bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rapid Quantification ofYersinia enterocoliticain Pork Samples by a Novel Sample Preparation Method, Flotation, Prior to Real-Time PCR
Authors: Wolffs Petra , Knutsson Rickard , Norling Börje , Rådström Peter 
Published: March 2004

ABSTRACTThe development of real-time PCR thermal cycles in the late 1990s has opened up the possibility of accurate quantification of microorganisms in clinical, environmental, and food samples. However, a lack of suitable sample preparation methods that allow rapid quantification of the nucleic acids, remove PCR inhibitors, and prevent false-positive results due to DNA originating from dead cells has limited the use of quantitative PCR. We have used for the first time a new variant of density gradient centrifugation, called flotation, as a user-friendly sample preparation method prior to PCR. This paper describes the use of this sample preparation method, without DNA purification, for direct detection and quantification ofYersinia enterocoliticain PCR-inhibitory meat juice from pork. Flotation combined with qPCR could overcome PCR interference in juice from pork, as was shown by amplification efficiencies of 1.006 ± 0.021 and 1.007 ± 0.025, which are comparable to the amplification efficiency obtained for purified DNA samples (1.005 ± 0.059). Applying flotation to meat juice samples containing natural background flora and spiked with different levels ofY. enterocoliticashowed that direct quantification ofY. enterocoliticawas possible down to a level of at least 4.2 × 103CFU per ml of meat juice, even in the presence of 106CFU of background flora per ml. Finally, the results showed that samples containing large amounts ofY. enterocoliticaDNA did not result in a positive PCR signal. This indicates that the risk of false-positive results due to detection of DNA originating from dead cells can be greatly reduced by using flotation prior to PCR.
Enteroaggregative
Escherichia coli
Virulence Factors Are Found To Be Associated with Infantile Diarrhea in Brazil
Authors: Zamboni Andresa , Fabbricotti Sandra H. , Fagundes-Neto Ulysses , Scaletsky Isabel C.A. 
Published: March 2004

ABSTRACT

We have previously shown that enteroaggregative
Escherichia coli
(EAEC) is an important pathogen among Brazilian infants. Most EAEC strains harbor a plasmid (pAA) from which a DNA fragment has been used as a probe (EAEC probe). To better understand the characteristics of EAEC in Brazil, 109 strains carrying and lacking the EAEC probe sequence were tested for the presence of pAA plasmid-borne and chromosomal factors. Common virulence factors of probe-positive and probe-negative isolates included the presence of the Pet, EAST-1, Shf, Irp2, ShET1/Pic, and Hly virulence markers. The presence of AggR or one other virulence factor (AAF/I, AAF/II, AAF/III, or Aap) was predominantly identified only in probe-positive strains. In EAEC probe-positive strains, the virulence marker Aap was found significantly more frequently (
P
= 0.023) in isolates from children with diarrhea (22%) than in isolates from controls (3%). EAST-1 and Shf were the markers most frequently detected (61%) in EAEC probe-negative strains and were found to be significantly associated with diarrhea (
P
= 0.003 and
P
= 0.020, respectively). Furthermore, our data suggest that AggR can be used as an important genetic marker for EAEC probe-positive strains.
Expanded Geographical Distribution of the N Family of
Mycobacterium tuberculosis
Strains within the United States
Authors: Milan S. Joy , Hauge Kirsten A. , Kurepina Natalia E. , Lofy Kathryn H. , Goldberg Stefan V. , Narita Masahiro , Nolan Charles M. , McElroy Peter D. , Kreiswirth Barry N. , Cangelosi Gerard A. 
Published: March 2004

ABSTRACT

The N and W-Beijing families of
Mycobacterium tuberculosis
are phylogenetically closely related. The ability of the W-Beijing family to rapidly cause widespread disease is well described; however, few outbreaks involving the N family have been reported outside the New York City, N.Y., area. During 2002 to 2003, Seattle, Wash., experienced a rapidly expanding tuberculosis outbreak involving 38 persons in a 23-month period. The outbreak strain, SBRI9, exhibited the genotypic properties of the N family. Its IS
6110
restriction fragment length polymorphism pattern was identical or nearly identical to those of two N family strains that were responsible for clusters of tuberculosis cases, including a large nosocomial outbreak, in New York City and New Jersey from 1989 to 1990. It was also identical to strains involved in late 1990s tuberculosis cases in Michigan, Maryland, and Arkansas. Further monitoring of the N family may show that it shares with the W-Beijing family the propensity to spread rapidly, suggesting that this characteristic evolved prior to the divergence of the two genetic lineages.
Rapid and Sensitive Detection ofMycobacterium aviumsubsp.paratuberculosisin Bovine Milk and Feces by a Combination of Immunomagnetic Bead Separation-Conventional PCR and Real-Time PCR
Authors: Khare Sangeeta , Ficht Thomas A. , Santos Renato L. , Romano Juan , Ficht Allison R. , Zhang Shuping , Grant Irene R. , Libal Melissa , Hunter David , Adams L. Garry 
Published: March 2004

ABSTRACTImmunomagnetic bead separation coupled with bead beating and real-time PCR was found to be a very effective procedure for the isolation, separation, and detection ofMycobacterium aviumsubsp.paratuberculosisfrom milk and/or fecal samples from cattle and American bison. Samples were spiked withM. aviumsubsp.paratuberculosisorganisms, which bound to immunomagnetic beads and were subsequently lysed by bead beating; then protein and cellular contaminants were removed by phenol-chloroform-isopropanol extraction prior to DNA precipitation. DNA purified by this sequence of procedures was then analyzed by conventional and real-time IS900-based PCR in order to detectM. aviumsubsp.paratuberculosisin feces and milk. By use of this simple and rapid technique, 10 or fewerM. aviumsubsp.paratuberculosisorganisms were consistently detected in milk (2-ml) and fecal (200-mg) samples, making this sensitive procedure very useful and cost-effective for the diagnosis of clinical and subclinical Johne's disease (paratuberculosis) compared to bacteriological culture, which is constrained by time, labor, and expense under diagnostic laboratory conditions.
Development of an Equine Herpesvirus Type 4-Specific Enzyme-Linked Immunosorbent Assay Using a B-Cell Epitope as an Antigen
Authors: Maeda Ken , Mizukoshi Fuminori , Hamano Masataka , Kai Kazushige , Iwata Hiroyuki , Kondo Takashi , Matsumura Tomio 
Published: March 2004

ABSTRACT

The equine herpesvirus type 4 (EHV-4)-specific region of glycoprotein G has served as an antigen for serodiagnosis and seroepizootic studies of EHV-4 infection (B. S. Crabb and M. J. Studdert, J. Virol.
67:
6332-6338, 1993; S. Yasunaga, K. Maeda, T. Matsumura, K. Kai, H. Iwata, and T. Inoue, J. Vet. Med. Sci.
60:
1133-137, 1998; S. Yasunaga, K. Maeda, T. Matsumura, T. Kondo, and K. Kai, J. Vet. Med. Sci.
62:
687-691, 2000). Here we identified a major B-cell epitope in the type-specific region of EHV-4 and applied it as an antigen in enzyme-linked immunosorbent assays (ELISAs). A 24-amino-acid repeat sequence expressed as a glutathione
S
-transferase fusion protein specifically reacted as well as the type-specific region with sera from foals infected with EHV-4. Five synthetic peptides (12-mer peptides) in the repeat sequence were included as ELISA antigens. The results indicated that the 12-mer peptide MKNNPIYSEGSL contained a major B-cell epitope specific for EHV-4 infection. Inclusion of this 12-mer peptide in ELISAs for an epidemiological study specifically detected EHV-4 infection in the field. These results indicated that the 12-mer epitope was responsible for the type-specific antibody response and therefore is useful for seroepizootic studies and serodiagnosis of EHV-4 infection.
Characterization of Shiga Toxin-Producing
Escherichia coli
Strains Isolated from Human Patients in Germany over a 3-Year Period
Authors: Beutin Lothar , Krause Gladys , Zimmermann Sonja , Kaulfuss Stefan , Gleier Kerstin 
Published: March 2004

ABSTRACT

We have investigated 677 Shiga toxin-producing
Escherichia coli
(STEC) strains from humans to determine their serotypes, virulence genes, and clinical signs in patients. Six different Shiga toxin types (1, 1c, 2, 2c, 2d, and 2e) were distributed in the STEC strains. Intimin (
eae
) genes were present in 62.6% of the strains and subtyped into intimins α1, β1, γ1, ε, θ, and η. Shiga toxin types 1c and 2d were present only in
eae
-negative STEC strains, and type 2 was significantly (
P
< 0.001) more frequent in
eae
-positive STEC strains. Enterohemorrhagic
E. coli
hemolysin was associated with 96.2% of the
eae
-positive strains and with 65.2% of the
eae
-negative strains. Clinical signs in the patients were abdominal pain (8.7%), nonbloody diarrhea (59.2%), bloody diarrhea (14.3%), and hemolytic-uremic syndrome (HUS) (3.5%), and 14.3% of the patients had no signs of gastrointestinal disease or HUS. Infections with
eae
-positive STEC were significantly (
P
< 0.001) more frequent in children under 6 years of age than in other age groups, whereas
eae
-negative STEC infections dominated in adults. The STEC strains were grouped into 74 O:H types by serotyping and by PCR typing of the flagellar (
fliC
) genes in 221 nonmotile STEC strains. Eleven serotypes (O157:[H7], O26:[H11], O103:H2, O91:[H14], O111:[H8], O145:[H28], O128:H2, O113:[H4], O146:H21, O118:H16, and O76:[H19]) accounted for 69% of all STEC strains. We identified 41 STEC strains belonging to 31 serotypes which had not previously been described as human STEC. Twenty-six of these were positive for intimins α1 (one serotype), β1 (eight serotypes), ε (two serotypes), and η (three serotypes). Our study indicates that different types of STEC strains predominate in infant and adult patients and that new types of STEC strains are present among human isolates.
Multiplex PCR Assay for Differentiation ofHelicobacter felis,H. bizzozeronii, andH. salomonis
Authors: Baele M. , Van den Bulck K. , Decostere A. , Vandamme P. , Hänninen M.-L. , Ducatelle R. , Haesebrouck F. 
Published: March 2004

ABSTRACTHelicobacter felis,Helicobacter bizzozeronii, andHelicobacter salomonisare frequently found in the gastric mucous membrane of dogs and cats. These large spiral organisms are phylogenetically highly related to each other. Their fastidious nature makes it difficult to cultivate them in vitro, hampering traditional identification methods. We describe here a multiplex PCR test based on the tRNA intergenic spacers and on the urease gene, combined with capillary electrophoresis, that allows discrimination of these three species. In combination with previously described 16S ribosomal DNA-based primers specific for the nonculturable “CandidatusHelicobacter suis,” our procedure was shown to be very useful in determining the species identity of “Helicobacter heilmannii”-like organisms observed in human stomachs and will facilitate research concerning their possible zoonotic importance.
Isolation and Characterization of
Borrelia hermsii
Associated with Two Foci of Tick-Borne Relapsing Fever in California
Authors: Fritz Curtis L. , Bronson Lawrence R. , Smith Charles R. , Schriefer Martin E. , Tucker James R. , Schwan Tom G. 
Published: March 2004

ABSTRACT

Relapsing fever, caused by the spirochete
Borrelia hermsii
and transmitted by the soft tick
Ornithodoros hermsi
, is endemic in many rural mountainous areas of California. Between 1996 and 1998, 12 cases of relapsing fever associated with two exposure sites in northern California were investigated. Follow-up at exposure sites included collection of soft ticks and serum specimens from sylvatic rodents. Attempts to cultivate spirochetes were made through inoculation of patient blood into mice and by feeding
Ornithodoros
ticks on mice. Three isolates of
B. hermsii
were recovered from two blood specimens and one pool of ticks. The protein and plasmid profiles of the three isolates were comparable to those of previous
B. hermsii
isolates from the western United States. Western immunoblotting of patient sera demonstrated an expanding immunologic response to antigens within four distinct molecular weight regions by 3 to 4 weeks postonset. Antibody to
B. hermsii
was detected in sera from 4 of 11 yellow-pine chipmunks (
Tamias amoenus
); no other rodent species collected were seropositive.
Rapid Serodiagnosis of Active Pulmonary
Mycobacterium tuberculosis
by Analysis of Results from Multiple Antigen-Specific Tests
Authors: Okuda Yoshinari , Maekura Ryoji , Hirotani Atsusi , Kitada Seigo , Yoshimura Kenji , Hiraga Touru , Yamamoto Yuoko , Itou Masami , Ogura Takeshi , Ogihara Toshio 
Published: March 2004

ABSTRACT
We have prospectively analyzed three antigens for serodiagnosis of tuberculosis (TB). These antigens were tuberculous glycolipid antigen, lypoarabinomannan polysaccharide antigen, and antigen 60 (A60), which was derived from purified protein derivatives. Of the 131 patients with active pulmonary TB, 57 were both smear and culture negative and 14 had chronic active pulmonary TB that remained smear positive for >12 months of chemotherapy. One hundred twenty healthy adults were controls. The percentages of patients positive in all three tests were 58.8% for smear-positive active pulmonary TB and 71.4% for chronic active pulmonary TB. When the results of the three serodiagnostic tests were evaluated in combination, the sensitivity increased to 91.5% in patients with active pulmonary TB and to 86.0% in smear- and culture-negative patients. The false-positive rate of the three-test combination was 12.5% in the healthy control groups. In conclusion, it was not possible to detect all of the antibodies against antigenic substances in the cell walls of the tuberculous bacilli in the sera of all TB patients by using available serodiagnostic tests. However, the combined use of tests with three separate antigens maximizes the effectiveness of serodiagnosis.
Optimization of Quantitative Detection of Cytomegalovirus DNA in Plasma by Real-Time PCR
Authors: Boeckh Michael , Huang MeeiLi , Ferrenberg James , Stevens-Ayers Terry , Stensland Laurence , Nichols W. Garrett , Corey Lawrence 
Published: March 2004

ABSTRACT

Previous studies have shown that detection of cytomegalovirus (CMV) DNA in plasma is less sensitive than the antigenemia assay for CMV surveillance in blood. In 1,983 blood samples, plasma PCR assays with three different primer sets (UL125 alone, UL126 alone, and UL55/UL123-exon 4) were compared to the pp65 antigenemia assay and blood cultures. Plasma PCR detected CMV more frequently in blood specimens than either the antigenemia assay or cultures, but of the three PCR assays, the double-primer assay (UL55/UL123-exon 4) performed best with regard to sensitivity, specificity, and predictive values compared to antigenemia: 122 of 151 antigenemia-positive samples were detected (sensitivity, 80.1%), and there were 122 samples that were PCR positive-antigenemia negative (specificity, 93%). Samples with discrepant results had a low viral load (median, 0.5 cells per slide; 1,150 copies per ml) and were often obtained from patients receiving antiviral therapy. CMV could be detected by other methods in 15 of 29 antigenemia positive-PCR negative samples compared to 121 of 122 PCR positive-antigenemia negative samples (
P
< 0.001). On a per-subject basis, 21 of 25 patients (antigenemia positive-PCR negative) and all 57 (PCR positive-antigenemia negative) could be confirmed at different time points during follow-up. The higher sensitivity of the double-primer assay resulted in earlier detection compared to antigenemia in a time-to-event analysis of 42 CMV-seropositive stem cell transplant recipients, and two of three patients with CMV disease who were antigenemia negative were detected by plasma PCR prior to the onset of disease. Interassay variability was low, and the dynamic range was >5 log
10
. Automated DNA extraction resulted in high reproducibility, accurate CMV quantitation (
R
= 0.87,
P
< 0.001), improved sensitivity, and increased speed of sample processing. Thus, primer optimization and improved DNA extraction techniques resulted in a plasma-based PCR assay that is significantly more sensitive than pp65 antigenemia and blood cultures for detection of CMV in blood specimens.
Translocation of
Enterococcus faecalis
Strains across a Monolayer of Polarized Human Enterocyte-Like T84 Cells
Authors: Zeng Jing , Teng Fang , Weinstock George M. , Murray Barbara E. 
Published: March 2004

ABSTRACT

We used a two-chamber system to study transcytosis of
Enterococcus faecalis
across monolayers of human colon carcinoma-derived T84 cells, which show structural resemblance to the native intestine. Among 16
E. faecalis
isolates from different sources, the well-characterized strain OG1RF and 8 other isolates (2 endocarditis isolates, 1 urine isolate, and all 5 fecal isolates) showed translocation in this assay, while 6 clinical isolates (3 endocarditis and 3 urine isolates), the recipient strain JH2-2, and the control,
Escherichia coli
DH5α, had no detectable translocation. Of two OG1RF mutants involving the previously studied
epa
(enterococcal polysaccharide antigen) gene cluster, known to be needed for virulence and resistance to killing by polymorphonuclear leukocytes, one
epa
mutant (TX5179) was unable to translocate, while TX5180, with an
epa
disruption farther downstream, showed a moderate decrease in translocation relative to that of the wild-type strain OG1RF (
P
< 0.01), indicating that the
epa
gene cluster is important for translocation across a T84 monolayer. This observation was confirmed by complementation of the
epa
mutant (TX5179) with
epa
genes and restoration of its translocation ability. In conclusion, we have demonstrated translocation of at least some strains of
E. faecalis
across T84 monolayers, although strains differ considerably in this ability, and we have demonstrated that
epa
mutations can cause marked changes in successful translocation. These results suggest that this model may be a useful in vitro system for studying the process of translocation from the intestinal tract.
Random Amplified Polymorphic DNA and Phenotyping Analysis of
Salmonella enterica
Serovar Enteritidis Isolates Collected from Humans and Poultry in Uruguay from 1995 to 2002
Authors: Betancor Laura , Schelotto Felipe , Martinez Araci , Pereira Marinela , Algorta Gabriela , Rodríguez M. Alejandra , Vignoli Rafael , Chabalgoity José A. 
Published: March 2004

ABSTRACT

Molecular and phenotyping techniques were applied to study
Salmonella enterica
serovar Enteritidis strains both from human cases of infection and of avian origin isolated in Uruguay from 1995 to 2002. A group of 62 isolates was subjected to random amplified polymorphic DNA (RAPD) assay and analysis of antibiotic resistance patterns. Twenty-one of these strains were further characterized by phage typing and analysis of their protein expression profiles. RAPD fingerprinting with five different primers discriminated 10 different genetic profiles. Of the 62 strains tested, 48 had a single major genetic profile, whereas the other nine profiles were evenly distributed among the other strains. The genetic diversity was greater among strains of animal origin than among isolates of human origin. Comparative examination of the results obtained by RAPD analysis and phenotypic analysis and by strain source provided evidence of the reliable discriminatory power of RAPD analysis in our study. Six avian isolates with antibiotic resistance were detected: two were nalidixic acid resistant and four had a particular β-lactam resistance pattern. The last four isolates all had the same unusual phage type (phage type 4b); however, RAPD analysis differentiated them into two groups. Two isolates with unique RAPD profiles were recovered from distinct human cases, suggesting that the technique differentiates unrelated strains. Overall, the results show the existence of a predominant genetic type that is present in poultry and that is transmitted to humans. There are also several other genotypes, but only a few of them could be recovered from human sources, suggesting the existence of different pathogenic traits among strains circulating in the country.
First Culture Isolation of
Borrelia lonestari
, Putative Agent of Southern Tick-Associated Rash Illness
Authors: Varela Andrea S. , Luttrell M. Page , Howerth Elizabeth W. , Moore Victor A. , Davidson William R. , Stallknecht David E. , Little Susan E. 
Published: March 2004

ABSTRACT

Southern tick-associated rash illness (STARI) is a Lyme disease-like infection described in patients in the southeastern and south-central United States, where classic Lyme disease is relatively rare. STARI develops following the bite of a lone star tick (
Amblyomma americanum
) and is thought to be caused by infection with an “uncultivable” spirochete tentatively named
Borrelia lonestari
. In this study, wild lone star ticks collected from an area where
B. lonestari
is endemic were cocultured in an established embryonic tick cell line (ISE6). The cultures were examined by dark-field microscopy for evidence of infection, and spirochete identity and morphology were evaluated by flagellin B and 16S rRNA gene sequence, by reaction to
Borrelia
-wide and
B. burgdorferi
-specific monoclonal antibodies, and by electron microscopy. Live spirochetes were first visualized in primary culture of
A. americanum
ticks by dark-field microscopy 14 days after the cell culture was inoculated. The sequences of the flagellin B and 16S rRNA genes of cultured spirochetes were consistent with previously reported sequences of
B. lonestari
. The cultured spirochetes reacted with a
Borrelia-
wide flagellin antibody, but did not react with an OspA antibody specific to
B. burgdorferi
, by indirect fluorescent antibody testing. Electron microscopy demonstrated organisms that were free and associated with ISE6 cells, with characteristic
Borrelia
sp. morphology. This study describes the first successful isolation of
B. lonestari
in culture, providing a much needed source of organisms for the development of diagnostic assays and forming a basis for future studies investigating the role of the organism as a human disease agent.