Identification of Glaesserella parasuis and Differentiation of Its 15 Serovars Using High-Resolution Melting Assays

Glaesserella parasuis is the etiological agent of Glässer’s disease, which is associated with polyserositis and arthritis and has a significant impact on the economy of the pig production industry. For the optimal surveillance of this pathogen, as well as for the investigation of G. parasuis-associated diseases, it is crucial to identify G. parasuis at the serovar level. In this work, we designed and developed new high-resolution melting (HRM) approaches, namely, the species-specific GPS-HRM1 and two serovar-specific HRM assays (GPS-HRM2 and GPS-HRM3), and evaluated the sensitivity and specificity of the assays. The HRM assays demonstrated good sensitivity, with 12.5 fg–1.25 pg of input DNA for GPS-HRM1 and 125 fg–12.5 pg for GPS-HRM2 and GPS-HRM3, as well as a specificity of 100% for the identification of all recognized 15 G. parasuis serovars. Eighteen clinical isolates obtained between 2014 and 2022 in Switzerland were tested by applying the developed HRM assays, which revealed a heterogeneous distribution of serovars 2, 7, 4, 13, 1, and 14. The combination with virulence marker vtaA (virulence-associated trimeric autotransporters) allows for the prediction of potentially virulent strains. The assays are simple to execute and enable a reliable low-cost approach, thereby refining currently available diagnostic tools

Strain diversity in Mycobacterium avium subsp. paratuberculosis-positive bovine fecal samples collected in Switzerland

Paratuberculosis or Johne’s disease is a chronic intestinal disease in domestic and wild ruminants. It affects global dairy economy and is caused by Mycobacterium avium subsp. paratuberculosis (MAP). The objective of this study was to analyze strain diversity in MAP-positive fecal samples by using a particular single nucleotide polymorphism (SNP) distinguishing between cattle (C-) and sheep (S-) type MAP and analysis of SNPs within gyrA and gyrB genes differentiating between Types I, II, and III. Moreover, mycobacterial interspersed repetitive unit and variable-number tandem repeat (MIRU-VNTR) analysis using eight established loci was performed. A total of 90 fecal samples from diseased animals presenting diarrhea and/or weight loss, originating from 59 bovine herds across 16 cantons of Switzerland were screened by PCR for the MAP-specific F57 and IS900 genes and were further subtyped. 96.7% and 3.3% of the samples contained C- and S-type MAP, respectively. Ten INRA Nouzilly MIRU-VNTR (INMV) profiles, with a discriminatory index of 0.802, calculated based on 65 epidemiological independent genotypes, were detected: INMV 1 (33.8%), INMV 2 (23.1%), INMV 6 (16.9%), INMV 9 (9.2%), INMV 116 (4.6%), INMV 3 (3.1%), INMV 5 (3.1%) and INMV 72 (1.5%), including two novel INMV profiles, namely INMV 253 (3.1%; S-type III) and INMV 252 (1.5%; C-type). INMV 1, INMV 2, and INMV 6 comprised almost 75% of the F57- and IS900-positive samples. Typing data from 11 herds suggest that there are some herds with intra-herd diversity of genotypes. The results of this study indicate a heterogeneity of MAP in Switzerland

Morphological and molecular characterization of a new mycobacterium avium Subsp. paratuberculosis S-type strain Genotype in goats

Paratuberculosis is a chronic bacterial disease of global importance mainly in domestic and wild ruminants, caused by Mycobacterium avium subsp. paratuberculosis (MAP). In goats, paratuberculosis is mostly caused by the "C-type" (cattle) and in a few cases by the "S-type" (sheep) strain of MAP. In 2017, a caprine S-type III isolate with a new VNTR profile was identified in a Swiss alpine region. In 2018, new caprine isolates with the same novel VNTR profile originating from a farm of a close by neighboring valley were analyzed. Here we report on this MAP S-type III outbreak in a Swiss dairy goat farm in which we investigated the pathological changes, distribution and genotype of MAP tissue homogenates. Full necropsy and histological examination were undertaken on two female adult goats with a history of weight loss and intermitting diarrhea. Routine and special stains were applied to characterize the morphological changes. DNA was extracted from 33 different tissue samples and tested for MAP by qPCR targeting IS900 and F57. Subtyping was performed, using the variable number tandem repeats (VNTR) and mycobacterial interspersed repetitive units (MIRU) approach. The goats showed moderate to marked emaciation and displayed typical clinical features of paratuberculosis. A moderate granulomatous enteritis and regional lymphadenitis with a small to moderate number of acid-fast bacteria within macrophages was detected. MAP detection was mainly restricted to the gastrointestinal tract, mesenteric and hepatic lymph nodes. Subtyping the S-type isolates using a panel of eight established MIRU-VNTR loci identified a new genotype, INMV 218

A novel multiplex qPCR targeting 23S rDNA for diagnosis of swine dysentery and porcine intestinal spirochaetosis

Figure S1. Consensus sequence alignment of the target DNA region within 23S ribosomal DNA. Primers (Brachy primer for. and Brachy primer rev.) on the target DNA are marked in grey. The probe for B. hyodysenteriae (Probe_hyo) is highlighted in yellow, the probe for B. pilosicoli (Probe_pilo) in purple, and the probe for the B. intermedia/B. innocens/B. murdochii (probe inter) in green. Differences in single residues are marked in red. (PDF 112 kb

Development of a novel high resolution melting assay for identification and differentiation of all known 19 serovars of Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a respiratory infectious disease responsible for global economic losses in the pig industry. From a monitoring perspective as well as due to the different courses of disease associated with the various serovars, it is essential to distinguish them in different herds or countries. In this study, we developed a novel high resolution melting (HRM) assay based on reference strains for each of the 19 known serovars and additional 15 clinical A. pleuropneumoniae isolates. The novel HRM comprises the species-specific APP-HRM1 and two serovar-specific HRM assays (APP-HRM2 and APP-HRM3). APP-HRM1 allowed polymerase chain reaction (PCR) amplification of apxIV resulting in an A. pleuropneumoniae specific melting curve, while nadV specific primers differentiated biovar 2 from biovar 1 isolates. Using APP-HRM2 and APP-HRM3, 13 A. pleuropneumoniae serovars can be determined by inspecting the assigned melting temperature. In contrast, serovar 3 and 14, serovar 9 and 11, and serovar 5 and 15 have partly overlapping melting temperatures and thus represent a challenge to accurately distinguish them. Consequently, to unambiguously ensure the correct assignment of the serovar, it is recommended to perform the serotyping HRM assay using a positive control for each serovar. This rapid and user-friendly assay showed high sensitivity with 1.25 fg-125 pg of input DNA and a specificity of 100% to identify A. pleuropneumoniae. Characteristic melting patterns of amplicons might allow detecting new serovars. The novel HRM assay has the potential to be implemented in diagnostic laboratories for better surveillance of this pathogen

Die Bedeutung des Berufsfeldbezugs in den Aufgaben und für Laufbahnen an Pädagogischen Hochschulen

Der Beitrag präsentiert ein «Kompetenzprofil des wissenschaftlichen Personals an Pädagogischen Hochschulen», in dem der Berufsfeldbezug des wissenschaftlichen Personals konzeptionell gefasst ist. Das Profil ist Reflexionsfolie für die Auseinandersetzung mit der Laufbahngestaltung und zugleich Referenz für einen zweifach pilotierten, im Herbst 2021 erstmals als CAS angebotenen Studiengang zur Stärkung des Berufsfeldbezugs. Dieser Studiengang wird anhand seines Konzepts, der Gruppe von Teilnehmenden und ihrer in einer Evaluation der Pilotdurchführungen erhobenen Erfahrungen beschrieben. Es werden formale und inhaltliche Aspekte des Berufsfeldbezugs in der Laufbahngestaltung an Pädagogischen Hochschulen besprochen.&nbsp

Development of a new High Resolution Melting (HRM) assay for identification and differentiation of Mycobacterium tuberculosis complex samples

The rapid identification and differentiation of members of the Mycobacterium tuberculosis complex (MTBC) is essential to assess the potential zoonotic risk. Different available molecular methods are time consuming since they depend on cultivation of mycobacteria. High Resolution Melting (HRM) is a low cost, rapid and easy to perform single-tube method not limited to cultured samples. In this study, a HRM assay specifically targeting gyrB was developed to simultaneously identify and differentiate Mycobacterium (M.) tuberculosis, M. microti and M. bovis/M. caprae. To evaluate the performance of this assay, 38 MTBC isolates and 25 directly extracted clinical specimens were analysed. HRM results of all 38 (100%) examined isolates correlated with the results obtained with the commercially available GenoType MTBC test (Hain Lifescience). From the 25 clinical specimens tested, species identification by HRM showed concordant results with the previously used identification methods in 23 samples (92%). The assay demonstrated a good analytical sensitivity, specificity and reproducibility and can be used directly on clinical specimens

Momo bewegt. Bewegungssequenzen in Bewegung und Sport eingebettet in den Kinderroman Momo von Michael Ende

Diese Bachelorarbeit befasst sich mit der Fragestellung, wie koordinative Fähigkeiten anhand verschiedener Bewegungssequenzen eingebettet in den Roman Momo gefördert werden können. Im Theorieteil wird analysiert, welchen Stellenwert Bewegung und Sport im Jugendalter besitzt und welche Bedeutung der Förderung der Koordination in der Primarschule zugeschrieben werden. Da diese Arbeit schlussendlich in einem Produkt endet, ist es ebenfalls zentral, gute Bewegungssequenzen zu definieren und Differenzierungsmöglichkeiten aufzuzeigen. Das Produkt besteht aus einer Zusammenstellung von Bewegungssequenzen, welche jede Lehrperson ohne grossen Aufwand in die Turnstunde mitnehmen und ausführen kann. Die Bewegungssequenzen orientieren sich an dem Buch Momo von Michael Ende, welches fortlaufend im Unterricht gelesen und behandelt werden soll. Eine Karte bezieht sich auf eine Sequenz, welche sich wiederum auf ein zu lesendes Kapitel im Kinderbuch Momo bezieht. Eben-falls basieren die Sequenzen auf einzelnen koordinativen Fähigkeiten, da diese zwingend im Primarschulalter gefördert werden sollen. Das Vorgehen bestand darin, sich die wichtigsten Schlüsselsequenzen aus dem Buch herauszuschreiben und dazu passende Sequenzen für den Bewegungs- und Sportunterricht zu erstellen. Durch die Ausführungen der Übungen werden die Schülerinnen und Schüler angeleitet, sich vertieft mit dem Inhalt des Buches auseinanderzusetzen

Novel multiplex TaqMan assay for differentiation of the four major pathogenic Brachyspira

A novel TaqMan 5-plex real-time PCR was developed for the identification of Brachyspira species in swine. B. hyodysenteriae, B. pilosicoli, and B. suanatina are identified using three hydrolysis probes targeting cpn60, while B. hampsonii is recognized by another nox specific probe. This highly sensitive and specific assay is easy to implement in routine veterinary diagnostic laboratories and enables rapid differentiation between the main four pathogenic Brachyspira species in pigs, using a single-tube approach. A novel TaqMan 5-plex real-time PCR using a combination of locked nucleic acid-modified (LNA)- and minor groove binding (MGB)-conjugated DNA probes was developed for identification and differentiation between the four main pathogenic Brachyspira species in swine. B. hyodysenteriae, B. pilosicoli, and B. suanatina are identified using three hydrolysis probes targeting cpn60, while B. hampsonii is recognized by another nox specific probe. The assay also includes an exogenous internal control simultaneously verifying the PCR competency of the DNA samples. Validation of the novel assay was performed using DNA samples from 18 Brachyspira reference strains and 477 clinical samples obtained from porcine rectal swabs by comparing them with different PCR-based methods targeting nox, 16S rDNA, and 23S rDNA. The specificity of the assay was 100% without cross-reactivity or detection of different pathogens. Depending on the Brachyspira species, the limit of detection was between 10 and 20 genome equivalents with a cut-off threshold cycle (Ct) value of 37. The developed highly sensitive and specific 5-plex real-time PCR assay is easy to implement in routine veterinary diagnostic laboratories and enables rapid differentiation between the main four pathogenic Brachyspira species recognized in pigs using a single-tube approach