Detection and Characterization of an Elm Yellows (16SrV) Group Phytoplasma Infecting Virginia Creeper Plants in Southern Florida

The polymerase chain reaction (PCR) employing phytoplasma-specific ribosomal RNA primer pair P1/P7 consistently amplified a product of expected size (1.8 kb) from 29 of 36 symptom-less Virginia creeper (Parthenocissus quinquefolia) plants growing in southern Florida. Restriction fragment length polymorphism analysis of P1/P7-primed PCR products indicated that most phytoplasmas detected in Virginia creeper were similar to phytoplasmas composing the elm yellows (16SrV) group. This relationship was verified by reamplification of P1/P7 products using an elm yellows (EY) group-specific rRNA primer pair fB1/rULWS1. rDNA products (1,571 bp) were generated by group-specific PCR from 28 phytoplasma-positive plants and 1 negatively testing plant identified by earlier P1/P7-primed PCR. Analysis of 16S rDNA sequences determined the Virginia creeper (VC) phytoplasma to be phylogenetically closest to the European alder yellows (ALY) agent, an established 16SrV-C subgroup strain. However, presence or absence of restriction sites for endonucleases AluI, BfaI, MspI, RsaI, and TaqI in the 16S rRNA and 16-23S rRNA intergenic spacer region of the VC phytoplasma collectively differentiated this strain from ALY and other 16SrV group phytoplasmas. Failure to detect the VC phytoplasma by PCR employing nonribosomal primer pair FD9f/FD9r suggests that this newly characterized agent varies from known European grapevine yellows (flavescence dorée) phyto-plasmas previously classified as 16SrV subgroup C or D strains.

Download Full-text

Simultaneous Detection and Identification of Common Cell Culture Contaminant and Pathogenic Mollicutes Strains by Reverse Line Blot Hybridization

Applied and Environmental Microbiology ◽

10.1128/aem.70.3.1483-1486.2004 ◽

2004 ◽

Vol 70 (3) ◽

pp. 1483-1486 ◽

Cited By ~ 33

Author(s):

Hui Wang ◽

Fanrong Kong ◽

Peter Jelfs ◽

Gregory James ◽

Gwendolyn L. Gilbert

Keyword(s):

Cell Culture ◽

16S Rrna ◽

Cell Line ◽

Intergenic Spacer ◽

23S Rrna ◽

Pcr Assay ◽

Intergenic Spacer Region ◽

Specific Pcr ◽

Specific Pcr Assay ◽

Species Specific

ABSTRACT We have developed a reverse line blot (RLB) hybridization assay to detect and identify the commonest mollicutes causing cell line contamination (Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma orale, and Acholeplasma laidlawii) and human infection (Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma parvum, and Ureaplasma urealyticum). We developed a nested PCR assay with “universal” primers targeting the mollicute 16S-23S rRNA intergenic spacer region. Amplified biotin-labeled PCR products were hybridized to membrane-bound species-specific oligonucleotide probes. The assay correctly identified reference strains of 10 mollicute species. Cell cultures submitted for detection of mollicute contamination, clinical specimens, and clinical isolates were initially tested by PCR assay targeting a presumed mollicute-specific sequence of the 16S rRNA gene. Any that were positive were assessed by the RLB assay, with species-specific PCR assay as the reference method. Initially, 100 clinical and 88 of 92 cell culture specimens gave concordant results, including 18 in which two or more mollicute species were detected by both methods. PCR and sequencing of the 16S-23S rRNA intergenic spacer region and subsequent retesting by species-specific PCR assay of the four cell culture specimens for which results were initially discrepant confirmed the original RLB results. Sequencing of amplicons from 12 cell culture specimens that were positive in the 16S rRNA PCR assay but negative by both the RLB and species-specific PCR assays failed to identify any mollicute species. The RLB hybridization assay is sensitive and specific and able to rapidly detect and identify mollicute species from clinical and cell line specimens.

Download Full-text

Use of 16S-23S rRNA Intergenic Spacer Region PCR and Repetitive Extragenic Palindromic PCR Analyses of Escherichia coli Isolates To Identify Nonpoint Fecal Sources

Applied and Environmental Microbiology ◽

10.1128/aem.69.8.4942-4950.2003 ◽

2003 ◽

Vol 69 (8) ◽

pp. 4942-4950 ◽

Cited By ~ 46

Author(s):

Sylvie Seurinck ◽

Willy Verstraete ◽

Steven D. Siciliano

Keyword(s):

Escherichia Coli ◽

Natural Waters ◽

Average Rate ◽

Intergenic Spacer ◽

23S Rrna ◽

Pcr Analysis ◽

Factors Affecting ◽

Intergenic Spacer Region ◽

E Coli ◽

Repetitive Extragenic Palindromic

ABSTRACT Despite efforts to minimize fecal input into waterways, this kind of pollution continues to be a problem due to an inability to reliably identify nonpoint sources. Our objective was to find candidate source-specific Escherichia coli fingerprints as potential genotypic markers for raw sewage, horses, dogs, gulls, and cows. We evaluated 16S-23S rRNA intergenic spacer region (ISR)-PCR and repetitive extragenic palindromic (rep)-PCR analyses of E. coli isolates as tools to identify nonpoint fecal sources. The BOXA1R primer was used for rep-PCR analysis. A total of 267 E. coli isolates from different fecal sources were typed with both techniques. E. coli was found to be highly diverse. Only two candidate source-specific E. coli fingerprints, one for cow and one for raw sewage, were identified out of 87 ISR fingerprints. Similarly, there was only one candidate source-specific E. coli fingerprint for horse out of 59 BOX fingerprints. Jackknife analysis resulted in an average rate of correct classification (ARCC) of 83% for BOX-PCR analysis and 67% for ISR-PCR analysis for the five source categories of this study. When nonhuman sources were pooled so that each isolate was classified as animal or human derived (raw sewage), ARCCs of 82% for BOX-PCR analysis and 72% for ISR-PCR analysis were obtained. Critical factors affecting the utility of these methods, namely sample size and fingerprint stability, were also assessed. Chao1 estimation showed that generally 32 isolates per fecal source individual were sufficient to characterize the richness of the E. coli population of that source. The results of a fingerprint stability experiment indicated that BOX and ISR fingerprints were stable in natural waters at 4, 12, and 28°C for 150 days. In conclusion, 16S-23S rRNA ISR-PCR and rep-PCR analyses of E. coli isolates have the potential to identify nonpoint fecal sources. A fairly small number of isolates was needed to find candidate source-specific E. coli fingerprints that were stable under the simulated environmental conditions.

Download Full-text

Longitudinal monitoring of the dynamics of infections due to Bartonella species in UK woodland rodents

Epidemiology and Infection ◽

10.1017/s095026880100526x ◽

2001 ◽

Vol 126 (2) ◽

pp. 323-329 ◽

Cited By ~ 60

Author(s):

R. J. BIRTLES ◽

S. M. HAZEL ◽

M. BENNETT ◽

K. BOWN ◽

D. RAOULT ◽

...

Keyword(s):

Sequence Comparison ◽

Intergenic Spacer ◽

23S Rrna ◽

Enzyme Analysis ◽

Restriction Enzyme Analysis ◽

Bartonella Species ◽

Intergenic Spacer Region ◽

Specific Pcr ◽

Time Specific ◽

Pcr Targeting

Blood samples were repeatedly collected from 12 sympatric woodland rodents over a 12-month period and DNA extracts from each were incorporated into a bartonella-specific PCR targeting a fragment of the 16S/23S rRNA intergenic spacer region (ISR). The composition of each amplicon was analysed using restriction enzyme analysis (REA) and base sequence comparison. Bartonella DNA was detected in 70 of 109 samples. Eleven samples contained DNA derived from more than one strain. Sequence analysis of 62 samples found 12 sequence variants (ISR genotypes) that were provisionally assigned to 5 different species, 2 of which were newly recognized. Up to five different species were detected in each animal. On about two-thirds of occasions, a species detected 1 month was not there the next, but never was a genotype superseded by another of the same species. However, a genotype could be re-encountered months later in the same animal, even if interim samples contained other genotypes. Our results suggest that although most animals are bacteraemic most of the time, specific infections are often superseded and that a complex and dynamic epidemiology of bartonella bacteraemias exists in woodland rodents.

Download Full-text

Revised Subgroup Classification of Group 16SrV Phytoplasmas and Placement of Flavescence Dorée-Associated Phytoplasmas in Two Distinct Subgroups

Plant Disease ◽

10.1094/pdis.2001.85.7.790 ◽

2001 ◽

Vol 85 (7) ◽

pp. 790-797 ◽

Cited By ~ 47

Author(s):

Robert E. Davis ◽

Ellen L. Dally

Keyword(s):

16S Rdna ◽

Rflp Analysis ◽

23S Rrna ◽

Nucleotide Substitutions ◽

Flavescence Dorée ◽

Rdna Sequences ◽

Restriction Sites ◽

Elm Yellows ◽

Subgroup Classification

The subgroup classification of phytoplasmas in 16S rRNA group 16SrV (elm yellows phytoplasma group) was revised and extended on the basis of enzymatic restriction fragment length polymorphism (RFLP) analysis of ribosomal (r) DNA and analysis of putative restriction sites in nucleotide sequences. A 1.85 kbp fragment of the rRNA operon from flavescence dorée (FD) phytoplasma strain FD70 from France was amplified and cloned, and its nucleotide sequence determined (GenBank acc. no. AF176319). Placement of FD70 in subgroup V-C was verified by analysis of amplified DNA and of the cloned sequence. Hemp dogbane phytoplasma HD1 (AF122912), a member of subgroup V-C, was distinguished from other subgroup V-C phytoplasmas by putative restriction site differences in the 16S-23S rRNA spacer region. A previously published FD phytoplasma sequence (GenBank accession no. X76560) differed from FD70 sequence AF176319 by at least eight nucleotide substitutions and differences in putative restriction sites. The X76560 FD phytoplasma was classified in a new subgroup (V-D). Based on analyses of 16S rDNA GenBank sequence Y16395, Rubus stunt phytoplasma was classified in new subgroup V-E. The revised classification was supported by sequence similarities, group 16SrV-characteristic sequences, and a phylogenetic tree constructed on the basis of 16S rDNA sequences.

Download Full-text

Genomic fingerprinting ofFrankiamicrosymbionts fromCeanothuscopopulations using repetitive sequences and polymerase chain reactions

Canadian Journal of Botany ◽

10.1139/b99-069 ◽

1999 ◽

Vol 77 (9) ◽

pp. 1220-1230 ◽

Cited By ~ 3

Author(s):

Soon-Chun Jeong ◽

David D Myrold

Keyword(s):

Dna Sequencing ◽

Dna Analysis ◽

Repetitive Sequences ◽

Intergenic Spacer ◽

Rrna Genes ◽

23S Rrna ◽

Genomic Fingerprinting ◽

Chain Reactions ◽

Intergenic Spacer Region ◽

Polymerase Chain

Specificity between Ceanothus species and their microsymbionts, Frankia, were investigated with nodules collected from three geographically separated copopulations of Ceanothus species. Nodules were analyzed using DNA sequencing and repetitive sequence polymerase chain reaction (rep-PCR) techniques. DNA sequencing of the intergenic spacer region between 16S and 23S rRNA genes suggested that Ceanothus-microsymbiotic Frankia are closely related at the intraspecific level. Diversity of the microsymbionts was further analyzed by genomic fingerprinting using repetitive sequences and PCR. A newly designed direct repeat (DR) sequence and a BOX sequence were used as PCR primers after justification that these primers can generate Frankia-specific fingerprints from nodule DNA. Analysis of the nodules using BOX- and DR-PCR showed that Ceanothus-microsymbiotic Frankia exhibited less diversity within each copopulation than among copopulations. These data suggested that geographic separation plays a more important role for divergence of Ceanothus-microsymbiotic Frankia than host plant.Key words: Frankia, Ceanothus, rep-PCR, diversity.

Download Full-text

Molecular Detection and Characterization of Borrelia garinii (Spirochaetales: Borreliaceae) in Ixodes nipponensis (Ixodida: Ixodidae) Parasitizing a Dog in Korea

Pathogens ◽

10.3390/pathogens8040289 ◽

2019 ◽

Vol 8 (4) ◽

pp. 289 ◽

Cited By ~ 2

Author(s):

Seung-Hun Lee ◽

Youn-Kyoung Goo ◽

Paul John L. Geraldino ◽

Oh-Deog Kwon ◽

Dongmi Kwak

Keyword(s):

Molecular Detection ◽

Single Gene ◽

Protein A ◽

Intergenic Spacer ◽

Surface Protein ◽

23S Rrna ◽

Borrelia Garinii ◽

Intergenic Spacer Region ◽

Individual Level ◽

Pcr Targeting

The present study aimed to detect and characterize Borrelia spp. in ticks attached to dogs in Korea. Overall, 562 ticks (276 pools) attached to dogs were collected and tested for Borrelia infection by PCR targeting the 5S-23S rRNA intergenic spacer region (rrf-rrl). One tick larva (pool level, 0.4%; individual level, 0.2%) was confirmed by sequencing Borrelia garinii, a zoonotic pathogen. For molecular characterization, the outer surface protein A (ospA) and flagellin genes were analyzed. Phylogenetic ospA analysis distinguished B. garinii from B. bavariensis, which has been recently identified as a novel Borrelia species. On the other hand, phylogenetic analysis showed that single gene analysis involving rrf-rrl or flagellin was not sufficient to differentiate B. garinii from B. bavariensis. In addition, the B. garinii-infected tick was identified as Ixodes nipponensis by sequencing according to mitochondrial 16S rRNA and the second transcribed spacer region. To our knowledge, this is the first study to report the molecular detection of B. garinii in I. nipponensis parasitizing a dog in Korea. Continuous monitoring of tick-borne pathogens in ticks attached to animals is required to avoid disease distribution and possible transmission to humans.

Download Full-text

Differentiation of group VIII Spiroplasma strains with sequences of the 16S–23S rDNA intergenic spacer region

Canadian Journal of Microbiology ◽

10.1139/w04-091 ◽

2004 ◽

Vol 50 (12) ◽

pp. 1061-1067 ◽

Cited By ~ 14

Author(s):

Laura B Regassa ◽

Kimberly M Stewart ◽

April C Murphy ◽

Frank E French ◽

Tao Lin ◽

...

Keyword(s):

Intergenic Spacer ◽

Analytical Models ◽

23S Rrna ◽

Rrna Gene ◽

Group Viii ◽

Similarity Coefficients ◽

Intergenic Spacer Region ◽

Intergenic Sequences ◽

The Stability ◽

The 16S Rrna Gene

Spiroplasma species (Mollicutes: Spiroplasmataceae) are associated with a wide variety of insects, and serology has classified this genus into 34 groups, 3 with subgroups. The 16S rRNA gene has been used for phylogenetic analysis of spiroplasmas, but this approach is uninformative for group VIII because the serologically distinct subgroups generally have similarity coefficients >0.990. Therefore, we investigated the utility of the 16S–23S rRNA spacer region as a means to differentiate closely related subgroups or strains. We generated intergenic sequences and detailed serological profiles for 8 group VIII Spiroplasma strains. Sequence analyses using Maximum Parsimony, Neighbor Joining, and Maximum Likelihood placed the strains into 2 clades. One clade consisted of strains BARC 2649 and GSU5367. The other clade was divided into clusters containing representatives of the 3 designated group VIII subgroups (EA-1, DF-1, and TAAS-1) and 3 previously unclassified strains. The stability of the positions of the strains in various analytical models and the ability to provide robust support for groupings tentatively supported by serology indicates that the 16S–23S intergenic rDNA sequence will prove useful in intragroup analysis of group VIII spiroplasmas.Key words: Mollicutes, Spiroplasma, phylogeny, Tabanidae.

Download Full-text

A putative transposase gene in the 16S–23S rRNA intergenic spacer region of Mycoplasma imitans

Microbiology ◽

10.1099/mic.0.26629-0 ◽

2004 ◽

Vol 150 (4) ◽

pp. 1023-1029 ◽

Cited By ~ 28

Author(s):

Ryô Harasawa ◽

David G. Pitcher ◽

Ana S. Ramírez ◽

Janet M. Bradbury

Keyword(s):

Relative Size ◽

Intergenic Spacer ◽

Its Region ◽

23S Rrna ◽

Mycoplasma Species ◽

Insertion Element ◽

Transposase Gene ◽

Intergenic Spacer Region ◽

Pcr Products ◽

Type Strains

Examination of the nucleotide sequences of the 16S–23S intergenic transcribed spacer (ITS) region of Mycoplasma imitans and Mycoplasma gallisepticum identified a putative transposase gene located only in the ITS of M. imitans, which can be used as a genetic marker to distinguish these two species. The relative size of the PCR products of the ITS region allowed a clear distinction to be made between strains of M. imitans and M. gallisepticum, both of which could be readily discriminated from the type strains of all the other recognized avian Mycoplasma species. In addition, the putative transposase gene assigned in the ITS of M. imitans was shown to include a sequence homologous to that of the P75 gene of M. gallisepticum. This is believed to be the first description of an insertion element in the rRNA operon region of a mycoplasma species.

Download Full-text