Isolation of free-living amoebae from sarein hot springs in ardebil province, iran.

Abstract

Free-living amoebae (FLA) are a group of ubiquitous protozoan, which are distributed in the natural and artificial environment sources. The main aim of the current study was to identify the presence of FLA in the recreational hot springs of Sarein in Ardebil Province of Iran.
Seven recreational hot springs were selected in Sarein City and 28 water samples (four from each hot spring) were collected using 500 ml sterile plastic bottles during three month. Filtration of water samples was performed, and culture was done in non-nutrient agar medium enriched with Escherichia coli. Identification of the FLA was based on morphological criteria of cysts and trophozoites. Genotype identification of Acanthamoeba positive samples were also performed using sequencing based method.
Overall, 12 out of 28 (42.9%) samples were positive for FLA which Acanthamoeba and Vahlkampfiid amoebae were found in one (3.6%) and 11 (39.3%) samples, respectively. Sequence analysis of the single isolate of Acanthamoeba revealed potentially pathogenic T(4) genotype corresponding to A. castellanii.
Contamination of hot springs to FLA, such as Acanthamoeba T(4) genotype (A. castellanii) and Vahlkampfiid amoebae, could present a sanitary risk for high risk people, and health authorities must be aware of FLA presence.

Full text

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Iranian J Parasitol: Vol. 6, No.2, 2011, pp.1-8

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Corresponding Author: Email: rezaiian@sina.tums.ac.ir. Fax: +98 21-88951392









Original Article

Isolation of Free-Living Amoebae from Sarein Hot Springs in Ardebil
Province, Iran

A Badirzadeh1,2, M Niyyati3, Z Babaei4, H Amini5, H Badirzadeh6, *M Rezaeian1,2

1 Department of Parasitology and Mycology, School of Public Health, Tehran University of Medical
Sciences, Tehran, Iran
2 Center for Research of Endemic Parasites of Iran (CREPI), Tehran University of Medical Sciences,
Tehran, Iran
3 Department of Parasitology and Mycology, School of Medicine, Shahid Beheshti University of Medical
Sciences, Tehran, Iran
4 Department of Parasitology and Mycology, Faculty of Medicine, Kerman University of Medical Sciences,
Tehran, Iran
5 Department of Environmental Health Engineering, School of Public Health, Tehran University of
Medical Sciences, Tehran, Iran
6 Laboratory of Microbiology and Parasitology, School of Medicine, Ardebil University of Medical
Sciences, Ardebil, Iran

(Received 12 Nov 2010; accepted 18 May 2011)

ABSTRACT
Background: Free-living amoebae (FLA) are a group of ubiquitous protozoan, which are distributed in
the natural and artificial environment sources. The main aim of the current study was to identify the
presence of FLA in the recreational hot springs of Sarein in Ardebil Province of Iran.
Methods: Seven recreational hot springs were selected in Sarein City and 28 water samples (four from
each hot spring) were collected using 500 ml sterile plastic bottles during three month. Filtration of water
samples was performed, and culture was done in non-nutrient agar medium enriched with Escherichia coli.
Identification of the FLA was based on morphological criteria of cysts and trophozoites. Genotype
identification of Acanthamoeba positive samples were also performed using sequencing based method.
Results: Overall, 12 out of 28 (42.9%) samples were positive for FLA which Acanthamoeba and
Vahlkampfiid amoebae were found in one (3.6%) and 11 (39.3%) samples, respectively. Sequence
analysis of the single isolate of Acanthamoeba revealed potentially pathogenic T4 genotype corresponding
to A. castellanii.
Conclusion: Contamination of hot springs to FLA, such as Acanthamoeba T4 genotype (A. castellanii)
and Vahlkampfiid amoebae, could present a sanitary risk for high risk people, and health authorities must
be aware of FLA presence.
Keywords: Hot spring, Free-living amoebae, Iran, Acanthamoeba castellanii, Vahlkampfiidae







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Badirzadeh et al : Isolation of Free-Living Amoebae from Sarein Hot Springs …
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Introduction


ree- living amoebae (FLA) include
many genera with high distribution in
natural and artificial environment,
such as air, water, dust, and soil (1). Several
genera of FLA have been concerned in
medical sciences, such as Acanthamoeba
spp., Balamuthia, and Naegleria (1-2). On
the other hand, these FLA could act as a
Potential pathogen leading to fatal disease in
domestic animals and humans by invasion to
central nerve system (CNS) and other organs,
especially in those with higher risk (3-4).
Amphizoic nature of FLA lead to their pres-
ence in different water types, such as fresh,
coastal, sea, mineral waters, wastewater,
swimming pools, and hot springs. High tem-
perature of hot springs (37-45 ºC) can sup-
port the growth and distribution of some
potentially pathogenic thermophilic FLA (5).
To date, Acanthamoeba spp., Naegleria
fowleri, Balamuthia mandrillaris, and Sap-
pinia pedata are known as causal agents for
human disease (6-7).
Acanthamoeba is the most prevalent amoe-
bae in the environmental sources, and this
genus has been isolated from different water
types (5, 7). To date, Acanthamoeba have
been classified to 17 genotypes (T1-T17)
based on the variable region of 18S rRNA
gene (8-10). However, strains belong to the
T4 genotype are the most important causa-
tive agent in Acanthamoeba-related disease,
such as amoebic keratitis (AK), and granulo-
matous encephalitis (9). Interestingly, most
of AK cases showed a history of wearing
contact lenses and water activity before the
onset of disease (9). In Iran genotypes be-
longed to T2, T4 and T6 have been identified
in stagnant waters thus far (11-12). However,
there are no reports regarding the presence
of Acanthamoeba spp. in hot springs in Iran.
Among many genera of Vahlkampfiidae,
Naegleria and Vahlkampfia are of medical
importance (1, 7, 13). These thermophilic
amoebae have global distribution in water
sources and can cause FLA-related disease,
such as corneal, and brain involvement (1).
Naegleria fowleri can cause primary amoe-
bic meningoencephalitis (PAM) in young
and healthy people with a history of water
activity (7, 14-15).
It is worthy to mention that Acanthamoeba
and Vahlkampfiidae can also act as a carrier
of pathogenic bacteria, such as Legionella,
Pseudomonas, and Helicobacteria, and this
can lead to their expanded threat for human
being (16-17).
Sarein is a town of Ardebil Province, which
is located in the northwest of Iran, and it is
famous for various hot springs and spas as
recreation areas and health purposes (Fig.1).
Indeed, local people and tourists enjoy water
activity in this region through all months of
year. The main goal of the current study was
to identify the presence of FLA in the hot
springs of Sarein town using culturing
method and morphological key.

Materials and Methods

Sampling
During August, September, and October of
2010, 28 water samples were collected from
seven hot springs of Sarein in Ardebil Prov-
ince. Fig.1 shows the map of Ardebil Prov-
ince and Sarein City (created by Arc GIS
version 9.3). Four samples were collected
from each water sources including two sam-
ples from margin, and two from center of the
hot springs. Water temperature was meas-
ured using standard thermometer. Samples
were transmitted to the Department of
F

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Parasitology and Mycology, Tehran univer-
sity of Medical Sciences, Tehran, Iran.

Filtration, cultivation, and cloning
Samples were filtered using sterile mem-
brane filters (Millipore, pore size 0.45 μm).
Each filters were cultured on non-nutrient
agar medium enriched with Escherichia coli
(NNA medium), and incubation was per-
formed at room temperature for one month.
All mentioned plates surveyed daily under
light microscope (Carl Zeizz Axiolab) with
the magnification of 100X and 400X. Posi-
tive samples were then cloned in order to
achieve a free-bacteria and fungi plates (12).

Identification of the cloned amoebae
The criteria for identification of amoebae
were based on morphology of cysts and
trophozoites according to the Page, FC
(1988) key (18). Acanthamoeba were de-
tected based on double wall and wrinkled or
wavy ectocyst and characteristic acantopodia
of trophozoites. Molecular identification was
performed for Acanthamoeba positive sam-
ple using primers JDP1-2 (JDP1 5'-
GGCCCAGATCGTTTACCGTGAA-3' and
JDP2 5'-TCTCACAAGCTGCTAGGGAG-
TCA-3') (19). DNA extraction was per-
formed using Phenol chloroform method ac-
cording to our previous study (12) and PCR
was achieved in 30 µl Ampliqone (Taq DNA
Polymerase Master Mix RED, Denmark) as
a ready-made mixture. Twenty-five µl of
Taq Master Mix were used with 5 ng tem-
plate DNA, 0.1 µm of each primer and dis-
tilled water. The thermal cycling conditions
were an initial denaturing step of 94 oC for 1
min and 35 repetitions at 94 oC for 35 s, an-
nealing step were 56 oC for 45 s, and 72 oC
for 1 min. PCR products were electrophore-
sed on a 2% agarose gel stained with a solu-
tion of ethidium bromide and detected under
UV light. Sequencing analysis was per-
formed using BLASTn in the genbank data-
base. Vahlkampfiid amoebae were detected
according to spherical and smooth wall cysts
and eruptive trophozoites in plates (18).

Results

Overall, 12 out of 28 (42.9%) samples were
positive for outgrowth of FLA (Table 1).
Acanthamoeba castellanii and Vahlkampfiid
amoebae were found in one (3.6%) and 11
(39.3%) samples, respectively (Fig.2, 3).
Only one A. castellanii strain was isolated in
Pehenloo hot spring from the margin site,
which was further confirmed using PCR
based method. Sequencing analysis of this
isolate in the GenBank database was re-
vealed a 100% similarity with the T4 geno-
type corresponding to A. castellanii species.
Vahlkampfiid amoebae
according to spherical and smooth wall cysts
and eruptive trophozoites
Contamination of hot springs to Vahlkamp-
fiid amoebae where detected in Pehenloo,
Ghavmishgholi, General, Abdarmanieh Sa-
balan, Sarisuei, Beshbajilar, and Ghahvesuei.
The mean temperature of surveyed hot
springs was 43.6ºC (Table 1). Both collected
samples of margin and center of the springs
were positive.

were detected
(Fig. 3).

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Badirzadeh et al : Isolation of Free-Living Amoebae from Sarein Hot Springs …
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Table 1: Frequency of free-living amoebae in Sarein Hot Springs, and Samples Sites

Sampling Site Temp
(ºC)
Oct Sep Aug
No. of Samples Total
Sam-
ples
4
4
4
4
Acanthamoeba
castellanii
Vahlkampfiid
amoebae
Pehenloo
General
Ghavmishgholi
Abdarmanieh
Sabalan
Sarisuei
Beshbajilar
Ghahvesuei
Total
*Mean temperature of 7 hot springs


43
43
46
41
2
2
2
2
1
1
1
1
1
1
1
1
1
-
-
-
2
2
1
1
46
41
43
43.6*
2
2
2
14
1
1
1
7
1
1
1
7
4
4
4
28
-
-
-
1
2
2
1
11


Fig. 1: Map of the Ardebil Province and Sarein city, Iran (Created by Arc GIS version 9.3)

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Fig. 2: Acanthamoeba castellanii cysts (400 X)



Fig. 3: Vahlkampfiidae cysts (400 X)

Discussion

This study is the first survey regarding the
presence of FLA in hot springs of Iran. To
date, hot springs are famous for health pur-
poses and traditional treatment option,
worldwide. Sarein in northwest of Iran con-
tain numerous hot springs and this region
attract many tourists annually.
The results of the present study revealed, hot
springs of Sarein were contaminated with
opportunistic FLA, such as Acanthamoeba,

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Badirzadeh et al : Isolation of Free-Living Amoebae from Sarein Hot Springs …
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and Vahlkampfiid amoebae, and this may
lead to severe disease in high-risk people,
such as contact lens wearers. This is in sup-
port with researches conducted worldwide
(15, 17, 20-22). In a study in Spain, 12 natu-
ral hot springs examined in which 8 springs
were contaminated to different taxa includ-
ing Acanthamoeba (3) and Vahlkampfiid
amoebae (7, 20). In another survey in Thai-
land, 28 out of 69 samples were found posi-
tive for FLA, which 13.0% (9/69) were
Acanthamoeba, and 34.8% (24/69) were
Naegleria (5). Moreover, in another study in
Thailand, 56 samples were collected from 28
natural water resources that six samples
were Naegleria spp., 3 samples were Acan-
thamoeba, 5 were mixed, and 2 samples
were unidentified (22).
Our results confirmed that Acanthamoeba is
more sensitive to growth in high temperature
in contrast to Vahlkampfiid amoebae which
hot water may lead to the favorable condi-
tion for their proliferation. This is in support
with other studies, which showed higher
isolation of Vahlkampfiids compared to
Acanthamoeba in hot springs (5, 22). More
researches are needed for identification of
different vahlkamfiids in Iran using molecu-
lar based methods.
Another interesting finding of the present
study was the isolation of potentially patho-
genic A. castellanii belonged to the T4 geno-
type. T4 strains have been proved to contain
certain properties that render them to be
more virulent (11). T4 genotype was also a
predominant type found in AK patients in
Iran (12). In another study by Rezaeian et al,
354 samples of water and soil were collected
from rivers and lakes of Parishan and Dashte
Arzhan in Kazerun city. They found 10
cases of FLA based on morphological crite-
ria (2). Furthermore, the number of AK
cases, which have been diagnosed up to
2004, were more than 3000 cases worldwide
and more than 70 percent were contact lens
wearers (23). In Iran, 45 AK cases have been
diagnosis during 1995-2005 in School of
Public Health at Tehran University of Medi-
cal Sciences (24).
In conclusion, contamination of hot springs
to FLA, such as Acanthamoeba and
Vahlkampfiid amoebae, could present a
sanitary risk for high-risk people, and health
authorities must be aware of FLA presence.
Education to high-risk people, and improved
disinfection methods must apply for prevent-
ing FLA-related disease in this region.

Acknowledgments

The authors acknowledge wholehearted
cooperation of the staff of Department of
Parasitology and Mycology of Tehran Univ-
ersity of Medical Sciences. Likewise, the
authors declare that they have no conflicts of
interest.

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Erratum
The Problem of Mixing up of Leishmania Isolates in the
Laboratory: Suggestion of ITS1 Gene Sequencing for Verification of
Species (Iranian J Parasitol: Vol. 6, No.1, 2011, pp. 41-48)


There was an error in a paper published recently in Iranian J Parasitol: Vol. 6, No.1,
2011, pp. 41-48 entitled "The Problem of Mixing up of Leishmania Isolates in the
Laboratory: Suggestion of ITS1 Gene Sequencing for Verification of Species".
The error is the international code of the parasite No. 1 in the Table 1 in Page 44. The
incorrect form is: “MHOM/IR/75/ER” and the correct form is “MRHO/IR/75/ER”. In
other words, "HOM" should be replaced by "RHO".


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