scholarly journals Detection and Molecular Characterization of Potentially Pathogenic Free-Living Amoebae from Recreational and Public Soils in Mazandaran, Northern Iran

2021 ◽  
Author(s):  
Asal Tanzifi ◽  
Yousef Moghaddam ◽  
Samira Dodangeh ◽  
Ahmad Daryani ◽  
Shahabeddin Sarvi ◽  
...  
Keyword(s):  
18S Rrna ◽  
Taxonomic Status ◽  
Soil Samples ◽  
Rrna Gene ◽  
Its Sequencing ◽  
Sequencing Data ◽  
Mazandaran Province ◽  
Free Living ◽  
Northern Iran ◽  
Public Places

Background: Free-living amoeba (FLA) belonging to Acanthamoeba spp., Naegleria, and Balamuthia mandrillaris are the soil-born protozoa. This study aimed to survey the occurrence of FLA, including Acanthamoeba spp., B. mandrillaris, Vermamoeba spp., and Naegleria spp., in soil samples collected from various districts of Mazandaran Province (Northern Iran) from July to December 2018. Methods: Overall, 118 soil samples from the recreational and public places were surveyed for the existence of Acanthamoeba spp., Vermamoeba, Naegleria, and B. mandrillaris using both morphological key and molecular tools with genus-specific primers of JDP1, NA, ITS1, and Bal, respectively. To verify the taxonomic status of isolated amoeba, the phylogenetic tree was made based on sequences of 18S rRNA by MEGA (5.05) software with the maximum likelihood model. Results: Overall, 61/118 samples (51.6%) were contaminated with FLA, and based on the sequencing data, 29 isolates were successfully sequenced. Among the samples, all isolated Acanthamoeba (52.4%) belonged to the T4 genotype with amplification of the DF3 region (18S rRNA gene). Internal transcribed spacer (ITS) sequencing revealed the presence of one strain of Naegleria americana. Twenty-eight V. vermiformis were also confirmed based on Nuclear SSU rDNA. Morphological survey and PCR assay did not show any positive samples for B. mandrillaris. Conclusion: The present study indicates the occurrence of FLA in soil sources of the recreational and public places in Mazandaran province that it can be a severe risk to human health. Thus, more studies are expected to survey the infection source in patients with FLA-related diseases.

scholarly journals Isolation and molecular identification of Acanthamoeba spp. from hot springs in Mazandaran province, northern Iran

2018 ◽  
Vol 16 (5) ◽  
pp. 807-813 ◽  
Author(s):  
Samira Dodangeh ◽  
Elham Kialashaki ◽  
Ahmad Daryani ◽  
Mehdi Sharif ◽  
Shahabeddin Sarvi ◽  
...  
Keyword(s):  
Hot Springs ◽  
Warning Signs ◽  
Rrna Gene ◽  
Sequencing Analysis ◽  
Mazandaran Province ◽  
Northern Iran ◽  
Pathogenic Potential ◽  
Granulomatous Amoebic Encephalitis ◽  
Amoebic Keratitis ◽  
Hot Tubs

Abstract Acanthamoeba is a free-living protozoan that can be found in natural and artificial environments such as hot tubs, surface water and springs and can cause severe diseases including amoebic keratitis and granulomatous amoebic encephalitis. The present study was conducted owing to the lack of research regarding genotypes of Acanthamoeba in hot springs of Mazandaran province in northern Iran. Twenty-four water samples were collected from all hot springs in Mazandaran province. After filtration through nitrocellulose membrane, samples were cultured on non-nutrient agar medium enriched with TYIS-33. The cultures were microscopically examined for the presence of Acanthamoeba. Positive cultures were analysed by polymerase chain reaction (PCR) and genotypes were determined by targeting the 18 S rRNA gene. The pathogenic potential of all positive isolates was identified using thermotolerance and osmotolerance tests. Eleven (47.8%) samples were positive for Acanthamoeba. Based on sequencing analysis, 100% of isolates belonged to the T4 genotype. Thermo- and osmo-tolerance tests showed that four (36.3%) Acanthamoeba strains were highly pathogenic. According to our research, the occurrence of Acanthamoeba in recreational hot springs could be a hazard for high risk persons. Posting warning signs and regular monitoring of these waters by health planners may therefore be useful for decreasing Acanthamoeba spp. infections.

Phylogenetic analysis based on 18S rRNA gene sequences ofSchellackiaparasites (Apicomplexa: Lankesterellidae) reveals their close relationship to the genusEimeria

Parasitology ◽  
2013 ◽  
Vol 140 (9) ◽  
pp. 1149-1157 ◽  
Author(s):  
R. MEGÍA-PALMA ◽  
J. MARTÍNEZ ◽  
S. MERINO
Keyword(s):  
Phylogenetic Analysis ◽  
Blood Cells ◽  
18S Rrna ◽  
Taxonomic Status ◽  
Refractile Body ◽  
18S Rrna Gene ◽  
Blood Parasites ◽  
Recent Common Ancestor ◽  
Rrna Gene ◽  
Amphibian Species

SUMMARYIn the present study we detectedSchellackiahaemoparasites infecting the blood cells ofLacerta schreiberiandPodarcis hispanica, two species of lacertid lizards from central Spain. The parasite morphometry, the presence of a refractile body, the type of infected blood cells, the kind of host species, and the lack of oocysts in the fecal samples clearly indicated these blood parasites belong to the genusSchellackia. Until now, the species of this genus have never been genetically characterized and its taxonomic position under the Lankesterellidae family is based on the lack of the exogenous oocyst stage. However, the phylogenetic analysis performed on the basis of the 18S rRNA gene sequence revealed that species of the genusSchellackiaare clustered withEimeriaspecies isolated from a snake and an amphibian species but not withLankesterellaspecies. The phylogenetic analysis rejects that both genera share a recent common ancestor. Based on these results we suggest a revision of the taxonomic status of the family Lankesterellidae.

Identification of Acanthamoeba genotype T4 and Paravahlkampfia sp. from two clinical samples

2008 ◽  
Vol 57 (3) ◽  
pp. 392-396 ◽  
Author(s):  
Soykan Ozkoc ◽  
Sema Tuncay ◽  
Songul Bayram Delibas ◽  
Ciler Akisu ◽  
Zeynep Ozbek ◽  
...  
Keyword(s):  
18S Rrna ◽  
Single Agent ◽  
18S Rrna Gene ◽  
Maximum Temperature ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Free Living ◽  
History Of ◽  
Simplex Virus ◽  
Corneal Trauma

In this study, two free-living amoebae strains, Acanthamoeba genotype T4 and Paravahlkampfia sp., which were isolated from keratitis cases are presented. While the Acanthamoeba strain was isolated as a single agent, the Paravahlkampfia strain was found together with herpes simplex virus. Neither of the patients were contact lens wearers, but they did have a history of minor corneal trauma. Amoebae were detected on non-nutrient agar covered with Escherichia coli. Based on PCR-amplified 18S rRNA-gene analysis the first isolate was identified as Acanthamoeba genotype T4 and the second as Paravahlkampfia sp. In thermotolerance tests, the maximum temperature at which trophozoites continued to divide was determined as 37 °C for this Acanthamoeba strain and 35 °C for the Paravahlkampfia strain. To the best of our knowledge, the Acanthamoeba strain described herein is the second molecularly identified Acanthamoeba strain in an Acanthamoeba keratitis patient in Turkey. However, the Paravahlkampfia isolate is believed to be the first strain that has been isolated from a keratitis patient and has been molecularly differentiated from Vahlkampfia.

scholarly journals Genotyping and phylogenetic analysis of free-living amoeba (Acanthamoeba and Naegleria) in treated and untreated water in the northeastern provinces of Iran

2021 ◽  
Author(s):  
Yousef Sharifi ◽  
Omid Ahmadi ◽  
Bibi Razieh Hossini Farash ◽  
Nazgol Khosravinia ◽  
Reza Fotouhi-Ardakani ◽  
...  
Keyword(s):  
Water Quality ◽  
Water Samples ◽  
Taxonomic Status ◽  
Water Quality Monitoring ◽  
Pcr Analysis ◽  
Growth Media ◽  
Genotype Distribution ◽  
Free Living ◽  
Northern Iran ◽  
Pcr Products

Abstract Free-living amoebae (FLA) are widely distributed protozoa in natural or man-made aquatic environments without the need for a host organism for survival. Several strains of FLA are known to be pathogenic. As of date, there is inadequate data on the geographical distribution of FLA in northeastern and northern Iran. This study aimed to investigate the prevalence and genotype distribution of Acanthamoeba and Naegleria in drinking water and surface water samples in northern and northeastern Iran. A total of 60 water samples were collected and filtered from various sources for the presence of amoebae. DNA extraction was performed, and PCR confirmed the presence of FLA. PCR products were sequenced to identify the species/genotype. Phylogenetic relationships and taxonomic status constructed using MEGA X software. The findings on growth media showed 35% (21/60) and 26% (16/60) were positive for Acanthamoeba and Naegleria, respectively, while PCR analysis also obtained similar results. All isolates of Acanthamoeba were identified as T4 genotype. Poor water quality, as well as insufficient preservation and treatment, might indicate that chlorine disinfection is ineffective in removing contamination of amoebas in treated water samples. Therefore, regular water quality monitoring is essential to control amoeba's growth, reducing the risk of human infections with FLA.

scholarly journals Free-Living Protozoa in Two Unchlorinated Drinking Water Supplies, Identified by Phylogenic Analysis of 18S rRNA Gene Sequences

2009 ◽  
Vol 75 (14) ◽  
pp. 4736-4746 ◽  
Author(s):  
Rinske M. Valster ◽  
Bart A. Wullings ◽  
Geo Bakker ◽  
Hauke Smidt ◽  
Dick van der Kooij
Keyword(s):  
Drinking Water ◽  
Legionella Pneumophila ◽  
Distribution System ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Free Living ◽  
Water Supplies ◽  
Molecular Approaches ◽  
High Level

ABSTRACT Free-living protozoan communities in water supplies may include hosts for Legionella pneumophila and other undesired bacteria, as well as pathogens. This study aimed at identifying free-living protozoa in two unchlorinated groundwater supplies, using cultivation-independent molecular approaches. For this purpose, samples (<20°C) of treated water, distributed water, and distribution system biofilms were collected from supply A, with a low concentration of natural organic matter (NOM) (<0.5 ppm of C), and from supply B, with a high NOM concentration (7.9 ppm of C). Eukaryotic communities were studied using terminal restriction fragment length polymorphism and clone library analyses of partial 18S rRNA gene fragments and a Hartmannella vermiformis-specific quantitative PCR (qPCR). In both supplies, highly diverse eukaryotic communities were observed, including free-living protozoa, fungi, and metazoa. Sequences of protozoa clustered with Amoebozoa (10 operational taxonomic units [OTUs]), Cercozoa (39 OTUs), Choanozoa (26 OTUs), Ciliophora (29 OTUs), Euglenozoa (13 OTUs), Myzozoa (5 OTUs), and Stramenopiles (5 OTUs). A large variety of protozoa were present in both supplies, but the estimated values for protozoan richness did not differ significantly. H. vermiformis was observed in both supplies but was not a predominant protozoan. One OTU with the highest similarity to Acanthamoeba polyphaga, an opportunistic human pathogen and a host for undesired bacteria, was observed in supply A. The high level of NOM in supply B corresponded with an elevated level of active biomass and with elevated concentrations of H. vermiformis in distributed water. Hence, the application of qPCR may be promising in elucidating the relationship between drinking water quality and the presence of specific protozoa.

scholarly journals Quantitative Detection of the Free-Living Amoeba Hartmannella vermiformis in Surface Water by Using Real-Time PCR

2006 ◽  
Vol 72 (9) ◽  
pp. 5750-5756 ◽  
Author(s):  
Melanie W. Kuiper ◽  
Rinske M. Valster ◽  
Bart A. Wullings ◽  
Harry Boonstra ◽  
Hauke Smidt ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Quantitative Detection ◽  
Rrna Gene ◽  
Free Living ◽  
Copy Numbers ◽  
Free Living Amoeba ◽  
Hartmannella Vermiformis

ABSTRACT A real-time PCR-based method targeting the 18S rRNA gene was developed for the quantitative detection of Hartmannella vermiformis, a free-living amoeba which is a potential host for Legionella pneumophila in warm water systems and cooling towers. The detection specificity was validated using genomic DNA of the closely related amoeba Hartmannella abertawensis as a negative control and sequence analysis of amplified products from environmental samples. Real-time PCR detection of serially diluted DNA extracted from H. vermiformis was linear for microscopic cell counts between 1.14 � 10−1 and 1.14 � 104 cells per PCR. The genome of H. vermiformis harbors multiple copies of the 18S rRNA gene, and an average number (with standard error) of 1,330 � 127 copies per cell was derived from real-time PCR calibration curves for cell suspensions and plasmid DNA. No significant differences were observed between the 18S rRNA gene copy numbers for trophozoites and cysts of strain ATCC 50237 or between the copy numbers for this strain and strain KWR-1. The developed method was applied to water samples (200 ml) collected from a variety of lakes and rivers serving as sources for drinking water production in The Netherlands. Detectable populations were found in 21 of the 28 samples, with concentrations ranging from 5 to 75 cells/liter. A high degree of similarity (≥98%) was observed between sequences of clones originating from the different surface waters and between these clones and the reference strains. Hence, H. vermiformis, which is highly similar to strains serving as hosts for L. pneumophila, is a common component of the microbial community in fresh surface water.

scholarly journals A novel metabarcoding strategy for studying nematode communities

2020 ◽  
Author(s):  
Md. Maniruzzaman Sikder ◽  
Mette Vestergård ◽  
Rumakanta Sapkota ◽  
Tina Kyndt ◽  
Mogens Nicolaisen
Keyword(s):  
18S Rrna ◽  
High Throughput Sequencing ◽  
Soil Samples ◽  
Coi Gene ◽  
Rrna Gene ◽  
Nematode Communities ◽  
Sequencing Platform ◽  
Spiked Soil ◽  
Primer Sets ◽  
Mock Communities

AbstractNematodes are widely abundant soil metazoa and often referred to as indicators of soil health. While recent advances in next-generation sequencing technologies have accelerated research in microbial ecology, the ecology of nematodes remains poorly elucidated, partly due to the lack of reliable and validated sequencing strategies. Objectives of the present study were (i) to compare commonly used primer sets and to identify the most suitable primer set for metabarcoding of nematodes; (ii) to establish and validate a high-throughput sequencing strategy for nematodes using Illumina paired-end sequencing. In this study, we tested four primer sets for amplicon sequencing: JB3/JB5 (mitochondrial, I3-M11 partition); SSU_04F/SSU_22R (18S rRNA, V1-V2 region); Nemf/18Sr2b (18S rRNA, V6-V8 region) from earlier studies; and MMSF/MMSR (18S rRNA, V4-V5 region), a newly developed primer set from this study. In order to test the primer sets, we used 22 samples of individual nematode species, 20 mock communities, 20 soil samples, 20 spiked soil samples (mock communities in soil), and 4 root/rhizosphere soil samples. We successfully amplified the target regions (I3-M11 partition of the COI gene; V1-V2, V4-V8 region of 18S rRNA gene) from these 86 DNA samples with the four different primer combinations and sequenced the amplicons on an Illumina MiSeq sequencing platform. We found that the MMSF/MMSR and Nemf/18Sr2b were efficient in detecting nematode compared to JB and SSU primer sets based on annotation of sequence reads at genus and in some cases at species level. Therefore, these primer sets are suggested for studies of nematode communities in agricultural environments.

scholarly journals DNA of free-living bodonids (Euglenozoa: Kinetoplastea) in bat ectoparasites: potential relevance to the evolution of parasitic trypanosomatids

2017 ◽  
Vol 65 (4) ◽  
pp. 531-540 ◽  
Author(s):  
Krisztin Szőke ◽  
Attila D. Sándor ◽  
Sándor A. Boldogh ◽  
Tamás Görföl ◽  
Jan Votýpka ◽  
...  
Keyword(s):  
Preliminary Data ◽  
Related Species ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Natural Habitats ◽  
Free Living ◽  
Three Samples ◽  
Freshwater Bodies ◽  
Pass Through

Kinetoplastids are flagellated protozoa, including principally free-living bodonids and exclusively parasitic trypanosomatids. In the most species-rich genus, Trypanosoma, more than thirty species were found to infect bats worldwide. Bat trypanosomes are also known to have played a significant role in the evolution of T. cruzi, a species with high veterinary medical significance. Although preliminary data attested the occurrence of bat trypanosomes in Hungary, these were never sought for with molecular methods. Therefore, amplification of an approx. 900-bp fragment of the 18S rRNA gene of kinetoplastids was attempted from 307 ixodid and 299 argasid ticks collected from bats, and from 207 cimicid bugs collected from or near bats in Hungary and Romania. Three samples, one per each bat ectoparasite group, were PCR positive. Sequencing revealed the presence of DNA from free-living bodonids (Bodo saltans and neobodonids), but no trypanosomes were detected. The most likely source of bodonid DNA detected here in engorged bat ectoparasites is the blood of their bat hosts. However, how bodonids were acquired by bats, can only be speculated. Bats are known to drink from freshwater bodies, i.e. the natural habitats of B. saltans and related species, allowing bats to ingest bodonids. Consequently, these results suggest that at least the DNA of bodonids might pass through the alimentary mucosa of bats into their circulation. The above findings highlight the importance of studying bats and other mammals for the occurrence of bodonids in their blood and excreta, with potential relevance to the evolution of free-living kinetoplastids towards parasitism.

scholarly journals Presence of Balamuthia mandrillaris in hot springs from Mazandaran province, northern Iran

2016 ◽  
Vol 144 (11) ◽  
pp. 2456-2461 ◽  
Author(s):  
A. R. LATIFI ◽  
M. NIYYATI ◽  
J. LORENZO-MORALES ◽  
A. HAGHIGHI ◽  
S. J. SEYYED TABAEI ◽  
...  
Keyword(s):  
Water Samples ◽  
Hot Springs ◽  
Hot Spring ◽  
Rare Event ◽  
Morphological Data ◽  
Rrna Gene ◽  
Mazandaran Province ◽  
Northern Iran ◽  
Environmental Distribution ◽  
Balamuthia Mandrillaris

SUMMARYBalamuthia mandrillaris is an opportunistic free-living amoeba that has been reported to cause cutaneous lesions and Balamuthia amoebic encephalitis. The biology and environmental distribution of B. mandrillaris is still poorly understood and isolation of this pathogen from the environment is a rare event. Previous studies have reported that the presence of B. mandrillaris in the environment in Iran may be common. However, no clinical cases have been reported so far in this country. In the present study, a survey was conducted in order to evaluate the presence of B. mandrillaris in hot-spring samples of northern Iran. A total of 66 water samples were analysed using morphological and molecular tools. Positive samples by microscopy were confirmed by performing PCR amplification of the 16S rRNA gene of B. mandrillaris. Sequencing of the positive amplicons was also performed to confirm morphological data. Two of the 66 collected water samples were positive for B. mandrillaris after morphological and molecular identification. Interestingly, both positive hot springs had low pH values and temperatures ranging from 32 °C to 42 °C. Many locals and tourists use both hot springs due to their medicinal properties and thus contact with water bodies containing the organism increases the likelihood of infection. To the best of our knowledge, this is the first report on the isolation of B. mandrillaris from hot-spring sources related to human activity. Therefore, B. mandrillaris should be considered as a possible causative agent if cases of encephalitis are suspected following immersion in hot springs in addition to Acanthamoeba and Naegleria.

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