Development and evaluation of molecular tools for detecting and differentiating intestinal amoebae in healthy individuals

Parasitology ◽  
2019 ◽  
Vol 146 (6) ◽  
pp. 821-827 ◽  
Author(s):  
Amal Chihi ◽  
Christen R. Stensvold ◽  
Imene Ben-abda ◽  
Rania Ben-Romdhane ◽  
Karim Aoun ◽  
...  
Keyword(s):  
Small Subunit ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Molecular Tools ◽  
Entamoeba Dispar ◽  
Accurate Identification ◽  
Chain Reactions ◽  
E Coli ◽  
Polymerase Chain Reactions

AbstractAmoebae are single-celled parasites frequently colonizing human gut. However, few molecular tools are available for accurate identification. Here, we evaluated a panel of polymerase chain reactions (PCRs) targeting Entamoeba histolytica, Entamoeba dispar, Entamoeba coli, Entamoeba hartmanni, Entamoeba polecki, Endolimax nana and Iodamoeba bütschlii. Thirty-six faecal samples (18 containing at least one amoeba species by microscopy and 18 microscopy negative for amoebae) were tested. Real-time PCRs were used for detection and differentiation of E. histolytica and E. dispar. Conventional PCR with Sanger sequencing were applied for detection and differentiation of E. coli, E. hartmanni, E. polecki, E. nana and I. bütschlii. All microscopy results were confirmed by DNA-based methods. However, more samples were positive for single and mixed amoebic species by DNA-based assays than by microscopy (22 vs 18 and 7 vs 1, respectively). DNA sequencing allowed identification of E. coli subtypes (ST1 and ST2), showed low intra-specific variation within E. hartmanni, identified two phylogenetically distinct groups within E. nana, and identified Iodamoeba at the ribosomal lineage level. Taking into account the high intra-genetic diversity within some of the species at the small subunit (SSU) rRNA gene level, amplification of SSU rRNA genes with subsequent sequencing represents a useful method for detecting, differentiating and subtyping intestinal amoebae.

Redescriptions of three trachelocercid ciliates (Protista, Ciliophora, Karyorelictea), with notes on their phylogeny based on small subunit rRNA gene sequences

2013 ◽  
Vol 63 (Pt_9) ◽  
pp. 3506-3514 ◽  
Author(s):  
Ying Yan ◽  
Yuan Xu ◽  
Zhenzhen Yi ◽  
Alan Warren
Keyword(s):  
Sequence Data ◽  
Phylogenetic Analyses ◽  
Small Subunit ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Small Subunit Rrna ◽  
Small Subunit Rrna Gene ◽  
Ssu Rrna Gene Sequence ◽  
First Time

Three trachelocercid ciliates, Kovalevaia sulcata (Kovaleva, 1966) Foissner, 1997, Trachelocerca sagitta (Müller, 1786) Ehrenberg, 1840 and Trachelocerca ditis (Wright, 1982) Foissner, 1996, isolated from two coastal habitats at Qingdao, China, were investigated using live observation and silver impregnation methods. Data on their infraciliature and morphology are supplied. The small subunit rRNA (SSU rRNA) genes of K. sulcata and Trachelocerca sagitta were sequenced for the first time. Phylogenetic analyses based on SSU rRNA gene sequence data indicate that both organisms, and the previously sequenced Trachelocerca ditis, are located within the trachelocercid assemblage and that K. sulcata is sister to an unidentified taxon forming a clade that is basal to the core trachelocercids.

Detection of phytoplasmas in dieback, yellow crinkle, and mosaic diseases of papaya using polymerase chain reaction techniques

1996 ◽  
Vol 47 (3) ◽  
pp. 387 ◽  
Author(s):  
B Liu ◽  
DT White ◽  
KB Walsh ◽  
PT Scott
Keyword(s):  
Rrna Genes ◽  
23S Rrna ◽  
Rrna Gene ◽  
Tissue Samples ◽  
Chain Reactions ◽  
Polymerase Chain Reactions ◽  
Pcr Product ◽  
Polymerase Chain ◽  
23S Rrna Genes ◽  
The 16S Rrna Gene

Oligonucleotide primers complementary to regions specific to plant-pathogenic mycoplasma-like organisms (phytoplasmas) were used in polymerase chain reactions on tissue samples from dieback, yellow crinkle, and mosaic affected papaya plants. The primer pair P068/P069, which hybridise to internal regions of the 16s rRNA gene, amplified an approximately 560 bp product in dieback, yellow crinkle and mosaic affected papaya. The primer pair P3/P7, which hybridise to the spacer region between the 16s and 23s rRNA genes, amplified an approximately 300 bp fragment in yellow crinkle and mosaic affected papaya, with no product from dieback affected plants. No PCR product was obtained with either set of primers from healthy plants. An identical Alu I restriction enzyme profile was obtained with all three 560 bp products. This study provides the first evidence for the association of phytoplasmas with papaya mosaic and Australian papaya dieback.

scholarly journals Detection and subtype identification of Blastocystis isolates from wastewater samples in the Philippines

2011 ◽  
Vol 9 (1) ◽  
pp. 128-137 ◽  
Author(s):  
Jan Ervin G. Banaticla ◽  
Windell L. Rivera
Keyword(s):  
Wastewater Treatment Plants ◽  
Small Subunit ◽  
The Philippines ◽  
Rrna Genes ◽  
Bootstrap Support ◽  
In Vitro Cultivation ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Wastewater Samples

To provide further evidence of waterborne transmission of Blastocystis, a total of 31 wastewater treatment plants from geographically distinct locations across the Philippines were sampled for influent and effluent sewage samples. In vitro cultivation was the method of choice to increase sensitivity of detection. Blastocystis cysts were detected in 15% (9/62) of the samples using in vitro culture. Moreover, influent and effluent samples were 23% (7/31) and 7% (2/31) positive for the parasite, respectively. The presence of viable cysts in treated samples may be an indication of the inefficiency of the treatment process in preventing Blastocystis from entering the environment. Polymerase chain reaction and sequencing of the full-length small subunit ribosomal RNA (SSU rRNA) genes of the nine wastewater isolates were performed. The SSU rRNA gene sequences of the isolates showed very high similarity (98 to 99%) to homologous sequences of Blastocystis described previously. The phylogenetic tree constructed showed that the wastewater isolates clustered with each other with good bootstrap support and belonged to two subtypes (ST) – ST1 and ST2. This is the first report of subtyping Blastocystis isolates from wastewater samples and gives further emphasis to the remarkable genetic diversity of the parasite.

scholarly journals Characterization of X-Disease Phytoplasmas in Chokecherry from North Dakota by PCR-RFLP and Sequence Analysis of the rRNA Gene Region

Plant Disease ◽  
2000 ◽  
Vol 84 (11) ◽  
pp. 1235-1240 ◽  
Author(s):  
Y. H. Guo ◽  
Z.-M. Cheng ◽  
J. A. Walla
Keyword(s):  
16S Rrna ◽  
North Dakota ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Rrna Genes ◽  
23S Rrna ◽  
Rrna Gene ◽  
Chain Reactions ◽  
Polymerase Chain Reactions ◽  
Pcr Rflp

Genetic variation of X-disease phytoplasma strains from chokecherry (ChX) in North Dakota and nearby sites, and their relatedness with three standard strains of the X-disease phytoplasma group, eastern X-disease (CX), western X-disease (WX), and goldenrod yellows (GR1) phyto-plasmas, were studied. Primer pairs were developed to amplify the 23S ribosomal RNA (rRNA) gene and the 16S/23S spacer region. The rRNA genes (16S rRNA, 23S rRNA, and two ribosomal protein [rp] genes) and the 16S/23S spacer region were amplified by polymerase chain reactions. The restriction fragment length polymorphism (RFLP) patterns of 16S rRNA, 23S rRNA, and rp genes, generated by digestion with four restriction enzymes (AluI, HpaII, MseI, and RsaI), showed no difference among 43 ChX phytoplasma isolates. Sequencing of the 441-bp 16S/23S spacer region revealed variation at four positions among 12 ChX phytoplasma strains. A tRNAIle and other conserved sequences were identified in the spacer region. Among X-disease subgroups, RFLP analysis indicated that ChX is similar to WX, closely related to CX, and easily distinguished from GR1. Sequencing indicated that ChX is closer to CX than to WX. Together, the analyses indicated that ChX phytoplasmas are genetically different from the standard strains of other X-disease phytoplasma subgroups.

Phylogenetic analysis of freshwater fish trypanosomes from Europe using ssu rRNA gene sequences and random amplification of polymorphic DNA

Parasitology ◽  
2004 ◽  
Vol 130 (4) ◽  
pp. 405-412 ◽  
Author(s):  
W. C. GIBSON ◽  
J. LOM ◽  
H. PECKOVÁ ◽  
V. R. FERRIS ◽  
P. B. HAMILTON
Keyword(s):  
Phylogenetic Analysis ◽  
Freshwater Fish ◽  
Genetic Relationships ◽  
Small Subunit ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Gene Sequences ◽  
Ssu Rrna Gene ◽  
Random Amplification

The taxonomy and phylogenetic relationships of fish trypanosomes are uncertain. A collection of 22 cloned trypanosome isolates from 14 species of European freshwater fish and 1 species of African freshwater fish were examined by molecular phylogenetic analysis. The small subunit ribosomal RNA (ssu rRNA) genes of 8 clones were sequenced and compared with ssu rRNA gene sequences from a wider selection of vertebrate trypanosome isolates by phylogenetic analysis. All trypanosomes from freshwater fish fell in a single clade, subdivided into 3 groups. This clade sits within a larger, robust clade containing trypanosomes from marine fish and various amphibious vertebrates. All 22 trypanosome clones were analysed by random amplification of polymorphic DNA. The resulting dendrogram shows 3 groups, which are congruent with the groups identified in the ssu rRNA gene phylogeny. Two of the groups contain the majority of trypanosome isolates and within-group variation is slight. These groups do not separate purported trypanosome species distinguished by morphology or host origin, and thus these criteria do not appear to be reliable guides to genetic relationships among fish trypanosomes. However, we suggest that the 2 groups themselves may represent different species of fish trypanosomes. The polymorphic DNA markers we have identified will facilitate future comparisons of the biology of these 2 groups of fish trypanosomes.

scholarly journals Theileria sp. Infections Associated with Bovine Fatalities in the United States Confirmed by Small-Subunit rRNA Gene Analyses of Blood and Tick Samples

1999 ◽  
Vol 37 (9) ◽  
pp. 3037-3040 ◽  
Author(s):  
Joon-seok Chae ◽  
Michael Levy ◽  
John Hunt ◽  
Jack Schlater ◽  
Glen Snider ◽  
...  
Keyword(s):  
Type A ◽  
The United States ◽  
Dermacentor Variabilis ◽  
Small Subunit ◽  
Amblyomma Americanum ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Ssu Rrna Gene ◽  
Type D

Theileria sp.-specific small subunit (SSU) rRNA gene amplification confirmed the presence of the organism in cattle and inAmblyomma americanum and Dermacentor variabilisticks collected from a cattle herd in Missouri. Blood from the index animal had type A and type D Theileria SSU rRNA genes. The type D gene was also found in blood from two cohort cattle and tick tissues. The type A SSU rRNA gene was previously reported from bovineTheileria isolates from Texas and North Carolina; the type D gene was reported from a Texas cow with theileriosis.

Phylogenetic Diversity of Ultraplankton Plastid Small-Subunit rRNA Genes Recovered in Environmental Nucleic Acid Samples from the Pacific and Atlantic Coasts of the United States

1998 ◽  
Vol 64 (1) ◽  
pp. 294-303 ◽  
Author(s):  
Michael S. Rappé ◽  
Marcelino T. Suzuki ◽  
Kevin L. Vergin ◽  
Stephen J. Giovannoni
Keyword(s):  
United States ◽  
The United States ◽  
Small Subunit ◽  
Rrna Genes ◽  
Gene Clone ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Phytoplankton Diversity ◽  
Ssu Rrna Gene ◽  
The Pacific

ABSTRACT The scope of marine phytoplankton diversity is uncertain in many respects because, like bacteria, these organisms sometimes lack defining morphological characteristics and can be a challenge to grow in culture. Here, we report the recovery of phylogenetically diverse plastid small-subunit (SSU) rRNA gene (rDNA) clones from natural plankton populations collected in the Pacific Ocean off the mouth of Yaquina Bay, Oreg. (OCS clones), and from the eastern continental shelf of the United States off Cape Hatteras, N.C. (OM clones). SSU rRNA gene clone libraries were prepared by amplifying rDNAs from nucleic acids isolated from plankton samples and cloning them into plasmid vectors. The PCR primers used for amplification reactions were designed to be specific for bacterial SSU rRNA genes; however, plastid genes have a common phylogenetic origin with bacteria and were common in both SSU rRNA gene clone libraries. A combination of restriction fragment length polymorphism analyses, nucleic acid sequencing, and taxon-specific oligonucleotide probe hybridizations revealed that 54 of the 116 OCS gene clones were of plastid origin. Collectively, clones from the OCS and OM libraries formed at least eight unique lineages within the plastid radiation, including gene lineages related to the classesBacillariophyceae, Cryptophyceae,Prymnesiophyceae, Chrysophyceae, andPrasinophyceae; for a number of unique clones, no close phylogenetic neighbors could be identified with confidence. Only a group of two OCS rRNA gene clones showed close identity to the plastid SSU rRNA gene sequence of a cultured organism [Emiliania huxleyi (Lohmann) Hay and Mohler; 99.8% similar]. The remaining clones could not be identified to the genus or species level. Although cryptic species are not as prevalent among phytoplankton as they are among their bacterial counterparts, this genetic survey nonetheless uncovered significant new information about phytoplankton diversity.

scholarly journals Evidence of independent acquisition and adaption of ultra-small bacteria to human hosts across the highly diverse yet reduced genomes of the phylum Saccharibacteria

2018 ◽  
Author(s):  
Jeffrey S. McLean ◽  
Batbileg Bor ◽  
Thao T. To ◽  
Quanhui Liu ◽  
Kristopher A. Kerns ◽  
...  
Keyword(s):  
Oral Cavity ◽  
De Novo ◽  
Gc Content ◽  
Single Copy ◽  
Small Subunit ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Ssu Rrna Gene ◽  
Human Oral Cavity

ABSTRACTRecently, we discovered that a member of the Saccharibacteria/TM7 phylum (strain TM7x) isolated from the human oral cavity, has an ultra-small cell size (200-300nm), a highly reduced genome (705 Kbp) with limited de novo biosynthetic capabilities, and a very novel lifestyle as an obligate epibiont on the surface of another bacterium 1. There has been considerable interest in uncultivated phyla, particularly those that are now classified as the proposed candidate phyla radiation (CPR) reported to include 35 or more phyla and are estimated to make up nearly 15% of the domain Bacteria. Most members of the larger CPR group share genomic properties with Saccharibacteria including reduced genomes (<1Mbp) and lack of biosynthetic capabilities, yet to date, strain TM7x represents the only member of the CPR that has been cultivated and is one of only three CPR routinely detected in the human body. Through small subunit ribosomal RNA (SSU rRNA) gene surveys, members of the Saccharibacteria phylum are reported in many environments as well as within a diversity of host species and have been shown to increase dramatically in human oral and gut diseases. With a single copy of the 16S rRNA gene resolved on a few limited genomes, their absolute abundance is most often underestimated and their potential role in disease pathogenesis is therefore underappreciated. Despite being an obligate parasite dependent on other bacteria, six groups (G1-G6) are recognized using SSU rRNA gene phylogeny in the oral cavity alone. At present, only genomes from the G1 group, which includes related and remarkably syntenic environmental and human oral associated representatives1, have been uncovered to date. In this study we systematically captured the spectrum of known diversity in this phylum by reconstructing completely novel Class level genomes belonging to groups G3, G6 and G5 through cultivation enrichment and/or metagenomic binning from humans and mammalian rumen. Additional genomes for representatives of G1 were also obtained from modern oral plaque and ancient dental calculus. Comparative analysis revealed remarkable divergence in the host-associated members across this phylum. Within the human oral cavity alone, variation in as much as 70% of the genes from nearest oral clade (AAI 50%) as well as wide GC content variation is evident in these newly captured divergent members (G3, G5 and G6) with no environmental relatives. Comparative analyses suggest independent episodes of transmission of these TM7 groups into humans and convergent evolution of several key functions during adaptation within hosts. In addition, we provide evidence from in vivo collected samples that each of these major groups are ultra-small in size and are found attached to larger cells.

Redescription and SSU rRNA gene-based phylogeny of an anaerobic ciliate, Plagiopyla ovata Kahl, 1931 (Ciliophora, Plagiopylea)

2021 ◽  
Vol 71 (8) ◽  
Author(s):  
Ran Li ◽  
Wenbao Zhuang ◽  
Congcong Wang ◽  
Hamed El-Serehy ◽  
Saleh A. Al-Farraj ◽  
...  
Keyword(s):  
Sequence Data ◽  
Phylogenetic Analyses ◽  
Small Subunit ◽  
The Yellow Sea ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Gene Trees ◽  
Ssu Rrna Gene ◽  
Anaerobic Ciliate ◽  
Pr China

The morphology and molecular phylogeny of Plagiopyla ovata Kahl, 1931, a poorly known anaerobic ciliate, were investigated based on a population isolated from sand samples collected from the Yellow Sea coast at Qingdao, PR China. Details of the oral ciliature are documented for the first time to our knowledge and an improved species diagnosis is given. The small subunit ribosomal RNA (SSU rRNA) gene was newly sequenced and phylogenetic analyses revealed that P. ovata clusters within the monophyletic family Plagiopylidae. However, evolutionary relationships within both the family Plagiopylidae and the genus Plagiopyla remain obscure owing to undersampling, the lack of sequence data from known species and low nodal support or unstable topologies in gene trees. A key to the identification of the species of the genus Plagiopyla with validly published names is also supplied.

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