Publications scientifiques de l'EnvA
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Les hydrogels synthétiques disponibles dans le commerce ne peuvent pas remplacer le Matrigel pour promouvoir la polarisation et la lumenogenèse d’agrégats 3D de cellules souches embryonnaires
No full textPluripotency is defined as the ability of cells to differentiate into all cell types. In vivo, this capacity is gradually acquired by pluripotent cells in the epiblast as they polarize and arrange themselves into a three-dimensional structure known as a 'rosette', which then forms a lumen. Major epigenetic and metabolic changes occur during this crucial period of early development. Embryonic stem cells (ESCs), an in vitro model for early epiblast cells, can self-organise into rosettes when cultured in 3D, making them an ideal model for studying the events that accompany this morphogenesis. While this model is relevant to the 3Rs in terms of reducing the use of embryos, there is a significant drawback: the hydrogel commonly used for 3D cultures, Matrigel, is derived from animals. Furthermore, some rosette formation protocols use a serum-containing medium. The aim of this study was therefore to test alternative, commercially available, synthetic hydrogels and to compare the use of serum-containing and serumfree medium for modelling epiblast morphogenesis. Our results demonstrate that morphogenesis is more efficient in serum-free medium but did not occur in the synthetic hydrogels tested. This underscores the need for further optimisation of these systems.La pluripotence est définie comme la capacité des cellules à se différencier en tous les types cellulaires. In vivo, cette capacité est progressivement acquise par les cellules pluripotentes de l’épiblaste lorsqu’elles se polarisent et s’organisent en une structure tridimensionnelle appelée « rosette », qui forme ensuite une lumière. Des modifications épigénétiques et métaboliques majeures surviennent durant cette période clé du développement précoce. Les cellules souches embryonnaires (CSE), modèle in vitro des cellules de l’épiblaste précoce, peuvent s’auto-organiser en rosettes en culture 3D, constituant ainsi un modèle pertinent pour étudier les événements associés à cette morphogenèse. Bien que ce modèle contribue aux principes des 3R en réduisant l’utilisation d’embryons, il présente une limite importante : l’hydrogel couramment utilisé pour les cultures 3D, le Matrigel, est d’origine animale. De plus, certains protocoles de formation de rosettes utilisent des milieux contenant du sérum. L’objectif de cette étude était donc de tester des hydrogels synthétiques commerciaux alternatifs et de comparer l’utilisation de milieux avec ou sans sérum pour modéliser la morphogenèse de l’épiblaste. Nos résultats montrent que la morphogenèse est plus efficace en milieu sans sérum, mais qu’elle ne se produit pas dans les hydrogels synthétiques testés, soulignant la nécessité d’optimiser davantage ces systèmes
Distribution of tick-borne microorganisms in human-biting ticks in France collected through a Citizen-science program
No full textInternational audienceTicks occupy diverse habitats, increasing the risk of human exposure. Assessing the public health threat posed by ticks requires rigorous monitoring of their distribution and of the prevalence of tick-borne pathogens. In France since 2017, the citizen science program CiTIQUE monitors human tick bites through multiple complementary approaches. Citizens can report bites and submit biting ticks to a national tick bank for research and surveillance. This study aimed to investigate human exposure to tick-borne microorganisms including pathogens across France, using ticks submitted through the CiTIQUE program. In total, 2009 ticks were selected from the CiTIQUE tick bank, identified, and screened for microorganisms using a real-time microfluidic PCR method. Most bites involved Ixodes ricinus nymphs except in Mediterranean regions where Dermacentor and Rhipicephalus ticks were more common. Twenty-six microorganisms were detected, eighteen of which are potentially pathogenic to humans. These pathogens were widely distributed across the country. Borrelia spp. were the most frequently detected pathogens with spatial variation among regions. Anaplasma phagocytophilum infection rates varied from region to region. Neoehrlichia mikurensis was found in seven out of twelve French regions. Rickettsia species diversity was highest in the southeast, associated with a greater diversity of vectors. Five percent of ticks were infected with more than one pathogen. Although spatial heterogeneity was observed, no region was free of infected ticks. This study demonstrates the power of citizen science for nationwide surveillance of tick-borne pathogens, providing a large-scale overview of pathogen diversity and distribution across France from crowdsourced tick data
Characterization of extracellular vesicles at parturition in dairy cows with lategestation heat stress
No full textInternational audienceThis study investigated how late-gestation heat stress (HT) affects extracellular vesicle (EV) protein profiles in dairy cows at parturition. Plasma EV were isolated from heat-stressed and cooled (CL) cows and analyzed by mass spectrometry. A total of 684 proteins were identified, of which 20 differed significantly between treatments. The EV from cooled cows were enriched in extracellular matrix and coagulation proteins, including laminins, collagen IV, fibrinogen, and von Willebrand factor. The presence of these proteins in CL cows suggests that cooling enhances molecular pathways involved in tissue repair and postpartum recovery. Conversely, immunoglobulin-related proteins and specific receptors were reduced in cooled cows, potentially reflecting lower immune stress. These findings indicate that HT during late gestation alters EV-mediated signaling related to metabolism, immune modulation, and tissue remodeling at parturition. The identified proteins may serve as potential biomarkers for assessing maternal adaptation and recovery, emphasizing the importance of environmental cooling during the dry period. NTA = nanoparticle tracking analysis; trt = treatment.Highlights• Heat stress during late gestation significantly alters EV content.• Cooling increased extracellular matrix and coagulation proteins, perhaps for tissue repair and postpartum recovery.• Cooling decreased immunoglobulin-related proteins and certain receptors in EV.• Changes in EV proteins may reflect improved recovery and lower immune stress with cooling
Persistence of vegetative and sporulated forms of Clostridium perfringens exposed to air at different relative humidities
No full textInternational audienceC. perfringens, an anaerobic bacterium, is a common cause of food poisoning that can persist on surfaces in slaughterhouses. However, the mechanisms governing its survival in such environments – characterised by variations in relative air humidity (RAH) – remain poorly understood. This study aimed to evaluate the effect of air exposure on C. perfringens survival and to identify the mechanisms responsible for its inactivation. Vegetative cells and spores of C. perfringens were deposited on inert surfaces and exposed to different RAH (11 %, 43 %, 75 %, 100 %) under both aerobic and anaerobic conditions, to assess the contributions of osmotic and oxidative effects induced by dehydration to cell death. At low RAH, more than 99 % of vegetative cells were inactivated within one day, regardless of oxygen presence. Epifluorescence microscopy and flow cytometry analyses revealed that dehydration and rehydration disrupted membrane integrity, contributing to inactivation through lethal mechanical damage. At 100 % RAH, vegetative cells survived over 3 days under aerobic conditions (>1 %) and over 30 days under anaerobic conditions (>0.003 %). The composition of the dehydration medium had little effect on cell survival. In contrast, spores were much more resistant, with around 10 % survival after two months of stress in presence of oxygen, without any significant effect of dehydration. These results highlight the potential of exploiting RAH fluctuations to develop control strategies targeting C. perfringens vegetative cells. However, the extreme resilience of spores confirms the need for specific and targeted decontamination methods to eliminate them effectively
Microbial network stability, not diversity, drives colonization resistance against Borrelia afzelii in Ixodes ricinus ticks
No full textInternational audienceMost tick-borne pathogens (TBPs) are acquired secondarily, when ticks feed on infected hosts, meaning the pathogen must establish itself within an already assembled microbiota. These scenarios are subject to "priority effects," where the order of microbial arrival influences the success of later colonizers. Microbial interactions within arthropod vectors can therefore shape infection outcomes, producing either infection-refractory states, where resident microbes and their interactions reduce the likelihood of pathogen establishment, or infectionpermissive states, where such barriers are absent or weakened and the pathogen establishes infection successfully. Hamilton et al. (2021) assessed larval microbiota before pathogen exposure and sequenced the microbiota of fed nymphs, both exposed or not to Borrelia afzelii, enabling priority-effect hypotheses to be tested. Despite uniform exposure to the highly infectious B. afzelii strain NE4049, only a subset of ticks became Borrelia-positive, suggesting refractory and permissive microbiota states. We reanalyzed the original dataset to test whether differences in microbiome community assembly and co-occurrence network features, beyond diversity metrics, were associated with these states. Refractory nymph networks exhibited higher connectivity and structural resilience, with Staphylococcus emerging as a central taxon already present in unfed larvae. In contrast, permissive networks showed reduced robustness and a marginal role for Staphylococcus. Notably, dysbiosis altered microbial assembly but did not prevent network reconfiguration in refractory ticks. Our findings suggest that colonization resistance is better explained by microbial network integrity than by diversity alone. Methodologically, they show that integrating community assembly theory and network analyses can reveal key features of the tick microbiota associated with vector competence.</div
Characterizing the Bacterial Microbiome of the Invasive Vector Aedes albopictus in Hungary: A Pilot Study Using Oxford Nanopore Sequencing
No full textInternational audienceAedes albopictus has recently established self‐sustaining populations in Hungary, but its microbiota—which may influence vector competence—remains poorly understood. We used Oxford Nanopore long‐read sequencing for full‐length 16S rRNA gene profiling of adult Ae. albopictus from two urban sites, Pécs and Barcs. Each location contributed 10 specimens, with contamination controls rigorously applied. Diversity metrics and co‐occurrence network analyses were performed using QIIME2, SparCC, and NetCoMi, with robustness assessed via simulated node removal and addition. Sequencing depth was sufficient to saturate rarefaction curves. Although alpha and beta diversity did not differ significantly between sites, the Pécs population exhibited greater taxonomic richness (100 unique taxa vs. 61 in Barcs) and denser, more clustered networks. Only 15 genera were shared, with Wolbachia dominating both communities. Networks differed in central taxa and structural properties: Pécs retained higher connectivity and shorter paths under perturbation, suggesting greater resilience. Removal of conserved taxa revealed location‐specific impacts on network stability, with Pécs more vulnerable to the loss of key genera. Negative interactions and compensatory taxa emerged post‐removal, indicating distinct reconfiguration strategies. Our findings highlight marked local variation in microbiome structure and robustness, even across a 65‐km gradient. These results establish a high‐resolution baseline for assessing how microbiota shape Ae. albopictus vector potential, informing microbiome‐based control strategies tailored to regional contexts
Optimiser la production du génome d'Anaplasma phagocytophilum, une étape cruciale pour comprendre la virulence et le tropisme d'hôte
No full textInternational audienceAnaplasma phagocytophilum (Aph) est une bactérie intracellulaire stricte transmise par les tiques du genre Ixodes, responsable de l’anaplasmose granulocytaire chez l’Homme et les ruminants. La variabilité de la virulence et la diversité des hôtes suggèrent une diversité génétique encore mal caractérisée. L’analyse génomique d’Aph demeure complexe en raison de la difficulté à isoler et cultiver la bactérie, ainsi qu’à séparer son ADN de celui de l’hôte vertébré. Ce projet vise (i) à évaluer la qualité des génomes disponibles dans les bases publiques et (ii) à développer une stratégie de séquençage long-read Oxford Nanopore Technologies (ONT®) permettant d’obtenir des génomes complets et d’origine plus diversifiée. En 2025, nous avons évalué 33 génomes Aph disponibles sur NCBI pour leur fragmentation, leur taille (QUAST) et leur pureté (BLAST, Kraken2). Vingt-neuf présentaient une qualité et une complétude satisfaisantes, tandis que 4 présentaient une contamination importante par des séquences hôtes.Un séquençage ONT d’une souche de source humaine enrichie en culture a permis d’obtenir, après sous-échantillonnage d’une profondeur de séquençage de 100X, un génome complet en un seul contig (~1,5 Mb). Des simulations de profondeurs variables (10X à 100X, 10 réplicas) ont montré qu’une couverture de 30X permettait l’assemblage d’un génome complet équivalent au contrôle. Nous avons également mis en évidence qu’à 20X les génomes étaient de taille attendue malgré une fragmentation plus importante (>5 contigs), et qu’à 10X le génome était fragmenté et incomplet. Les analyses de clustering par k-mer ont révélé que dès 20X, les topologies d’arbre étaient stables, indiquant que cette profondeur est suffisante pour des comparaisons robustes de génomes. Des essais de séquençage réel utilisant la technologie ONT® sur des cultures présentant différents ratios hôte/Aph (1 et 10 %) et utilisant « l’adaptive sampling » pour enrichir en temps réel la fraction d’ADN bactérien ont permis d’atteindre des profondeurs de 7X et 65X. Des optimisations restent nécessaires pour améliorer l’enrichissement et le multiplexage des échantillons. Notre preuve de concept démontre la faisabilité du séquençage ONT® d’Aph à partir d’échantillons faiblement enrichis et ouvre la voie au séquençage de génomes plus diversifiés qui permettront d’approfondir les connaissances épidémiologiques sur Aph
Subacute loss of olfactory neurons following SARS-CoV-2 infection in hamsters
No full textInternational audienceThe loss of smell has been a hallmark of the COVID-19 pandemic. Odorant detection relies on neurons present in the olfactory epithelium supported by sustentacular cells. The latter are massively infected by SARS-CoV-2 along with infiltration of innate immune cells and desquamation of the olfactory epithelium. This destruction leads to release of olfactory epithelium cells into the lumen of the nasal cavity, but the extent of the loss of mature olfactory neurons remains to be clarified. In this study, we compared the spatiotemporal evolution of the olfactory epithelium during SARS-CoV-2 infection with associated smell impairment in hamsters. The olfactory performance of infected hamsters decreases as early as 2 days post-infection (dpi), then gradually recovers through 17 dpi. While the infection is mostly resolved after 4 dpi in the nasal cavity, we observed a subacute decrease of the mature olfactory neuron population which almost completely disappear at 11 dpi. Furthermore, regeneration of the olfactory epithelium does not start until 8 dpi and leads to a high fraction of immature olfactory neurons. The delayed regeneration and persistent alteration of the olfactory neuron population was correlated with a prolonged expression of inflammatory cytokines and a rapid decrease of the levels of anti-inflammatory markers linked to regeneration. Overall, our results suggest that the regeneration process is altered in some areas of the olfactory epithelium leading to delayed recovery of the epithelium. The later may explain the prolonged smell alteration linked to SARS-CoV-2 infection
Citrulline supplementation does not reverse the effects of late gestation heat stress in ewes on feto-placental development
No full textInternational audienceHeat stress reduces fetal growth in late gestation in cattle, driven by shifts in placental form and function. Sheep are short-day breeders, but they are also bred off-season, allowing late summer lambing, which is associated with heat stress exposure in late pregnancy. Citrulline is known to induce nitric oxide release and vasodilatation and may not be degraded by the rumen, but its impacts on placental function are unknown. This study aimed to: 1) evaluate the effects of late gestation heat stress on placental/fetal development in ewes; and 2) evaluate if citrulline supplementation mitigates heat stress impacts. To that end, 28 pregnant ewes were randomly assigned to each treatment in environmental chambers: control (CN; n = 14, 18 degrees C) or heat stress (HT; n = 14: 28 degrees C daytime and 25 degrees C nighttime) during their last month of gestation. Within temperatures, animals received citrulline (0.5 % dry matter intake (DMI), CT) or not (NO), resulting in a 2 x 2 factorial design: CNCT, CNNO, HTCT, and HTNO (n = 7/trt). Respiration rates (RR) and rectal temperature (RT) were measured once weekly and placental perfusion was estimated by quantitative power Doppler ultrasonography at the beginning and 15 days after the start of the experiment. Gestation length (GL) and lamb birth weight (BW) were recorded. Placentas were collected at spontaneous delivery (3 +/- 0.66 h postpartum). Data analysis used the GLIMMIX procedure in SAS with fixed effects of ewe, lamb sex, category, treatments, and their interaction. In ewes, HT increased RR, whereas CT decreased RT in both treatments. HT decreased GL and lamb BW tended to be lower in the HTCT group. Placental morphology did not differ among treatments, but female twins had greater cotyledon number and lower placental efficiency. In conclusion, exposure of ewes to HT during late gestation reduced GL, confirming observations in cattle, and CT tended to reduce BW in heat stress conditions. Further investigations are ongoing in placental function and transcriptomics are currently being evaluated
Transcriptomic profiling reveals similarities between equine IVF and ICSI embryos
No full textInternational audienceIn vitro production of equine embryos has been performed using intracytoplasmic sperm injection (ICSI) for the last two decades. Since 2022, a repeatable protocol for conventional in vitro fertilization (IVF) provides a successful alternative. However, little is known about the influence of the fertilization method on embryo quality and the transcriptomic profile. In this study, we aimed to examine differentially expressed genes (DEGs) between ICSI and IVF embryos in the horse. Therefore, ten equine sibling blastocysts, produced in vitro by either ICSI or IVF from three different mares, were subjected to Full-Length Single-Cell RNA-Sequencing (FLASH-sequencing). As such, 11,518 genes were identified, with no DEGs between ICSI and IVF embryos. Cleavage rates, calculated on collected COCs, of IVF zygotes (55.0 %) were similar to those of ICSI zygotes (51.9 %; P = 0.74), but blastocyst rates were higher following ICSI (37.0 % vs 22.5 % calculated on collected COCs and 71.4 % vs 40.9 % calculated on cleaved zygotes; P = 0.04 and 0.004, respectively). The average day of blastocyst development did not differ (P = 0.55). In conclusion, gene expression was similar for the two fertilization techniques, supporting the safety of equine IVF for further clinical studies. Overall, the horse provides a valuable model to study longterm effects of assisted reproductive technologies with potential extrapolation to human medicine