Emerg’in, Infrastructure Nationale de Recherche pour la lutte contre les maladies infectieuses animales émergentes ou zoonotiques par l'exploration in vivo.
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HAL : Dernières publications

  • [anses-04261882] Investigations into SARS-CoV-2 and other coronaviruses on mink farms in France late in the first year of the COVID-19 pandemic

    Soon after the beginning of the COVID-19 pandemic in early 2020, the Betacoronavirus SARS-CoV-2 infection of several mink farms breeding American minks ( Neovison vison ) for fur was detected in various European countries. The risk of a new reservoir being formed and of a reverse zoonosis from minks quickly became a major concern. The aim of this study was to investigate the four French mink farms to see whether SARS-CoV-2 was circulating there in late 2020. The investigations took place during the slaughtering period, thus facilitating different types of sampling (swabs and blood). On one of the four mink farms, 96.6% of serum samples were positive when tested with a SARS-CoV-2 ELISA coated with purified N protein recombinant antigen, and 54 out of 162 (33%) pharyngo-tracheal swabs were positive by RT-qPCR. The genetic variability among 12 SARS-CoV-2 genomes sequenced from this farm indicated the co-circulation of several lineages at the time of sampling. All the SARS-CoV-2 genomes detected were nested within the 20A clade (Nextclade), together with SARS-CoV-2 genomes from humans sampled during the same period. The percentage of SARS-CoV-2 seropositivity by ELISA varied between 0.3 and 1.1% on the other three farms. Interestingly, among these three farms, 11 pharyngo-tracheal swabs and 3 fecal pools from two farms were positive by end-point RT-PCR for an Alphacoronavirus very similar to a mink coronavirus sequence observed on Danish farms in 2015. In addition, a mink Caliciviridae was identified on one of the two farms positive for Alphacoronavirus . The clinical impact of these inapparent viral infections is not known. The co-infection of SARS-CoV-2 with other viruses on mink farms could help explain the diversity of clinical symptoms noted on different infected farms in Europe. In addition, the co-circulation of an Alphacoronavirus and SARS-CoV-2 on a mink farm would potentially increase the risk of viral recombination between alpha and betacoronaviruses as already suggested in wild and domestic animals, as well as in humans.

    ano.nymous@ccsd.cnrs.fr.invalid (Marine Wasniewski) 27 Oct 2023

    https://anses.hal.science/anses-04261882v1
  • [anses-04228409] Controlled Experimental Infection in Pigs with a Strain of Yersinia enterocolitica Harboring Genetic Markers for Human Pathogenicity: Colonization and Stability

    Yersinia enterocolitica (Ye) is one of the major causes of foodborne zoonosis. The BT4/O:3 bioserotype is most commonly isolated in human infections. Pigs are considered the main reservoir of Ye, and hence, understanding the dynamics of infection by this pathogen at the individual and group levels is crucial. In the present study, an experimental model was validated in Large White pigs infected with a BT4/O:3 strain. This study showed that Ye contamination in pigs may occur via the introduction of the bacteria not only by mouth but also by snout, with a colonization process consisting of three periods corresponding to three contamination statuses of pigs: P1, corresponding to the 24 h following ingestion or inhalation of Ye with the appearance of bacteria in tonsils or in feces; P2, from 2 days postinoculation (dpi), corresponding to expansion of Ye and colonization of the digestive system and extraintestinal organs associated with an IgG serological response; and P3, after 21 dpi, corresponding to regression of colonization with intermittent Ye detection in tonsils and feces. Although the inoculated strain persisted up to 56 dpi in all pigs, genetic variations with the loss of the gene yadA (a gene involved in human infection) and the emergence of two new multilocus variable-number tandemrepeat analysis (MLVA) profiles were observed in 33% of the 30 isolates studied. This experimental infection model of pigs by Ye provides new insights into the colonization steps in pigs in terms of bacterial distribution over time and bacterial genetic stability.

    ano.nymous@ccsd.cnrs.fr.invalid (Emilie Esnault) 04 Oct 2023

    https://anses.hal.science/anses-04228409v1
  • [hal-04416438] Microbiota promotes recruitment and pro-inflammatory response of caecal macrophages during E. tenella infection

    Background: Eimeria genus belongs to the apicomplexan parasite phylum and is responsible for coccidiosis, an intestinal disease with a major economic impact on poultry production. Eimeria tenella is one of the most virulent species in chickens. In a previous study, we showed a negative impact of caecal microbiota on the physiopathology of this infection. However, the mechanism by which microbiota leads to the physiopathology remained undetermined. Macrophages play a key role in inflammatory processes and their interaction with the microbiota during E. tenella infection have never been investigated. We therefore examined the impact of microbiota on macrophages during E. tenella infection. Macrophages were monitored in caecal tissues by immunofluorescence staining with KUL01 antibody in non-infected and infected germ-free and conventional chickens. Caecal cells were isolated, stained, analyzed and sorted to examine their gene expression using high-throughput qPCR. Results: We demonstrated that microbiota was essential for caecal macrophage recruitment in E. tenella infection. Furthermore, microbiota promoted a pro-inflammatory transcriptomic profile of macrophages characterized by increased gene expression of NOS2, ACOD1, PTGS2, TNFα, IL1β, IL6, IL8L1, IL8L2 and CCL20 in infected chickens. Administration of caecal microbiota from conventional chickens to germ-free infected chickens partially restored macrophage recruitment and response. Conclusions: Taken together, these results suggest that the microbiota enhances the physiopathology of this infection through macrophage recruitment and activation. Consequently, strategies involving modulation of the gut microbiota may lead to attenuation of the macrophage-mediated inflammatory response, thereby limiting the negative clinical outcome of the disease.

    ano.nymous@ccsd.cnrs.fr.invalid (Florian Tomal) 25 Jan 2024

    https://hal.inrae.fr/hal-04416438v1
  • [hal-04067308] Vector competence of sterile male Glossina fuscipes fuscipes for Trypanosoma brucei brucei: implications for the implementation of the sterile insect technique in a sleeping sickness focus in Chad

    Background Human African trypanosomiasis (HAT) is a neglected tropical disease caused by Trypanosoma brucei gambiense transmitted by tsetse flies in sub-Saharan West Africa. In southern Chad the most active and persistent focus is the Mandoul focus, with 98% of the reported human cases, and where African animal trypanosomosis (AAT) is also present. Recently, a control project to eliminate tsetse flies ( Glossina fuscipes fuscipes ) in this focus using the sterile insect technique (SIT) was initiated. However, the release of large numbers of sterile males of G. f. fuscipes might result in a potential temporary increase in transmission of trypanosomes since male tsetse flies are also able to transmit the parasite. The objective of this work was therefore to experimentally assess the vector competence of sterile males treated with isometamidium for Trypanosoma brucei brucei . Methods An experimental infection was set up in the laboratory, mimicking field conditions: the same tsetse species that is present in Mandoul was used. A T. b. brucei strain close to T. b. gambiense was used, and the ability of the sterile male tsetse flies fed on blood with and without a trypanocide to acquire and transmit trypanosomes was measured. Results Only 2% of the experimentally infected flies developed an immature infection (midgut) while none of the flies developed a metacyclic infection of T. b. brucei in the salivary glands. We did not observe any effect of the trypanocide used (isometamidium chloride at 100 mg/l) on the development of infection in the flies. Conclusions Our results indicate that sterile males of the tested strain of G. f. fuscipes were unable to cyclically transmit T. b. brucei and might even be refractory to the infection. The data of the research indicate that the risk of cyclical transmission of T. brucei by sterile male G. f. fuscipes of the strain colonized at IAEA for almost 40 years appears to be small. Graphical Abstract

    ano.nymous@ccsd.cnrs.fr.invalid (Mahamat Hissene Mahamat) 13 Apr 2023

    https://hal.inrae.fr/hal-04067308v1
  • [hal-04174029] Flying syringes for emerging enzootic virus screening: proof of concept for the devlopment of noninvasive xenosurveillance tools based on Tsetse flies

    Pathogen transfers between wild and domestic animals and between animals and humans are increasing. Their dramatic consequences for public and veterinary health as well as for conservation call for innovative and user-friendly methods for pathogen surveillance in wildlife. Xenosurveillance, a method based on the use of invertebrates (e.g., mosquitoes, hematophagous flies, leeches, cadaveric arthropods) to sample animal tissues (e.g., blood) and the associated pathogens, is one of these tools. Previously, we demonstrated that hematophagous flies, such as tsetse flies, could be useful to detect and identify the etiological agents of malaria in a diverse range of mammals in Gabon. However, we did not assess whether this method can be also used to detect viruses. In the present study, we experimentally fed tsetse flies (Glossina fuscipes fuscipes) rabbit blood containing different viruses of medical or veterinary importance (Zika, Dengue, Chikungunya, African swine fever, Bluetongue, and peste des petits ruminants viruses). Then, we used quantitative PCR (i) to determine for how long viral nucleic acid fragments remained detectable in the tsetse midgut during blood digestion and (ii) to compare two blood meal preservation methods (i.e., FTA cards and RNAlater solution) tested using tsetse flies engorged with blood and dengue-2 virus. All viruses remained detectable for 6 days after feeding, although the detection probability significantly decreased over time. FTA cards and RNAlater solution gave similar results in terms of virus detection. Our results demonstrate that xenosurveillance using blood-engorged tsetse flies is a valuable tool to track and survey viruses in wildlife in Sub-Saharan Africa.

    ano.nymous@ccsd.cnrs.fr.invalid (Adeline Valente) 25 Sep 2023

    https://hal.science/hal-04174029v1
  • [hal-04116796] DNA methylation and gene expression changes in mouse mammary tissue during successive lactations: part II – the impact of lactation rank

    Mastitis is among the main reasons women cease breastfeeding. In farm animals, mastitis results in significant economic losses and the premature culling of some animals. Nevertheless, the effect of inflammation on the mammary gland is not completely understood. This article discusses the changes to DNA methylation in mouse mammary tissue caused by lipopolysaccharide-induced inflammation after in vivo intramammary challenges and the differences in DNA methylation between 1st and 2nd lactations. Lactation rank induces 981 differential methylations of cytosines (DMCs) in mammary tissue. Inflammation in 1st lactation compared to inflammation in 2nd lactation results in the identification of 964 DMCs. When comparing inflammation in 1st vs. 2nd lactations with previous inflammation history, 2590 DMCs were identified. Moreover, Fluidigm PCR data show changes in the expression of several genes related to mammary function, epigenetic regulation, and the immune response. We show that the epigenetic regulation of two successive physiological lactations is not the same in terms of DNA methylation and that the effect of lactation rank on DNA methylation is stronger than that of the onset of inflammation. The conditions presented here show that few DMCs are shared between comparisons, suggesting a specific epigenetic response depending on lactation rank, the presence of inflammation, and even whether the cells had previously suffered inflammation. In the long term, this information could lead to a better understanding of the epigenetic regulation of lactation in both physiological and pathological conditions.

    ano.nymous@ccsd.cnrs.fr.invalid (E Ivanova) 05 Jun 2023

    https://hal.inrae.fr/hal-04116796v1
  • [hal-04083477] Study of the effect of administration of narasin or antibiotics on in vivo selection of a narasin- and multidrug-resistant Enterococcus cecorum strain

    Enterococcus cecorum is a member of the normal poultry gut microbiota and an emerging poultry pathogen. Some strains are resistant to key antibiotics and coccidiostats. We evaluated the impact on chicken excretion and persistence of a multidrug-resistant E. cecorum of administering narasin or antibiotics. E. cecorum CIRMBP-1294 (Ec1294) is non-wild-type to many antimicrobials, including narasin, levofloxacin, oxytetracycline and glycopeptides, it has a low susceptibility to amoxicillin, and carries a chromosomal vanA operon. Six groups of 15 chicks each were orally inoculated with Ec1294 and two groups were left untreated. Amoxicillin, oxytetracycline or narasin were administered orally to one group each, either at the recommended dose for five days (amoxicillin, oxytetracycline) or continuously (narasin). Faecal samples were collected weekly and caecal samples were obtained from sacrificed birds on day 28. Ec1294 titres were evaluated by culture on vancomycin- and levofloxacin-supplemented media in 5 % CO2. For inoculated birds given narasin, oxytetracycline or no antimicrobials, vancomycin-resistant enterococci were searched by culture on vancomycin-supplemented media incubated in air, and a PCR was used to detect the vanA gene. Ec1294 persisted in inoculated chicks up to day 28. Compared to the control group, the Ec1294 titre was significantly lower in the amoxicillin- and narasin-receiving groups on days 21 and 28, but was unexpectedly higher in the oxytetracycline-receiving group before and after oxytetracycline administration, preventing a conclusion for this group. No transfer of the vanA gene to other enterococci was detected. Other trials in various experimental conditions should now be conducted to confirm this apparent absence of co-selection of the multi-drug-resistant E. cecorum by narasin or amoxicillin administration.

    ano.nymous@ccsd.cnrs.fr.invalid (Jeanne Laurentie) 27 Apr 2023

    https://hal.inrae.fr/hal-04083477v1
  • [anses-04188278] Antimicrobial resistance of Enterococcus cecorum: ECOFF determination

    Enterococcus cecorum, a commensal Gram-positive bacterium of the chicken gut, has emerged as a worldwide cause of lameness in poultry, particularly in fast-growing broilers. It is responsible for osteomyelitis, spondylitis and femoral head necrosis, causing animal suffering, mortality and antimicrobial use. Research on the antimicrobial resistance of E. cecorum clinical isolates in France is scarce, and epidemiological cut-off (ECOFF) values unknown. To determine tentative ECOFF (COWT) values for E. cecorum and to investigate the antimicrobial resistance patterns of isolates from mainly French broilers, we tested the susceptibility of a collection of commensal and clinical isolates (n=208) to 29 antimicrobials by the disc diffusion (DD) method. We also determined the minimum inhibitory concentrations (MICs) of 23 antimicrobials by the broth micro-dilution method. To detect chromosomal mutations conferring antimicrobial resistance, we investigated the genomes of 118 E. cecorum isolates mainly obtained from infectious sites and previously described in the literature. We determined the COWT values for more than 20 antimicrobials and identified two chromosomal mutations explaining fluoroquinolone resistance. The DD method appears better suited for detecting E. cecorum antimicrobial resistance. Although tetracycline and erythromycin resistances were persistent in clinical and non-clinical isolates, we found little or no resistance to medically important antimicrobials.

    ano.nymous@ccsd.cnrs.fr.invalid (Jeanne Laurentie) 25 Aug 2023

    https://anses.hal.science/anses-04188278v1
  • [hal-04116035] Whole‐genome sequencing identifies interferon-induced protein IFI6/IFI27-like as a strong candidate gene for VNN resistance in European sea bass

    Background Viral nervous necrosis (VNN) is a major disease that affects European sea bass, and understanding the biological mechanisms that underlie VNN resistance is important for the welfare of farmed fish and sustainability of production systems. The aim of this study was to identify genomic regions and genes that are associated with VNN resistance in sea bass. Results We generated a dataset of 838,451 single nucleotide polymorphisms (SNPs) identified from whole-genome sequencing (WGS) in the parental generation of two commercial populations (A: 2371 individuals and B: 3428 individuals) of European sea bass with phenotypic records for binary survival in a VNN challenge. For each population, three cohorts were submitted to a red-spotted grouper nervous necrosis virus (RGNNV) challenge by immersion and genotyped on a 57K SNP chip. After imputation of WGS SNPs from their parents, quantitative trait loci (QTL) were mapped using a Bayesian sparse linear mixed model (BSLMM). We found several QTL regions that were specific to one of the populations on different linkage groups (LG), and one 127-kb QTL region on LG12 that was shared by both populations and included the genes ZDHHC14, which encodes a palmitoyltransferase, and IFI6/IFI27-like, which encodes an interferon-alpha induced protein. The most significant SNP in this QTL region was only 1.9 kb downstream of the coding sequence of the IFI6/IFI27-like gene. An unrelated population of four large families was used to validate the effect of the QTL. Survival rates of susceptible genotypes were 40.6% and 45.4% in populations A and B, respectively, while that of the resistant genotype was 66.2% in population B and 78% in population A. Conclusions We have identified a genomic region that carries a major QTL for resistance to VNN and includes the ZDHHC14 and IFI6/IFI27-like genes. The potential involvement of the interferon pathway, a well-known anti-viral defense mechanism in several organisms (chicken, human, or fish), in survival to VNN infection is of particular interest. Our results can lead to major improvements for sea bass breeding programs through marker-assisted genomic selection to obtain more resistant fish.

    ano.nymous@ccsd.cnrs.fr.invalid (Emilie Delpuech) 02 Jun 2023

    https://hal.inrae.fr/hal-04116035v1
  • [hal-04197332] EcoXtract® green solvent increases rainbow trout performances and decreases susceptibility to VHSV

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    ano.nymous@ccsd.cnrs.fr.invalid (Dimitri Rigaudeau) 06 Sep 2023

    https://hal.inrae.fr/hal-04197332v1
  • [hal-04229569] The Smallest Infectious Substructure Encoding the Prion Strain Structural Determinant Revealed by Spontaneous Dissociation of Misfolded Prion Protein Assemblies

    It is commonly accepted that the prion replicative propensity and strain structural determinant (SSD) are encoded in the fold of PrPSc amyloid fibril assemblies. By exploring the quaternary structure dynamicity of several prion strains, we revealed that all mammalian prion assemblies exhibit the generic property of spontaneously generating two sets of discreet infectious tetrameric and dimeric species differing significantly by their specific infectivity. By using perturbation approaches such as dilution and ionic strength variation, we demonstrated that these two oligomeric species were highly dynamic and evolved differently in the presence of chaotropic agents. In general, our observations of seven different prion strains from three distinct species highlight the high dynamicity of PrPSc assemblies as a common and intrinsic property of mammalian prions. The existence of such small infectious PrPSc species harboring the SSD indicates that the prion infectivity and the SSD are not restricted only to the amyloid fold but can also be encoded in other alternative quaternary structures. Such diversity in the quaternary structure of prion assemblies tends to indicate that the structure of PrPSc can be divided into two independent folding domains: a domain encoding the strain structural determinant and a second domain whose fold determines the type of quaternary structure that could adopt PrPSc assemblies.

    ano.nymous@ccsd.cnrs.fr.invalid (Jan Bohl) 23 Oct 2023

    https://hal.science/hal-04229569v1
  • [hal-04107725] Investigation of the Genus Flavobacterium as a Reservoir for Fish-Pathogenic Bacterial Species: the Case of Flavobacterium collinsii

    Bacteria of the genus Flavobacterium are recovered from a large variety of environments. Among the described species, Flavobacterium psychrophilum and Flavobacterium columnare cause considerable losses in fish farms. Alongside these well-known fish-pathogenic species, isolates belonging to the same genus recovered from diseased or apparently healthy wild, feral, and farmed fish have been suspected to be pathogenic. Here, we report the identification and genomic characterization of a Flavobacterium collinsii isolate (TRV642) retrieved from rainbow trout spleen. A phylogenetic tree of the genus built by aligning the core genome of 195 Flavobacterium species revealed that F. collinsii stands within a cluster of species associated with diseased fish, the closest one being F. tructae, which was recently confirmed as pathogenic. We evaluated the pathogenicity of F. collinsii TRV642 as well as of Flavobacterium bernardetii F-372T, another recently described species reported as a possible emerging pathogen. Following intramuscular injection challenges in rainbow trout, no clinical signs or mortalities were observed with F. bernardetii. F. collinsii showed very low virulence but was isolated from the internal organs of survivors, indicating that the bacterium is able to survive inside the host and may provoke disease in fish under compromised conditions such as stress and/or wounds. Our results suggest that members of a phylogenetic cluster of fish-associated Flavobacterium species may be opportunistic fish pathogens causing disease under specific circumstances. IMPORTANCE Aquaculture has expanded significantly worldwide in the last decades and accounts for half of human fish consumption. However, infectious fish diseases are a major bottleneck for its sustainable development, and an increasing number of bacterial species from diseased fish raise a great concern. The current study revealed phylogenetic associations with ecological niches among the Flavobacterium species. We also focused on Flavobacterium collinsii, which belongs to a group of putative pathogenic species. The genome contents revealed a versatile metabolic repertoire suggesting the use of diverse nutrient sources, a characteristic of saprophytic or commensal bacteria. In a rainbow trout experimental challenge, the bacterium survived inside the host, likely escaping clearance by the immune system but without provoking massive mortality, suggesting opportunistic pathogenic behavior. This study highlights the importance of experimentally evaluating the pathogenicity of the numerous bacterial species retrieved from diseased fis

    ano.nymous@ccsd.cnrs.fr.invalid (Bo-Hyung Lee) 05 Jun 2023

    https://hal.science/hal-04107725v1
  • [hal-04172998] Deficiency in hereditary hemorrhagic telangiectasia-associated Endoglin elicits hypoxia-driven heart failure in zebrafish

    Hereditary hemorrhagic telangiectasia (HHT) is a rare genetic disease caused by mutations affecting components of bone morphogenetic protein (BMP)/transforming growth factor-β (TGF-β) signaling in endothelial cells. This disorder is characterized by arteriovenous malformations that are prone to rupture, and the ensuing hemorrhages are responsible for iron-deficiency anemia. Along with activin receptor-like kinase (ALK1), mutations in endoglin are associated with the vast majority of HHT cases. In this study, we characterized the zebrafish endoglin locus and demonstrated that it produces two phylogenetically conserved protein isoforms. Functional analysis of a CRISPR/Cas9 zebrafish endoglin mutant revealed that Endoglin deficiency is lethal during the course from juvenile stage to adulthood. Endoglin-deficient zebrafish develop cardiomegaly, resulting in heart failure and hypochromic anemia, which both stem from chronic hypoxia. endoglin mutant zebrafish display structural alterations of the developing gills and underlying vascular network that coincide with hypoxia. Finally, phenylhydrazine treatment demonstrated that lowering hematocrit/blood viscosity alleviates heart failure and enhances the survival of Endoglin-deficient fish. Overall, our data link Endoglin deficiency to heart failure and establish zebrafish as a valuable HHT model.

    ano.nymous@ccsd.cnrs.fr.invalid (Etienne Lelièvre) 21 May 2024

    https://hal.inrae.fr/hal-04172998v1
  • [hal-04177128] Staphylococcus epidermidis isolates from atopic or healthy skin have opposite effect on skin cells: potential implication of the AHR pathway modulation

    Introduction S taphylococcus epidermidis is a commensal bacterium ubiquitously present on human skin. This species is considered as a key member of the healthy skin microbiota, involved in the defense against pathogens, modulating the immune system, and involved in wound repair. Simultaneously, S. epidermidis is the second cause of nosocomial infections and an overgrowth of S. epidermidis has been described in skin disorders such as atopic dermatitis. Diverse isolates of S. epidermidis co-exist on the skin. Elucidating the genetic and phenotypic specificities of these species in skin health and disease is key to better understand their role in various skin conditions. Additionally, the exact mechanisms by which commensals interact with host cells is partially understood. We hypothesized that S. epidermidis isolates identified from different skin origins could play distinct roles on skin differentiation and that these effects could be mediated by the aryl hydrocarbon receptor (AhR) pathway. Methods For this purpose, a library of 12 strains originated from healthy skin (non-hyperseborrheic (NH) and hyperseborrheic (H) skin types) and disease skin (atopic (AD) skin type) was characterized at the genomic and phenotypic levels. Results and discussion Here we showed that strains from atopic lesional skin alter the epidermis structure of a 3D reconstructed skin model whereas strains from NH healthy skin do not. All strains from NH healthy skin induced AhR/OVOL1 path and produced high quantities of indole metabolites in co-culture with NHEK; especially indole-3-aldehyde (IAld) and indole-3-lactic acid (ILA); while AD strains did not induce AhR/OVOL1 path but its inhibitor STAT6 and produced the lowest levels of indoles as compared to the other strains. As a consequence, strains from AD skin altered the differentiation markers FLG and DSG1. The results presented here, on a library of 12 strains, showed that S. epidermidis originated from NH healthy skin and atopic skin have opposite effects on the epidermal cohesion and structure and that these differences could be linked to their capacity to produce metabolites, which in turn could activate AHR pathway. Our results on a specific library of strains provide new insights into how S. epidermidis may interact with the skin to promote health or disease.

    ano.nymous@ccsd.cnrs.fr.invalid (Leslie Landemaine) 04 Aug 2023

    https://hal.inrae.fr/hal-04177128v1
  • [hal-04294675] Identification of a pharyngeal mucosal lymphoid organ in zebrafish and other teleosts: Tonsils in fish?

    The constant exposure of the fish branchial cavity to aquatic pathogens causes local mucosal immune responses to be extremely important for their survival. Here, we used a marker for T lymphocytes/natural killer (NK) cells (ZAP70) and advanced imaging techniques to investigate the lymphoid architecture of the zebrafish branchial cavity. We identified a sub-pharyngeal lymphoid organ, which we tentatively named "Nemausean lymphoid organ" (NELO). NELO is enriched in T/NK cells, plasma/B cells, and antigen-presenting cells embedded in a network of reticulated epithelial cells. The presence of activated T cells and lymphocyte proliferation, but not V(D)J recombination or hematopoiesis, suggests that NELO is a secondary lymphoid organ. In response to infection, NELO displays structural changes including the formation of T/NK cell clusters. NELO and gill lymphoid tissues form a cohesive unit within a large mucosal lymphoid network. Collectively, we reveal an unreported mucosal lymphoid organ reminiscent of mammalian tonsils that evolved in multiple teleost fish familie

    ano.nymous@ccsd.cnrs.fr.invalid (Julien Resseguier) 20 Nov 2023

    https://hal.science/hal-04294675v1
  • [hal-04271263] Non-Steroidal Estrogens Inhibit Influenza Virus by Interacting with Hemagglutinin and Preventing Viral Fusion

    Influenza virus is one of the main causes of respiratory infections worldwide. Despite the availability of seasonal vaccines and antivirals, influenza virus infections cause an important health and economic burden. Therefore, the need to identify alternative antiviral strategies persists. In this study, we identified non-steroidal estrogens as potent inhibitors of influenza virus due to their interaction with the hemagglutinin protein, preventing viral entry. This activity is maintained in vitro, ex vivo, and in vivo. Therefore, we found a new domain to target on the hemagglutinin and a class of compounds that could be further optimized for influenza treatment.

    ano.nymous@ccsd.cnrs.fr.invalid (Elisa Franzi) 10 Nov 2023

    https://hal.science/hal-04271263v1
  • [hal-04344510] Gut barrier-microbiota imbalances in early life lead to higher sensitivity to inflammation in a murine model of C-section delivery

    Background Most interactions between the host and its microbiota occur at the gut barrier, and primary colonizers are essential in the gut barrier maturation in the early life. The mother–offspring transmission of microorganisms is the most important factor influencing microbial colonization in mammals, and C-section delivery (CSD) is an important disruptive factor of this transfer. Recently, the deregulation of symbiotic host-microbe interactions in early life has been shown to alter the maturation of the immune system, predisposing the host to gut barrier dysfunction and inflammation. The main goal of this study is to decipher the role of the early-life gut microbiota-barrier alterations and its links with later-life risks of intestinal inflammation in a murine model of CSD. Results The higher sensitivity to chemically induced inflammation in CSD mice is related to excessive exposure to a too diverse microbiota too early in life. This early microbial stimulus has short-term consequences on the host homeostasis. It switches the pup’s immune response to an inflammatory context and alters the epithelium structure and the mucus-producing cells, disrupting gut homeostasis. This presence of a too diverse microbiota in the very early life involves a disproportionate short-chain fatty acids ratio and an excessive antigen exposure across the vulnerable gut barrier in the first days of life, before the gut closure. Besides, as shown by microbiota transfer experiments, the microbiota is causal in the high sensitivity of CSD mice to chemical-induced colitis and in most of the phenotypical parameters found altered in early life. Finally, supplementation with lactobacilli, the main bacterial group impacted by CSD in mice, reverts the higher sensitivity to inflammation in ex-germ-free mice colonized by CSD pups’ microbiota. Conclusions Early-life gut microbiota-host crosstalk alterations related to CSD could be the linchpin behind the phenotypic effects that lead to increased susceptibility to an induced inflammation later in life in mice.

    ano.nymous@ccsd.cnrs.fr.invalid (M. Barone) 30 May 2024

    https://hal.inrae.fr/hal-04344510v1
  • [hal-04189068] Modulation of gut microbiota by antibiotics did not affect anhedonia in a high-fat diet-induced model of depression in male mice

    Background: Long-term consumption of a high-fat diet (HFD) causes obesity and is a risk factor for depression. The HFD has a significant impact on the gut microbiota, and dysbiosis of the microbiota is now associated with certain psychiatric disorders such as anxiety and depression. We aimed to investigate whether modulation by antibiotic treatment of the composition of the gut microbiota in diet-induced obese (DIO) C57BL/6J male mice has an impact on depressive-like behaviour. Methods: In this study, we have analysed the effects of a 15 weeks HFD on helplessness assessed in the forced swim test and anhedonia assessed in the sucrose preference test. Two weeks before the start of the behavioural tests, a group of HFD mice were given a combination of two non-absorbable antibiotics, neomycin and polymyxin B. Results: In DIO mice, anhedonia and significant changes in the composition of the gut microbiota at the phyla and family level were observed. On the other hand, there was no significant effect of HFD on the peripheral inflammatory profile. In DIO mice, antibiotic treatment resulted in very pronounced alteration in the composition of the gut microbiota, without any change in anhedonia behaviour. Conclusion: In DIO mice, only four families of bacteria were not affected in their relative abundance by the antibiotic treatment, the Bifidobacteriaceae, Erysipelotrichaceae, Rikenellaceae and Streptococcaceae. This stability concomitant with that of anhedonia suggests that these families may be involved in anhedonia in DIO mice.

    ano.nymous@ccsd.cnrs.fr.invalid (Magali Monnoye) 28 Aug 2023

    https://hal.inrae.fr/hal-04189068v1
  • [hal-03943007] An Isolate of Streptococcus mitis Displayed In Vitro Antimicrobial Activity and Deleterious Effect in a Preclinical Model of Lung Infection

    Microbiota studies have dramatically increased over these last two decades, and the repertoire of microorganisms with potential health benefits has been considerably enlarged. The development of next generation probiotics from new bacterial candidates is a long-term strategy that may be more efficient and rapid with discriminative in vitro tests. Streptococcus strains have received attention regarding their antimicrobial potential against pathogens of the upper and, more recently, the lower respiratory tracts. Pathogenic bacterial strains, such as non-typable Haemophilus influenzae (NTHi), Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus), are commonly associated with acute and chronic respiratory diseases, and it could be interesting to fight against pathogens with probiotics. In this study, we show that a Streptococcus mitis (S. mitis) EM-371 strain, isolated from the buccal cavity of a human newborn and previously selected for promising anti-inflammatory effects, displayed in vitro antimicrobial activity against NTHi, P. aeruginosa or S. aureus. However, the anti-pathogenic in vitro activity was not sufficient to predict an efficient protective effect in a preclinical model. Two weeks of treatment with S. mitis EM-371 did not protect against, and even exacerbated, NTHi lung infection.

    ano.nymous@ccsd.cnrs.fr.invalid (Elliot Mathieu) 06 Dec 2023

    https://hal.science/hal-03943007v1
  • [hal-04057783] From In Vitro to In Vivo: A Rational Flowchart for the Selection and Characterization of Candidate Probiotic Strains in Intestinal Disorders

    Experimental and clinical evidence has demonstrated the potential of probiotic strains in the prevention or treatment of inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). However, there is little data on what the methodology leading to the identification of such strains should be. In this work, we propose a new flowchart to identify strains with probiotic potential for the management of IBS and IBD, which we tested on a collection of 39 lactic acid bacteria and Bifidobacteria strains. This flowchart included in vitro tests of immunomodulatory properties on intestinal and peripheral blood mononuclear cells (PBMCs), assessment of the barrier-strengthening effect by measuring transepithelial electric resistance (TEER) and quantification of short-chain fatty acids (SCFAs) and aryl hydrocarbon receptor (AhR) agonists produced by the strains. The in vitro results were then combined in a principal component analysis (PCA) to identify strains associated with an anti-inflammatory profile. To validate our flowchart, we tested the two most promising strains identified in the PCA in mouse models of post-infectious IBS or chemically induced colitis to mimic IBD. Our results show that this screening strategy allows the identification of strains with potential beneficial effects on colonic inflammation and colonic hypersensitivity.

    ano.nymous@ccsd.cnrs.fr.invalid (Flore Maillard) 06 Dec 2023

    https://uca.hal.science/hal-04057783v1
  • [hal-04197473] Technological developments and 3D imaging for the diagnosis of aquaculture fish diseases

    [...]

    ano.nymous@ccsd.cnrs.fr.invalid (M. Merhaz) 06 Sep 2023

    https://hal.inrae.fr/hal-04197473v1
  • [hal-04175302] Recombinant viral hemorrhagic septicemia virus with rearranged genomes as vaccine vectors to protect against lethal betanodavirus infection

    The outbreaks of viral hemorrhagic septicemia (VHS) and viral encephalopathy and retinopathy (VER) caused by the enveloped novirhabdovirus VHSV, and the non-enveloped betanodavirus nervous necrosis virus (NNV), respectively, represent two of the main viral infectious threats for aquaculture worldwide. Non-segmented negative-strand RNA viruses such as VHSV are subject to a transcription gradient dictated by the order of the genes in their genomes. With the goal of developing a bivalent vaccine against VHSV and NNV infection, the genome of VHSV has been engineered to modify the gene order and to introduce an expression cassette encoding the major protective antigen domain of NNV capsid protein. The NNV Linker-P specific domain was duplicated and fused to the signal peptide (SP) and the transmembrane domain (TM) derived from novirhabdovirus glycoprotein to obtain expression of antigen at the surface of infected cells and its incorporation into viral particles. By reverse genetics, eight recombinant VHSVs (rVHSV), termed NxGyCz according to the respective positions of the genes encoding the nucleoprotein (N) and glycoprotein (G) as well as the expression cassette (C) along the genome, have been successfully recovered. All rVHSVs have been fully characterized in vitro for NNV epitope expression in fish cells and incorporation into VHSV virions. Safety, immunogenicity and protective efficacy of rVHSVs has been tested in vivo in trout ( Oncorhynchus mykiss) and sole ( Solea senegalensis ). Following bath immersion administration of the various rVHSVs to juvenile trout, some of the rVHSVs were attenuated and protective against a lethal VHSV challenge. Results indicate that rVHSV N2G1C4 is safe and protective against VHSV challenge in trout. In parallel, juvenile sole were injected with rVHSVs and challenged with NNV. The rVHSV N2G1C4 is also safe, immunogenic and efficiently protects sole against a lethal NNV challenge, thus presenting a promising starting point for the development of a bivalent live attenuated vaccine candidate for the protection of these two commercially valuable fish species against two major diseases in aquaculture.

    ano.nymous@ccsd.cnrs.fr.invalid (Sandra Souto) 02 Aug 2023

    https://hal.inrae.fr/hal-04175302v1
  • [hal-04172440] Conserved and divergent arms of the antiviral response in the duplicated genomes of salmonid fishes

    Antiviral innate immunity is orchestrated by the interferon system, which appeared in ancestors of jawed vertebrates. Interferon upregulation induces hundreds of interferon-stimulated-genes (ISGs) with effector or regulatory functions. Here we investigated the evolutionary diversification of ISG responses through comparison of two salmonid fishes, accounting for the impact of sequential whole genome duplications ancestral to teleosts and salmonids. We analysed the transcriptomic response of the IFN pathway in the head kidney of rainbow trout and Atlantic salmon, which separated 25-30 Mya. We identified a large set of ISGs conserved in both species and cross-referenced them with zebrafish and human ISGs. In contrast, around one-third of salmonid ISG lacked orthologs in human, mouse, chicken or frog, and often between rainbow trout and Atlantic salmon, revealing a fast-evolving, lineage-specific arm of the antiviral response. This study also provides a key resource for in-depth functional analysis of ISGs in salmonids of commercial significance.

    ano.nymous@ccsd.cnrs.fr.invalid (Thomas C Clark) 27 Jul 2023

    https://hal.inrae.fr/hal-04172440v1
  • [hal-04127637] DNA methylation and gene expression changes in mouse mammary tissue during successive lactations: part I – the impact of inflammation

    Mastitis is among the main reasons women cease breastfeeding, which leads to them supplementing breast milk with artificial formula. In farm animals, mastitis results in significant economic losses and the premature culling of some animals. Nevertheless, researchers do not know enough about the effect of inflammation on the mammary gland. This article discusses the changes to DNA methylation in mouse mammary tissue caused by lipopolysaccharide-induced inflammation (4 h post-injection of lipopolysaccharide). We analysed the expression of some genes related to mammary gland function, epigenetic regulation, and the immune response. The analysis focused on three comparisons: inflammation during the first lactation, inflammation during second lactation with no history of inflammation, and inflammation during second lactation with previous inflammation. We identified differentially methylated cytosines (DMCs), differentially methylated regions (DMRs), and some differentially expressed genes (DEGs) for each comparison. The three comparisons shared some DEGs; however, few DMCs and only one DMR were shared. These observations suggest that inflammation is one of several factors affecting epigenetic regulation during successive lactations. Furthermore, the comparison between animals in second lactation with and without inflammation, with no inflammation history during first lactation showed a different pattern compared to the other conditions in this experiment. This indicates that inflammation history plays an important role in determining epigenetic changes. The data presented in this study suggest that lactation rank and previous inflammation history are equally important when explaining mammary tissue gene expression and DNA methylation changes.

    ano.nymous@ccsd.cnrs.fr.invalid (E. Ivanova) 14 Jun 2023

    https://hal.inrae.fr/hal-04127637v1
  • [hal-04176764] Interplay between a bacterial pathogen and its host in rainbow trout isogenic lines with contrasted susceptibility to Cold Water Disease

    Infectious diseases are a major constraint on aquaculture. Genetic lines with different susceptibilities to diseases are useful models to identify resistance mechanisms to pathogens and to improve prophylaxis. Bacterial cold-water disease (BCWD) caused by Flavobacterium psychrophilum represents a major threat for freshwater salmonid farming worldwide. A collection of rainbow trout (Oncorhynchus mykiss) isogenic lines was previously produced from a French domestic population. Here, we compared BCWD resistance phenotypes using a subset of isogenic lines chosen for their contrasted susceptibilities to F. psychrophilum. We applied individual monitoring to document the infection process, including time-course quantification of bacteremia and innate immune response. Strikingly, BCWD resistance was correlated with a lower bacterial growth rate in blood. Several immune genes were expressed at higher levels in resistant fish regardless of infection: the Type II arginase (arg2), a marker for M2 macrophages involved in anti-inflammatory responses and tissue repair, and two Toll-like receptors (tlr2/tlr7), responsible for pathogen detection and inflammatory responses. This study highlights the importance of innate and intrinsic defense mechanisms in determining the outcome of F. psychrophilum infections, and illustrates that non-lethal time-course blood sampling for individual monitoring of bacteremia is a powerful tool to resolve within-host pathogen behavior in bacterial fish diseases.

    ano.nymous@ccsd.cnrs.fr.invalid (Bo-Hyung Lee) 03 Aug 2023

    https://hal.inrae.fr/hal-04176764v1
  • [anses-04145238] OVX033, a nucleocapsid-based vaccine candidate, provides broad-spectrum protection against SARS-CoV-2 variants in a hamster challenge model

    Spike-based COVID-19 vaccines induce potent neutralizing antibodies but their efficacy against SARS-CoV-2 variants decreases. OVX033 is a recombinant protein composed of the full-length nucleocapsid (N) protein of SARS-CoV-2 genetically fused to oligoDOM ® , a self-assembling domain which improves antigen immunogenicity. OVX033 including N as an antigenic target is proposed as new vaccine candidate providing broad-spectrum protection against sarbecoviruses. OVX033 demonstrated its ability to trigger cross-reactive T cell responses and cross-protection against three variants of SARS-CoV-2 (B.1 Europe, Delta B.1.617.2, and Omicron B.1.1.529) in a hamster challenge model, as evidenced by lower weight loss, lower lung viral loads, and reduced lung histopathological lesions.

    ano.nymous@ccsd.cnrs.fr.invalid (Charlotte Primard) 29 Jun 2023

    https://anses.hal.science/anses-04145238v1
  • [anses-03966233] High antigenic diversity of serotype 1 infectious bursal disease virus revealed by antigenic cartography

    The antigenic characterization of IBDV, a virus that causes an immunosuppressive disease in young chickens, has been historically addressed using cross virus neutralization (VN) assay and antigen-capture enzyme-linked immunosorbent (AC-ELISA). However, VN assay has been usually carried out either in specific antibody negative embryonated eggs, for non-cell culture adapted strains, which is tedious, or on chicken embryo fibroblasts (CEF), which requires virus adaptation to cell culture. AC-ELISA has provided crucial information about IBDV antigenicity, but this information is limited to the epitopes included in the tested panel with a lack of information of overall antigenic view. The present work aimed at overcoming those technical limitations and providing an extensive antigenic landscape based on original cross VN assays employing primary chicken B cells, where no previous IBDV adaptation is required. Sixteen serotype 1 IBDV viruses, comprising both reference strains and documented antigenic variants were tested against eleven chicken post-infectious sera. The VN data were analysed by antigenic cartography, a method which enables reliable high-resolution quantitative and visual interpretation of large binding assay datasets. The resulting antigenic cartography revealed i) the existence of several antigenic clusters of IBDV, ii) high antigenic relatedness between some genetically unrelated viruses, iii) a highly variable contribution to global antigenicity of previously identified individual epitopes and iv) broad reactivity of chicken sera raised against antigenic variants. This study provides an overall view of IBDV antigenic diversity. Implementing this approach will be instrumental to follow the evolution of IBDV antigenicity and control the disease.

    ano.nymous@ccsd.cnrs.fr.invalid (Liliana L Cubas-Gaona) 15 Mar 2023

    https://anses.hal.science/anses-03966233v1
  • [hal-04158032] Expérimentation de vaccination des canards mulards en élevage contre un virus influenza aviaire hautement pathogène A(H5N1) clade 2.3.4.4b

    [...]

    ano.nymous@ccsd.cnrs.fr.invalid (Béatrice Grasland) 10 Jul 2023

    https://hal.inrae.fr/hal-04158032v1
  • [hal-04272844] Infectious bursal disease virus: predicting viral pathotype using machine learning models focused on early changes in total blood cell counts

    Infectious bursal disease (IBD) is an avian viral disease caused in chickens by infectious bursal disease virus (IBDV). IBDV strains (Avibirnavirus genus, Birnaviridae family) exhibit different pathotypes, for which no molecular marker is available yet. The different pathotypes, ranging from sub-clinical to inducing immunosuppression and high mortality, are currently determined through a 10-day-long animal experiment designed to compare mortality and clinical score of the uncharacterized strain with references strains. Limits of this protocol lie within standardization and the extensive use of animal experimentation. The aim of this study was to establish a predictive model of viral pathotype based on a minimum number of early parameters measured during infection, allowing faster pathotyping of IBDV strains with improved ethics. We thus measured, at 2 and 4 days post-infection (dpi), the blood concentrations of various immune and coagulation related cells, the uricemia and the infectious viral load in the bursa of Fabricius of chicken infected under standardized conditions with a panel of viruses encompassing the different pathotypes of IBDV. Machine learning algorithms allowed establishing a predictive model of the pathotype based on early changes of the blood cell formula, whose accuracy reached 84.1%. Its accuracy to predict the attenuated and strictly immunosuppressive pathotypes was above 90%. The key parameters for this model were the blood concentrations of B cells, T cells, monocytes, granulocytes, thrombocytes and erythrocytes of infected chickens at 4 dpi. This predictive model could be a second option to traditional IBDV pathotyping that is faster, and more ethical.

    ano.nymous@ccsd.cnrs.fr.invalid (Annonciade Molinet) 06 Nov 2023

    https://hal.inrae.fr/hal-04272844v1
  • [hal-04417714] HTLV-1 biofilm polarization maintained by tetraspanin CD82 is required for efficient viral transmission

    The human T-lymphotropic virus type 1 (HTLV-1) is an oncogenic retrovirus whose transmission relies primarily on cell-to-cell contacts as cell-free viruses are poorly infectious. Among the intercellular transmission routes described, HTLV-1 biofilms are adhesive structures polarized at the cell surface that confine virions in a protective environment, which is believed to promote their simultaneous delivery during infection. Here, we show that several tetraspanins are enriched in HTLV-1 biofilms and incorporated into the viral envelope. However, we report that only the tetraspanin CD82 interacts with HTLV-1 Gag proteins which initiates their polarization into viral biofilms. Also, we demonstrate that CD82 maintains HTLV-1 biofilm polarization and favors viral transmission, as its silencing induces a complete reorganization of viral clusters at the cell surface and reduces the ability of infected T-cells to transmit the virus. Our results highlight the crucial role of CD82 and its glycosylation state in the architectural organization of HTLV-1 biofilms and their subsequent transfer through intercellular contacts. IMPORTANCE In the early stages of infection, human T-lymphotropic virus type 1 (HTLV-1) dissemination within its host is believed to rely mostly on cell-to-cell contacts. Past studies unveiled a novel mechanism of HTLV-1 intercellular transmission based on the remodeling of the host-cell extracellular matrix and the generation of cell-surface viral assemblies whose structure, composition, and function resemble bacterial biofilms. These polarized aggregates of infectious virions, identified as viral biofilms, allow the bulk delivery of viruses to target cells and may help to protect virions from immune attacks. However, viral biofilms’ molecular and functional description is still in its infancy, although it is crucial to fully decipher retrovirus pathogenesis. Here, we explore the function of cellular tetraspanins (CD9, CD81, CD82) that we detect inside HTLV-1 particles within biofilms. Our results demonstrate specific roles for CD82 in the cell-surface distribution and intercellular transmission of HTLV-1 biofilms, which we document as two essential parameters for efficient viral transmission. At last, our findings indicate that N-glycosylation of cell-surface molecules, including CD82, is required for the polarization of HTLV-1 biofilms and for the efficient transmission of HTLV-1 between T-lymphocytes.

    ano.nymous@ccsd.cnrs.fr.invalid (Coline Arone) 25 Jan 2024

    https://hal.inrae.fr/hal-04417714v1
  • [hal-04320097] Caractérisation des particules exhalées par des porcs infectes par un virus influenza porcin

    L’objectif principal de ce travail expérimental est la caractérisation physique et biologique des exhalaisons de modèles porcins infectés par un virus influenza porcin. Ces expérimentations sont conduites sur le site de Ploufragan-Plouzané-Niort de l’ANSES dans l’animalerie expérimentale de niveau de biosécurité A3. Un banc expérimental a été développé de manière à pouvoir isoler, collecter et caractériser les émissions respiratoires. La description technique du banc, la procédure expérimentale employée ainsi que des résultats préliminaires de caractérisation physique sont présentés.

    ano.nymous@ccsd.cnrs.fr.invalid (A Boulbair) 04 Dec 2023

    https://hal.u-pec.fr/hal-04320097v1
  • [hal-04303778] Characterization of particles exhaled by swine infected with an Influenza virus

    [...]

    ano.nymous@ccsd.cnrs.fr.invalid (Lyes Ait Ali Yahia) 23 Nov 2023

    https://hal.u-pec.fr/hal-04303778v1
  • [hal-04320100] Conception d'un banc expérimental pour l'étude des émissions d'aérosols chez le porc

    Dans ce travail, nous avons conçu et testé un premier banc expérimental pour l'estimation des exhalaisons d'aérosols chez le porc. Ce nouveau banc, modulable et transportable, a été adapté à la manipulation dans des installations expérimentales protégées de niveau de biosécurité A3. Il permet de réaliser des mesures d'émission dans un environnement contrôlé. Outre la caractérisation des bioaérosols produits par l'animal, ce banc permet de contrôler la concentration en particules en utilisant un Aerosol Particle sizer (APS). Une première caractérisation pour déterminer le taux d'épuration et de dépôt du banc a été réalisée ainsi que des primomesures en animalerie sur des porcs exempts d'organismes pathogènes spécifiés de l'espèce ou EOPS.

    ano.nymous@ccsd.cnrs.fr.invalid (A Boulbair) 04 Dec 2023

    https://hal.u-pec.fr/hal-04320100v1
  • [hal-04429897] Et si le SARS-CoV-2 se transmettait sous forme de biofilm viral ?

    [...]

    ano.nymous@ccsd.cnrs.fr.invalid (Marie-Isabelle Thoulouze) 01 Feb 2024

    https://hal.inrae.fr/hal-04429897v1
  • [hal-04431895] What about a transmission of SARS-CoV-2 through a viral biofilm?

    [...]

    ano.nymous@ccsd.cnrs.fr.invalid (Marie-Isabelle Thoulouze) 01 Feb 2024

    https://hal.inrae.fr/hal-04431895v1
  • [hal-04175595] Evaluation of a Lateral Flow Immunochromatography Assay (LFIA) for Diagnosis and Surveillance of Brucellosis in French Alpine Ibex (Capra ibex)

    France has been officially free of bovine brucellosis since 2005. Nevertheless, in 2012, as the source of two human cases, a bovine outbreak due to B. melitensis biovar 3 was confirmed in the French Alpine Bargy massif, due to a spillover from wild, protected Alpine ibex (Capra ibex). In order to reduce high Brucella prevalence in the local ibex population, successive management strategies have been implemented. Lateral flow immunochromatography assay (LFIA) was thus identified as a promising on-site screening test, allowing for a rapid diagnosis far from the laboratory. This study compared a commercial LFIA for brucellosis diagnosis with the WOAH-recommended tests for small ruminants (i.e., Rose Bengal test (RBT), Complement fixation test, (CFT) and Indirect ELISA, (iELISA)). LFIA showed the same analytical sensitivity as iELISA on successive dilutions of the International Standard anti-Brucella melitensis Serum (ISaBmS) and the EU Goat Brucella Standard Serum (EUGBSS). Selectivity was estimated at 100% when vaccinated ibex sera were analyzed. When used on samples from naturally infected ibex, LFIA showed high concordance, as well as relative sensitivity and specificity (>97.25%) in comparison with RBT and CFT. This work shows high reliability and ensures a better standardization of LFIA testing for wild ruminants.

    ano.nymous@ccsd.cnrs.fr.invalid (Luca Freddi) 02 Aug 2023

    https://hal.inrae.fr/hal-04175595v1
  • [anses-04127004] Analysis of a multi-type resurgence of Mycobacterium bovis in cattle and badgers in Southwest France, 2007-2019

    Although control measures to tackle bovine tuberculosis (bTB) in cattle have been successful in many parts of Europe, this disease has not been eradicated in areas where Mycobacterium bovis circulates in multi-host systems. Here we analyzed the resurgence of 11 M. bovis genotypes (defined based on spoligotyping and MIRU-VNTR) detected in 141 farms between 2007 and 2019, in an area of Southwestern France where wildlife infection was also detected from 2012 in 65 badgers. We used a spatially-explicit model to reconstruct the simultaneous diffusion of the 11 genotypes in cattle farms and badger populations. Effective reproduction number R was estimated to be 1.34 in 2007-2011 indicating a self-sustained M. bovis transmission by a maintenance community although within-species Rs were both < 1, indicating that neither cattle nor badger populations acted as separate reservoir hosts. From 2012, control measures were implemented, and we observed a decrease of R below 1. Spatial contrasts of the basic reproduction ratio suggested that local field conditions may favor (or penalize) local spread of bTB upon introduction into a new farm. Calculation of generation time distributions showed that the spread of M. bovis has been more rapid from cattle farms (0.5-0.7 year) than from badger groups (1.3-2.4 years). Although eradication of bTB appears possible in the study area (since R < 1), the model suggests it is a long-term prospect, because of the prolonged persistence of infection in badger groups (2.9-5.7 years). Supplementary tools and efforts to better control bTB infection in badgers (including vaccination for instance) appear necessary.

    ano.nymous@ccsd.cnrs.fr.invalid (Malika Bouchez-Zacria) 13 Jun 2023

    https://anses.hal.science/anses-04127004v1
  • [anses-04830377] High-throughput mapping of virus-host interactions to identify new factors of virulence and pathogenicity for ASFV

    African swine fever (ASF) is a highly pathogenic disease causing hemorrhagic fever in domestic pigs and wild boar. It is responsible for numerous epizootics, particularly in Europe and Asia, causing major economic losses to the swine industry. The African Swine Fever virus (ASFV) is the etiological agent responsible for this disease. It is a large double-stranded DNA virus encoding for more than 150 proteins. Different works have shown that there is a close relationship between the ability of some viral proteins to modulate the host antiviral response and the attenuation and virulence processes of ASFV. However, only few protein-protein interactions have been described so far to explain how ASFV escapes the host immunity, notably by inhibiting the type I interferon (IFN-I) response. First, we used an unbiased screen to search for cellular partners of 100 viral proteins. We performed yeast two-hybrid screens using these viral proteins of the Georgia 2007/1 strain as baits and identified more than 50 new virus-host interactions. The global analysis of these interactions clearly shows an enrichment for cellular factors involved in the cytoskeleton (KIF15, FNLB, CENPF) and the innate immunity (COPA, TNIP2, TRIM7, CALCOCO2, BANF1). In parallel, we were interested in the ability of ASFV proteins to individually inhibit the IFN-I pathway. For this purpose, we have screened 100 ASFV proteins using an IFN-luciferase reporter gene system. We showed that at least seven viral proteins (I267L, MGF360-11L, DP96R, MGF505-3R, R298L, DP71L, C962R) contribute to the inhibition of the IFN-I induction pathway. In order to characterize their antagonist effect, a split-nanoluciferase approach was used to screen these viral proteins with a library of 16 major proteins of the IFN-I response. This approach led us to identify IRF3, IRF7, NEMO as new putative targets of ASFV proteins. By combining different screening approaches, we have already highlighted new mechanisms by which ASFV hijacks cellular pathway for replication and escapes the vigilance of the immune system. Later on, by comparing virus-host interactions that have been obtained with attenuated strains of ASFV, we should identify specific targets that could explain the attenuation process at the molecular level.

    ano.nymous@ccsd.cnrs.fr.invalid (Juliette Dupré) 11 Dec 2024

    https://anses.hal.science/anses-04830377v1
  • [anses-03868510] Evaluation of three hemagglutinin-based vaccines for the experimental control of a panzootic clade 2.3.4.4b A(H5N8) high pathogenicity avian influenza virus in mule ducks

    In France during winter 2016–2017, 487 outbreaks of clade 2.3.4.4b H5N8 subtype high pathogenicity (HP) avian influenza A virus (AIV) infections were detected in poultry and captive birds. During this epizootic, HPAIV A/decoy duck/France/161105a/2016 (H5N8) was isolated and characterized in an experimental infection transmission model in conventional mule ducks. To investigate options to possibly protect such ducks against this HPAIV, three vaccines were evaluated in controlled conditions. The first experimental vaccine was derived from the hemagglutinin gene of another clade 2.3.4.4b A(H5N8) HPAIV. It was injected at three weeks of age, either alone (Vac1) or after a primer injection at day-old (Vac1 + boost). The second vaccine (Vac2) was a commercial bivalent adjuvanted vaccine containing an expressed hemagglutinin modified from a clade 2.3.2 A(H5N1) HPAIV. Vac2 was administered as a single injection at two weeks of age. The third experimental vaccine (Vac3) also incorporated a homologous 2.3.4.4b H5 HA gene and was administered as a single injection at three weeks of age. Ducks were challenged with HPAIV A/decoy duck/France/161105a/2016 (H5N8) at six weeks of age. Post-challenge virus excretion was monitored in vaccinated and control birds every 2–3 days for two weeks using real-time reverse-transcription polymerase chain reaction and serological analyses (haemagglutination inhibition test against H5N8, H5 ELISA and AIV ELISA) were performed. Vac1 abolished oropharyngeal and cloacal shedding to almost undetectable levels, whereas Vac3 abolished cloacal shedding only (while partially reducing respiratory shedding) and Vac2 only partly reduced the respiratory and intestinal excretion of the challenge virus. These results provided relevant insights in the immunogenicity of recombinant H5 vaccines in mule ducks, a rarely investigated hybrid between Pekin and Muscovy duck species that has played a critical role in the recent H5 HPAI epizootics in France.

    ano.nymous@ccsd.cnrs.fr.invalid (Éric Niqueux) 23 Nov 2022

    https://anses.hal.science/anses-03868510v1
  • [hal-04343291] Potential of Marine Strains of Pseudoalteromonas to Improve Resistance of Juvenile Sea Bass to Pathogens and Limit Biofilm Development

    The European sea bass (Dicentrarchus labrax), one of the most produced marine fish species in Europe, is acutely vulnerable to multiple infectious hazards. In this study, we investigated the potential probiotic effect of some marine Pseudoalteromonas bacterial strains against two major pathogens of this species, Vibrio harveyi and the nervous necrosis virus (NNV), and examined their antibiofilm effect. Impregnation phase was done by repeated immersion of juvenile’s sea bass during 8 to 12 weeks in seawater containing the probiotic candidates at a concentration of 106 CFU/mL. Four candidates were tested: (1) a combination of two strains producing antimicrobial compounds, hCg-42 and hOe-125; (2) strain 3J6, with known antibiofilm properties; (3) strain RA15, from the same genus, but with no identified probiotic effect; and (4) a control group without probiotics. At the end of the impregnation phase, fish underwent an infection challenge with V. harveyi or with a pathogenic strain of NNV and mortality was monitored. For the V. harveyi challenge, improved survival rates of 10 and 25% were obtained for the RA15 and the mix hCg-42 + hOe-125-impregnated groups, respectively. For the NNV challenge, no significant benefic effect of the probiotics on infection kinetics or cumulative mortality was observed. At the end of the impregnation phase, the maximal thickness of biofilm was significantly lower in the 3J6, double strain, and RA15 groups, compared with the non-impregnated control group. This study highlights the interesting probiotic potential of marine bacteria to limit mortalities induced by bacterial pathogens as well as biofilm development.

    ano.nymous@ccsd.cnrs.fr.invalid (Alexandra Rahmani) 22 Feb 2024

    https://hal.science/hal-04343291v1
  • [hal-04149717] Combining two genetic sexing strains allows sorting of non-transgenic males for Aedes genetic control

    Chemical control of disease vectoring mosquitoes Aedes albopictus and Aedes aegypti is costly, unsustainable, and increasingly ineffective due to the spread of insecticide resistance. The Sterile Insect Technique is a valuable alternative but is limited by slow, error-prone, and wasteful sex-separation methods. Here, we present four Genetic Sexing Strains (two for each Aedes species) based on fluorescence markers linked to the m and M sex loci, allowing for the isolation of transgenic males. Furthermore, we demonstrate how combining these sexing strains enables the production of non-transgenic males. In a mass-rearing facility, 100,000 first instar male larvae could be sorted in under 1.5 h with an estimated 0.01-0.1% female contamination on a single machine. Cost-efficiency analyses revealed that using these strains could result in important savings while setting up and running a mass-rearing facility. Altogether, these Genetic Sexing Strains should enable a major upscaling in control programmes against these important vectors.

    ano.nymous@ccsd.cnrs.fr.invalid (Célia Lutrat) 04 Jul 2023

    https://hal.science/hal-04149717v1
  • [hal-04175372] Differential Salmonella Typhimurium intracellular replication and host cell responses in caecal and ileal organoids derived from chicken

    Chicken infection with Salmonella Typhimurium is an important source of foodborne human diseases. Salmonella colonizes the avian intestinal tract and more particularly the caecum, without causing symptoms. This thus poses a challenge for the prevention of foodborne transmission. Until now, studies on the interaction of Salmonella with the avian gut intestine have been limited by the absence of in vitro intestinal culture models. Here, we established intestinal crypt‐derived chicken organoids to better decipher the impact of Salmonella intracellular replication on avian intestinal epithelium. Using a 3D organoid model, we observed a significantly higher replication rate of the intracellular bacteria in caecal organoids than in ileal organoids. Our model thus recreates intracellular environment, allowing Salmonella replication of avian epithelium according to the intestinal segment. Moreover, an inhibition of the cellular proliferation was observed in infected ileal and caecal organoids compared to uninfected organoids. This appears with a higher effect in ileal organoids, as well as a higher cytokine and signaling molecule response in infected ileal organoids at 3 h post-infection (hpi) than in caecal organoids that could explain the lower replication rate of Salmonella observed later at 24 hpi. To conclude, this study demonstrates that the 3D organoid is a model allowing to decipher the intracellular impact of Salmonella on the intestinal epithelium cell response and illustrates the importance of the gut segment used to purify stem cells and derive organoids to specifically study epithelial cell - Salmonella interaction.

    ano.nymous@ccsd.cnrs.fr.invalid (Sonia Lacroix-Lamandé) 02 Aug 2023

    https://hal.inrae.fr/hal-04175372v1
  • [hal-04230480] Tetraspanin CD82 maintains HTLV-1 biofilm polarization and is required for efficient viral transmission

    The human T-lymphotropic virus type-1 (HTLV-1) is an oncogenic retrovirus whose transmission relies primarily on cell-to-cell contacts as cell-free viruses are poorly infectious. Among the intercellular transmission routes described, HTLV-1 biofilms are adhesive structures polarized at the cell surface that confine virions in a protective environment, which is believed to promote their simultaneous delivery during infection. Here, we show that several tetraspanins are enriched in HTLV-1 biofilms and incorporated into the viral envelope. However, we report that only tetraspanin CD82 interacts with HTLV-1 Gag which initiates its polarization into viral biofilms. Also, we demonstrate that CD82 maintains HTLV-1 biofilm polarization and favors viral transmission, as its silencing induces a complete reorganization of viral clusters at the cell surface and reduces the ability of infected T-cells to transmit the virus. Our results highlight the crucial role of CD82 in the architectural organization of HTLV-1 biofilms and their transfer through intercellular contacts.

    ano.nymous@ccsd.cnrs.fr.invalid (Coline Arone) 06 Oct 2023

    https://hal.inrae.fr/hal-04230480v1
  • [hal-04129892] The Brown Alga Bifurcaria bifurcata Presents an Anthelmintic Activity on All Developmental Stages of the Parasitic Nematode Heligmosomoides polygyrus bakeri

    The current control of gastrointestinal (GI) parasitic nematodes mainly relies on the widespread use of anthelmintics, which has inevitably led to resistance. Therefore, there is an urgent need to find new sources of antiparasitic compounds. Macroalgae represent a rich source of active molecules and are widely described as having medicinal properties. In the present study, we investigated the potential anthelmintic activity of aqueous extracts from three species of algae (Bifurcaria bifurcata, Grateloupia turuturu and Osmundea pinnatifida) on the murine parasite Heligmosomoides polygyrus bakeri. Using a set of complementary in vitro tests, including larval development assays, egg hatching tests and nematicidal activity assays on larvae and adults, we report the nematicidal activity of aqueous extracts of B. bifurcata. In addition, aqueous extract fractionation using liquid/liquid partitioning with a solvent of increasing polarity was performed in order to identify the groups of active molecules underlying the anthelmintic activity. Non-polar extracts (heptane, ethyl acetate) demonstrated high anthelmintic potential, highlighting the role of non-polar metabolites such as terpenes. Here, we highlight the strong anthelmintic potential of the brown alga B. bifurcata on a mouse model of GI parasites, thus confirming the strong interest in algae as natural alternatives for the control of parasitic nematodes.

    ano.nymous@ccsd.cnrs.fr.invalid (Morgane Miclon) 15 Jun 2023

    https://hal.inrae.fr/hal-04129892v1
  • [hal-04168152] Overexpression of Eimeria tenella Rhoptry Kinase 2 Induces Early Production of Schizonts

    Eimeria tenella is an obligate intracellular parasite responsible for avian coccidiosis. Like other apicomplexan parasites, such as Toxoplasma gondii, cell invasion and intracellular development rely on apical organelle content discharge, named micronemes and rhoptries. Some rhoptry (ROP) kinases (ROPK) are key virulence factors in T. gondii. To date, among the 28 ropk genes carried by E. tenella, only two to four were confirmed by proteomic analysis or immunostaining to be expressed at the sporozoite stage. We have previously shown that EtROP1 is implicated in the inhibition of host cell apoptosis by interacting with the cellular p53. This work functionally described the second ROP kinase expressed at the sporozoite stage in E. tenella. EtROP2 is an active kinase that phosphorylates cell substrates of approximately 50 kDa. Its overexpression leads to the shortening of the prepatent period and to the early development of first-generation schizonts. Conduction of RNA sequencing analysis and reverse transcriptase quantitative PCR (RT-qPCR) on the host cell allowed us to identify the mitogen-activated protein kinase (MAPK) pathway and the transcription factor cFos to be upregulated by EtROP2. We also showed by immunofluorescence assay that the active kinase EtROP2 is implicated in the p38 MAPK pathway activation. We established here that EtROP2 activates the p38 MAPK pathway through a direct or indirect phosphorylation, leading to the overexpression of the master transcription factor cFos known to be implicated in E. tenella development. IMPORTANCE Rhoptries are specialized secretory organelles found in zoite stages of apicomplexan parasites. In addition to well-conserved rhoptry neck proteins, their protein consists mostly of kinase proteins, highly divergent from eukaryotic kinases. Some of those kinases are described as major virulence factors in Toxoplasma gondii, secreted into the host cell to hijack signaling pathways. Most of those kinases remain to be characterized in Eimeria tenella. Deciphering their cellular function is a prerequisite to supporting their relevance as a druggable target in development of new means of Eimeria tenella control. Secreted divergent kinases that interact with host cell partners to modulate pathways are good candidates, as they coevolve with their host targets to ensure their function within the host and are less prone to mutations that would lead to drug resistance. The absence of any orthologous kinase in host cells makes these parasite kinases a promising drug target candidate.

    ano.nymous@ccsd.cnrs.fr.invalid (Adeline Ribeiro E Silva) 21 Jul 2023

    https://hal.inrae.fr/hal-04168152v1
  • [hal-04411441] Use of porcine intestinal organoids to study the transmissible gastroenteritis virus

    To date, host-virus interactions have been studied mainly in cell cultures and/or animal models. These approaches come up against two problems: i) methodological, usually related to using immortalized cell lines, which can differ greatly from the target cells of the virus, and ii) ethical, related to experimenting with animals, which can induce varying degrees of symptoms and cause suffering and death. The recent development of organoids has made it possible to develop ex vivo models whose experimental conditions are significantly closer to physiological conditions. Using organoids makes it possible to plan to decrease animal experimentation greatly, in line with the 3Rs principle (Reduction, Refinement, Replacement), and each animal can potentially produce thousands of organoids from different tissues. The Viral Genetics and Biosafety Unit applies the porcine organoid system developed locally as part of the PigOrg project (of INRAE, ANSES and INSERM, funded by the Carnot Agrifood Transition Institute) to models of enteric coronavirosis in piglets, which has a strong impact on the pig industry. Here, we used the porcine transmissible gastroenteritis virus (TGEv) as a model, for which several strains of varying virulence exist and which can be cultivated on immortalized cells, to establish protocols of infections of different organoids (jejunum, duodenum, and ileum). Infections seem more effective for the jejunum than for the duodenum or ileum, and for viruses isolated on cells than on organ homogenate. This organoid system, which connects in vitro and in vivo conditions, will open novel and original perspectives into understanding the physiopathology of virus infections, especially deciphering hostpathogen interactions, without always needing to rely on extensive animal experiments.

    ano.nymous@ccsd.cnrs.fr.invalid (Maud Contrant) 23 Jan 2024

    https://hal.science/hal-04411441v1
  • [hal-04258691] Monophasic Variant of Salmonella Typhimurium Infection Affects the Serum Metabolome in Swine

    Salmonella is the most relevant foodborne zoonotic agent found in swine, and its presence in French herds is significant. Its carriage is asymptomatic, which makes it difficult to detect during rearing, thus increasing the risk of its presence on pork meat. Studies have shown that enteric infection in animals could be associated with changes in the serum metabolome composition, through the immune response or changes in the digestive microbiota composition. We hypothesized that these changes in the serum metabolome composition could be used as markers for the detection of asymptomatic animals infected by Salmonella. Using untargeted analysis by liquid chromatography coupled with mass spectrometry, we showed that significant differences in the composition of the serum metabolome could be detected between infected or noninfected animals both 1 and 21 days after experimental infection. This serum metabolome composition significantly changed during the 21 days postinfection in the infected animal groups, suggesting an evolution of the impact of infection with time. Despite this evolution, differences in the serum metabolome composition persisted between infected and noninfected animals 21 days after the initial infection. We also showed a possible difference between high-shedding and low-shedding animals 21 days postinfection. Finally, some of the variations in the metabolome were found to be significantly associated with variations of specific members of the fecal microbiota. Thus, excreting and asymptomatic animals, but also high-shedding animals, could be identified on the basis of their serum metabolome composition.

    ano.nymous@ccsd.cnrs.fr.invalid (Guillaume Larivière-Gauthier) 13 Feb 2024

    https://hal.science/hal-04258691v1
  • [anses-04509732] Bacteroides fragilis : un probiotique nouvelle génération pour maîtriser Salmonella dans la filière porcine ?

    [...]

    ano.nymous@ccsd.cnrs.fr.invalid (Valérie Rose) 18 Mar 2024

    https://anses.hal.science/anses-04509732v1
  • [hal-04484150] Experimental study of infection with a new genotype of swine influenza virus that has spread in France and evaluation of vaccine protection

    [...]

    ano.nymous@ccsd.cnrs.fr.invalid (Céline Deblanc) 29 Feb 2024

    https://hal.science/hal-04484150v1
  • [hal-03947262] Inflammatory Responses Induced by the Monophasic Variant of Salmonella Typhimurium in Pigs Play a Role in the High Shedder Phenotype and Fecal Microbiota Composition

    Pigs infected with Salmonella may excrete large amounts of Salmonella, increasing the risk of spread of this pathogen in the food chain. Identifying Salmonella high shedder pigs is therefore required to mitigate this risk. We analyzed immune-associated markers and composition of the gut microbiota in specific-pathogen-free pigs presenting different shedding levels after an oral infection with Salmonella. Immune response was studied through total blood cell counts, production of anti-Salmonella antibodies and cytokines, and gene expression quantification. Total Salmonella shedding for each pig was estimated and hierarchical clustering was used to cluster pigs into high, intermediate, and low shedders. Gut microbiota compositions were assessed using 16S rRNA microbial community profiling. Comparisons were made between control and inoculated pigs, then between high and low shedders pigs. Prior to infection, high shedders had similar immunological profiles compared to low shedders. As soon as 1 day postinoculation (dpi), significant differences on the cytokine production level and on the expression level of several host genes related to a proinflammatory response were observed between high and low shedders. Infection with Salmonella induced an early and profound remodeling of the immune response in all pigs, but the intensity of the response was stronger in high shedders. In contrast, low shedders seroconverted earlier than high shedders. Just after induction of the proinflammatory response (at 2 dpi), some taxa of the fecal microbiota were specific to the shedding phenotypes. This was related to the enrichment of several functional pathways related to anaerobic respiration in high shedders. In conclusion, our data show that the immune response to Salmonella modifies the fecal microbiota and subsequently could be responsible for shedding phenotypes. Influencing the gut microbiota and reducing intestinal inflammation could be a strategy for preventing Salmonella high shedding in livestock. IMPORTANCE Salmonellosis remains the most frequent human foodborne zoonosis after campylobacteriosis and pork meat is considered one of the major sources of human foodborne infections. At the farm, host heterogeneity in pig infection is problematic. High Salmonella shedders contribute more significantly to the spread of this foodborne pathogen in the food chain. The identification of predictive biomarkers for high shedders could help to control Salmonella in pigs. The purpose of the present study was to investigate why some pigs become super shedders and others low shedders. We thus investigated the differences in the fecal microbial composition and the immune response in orally infected pigs presenting different Salmonella shedding patterns. Our data show that the proinflammatory response induced by S. Typhimurium at 1 dpi could be responsible for the modification of the fecal microbiota composition and functions observed mainly at 2 and 3 dpi and to the low and super shedder phenotypes.

    ano.nymous@ccsd.cnrs.fr.invalid (Florent Kempf) 19 Apr 2023

    https://hal.inrae.fr/hal-03947262v1