Abstract
A survey was carried out in Bulgaria to determine the presence of free-living amoebae (FLA) from environmental sources. In 171 (61.1%) of 280 samples, isolates of Acanthamoeba with group II or III morphology, as well as Hartmannella spp. were recovered. Five isolates named “6” (artificial lake), Ep (lake), G2 (soil), R4* (river) and PK (spring water)—all exhibiting a highly efficient proliferation in axenic cultures—were subsequently cloned and subjected to molecular analyses for identification and genoty** In accordance with morphological findings, PCR-based analyses identified four isolates (6, Ep, G2, R4*) belonging to the genus Acanthamoeba. Confirmation of these findings was obtained by phylogenetic analysis using partial sequencing of the 18S rDNA (ASA.S1) Acanthamoeba-gene. Comparison of these sequences with corresponding regions from other Acanthamoeba strains available from GenBank sorted all four isolates into the sequence type group T4 that contains most of the pathogenic Acanthamoeba strains already identified. The fifth isolate (PK) exhibited morphological characteristics matching those of Hartmannella, and scored negative in the Naegleria fowleri and Acanthamoeba PCRs.
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Aitken D, Hay J, Kinnear FB, Kirkness, CM, Lee WR, Seal DV (1996) Amebic keratitis in a wearer of disposable contact lenses due to a mixed Vahlkampfia and Hartmannella infection. Ophthalmology 103:485–494
Booton GC, Kelly DJ, Chu Y-W, Seal DV, Houang E, Lam DSM, Byers TJ, Fuerst PA (2002) 18S ribosomal DNA ty** and tracking of Acanthamoeba species isolates from corneal scrape specimens, contact lenses, lens cases, and home water supplies of Acanthamoeba keratitis patients in Hong Kong. J Clin Microbiol 40:1621–1625
Cerva L (1980) Laboratory diagnosis of primary amoebic mining-encephalitis and methods for the detection of Limax amoebae in the environment. Folia Parasitol 97:1–9
Clayton A, Wiley A (1987) Acanthamoeba meningoencephalitis in a patient with AIDS. J Infect Dis 155:130–133
De Jonckheere JF (1977) Use of an axenic medium for differentiation between pathogenic and nonpathogenic Naegleria fowleri isolates. Appl Environm Microbiol 33:751–757
De Jonckheere JF (1979a) Studies on pathogenic free-living amoebae in swimming pools. Bull Inst Pasteur 77:385–392
De Jonckheere JF (1979b) Pathogenic free-living amoebae in swimming pools: a survey in Belgium. Ann Microbiol (Paris) 130B:205–212
De Jonckheere JF (1991) Ecology of Acanthamoeba. Rev Infect Dis 13:[Suppl 5]:S385–7
De Jonckheere JF, Michel R (1988) Species identification and virulence of Acanthamoeba strains from human nasal mucosa. Parasitol Res 74:314–316
Fields BS, Nerad TA, Sawer TK, King CH, Barberee JM, Martin WT, Morrill WE, Sanden GN (1990) Characterization of an axenic strain of Hartmannella vermiformis obtained from investigation of nosocomial legionellosis. J Protozool 37:581–583
Friedland LR, Raphael SA, Deutsch ES, Johal J, Martyn LJ, Visvesvara GS, Lischner HW (1992) Disseminated Acanthamoeba infection in a child with symptomatic human immunodeficiency virus infection. Pediatr Infect Dis J 11:404–407
Gonzalez MM, Gould E, Martinez AJ, Visvesvara GS, Cleary TJ, Hensley GT (1986) Acquired immunodeficiency syndrome associated with Acanthamoeba infection and other opportunistic organisms. Arch Pathol Lab Med 110:749–751
Gregorio CD, Rivasi F, Mongiardo N, Rienzo BD, Wallace S, Visvesvara GS (1992) Acanthamoeba meningoencephalitis in a patient with acquired immunodeficiency syndrome. Arch Pathol Lab Med 116:1363–1365
Herbst R, Ott C, Jacobs T, Marti T, Marciano-Cabral F, Leippe M (2002) Pore-forming polypeptides of the pathogenic protozoon Naegleria fowleri. J Biol Chem 277:22353–22360
Howe DK, Vodkin MH, Novak RJ, Visvesvara G, McLaughlin GL (1997) Identification of two genetic markers that distinguish pathogenic and nonpathogenic strains of Acanthamoeba spp. Parasitol Res 83:345–348
Inoue T, Asari S, Tahara K, Hayashi K, Kiritoshi A, Shimomura Y (1998) Acanthamoeba keratitis with symbiosis of Hartmannella ameba. Am J Ophthalmol 125:721–723
Janitschke K, Werner H, Muller G (1980) Examination on the occurrence of free-living amoebae with possible pathogenic straits in swimming pools. Zentralbl Bakteriol I. Abt Orig B 170:108–122
Johan DT, De Jonckheere JF (1985) Isolation of Naegleria australiensis from an Oklahoma lake. J Protozool 32:571–575
Jones DB, Visvesvara GS, Robinson NR (1975) Acanthamoebae polyphaga keratitis and Acanthamoeba uveitis associated with fatal meningoencephalitis. Trans Ophthalmol Soc U K 1075:221–232
Kadlec K (1981) Different virulence of Naegleria fowleri strains isolated from a swimming pool. Folia Parasitol 28:97–103
Kadlek V (1978) The occurrence of amphizoic amebae in domestic animals. J Protozool 25:235–237
Kennedy S, Devine M, Hurloy C, Ooi YS, Collum LM (1995) Corneal infection associated with Hartmannella vermiformis in contact lens wearer. Lancet 346:637–638
Kilvington S, Beeching J (1995) Development of a PCR for identification of Naegleria fowleri from the environment. Appl Environ Microbiol 61:3764–3767
Kumar S, Tamura K, Jakobsen IB, Nei M (2001) MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17:1244–1245
Kurdova-Mintcheva R (1979) Study of Limax amoebae as potential agents of human diseases (in Russian). PhD thesis, Moscow
Kurdova-Mintcheva R (1984) Possible sources of exogenic amoebiasis in Bulgaria (in Bulgarian). Epidem Microbiol Infect Dis 1:62–69
Kurdova-Mintcheva R, Petrov P, Bradvarova I, Vinarova M, Tzvetanov I (1979) Investigations of free-living amebae group limax in tissue culture. Problems of infections and parasitic diseases. Med Fiskult 8:100–105
Ledee DR, Hay J, Byers TJ, Seal DV, Kirkness CM (1996) Acanthamoeba griffini: molecular characterization of a new corneal epithelial and tear samples in the diagnosis of Acanthamoeba keratitis. Invest Ophthalmol Vis Sci 39:1261–1265
Ledee DR, Seal DV, Byers TJ (1998) Confirmatory evidence from 18S r RNA gene analysis for in vivo development of propamidine resistance in a temporal series of Acanthamoeba ocular isolates from a patient. Antimicrob Agents Chemother 42:2144–2145
Ma P, Visvesvara GS, Martinez AJ, Frederick HT, Daggett PM, Sawyer TK (1990) Naegleria and Acanthamoeba infection. Rev Infect Dis 12:490–513
Martinez AJ (1985) Free-living amebas: natural history, prevention, diagnosis, pathology and treatment of disease. CRC Press, Boca Raton
Mathers WD, Nelson SE, Lane JL, Wilson ME, Allen RC, Folberg R (2000) Confirming of confocal microscopy diagnosis of Acanthamoeba keratitis using polymerase chain reaction analysis. Arch Ophthalmol 118:178–183
McLaughlin GL, Vodkin MN, Huizinga HW (1991) Amplification of repetitive DNA for the specific detection of Naegleria fowleri. J Clin Microbiol 29:227–230
Michel R, Röhl R, Schneider H (1982) Isolation of free-living amoebae from nasal mucosa of healthy individuals. Zentralbl Bakteriol Hyg 176:155–159
Page FC (1988) A new key to freshwater and soil gymnamoebae with instructions for culture. Freshwater Biological Association, Ambleside
Pussard M, Pons R (1977) Morpholofie de la paroi kystique et taxonomie du genre Acanthamoeba (Protozoa, Amoebida). Protistologica 13:557–598
Rivera F, Cerva L, Martinez J, Keleti G, Lares F, Ramirez E, Bonilla P, Graner SR, Saha AK, Glew RH (1990) Naegleria lovaniensis tarasca new subspecies, and the purepecha strain, a morphological variant of N. lovaniensis, isolated from natural thermal waters in Mexico. J Protozool 37:301–310
Rodriguez-Zaragoza S, Magana-Becerrs A (1997) Prevalence of pathogenic Acanthamoeba (protozoa: Amoebidae) in the atmosphere of the city of San Luis Potosi, Mexico. Toxicol Ind Health 13:519–526
Schroeder JM, Booton GC, Hay J, Niszl IA, Seal DV, Markus MB, Fuerst PA, Byers TJ (2001) Use of subgenic 18S ribosomal DNA PCR and sequencing for genus and genotype identification of Acanthamoeba from humans with keratitis and from sewage sludge. J Clin Microbiol 39:1903–1911
Sepetliev D (1972) Principles of medical statistics (in Bulgarian). Med Fizkult
Slater C, Sickel JZ, Visvesvara GS, Pabico RC, Gaspari AA (1994) Brief report: successful treatment of disseminated Acanthamoeba infection in an immunocompromised patient. N Engl J Med 331:85–87
Smirnov AV, Michel R (1999) New data on the cyst structure of Hartmannella vermiformis Page, 1967 (Lobosea, Gymnamoebia). Protistology 1:82–85
Stevens AR, DeJonckheere J, Willaert E (1980) Naegleria lovaniensis new species: isolation and identification of six thermophilic strains of a new species found in association with Naegleria fowleri. Int J Parasitol 10:51–64
Stothard DR, Shroeder-Diedrich JM, Awward MH, Gast RJ, Ledee DR, Rodriguez-Zaragoza S, Dean CL, Fuerst PA, Byers TJ (1998) The evolutionary history of eight new 18S rRNA gene sequence types. J Eukaryot Microbiol 45:45–54
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24:4876–4882
Tsvetkova N, Kurdova R (1998) Study of pathogenic features of Acanthamoeba isolated from the environment in Bulgaria in cell culture. Exp Pathol Parasitol 1:36–45
Visvesvara GS (1991) Classification of Acanthamoeba. Rev Infect Dis 13:369–372
Visvesvara GS, Stehr-Green JK (1990): Epidemiology of free-living ameba infections. J Protozool 37:25–33
Walochnik J, Haller-Schober EM, Kolli H, Picher O, Obwaller A, Aspöck H (2000a) Discrimination between clinically relevant and nonrelevant Acanthamoeba strains isolated from contact lens-wearing keratitis patients in Austria. J Clin Microbiol 38:3932–3936
Walochnik J, Obwaller A, Aspöck H (2000b) Correlations between morphological, molecular biological, and physiological characteristics in clinical and nonclinical isolates of Acanthamoeba spp. Appl Environ Microbiol 66:4408–4413
Walochnik J, Obwaller A, Aspöck H (2001) Immunological inter-strain cross-reactivity correlated to 18S rDNA sequence types in Acanthamoeba spp. Int J Parasitol 31:163–167
Walochnik J, Michel R, Aspöck H (2002) Discrepancy between morphological and molecular biological characters in a strain of Hartmannella vermiformis Page, 1967 (Lobosea, Gymnamoebia). Protistology 2:185–188
Walochnik J, Michel R, Aspöck H (2003) New insights into amoebozoan phylogeny. 10th International Meeting on the Biology and Pathogenicity of Free-Living Amoebae Proceedings (in press)
Willaert E, Stevens AR, Tyndall RL (1978) Acanthamoeba royreba sp. n. from a human tumor cell culture. J Protozool 25:1–14
Acknowledgements
This work was supported by the Swiss National Science Foundation (SCOPES No. 7IP062584), the Federal Office For Civil Protection and by the “Gesellschaft zur Ober Gerwern”, Berne. We would like to thank Prof. Andrew Hemphill from the Institute of Parasitology, University of Berne, Berne, Switzerland for his help in microscopy of the amoebae and Dr. Rolf Michel from the Central Institute of the Federal Armed Forces Medical Services, Koblenz, Germany for providing the strains 72/2, Pb40, De610 and Rhodos. We are indebted to Dr. Nadia Schürch and Dr. Martin Schütz from the Spiez Laboratory for their valuable support and logistic contribution to the work.
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Tsvetkova, N., Schild, M., Panaiotov, S. et al. The identification of free-living environmental isolates of amoebae from Bulgaria. Parasitol Res 92, 405–413 (2004). https://doi.org/10.1007/s00436-003-1052-x
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DOI: https://doi.org/10.1007/s00436-003-1052-x