Extraktion und Amplifikation von Pilz-DNA aus Kleinsäugerkotproben

Das Sammeln von Kotproben von Kleinsäugern und deren genetische Analyse stellt eine nicht invasive Art der Beprobung dar. Diese kommt vor allem dann zum Einsatz, wenn es sich um seltene, unauffällige oder störungsanfällige Arten handelt, oder wenn invasive Probenentnahmen nicht erlaubt bzw. nicht durchführbar sind. Solche Proben bergen eine Reihe von technischen Problemen. Der geringe Probenumfang von wenigen Milligramm Kot kann die molekularen Analysen beeinträchtigen. Die Proben enthalten unter Umständen nur sehr geringe Mengen der zu extrahierenden und amplifizierenden DNA. Es wurde ein gegenüber existierenden Methoden verbessertes Extraktionsprotokoll entwickelt, optimiert für die Beurteilung von Mykophagie unter Kleinsäugern. Kotproben von vier Kleinsäugerarten (Myodes glareolus, Apodemus flavicollis, Microtus agrestis und Sorex minutus), mit unterschiedlich enthaltener Menge an Pilzsporen wurden verwendet, um die Extraktionsleistung zweier kommerzieller Extraktionskits und zweier selbsthergestellter Extraktionspuffer mit modifiziertem Extraktionsprotokoll, zu vergleichen. Die besten Resultate bezüglich Menge und Amplifikation der extrahierten Ziel-DNA erzielte der einfachere der beiden selbstgemachten Extraktionspuffern in Kombination mit mechanischem Aufschluss und Hitzebehandlung. Die Puffersimplizität, einfache Extraktions- und Aufreinigungsschritte und vor allem die Möglichkeit der Arbeit mit sehr kleinen Probenmengen lassen den Schluss zu, dass das neu entwickelte Extraktionsprotokoll neben der Extraktion von (Pilz) DNA aus Säugetierkot möglicherweise auch passend für andere, ähnliche Studien wäre (andere Vektorart, andere DNA-Quellen). Weiters sollte die Frage geklärt werden, welche Pilze die Kleinsäuger bevorzugt konsumieren. Nach Klonierung und Sequenzierung von elf Proben konnten in neun Proben Ektomykorrhizapilze nachgewiesen werden.DNA-based analysis of faecal samples is essential for research about ecology of rare, elusive or endangered species, where invasive sampling is impossible or not practicable. When investigating small species, sample size constraints risk impairing molecular investigations, because of low DNA amounts present in sample sizes of a few milligrams. Comparison of existing methods led to the development of an improved protocol for DNA extraction optimised for the assessment of mycophagy among small mammals. Stool samples from four different species (Myodes glareolus, Apodemus flavicollis, Microtus agrestis and Sorex minutus) containing varying amounts of fungal spores were used to assess the extraction ability of two commercial stool DNA extraction kits and of two different in-house extraction protocols. The simplest extraction buffer in combination with an adequate mechanical and heat lysis treatment yielded best results regarding amount and amplification of the extracted DNA. Because of the possibility to work with very small samples, buffer simplicity, and easy extraction and purification procedure, we regard our newly developed extraction protocol for (fungal) DNA from mammalian faeces as very suitable for use in other studies, possibly involving different vector species as well as other sources of DNA, with probably only minor modifications necessary for adaptation. Further molecular work was done with the faecal samples extracted with the improved in-house protocols to find out which fungi were preferably eaten by small mammals. In nine out of eleven samples ectomycorrhizal fungi were identified

Is small mammal mycophagy relevant for truffle cultivation?

The role of small mammal mycophagy as vectors of hypogeous fungi is well established. However, little is known about dispersal of gourmet truffle species by mammal vectors, or about the potential role of mycophagy in truffle plantations. We hypothesize that small mammal mycophagy contributes to the productivity of truffle plantations by providing inoculum for truffle mycelium establishment and mating. Spread of non-desired competitors of gourmet truffles is a potential adverse effect of small mammal mycophagy

Is small mammal mycophagy relevant for truffle cultivation?

The role of mycophagous small mammals as vectors of hypogeous fungi is well established. However, little is known about dispersal of gourmet truffle species by mammal vectors, or about the potential role of mycophagy in truffle plantations. We hypothesize that small mammal mycophagy contributes to the productivity of truffle plantations by providing inoculum for truffle mycelium establishment and mating. Spread of non-desired competitors of gourmet truffles is a potential adverse effect of small mammal mycophagy

Chronic virus infection compromises memory bystander T cell function in an IL-6/STAT1-dependent manner

Barnstorf et al. demonstrate that chronic viral infections numerically reduce memory non–virus-specific (bystander) cytotoxic T lymphocytes and alter their phenotype and function. Phenotypic changes are induced by the inflammatory cytokine IL-6, and functional impairment is not cell-intrinsic but inferred by the chronically infected host

Chronic virus infection compromises memory bystander T cell function in an IL-6/STAT1-dependent manner

Chronic viral infections are widespread among humans, with ∼8–12 chronic viral infections per individual, and there is epidemiological proof that these impair heterologous immunity. We studied the impact of chronic LCMV infection on the phenotype and function of memory bystander CD8+ T cells. Active chronic LCMV infection had a profound effect on total numbers, phenotype, and function of memory bystander T cells in mice. The phenotypic changes included up-regulation of markers commonly associated with effector and exhausted cells and were induced by IL-6 in a STAT1-dependent manner in the context of chronic virus infection. Furthermore, bystander CD8 T cell functions were reduced with respect to their ability to produce inflammatory cytokines and to undergo secondary expansion upon cognate antigen challenge with major cell-extrinsic contributions responsible for the diminished memory potential of bystander CD8+ T cells. These findings open new perspectives for immunity and vaccination during chronic viral infections.ISSN:0022-1007ISSN:1540-0069ISSN:1540-953