Leseförderung im Vorschulalter - Möglichkeiten im Rahmen der Büchereiarbeit des Sankt Michaelsbundes

Der Sankt Michaelsbund betreut im Rahmen seiner Arbeit eine Vielzahl von kirchlichen Bibliotheken in ganz Bayern. In einer Zeit, in der das Thema Leseförderung in aller Munde ist, beschäftigt sich diese Diplomarbeit mit der Frage, welche Faktoren Einfluß auf das Lesenlernen haben, wie diese durch Förderung optimiert und konkrete Aktionen durchgeführt werden können. Das Hauptaugenmerk liegt dabei jeweils auf dem Vorschulalter. Anschauliche Beispiele aus der Praxis berücksichtigen die Möglichkeiten der Kirchlichen Öffentlichen Bibliotheken, die – oftmals ehrenamtlich geleitet – durch den Sankt Michaelsbund fachlich und methodisch angeleitet werden. Abschließend folgen Vorschläge zur konkreten Anleitung und Unterstützung bei der Durchführung von Veranstaltungen zur Leseförderung sowie Verbesserungsvorschläge der derzeitigen Aktivitäten des Sankt Michaelsbundes im Hinblick auf Leseförderung

Pentaplexed quantitative real-time PCR assay for the simultaneous detection and quantification of botulinum neurotoxin-producing clostridia in food and clinical samples

Botulinum neurotoxins are produced by the anaerobic bacterium Clostridium botulinum and are divided into seven distinct serotypes (A to G) known to cause botulism in animals and humans. In this study, a multiplexed quantitative real-time PCR assay for the simultaneous detection of the human pathogenic C. botulinum serotypes A, B, E, and F was developed. Based on the TaqMan chemistry, we used five individual primer-probe sets within one PCR, combining both minor groove binder- and locked nucleic acid-containing probes. Each hydrolysis probe was individually labeled with distinguishable fluorochromes, thus enabling discrimination between the serotypes A, B, E, and F. To avoid false-negative results, we designed an internal amplification control, which was simultaneously amplified with the four target genes, thus yielding a pentaplexed PCR approach with 95% detection probabilities between 7 and 287 genome equivalents per PCR. In addition, we developed six individual singleplex real-time PCR assays based on the TaqMan chemistry for the detection of the C. botulinum serotypes A, B, C, D, E, and F. Upon analysis of 42 C. botulinum and 57 non-C. botulinum strains, the singleplex and multiplex PCR assays showed an excellent specificity. Using spiked food samples we were able to detect between 10(3) and 10(5) CFU/ml, respectively. Furthermore, we were able to detect C. botulinum in samples from several cases of botulism in Germany. Overall, the pentaplexed assay showed high sensitivity and specificity and allowed for the simultaneous screening and differentiation of specimens for C. botulinum A, B, E, and F

An international proficiency test to detect, identify and quantify ricin in complex matrices

While natural intoxications with seeds of Ricinus communis have long been known, the toxic protein ricin contained in the seeds is of major concern due to its history of criminal, terrorist and military use. In order to harmonize detection capabilities in expert laboratories an international proficiency test was organized that aimed at identifying good analytical practices (qualitative measurements) and determining a consensus concentration on a highly pure ricin reference material (quantitative measurements). Sample materials included highly pure ricin as well as the related R. communis agglutinin spiked into buffer, milk and meat extract; additionally, an organic fertilizer naturally contaminated with R. communis shred was investigated in the proficiency test. The qualitative results showed that either a suitable combination of immunological, MS-based and functional approaches or sophisticated MS-based approaches alone successfully allowed to detect and identify ricin in all samples. In terms of quantification, it was possible to determine a consensus concentration for the highly pure ricin reference material. The results provide a basis for further steps in quality assurance and improve biopreparedness in expert laboratories worldwide.JRC.D.2-Standards for Innovation and sustainable Developmen

Qualitative and Quantitative Detection of Botulinum Neurotoxins from Complex Matrices: Results of the First International Proficiency Test

In the framework of the EU project EQuATox a first international proficiency test (PT) on the detection and quantification of BoNT was conducted. Sample materials included BoNT serotypes A, B and E spiked into buffer, milk, meat extract and serum. A variety of methods was applied by the participants combining different principles of detection, identification and quantification. Based on qualitative assays, 95% of all results reported were correct. Successful strategies for BoNT detection were based on a combination of complementary immunological, MS-based and functional methods or on suitable functional in vivo / in vitro approaches (mouse bioassay, hemidiaphrama assay, Endopep-MS assay). Quantification of BoNT/A, BoNT/B and BoNT/E was performed by 48% of participating laboratories. It turned out that precise quantification of BoNT was difficult resulting in a substantial scatter of quantitative data. This was especially true for results obtained by the mouse bioassay which is currently seen as “gold standard” for BoNT detection. The results clearly demonstrate the urgent need of certified BoNT reference materials and the development of methods replacing animal testing. In this context, the BoNT PT provided the valuable information that both the Endopep-MS assay and the hemidiaphrama assay delivered quantitative results superior to the mouse bioassay.JRC.D.2-Standards for Innovation and sustainable Developmen

Genomic and Phenotypic Characterization of Clostridium botulinum Isolates from an Infant Botulism Case Suggests Adaptation Signatures to the Gut

In early life, the immature human gut microbiota is prone to colonization by pathogens that are usually outcompeted by mature microbiota in the adult gut. Colonization and neurotoxin production by a vegetative Clostridium botulinum culture in the gut of an infant can lead to flaccid paralysis, resulting in a clinical outcome known as infant botulism, a potentially life-threatening condition. Beside host factors, little is known of the ecology, colonization, and adaptation of C. botulinum to the gut environment. In our previous report, an infant with intestinal botulism was shown to be colonized by neurotoxigenic C. botulinum culture for 7 months. In an effort to gain ecological and evolutionary insights into this unusually long gut colonization by C. botulinum, we analyzed and compared the genomes of C. botulinum isolates recovered from the infant feces during the course of intoxication and isolates from the infant household dust. A number of observed mutations and genomic alterations pinpointed at phenotypic traits that may have promoted colonization and adaptation to the gut environment and to the host. These traits include motility, quorum-sensing, sporulation, and carbohydrate metabolism. We provide novel perspectives and suggest a tentative model of the pathogenesis of C. botulinum in infant botulism. IMPORTANCE While the clinical aspects of infant botulism and the mode of action of BoNT have been thoroughly investigated, little is known on the pathogenesis and adaptive mechanisms of C. botulinum in the gut. Here, we provide for the first time a comprehensive view on the genomic dynamics and plasticity of C. botulinum over time in a case of infant botulism. The genomic and phenotypic analysis of C. botulinum isolates collected during the disease course offers an unprecedented view of C. botulinum ecology, evolution, and pathogenesis and may be instrumental in developing novel strategies for prevention and treatment of toxicoinfectious botulism. While the clinical aspects of infant botulism and the mode of action of BoNT have been thoroughly investigated, little is known on the pathogenesis and adaptive mechanisms of C. botulinum in the gut. Here, we provide for the first time a comprehensive view on the genomic dynamics and plasticity of C. botulinum over time in a case of infant botulism.Peer reviewe

Differentiation, Quantification and Identification of Abrin and Abrus precatorius Agglutinin

Abrin, the toxic lectin from the rosary pea plant Abrus precatorius, has gained considerable interest in the recent past due to its potential malevolent use. However, reliable and easy-to-use assays for the detection and discrimination of abrin from related plant proteins such as Abrus precatorius agglutinin or the homologous toxin ricin from Ricinus communis are sparse. To address this gap, a panel of highly specific monoclonal antibodies was generated against abrin and the related Abrus precatorius agglutinin. These antibodies were used to establish two sandwich ELISAs to preferentially detect abrin or A. precatorius agglutinin (limit of detection 22 pg/mL for abrin; 35 pg/mL for A. precatorius agglutinin). Furthermore, an abrin-specific lateral flow assay was developed for rapid on-site detection (limit of detection ~1 ng/mL abrin). Assays were validated for complex food, environmental and clinical matrices illustrating broad applicability in different threat scenarios. Additionally, the antibodies turned out to be suitable for immuno-enrichment strategies in combination with mass spectrometry-based approaches for unambiguous identification. Finally, we were able to demonstrate for the first time how the developed assays can be applied to detect, identify and quantify abrin from a clinical sample derived from an attempted suicide case involving A. precatorius.Peer Reviewe

Development of a Genus-Specific Antigen Capture ELISA for Orthopoxviruses – Target Selection and Optimized Screening

Orthopoxvirus species like cowpox, vaccinia and monkeypox virus cause zoonotic infections in humans worldwide. Infections often occur in rural areas lacking proper diagnostic infrastructure as exemplified by monkeypox, which is endemic in Western and Central Africa. While PCR detection requires demanding equipment and is restricted to genome detection, the evidence of virus particles can complement or replace PCR. Therefore, an easily distributable and manageable antigen capture enzyme-linked immunosorbent assay (ELISA) for the detection of orthopoxviruses was developed to facilitate particle detection. By comparing the virus particle binding properties of polyclonal antibodies developed against surface-exposed attachment or fusion proteins, the surface protein A27 was found to be a well-bound, highly immunogenic and exposed target for antibodies aiming at virus particle detection. Subsequently, eight monoclonal anti-A27 antibodies were generated and characterized by peptide epitope mapping and surface plasmon resonance measurements. All antibodies were found to bind with high affinity to two epitopes at the heparin binding site of A27, toward either the N- or C-terminal of the crucial KKEP-segment of A27. Two antibodies recognizing different epitopes were implemented in an antigen capture ELISA. Validation showed robust detection of virus particles from 11 different orthopoxvirus isolates pathogenic to humans, with the exception of MVA, which is apathogenic to humans. Most orthopoxviruses could be detected reliably for viral loads above 1 × 103 PFU/mL. To our knowledge, this is the first solely monoclonal and therefore reproducible antibody-based antigen capture ELISA able to detect all human pathogenic orthopoxviruses including monkeypox virus, except variola virus which was not included. Therefore, the newly developed antibody-based assay represents important progress towards feasible particle detection of this important genus of viruses

Construction and validation of safe Clostridium botulinum Group II surrogate strain producing inactive botulinum neurotoxin type E toxoid

Botulinum neurotoxins (BoNTs), produced by the spore-forming bacterium Clostridium botulinum, cause botulism, a rare but fatal illness affecting humans and animals. Despite causing a life-threatening disease, BoNT is a multipurpose therapeutic. Nevertheless, as the most potent natural toxin, BoNT is classified as a Select Agent in the US, placing C. botulinum research under stringent governmental regulations. The extreme toxicity of BoNT, its impact on public safety, and its diverse therapeutic applications urge to devise safe solutions to expand C. botulinum research. Accordingly, we exploited CRISPR/Cas9-mediated genome editing to introduce inactivating point mutations into chromosomal bont/e gene of C. botulinum Beluga E. The resulting Beluga Ei strain displays unchanged physiology and produces inactive BoNT (BoNT/Ei) recognized in serological assays, but lacking biological activity detectable ex- and in vivo. Neither native single-chain, nor trypsinized di-chain form of BoNT/Ei show in vivo toxicity, even if isolated from Beluga Ei sub-cultured for 25 generations. Beluga Ei strain constitutes a safe alternative for the BoNT research necessary for public health risk management, the development of food preservation strategies, understanding toxinogenesis, and for structural BoNT studies. The example of Beluga Ei generation serves as template for future development of C. botulinum producing different inactive BoNT serotypes.Peer reviewe

Crystal structures of OrfX1, OrfX2, and the OrfX1–OrfX3 complex from the orfX gene cluster of botulinum neurotoxin E1

Botulinum neurotoxins (BoNTs) are among the most lethal toxins known to humans, comprising seven established serotypes termed BoNT/A–G encoded in two types of gene clusters (ha and orfX) in BoNT-producing clostridia. The ha cluster encodes four non-toxic neurotoxin-associated proteins (NAPs) that assemble with BoNTs to protect and enhance their oral toxicity. However, the structure and function of the orfX-type NAPs remain largely unknown. Here, we report the crystal structures for OrfX1, OrfX2, and an OrfX1–OrfX3 complex, which are encoded in the orfX cluster of a BoNT/E1-producing Clostridium botulinum strain associated with human foodborne botulism. These structures lay the foundation for future studies on the potential roles of OrfX proteins in oral intoxication and pathogenesis of BoNTs.Peer reviewe