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Immunologic Research 05/2013; · 3.03 Impact Factor
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ABSTRACT: Antibodies executing catalytic activity are referred to as antibody enzymes or short "abzymes" and may have diagnostic relevance. Abzymes with deoxyribonuclease (DNase) activity have been demonstrated in patients with autoimmune and infectious diseases. Despite several reports on the occurrence of DNase abzymes in systemic autoimmune rheumatic diseases, conclusive data about DNase activity of antibodies in patients with spondyloarthritides (SpAs) are lacking. In recent cross-sectional studies evaluating levels of IgG DNase activity in patients with psoriatic arthritis (PsA), reactive arthritis (ReA), and ankylosing spondylitis (AS), DNase activity of IgG has been assessed by the rivanol clot method and confirmed by agarose gel electrophoresis. Remarkably, levels of IgG DNase activity were significantly higher in sera of SpA patients than those in control subjects. In patients with PsA, ReA, and AS, a positive correlation of DNase IgG activity with synovitis, disease activity, and stage of spondylitis was observed, respectively. Given the involvement of autoimmune reactions in cytolysis and connective tissue degradation in PsA, ReA, and to a lesser extent in AS, abzymes might have an impact on the pathophysiology of SpAs. Detection of IgG DNase activity in patients suffering from SpA represents an exciting new research field and may assist in the differential diagnosis of SpA.
Immunologic Research 04/2013; · 3.03 Impact Factor
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Peter Schierack,
Stefan Rödiger,
Christoph Kuhl,
Rico Hiemann,
Dirk Roggenbuck,
Ganwu Li,
Jörg Weinreich,
Enrico Berger,
Lisa K Nolan,
Bryon Nicholson,
Antje Römer,
Ulrike Frömmel,
Lothar H Wieler,
Christian Schröder
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ABSTRACT: textlessptextgreaterWe established an automated screening method to characterize adhesion of textlessitalictextgreaterEscherichia colitextless/italictextgreater to intestinal porcine epithelial cells (IPEC-J2) and their probiotic activity against infection by enteropathogenic textlessitalictextgreaterE. colitextless/italictextgreater (EPEC). 104 intestinal textlessitalictextgreaterE. colitextless/italictextgreater isolates from domestic pigs were tested by PCR for the occurrence of virulence-associated genes, genes coding for resistances to antimicrobial agents and metals, and for phylogenetic origin by PCR. Adhesion rates and probiotic activity were examined for correlation with the presence of these genes. Finally, data were compared with those from 93 textlessitalictextgreaterE. colitextless/italictextgreater isolates from wild boars.textless/ptextgreatertextlessptextgreaterIsolates from domestic pigs carried a broad variety of all tested genes and showed great diversity in gene patterns. Adhesions varied with a maximum of 18.3 or 24.2 mean bacteria adherence per epithelial cell after 2 or 6 hours respectively. Most isolates from domestic pigs and wild boars showed low adherence, with no correlation between adhesion/probiotic activity and textlessitalictextgreaterE. colitextless/italictextgreater genes or gene clusters. The gene textlessitalictextgreatersfa/foctextless/italictextgreater, encoding for a subunit of F1C fimbriae did show a positive correlative association with adherence and probiotic activity; however textlessitalictextgreaterE. colitextless/italictextgreater isolates from wild boars with the textlessitalictextgreatersfa/foctextless/italictextgreater gene showed less adhesion and probiotic activity than textlessitalictextgreaterE. colitextless/italictextgreater with the textlessitalictextgreatersfa/foctextless/italictextgreater gene isolated from domestic pigs after 6 hour incubation.textless/ptextgreatertextlessptextgreaterIn conclusion, screening porcine textlessitalictextgreaterE. colitextless/italictextgreater for virulence associated genes genes, adhesion to intestinal epithelial cells, and probiotic activity revealed a single important adhesion factor, several probiotic candidates, and showed important differences between textlessitalictextgreaterE. colitextless/italictextgreater of domestic pigs and wild boars.textless/ptextgreatertextless/sectextgreater
PLoS ONE 04/2013; 8(4):e59242. · 4.09 Impact Factor
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ABSTRACT: [Article in German] PCR is a simplistic and robust laboratory technology for nucleic acid detection. However, for research and diagnostics processing multiple tar- gets within one reaction in an automatic fashion is a demanded feature. Combining two multiplex read out technologies, such as microarray and microbeads, the VideoScan platform was designed. This microscope imaging technology enables an automatable high throughput multiplex measurement of genetic material from biological and patient samples.
http://biospektrum.de/sixcms/media.php/1093/12268-013-0287-z.pdf
BioSpektrum 03/2013; 19(2):153-156.
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ABSTRACT: The pathogenesis of Crohn's disease (CrD) and ulcerative colitis (UC), the two major inflammatory bowel diseases (IBD), remains poorly understood. Autoimmunity is considered to be involved in the triggering and perpetuation of inflammatory processes leading to overt disease. Approximately 30% of CrD patients and less than 8% of UC patients show evidence of humoral autoimmunity to exocrine pancreas, detected by indirect immunofluorescence. Pancreatic autoantibodies (PAB) were described for the first time in 1984, but the autoantigenic target(s) of PABs were identified only in 2009. Utilizing immunoblotting and matrix-assisted laser desorption ionization time-of-flight mass spectrometry, the major zymogen granule membrane glycoprotein 2 (GP2) has been discovered as the main PAB autoantigen. The expression of GP2 has been demonstrated at the site of intestinal inflammation, explaining the previously unaddressed contradiction of pancreatic autoimmunity and intestinal inflammation. Recent data demonstrate GP2 to be a specific receptor on microfold (M) cells of intestinal Peyer's patches, which are considered to be the original site of inflammation in CrD. Novel ELISAs, employing recombinant GP2 as the solid phase antigen, have confirmed the presence of IgA and IgG anti-GP2 PABs in CrD patients and revealed an association of anti-GP2 IgA as well as IgG levels with a specific clinical phenotype in CrD. Also, GP2 plays an important role in modulating innate and acquired intestinal immunity. Its urinary homologue, Tamm-Horsfall protein or uromodulin, has a similar effect in the urinary tract, further indicating that GP2 is not just an epiphenomenon of intestinal destruction. This review discusses the role of anti-GP2 autoantibodies as novel CrD-specific markers, the quantification of which provides the basis for further stratification of IBD patients. Given the association with a disease phenotype and the immunomodulating properties of GP2 itself, an important role for GP2 in the immunopathogenesis of IBD cannot be excluded.
Advances in clinical chemistry 01/2013; 60:187-208. · 3.20 Impact Factor
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ABSTRACT: To test the hypothesis that Escherichia coli populations have adapted to conventional pig production practices, we comparatively tested intestinal commensal E. coli from wild boars versus isolates from domestic pigs by analyzing virulence-associated factors, adhesion, and metabolic activities. Virulence-associated genes typical for intestinal pathogenic E. coli (inVAGs) were sporadically detected among E. coli from wild boars except the adhesion-related gene paa and the enterotoxin-encoding gene astA. In contrast, several VAGs typical for extraintestinal pathogenic E. coli (exVAGs) were common in E. coli from wild boars. The exVAG chuA occurred more often in E. coli from wild boars compared to E. coli from domestic pigs. 23.5% of E. coli from wild boars belonged to EcoR group B2 which is higher than observed for E. coli from clinically healthy domestic pigs. Furthermore, E. coli from wild boars were more efficient in fermentation of carbohydrate sources (dulcitol, inositol, d-sucrose, d-tagatose), and adhered better to the intestinal porcine epithelial cell line IPEC-J2. In conclusion, our findings point towards an adaptation of porcine intestinal E. coli to a specific intestinal milieu caused by different animal living conditions.
Veterinary Microbiology 07/2012; · 3.33 Impact Factor
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Stefan Rödiger, Peter Schierack,
Alexander Böhm,
Jörg Nitschke,
Ingo Berger,
Ulrike Frömmel,
Carsten Schmidt,
Mirko Ruhland,
Ingolf Schimke,
Dirk Roggenbuck,
Werner Lehmann,
Christian Schröder
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ABSTRACT: The analysis of different biomolecules is of prime importance for life science research and medical diagnostics. Due to the discovery of new molecules and new emerging bioanalytical problems, there is an ongoing demand for a technology platform that provides a broad range of assays with a user-friendly flexibility and rapid adaptability to new applications. Here we describe a highly versatile microscopy platform, VideoScan, for the rapid and simultaneous analysis of various assay formats based on fluorescence microscopic detection. The technological design is equally suitable for assays in solution, microbead-based assays and cell pattern recognition. The multiplex real-time capability for tracking of changes under dynamic heating conditions makes it a useful tool for PCR applications and nucleic acid hybridization, enabling kinetic data acquisition impossible to obtain by other technologies using endpoint detection. The paper discusses the technological principle of the platform regarding data acquisition and processing. Microbead-based and solution applications for the detection of diverse biomolecules, including antigens, antibodies, peptides, oligonucleotides and amplicons in small reaction volumes, are presented together with a high-content detection of autoimmune antibodies using a HEp-2 cell assay. Its adaptiveness and versatility gives VideoScan a competitive edge over other bioanalytical technologies.
Advances in biochemical engineering/biotechnology 03/2012; · 1.64 Impact Factor
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Annika Willitzki,
Rico Hiemann,
Vanessa Peters,
Ulrich Sack, Peter Schierack,
Stefan Rödiger,
Ursula Anderer,
Karsten Conrad,
Dimitrios P Bogdanos,
Dirk Reinhold,
Dirk Roggenbuck
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ABSTRACT: Antibody assessment is an essential part in the serological diagnosis of autoimmune diseases. However, different diagnostic strategies have been proposed for the work up of sera in particular from patients with systemic autoimmune rheumatic disease (SARD). In general, screening for SARD-associated antibodies by indirect immunofluorescence (IIF) is followed by confirmatory testing covering different assay techniques. Due to lacking automation, standardization, modern data management, and human bias in IIF screening, this two-stage approach has recently been challenged by multiplex techniques particularly in laboratories with high workload. However, detection of antinuclear antibodies by IIF is still recommended to be the gold standard method for antibody screening in sera from patients with suspected SARD. To address the limitations of IIF and to meet the demand for cost-efficient autoantibody screening, automated IIF methods employing novel pattern recognition algorithms for image analysis have been introduced recently. In this respect, the AKLIDES technology has been the first commercially available platform for automated interpretation of cell-based IIF testing and provides multiplexing by addressable microbead immunoassays for confirmatory testing. This paper gives an overview of recently published studies demonstrating the advantages of this new technology for SARD serology.
Clinical and Developmental Immunology 01/2012; 2012:284740. · 1.84 Impact Factor
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ABSTRACT: Testing 1,666 fecal or intestinal samples from healthy and diarrheic pigs, we obtained hemolytic Escherichia coli isolates from 593 samples. Focusing on hemolytic E. coli isolates without virulence-associated genes (VAGs) typical for enteropathogens, we found that such isolates carried a broad variety of VAGs typical for extraintestinal pathogenic E. coli.
Applied and environmental microbiology 09/2011; 77(23):8451-5. · 3.69 Impact Factor
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ABSTRACT: Microbead-based assays have evolved into powerful tools for the multiplex detection of biomolecules. Analytes are captured by DNA or protein capture molecules which are coupled on microbead surfaces. A homogeneous carboxylation of microbeads is essential for the optimal and reproducible coupling of capture molecules and thus a prerequisite for an optimal multiplex microbead-based assay performance. We developed a simple fluorescence dye adsorption assay for the description of microbead carboxylation and for the prediction of coupling successes of capture molecules. Using the fluorescence dye SYTO-62 it is possible to quantify the degree of carboxylation of poly(methyl methacrylate) (PMMA) microbeads within 1 h in a multiplex format by fluorescence microscopy or flow cytometry. Compared to conventional bulk assays which only provide an average degree of carboxylation the main advantage of the SYTO-62 assay is the single microbead analysis and therefore the description of the qualitative distribution of carboxylation in microbead populations. The SYTO-62 assay is sensitive enough to even determine weak carboxylation. Also, the quality of microbeads can be evaluated. To our knowledge this is the first report which applies a reversible noncovalent fluorescent dye adsorption assay to quantify the degree of carboxylation on surfaces.
Analytical Chemistry 03/2011; 83(9):3379-85. · 5.86 Impact Factor
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ABSTRACT: Im Zeitalter der sequenzierten Genome ist der spezifische Nachweis von DNA und RNA Sequenzen eine zentrale Aufgabe molekularer Diagnostik. Die qualitative und quantitative Analyse von Nukleinsäuresequenzen findet unter anderem Anwendung in der Krankheitserregerdetektion, der medizinischen Diagnostik, dem Wirkstoff-Screening in der Pharmazie und im Bereich der Systembiologie. Für den Nachweis von Nukleinsäureanalyten existieren grundlegend zwei Ansätze. Zum einen ist es möglich Methoden zu verwenden, die nicht auf einer Analytvervielfältigung beruhen, sondern den Analyt direkt detektieren. Allerdings erfordern solche Verfahren eine Signalverstärkung. Hier ist die Hybridisierung markierter Nachweissonden an die Zielsequenz zu nennen. Zum anderen können spezifisch Analyten in einem Reaktionsraum amplifiziert und nachgewiesen werden. Die Amplifikation erfolgt in der Regel enzymatisch, sodass eine indirekte Quantifizierung des Analyten möglich ist. Als Erweiterung solcher Einzelparametermessungen sind Multiparameteranalysen zu betrachten, die simultan mehrere unabhängige Analyten in einem Reaktionsraum sowohl qualitativ als auch quantitativ erfassen. Gegenüber Einzelmessungen bieten Multiparameteranalysen eine höhere Wirtschaftlichkeit, eine Steigerung der Bearbeitungsgeschwindigkeit, eine Reduktion des notwendigen Probenvolumens und eine Steigerung der Vergleichbarkeit der ermittelten Parameter untereinander. Der modernen Multiparameteranalytik stehen diverse Amplifikationsmethoden zur Verfügung. Eine zentrale Rolle nimmt die PCR mit ihren zahlreichen Variationen ein. Daneben haben sich weitere Technologien etabliert, die sich grundsätzlich von der konventionellen PCR bezüglich Reaktionsprinzip und Reaktionsbedingungen unterscheiden. Eine vergleichende Betrachtung der aktuellen Methoden ist Gegenstand dieser Abhandlung.
03/2011: pages 133-146; , ISBN: 978-3-89967-703-4
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ABSTRACT: Advances in immunofluorescence assay development paved the way for the simultaneous detection of several antibodies in one sample, for the serological diagnosis of systemic rheumatic diseases. Standardized automated screening of such antibodies can be achieved by HEp-2 cell-based indirect immunofluorescence (IIF) using a multicolor fluorescence imaging technical platform. To create a common platform for both screening and specific antibody assessment, multiplex measurement of antibodies using fluorescence-coded immobilized microbeads was employed on the same platform. The multicolor fluorescence detection system VideoScan (AKLIDES®) was used for the fluorescence analysis of a multiplex microbead-based immunoassay (MIA). First, immunoglobulin G (IgG) was covalently coupled to one microbead population in duplicate and in three independent experiments. The coupled IgG was detected by a Cy™5-conjugated secondary antibody. Thus, intra- and interassay coefficients of variation (CV) were obtained. Second, a multiplex determination of antinuclear autoantibodies (ANA) to Scl-70, Sm, dsDNA, SS-A (Ro60), CENP-B, and La/SS-B by solid-phase MIA was investigated, using 72 sera from patients with autoimmune diseases such as systemic lupus erythematosus and systemic sclerosis (SS). The reproducibility study revealed intra-assay CVs ranging from 3.2% to 9.9%, and interassay CVs ranging from 9.6% to 14.7%. The detection of Scl-70-, Sm-, CENP-B-, and La/SS-B-ANA with MIA showed very good agreement with the ELISA results (kappa = 1.0). The resulting relative sensitivities and specificities for Scl-70-, Sm-, CENP-B-, dsDNA-, and La/SS-B-ANA were 100%, respectively, with the exception of dsDNA (specificity 97%). Multiplex detection by immobilized fluorescence-coded microbeads using multicolor fluorescence is a reliable method for the assessment of rheumatic-disease-specific antibodies. Multicolor fluorescence analyses with pattern detection algorithms provide a common platform technique for both the screening of ANA by cell-based IIF and specific antibody assessment by multiplex detection.
Cytometry Part A 02/2011; 79(2):118-25. · 3.73 Impact Factor
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ABSTRACT: The probiotic Escherichia coli strain Nissle 1917 (EcN) has been shown to interfere in a human in vitro model with the invasion of several bacterial pathogens into epithelial cells, but the underlying molecular mechanisms are not known.
In this study, we investigated the inhibitory effects of EcN on Salmonella Typhimurium invasion of porcine intestinal epithelial cells, focusing on EcN effects on the various stages of Salmonella infection including intracellular and extracellular Salmonella growth rates, virulence gene regulation, and adhesion. We show that EcN affects the initial Salmonella invasion steps by modulating Salmonella virulence gene regulation and Salmonella SiiE-mediated adhesion, but not extra- and intracellular Salmonella growth. However, the inhibitory activity of EcN against Salmonella invasion always correlated with EcN adhesion capacities. EcN mutants defective in the expression of F1C fimbriae and flagellae were less adherent and less inhibitory toward Salmonella invasion. Another E. coli strain expressing F1C fimbriae was also adherent to IPEC-J2 cells, and was similarly inhibitory against Salmonella invasion like EcN.
We propose that EcN affects Salmonella adhesion through secretory components. This mechanism appears to be common to many E. coli strains, with strong adherence being a prerequisite for an effective reduction of SiiE-mediated Salmonella adhesion.
PLoS ONE 01/2011; 6(2):e14712. · 4.09 Impact Factor
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01/2011; , ISBN: 978-3-89967-703-4
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ABSTRACT: Mallard ducks may pose a so far underestimated risk to human and animal health by transmitting pathogenic Escherichia coli via their faecal deposits to various environmental sources. We processed Mallard duck faecal samples for E. coli and unique clones, as defined by pulsed-field gel electrophoresis (PFGE), were subsequently investigated for their virulence genotype and phylogenetic background. Multilocus sequence typing and in vivo experiments were performed for selected clones. Of 60 clones identified among 142 E. coli isolated from 175 samples, 15 (25%) were recovered from multiple individuals (up to 23 per clone). None of the clones harboured stx1 and stx2 genes and other intestinal pathogenic E. coli virulence-associated genes were only occasionally identified in the collection. In contrast, the clones possessed considerable numbers of VAGs (up to 30) linked with extraintestinal pathogenic E. coli (ExPEC). Their pathogenic potential was confirmed in chicken infection experiments. Moreover, selected clones were assigned to sequence types (STs) being most prominent for human ExPEC strains, including ST95 and ST73. One clone exhibited a multi-resistant phenotype against several antibiotics including beta-lactams, tetracyclines and sulfonamides. Mallard ducks have therefore to be considered as an important reservoir for zoonotic E. coli strains, thus serving as a substantial non-point source especially of strains capable of causing extraintestinal diseases.
Environmental Microbiology Reports 12/2009; 1(6):510-7. · 3.23 Impact Factor
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ABSTRACT: Although antimicrobial resistance and persistence of resistant bacteria in humans and animals are major health concerns worldwide, the impact of antimicrobial resistance on bacterial intestinal colonization in healthy domestic animals has only been rarely studied. We carried out a retrospective analysis of the antimicrobial susceptibility status and the presence of resistance genes in intestinal commensal E. coli clones from clinically healthy pigs from one production unit with particular focus on effects of pheno- and/or genotypic resistance on different nominal and numerical intestinal colonization parameters. In addition, we compared the occurrence of antimicrobial resistance phenotypes and genotypes with the occurrence of virulence associated genes typical for extraintestinal pathogenic E. coli.
In general, up to 72.1% of all E. coli clones were resistant to ampicillin, chloramphenicol, kanamycin, streptomycin, sulfamethoxazole or tetracycline with a variety of different resistance genes involved. There was no significant correlation between one of the nominal or numerical colonization parameters and the absence or presence of antimicrobial resistance properties or resistance genes. However, there were several statistically significant associations between the occurrence of single resistance genes and single virulence associated genes.
The demonstrated resistance to the tested antibiotics might not play a dominant role for an intestinal colonization success in pigs in the absence of antimicrobial drugs, or cross-selection of other colonization factors e.g. virulence associated genes might compensate "the cost of antibiotic resistance". Nevertheless, resistant strains are not outcompeted by susceptible bacteria in the porcine intestine.
Gut Pathogens 10/2009; 1(1):18. · 2.11 Impact Factor
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ABSTRACT: Salmonella infection might affect other intestinal Enterobacteriaceae populations and possible correlations between single Enterobacteriaceae populations would help to predict subclinical Salmonella infections in pigs. In one experimental setup, weaned piglets (n=40) were infected with Salmonella and sacrificed at 3h, 24h, 72 h or 28 days post-infection (p.i.). Dilutions of intestinal contents and mucosal tissues were plated on agar plates for determinations of Enterobacteriaceae. In another experimental setup, weaned piglets (n=12) were infected with Salmonella and probed over a period of 28 days p.i., and dilutions of rectal contents were also tested for Enterobacteriaceae populations. The occurrence of single Enterobacteriaceae populations was correlated with the occurrence of other tested Enterobacteriaceae populations as well as with clinical parameters of the piglets. Salmonella (infection strain), Escherichia coli (hemolytic and non-hemolytic) and another six non-E. coli/non-Salmonella Enterobacteriaceae (NENSE) genera with eight species were identified. In general, the absolute numbers of E. coli, Salmonella and NENSE populations decreased with increasing age of the animals. In the jejunum, the numbers of NENSE, E. coli and Salmonella were all highly positively correlated with each other. The occurrence of hemolytic E. coli had no obvious effects on the occurrence of other Enterobacteriaceae. Furthermore, only few associations of Enterobacteriaceae populations with clinical parameters were observed. In conclusion, we did not observe evidences for either a competition between or benefits for specific Enterobacteriaceae populations during Salmonella infection indicating that changes in the composition of the intestinal Enterobacteriaceae microflora are not useful indicators of subclinical Salmonella infections.
Veterinary Microbiology 10/2009; 142(3-4):352-60. · 3.33 Impact Factor
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Environmental Microbiology Reports. 09/2009; DOI: 10.1111/j.1758-2229.2009.00058.x.
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ABSTRACT: Our understanding of the composition of Escherichia coli populations in wild boars is very limited. In order to obtain insight into the E. coli microflora of wild boars, we studied E. coli isolates from the jejunums, ileums, and colons of 21 wild boars hunted in five geographic locations in Germany. Ten isolates per section were subjected to clonal determination using pulsed-field gel electrophoresis. One representative isolate per clone was further investigated for virulence traits, phylogenetic affiliation, and antimicrobial susceptibility. Macrorestriction analysis of 620 isolates revealed a range of clone diversity among the sections and animals, with up to 9 and 16 different clones per section and animal, respectively. Most of the clones for a given animal were shared between two adjacent intestinal sections. The overall highest clonal diversity was observed within the colon. While the astA gene was present in a large number of clones, other virulence genes and hemolytic ability were detected only sporadically. Clones of all four ECOR groups dominated the intestinal sections. Phylogenetic analysis and the occurrence of virulence genes correlated with the isolation frequencies for clones. All E. coli clones from wild boars were susceptible to all antimicrobial agents tested. In conclusion, though several parameters (including an animal-specific and highly diverse E. coli clone composition, the simultaneous occurrence of single clones in two adjacent intestinal sections of a given animal, and a higher E. coli diversity in the large intestine than in the small intestine) of E. coli populations of wild boars were similar to those of previously described E. coli populations of conventionally reared domestic pigs, our data also indicate possible differences, as seen for the E. coli diversity in the large intestine, the occurrence of certain virulence genes and phylogenetic groups, and antimicrobial susceptibilities.
Applied and environmental microbiology 01/2009; 75(3):695-702. · 3.69 Impact Factor
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ABSTRACT: Infections caused by Trichinella species occur throughout the world in many wild and domestic animals resulting in trichinellosis in men. In Europe, domestic pigs are predominantly infected by three Trichinella species: T. spiralis, T. britovi and T. pseudospiralis. Present methods for detection of Trichinella spp. (compressorium method, artificial digestion) do not always sufficiently recognize Trichinella larvae and these techniques are labor-intensive, time consuming and do not differentiate isolates on the species level since there are no distinguishing morphological features. Additionally, conventional PCRs cannot quantify numbers of larvae in infectious material. In order to better meet these requirements, we developed a real-time PCR assay for the accurate, rapid and specific identification of the three common European species of the genus Trichinella. The assay targets the large subunit of the mitochondrial rRNA (rrnL) and enables sensitive determination and discrimination of larvae in muscle tissue samples. The real-time PCR assay was developed and validated using reference and field strains from T. spiralis, T. britovi and T. pseudospiralis. In the described real-time PCR assay, the melting points of specific amplificates were always discernable via the melting curve from melting points of unspecific amplificates. This is important for the methods workflow because only C(T) values connected with the additional melting curve analysis allow a distinction of the individual species with confidence. The sensitivity of the technique enabled detection down to 0.1 Trichinella larva per gram meat sample. High disruption levels of tissues by mincing generally resulted in higher sensitivities than protocols without mincing. With its short completion time as well as accurate and specific detection of selected species this assay could become a convenient tool for the fast detection of Trichinella larvae in meat.
Journal of Microbiological Methods 10/2008; 75(2):287-92. · 2.09 Impact Factor
