Introduction

Acanthamoeba spp. are free-living opportunistic protozoan parasites that pervade the entire environment, and they can be found in tap, fresh, coastal and bottled mineral water, sewage, soil, dust and air, heating or air-conditioning units and contact lens solutions, eyewash stations, and gastrointestinal washings (Mergeryan 1991; Marciano-Cabral et al. 2000; Khan 2003). These amoebae are the causative agents of multifocal encephalitis called granulomatous amebic encephalitis, a chronic central nervous system disease that usually occurs in immunocompromised hosts, amoebic keratitis (AK) and pneumonitis. AK is a corneal infection mainly associated with contact lenses use (Visvesvara et al. 2007). Most episodes of keratitis occur after water exposure or a history of swimming in lakes and ponds while wearing contact lenses and the infection is also linked to non-sterile home-made saline solutions for contact lenses (Radford et al. 1998; Khan 2003). In addition to their own pathogenicitiy, Acanthamoeba are known to be a reservoir of bacterial pathogens such as Legionella pneumophila, Mycobacterium avium, and other “amoeba-resistant microorganisms” (Greub and Raoult 2004). Acanthamoeba feed on bacteria by phagocytosis, but some bacteria are able to survive and sometimes multiply in the host, resulting in new properties of the bacteria (Sandström et al. 2011). Recently, the ability of free-living amoebae (FLA) to serve as vectors of Cryptosporidia has been reported for the first time (Scheid and Schwarzenberger 2011). FLA appear able to act as carriers or vectors of the oocysts and thus may play a certain role in the transmission of Cryptosporidium parvum oocysts.

As a co-investigation of our current research for Giardia and Cryptosporidium distribution in water supplies in Iran, we carried out a survey to determine the prevalence of Acanthamoeba spp. in drinking water sources of Iran.

Materials and methods

General information on geography

Gilan is one of the provinces of Iran, and it lies along the Caspian Sea. Gilan has a humid temperate climate with plenty of annual rainfall and is known for its moderate, mild, and Mediterranean-like climate. Large parts of the province are mountainous, green, and forested. Thousands of domestic and foreign tourists come to the seashore river for swimming and camping.

Sampling

During March to November 2009, 27 surface water samples were collected from environmental sources, including natural (rivers, lakes, springs, and lagoon) source from different parts of the Gilan province, north of Iran (Table 1; Fig. 1). From each sampling point, one to three water samples were collected in 500-ml sterile bottles and transported immediately to the laboratory for further processing. The samples were examined for the presence of Acanthamoeba in the laboratory of Protozoology Unit, Department of Parasitology and Mycology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

Table 1 Surface water samples collected from various location in Gilan, Iran and investigated for Acanthamoeba
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Fig. 1
figure 1

Map of the investigated Gilan greater area (right) and its location in Iran (left)

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Isolation of Acanthamoeba species and culture

For the isolation of Acanthamoeba species, approximately 500 ml of the collected water samples were filtered through a cellulose nitrate membrane with pore size 0.45 μ. Filter was transferred on non-nutrient agar plates seeded with Gram-negative bacteria (E. coli) as a food source. Plates were incubated at room temperature, and 3 days later, they were microscopically examined for the presence of Acanthamoeba trophozoites. However, in the absence of amoebae, plates were monitored for up to 14 days. Acanthamoeba were identified at the genus level, based on morphological characteristics of trophozoites and cysts using phase-contrast microscopy (Fig. 2).

Fig. 2
figure 2

Acanthamoeba species after isolation from water samples and cultivation. a Trophozoites (thin arrow) and a cyst (big arrow) after trichrome stain; ×400; b cyst form ×400

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DNA extraction and PCR

Amoeba cells were harvested from culture plates, concentrated by centrifugation, and then lysed by treatment with lysozyme (100 mg/ml) and freeze-thawing (using liquid nitrogen and heating to 96°C). The samples were then treated with 2–5 μl proteinase K (18.9 mg/ml), and DNA extraction was performed by phenol–chloroform method. PCR assay: the Acanthamoeba-specific primer pairs JDP1 (5′-GGCCCAGATCGTTTACCGTGAA) and JDP2 (5′-TCTCACAAGCTGCTAGGGAGTCA) as described by Schroeder et al. (2001) were used for the amplification of the 500 bp of 18S rDNA gene (Edagawa et al. 2009).

Standard PCRs were performed in 50-μl volumes, containing 5 μl of 10× PCR buffer, 20 pM of each of the primers, 4 mM MgCl2, 0.2 mM dNTP, 1.25 U Taq polymerase (Cinnagen), and 1–10 ng of template DNA. Thermal cycling conditions were 94°C for 5 min; 32 cycles of 94°C for 30 s, 57°C for 30 s, 72°C for 40 s; followed by a final extension at 72°C for 5 min.

Results and discussion

Culture

Based on the morphological criteria, 19 out of 27 (70.3%) surface waters contained Acanthamoeba spp. (Table 1). Some of the samples contain other free-living organisms. Acanthamoeba can be identified at the genus level based on distinctive features of trophozoites and cysts, especially the double-walled cyst shape that is unique to the genus. Acanthamoeba species have been classified into three distinct morphological groups (I, II, and III). However, this division is inconsistent (Stothard et al. 1998; Alves et al. 2000). The taxonomy and classification of these parasites are currently under revision, following the successful application of molecular techniques (Booton et al. 2002; Kong et al. 2002; Khan 2003).

PCR

Further detection and identification of Acanthamoeba species from the culture plates has been approved by the PCR (Fig. 3). The primers used in this study are specific for Acanthamoeba spp.. From 19 microscopically positive culture samples, 14 (73.7%) were also positive by PCR (Tables 1 and 2). Five negative PCR amplifications may include other FLA, like Thecamoebidae family (Table 2). According to Khan and Paget (2002) DNA sequencing is probably the most detailed and accurate technique for Acanthamoeba species differentiation.

Fig. 3
figure 3

PCR products on a 1.5% agarose gel. Lanes 1–10 Acanthamoeba positive isolates with JDP primers. Lane NC Acanthamoeba negative control, lane PC positive control Acanthamoeba isolate NHE (Ac) 14-IR (Accession no.AB525824.1), lane M 100-bp marker

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Table 2 Comparative results on the identification of Acanthamoeba species in surface water samples using culture and PCR
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Current situation in Iran: A 10-year study in Iran reveals that there is a considerable increase in the incidence of amoebic keratitis during the recent years (Rezeaian et al. 2007). Bagheri et al. (2010) showed that about half of the investigated samples collected from cold and warm tap water in Iran were infected with Acanthamoeba species. Maghsood et al. (2005) examined 12 water samples in Iran by molecular methods and revealed that T2 genotype was the most frequent genotype in their samples and also isolated, T2 genotype from three keratitis patients although to date, only isolates belonging to the T3, T4, T6, and T11 genotypes have been associated with Acanthamoeba keratitis (Maghsood et al. 2005). Also Rezaeian et al. (2008) recovered Acanthamoeba spp. from 58% collected samples from a variety of ecological habitats such as tap water, soil, and dust. However, systematic information on the occurrence and distribution of potentially pathogenic species of FLA in environmental waters in different parts of Iran remain unclear.

According to Karanis et al. (2007), Acanthamoeba spp. was the causative agent of an outbreak associated with a contaminated municipal water supply in the USA and FLA has been commonly found in various environmental water sources throughout the world. Edagawa et al. (2009) reported the prevalence of FLA in tap water sources from rivers and water treatment plants located in Osaka Prefecture, Japan. A total of 257 of 374 samples (68.7%) were positive for FLA by microscopy. Tsvetkova et al. (2004) in Bulgaria environmental sources, determine FLA from 171 (61.1%) of 280 samples. There are also many cases of amoebic keratitis, which occurs after swimming in contaminated pools, usage of homemade saline for washing contact lenses, and exposure to dirt and dust (Khan 2006).

In conclusion, we found that nearly 70% of the investigated water samples in Gilan greater area in Iran were contaminated by potentially pathogenic Acanthamoeba species. The investigated surface waters, including natural sources (rivers, lakes, springs, lagoons), are used for agriculture and swimming activities, and in case of Emamzadeh Hashem Dam water, using for drinking water source after treatment. We suggest that the real risk of Acanthamoeba pathogenesis, especially immunocompromised patients, should be considered, and more attention must be paid on the distribution of Acanthamoeba species in surface waters to this organism in Iran. The presented work can serve as a platform for future investigations.