares B, Mas-Coma S (2006) Acanthamoeba isolates belonging to T1, T2, T3, T4 and T7 genotypes from environmental freshwater samples in the Nile Delta region, Egypt. Acta Trop 100:63–69
Lorenzo-Morales J, Martinez-Carretero E, Batista N, Alvarez-Marin J, Bahaya Y, Walochnik J, Valladares B (2007) Early diagnosis of amoebic keratitis due to a mixed infection with Acanthamoeba and Hartmannella. Parasitol Res 102:167–169
Martinez AJ, Visvesvara GS (1997) Free-living, amphizoic and opportunistic amebas. Brain Pathol 7:583–598
Nacapunchai D, Kino H, Ruangsitticha C, Sriwichai P, Ishih A, Terada M (2001) A brief survey of free-living amebae in Thailand and Hamamatsu District Japan. Southeast Asian J Trop Med Pub Health 32(Suppl 2):179–182
Page FC (1988) A new key to freshwater and soil Gymnamoebae. Ferry House, London
Pelandakis M, Pernin P (2002) Use of multiplex PCR and PCR restriction enzyme analysis for detection and exploration of the variability in the free-living amoeba Naegleria in the environment. Appl Environ Microbiol 68:2061–2065
Scaglia M, Strosselli M, Grazioli V, Gatti S, Bernuzzi AM, de Jonckheere JF (1983) Isolation and identification of pathogenic Naegleria australiensis (Amoebida, Vahlkampfiidae) from a spa in northern Italy. Appl Environment Microbiol 46:1282–1285
Scaglia M, Gatti S, Cevini C, Bernuzzi AM, Martinez AJ (1989) Naegleria australiensis ssp. italica: experimental study in mice. Exp Parasitol 69:294–299
Schroeder J, Booton GC, Hay J, Niszl IA, Seal DV, Markus MB, Fuerst PA, Byers TJ (2001) Use of subgenic 18 S ribosomal DNA PCR and sequencing for genus and genotype identification of Acanthamoebae from humans with keratitis and from sewage sludge. J Clin Microbiol 39:1903–1911
Schuster FL, Visvesvara GS (2004) Free-living amoebae as opportunistic and non-opportunistic pathogens of humans and animals. Int J Parasitol 34:1001–1027
Seal DV (2003) Acanthamoeba keratitis update-incidence, molecular epidemiology and new drugs for treatment. Eye 17:893–905
Sheehan KB, Ferris MJ, Henson JM (2003) Detection of Naegleria sp. in a thermal, acidic stream in Yellowstone National Park. J Eukaryot Microbiol 50:263–265
Shoff ME, Rogerson A, Kessler K, Schatz S, Seal DV (2008) Prevalence of Acanthamoeba and other naked amoebae in South Florida domestic water. J Water Health 6:99–104
Spanakos G, Tzanetou K, Miltsakakis D, Patsoula E, Malamou-Lada E, Vakalis NC (2006) Genotyping of pathogenic Acanthamoebae isolated from clinical samples in Greece—report of a clinical isolate presenting T5 genotype. Parasitol Internat 55:147–149
Sugita Y, Fujii T, Hayashi I, Aoki T, Yokoyama T, Morimatsu M, Fukuma T, Takamiya Y (1999) Primary amebic meningoencephalitis due to Naegleria fowleri: an autopsy case in Japan. Pathol Internat 49:468–470
Thomas V, Loret JF, Jousset M, Greub G (2008) Biodiversity of amoebae and amoebae-resisting bacteria in a drinking water treatment plant. Environ Microbiol 10:2728–2745
Tsvetkova N, Schild M, Panaiotov S, Kurdova-Mintcheva R, Gottstein B, Walochnik J, Aspock H, Lucas MS, Muller N (2004) The identification of free-living environmental isolates of amoebae from Bulgaria. Parasitol Res 92:405–413
Visvesvara GS, De Jonckheere JF, Sriram R, Daft B (2005) Isolation and molecular typing of Naegleria fowleri from the brain of a cow that died of primary amebic meningoencephalitis. J Clin Microbiol 43:4203–4204
Visvesvara GS, Moura H, Schuster FL (2007) Pathogenic and opportunistic free-living amoebae: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, and Sappinia diploidea. FEMS Immunol Med Microbiol 50:1–26
Yamada T (2002) Advanced water treatment in Osaka. In: Osaka and Its Technology 41. Osaka Municipal Government, Osaka, pp 43–49
Acknowledgments
We thank Mr. H. Doi and Ms. T. Hizuka for technical assistance. We are grateful to Dr. T. Endo of the National Institute of Infectious Diseases, Japan for providing the DNA of N. lovaniensis. We are also grateful to Mr. B. Sheridan and Dr. G. Harris of DMC Corporation for their review of the manuscript during its preparation. This work was supported in part by grants to A. E from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.
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Edagawa, A., Kimura, A., Kawabuchi-Kurata, T. et al. Isolation and genotyping of potentially pathogenic Acanthamoeba and Naegleria species from tap-water sources in Osaka, Japan. Parasitol Res 105, 1109–1117 (2009). https://doi.org/10.1007/s00436-009-1528-4
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DOI: https://doi.org/10.1007/s00436-009-1528-4