scholarly journals Identity of Ehrlichial DNA Sequences Derived from Ixodes ricinus Ticks with Those Obtained from Patients with Human Granulocytic Ehrlichiosis in Slovenia

1999 ◽  
Vol 37 (1) ◽  
pp. 209-210 ◽  
Author(s):  
M. Petrovec ◽  
J. W. Sumner ◽  
W. L. Nicholson ◽  
J. E. Childs ◽  
F. Strle ◽  
...  
Keyword(s):  
Heat Shock ◽  
16S Rrna ◽  
Ixodes Ricinus ◽  
Dna Sequences ◽  
Rrna Gene ◽  
Granulocytic Ehrlichiosis ◽  
Human Granulocytic Ehrlichiosis ◽  
Ixodes Ricinus Ticks ◽  
Pcr Assays ◽  
The 16S Rrna Gene

Adult Ixodes ricinus (Acari: Ixodidae) ticks collected near Ljubljana, Slovenia, were tested for the agent of human granulocytic ehrlichiosis (HGE) by using PCR assays based on the 16S rRNA gene. Three (3.2%) of 93 ticks were found to contain granulocytic ehrlichiae. Nucleotide sequences of portions of the bacterialgroESL heat shock operon amplified from these ticks were identical or nearly (99.8%) identical to those previously determined for human patients with HGE from Slovenia, providing additional evidence that the ticks were infected with the HGE agent. This study identified I. ricinus as the likely vector for these ehrlichial pathogens of humans in this part of Europe.

scholarly journals Quantitative Real-Time PCR for Detection of Members of the Ehrlichia phagocytophila Genogroup in Host Animals and Ixodes ricinus Ticks

1999 ◽  
Vol 37 (5) ◽  
pp. 1329-1331 ◽  
Author(s):  
Nicola Pusterla ◽  
Jon B. Huder ◽  
Christian M. Leutenegger ◽  
Ueli Braun ◽  
John E. Madigan ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Ixodes Ricinus ◽  
Nested Pcr ◽  
Rrna Gene ◽  
Granulocytic Ehrlichiosis ◽  
Ehrlichia Phagocytophila ◽  
Ixodes Ricinus Ticks ◽  
Taqman Pcr ◽  
The 16S Rrna Gene

A TaqMan PCR was established for identification and quantitation of members of the Ehrlichia phagocytophila group in experimentally infected cows and in Ixodes ricinus ticks. The TaqMan PCR identified a 106-bp section of the 16S rRNA gene by use of a specific fluorogenic probe and two primers. This technique was specific for members of the E. phagocytophila group, which include E. phagocytophila, Ehrlichia equi, and the agent of human granulocytic ehrlichiosis. The TaqMan system identified 10 copies of a cloned section of the 16S rRNA gene ofE. phagocytophila. The sensitivity and specificity of the TaqMan PCR were similar to those of conventional nested PCR. The numbers of ehrlichiae in leukocytes of the two cows experimentally infected with E. phagocytophila were measured daily by TaqMan PCR and had a course similar to that of the percentages of infected leukocytes determined daily by light microscopy. The prevalence of infected free-living ticks, which were collected from areas where bovine ehrlichiosis is endemic and from regions with sporadic occurrences of granulocytic ehrlichiosis in dogs and horses, was identical as determined by nested PCR and TaqMan PCR.

scholarly journals Metagenomic 16S rRNA gene sequencing survey of Borrelia species in Irish samples of Ixodes ricinus ticks

2018 ◽  
Author(s):  
John S Lambert ◽  
Michael John Cook ◽  
John Eoin Healy ◽  
Ross Murtagh ◽  
Gordana Avramovic ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Ixodes Ricinus ◽  
Sanger Sequencing ◽  
Reference Sequence ◽  
Borrelia Burgdorferi Sensu Lato ◽  
Tick Population ◽  
Rrna Gene ◽  
Ixodes Ricinus Ticks ◽  
The 16S Rrna Gene

Lyme borreliosis is a systemic infection caused by tick-borne pathogenic borreliae of the Borrelia burgdorferi sensu lato complex or of the more heterogeneous relapsing fever borrelia group. Clinical distinction of the infections due to different borrelia species is difficult. Accurate knowledge of the prevalence and the species of borreliae in the infected ticks in the endemic areas is valuable for formulating appropriate guidelines for proper management of this infectious disease. The purpose of this research was to design a readily implementable protocol to detect the divergent species of borreliae known to exist in Europe, using Irish samples of Ixodes ricinus ticks as the subject for study. Questing I. ricinus nymph samples were taken at six localities within Ireland. The crude DNA of each dried tick was extracted by hot NH4OH and used to initiate a same-nested PCR with a pair of borrelial genus-specific primers to amplify a highly conserved 357/358 bp segment of the 16S rRNA gene for detection and as the template for Sanger sequencing. To distinguish B. garinii from B. burgdorferi and to discriminate the various strains of B. garinii, a second 282 bp segment of the 16S rRNA gene was amplified for Sanger sequencing. A signature segment of the DNA sequence excised from the computer-generated electropherogram was submitted to the GenBank for BLAST alignment analysis. A 100% ID match with the unique reference sequence in the GenBank was required for the molecular diagnosis of the borrelial species or strain. We found the overall rate of borrelial infection in the Irish tick population to be 5%, with a range from 2% to 12% depending on the locations of tick collection. At least 3 species, namely B. garinii, B. valaisiana and B. miyamotoi, are infecting the ticks collected in Ireland. The isolates of B. garinii were confirmed to be strain BgVir, strain Bernie or strain T25. Since antigens for diagnostic serology tests may be species- or even strain-specific, expanded surveillance of the species and strains of the borreliae among human-biting ticks in Ireland is needed to ensure that the antigens used for the serology tests do contain the epitopes matching the antibodies elicited by the borrelial species and strains in the ticks cohabitating in the same environment.

scholarly journals Evidence of the Human Granulocytic Ehrlichiosis Agent in Ixodes ricinus Ticks in Switzerland

1999 ◽  
Vol 37 (5) ◽  
pp. 1332-1334 ◽  
Author(s):  
Nicola Pusterla ◽  
Christian M. Leutenegger ◽  
Jon B. Huder ◽  
Rainer Weber ◽  
Ueli Braun ◽  
...  
Keyword(s):  
Ixodes Ricinus ◽  
Nucleotide Sequencing ◽  
Rrna Gene ◽  
Adult Males ◽  
Granulocytic Ehrlichiosis ◽  
Ehrlichia Phagocytophila ◽  
Stage Of Development ◽  
Human Granulocytic Ehrlichiosis ◽  
Pcr Products ◽  
Ixodes Ricinus Ticks

A total of 1,667 Ixodes ricinus ticks were collected from five regions in Switzerland where there have been sporadic occurrences of granulocytic ehrlichiosis in dogs and horses. The ticks were examined for rickettsiae of the Ehrlichia phagocytophila group via nested PCR. Twenty-one ticks (1.3%) were positive; 3 (0.5%) were nymphs, 6 (1.3%) were adult males, and 12 (1.9%) were adult females. The number of positive ticks varied with the stage of development and with the geographical origin. Nucleotide sequencing of the isolated PCR products identified these products as part of the 16S rRNA gene of Ehrlichia. In addition, these products had 100% homology with the agent of human granulocytic ehrlichiosis. The occurrence of this agent in I. ricinus in Switzerland presents a potential danger of transmission of granulocytic ehrlichiosis to dogs, horses, and humans.

scholarly journals Isolation and Characterization of Two European Strains of Ehrlichia phagocytophila of Equine Origin

2002 ◽  
Vol 9 (2) ◽  
pp. 341-343 ◽  
Author(s):  
Anneli Bjöersdorff ◽  
Bodil Bagert ◽  
Robert F. Massung ◽  
Asiya Gusa ◽  
Ingvar Eliasson
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Dna Sequences ◽  
Rrna Gene ◽  
Granulocytic Ehrlichiosis ◽  
Ehrlichia Phagocytophila ◽  
Isolation And Characterization ◽  
Operon Gene ◽  
The 16S Rrna Gene

ABSTRACT We report the isolation and partial genetic characterization of two equine strains of granulocytic Ehrlichia of the genogroup Ehrlichia phagocytophila. Frozen whole-blood samples from two Swedish horses with laboratory-verified granulocytic ehrlichiosis were inoculated into HL-60 cell cultures. Granulocytic Ehrlichia was isolated and propagated from both horses. DNA extracts from the respective strains were amplified by PCR using primers directed towards the 16S rRNA gene, the groESL heat shock operon gene, and the ank gene. The amplified gene fragments were sequenced and compared to known sequences in the GenBank database. With respect to the 16S rRNA gene, the groESL gene, and the ank gene, the DNA sequences of the two equine Ehrlichia isolates were identical to sequences found in isolates from clinical cases of granulocytic ehrlichiosis in humans and domestic animals in Sweden. However, compared to amplified DNA from an American Ehrlichia strain of the E. phagocytophila genogroup, differences were found in the groESL gene and ank gene sequences.

scholarly journals Comparison of the Nucleotide Sequences of 16S rRNA, 444Ep-ank, and groESL Heat Shock Operon Genes in Naturally Occurring Ehrlichia equi and Human Granulocytic Ehrlichiosis Agent Isolates from Northern California

2000 ◽  
Vol 38 (4) ◽  
pp. 1364-1369 ◽  
Author(s):  
Joon-seok Chae ◽  
Janet E. Foley ◽  
J. Stephen Dumler ◽  
John E. Madigan
Keyword(s):  
Heat Shock ◽  
16S Rrna ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Northern California ◽  
Eastern United States ◽  
Granulocytic Ehrlichiosis ◽  
Naturally Occurring ◽  
Human Granulocytic Ehrlichiosis ◽  
Ehrlichia Equi

We examined 11 naturally occurring isolates of Ehrlichia equi in horses and two human granulocytic ehrlichiosis agent isolates in California for sequence diversity in three genes.Ehrlichia equi isolates were from Sierra (n = 6), Mendocino (n = 3), Sonoma (n = 1), and Marin (n = 1) counties, and human granulocytic ehrlichiosis (HGE) agent isolates were obtained from Humboldt county. PCR with specific primers for 16S rRNA, 444Ep-ank and groESL heat shock operon genes successfully produced amplicons for all 13 clinical samples. The 444Ep-ank gene of the HGE agent and E. equiisolates from northern California is different from the eastern U.S. isolates BDS and USG3. The translated amino acid sequence of thegroESL heat shock operon gene fragment is identical amongE. equi, the HGE agent, and E. phagocytophila, with the exception of the northern Californian equine CASOLJ isolate. Microheterogeneity was observed in the 16S rRNA gene sequences of HGE agent and E. equi isolates from northern California. These results suggest that E. equi and the HGE agent found in California are similar or identical but may differ from the isolates of equine and human origin found in the eastern United States.

scholarly journals Highly Specific Sewage-DerivedBacteroidesQuantitative PCR Assays Target Sewage-Polluted Waters

2019 ◽  
Vol 85 (6) ◽  
Author(s):  
Shuchen Feng ◽  
Sandra L. McLellan
Keyword(s):  
Population Structure ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Water Samples ◽  
Rrna Gene ◽  
Content Type ◽  
Hypervariable Regions ◽  
Animal Hosts ◽  
Pcr Assays ◽  
The 16S Rrna Gene

ABSTRACTThe identification of sewage contamination in water has primarily relied on the detection of human-associatedBacteroidesusing markers within the V2 region of the 16S rRNA gene. Despite the establishment of multiple assays that target the HF183 cluster (i.e.,Bacteroides dorei) and otherBacteroidesorganisms (e.g.,Bacteroides thetaiotaomicron), the potential for more human-associated markers in this genus has not been explored in depth. We examined theBacteroidespopulation structure in sewage and animal hosts across the V4V5 and V6 hypervariable regions. Using near-full-length cloned sequences, we identified the sequences in the V4V5 and V6 hypervariable regions that are linked to the HF183 marker in the V2 region and found these sequences were present in multiple animals. In addition, the V4V5 and V6 regions contained human fecal marker sequences for organisms that were independent of the HF183 cluster. The most abundantBacteroidesin untreated sewage was not human associated but pipe derived. Two TaqMan quantitative PCR (qPCR) assays targeting the V4V5 and V6 regions of this organism were developed. Validation studies using fecal samples from seven animal hosts (n = 76) and uncontaminated water samples (n = 30) demonstrated the high specificity of the assays for sewage. FreshwaterBacteroideswere also identified in uncontaminated water samples, demonstrating that measures of totalBacteroidesdo not reflect fecal pollution. A comparison of two previously described humanBacteroidesassays (HB and HF183/BacR287) in municipal wastewater influent and sewage-contaminated urban water samples revealed identical results, illustrating the assays target the same organism. The detection of sewage-derivedBacteroidesprovided an independent measure of sewage-impacted waters.IMPORTANCEBacteroidesare major members of the gut microbiota, and host-specific organisms within this genus have been used extensively to gain information on pollution sources. This study provides a broad view of the population structure ofBacteroideswithin sewage to contextualize the well-studied HF183 marker for a human-associatedBacteroides. The study also delineates host-specific sequence patterns across multiple hypervariable regions of the 16S rRNA gene to improve our ability to use sequence data to assess water quality. Here, we demonstrate that regions downstream of the HF183 marker are nonspecific but other potential human-associated markers are present. Furthermore, we show the most abundantBacteroidesin sewage is free living, rather than host associated, and specifically found in sewage. Quantitative PCR assays that target organisms specific to sewer pipes offer measures that are independent of the human microbiome for identifying sewage pollution in water.

A new species of Leptolalax (Anura: Megophryidae) from northern Vietnam

Zootaxa ◽  
2017 ◽  
Vol 4243 (3) ◽  
pp. 544 ◽  
Author(s):  
JODI J. L. ROWLEY ◽  
VINH Q. DAU ◽  
HUY D. HOANG ◽  
DUONG T. T. LE ◽  
TIMOTHY P. CUTAJAR ◽  
...  
Keyword(s):  
New Species ◽  
16S Rrna ◽  
Dna Sequences ◽  
Dominant Frequency ◽  
Rrna Gene ◽  
Adult Males ◽  
Skin Texture ◽  
Adult Females ◽  
A New Species ◽  
The 16S Rrna Gene

We describe a new, medium-sized Leptolalax species from Vietnam. Leptolalax petrops sp. nov. is distinguished from its congeners by a combination of having a medium-sized body (23.6–27.6 mm in 21 adult males, 30.3–47.0 mm in 17 adult females), immaculate white chest and belly, no distinct black markings on the head, highly tuberculate skin texture, toes lacking webbing and with narrow lateral fringes, and a call consisting of an average of four notes and a dominant frequency of 5.6–6.4 kHz (at 24.5–25.3 °C). Uncorrected sequence divergences between L. petrops sp. nov. and all homologous DNA sequences available for the 16S rRNA gene are >8%. 

scholarly journals Analytical Sensitivity, Reproducibility of Results, and Clinical Performance of Five PCR Assays for Detecting Chlamydia pneumoniae DNA in Peripheral Blood Mononuclear Cells

2000 ◽  
Vol 38 (7) ◽  
pp. 2622-2627 ◽  
Author(s):  
J. B. Mahony ◽  
S. Chong ◽  
B. K. Coombes ◽  
M. Smieja ◽  
A. Petrich
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Nested Pcr ◽  
Chlamydia Pneumoniae ◽  
Mononuclear Cells ◽  
Rrna Gene ◽  
Analytical Sensitivity ◽  
Infected Cells ◽  
Pcr Assays ◽  
The 16S Rrna Gene

Chlamydia pneumoniae has been associated with atherosclerosis and coronary artery disease (CAD), and its DNA has been detected in atheromatous lesions of the aorta, carotid, and coronary arteries by a variety of PCR assays. The objective of this study was to compare the performances of five published PCR assays in the detection of C. pneumoniae in peripheral blood mononuclear cells (PBMCs) from patients with coronary artery disease. The assays included two conventional PCRs, one targeting a cloned PstI fragment and one targeting the 16S rRNA gene; two nested PCRs, one targeting the 16S rRNA gene and one targeting ompA; and a touchdown enzyme time release (TETR) PCR, targeting the 16S rRNA gene. All PCRs had similar analytical sensitivities and detected a minimum of 0.005 inclusion-forming units (IFU) of C. pneumoniae; the ompA nested PCR and the TETR PCR were slightly more sensitive and detected 0.001 IFU. Assay reproducibility was examined by testing 10 replicates of C. pneumoniae DNA by each assay. All five assays showed excellent reproducibility at high levels of DNA, with scores of 10 out of 10 for 0.01 IFU, but exhibited decreased reproducibility for smaller numbers of C. pneumoniae IFU for all tests. Pairwise comparison of test results indicated that there was a significant difference between tests (Cochran Q = 32.0, P < 0.001), with thePstI fragment (P < 0.001) and 16S rRNA (P = 0.002) assays having lower reproducibility than the nested ompA and TETR assays. To further analyze assay sensitivity, C. pneumoniae-infected U-937 mononuclear cells were added to whole blood, and extracted mononuclear-cell DNA was tested by each assay. All five assays showed similar sensitivities, detecting 15 infected cells; three assays detected 3 infected cells, while all assays were negative at the next dilution (1.5 infected cells). A striking difference in performance of the five assays was seen, however, when PBMCs from CAD patients were tested for C. pneumoniae DNA. The ompA nested PCR detected C. pneumoniae DNA in 11 of 148 (7.4%) specimens, the 16S rRNA nested PCR detected 2 positives among the 148 specimens (1.4%) (P < 0.001), and the other 3 assays detected no positive specimens (P < 0.001, compared with theompA assay). These results indicate that analytical sensitivity alone does not predict the ability of an assay to detectC. pneumoniae in whole-blood-derived PBMCs. Before standardized assays can be used in wide-scale epidemiological studies, further characterization of these assays will be required to improve our understanding of their performance in the detection of C. pneumoniae in clinical material.

scholarly journals Seasonal changes in bacterial communities associated with healthy and diseasedPoritescoral in southern Taiwan

2016 ◽  
Vol 62 (12) ◽  
pp. 1021-1033 ◽  
Author(s):  
Chorng-Horng Lin ◽  
Chih-Hsiang Chuang ◽  
Wen-Hung Twan ◽  
Shu-Fen Chiou ◽  
Tit-Yee Wong ◽  
...  
Keyword(s):  
Health Status ◽  
Bacterial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Dna Sequences ◽  
Bacterial Communities ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Rrna Gene ◽  
Southern Taiwan ◽  
The 16S Rrna Gene

We compared the bacterial communities associated with healthy scleractinian coral Porites sp. with those associated with coral infected with pink spot syndrome harvested during summer and winter from waters off the coast of southern Taiwan. Members of the bacterial community associated with the coral were characterized by means of denaturing gradient gel electrophoresis (DGGE) of a short region of the 16S rRNA gene and clone library analysis. Of 5 different areas of the 16S rRNA gene, we demonstrated that the V3 hypervariable region is most suited to represent the coral-associated bacterial community. The DNA sequences of 26 distinct bands extracted from DGGE gels and 269 sequences of the 16S rRNA gene from clone libraries were determined. We found that the communities present in diseased coral were more heterogeneous than the bacterial communities of uninfected coral. In addition, bacterial communities associated with coral harvested in the summer were more diverse than those associated with coral collected in winter, regardless of the health status of the coral. Our study suggested that the compositions of coral-associated bacteria communities are complex, and the population of bacteria varies greatly between seasons and in coral of differing health status.

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