Abstract

Free-living amoebae are ubiquitous protozoa, frequently found in the aquatic environment. Acanthamoeba spp., in some conditions, causes amoebic keratitis. Our research project aimed at studying in vivoAcanthamoeba spp. that are possibly present in water destined for human consumption. Thus, we can evaluate the rate of water contamination by determining the critical areas of its presence. In total, 150 water samples were analysed from Rabat. All the samples were collected from five different sources: river, fountain water, seawater, public bath water and tap water. The samples were distributed over three seasons: spring, summer and autumn. The positive samples by culture method were confirmed by polymerase chain reaction (PCR) method. The obtained results by microscopic identification and PCR method showed a high percentage of the presence of Acanthamoeba spp. in water in Rabat. However, during the sampling period, we noticed a non-uniform division of the positive samples with a remarkably high rate during summer. Our study showed that water contamination by Acanthamoeba spp. in Rabat, Morocco is at high risk of having a negative impact on public health. It is necessary to do a follow-up and study the health impacts to better evaluate the risk associated with this contamination by Acanthamoeba spp.

INTRODUCTION

Free-living amoebae are unicells, eukaryotic and ubiquitous, which are present in the aquatic environment contrary to pathogenic amoebae (associated with a host). Most of the genera are not pathogenic, some others are opportunists: Acanthamoeba spp. can cause, in certain conditions, Acanthamoeba keratitis. This disease becomes more frequent when using contact lenses (more than 80% of the cases) (Cabral & Cabral 2003; Lorenzo-Morales et al. 2015). Acanthamoeba, Naegleria and Vahlkampfia are the main genera which represent the aquatic free-living amoebae (Kuiper et al. 2006; Thomas et al. 2006; Corsaro et al. 2007). Moreover, the amoebae population is regulated by the presence of nutriments, physicochemical factors, pH and annual temperature fluctuations (Kyle & Noblet 1986; Rohr et al. 1998; Pumidonming et al. 2010). The source of keratitis infection is highly related to the use of water contaminated by Acanthamoeba spp. In Morocco, studies associated with water contaminated by free-living amoebae have never been done and there are no related statistical data. Our research project suggests studying, in vivo, free-living amoebae (Acanthamoeba spp.) that are present in water destined for human consumption; thereby, we can assess the degree of contamination of these waters by determining the critical areas.

MATERIALS AND METHODS

During the period from April to November 2016, 150 water samples were collected from Bouregreg river, fountains, taps, public baths and ocean from the Rabat region, Morocco. The samples were collected weekly from different points, depending on the water category. Subsequently, 30 samples were divided according to the three seasons: spring, summer and autumn. The samples were collected using plastic bottles with a capacity of 2 L and these were examined by parasitological analysis in the General Department of Parasitology of the National Institute of Hygiene, Rabat, Morocco.

Culture method

The culture method used was non-nutritive agar with attenuated bacterial substrate (NNE) Escherichia coli ATCC 25922. The samples were filtered by a pumping aspiration system using a filter (3 μm pore size) of nitrocellulose. The volume of filtration according to the type of samples was: clean water box 1 L and raw water box 1 L 500 mL. The filter side of the filter was deposited on the non-nutrient agar surface. Petri dishes were incubated at two different temperatures (20 °C and 30 °C) for 15 days. Two different temperatures were used to increase the possibility of detecting other free-living amoebae in the water. Observation of the Petri dishes was done through an inverted microscope (this observation was made using magnification of 40 ×). Positive Petri dishes were analysed using the conventional polymerase chain reaction (PCR) method.

Genomic method

The cysts of the amoebae and their possible migration traces observed in the Petri dishes were suspended in 300 μL of the lysis buffer (0.1 M Tris-HCl, 0.1 M EDTA, 0.1 M NaCl, 2% SDS). Before DNA extraction, several steps were used to lyse the double cellulose wall of amoeba cysts, where five cycles of thermal shock were used, alternating with 1 minute of incubation at a temperature of 95 °C and 1 minute in a bath of liquid nitrogen, mechanical lysis by glass beads, and enzymatic digestion with proteinase K for 1 hour at 37 °C. Subsequently, DNA extractions were performed using a Bioline kit according to the supplier's recommendations (Fields 1996; Dykova et al. 2005; Fouque et al. 2012; Trabelsi et al. 2012). In the PCR reaction (25 μL) each sample was prepared as follows: DNA template (1 μL), forward primer (1.5 μL) (ACA-F (5′–3′): TGG CAG CGC GAG GAC TAG GG) reverse primer (1.5 μL) (ACA-R (3′–5′): ACC GCA CCG ATG GTG GTG TTT) (Leduc et al. 2012), Taq buffer and Taq DNA polymerase (12 μL), and pure water (9 μL). The amplification of DNA was performed by using a PCR thermal cycler. The programme included an initial denaturation at 95 °C for 5 min followed by 35 cycles; each cycle consisted of denaturation at 95 °C for 1 min, annealing at 63 °C for 1 min and extension at 72 °C for 1 min. The programme included a final extension step at 72 °C for 10 min. The validation process of the 18S rDNA gene was performed by electrophoresis on agar-agar (1%), and the amplification of the fragment was visualized by using ethidium bromide.

RESULTS

Of the total 150 samples from the five sampling areas in Rabat, Morocco, the microscopic presence of Acanthamoeba spp. cysts was observed in 20 samples (13.33%). By means of the PCR method, 18 samples were positively obtained (Table 1). However, the distribution of positive samples during the three seasons has revealed an abundance of Acanthamoeba spp. in the summer period (Table 2). Table 2 shows the percentage of contamination of the waters of Rabat by Acanthamoeba spp. during the three seasons.

Table 1

Distribution of positive samples at the different sampling sites

Sites of samplesSample numberPositive numbers% of positivity
Sea 30 23% 
Bouregreg river 30 11 36% 
Fountains 30 0% 
Public bath wáter 30 0% 
Tap 30 0% 
Total 150 20 13% 
Sites of samplesSample numberPositive numbers% of positivity
Sea 30 23% 
Bouregreg river 30 11 36% 
Fountains 30 0% 
Public bath wáter 30 0% 
Tap 30 0% 
Total 150 20 13% 
Table 2

Distribution of Achanthamoeba spp. in three seasons

SeasonsSample numberPositive numbers
Spring 50 
Summer 50 15 
Autumn 50 
Total 150 18 
SeasonsSample numberPositive numbers
Spring 50 
Summer 50 15 
Autumn 50 
Total 150 18 

DISCUSSION

The results obtained during sampling of the water samples revealed the presence of a high percentage of Acanthamoeba spp. in the Rabat area (Table 1), which may be due to bacterial diversity (Greub & Raoult 2004; Coleman et al. 2009). Bouregreg river has the highest pollution rate, unlike the samples collected from seawater (36.6% vs. 26.66%), respectively. The presence of Acanthamoeba spp. can be due to several factors, such as wastes and excretion products of livestock activity near tributaries, the presence of wildlife in rivers, as well as discharges of untreated wastewater and industrial emissions, especially from agriculture through the Bouregreg river (Laouina et al. 2010), which makes it the ideal environment for bacterial growth and development, particularly free-living amoebae (Adekambi et al. 2006; Pickup et al. 2007). The low pollution rate of seawater is related to the salinity of the Atlantic Ocean (33.5–37.4). Researchers have focused less on the study of Acanthamoeba spp. in seawater. This high salinity generates a limiting factor for the development of Acanthamoeba spp. (Booton et al. 2004; Steinum et al. 2008). Even so, water exposure to the air and soil contamination helps Acanthamoeba live and spread (Trabelsi et al. 2012). In Iran, researchers have indicated that Acanthamoeba spp. is present in 73.53% of environmental water samples (Mohammadi Manesh et al. 2016). Forty-eight per cent of the water collected from 14 cities is contaminated by Acanthamoeba spp. (Bagheri et al. 2010). In Taiwan, in 2014, 39.5% of the waters were contaminated by free-living amoebae (Kao et al. 2014). The increase in summer contaminated samples shows the impact of temperature on the development of the amoeba population (Niederkorn et al. 1999; Douglas-Helders et al. 2001; McAllum et al. 2009).

CONCLUSIONS

The number of samples and the limited filtration method are the two notable factors about uncontaminated waters in this study. Water pollution in the Rabat region can be considered a potential source of infection for people, and the use of contaminated water to wash the eyes can put people at risk of contracting Acanthamoeba keratitis. Therefore, public education, as well as prevention measures regarding Acanthamoeba spp. seems to be necessary in Rabat.

ACKNOWLEDGEMENTS

This study was supported by Mohammed V University and National Institute of Hygiene.

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