Characterization of intestinal mononuclear phagocyte subsets in young ruminants at homeostasis and during Cryptosporidium parvum infection

IntroductionCryptosporidiosis is a poorly controlled zoonosis caused by an intestinal parasite, Cryptosporidium parvum, with a high prevalence in livestock (cattle, sheep, and goats). Young animals are particularly susceptible to this infection due to the immaturity of their intestinal immune system. In a neonatal mouse model, we previously demonstrated the importance of the innate immunity and particularly of type 1 conventional dendritic cells (cDC1) among mononuclear phagocytes (MPs) in controlling the acute phase of C. parvum infection. These immune populations are well described in mice and humans, but their fine characterization in the intestine of young ruminants remained to be further explored.MethodsImmune cells of the small intestinal Peyer’s patches and of the distal jejunum were isolated from naive lambs and calves at different ages. This was followed by their fine characterization by flow cytometry and transcriptomic analyses (q-RT-PCR and single cell RNAseq (lamb cells)). Newborn animals were infected with C. parvum, clinical signs and parasite burden were quantified, and isolated MP cells were characterized by flow cytometry in comparison with age matched control animals.ResultsHere, we identified one population of macrophages and three subsets of cDC (cDC1, cDC2, and a minor cDC subset with migratory properties) in the intestine of lamb and calf by phenotypic and targeted gene expression analyses. Unsupervised single-cell transcriptomic analysis confirmed the identification of these four intestinal MP subpopulations in lamb, while highlighting a deeper diversity of cell subsets among monocytic and dendritic cells. We demonstrated a weak proportion of cDC1 in the intestine of highly susceptible newborn lambs together with an increase of these cells within the first days of life and in response to the infection.DiscussionConsidering cDC1 importance for efficient parasite control in the mouse model, one may speculate that the cDC1/cDC2 ratio plays also a key role for the efficient control of C. parvum in young ruminants. In this study, we established the first fine characterization of intestinal MP subsets in young lambs and calves providing new insights for comparative immunology of the intestinal MP system across species and for future investigations on host–Cryptosporidium interactions in target species

Schmallenberg virus experimental infection of sheep

International audienceSince late 2011, a novel orthobunyavirus, named Schmallenberg virus (SBV), has been implicated in many cases of severely malformed bovine and ovine offspring in Europe. In adult cattle, SBV is known to cause a mild transient disease; clinical signs include short febrile episodes, decreased milk production and diarrhoea for a few days. However, the knowledge about clinical signs and pathogenesis in adult sheep is limited. In the present study, adult sheep of European domestic breeds were inoculated with SBV either as cell culture grown virus or as virus with no history of passage in cell cultures. Various experimental set-ups were used. Sampling included blood collection at different time points during the experimental period and selected organ material at autopsy. Data from this study showed, that the RNAemic period in sheep was as short as reported for cattle; viral genome was detectable for about 3-5 days by real-time RT-PCR. In total, 13 out of 30 inoculated sheep became RNAemic, with the highest viral load in animals inoculated with virus from low cell culture passaged or the animal passaged material. Contact animals remained negative throughout the study. One RNAemic sheep showed diarrhoea for several days, but fever was not recorded in any of the animals. Antibodies were first detectable 10-14 days post inoculation. Viral RNA was detectable in spleen and lymph nodes up to day 44 post inoculation. In conclusion, as described for cattle, SBV-infection in adult sheep predominantly results in subclinical infection, transient RNAemia and a specific antibody response. Maintenance of viral RNA in the lymphoreticular system is observed for an extended period

CIMAC. Chirurgie - Imagerie médicale - Anesthésie en Confinement

Présentation du poster en 180 sec de l'activité des équipes expérimentation / élevage de la PFIENational audienc

Le système de management de la qualité : un outil précieux pour la gestion du risque biologique dans les installations de haute sécurité utilisées en infectiologie expérimentale

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Effect of Amoxicillin-Clavulanic Acid on Human Fecal Flora in a Gnotobiotic Mouse Model Assessed with Fluorescence Hybridization Using Group-Specific 16S rRNA Probes in Combination with Flow Cytometry

Predominant groups of bacteria from a human fecal flora-associated mouse model challenged with amoxicillin-clavulanic acid were quantified with fluorescence in situ hybridization combined with flow cytometry using specific 16S rRNA targeted oligonucleotide probes. This approach provides a useful tool with high throughput to evaluate fecal microflora under antibiotic treatment

Characterization of intestinal macrophages and dendritic cell subsets in neonatal lambs at homeostasis and following Cryptosporidium parvum infection

International audienceCryptosporidiosis is a poorly controlled zoonosis caused by an intestinal parasite, Cryptosporidium parvum (Cp), with a high prevalence in livestock (cattle, sheep, goats). Young animals are particularly susceptible to this infection due to the immaturity of their intestinal immune system. In a neonatal mouse model, we previously demonstrated the importance of the innate immunity and in particular of CD11c+CD103+CD11b-conventional dendritic cells (cDC) among mononuclear phagocytes (MP) in controlling the acute phase of Cp infection. During infection, in response to chemokine production by infected epithelial cells, newly recruited cDCs produce IL12 and IFNγ contributing to the elimination of the parasite. According to the well-established mouse cDC classification, this Batf3+DC subpopulation corresponds to the cDC1 subset. The aim of this project was to better characterize intestinal MP subpopulations in neonatal lamb and calf at homeostasis and during Cp infection. As in the mouse model, the parasite invades and multiplies mainly in the ileum of animals. However, a peculiarity of young ruminants is the presence of a large ileal Peyer’s patch (lymphoid tissue) that extends all along the ileum. MP were therefore analyzed in lymphoid and non-lymphoid intestinal tissues of lambs and claves. We performed phenotypic and functional analyses of mononuclear phagocytes by flow cytometry and by transcriptomic methods (FLUIDIGM®) respectively, in the distal jejunum, jejunal and ileal Peyer’s patches. We characterized a population of macrophages and three subpopulations of cDC. We demonstrated that the subset identified as cDC1, according to the current common classification of cDC in different species (human, mouse, pig, sheep and chicken), increases with the age of animal. This might be linked with the decrease in sensitivity to C. parvum observed with the age. We are currently investigating the evolution of cDC1 subset during C. parvum infection

Characterization of Intestinal mononuclear phagocyte subsets of young lamb at homeostasis by single cell RNA-Seq and during Cryptosporidium parvum infection by flow cytometry.

International audienceIntestinal mononuclear phagocytes are key immune cells that maintain intestinal homeostasis and participate in the protective immune responses toward pathogens. Cryptosporidiosis is a poorly controlled zoonosis caused by an intestinal parasite, Cryptosporidium parvum (Cp), with a high prevalence in ruminant farms. Young animals are particularly susceptible to this infection due to the immaturity of their intestinal immune system. In a neonatal mouse model, we previously demonstrated the importance of the innate immunity in controlling the acute phase of Cp infection and deciphered the role of different subsets of intestinal mononuclear phagocytes in this protective immune response. The aim of this project was to better characterize intestinal mononuclear phagocytes in lamb at homeostasis and during Cp infection. The parasite invades and multiplies mainly in the ileum of animals. However, a peculiarity of young ruminants is the presence of a large ileal Peyer’s patch (IPP) (lymphoid tissue) that extends all along the ileum. We first performed a characterization of mononuclear phagocytes present in the IPP of a non-infected 10-day-old lamb by a single cell transcriptomic approach on CD11c+ MHCII+ sorted cells. This global approach allowed us to identify in the IPP of young lamb two main populations of macrophages and at least three different population of dendritic cells. Then, we carried out phenotypic and functional analyses of these different cell subsets by flow cytometry and transcriptomic methods in various compartments of the small intestine at homeostasis and during infection

Établissement d'un modèle d'anse intestinale chez l'agneau nouveau-né afin d'évaluer de nouvelles méthodes de lutte contre les maladies entériques et de réduire l'utilisation d'animaux de laboratoire

National audienceDans le but d'évaluer des alternatives naturelles pour contrôler la cryptosporidiose, infection entérique parasitaire affectant principalement les jeunes ruminants d'espèce de rente mais également l'être humain (zoonose), nous utilisons les agneaux comme espèce cible et comme modèle pour les animaux de plus grande taille comme les veaux. Nous avons pour objectif de stimuler les réponses immunitaires des animaux dès la naissance avec du colostrum supplémenté avec des produits naturels tels que des produits dérivés de levures qui contiennent des ligands des récepteurs de l'immunité innée. Cependant, la tolérance immunitaire intestinale s'installe rapidement après la naissance en réponse à la colonisation microbienne débutant dès la mise-bas par voie naturelle et par la suite via le colostrum, le lait et les multiples contacts avec l'environnement, et se caractérise par une hypo-réactivité aux antigènes microbiens (cf. modèles murins). Nous avons donc développé le modèle de chirurgie des anses intestinales sur agneaux nouveau-nés mis au monde par césarienne (et donc sans flore commensale) adapté à l'étude des interactions hôte-agent pathogène dans un environnement contrôlé et à l'évaluation de nouveaux composés antiparasitaires naturels in vivo. La possibilité de comparer différents produits naturels simultanément ainsi que des effets doses sur ce modèle, permet de réduire considérablement le nombre d’animaux expérimentaux à utiliser.Pour faciliter la réalisation de ces protocoles expérimentaux, nous utilisons notre troupeau d'ovins qui synchronise les mises-bas.Le jour de la chirurgie, la brebis est transférée dans la salle de chirurgie (confinement de niveau 2). Une césarienne est pratiquée et les agneaux sont réanimés. Après une courte phase de récupération, un agneau est choisi et transféré en salle de chirurgie. Il est installé sur une table de réanimation néonatale.Le protocole d'anesthésie / analgésie comprend : induction au masque (isoflurane), pose d'une sonde endotrachéale sous anesthésie locale (xylocaïne), maintien sous isoflurane (ventilation contrôlée), analgésie parentérale (buprénorphine), pose d'un cathéter veineux et mise sous perfusion, anesthésie locale traçante (lidocaïne).Pendant toute la procédure, les paramètres suivants sont surveillés : fréquences cardiaque et respiratoire, température rectale, SaO2, ETCO2.Une chirurgie à 4 mains est ensuite réalisée :- Extériorisation de la jonction iléo-caecale (IC) et de la portion distale de l'intestin grêle (IG)) ;- Ligatures avec création en général de triplicats - séparés par un inter-segment vide de "sécurité", y compris en amont et en aval de l'anse - (9 triplicats pour une anse de 50 cm environ) ;- Entérotomie en aval de l'anse ainsi créée, à proximité de la jonction IC, suture de l'intestin du côté de l'anse, puis idem en amont ;- Anastomose termino-terminale ;- Injection des préparations d'intérêt dans chaque segment.Pendant la chirurgie, une irrigation régulière de la portion externalisée de l'intestin est réalisée au sérum physiologique tiédi

Intestinal mononuclear phagocyte subsets of young ruminants : Characterization at homeostasis and during Cryptosporidium parvum infection

International audienceCryptosporidiosis is a poorly controlled zoonosis caused by an intestinal parasite, Cryptosporidium parvum (Cp), with a high prevalence in ruminant farms. Young animals are particularly susceptible to this infection due to the immaturity of their intestinal immune system. In a neonatal mouse model, we demonstrated the importance of the innate immunity and in particular of CD11c+CD103+CD11b- dendritic cells (DC) in controlling the acute phase of Cp infection. During infection, in response to chemokine production by infected epithelial cells, newly recruited CD11c+CD103+CD11b- DC produce IL12 and IFNγ contributing to the parasite elimination process. According to the common classification of DC in different species (human, mouse, pig, sheep), this subpopulation can be identified as the cDC1 subset of conventional DC. The aim of this project is to better characterize intestinal DC subpopulations in lamb and calf at homeostasis and during Cp infection. The parasite invades and multiplies mainly in the ileum of animals. However, a peculiarity of young ruminants is the presence of a large ileal Peyer’s patch (lymphoid tissue) that extends all along the ileum. We therefore performed phenotypic and functional analyses of mononuclear phagocytes by flow cytometry and transcriptomic methods in various compartments of the small intestine. We characterized one population of macrophages and three subpopulations of DC. We demonstrated that the subset identified as cDC1 increases according to the age of animal, and this might be linked with the decrease in sensitivity to C. parvum observed with the age. We are currently investigating cDC1 subset during C. parvum infection

A Rift Valley fever virus Gn ectodomain-based DNA vaccine induces a partial protection not improved by APC targeting

Rift Valley fever: DNA vaccines hold promise, but need work A vaccine made from the genome of Rift Valley fever virus (RVFV) offers partial protection, but pieces of the puzzle are missing, say scientists. French and Spanish researchers, led by the French National Institute for Agricultural Research’s Isabelle Schwartz-Cornil, tested in sheep three slightly-differing vaccine candidates using RVFV genes. Such DNA vaccines are designed to generate proteins which a host’s immune system can use to arm itself against a genuine viral infection. Two of the candidates, designed to target cells that would present the viral proteins to the host’s immune system, provided some benefit to the vaccinated sheep. However, the third untargeted candidate, was the most efficient at protecting sheep, although not completely, and at boosting antibody levels despite not neutralizing the virus. These results provide hope for DNA vaccines against RVFV, and offer direction for future research effort