bluuebunny/crossref_metadata_2025_split huggingface dataset:
Application of tRNA Intergenic Spacer PCR for Identification of
Enterococcus
Species
Authors: Baele Margo , Baele Paul , Vaneechoutte Mario , Storms Virginie , Butaye Patrick , Devriese Luc A. , Verschraegen Gerda , Gillis Monique , Haesebrouck Freddy 
Published: December 2019

ABSTRACT

tRNA intergenic spacer PCR (tDNA-PCR) was evaluated for its usefulness in the differentiation of enterococcal species of human and animal origin. This technique was carried out for 124 strains belonging to 17 enterococcal species and generated DNA fragments, which were separated by capillary electrophoresis. tDNA-PCR enabled us to discriminate for all species tested.
Enterococcus faecium
showed minor but reproducible differences with
Enterococcus durans
, while
Enterococcus hirae
was easily distinguishable.
Enterococcus avium
,
Enterococcus malodoratus
, and
Enterococcus raffinosus
generated highly similar though distinctive patterns.
Granulocytic Ehrlichiae in
Ixodes persulcatus
Ticks from an Area in China Where Lyme Disease Is Endemic
Authors: Cao Wu-Chun , Zhao Qiu-Min , Zhang Pan-He , Dumler J. Stephen , Zhang Xi-Tan , Fang Li-Qun , Yang Hong 
Published: December 2019

ABSTRACT

A total of 372 adult
Ixodes persulcatus
ticks were collected from vegetation in a forest area of Heilongjiang Province in northeastern China, where Lyme disease is known to be endemic. The ticks were examined for the presence of granulocytic ehrlichiae by heminested PCR with primers derived from the 16S rRNA gene. Of 310 ticks obtained from the Dahe forestry farm, two pools (each containing 5 ticks) were found positive, with a minimum infection rate of 0.6%. Ehrlichial DNA was also detected in one female (1.6%) of 62 ticks collected from the Yulin forestry farm. The overall minimum infection rate of the 372
I. persulcatus
adults was 0.8%. The nucleotide sequences of 919-bp PCR products from the three positive tick specimens were identical to each other and very closely related to the members of the
Ehrlichia phagocytophila
genogroup. This is the first identification of granulocytic ehrlichiae in ticks in Asia and the first report of infection in
I. persulcatus
anywhere.
Frequency of Isolation of
Staphylococcus lugdunensis
among Staphylococcal Isolates Causing Endocarditis: a 20-Year Experience
Authors: Patel Robin , Piper Kerryl E. , Rouse Mark S. , Uhl James R. , Cockerill Franklin R. , Steckelberg James M. 
Published: December 2019

ABSTRACT

Eighty-nine staphylococcal isolates recovered from patients with bacterial endocarditis at the Mayo Clinic from 1980 to 1999 were studied to determine the prevalence of
Staphylococcus lugdunensis
among clinical isolates of staphylococci causing endocarditis. Four isolates, all from patients with native mitral valve endocarditis, were identified as
S. lugdunensis
.
Can Pooling Be Used for Seroprevalence Studies of Hepatitis C?
Authors: Stephens G. M. , Raboud J. M. , Karakas L. , Sherlock C. H. 
Published: December 2019

ABSTRACT
Two hepatitis C antibody assays were used to test diluted positive sera. Dilutions of 1 in 5, 1 in 10, and 1 in 20 all resulted in loss of reactivity, with the greatest losses occurring in samples with low and moderate reactivities. These results disqualify pooling as a strategy for seroprevalence studies and screening programs.
Improved Genotyping Vaccine and Wild-Type Poliovirus Strains by Restriction Fragment Length Polymorphism Analysis: Clinical Diagnostic Implications
Authors: Georgopoulou Amalia , Markoulatos Panayotis , Spyrou Niki , Vamvakopoulos Nicholas C. 
Published: December 2019

ABSTRACT

The combination of preventive vaccination and diagnostic typing of viral isolates from patients with clinical poliomyelitis constitutes our main protective shield against polioviruses. The restriction fragment length polymorphism (RFLP) adaptation of the reverse transcriptase (RT)-PCR methodology has advanced diagnostic genotyping of polioviruses, although further improvements are definitely needed. We report here on an improved RFLP procedure for the genotyping of polioviruses. A highly conserved segment within the 5′ noncoding region of polioviruses was selected for RT-PCR amplification by the UC
53
-UG
52
primer pair with the hope that it would be most resistant to the inescapable genetic alteration-drift experienced by the other segments of the viral genome. Complete inter- and intratypic genotyping of polioviruses by the present RFLP method was accomplished with a minimum set of four restriction endonucleases (
Hae
III,
Dde
I,
Nco
I, and
Ava
I). To compensate for potential genetic drift within the recognition sites of
Hae
III,
Dde
I, or
Nco
I in atypical clinical samples, the RFLP patterns generated with
Hpa
II and
Sty
I as replacements were analyzed. The specificity of the method was also successfully assessed by RFLP analysis of 55 reference nonpoliovirus enterovirus controls. The concerted implementation of these conditional protocols for diagnostic inter- and intratypic genotyping of polioviruses was evaluated with 21 clinical samples with absolute success.
Glyceraldehyde-3-Phosphate Dehydrogenase-Encoding Gene as a Useful Taxonomic Tool for
Staphylococcus
spp
Authors: Yugueros Javier , Temprano Alejandro , Berzal Beatriz , Sánchez Marı́a , Hernanz Carmen , Luengo José Marı́a , Naharro Germán 
Published: December 2019

ABSTRACT

The
gap
gene of
Staphylococcus aureus
, encoding glyceraldehyde-3-phosphate dehydrogenase, was used as a target to amplify a 933-bp DNA fragment by PCR with a pair of primers 26 and 25 nucleotides in length. PCR products, detected by agarose gel electrophoresis, were also amplified from 12
Staphylococcus
spp. analyzed previously. Hybridization with an internal 279-bp DNA fragment probe was positive in all PCR-positive samples. No PCR products were amplified when other gram-positive and gram-negative bacterial genera were analyzed using the same pair of primers.
Alu
I digestion of PCR-generated products gave 12 different restriction fragment length polymorphism (RFLP) patterns, one for each species analyzed. However, we could detect two intraspecies RFLP patterns in
Staphylococcus epidermidis
,
Staphylococcus hominis
, and
Staphylococcus simulans
which were different from the other species. An identical RFLP pattern was observed for 112
S. aureus
isolates from humans, cows, and sheep. The sensitivity of the PCR assays was very high, with a detection limit for
S. aureus
cells of 20 CFU when cells were suspended in saline. PCR amplification of the
gap
gene has the potential for rapid identification of at least 12 species belonging to the genus
Staphylococcus
, as it is highly specific.
Evaluation of Three Newly Developed Enzyme-Linked Immunosorbent Assays and Two Agglutination Tests for DetectingSalmonella entericasubsp.entericaSerovar Dublin Infections in Dairy Cattle
Authors: Veling J. , van Zijderveld F. G. , van Zijderveld-van Bemmel A. M. , Barkema H. W. , Schukken Y. H. 
Published: December 2019

ABSTRACTIn this study test characteristics of three newly developed enzyme-linked immunosorbent assays (ELISAs) forSalmonella entericasubsp.entericaserovar Dublin were evaluated and compared with two agglutination tests. The ELISAs involved were an indirect ELISA with serovar Dublin lipopolysaccharide (LPS ELISA), an indirect ELISA with serovar Dublin flagellar antigen (GP ELISA), and a double-antibody sandwich blocking ELISA that uses monoclonal antibodies againstS. entericasubsp.entericaserovar Enteritidis flagellin (GM-DAS ELISA). The agglutination tests involved were two routine serum agglutination tests with either somatic (O) or flagellar (H) antigen. Diagnostic specificity of the three ELISAs was determined using 840 serum samples from seven dairy herds without any history of serovar Dublin infection. Cutoff values at a titer of 100, 100, and 10, respectively, for the LPS ELISA, GP ELISA, and GM-DAS blocking ELISA resulted in a specificity of 99.3, 100, and 100%, respectively. Using these cutoff values the LPS ELISA, GP ELISA, and GM-DAS ELISA were able to detect, respectively, 30, 46, and 38% of 50 fecal culture-positive animals from 13 herds with a recent serovar Dublin infection. With the same cutoff values, active carriers (n= 18) were detected for 94.4% with the LPS ELISA and for 100% with the GP and GM-DAS ELISAs. Kappa values determined on the results of all tests from 8 of the 13 serovar Dublin-infected herds and the 7 control herds demonstrated a good correlation between the results of all ELISAs and the H-agglutination test. The results of the O-agglutination test failed to correlate with those of the other tests. Using a set of sera from 170 aborting cows (with 25 abortions due to serovar Dublin), test results of the ELISAs and the H-agglutination test were comparable. The H-agglutination test may be used successfully for single sample testing, especially to diagnose abortion due to serovar Dublin. It is concluded that the ELISAs are useful diagnostic tools in serovar Dublin control programs and that they are preferred to agglutination tests for reasons of automation and costs.
Detection of
Chlamydia pneumoniae
and
Helicobacter pylori
DNA in Human Atherosclerotic Plaques by PCR
Authors: Farsak Bora , Yildirir Aylin , Akyön Yakut , Pinar Ahmet , Öç Mehmet , Böke Erkmen , Kes Sirri , Tokgözoğlu Lale 
Published: December 2019

ABSTRACT

Chlamydia pneumoniae
and
Helicobacter pylori
can cause persistent infections of the respiratory and gastrointestinal tract, respectively. It has been suggested that persistent infection of arteries with these bacteria can contribute to the development of atherosclerosis. The aims of this study were to determine the presence of
C. pneumoniae
and
H. pylori
DNA in atherosclerotic plaque samples by PCR and to evaluate the correlation between clinical status and DNA positivity of these bacteria. Eighty-five consecutive patients (mean age, 59 ± 10; 75 male, 10 female) undergoing coronary artery bypass grafting, carotid endarterectomy, and surgery of the abdominal aorta for atherosclerotic obstructive lesions were included in the study. Forty-six endarterectomy specimens from the atherosclerotic lesions and 39 specimens from healthy regions of the ascending aorta, which were accepted as the control group, were excised. The presence of microorganism DNA in endarterectomy specimens was assessed by PCR.
C. pneumoniae
DNA was found in 12 (26%) of 46 endarterectomy specimens and none of the healthy vascular-wall specimens (
P
< 0.001), while
H. pylori
DNA was found in 17 (37%) of 46 endarterectomy specimens and none of the controls (
P
< 0.001). Either
C. pneumoniae
or
H. pylori
DNA was positive in 23 (50%) of 46 patients and none of the controls (
P
< 0.001). Six of the atherosclerotic lesions showed coexistence of both of the microorganism DNAs. The presence of
C. pneumoniae
and
H. pylori
DNA in a considerable number of atherosclerotic plaques but their absence in healthy vascular wall supports the idea that they may have a role in the development of atherosclerosis, especially in countries where infection is prevalent and where conventional risk factors fail to explain the high prevalence of atherosclerotic vascular disease.
Prevalence of Diarrheagenic
Escherichia coli
in Finns with or without Diarrhea during a Round-the-World Trip
Authors: Keskimäki Markku , Mattila Leena , Peltola Heikki , Siitonen Anja 
Published: December 2019

ABSTRACT

The incidence of diarrhea and the prevalence of bacterial enteropathogens, viruses, and parasites in feces of subjects with and without diarrhea were evaluated in 204 Finns traveling round the world (from Finland to China, Malaysia, Australia, Fiji, Chile, and Brazil and back to Finland). Special emphasis was placed on the finding of diarrheagenic
Escherichia coli
(enterotoxigenic, enteropathogenic, Shiga toxin-producing, and enteroaggregative strains) by PCR from growth on primary culture plates. From the PCR-positive samples, corresponding strains were isolated, confirmed as
E. coli
, and O serotyped. Of all the subjects, 37% experienced a total of 90 episodes of diarrhea. No adenoviruses or rotaviruses were detected, and findings of parasites were insignificant. In contrast, enteropathogenic bacteria were present in 62% of the 65 diarrheal and in 33% of the 127 nondiarrheal samples (
P
< 0.001); diarrheagenic
E. coli
strains were found in 35 and 26% of these, respectively (not statistically significant). As a single pathogen,
E. coli
was found in 20 and 24% of samples (not significant). Of all diarrheagenic
E. coli
strains, enteropathogenic strains were the most commonly found independently of the clinical picture of the subjects, whereas
Salmonella enterica
as a single pathogen was the most common non-
E. coli
organism found in diarrheal samples. Multiple bacterial pathogens were found 10 times more commonly in diarrheal than in nondiarrheal samples (20 versus 2%;
P
< 0.001).
NSP4 Gene Analysis of Rotaviruses Recovered from Infected Children with and without Diarrhea
Authors: Lee Chun-Nan , Wang Yu-Lan , Kao Chuan-Liang , Zao Chih-Ling , Lee Chin-Yun , Chen Hsiao-Neng 
Published: December 2019

ABSTRACT
The transmembrane glycoprotein NSP4 functions as a viral enterotoxin capable of inducing diarrhea in young mice. It has been suggested that NSP4 may be a key determinant of rotavirus pathogenicity and a target for vaccine development. Twenty two G1P[6] rotaviruses from babies with and without diarrhea were comparatively analyzed along with reference strains and another 22 Taiwanese human rotaviruses of G and P combination types different from the G1P[6] type. The sequence variations in the NSP4 genes were studied by direct sequencing analysis of the amplicons of reverse transcription-PCR. Two genetic groups could be identified in this analysis. While the majority of these strains were closely related to the Wa strain, the G2 viruses were closely related to the S2 strain. Furthermore, phylogenetic analysis of the NSP4 gene among the G2 rotaviruses revealed three distinct lineages associated with DS-1, S2, and E210, respectively, as has been reported previously for the VP7 gene. However, we found no apparent correlation in the deduced amino acid sequences corresponding to the proposed enterotoxic peptide region between the rotaviruses recovered from individuals with and without diarrhea. The NSP4 gene product being a pathogenic determinant may not be a generalized phenomenon.
Rapid-Cycle PCR Method To Detect
Bordetella pertussis
That Fulfills All Consensus Recommendations for Use of PCR in Diagnosis of Pertussis
Authors: Farrell D. J. , McKeon M. , Daggard G. , Loeffelholz M. J. , Thompson C. J. , Mukkur T. K. S. 
Published: December 2019

ABSTRACT

No standardized PCR method is available for the laboratory diagnosis of the pertussis syndrome. Consensus recommendations for the use of PCR in the diagnosis of
Bordetella pertussis
infections have been proposed, and the aim of this study was to develop a method that fulfills all of these criteria. A rapid-cycle shared-primer PCR method with a microwell format and probe hybridization detection step (POR) was developed using novel oligonucleotides targeted to the outer membrane porin gene (
Bordetella
spp.). In specimens positive for
Bordetella
spp.,
B. pertussis
was differentiated from
Bordetella parapertussis
and
Bordetella bronchiseptica
by hybridization with organism-specific oligonucleotide probes. An internal control was developed using overlap extension PCR and mouse β-actin DNA. The analytical specificity was 100%. The analytical sensitivity was comparable to that of nested IS
481
and IS
1001
PCR (∼1 organism per reaction). The clinical sensitivity and specificity were ascertained using 705 specimens (from 705 patients). The results were compared to those of a nested-PCR method targeting the insertion sequences IS
481
and IS
1001
. Fifty-one specimens were positive for
B. pertussis
by POR and IS
481
PCR. Two specimens which fulfilled a clinical definition of pertussis were positive by POR and negative by IS
481
PCR. A total of 652 specimens were negative by both methods.
B. parapertussis
was not detected in any specimens. PCR inhibition was detected in 21 out of 705 specimens (2.98%). Thus, a rapid (4 h, including specimen preparation) PCR method which fulfills all of the consensus recommendations was developed and validated for the detection of
B. pertussis
.
Identification and Phylogenetic Relationship of the Most Common Pathogenic
Candida
Species Inferred from Mitochondrial Cytochrome
b
Gene Sequences
Authors: Yokoyama Koji , Biswas Swarajit Kumar , Miyaji Makoto , Nishimura Kazuko 
Published: December 2019

ABSTRACT

We sequenced a 396-bp region of the mitochondrial cytochrome
b
gene of the most common clinically important
Candida
species:
Candida albicans
,
C. glabrata
,
C. parapsilosis
,
C. tropicalis
,
C. krusei
, and
C. lusitaniae
. The recently described species of
Candida
,
C. dubliniensis
, associated with mucosal candidiasis in human immunodeficiency virus-infected individuals, was also included. Two to five strains of each species were examined. Some species represented intraspecies variation, which was not more than 1.8% (DNA). However, interspecies variations were more than 10 and 7%, respectively, for DNA and amino acid sequences. Multiple alignments of nucleotide and deduced amino acid sequences revealed species-specific nucleotides and amino acids. Nucleotide- and amino acid-based phylogenetic trees were constructed and are discussed. Using the database, it is possible to identify presumptive
Candida
species within a working day.
Conservation of Type-Specific B-Cell Epitopes of Glycoprotein G in Clinical Herpes Simplex Virus Type 2 Isolates
Authors: Liljeqvist Jan-Åke , Svennerholm Bo , Bergström Tomas 
Published: December 2019

ABSTRACTGlycoprotein G (gG-2) of herpes simplex virus type 2 (HSV-2) is cleaved to a secreted amino-terminal portion and to a cell-associated, heavily O-glycosylated carboxy-terminal portion that constitutes the mature gG-2 (mgG-2). The mgG-2 protein is commonly used as a type-specific antigen in the serodiagnosis of HSV-2 infection. As the amino acid sequence variability of mgG-2 in clinical isolates may affect the performance of such assays, the gG-2 gene was sequenced from 15 clinical HSV-2 isolates. Few mutations were identified, and these were mostly localized outside the epitope regions described earlier. Five isolates were identical to different laboratory strains, indicating that the gG-2 gene is highly conserved over time. In the search for HSV-2 isolates harboring mutations within the immunodominant region of mgG-2, a pool of 2,400 clinical HSV-2 isolates was tested for reactivity with two anti-mgG-2 monoclonal antibodies (MAbs). Ten MAb escape HSV-2 mutants, which all harbored structurally restricted single- or dual-point mutations within the respective epitopes explaining the loss of binding, were identified. Sera from corresponding patients were reactive to mgG-2, as well as to a peptide representing the immunodominant region, suggesting that the point mutations detected did not diminish seroreactivity to mgG-2. The conservation of the gG-2 gene reported here further supports the use of mgG-2 as a type-specific antigen in the diagnosis of HSV-2 infections.
Characterization of a Coronavirus Isolated from a Diarrheic Foal
Authors: Guy James S. , Breslin Jamie J. , Breuhaus Babetta , Vivrette Sally , Smith Lynda G. 
Published: December 2019

ABSTRACT
A coronavirus was isolated from feces of a diarrheic foal and serially propagated in human rectal adenocarcinoma (HRT-18) cells. Antigenic and genomic characterizations of the virus (isolate NC99) were based on serological comparison with other avian and mammalian coronaviruses and sequence analysis of the nucleocapsid (N) protein gene. Indirect fluorescent-antibody assay procedures and virus neutralization assays demonstrated a close antigenic relationship with bovine coronavirus (BCV) and porcine hemagglutinating encephalomyelitis virus (mammalian group 2 coronaviruses). Using previously described BCV primers, the N protein gene of isolate NC99 was amplified by a reverse transcriptase PCR (RT-PCR) procedure. The RT-PCR product was cloned into pUC19 and sequenced; the complete N protein of NC99 (446 amino acids) was then compared with published N protein sequences of other avian and mammalian coronaviruses. A high degree of identity (89.0 to 90.1%) was observed between the N protein sequence of NC99 and published sequences of BCV (Mebus and F15 strains) and human coronavirus (strain OC43); only limited identity (<25%) was observed with group 1 and group 3 coronaviruses. Based on these findings, the virus has been tentatively identified as equine coronavirus (ECV). ECV NC99 was determined to have close antigenic and/or genetic relationships with mammalian group 2 coronaviruses, thus identifying it as a member of this coronavirus antigenic group.
Fluorescent Amplified-Fragment Length Polymorphism Genotyping of
Neisseria meningitidis
Identifies Clones Associated with Invasive Disease
Authors: Goulding Jonathan N. , Hookey John V. , Stanley John , Olver Will , Neal Keith R. , Ala'Aldeen Dlawer A. A. , Arnold Catherine 
Published: December 2019

ABSTRACT

Fluorescent amplified-fragment length polymorphism (FAFLP), a genotyping technique with phylogenetic significance, was applied to 123 isolates of
Neisseria meningitidis
. Nine of these were from an outbreak in a British university; 9 were from a recent outbreak in Pontypridd, Glamorgan; 15 were from sporadic cases of meningococcal disease; 26 were from the National Collection of Type Cultures; 58 were carrier isolates from Ironville, Derbyshire; 1 was a disease isolate from Ironville; and five were representatives of invasive clones of
N. meningitidis
. FAFLP analysis results were compared with previously published multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) results. FAFLP was able to identify hypervirulent, hyperendemic lineages (invasive clones) of
N. meningitidis
as well as did MLST. PFGE did not discriminate between two strains from the outbreak that were classified as similar but distinct by FAFLP. The results suggest that high resolution of
N. meningitidis
for outbreak and other epidemiological analyses is more cost efficient by FAFLP than by sequencing procedures.
PCR Detection and Evidence of Shedding of Porcine Circovirus Type 2 in Boar Semen
Authors: Larochelle Renée , Bielanski Andrzej , Müller Peter , Magar Ronald 
Published: December 2019

ABSTRACT
An experimental study was conducted to evaluate the potential presence of porcine circovirus type 2 (PCV2) in the semen of infected boars. Four mature boars were inoculated intranasally with PCV2 isolate LHVA-V53 propagated on PK15 cells. Two boars inoculated with the supernatant of noninfected PK15 cells were kept as controls. Serum samples were collected from all boars at 4, 7, 11, 13, 18, 21, 25, 28, 35, and 55 days postinoculation (dpi) and from the four PCV2-infected boars at 90 dpi. Samples were tested for the presence of antibodies to PCV2 by an indirect immunofluorescence assay and for the presence of PCV2 DNA by PCR and nested PCR. Semen samples were collected from all six boars at 5, 8, 11, 13, 18, 21, 25, 28, 33, and 47 dpi and tested for the presence of PCV2 DNA by a nested PCR assay. Antibodies to PCV2 could be detected as early as 11 dpi in one boar, and all four infected boars were found positive for PCV2 antibodies by 18 dpi. Thereafter all infected boars remained positive for antibodies to PCV2 until 90 dpi. Analysis of serum samples by nested PCR demonstrated the presence of PCV2 DNA as early as 4 dpi in three of four infected boars. Serum samples from all infected boars were positive for PCV2 DNA from 11 dpi until 35 dpi but were negative at 90 dpi. PCV2 DNA was detected as soon as 5 dpi in the semen of two infected boars and intermittently thereafter in the semen of all four infected boars. The semen of two infected boars was positive for PCV2 DNA at 47 dpi. Following infection, PCV2 DNA can be detected in semen concurrently with the presence of PCV2 DNA and antibodies in the serum. The present study suggests that PCV2 may be shed intermittently in the semen of infected boars.