scholarly journals Species, Sequence Types and Alleles: Dissecting Genetic Variation in Acanthamoeba

Pathogens ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 534
Author(s):  
Paul A. Fuerst ◽  
Gregory C. Booton
Keyword(s):  
Genetic Variation ◽  
18S Rrna ◽  
Hypervariable Region ◽  
18S Rrna Gene ◽  
Sequence Type ◽  
Rrna Gene ◽  
Dna Databases ◽  
Molecular Approaches ◽  
History Of ◽  
Sequence Types

Species designations within Acanthamoeba are problematic because of pleomorphic morphology. Molecular approaches, including DNA sequencing, hinted at a resolution that has yet to be fully achieved. Alternative approaches were required. In 1996, the Byers/Fuerst lab introduced the concept of sequence types. Differences between isolates of Acanthamoeba could be quantitatively assessed by comparing sequences of the nuclear 18S rRNA gene, ultimately producing 22 sequence types, designated T1 through T22. The concept of sequence types helps our understanding of Acanthamoeba evolution. Nevertheless, substantial variation in the 18S rRNA gene differentiates many isolates within each sequence type. Because the majority of isolates with sequences in the international DNA databases have been studied for only a small segment of the gene, designated ASA.S1, genetic variation within this hypervariable region of the 18S rRNA gene has been scrutinized. In 2002, we first categorized variation in this region in a sample of T3 and T4 isolates from Hong Kong, observing ten “alleles” within type T4 and five “alleles” within T3. Subsequently, confusion occurred when different labs applied redundant numerical labels to identify different alleles. A more unified approach was required. We have tabulated alleles occurring in the sequences submitted to the international DNA databases, and determined their frequencies. Over 150 alleles have occurred more than once within 3500+ isolates of sequence type T4. Results from smaller samples of other sequence types (T3, T5, T11 and T15, and supergroup T2/6) have also been obtained. Our results provide new insights into the evolutionary history of Acanthamoeba, further illuminating the degree of genetic separation between significant taxonomic units within the genus, perhaps eventually elucidating what constitutes a species of Acanthamoeba.

scholarly journals The Evolutionary History of the Genus Acanthamoeba and the Identification of Eight New 18S rRNA Gene Sequence Types

1998 ◽  
Vol 45 (1) ◽  
pp. 45-54 ◽  
Author(s):  
Diane R. Stothard ◽  
Jill M. Schroeder-Diedrich ◽  
Mohammad H. Awwad ◽  
Rebecca J. Gast ◽  
Dolena R. Ledee ◽  
...  
Keyword(s):  
Evolutionary History ◽  
18S Rrna ◽  
Gene Sequence ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
History Of ◽  
Sequence Types

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 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 Design and Evaluation of Illumina MiSeq-Compatible, 18S rRNA Gene-Specific Primers for Improved Characterization of Mixed Phototrophic Communities

2016 ◽  
Vol 82 (19) ◽  
pp. 5878-5891 ◽  
Author(s):  
Ian M. Bradley ◽  
Ameet J. Pinto ◽  
Jeremy S. Guest
Keyword(s):  
Community Structure ◽  
18S Rrna ◽  
Target Genes ◽  
Hypervariable Region ◽  
Illumina Miseq ◽  
Amplicon Sequencing ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Sequencing Bias ◽  
Primer Sets

ABSTRACTThe use of high-throughput sequencing technologies with the 16S rRNA gene for characterization of bacterial and archaeal communities has become routine. However, the adoption of sequencing methods for eukaryotes has been slow, despite their significance to natural and engineered systems. There are large variations among the target genes used for amplicon sequencing, and for the 18S rRNA gene, there is no consensus on which hypervariable region provides the most suitable representation of diversity. Additionally, it is unclear how much PCR/sequencing bias affects the depiction of community structure using current primers. The present study amplified the V4 and V8-V9 regions from seven microalgal mock communities as well as eukaryotic communities from freshwater, coastal, and wastewater samples to examine the effect of PCR/sequencing bias on community structure and membership. We found that degeneracies on the 3′ end of the current V4-specific primers impact read length and mean relative abundance. Furthermore, the PCR/sequencing error is markedly higher for GC-rich members than for communities with balanced GC content. Importantly, the V4 region failed to reliably capture 2 of the 12 mock community members, and the V8-V9 hypervariable region more accurately represents mean relative abundance and alpha and beta diversity. Overall, the V4 and V8-V9 regions show similar community representations over freshwater, coastal, and wastewater environments, but specific samples show markedly different communities. These results indicate that multiple primer sets may be advantageous for gaining a more complete understanding of community structure and highlight the importance of including mock communities composed of species of interest.IMPORTANCEThe quantification of error associated with community representation by amplicon sequencing is a critical challenge that is often ignored. When target genes are amplified using currently available primers, differential amplification efficiencies result in inaccurate estimates of community structure. The extent to which amplification bias affects community representation and the accuracy with which different gene targets represent community structure are not known. As a result, there is no consensus on which region provides the most suitable representation of diversity for eukaryotes. This study determined the accuracy with which commonly used 18S rRNA gene primer sets represent community structure and identified particular biases related to PCR amplification and Illumina MiSeq sequencing in order to more accurately study eukaryotic microbial communities.

Phylogenetic perspectives on basidiomycete systematics: evidence from the 18S rRNA gene

1995 ◽  
Vol 73 (S1) ◽  
pp. 862-868 ◽  
Author(s):  
Eric C. Swann ◽  
John W. Taylor
Keyword(s):  
18S Rrna ◽  
Spindle Pole Body ◽  
Primary Source ◽  
Spindle Pole ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
New Class ◽  
Class Level ◽  
History Of ◽  
Made In

Basidiomycete systematics has a history of dramatic change with the introduction of new forms of information. We present a new class level taxonomy for basidiomycetes consisting of the Urediniomycetes emend., Ustilaginomycetes, and Hymenomycetes. The primary source of characters for this analysis is the 18S rRNA gene. Other characters such as cellular carbohydrate composition, major ubiquinone system, 5S rRNA sequence, basidium morphology, and septum and spindle pole body ultrastructure are considered. Comparison of the new class system to other basidiomycete taxonomies is made. Use of 18S sequence to test the monophyly of an order is made in an examination of the Filobasidiales. Key words: basidiomycetes, molecular systematics, taxonomy, Filobasidiales, basidiomycetous yeasts.

Novel Acanthamoeba 18S rRNA gene sequence type from an environmental isolate

2014 ◽  
Vol 113 (8) ◽  
pp. 2845-2850 ◽  
Author(s):  
A. Magnet ◽  
N. Henriques-Gil ◽  
A. L. Galván-Diaz ◽  
F. Izquiedo ◽  
S. Fenoy ◽  
...  
Keyword(s):  
18S Rrna ◽  
Gene Sequence ◽  
18S Rrna Gene ◽  
Sequence Type ◽  
Environmental Isolate ◽  
Rrna Gene ◽  
Rrna Gene Sequence

scholarly journals Morphology, 18S rRNA gene sequence and life history of a new Polydora species (Polychaeta: Spionidae) from northeastern Japan

2013 ◽  
Vol 18 (1) ◽  
pp. 31-45 ◽  
Author(s):  
W Teramoto ◽  
W Sato-Okoshi ◽  
H Abe ◽  
G Nishitani ◽  
Y Endo
Keyword(s):  
Life History ◽  
18S Rrna ◽  
Gene Sequence ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
Northeastern Japan ◽  
History Of

scholarly journals Phylogenetic Analysis of the Hypervariable Region of the 18S rRNA Gene of Cryptosporidium Oocysts in Feces of Canada Geese (Branta canadensis): Evidence for Five Novel Genotypes

2004 ◽  
Vol 70 (1) ◽  
pp. 452-458 ◽  
Author(s):  
Kristen L. Jellison ◽  
Daniel L. Distel ◽  
Harold F. Hemond ◽  
David B. Schauer
Keyword(s):  
Genetic Diversity ◽  
Phylogenetic Analysis ◽  
18S Rrna ◽  
Hypervariable Region ◽  
Pcr Amplification ◽  
The United States ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Canada Geese ◽  
Pcr Products

ABSTRACT To assess genetic diversity in Cryptosporidium oocysts from Canada geese, 161 fecal samples from Canada geese in the United States were analyzed. Eleven (6.8%) were positive for Cryptosporidium spp. following nested PCR amplification of the hypervariable region of the 18S rRNA gene. Nine PCR products from geese were cloned and sequenced, and all nine diverged from previously reported Cryptosporidium 18S rRNA gene sequences. Five sequences were very similar or identical to each other but genetically distinct from that of Cryptosporidium baileyi; two were most closely related to, but genetically distinct from, the first five; and two were distinct from any other sequence analyzed. One additional sequence in the hypervariable region of the 18S rRNA gene isolated from a cormorant was identical to that of C. baileyi. Phylogenetic analysis provided evidence for new genotypes of Cryptosporidium species in Canada geese. Results of this study suggest that the taxonomy of Cryptosporidium species in geese is complex and that a more complete understanding of genetic diversity among these parasites will facilitate our understanding of oocyst sources and species in the environment.

scholarly journals Fluorescent Oligonucleotide Probes for Clinical and Environmental Detection of Acanthamoeba and the T4 18S rRNA Gene Sequence Type

1999 ◽  
Vol 37 (8) ◽  
pp. 2687-2693 ◽  
Author(s):  
Diane R. Stothard ◽  
John Hay ◽  
Jill M. Schroeder-Diedrich ◽  
David V. Seal ◽  
Thomas J. Byers
Keyword(s):  
18S Rdna ◽  
18S Rrna ◽  
Sequence Type ◽  
Rdna Sequence ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Oligonucleotide Probes ◽  
Specific Probe ◽  
Sequence Types ◽  
18S Rdna Sequence

The first genus- and subgenus-specific fluorescent oligonucleotide probes for in situ staining of Acanthamoeba are described. Sequences of these phylogeny-based probes complement the 18S rRNA and the gene encoding it (18S rDNA). The genus-specific probe (GSP) is a fluorescein-labeled 22-mer specific forAcanthamoeba as shown here by its hybridization to growing trophozoites of all 12 known Acanthamoeba 18S rDNA sequence types and by its failure to hybridize with amoebae of two other genera (Hartmannella vermiformis and Balamuthia mandrillaris), two human cell lines, and two bacteria (Pseudomonas aeruginosa and Escherichia coli). The sequence type T4-specific probe (ST4P) is a rhodamine-labeled 30-mer specific for Acanthamoeba 18S rDNA sequence type T4, as shown here in hybridization tests with trophozoites of all 12 sequence types. T4 is the subgenus group associated most closely withAcanthamoeba keratitis (AK). GSP also was tested with corneal scrapings from 17 patients with a high index of clinical suspicion of AK plus 5 patient controls. GSP stained both trophozoites and cysts, although nonspecific cyst wall autofluorescence also was observed. Results could be obtained with GSP in 1 to 2 days, and based on results from cell culture tests, the probe correctly detected the presence or absence of Acanthamoeba in 21 of 24 specimens from the 22 patients. The use of GSP with cultured trophozoites and cysts from corneal scrapings has illustrated the suitability of using fluorescent oligonucleotide probes for identification of the genusAcanthamoeba in both environmental and clinical samples. In addition, the use of ST4P with cultured amoebae has indicated the potential of oligonucleotide probes for use in subgenus classification.

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