Advertisement

Parasitology Research

, Volume 92, Issue 5, pp 405–413 | Cite as

The identification of free-living environmental isolates of amoebae from Bulgaria

  • Nina TsvetkovaEmail author
  • Mark Schild
  • Stefan Panaiotov
  • Rossitza Kurdova-Mintcheva
  • Bruno Gottstein
  • Julia Walochnik
  • Horst Aspöck
  • Mar Siles Lucas
  • Norbert Müller
Original Paper

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 genotyping 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.

Keywords

Swimming Pool Axenic Culture Medium Mineral Spring Bottle Mineral Water Indoor Swimming Pool 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

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.

References

  1. 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 103:485–494PubMedGoogle Scholar
  2. Booton GC, Kelly DJ, Chu Y-W, Seal DV, Houang E, Lam DSM, Byers TJ, Fuerst PA (2002) 18S ribosomal DNA typing 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 40:1621–1625CrossRefPubMedGoogle Scholar
  3. Cerva L (1980) Laboratory diagnosis of primary amoebic mining-encephalitis and methods for the detection of Limax amoebae in the environment. Folia Parasitol 97:1–9Google Scholar
  4. Clayton A, Wiley A (1987) Acanthamoeba meningoencephalitis in a patient with AIDS. J Infect Dis 155:130–133Google Scholar
  5. De Jonckheere JF (1977) Use of an axenic medium for differentiation between pathogenic and nonpathogenic Naegleria fowleri isolates. Appl Environm Microbiol 33:751–757Google Scholar
  6. De Jonckheere JF (1979a) Studies on pathogenic free-living amoebae in swimming pools. Bull Inst Pasteur 77:385–392Google Scholar
  7. De Jonckheere JF (1979b) Pathogenic free-living amoebae in swimming pools: a survey in Belgium. Ann Microbiol (Paris) 130B:205–212Google Scholar
  8. De Jonckheere JF (1991) Ecology of Acanthamoeba. Rev Infect Dis 13:[Suppl 5]:S385–7Google Scholar
  9. De Jonckheere JF, Michel R (1988) Species identification and virulence of Acanthamoeba strains from human nasal mucosa. Parasitol Res 74:314–316PubMedGoogle Scholar
  10. 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 37:581–583PubMedGoogle Scholar
  11. 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 11:404–407PubMedGoogle Scholar
  12. 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 110:749–751PubMedGoogle Scholar
  13. 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 116:1363–1365PubMedGoogle Scholar
  14. 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 277:22353–22360CrossRefPubMedGoogle Scholar
  15. 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 83:345–348Google Scholar
  16. Inoue T, Asari S, Tahara K, Hayashi K, Kiritoshi A, Shimomura Y (1998) Acanthamoeba keratitis with symbiosis of Hartmannella ameba. Am J Ophthalmol 125:721–723CrossRefPubMedGoogle Scholar
  17. 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 170:108–122Google Scholar
  18. Johan DT, De Jonckheere JF (1985) Isolation of Naegleria australiensis from an Oklahoma lake. J Protozool 32:571–575PubMedGoogle Scholar
  19. Jones DB, Visvesvara GS, Robinson NR (1975) Acanthamoebae polyphaga keratitis and Acanthamoeba uveitis associated with fatal meningoencephalitis. Trans Ophthalmol Soc U K 1075:221–232Google Scholar
  20. Kadlec K (1981) Different virulence of Naegleria fowleri strains isolated from a swimming pool. Folia Parasitol 28:97–103PubMedGoogle Scholar
  21. Kadlek V (1978) The occurrence of amphizoic amebae in domestic animals. J Protozool 25:235–237PubMedGoogle Scholar
  22. Kennedy S, Devine M, Hurloy C, Ooi YS, Collum LM (1995) Corneal infection associated with Hartmannella vermiformis in contact lens wearer. Lancet 346:637–638CrossRefGoogle Scholar
  23. Kilvington S, Beeching J (1995) Development of a PCR for identification of Naegleria fowleri from the environment. Appl Environ Microbiol 61:3764–3767PubMedGoogle Scholar
  24. Kumar S, Tamura K, Jakobsen IB, Nei M (2001) MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17:1244–1245PubMedGoogle Scholar
  25. Kurdova-Mintcheva R (1979) Study of Limax amoebae as potential agents of human diseases (in Russian). PhD thesis, MoscowGoogle Scholar
  26. Kurdova-Mintcheva R (1984) Possible sources of exogenic amoebiasis in Bulgaria (in Bulgarian). Epidem Microbiol Infect Dis 1:62–69Google Scholar
  27. 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 8:100–105Google Scholar
  28. 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 39:1261–1265Google Scholar
  29. 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 42:2144–2145PubMedGoogle Scholar
  30. Ma P, Visvesvara GS, Martinez AJ, Frederick HT, Daggett PM, Sawyer TK (1990) Naegleria and Acanthamoeba infection. Rev Infect Dis 12:490–513PubMedGoogle Scholar
  31. Martinez AJ (1985) Free-living amebas: natural history, prevention, diagnosis, pathology and treatment of disease. CRC Press, Boca RatonGoogle Scholar
  32. 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 118:178–183PubMedGoogle Scholar
  33. McLaughlin GL, Vodkin MN, Huizinga HW (1991) Amplification of repetitive DNA for the specific detection of Naegleria fowleri. J Clin Microbiol 29:227–230PubMedGoogle Scholar
  34. Michel R, Röhl R, Schneider H (1982) Isolation of free-living amoebae from nasal mucosa of healthy individuals. Zentralbl Bakteriol Hyg 176:155–159Google Scholar
  35. Page FC (1988) A new key to freshwater and soil gymnamoebae with instructions for culture. Freshwater Biological Association, AmblesideGoogle Scholar
  36. Pussard M, Pons R (1977) Morpholofie de la paroi kystique et taxonomie du genre Acanthamoeba (Protozoa, Amoebida). Protistologica 13:557–598Google Scholar
  37. 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 37:301–310PubMedGoogle Scholar
  38. 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 13:519–526PubMedGoogle Scholar
  39. 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 39:1903–1911CrossRefPubMedGoogle Scholar
  40. Sepetliev D (1972) Principles of medical statistics (in Bulgarian). Med FizkultGoogle Scholar
  41. 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 331:85–87CrossRefPubMedGoogle Scholar
  42. Smirnov AV, Michel R (1999) New data on the cyst structure of Hartmannella vermiformis Page, 1967 (Lobosea, Gymnamoebia). Protistology 1:82–85Google Scholar
  43. 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 10:51–64PubMedGoogle Scholar
  44. 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 45:45–54PubMedGoogle Scholar
  45. 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 24:4876–4882CrossRefGoogle Scholar
  46. Tsvetkova N, Kurdova R (1998) Study of pathogenic features of Acanthamoeba isolated from the environment in Bulgaria in cell culture. Exp Pathol Parasitol 1:36–45Google Scholar
  47. Visvesvara GS (1991) Classification of Acanthamoeba. Rev Infect Dis 13:369–372Google Scholar
  48. Visvesvara GS, Stehr-Green JK (1990): Epidemiology of free-living ameba infections. J Protozool 37:25–33Google Scholar
  49. 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 38:3932–3936PubMedGoogle Scholar
  50. 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 66:4408–4413CrossRefPubMedGoogle Scholar
  51. Walochnik J, Obwaller A, Aspöck H (2001) Immunological inter-strain cross-reactivity correlated to 18S rDNA sequence types in Acanthamoeba spp. Int J Parasitol 31:163–167CrossRefPubMedGoogle Scholar
  52. 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 2:185–188Google Scholar
  53. 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)Google Scholar
  54. Willaert E, Stevens AR, Tyndall RL (1978) Acanthamoeba royreba sp. n. from a human tumor cell culture. J Protozool 25:1–14PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Nina Tsvetkova
    • 1
    • 2
    Email author
  • Mark Schild
    • 2
  • Stefan Panaiotov
    • 1
  • Rossitza Kurdova-Mintcheva
    • 1
  • Bruno Gottstein
    • 2
  • Julia Walochnik
    • 3
  • Horst Aspöck
    • 3
  • Mar Siles Lucas
    • 4
  • Norbert Müller
    • 2
  1. 1.Department of Parasitology and Tropical MedicineNational Center of Infectious and Parasitic DiseasesSofiaBulgaria
  2. 2.Institute of ParasitologyUniversity of BerneBerneSwitzerland
  3. 3.Department of Medical Parasitology, Clinical Institute of Hygiene and Medical MicrobiologyUniversity of ViennaViennaAustria
  4. 4.Unidad de Parasitologia, Facultat de FarmaciaUniversidad de SalamancaSalamancaSpain

Personalised recommendations