Peter Schierack

Characterization of a porcine intestinal epithelial cell line for in vitro studies of microbial pathogenesis in swine

In vitro studies on the pathogenesis in swine have been hampered by the lack of relevant porcine cell lines. Since many bacterial infections are swine-specific, studies on pathogenic mechanisms require appropriate cell lines of porcine origin. We have characterized the permanent porcine intestinal epithelial cell line, IPEC-J2, using a variety of methods in order to assess the usefulness of this cell line as an in vitro infection model. Electron microscopic analyses and histochemical staining revealed the cells to be enterocyte-like with microvilli, tight junctions and glycocalyx-bound mucin. The functional integrity of monolayers was determined by transepithelial electrical resistance (TEER) measurements. Both commensal bacteria and important bacterial pathogens were chosen for study based on their principally different infection mechanisms: obligate extracellular Escherichia coli, facultative intracellular Salmonella and obligate intracellular Chlamydia. We determined the colonization and proliferation of the bacteria on and within the host cells and monitored the host cell response. We verified the expression of mRNAs encoding the cytokines IL-1α, −6, −7, −8, −18, TNF-α and GM-CSF, but not TGF-β or MCP-1. IL-8 protein expression was enhanced by Salmonella invasion. We conclude that the IPEC-J2 cell line provides a relevant in vitro model system for porcine intestinal pathogen–host cell interactions.

Parasite-Specific Immunomodulatory Functions of Filarial Cystatin

ABSTRACT Cystatins of parasitic nematodes are well-described pathogenicity factors which contribute to downregulation of T-cell proliferation of their hosts and induce anti-inflammatory cytokine responses. We compared the immunomodulatory effects of two cystatins of the filarial nematodes Onchocerca volvulus and Acanthocheilonema viteae with two homologous proteins of the free-living nematode Caenorhabditis elegans. Like filarial cystatins, the C. elegans cystatins (rCysele1 and rCysele2) possessed domains relevant for inhibition of papain-like proteases and were biologically active inhibitors of human cathepsins B, L, and S. However, the inhibition of cathepsin B by C. elegans cystatin was much stronger. C. elegans cystatins lacked a domain involved in inhibition of legumain-like proteases that was present in O. volvulus cystatin. Filarial cystatins suppressed the proliferation of human peripheral blood mononuclear cells (PBMC) and murine spleen cells, while the C. elegans cystatins had this effect to a much lesser extent. Whereas filarial cystatins markedly increased the production of interleukin (IL)-10, C. elegans cystatins increased the production of IL-12 and gamma interferon (IFN-γ) by human PBMC. The cystatins of both the filariae and C. elegans induced an upregulation of inducible nitric oxide by IFN-γ-stimulated murine macrophages. These data suggest that filarial cystatins but not the C. elegans cystatins downregulate proliferative responses of host cells due to characteristics which might reflect an adaptation of filariae to their parasitic life style.

Shiga Toxin 2e-Producing Escherichia coli Isolates from Humans and Pigs Differ in Their Virulence Profiles and Interactions with Intestinal Epithelial Cells

ABSTRACT Thirteen Escherichia coli strains harboring stx2e were isolated from 11,056 human stools. This frequency corresponded to the presence of the stx2e allele in 1.7% of all Shiga toxin-producing E. coli (STEC) strains. The strains harboring stx2e were associated with mild diarrhea (n = 9) or asymptomatic infections (n= 4). Because STEC isolates possessing stx2e are porcine pathogens, we compared the human STEC isolates with stx2e-harboring E. coli isolated from piglets with edema disease and postweaning diarrhea. All pig isolates possessed the gene encoding the F18 adhesin, and the majority possessed adhesin involved in diffuse adherence; these adhesins were absent from all the human STEC isolates. In contrast, the high-pathogenicity island encoding an iron uptake system was found only in human isolates. Host-specific patterns of interaction with intestinal epithelial cells were observed. All human isolates adhered to human intestinal epithelial cell lines T84 and HCT-8 but not to pig intestinal epithelial cell line IPEC-J2. In contrast, the pig isolates completely lysed human epithelial cells but not IPEC-J2 cells, to which most of them adhered. Our data demonstrate that E. coli isolates producing Shiga toxin 2e have imported specific virulence and fitness determinants which allow them to adapt to the specific hosts in which they cause various forms of disease.

Virulence factor gene profiles of Escherichia coli isolates from clinically healthy pigs.

ABSTRACT Nonpathogenic, intestinal Escherichia coli (commensal E. coli) supports the physiological intestinal balance of the host, whereas pathogenic E. coli with typical virulence factor gene profiles can cause severe outbreaks of diarrhea. In many reports, E. coli isolates from diarrheic animals were classified as putative pathogens. Here we describe a broad variety of virulence gene-positive E. coli isolates from swine with no clinical signs of intestinal disease. The isolation of E. coli from 34 pigs from the same population and the testing of 331 isolates for genes encoding heat-stable enterotoxins I and II, heat-labile enterotoxin I, Shiga toxin 2e, and F4, F5, F6, F18, and F41 fimbriae revealed that 68.6% of the isolates were positive for at least one virulence gene, with a total of 24 different virulence factor gene profiles, implying high rates of horizontal gene transfer in this E. coli population. Additionally, we traced the occurrence of hemolytic E. coli over a period of 1 year in this same pig population. Hemolytic isolates were differentiated into seven clones; only three were found to harbor virulence genes. Hemolytic E. coli isolates without virulence genes or with only the fedA gene were found to be nontypeable by slide agglutination tests with OK antisera intended for screening live cultures against common pathogenic E. coli serogroups. The results appear to indicate that virulence gene-carrying E. coli strains are a normal part of intestinal bacterial populations and that high numbers of E. coli cells harboring virulence genes and/or with hemolytic activity do not necessarily correlate with disease.

The broader context of antibiotic resistance: Zinc feed supplementation of piglets increases the proportion of multi-resistant Escherichia coli in vivo

Following the Europe-wide ban of antimicrobial growth promoters, feed supplementation with zinc has increased in livestock breeding. In addition to possible beneficial effects on animal health, feed supplementation with heavy metals is known to influence the gut microbiota and might promote the spread of antimicrobial resistance via co-selection or other mechanisms. As Escherichia coli is among the most important pathogens in pig production and often displays multi-resistant phenotypes, we set out to investigate the influence of zinc feed additives on the composition of the E. coli populations in vivo focusing on phylogenetic diversity and antimicrobial resistance. In a piglet feeding trial, E. coli were isolated from ileum and colon digesta of high dose zinc-supplemented (2500ppm) and background dose (50ppm) piglets (control group). The E. coli population was characterized via pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) for the determination of the phylogenetic background. Phenotypic resistance screening via agar disk diffusion and minimum inhibitory concentration testing was followed by detection of resistance genes for selected clones. We observed a higher diversity of E. coli clones in animals supplemented with zinc compared to the background control group. The proportion of multi-resistant E. coli was significantly increased in the zinc group compared to the control group (18.6% vs. 0%). For several subclones present both in the feeding and the control group we detected up to three additional phenotypic and genotypic resistances in the subclones from the zinc feeding group. Characterization of these subclones suggests an increase in antimicrobial resistance due to influences on plasmid uptake by zinc supplementation, questioning the reasonability of zinc feed additives as a result of the ban of antimicrobial growth promoters.

New Platform Technology for Comprehensive Serological Diagnostics of Autoimmune Diseases

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.

Isolation and characterization of intestinal Escherichia coli clones from wild boars in Germany

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.

ExPEC-typical virulence associated genes correlate with successful colonization by intestinal E. coli in a small piglet group

Upon studying the transmission of Escherichia coli from a sow to five of her piglets, we observed domination of the coliform flora in piglets by a single E. coli clone, especially after weaning. This haemolytic cloneH1 did not harbour any virulence determinants typical for intestinal pathogenic E. coli isolates from swine but had a virulence gene profile very similar to extraintestinal E. coli (ExPEC), including genes coding for P fimbriae and several iron acquisition systems, besides having an affiliation to the phylogenetic B2 group. Overall, we show that the presence of higher numbers of ExPEC-typical virulence-associated genes (VAGs) in clones correlate with their successful colonization ability in piglets. We conclude that VAGs typical for ExPEC also support intestinal colonization in healthy pigs. Faeces of healthy domestic pigs can harbour high numbers of ExPEC-similar E. coli and are suggested to be a potential risk for the transmission of such bacteria to other hosts.

A Highly Versatile Microscope Imaging Technology Platform for the Multiplex Real-Time Detection of Biomolecules and Autoimmune Antibodies

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.

Detection and distribution of probiotic Escherichia coli Nissle 1917 clones in swine herds in Germany

Aims:  To verify the presence of Escherichia coli Nissle 1917 as a natural isolate in swine and to characterize in vitro probiotic properties as well as in vivo persistence in a feeding experiment.