Morpho-Physiological and Biochemical Criteria of Acanthamoeba spp. Isolated from the Egyptian Aquatic Environment.

  • A Al-Herrawy Parasitology Laboratory, Water Pollution Research Department, NRC, 12622 Dokki, Giza, Egypt.
  • M Bahgat Therapeutic Chemistry Department, NRC, 12622 Dokki, Giza, Egypt
  • A Mohammed Zoology Department, Faculty of Science, Ain Shams University, Cairo, Egypt
  • A Ashour Zoology Department, Faculty of Science, Ain Shams University, Cairo, Egypt
  • W Hikal Parasitology Laboratory, Water Pollution Research Department, NRC, 12622 Dokki, Giza, Egypt
Keywords: Acanthamoeba, Egypt, Heat-tolerance, Proteases, Swimming pools, Water


 Background: The free-living amoebae Acanthamoeba spp., have been recognized as etiologic agents of amoebic encephalitis, keratitis, otitis, lung lesions and other skin infections mainly in immuno-compromised individuals. In this study, morpho-physiological and biochemical characterization of Acanthamoeba strains isolated from the Egyptian aquatic environment were surveyed. Methods: some Acanthamoeba species were cultivated on non-nutrient agar. Isolated strains of Acanthamoeba were identification based on the morphology of trophic and cyst forms in addition to temperature and osmo-tolerance assays. Biochemical characterization of the isolated amoeba strains was performed using quantitative assay as well as qualitative determination of proteolytic activity in zymograph analysis. Results: Potentially pathogenic Acanthamoeba species were isolated from all of the examined water sources. Colorimetric assays showed protease activity in the heat-tolerant isolates of Acanthamoeba. All pathogenic isolates of Acanthamoeba exhibited higher protease activity than did the non-patho-genic ones. The zymographic protease assays showed various banding patterns for different strains of Acanthamoeba. Conclusion: The incidence and prevalence of the pathogenic Acanthamoeba species in the aquatic environment using parasitological and biochemical diagnostic tools will provide baseline data against which the risk factors associated with waterborne transmission can be identified.


Marciano-Cabral F, Cabral G. Acanthamoeba spp. as agents of disease in humans. Clin Microbiol Rev. 2003; 16: 273–307.

Khan NA. Pathogenesis of Acanthamoeba in-fections. Microb Pathog. 2003; 34:277-28.

Krusnell J, Linder E. Bacterial infections of free-living amoebae. Res Microbiol. 2001; 152:613-619.

Lorenzo-Morales J, Lopez-Darias M, Marti-nez-Carretero E, Valladares B. Isolation of po-tentially pathogenic strains of Acanthamoeba in wild squirrels from Canary Islands and Moroc-co. Exp Parasitol. 2007; 117:74-79.

Jager B, Stamm, W. Brain abscesses caused by free-living amoebae probably of the genus Hartmannella in a patient with Hodgkin's dis-ease. Lancet. 1972; ii: 1343-1345.

Al-Herrawy A. In vitro cultivation of agents of amoebic meningo-encephalitis isolated from water and sewage. Ph. D. thesis, Fac. Vet. Med. 1992; Alexandria Univ., Egypt.

Hamadto H, Aufy S, El-Hayawan I, Saleh M, Nagaty I. Study of free-living amoebae in Egypt. J Egypt Soci Parasitol. 1993; 23: 631-637.

Lorenzo-Morals J, Ortiga-Rivas A, Martinez E, Khoubbane M, Artigas P, Periago M, Foronda P, Abreu-Acosta N, Valladares B, Mas-Coma S. Acanthamoeba isolates belonging to T1, T2, T3, T4 and T7 genotypes from environmental freshwater samples in the Nile Delta region, Egypt. Acta Trop. 2006; 100: 63-69.

Ismail, M. General studies on free living amoeba isolated from the environment. MD. Thesis; 1995, Cairo University.

Sadaka H, El-Nassery S, Abou-Samra L, Awadalla H. Isolation and identification of free-living Amoebae from some water sources in Alexandria. J Egypt Soc Parasitol. 1994; 24(2):247-57.

El-Marhoumy S, Antonics S, Warhurst D. Iso-lation of soil amoebae from the nasal mucosa of children in Tanta - Egypt. J Egypt Soc Parasitol. 1988; 18(2):665.

El-Deeb MAB. Preliminary studies on free-living amoebae. M.Sc. Thesis; 1990, Cairo University.

Gradus M, Koenig S, Hyndiuk R, De Carlo J. Filter-culture technique using amoebae saline transport medium for the noninvasive diagno-sis of Acanthamoeba keratitis. Am J Clin Pathol. 1989; 92: 682-685.

De Jonckheere J, Van Dijk P, De Voord H. Evaluation of the indirect fluorescent antibody technique for identification of Naegleria species. Appl. Microbiol.1974; 28: 159-164.

Pussard M, Pons R. Morphologie de la paroi kystique et taxonomie du genre Acanthamoeba (Protozoa, Amoebida). Protistol. 1977;TXIII: 557-598.

Page F. A New Key to Freshwater and Soil Gymnamoebae. Freshwater Biol. Ass: Amble-side;1988

Khan NA, Jarroll E, Paget T. Molecular and physiological differentiation between patho-genic and non-pathogenic Acanthamoeba. Curr Microbiol. 2002; 45: 197-202.

Khan NA, Jarroll E, Panjwani N, Cao Z, Pag-er T. Proteases as markers of differentiation of pathogenic and non-pathogenic Acanthamoeba. J Clin Microbiol. 2000; 38: 2858-2861.

Iwanaga Y. Studies on host-parasite relation-ship between the Puerto Rican strain of Schistosoma mansoni and Biomphalaria snails. Southeast Asian J Trop Med Public Health. 1994; 25: 509-15.

Bahgat M, Ruppel A. Biochemical comparison of the serine protease (elastase) activities in cercarial secretions from Trichobilharzia ocellata and Schistosoma mansoni. Parasitol Res. 2002; 88: 495-500.

Bahgat M, Sorgho H, Ouédraogo J. Enzyme-linked immunosorbent assay with worm vomit and cercarial secretions of Schistosoma mansoni to detect infections in an endemic focus of Burkina Faso. J Helminthol. 2006, 80:19-23.

John D, Howard M. Seasonal distribution of pathogenic free-living amoebae in Oklahoma water. Parasitol Res. 1995; 81: 193-201.

John D, Howard M. Isolation of thermo-to-lerant free-living amoebae from Lake Tenkiller. Oklahoma Proc Okla Acad Sci. 1996; 76: 1-4.

Hilali M, Ashmawy K, Samaha H, Draz A, Abu El-Waf, S, Salem A. Preliminary studies on amoebic pathogens isolated from water and sewage with respect to Naegleria and Acanthamoeba. J Egypt Vet Med Ass. 1994; 53: 215-224.

Kilvington S, Gray T, Dart J, Morlet N, Beeching J, Frazer D, Matheson M. Acantha-moeba keratitis: the role of domestic tap water contamination in the United Kingdom. Invest. Ophthalmol Vis Sci. 2004; 45: 165-169.

Jeong H, Yu H. The role of domestic water in Acanthamoeba contamination in contact lens storage cases in Korea. Korean J Parasitol. 2005; 43: 47-50.

Gronik K, Kuzna-Grygiel W. Presence of virulent strains of amphizoic amoebae in swimming pools of the city of Szczecin. Ann Agric Environ Med. 2004; 11: 233-236.

Al-Herrawy A, Al-Rasheid K. Identification of Acanthamoeba strains isolated from a freshwater course in Saudi Arabia. J Egypt Pub Healt Ass. 1998; LXXIII: 621-633.

Nacapunchai D, Kino H, Ruangsitticha C, Sriwichai P, Ishih A, Terada M. A brief survey of free-living amoebae in Thailand and Ha-mamatsu district, Japan. Southeast Asian J Trop Med Public Health. 2001; 32: 179-82.

Ettinger M, Webb S, Harris S, McIninch S, Garman G, Brown B. Distribution of free-living amoebae in James River, Virginia, USA. Parasitol Res. 2003; 89: 6-15.

Lorenzo-Morals J, Ortiga-Rivas A, Martinez E, Foronda P, Valladares B. Isolation and identi-fication of pathogenisis Acanthamoeba strains in Tenerife, Canary Island, Spain from water sources. Parasitol Res. 2005; 95: 273-277.

Brown T. The occurrence and distribution of pathogenic free-living amoebae in thermal are-as of the North Island of New Zealand. New Zealand Journal of Marine and Freshwater Re-search. 1983; 17: 59-69.

Rivera F, Ramirez P, Vilaclara G, Robles E, Medina F. A survey of pathogenic and free-living amoebae inhabiting swimming pool wa-ter in Mexico City. Environ Res. 1983; 32: 205-211.

Rivera F, Ramirez E, Bonilla P, Calderon A, Gallegos E, Rodríguez S, Ortiz R, Zaldivar B, Ramirez P, Duran A. Pathogenic and free-living amoebae isolated from swimming and physiotherapy tubs in Mexico. Environ Res. 1993; 62: 43-52.

Lekkla A, Sutthikornchai C, Bovornkitti S, Sukthana Y. Free-living amoebae contamina-tion in natural hot springs in Thailand. South-east Asian J Trop Med Public Health. 2005; 36: 5-9.

Hsu B, Ma P, Liou T, Chen J, Shine F. Identi-fication of 18S ribosomal DNA genotype of Acanthamoeba from hot spring recreation areas in the central range, Taiwan. Journal of Hy-drobiology. 2009; 367: 249-254.

Khan N, Tareen N. Genotypic, phenotypic, biochemical, physiological and pathogenicity-based categorization of Acanthamoeba strain. Folia Parasitologica. 2003; 50: 97-104.

Kilic A, Tanyuksel M, Sissons J, Jayasekera S, Khan NIsolation of Acanthamoeba isolates be-longing to T2, T3, T4, and T7 genotypes from environmental samples in Ankara, Turkey. Acta Parasitol. 2004; 49: 246–252.

Mckerrow J, Sun E, Rosenthal P, Bouvier J. The proteases and pathogenicity of parasitic protozoa. Annu Rev Microbiol. 1993; 47: 821-853.

Serrano-Luna J, Cervantes-Sandoval I, Calde-ron J, Navarro-Garcia F, Tsutsumi V, Shibayama M. Protease activities of Aca-nthamoeba polyphaga and Acanthamoeba castellanii. Can J Microbiol. 2006; 52: 16-23.

Alfieri S, Correia C, Motegi S, Pral E. Protein-ase activities in total extracts and in medium conditiond by Acanthamoeba polyphaga trophozoites. J Parasitol. 2000; 86: 220-227.

Mitro K, Bhagavathiammai A, Zhou O, Bobbett G, Mckerrow J, Chokshi R, Chokshi B, James E. Partial characterization of the proteolytic secretions of Acanthamoeba polyphaga. Exp Parasitol. 1994; 78: 377-385.

Nashed N, Youssef F, Mansour N. Isoenzyme characterization of Acanthamoeba from water sites in Egypt as compared to other geograph-ical Acanthamoeba species and strains. Inverte-brate Zoology and Parasitology. 1993; 11: 43-54.

Cho J, Na B, Kim T, Song C. Purification and characterization of an extracellular serine pro-teinase from Acanthamoeba castellanii. IUBMB Life. 2000; 50: 209-214.

Alsam S, Sisson J, Jayasekera S, Khan N. Ex-tracellular proteases of Acanthamoeba castellanii (encephalitis isolate belonging to T1 genotype) contribute to increased permeability in an in vitro model of the human blood-brain barrier. J Inf. 2005; 51: 150-156.

Sissons J, Alsam S, Goldsworthy G, Lightfoot M, Jarroll E, Khan, N. Identification and properties of proteases from an Acanthamoeba isolate capable of producing granulomatous encephalitis. BMC Microbiology. 2006; 6: 42-49.

Kim W, Kong H, Ha Y, Hong Y, Jeong H, Yu H, Chung D. Comparison of specific activity and cytopathic effects of purified 33 kDa ser-ine proteinase from Acanthamoeba strains with different degree of virulence. Korean J Parasitol. 2006; 4: 321-330.

Kong H, Kim T, Chung D. Purification and characterization of secretory serine proteinase of Acanthamoeba healyi isolated from gave. J Parasitol. 2000; 43: 391-395.

Blaschitz M, Kohsler M, Aspock H, Walochnik J. Detection of a serine proteinase gene in Aca-nthamoeba genotype T6 (Amoebozoa: Lobosea). Exp Parasitol. 2006; 114: 26-33.

How to Cite
Al-Herrawy A, Bahgat M, Mohammed A, Ashour A, Hikal W. Morpho-Physiological and Biochemical Criteria of Acanthamoeba spp. Isolated from the Egyptian Aquatic Environment. Iran J Parasitol. 8(2):302-1.