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In the original publication [...].

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The use of wild animals in research is complicated due to the capture and housing conditions, as well as to legal aspects, making it difficult to develop in vivo and in vitro models for the study of pathologies that affect these species. Here we validate an in vitro model of tendon-derived mesenchymal cells (TDSC) from Eurasian blackbird (Turdus merula) cadaveric samples. Through the expression of surface markers and the ability to differentiate into multiple lineages, the nature of the cells was confirmed. We then evaluated Mesenchymal Stem Cells (MSCs) as an infection model for the Usutu Flavivirus. To this aim, blackbird TDSCs were compared to Vero E6 cells, commonly used in Flavivirus studies. Both cells showed permissiveness to USUV infection as confirmed by immunocytochemistry. Moreover, TDSCs exhibited replication kinetics similar to, although slightly lower than, Vero E6, confirming these cells as a pertinent study model for the study of the pathogenesis of USUV. In this work, we isolated and characterized tendon-derived mesenchymal stem cells, which represent an interesting and convenient in vitro model for the study of wildlife species in laboratories.

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When derived from chicken embryos, avian primordial germ cells (PGCs) have been reported to keep their germline-specific properties and proliferative potential even after long-term culture and genetic modifications. Few teams to date have reported such long-term expansion and engineering without differentiation of primary avian PGCs' cultures. We have developed original and robust methods that allow more than 1 year culture, expansion and cryobanking of primary cultures of PGCs without obvious effects on their biological properties, including their ability to colonise the genital ridges. Overall, 38% of embryonic samples gave rise to PGCs lines derived from three commercial layers and two Belgian endangered breeds. The lines kept their proliferative potential and their characteristic PGCs phenotype after 20 months in culture, whether or not interrupted by a cryopreservation step. All the resulting lines appeared devoid of female cells, although initially pooled from male and female embryos. Labelled PGCs from 12 long-term cultured lines colonised the genital ridges of recipient embryos. Thus, this procedure allows derivation, long-term expansion and cryobanking of primary cultures of PGCs without obvious changes to their original characteristics, providing an alternative access to applications in avian biotechnology and preservation of genetic resources.

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The pharyngeal tonsil has recently been identified as a new participant in airborne contamination by the ovine scrapie agent. In the context of scrapie pathogenesis, we conducted a three-dimensional reconstruction of the innervation pattern in the lymphoid compartments of this tonsil. This model confirmed that very few nerve fibres penetrated the lymphoid follicles and suggested that the nerve fibre distribution in the interfollicular and subepithelial areas is more suitable with neuro-invasion through direct contact between these nerve fibres and prion-transporting cells prior to or after prion amplification in the germinal centre of the pharyngeal tonsil lymphoid follicles.

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Recent studies have established the involvement of nasal-associated lymphoid tissues, mainly the pharyngeal tonsil, in prion pathogenesis. However, the mechanisms of the associated neuroinvasion are still debated. To determine potential sites for prion neuroinvasion inside the ovine pharyngeal tonsil, the topography of heavy (200 kDa) and light (70 kDa) neurofilaments and of glial fibrillar acidic protein has been semi-quantitatively analysed inside the various compartments of the tonsil. The results show that the most innervated areas are the interfollicular area and the connective tissue located beneath the respiratory epithelium. The existence of rare synapses between follicular dendritic cells and nerve fibres inside the germinal centre indicates that this mechanism of neuroinvasion is possible but, since germinal centres of lymphoid follicles are poorly innervated, other routes of neuroinvasion are likely. The host PRNP genotype does not influence the pattern of innervation in these various tonsil compartments, unlike ageing during which an increase of nerve endings occurs in a zone of high trafficking cells beneath the respiratory epithelium. A minimal age-related increase of innervation inside the lymphoid follicles has also been observed. An increase in nerve fibre density around the lymphoid follicles, in an area rich in mobile cells such as macrophages and dendritic cells capable of capturing and conveying pathogen prion protein (PrPd), might ensure more efficient infectivity, not in the early phase but in the advanced phase of lymphoinvasion after the amplification of PrPd; alternatively, this area might even act as a direct site of entry during neuroinvasion.

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Although the alimentary tract has been suggested as the most likely portal of entry in natural scrapie, a growing amount of data indicates that the respiratory system and more specifically the pharyngeal tonsils serve as a natural portal of entry for scrapie. This study describes for the first time the broad cell populations in the lymphoid compartment of pharyngeal tonsils and more specifically inside the lymphoid follicles where the scrapie agent accumulates during the period of latency. Follicular dendritic cells (FDCs), stromal cells located in the light zone of the germinal centre of lymphoid follicles, seem to be the principal causal factor in the accumulation of the infectious agent in transmissible spongiform encephalopathy (TSE) diseases. Knowing that efficient lymphoreticular prion propagation requires PrPc expression, we analysed the expression of PrPc with the mouse monoclonal antibody Pri 909 both in situ and on FDC-cluster-enriched cell suspensions. In situ, a positive staining was observed in the germinal centre of pharyngeal lymph follicles. The germinal centre labelling was due to the presence of a follicular dendritic network as revealed after immunogold staining of isolated FDC clusters. Our results suggest that the pharyngeal lymphoreticular system and more specifically PrPc expressing follicular dendritic cells could serve as a prion "reservoir" during the latency phase, thus playing a key role during the scrapie lymphoinvasion.

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Using a two-step procedure involving insertional gene targeting and recombinase-mediated cassette exchange in ES cells, we have produced two lines of transgenic mice expressing a dominant-negative latency-associated myostatin propeptide under control of the myosin light chain 1F promoter and 1/3 enhancer from the TSPY locus on the Y chromosome. Males of the corresponding lines are characterized by a 5-20% increase in skeletal muscle mass. This experiment demonstrates the feasibility of a more efficient cattle production system combining superior beef production abilities for bulls and dairy abilities for cows.

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By using a conditional gene targeting approach exploiting the cre-lox system, we show that postnatal inactivation of the myostatin gene in striated muscle is sufficient to cause a generalized muscular hypertrophy of the same magnitude as that observed for constitutive myostatin knockout mice. This formally demonstrates that striated muscle is the production site of functional myostatin and that this member of the TGFbeta family of growth and differentiation factors regulates muscle mass not only during early embryogenesis but throughout development. It indicates that myostatin antagonist could be used to treat muscle wasting and to promote muscle growth in man and animals.

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