Publications by authors named "Mickaël Riou"

26 Publications

  • Page 1 of 1

Airway Administration of Flagellin Regulates the Inflammatory Response to .

Am J Respir Cell Mol Biol 2021 Jun 8. Epub 2021 Jun 8.

INRAE, 27057, Infectiologie et Santé Publique, Nouzilly, France;

Excessive lung inflammation and airway epithelium damage are hallmarks of human inflammatory lung diseases, such as cystic fibrosis (CF). Enhancement of innate immunity provides protection against pathogens while reducing lung-damaging inflammation. However, the mechanisms underlying innate immunity-mediated protection in the lung remain mysterious, in part because of the lack of appropriate animal models for these human diseases. Toll-like receptor 5 (TLR5) stimulation by its specific ligand, the bacterial protein flagellin, has been proposed to enhance protection against several respiratory infectious diseases, although other cellular events, such as calcium signaling, may also control the intensity of innate immune response. Here, we investigated the molecular events prompted by stimulation with flagellin and its role in regulating innate immunity in the lung of the pig, which is anatomically and genetically more similar to humans than rodent models. We found that flagellin treatment modulated NF-κB signaling and intracellular calcium homeostasis in airway epithelial cells. Flagellin pre-treatment reduced the NF-κB nuclear translocation and the expression of pro-inflammatory cytokines to a second flagellin stimulus as well as to Pseudomonas aeruginosa infection. Moreover, in vivo administration of flagellin decreased the severity of P. aeruginosa-induced pneumonia. Then, we confirmed these beneficial effects of flagellin in a pathological model of CF by using ex vivo precision-cut lung slices from a CF pig model. These results provide evidence that flagellin treatment contribute to a better regulation of the inflammatory response in inflammatory lung diseases such as CF. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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http://dx.doi.org/10.1165/rcmb.2021-0125OCDOI Listing
June 2021

Inhaled bacteriophage therapy in a porcine model of pneumonia caused by Pseudomonas aeruginosa during mechanical ventilation.

Br J Pharmacol 2021 May 11. Epub 2021 May 11.

INSERM, Centre d'Etude des Pathologies Respiratoires, U1100, Tours, France.

Background And Purpose: Pseudomonas aeruginosa is a main cause of ventilator-associated pneumonia (VAP) with drug-resistant bacteria. Bacteriophage therapy has experienced resurgence to compensate for the limited development of novel antibiotics. However, phage therapy is limited to a compassionate use so far, resulting from lack of adequate studies in relevant pharmacological models. We used a pig model of pneumonia caused by P. aeruginosa that recapitulates essential features of human disease to study the antimicrobial efficacy of nebulized-phage therapy.

Experimental Approach: (i) Lysis kinetic assays were performed to evaluate in vitro phage antibacterial efficacy against P. aeruginosa and select relevant combinations of lytic phages. (ii) The efficacy of the phage combinations was investigated in vivo (murine model of P. aeruginosa lung infection). (iii) We determined the optimal conditions to ensure efficient phage delivery by aerosol during mechanical ventilation. (iv) Lung antimicrobial efficacy of inhaled-phage therapy was evaluated in pigs, which were anesthetized, mechanically ventilated and infected with P. aeruginosa.

Key Results: By selecting an active phage cocktail and optimizing aerosol delivery conditions, we were able to deliver high phage concentrations in the lungs, which resulted in a rapid and marked reduction in P. aeruginosa density (1.5 Log reduction, p<0.001). No infective phage was detected in the sera and urines throughout the experiment.

Conclusion And Implications: Our findings demonstrated: (i) the feasibility of delivering large amounts of active phages by nebulization during mechanical ventilation, (ii) rapid control of in situ infection by inhaled bacteriophage in an experimental model of P. aeruginosa pneumonia with high translational value.
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http://dx.doi.org/10.1111/bph.15526DOI Listing
May 2021

Transmission of Bluetongue Virus Serotype 8 by Artificial Insemination with Frozen-Thawed Semen from Naturally Infected Bulls.

Viruses 2021 04 9;13(4). Epub 2021 Apr 9.

Unit of Exotic and Particular Diseases, Scientific Directorate Infectious Diseases in Animals, Sciensano, 1180 Brussels, Belgium.

Transmission of bluetongue (BT) virus serotype 8 (BTV-8) via artificial insemination of contaminated frozen semen from naturally infected bulls was investigated in two independent experiments. Healthy, BT negative heifers were hormonally synchronized and artificially inseminated at oestrus. In total, six groups of three heifers received semen from four batches derived from three bulls naturally infected with BTV-8. Each experiment included one control heifer that was not inseminated and that remained BT negative throughout. BTV viraemia and seroconversion were determined in 8 out of 18 inseminated heifers, and BTV was isolated from five of these animals. These eight heifers only displayed mild clinical signs of BT, if any at all, but six of them experienced pregnancy loss between weeks four and eight of gestation, and five of them became BT PCR and antibody positive. The other two infected heifers gave birth at term to two healthy and BT negative calves. The BT viral load varied among the semen batches used and this had a significant impact on the infection rate, the time of onset of viraemia post artificial insemination, and the gestational stage at which pregnancy loss occurred. These results, which confirm unusual features of BTV-8 infection, should not be extrapolated to infection with other BTV strains without thorough evaluation. This study also adds weight to the hypothesis that the re-emergence of BTV-8 in France in 2015 may be attributable to the use of contaminated bovine semen.
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http://dx.doi.org/10.3390/v13040652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8069090PMC
April 2021

Effects of cholesterol content on activity of P-glycoproteins and membrane physical state, and consequences for anthelmintic resistance in the nematode Haemonchus contortus.

Parasite 2020 14;27. Epub 2020 Jan 14.

INRAE, Université de Tours, UMR-1282 Infectiologie et Santé Publique (ISP), Centre de Recherche Val de Loire, 37380 Nouzilly, France.

Eukaryote plasma membranes protect cells from chemical attack. Xenobiotics, taken up through passive diffusion, accumulate in the membranes, where they are captured by transporters, among which P-glycoproteins (Pgps). In nematodes such as Haemonchus contortus, eggshells and cuticles provide additional protective barriers against xenobiotics. Little is known about the role of these structures in the transport of chemical molecules. Pgps, members of the ABC transporter family, are present in eggshells and cuticles. Changes in the activity of these proteins have also been correlated with alterations in lipids, such as cholesterol content, in eggshells. However, the cellular mechanisms underlying these effects remain unclear. We show here that an experimental decrease in the cholesterol content of eggshells of Haemonchus contortus, with Methyl-beta-CycloDextrin (MβCD), results in an increase in membrane fluidity, favouring Pgp activity and leading to an increase in resistance to anthelmintics. This effect is modulated by the initial degree of anthelminthic resistance of the eggs. These results suggest that eggshell fluidity plays a major role in the modulation of Pgp activity. They confirm that Pgp activity is highly influenced by the local microenvironment, in particular sterols, as observed in some vertebrate models. Thus, eggshell barriers could play an active role in the transport of xenobiotics.
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http://dx.doi.org/10.1051/parasite/2019079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6959138PMC
September 2020

Brucella melitensis Rev.1 vaccination generates a higher shedding risk of the vaccine strain in Alpine ibex (Capra ibex) compared to the domestic goat (Capra hircus).

Vet Res 2019 Nov 27;50(1):100. Epub 2019 Nov 27.

Unité sanitaire de la Faune, Direction de la Recherche et de l'Expertise (DRE), Office National de la Chasse et de la Faune Sauvage (ONCFS), 5 rue de Saint-Thibaud, Saint-Benoît, 78610, Auffargis, France.

Epidemiological investigations implemented in wild and domestic ruminants evidenced a reservoir for Brucella in Capra ibex in the French Alps. Vaccination was considered as a possible way to control Brucella infection in this wildlife population. Twelve ibexes and twelve goats were allocated into four groups housed separately, each including six males or six non-pregnant females. Four to five animals were vaccinated and one or two animals were contact animals. Half of the animals were necropsied 45 days post-vaccination (pv), and the remaining ones at 90 days pv. Additional samples were collected 20 and 68 days pv to explore bacterial distribution in organs and humoral immunity. Neither clinical signs nor Brucella-specific lesions were observed and all vaccinated animals seroconverted. Brucella distribution and antibody profiles were highly contrasted between both species. Proportion of infected samples was significantly higher in ibex compared to goats and decreased between 45 and 90 days pv. Two male ibex presented urogenital excretion at 20 or 45 days pv. The bacterial load was higher 45 days in ibexes compared to goats, whereas it remained moderate to low 90 days pv in both species with large variability between animals. In this experiment, differences between species remained the main source of variation, with low impact of other individual factors. To conclude, multiplicative and shedding capacity of Rev.1 was much higher in ibex compared to goats within 90 days. These results provide initial information on the potential use in natura of a commercial vaccine.
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http://dx.doi.org/10.1186/s13567-019-0717-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6882340PMC
November 2019

A DNA Prime Immuno-Potentiates a Modified Live Vaccine against the Porcine Reproductive and Respiratory Syndrome Virus but Does Not Improve Heterologous Protection.

Viruses 2019 06 25;11(6). Epub 2019 Jun 25.

VIM, INRA, Université Paris-Saclay, Domaine de Vilvert, 78350 Jouy-en-Josas, France.

The porcine reproductive and respiratory syndrome virus (PRRSV), an RNA virus inducing abortion in sows and respiratory disease in young pigs, is a leading infectious cause of economic losses in the swine industry. Modified live vaccines (MLVs) help in controlling the disease, but their efficacy is often compromised by the high genetic diversity of circulating viruses, leading to vaccine escape variants in the field. In this study, we hypothesized that a DNA prime with naked plasmids encoding PRRSV antigens containing conserved T-cell epitopes may improve the protection of MLV against a heterologous challenge. Plasmids were delivered with surface electroporation or needle-free jet injection and European strain-derived PRRSV antigens were targeted or not to the dendritic cell receptor XCR1. Compared to MLV-alone, the DNA-MLV prime- boost regimen slightly improved the IFNγ T-cell response, and substantially increased the antibody response against envelope motives and the nucleoprotein N. The XCR1-targeting of N significantly improved the anti-N specific antibody response. Despite this immuno-potentiation, the DNA-MLV regimen did not further decrease the serum viral load or the nasal viral shedding of the challenge strain over MLV-alone. Finally, the heterologous protection, achieved in absence of detectable effective neutralizing antibodies, was not correlated to the measured antibody or to the IFNγ T-cell response. Therefore, immune correlates of protection remain to be identified and represent an important gap of knowledge in PRRSV vaccinology. This study importantly shows that a naked DNA prime immuno-potentiates an MLV, more on the B than on the IFNγ T-cell response side, and has to be further improved to reach cross-protection.
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http://dx.doi.org/10.3390/v11060576DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6631340PMC
June 2019

Tetrafunctional Block Copolymers Promote Lung Gene Transfer in Newborn Piglets.

Mol Ther Nucleic Acids 2019 Jun 26;16:186-193. Epub 2019 Feb 26.

Université Côte d'Azur, INSERM, CNRS, IPMC, Valbonne, France; FHU-OncoAge, Nice, France. Electronic address:

Tetrafunctional block copolymers are molecules capable of complexing DNA. Although ineffective in vitro, studies in mice have shown that the tetrafunctional block copolymer 704 is a more efficient lung gene transfer agent than the cationic liposome GL67A, previously used in a phase II clinical trial in cystic fibrosis patients. In the present study, we compared the gene transfer capacity of the 704-DNA formulation and a cationic liposome-DNA formulation equivalent to GL67A in a larger-animal model, the newborn piglet. Our results indicate an efficacy of the 704-DNA formulation well above one order of magnitude higher than that of the cationic liposome-DNA formulation, with no elevated levels of interleukin-6 (IL-6), taken as a marker of inflammation. Transgene expression was heterogeneous within lung lobes, with expression levels that were below the detection threshold in some samples, while high in other samples. This heterogeneity is likely to be due to the bolus injection procedure as well as to the small volume of injection. The present study highlights the potential of tetrafunctional block copolymers as non-viral vectors for lung gene therapy.
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http://dx.doi.org/10.1016/j.omtn.2019.02.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6426709PMC
June 2019

Host factors determine the evolution of infection with Staphylococcus aureus to gangrenous mastitis in goats.

Vet Res 2018 07 25;49(1):72. Epub 2018 Jul 25.

GABI, INRA, UMR 1313, Université Paris Saclay, 78350, Jouy-en-Josas, France.

Staphylococcus aureus is the major cause of very severe mastitis of dairy goats. The initial objective of our study was to fine-tune an experimental model of infection of the goat mammary gland with two strains of S. aureus and two lines of goats (low and high somatic cell score lines). Following the challenge, the 10 infected goats divided in two clear-cut severity groups, independently of the S. aureus strain and the goat line. Five goats developed very severe mastitis (of which four were gangrenous) characterized by uncontrolled infection (UI group), whereas the other five kept the infection under control (CI group). The outcome of the infection was determined by 18 h post-infection (hpi), as heralded by the bacterial milk concentration at 18 hpi: more than 10/mL in the UI group, about 10/mL in the CI group. Leukocyte recruitment and composition did not differ between the groups, but the phagocytic killing at 18 hpi efficiency did. Contributing factors involved milk concentrations of α-toxin and LukMF' leukotoxin, but not early expression of the genes encoding the pentraxin PTX3, the cytokines IL-1α and IL-1β, and the chemokines IL-8 and CCL5. Concentrations of TNF-α, IFN-γ, IL-17A, and IL-22 rose sharply in the milk of UI goats when infection was out of control. The results indicate that defenses mobilized by the mammary gland at an early stage of infection were essential to prevent staphylococci from reaching critical concentrations. Staphylococcal exotoxin production appeared to be a consequent event inducing the evolution to gangrenous mastitis.
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http://dx.doi.org/10.1186/s13567-018-0564-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6060506PMC
July 2018

Strongyle Infection and Gut Microbiota: Profiling of Resistant and Susceptible Horses Over a Grazing Season.

Front Physiol 2018 21;9:272. Epub 2018 Mar 21.

UMR 1313, Institut National de la Recherche Agronomique, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France.

Gastrointestinal strongyles are a major threat to horses' health and welfare. Given that strongyles inhabit the same niche as the gut microbiota, they may interact with each other. These beneficial or detrimental interactions are unknown in horses and could partly explain contrasted susceptibility to infection between individuals. To address these questions, an experimental pasture trial with 20 worm-free female Welsh ponies (10 susceptible (S) and 10 resistant (R) to parasite infection) was implemented for 5 months. Fecal egg counts (FEC), hematological and biochemical data, body weight and gut microbiological composition were studied in each individual after 0, 24, 43, 92 and 132 grazing days. R and S ponies displayed divergent immunological profiles and slight differences in microbiological composition under worm-free conditions. After exposure to natural infection, the predicted R ponies exhibited lower FEC after 92 and 132 grazing days, and maintained higher levels of circulating monocytes and eosinophils, while lymphocytosis persisted in S ponies. Although the overall gut microbiota diversity and structure remained similar during the parasite infection between the two groups, S ponies exhibited a reduction of bacteria such as XIVa and members of the family, which may have promoted a disruption of mucosal homeostasis at day 92. In line with this hypothesis, an increase in pathobionts such as and together with changes in several predicted immunological pathways, including pathogen sensing, lipid metabolism, and activation of signal transduction that are critical for the regulation of immune system and energy homeostasis were observed in S relative to R ponies. Moreover, S ponies displayed an increase in protozoan concentrations at day 92, suggesting that strongyles and protozoa may contribute to each other's success in the equine intestines. It could also be that S individuals favor the increase of these carbohydrate-degrading microorganisms to enhance the supply of nutrients needed to fight strongyle infection. Overall, this study provides a foundation to better understand the mechanisms that underpin the relationship between equines and natural strongyle infection. The profiling of horse immune response and gut microbiota should contribute to the development of novel biomarkers for strongyle infection.
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http://dx.doi.org/10.3389/fphys.2018.00272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5871743PMC
March 2018

Fetopathic effects of experimental Schmallenberg virus infection in pregnant goats.

Vet Microbiol 2017 Nov 14;211:141-149. Epub 2017 Oct 14.

Université Paris-Est, ANSES, Laboratoire de Santé Animale, UMR 1161 Virologie ANSES-INRA-ENVA, 14 rue Pierre et Marie Curie, 94704 Maisons-Alfort, France.

Schmallenberg virus (SBV) is an emerging virus responsible for congenital malformations in the offspring of domestic ruminants. It is speculated that infection of pregnant dams may also lead to a significant number of unrecognized fetal losses during the early period of gestation. To assess the pathogenic effects of SBV infection of goats in early pregnancy, we inoculated dams at day 28 or 42 of gestation and followed the animals until day 55 of gestation. Viremia in the absence of clinical signs was detected in all virus-inoculated goats. Fetal deaths were observed in several goats infected at day 28 or 42 of gestation and were invariably associated with the presence of viral genomic RNA in the affected fetuses. Among the viable fetuses, two displayed lesions in the central nervous system (porencephaly) in the presence of viral genome and antigen. All fetuses from goats infected at day 42 and the majority of fetuses from goats infected at day 28 of gestation contained viral genomic RNA. Viral genome was widely distributed in these fetuses and their respective placentas, and infectious virus could be isolated from several organs and placentomes of the viable fetuses. Our results show that fetuses of pregnant goats are susceptible to vertical SBV infection during early pregnancy spanning at least the period between day 28 and 42 of gestation. The outcomes of experimental SBV infection assessed at day 55 of gestation include fetal mortalities, viable fetuses displaying lesions of the central nervous system, as well as viable fetuses without any detectable lesion.
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http://dx.doi.org/10.1016/j.vetmic.2017.10.011DOI Listing
November 2017

The Pig: A Relevant Model for Evaluating the Neutrophil Serine Protease Activities during Acute Pseudomonas aeruginosa Lung Infection.

PLoS One 2016 16;11(12):e0168577. Epub 2016 Dec 16.

INSERM, Centre d'Etude des Pathologies Respiratoires, UMR 1100, Tours cedex, France.

The main features of lung infection and inflammation are a massive recruitment of neutrophils and the subsequent release of neutrophil serine proteases (NSPs). Anti-infectious and/or anti-inflammatory treatments must be tested on a suitable animal model. Mice models do not replicate several aspects of human lung disease. This is particularly true for cystic fibrosis (CF), which has led the scientific community to a search for new animal models. We have shown that mice are not appropriate for characterizing drugs targeting neutrophil-dependent inflammation and that pig neutrophils and their NSPs are similar to their human homologues. We induced acute neutrophilic inflammatory responses in pig lungs using Pseudomonas aeruginosa, an opportunistic respiratory pathogen. Blood samples, nasal swabs and bronchoalveolar lavage fluids (BALFs) were collected at 0, 3, 6 and 24 h post-insfection (p.i.) and biochemical parameters, serum and BAL cytokines, bacterial cultures and neutrophil activity were evaluated. The release of proinflammatory mediators, biochemical and hematological blood parameters, cell recruitment and bronchial reactivity, peaked at 6h p.i.. We also used synthetic substrates specific for human neutrophil proteases to show that the activity of pig NSPs in BALFs increased. These proteases were also detected at the surface of lung neutrophils using anti-human NSP antibodies. Pseudomonas aeruginosa-induced lung infection in pigs results in a neutrophilic response similar to that described for cystic fibrosis and ventilator-associated pneumonia in humans. Altogether, this indicates that the pig is an appropriate model for testing anti-infectious and/or anti-inflammatory drugs to combat adverse proteolytic effects of neutrophil in human lung diseases.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0168577PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5161375PMC
July 2017

Increase of efflux-mediated resistance in Pseudomonas aeruginosa during antibiotic treatment in patients suffering from nosocomial pneumonia.

Int J Antimicrob Agents 2016 Jan 26;47(1):77-83. Epub 2015 Nov 26.

Pharmacologie cellulaire et moléculaire & Louvain Drug Research Institute, Université catholique de Louvain, Brussels, Belgium. Electronic address:

Increases in antibiotic minimum inhibitory concentrations (MICs) for Pseudomonas aeruginosa during treatment are commonly observed but their relationship to efflux overexpression remains poorly documented. In this study, pairs of first [at time of diagnosis (D0)] and last [during treatment (DL)] P. aeruginosa isolates were obtained from patients treated for suspicion of nosocomial pneumonia. Pair clonality was determined by repetitive extragenic palindromic PCR. Overexpression of mexA and mexX was assessed by real-time PCR, and expression of mexC and mexE was assessed by PCR. Antibiotics received by patients before and during treatment were determined from clinical charts. For D0 isolates, 24% were from patients without antibiotics for 1 month and 64% were negative for mexA/mexX overexpression and mexC/mexE expression. For DL isolates, approximately one-half of the patients had received piperacillin/tazobactam, amikacin, meropenem and/or cefepime, and 17% had received ciprofloxacin (alone or in combination); 38% did not show changes in expression of the four genes, whereas 38% showed increased expression for one gene (mainly mexA or mexX), 19% for two genes (mainly mexA and mexX) and 5% for three or four genes. Isolates overexpressing mexA or mexX had median MICs above EUCAST clinical resistance breakpoints for ciprofloxacin, cefepime and meropenem, or for ciprofloxacin, amikacin, cefepime and meropenem, respectively. mexA or mexX overexpression was statistically significantly associated with patients' exposure to ciprofloxacin and meropenem or cefepime and meropenem, respectively. Overexpression of genes encoding antibiotic transporters in P. aeruginosa during treatment is frequent and is associated with increases in MICs above EUCAST clinical susceptibility breakpoints.
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http://dx.doi.org/10.1016/j.ijantimicag.2015.11.004DOI Listing
January 2016

Strong protection induced by an experimental DIVA subunit vaccine against bluetongue virus serotype 8 in cattle.

Vaccine 2014 Nov 11;32(49):6614-21. Epub 2014 Oct 11.

Swedish University of Agricultural Sciences, Host Pathogen Interaction Group, Department of Clinical Sciences, Almas allé 4 C, Uppsala, Sweden; National Veterinary Institute, Department of Virology, Immunology, and Parasitology, Ulls väg 2B, Uppsala, Sweden. Electronic address:

Bluetongue virus (BTV) infections in ruminants pose a permanent agricultural threat since new serotypes are constantly emerging in new locations. Clinical disease is mainly observed in sheep, but cattle were unusually affected during an outbreak of BTV seroype 8 (BTV-8) in Europe. We previously developed an experimental vaccine based on recombinant viral protein 2 (VP2) of BTV-8 and non-structural proteins 1 (NS1) and NS2 of BTV-2, mixed with an immunostimulating complex (ISCOM)-matrix adjuvant. We demonstrated that bovine immune responses induced by this vaccine were as good or superior to those induced by a classic commercial inactivated vaccine. In this study, we evaluated the protective efficacy of the experimental vaccine in cattle and, based on the detection of VP7 antibodies, assessed its DIVA compliancy following virus challenge. Two groups of BTV-seronegative calves were subcutaneously immunized twice at a 3-week interval with the subunit vaccine (n=6) or with adjuvant alone (n=6). Following BTV-8 challenge 3 weeks after second immunization, controls developed viremia and fever associated with other mild clinical signs of bluetongue disease, whereas vaccinated animals were clinically and virologically protected. The vaccine-induced protection was likely mediated by high virus-neutralizing antibody titers directed against VP2 and perhaps by cellular responses to NS1 and NS2. T lymphocyte responses were cross-reactive between BTV-2 and BTV-8, suggesting that NS1 and NS2 may provide the basis of an adaptable vaccine that can be varied by using VP2 of different serotypes. The detection of different levels of VP7 antibodies in vaccinated animals and controls after challenge suggested a compliancy between the vaccine and the DIVA companion test. This BTV subunit vaccine is a promising candidate that should be further evaluated and developed to protect against different serotypes.
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http://dx.doi.org/10.1016/j.vaccine.2014.09.066DOI Listing
November 2014

Schmallenberg virus experimental infection of sheep.

Vet Microbiol 2013 Oct 6;166(3-4):461-6. Epub 2013 Jul 6.

Institute of Diagnostic Virology, Friedrich-Loeffler-Institut (FLI), Suedufer 10, 17493 Greifswald - Insel Riems, Germany.

Since 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.
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http://dx.doi.org/10.1016/j.vetmic.2013.06.030DOI Listing
October 2013

Epizootic hemorrhagic disease virus serotype 6 experimentation on adult cattle.

Res Vet Sci 2013 Oct 27;95(2):794-8. Epub 2013 Jul 27.

ANSES, UMR 1161 Virologie ANSES-INRA-ENVA, 23 avenue du Général de Gaulle, 94704 Maisons-Alfort, France.

Epizootic hemorrhagic disease virus (EHDV), an arthropod-borne orbivirus (family Reoviridae), is an emerging pathogen of wild and domestic ruminants closely related to bluetongue virus (BTV). EHDV serotype 6 (EHDV6) has recently caused outbreaks close to Europe in Turkey and Morocco and a recent experimental study performed on calves inoculated with these two EHDV6 strains showed that the young animals have remained clinically unaffected. The aim of this study was to investigate the pathogenicity of an EHDV6 strain from La Reunion Island in adult Holstein (18-month-old heifers). This EHDV6 strain has induced clinical signs in cattle in the field. Samples taken throughout the study were tested with commercially available ELISA and real-time RT-PCR kits. Very mild clinical manifestations were observed in cattle during the experiment although high levels of viral RNA and virus were found in their blood. EHDV was isolated from the blood of infected animals at 8 dpi. Antibodies against EHDV were first detected by 7 dpi and persisted up to the end of the study. Virus was detected in various tissue samples until 35 dpi, but was not infectious. In view of the recent circulation of different arboviruses in Europe, this study demonstrates what the EHD induces a strong viraemia in adult Holstein cattle and shows that a spread of EHD on European livestock cattle is possible.
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http://dx.doi.org/10.1016/j.rvsc.2013.06.026DOI Listing
October 2013

Host-seeking activity of bluetongue virus vectors: endo/exophagy and circadian rhythm of Culicoides in Western Europe.

PLoS One 2012 29;7(10):e48120. Epub 2012 Oct 29.

CIRAD, UMR Contrôle des Maladies, Montpellier, France.

Feeding success of free-living hematophagous insects depends on their ability to be active when hosts are available and to reach places where hosts are accessible. When the hematophagous insect is a vector of pathogens, determining the components of host-seeking behavior is of primary interest for the assessment of transmission risk. Our aim was to describe endo/exophagy and circadian host-seeking activity of Palaearctic Culicoides species, which are major biting pests and arbovirus vectors, using drop traps and suction traps baited with four sheep, as bluetongue virus hosts. Collections were carried out in the field, a largely-open stable and an enclosed stable during six collection periods of 24 hours in April/May, in late June and in September/October 2010 in western France. A total of 986 Culicoides belonging to 13 species, mainly C. brunnicans and C. obsoletus, was collected on animal baits. Culicoides brunnicans was clearly exophagic, whereas C. obsoletus was able to enter stables. Culicoides brunnicans exhibited a bimodal pattern of host-seeking activity with peaks just after sunrise and sunset. Culicoides obsoletus was active before sunset in spring and autumn and after sunset in summer, thus illustrating influence of other parameters than light, especially temperature. Description of host-seeking behaviors allowed us to discuss control strategies for transmission of Culicoides-borne pathogens, such as bluetongue virus. However, practical vector-control recommendations are difficult to provide because of the variation in the degree of endophagy and time of host-seeking activity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0048120PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3483221PMC
August 2014

Membrane lipidomics for the discovery of new antiparasitic drug targets.

Trends Parasitol 2011 Nov 22;27(11):496-504. Epub 2011 Aug 22.

Laboratoire de Physiologie Cellulaire Végétale, UMR 5168, CNRS-CEA-INRA-Université Joseph Fourier, Institut de Recherches en Sciences et Technologies pour le Vivant, CEA Grenoble, 17 rue des Martyrs, Grenoble, France.

Advances in lipid separation methods and mass spectrometry technologies allow the fine characterization of the lipidome of parasites, ranging from unicellular protists to worms, which cause threatening infections in vertebrates, including humans. Specific lipid structures or lipid metabolic pathways can inspire the development of novel antiparasitic drugs. Changes in the lipid balance in membranes of parasites can also provide clues on the dynamics of drugs and some mechanisms of drug resistance. This review highlights recent trends in parasite lipidomics, combined with functional analyses, for the discovery of novel targets and the development of novel drugs.
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http://dx.doi.org/10.1016/j.pt.2011.07.002DOI Listing
November 2011

The Pseudomonas aeruginosa membranes: a target for a new amphiphilic aminoglycoside derivative?

Biochim Biophys Acta 2011 Jun 1;1808(6):1716-27. Epub 2011 Feb 1.

Université catholique de Louvain, Louvain Drug Research Institute, Unité de Pharmacologie Cellulaire et Moléculaire, UCL 73.70, avenue E. Mounier 73, B-1200 Bruxelles, Belgium.

Aminoglycosides are among the most potent antimicrobials to eradicate Pseudomonas aeruginosa. However, the emergence of resistance has clearly led to a shortage of treatment options, especially for critically ill patients. In the search for new antibiotics, we have synthesized derivatives of the small aminoglycoside, neamine. The amphiphilic aminoglycoside 3',4',6-tri-2-naphtylmethylene neamine (3',4',6-tri-2NM neamine) has appeared to be active against sensitive and resistant P. aeruginosa strains as well as Staphylococcus aureus strains (Baussanne et al., 2010). To understand the molecular mechanism involved, we determined the ability of 3',4',6-tri-2NM neamine to alter the protein synthesis and to interact with the bacterial membranes of P. aeruginosa or models mimicking these membranes. Using atomic force microscopy, we observed a decrease of P. aeruginosa cell thickness. In models of bacterial lipid membranes, we showed a lipid membrane permeabilization in agreement with the deep insertion of 3',4',6-tri-2NM neamine within lipid bilayer as predicted by modeling. This new amphiphilic aminoglycoside bound to lipopolysaccharides and induced P. aeruginosa membrane depolarization. All these effects were compared to those obtained with neamine, the disubstituted neamine derivative (3',6-di-2NM neamine), conventional aminoglycosides (neomycin B and gentamicin) as well as to compounds acting on lipid bilayers like colistin and chlorhexidine. All together, the data showed that naphthylmethyl neamine derivatives target the membrane of P. aeruginosa. This should offer promising prospects in the search for new antibacterials against drug- or biocide-resistant strains.
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http://dx.doi.org/10.1016/j.bbamem.2011.01.014DOI Listing
June 2011

In vivo development of antimicrobial resistance in Pseudomonas aeruginosa strains isolated from the lower respiratory tract of Intensive Care Unit patients with nosocomial pneumonia and receiving antipseudomonal therapy.

Int J Antimicrob Agents 2010 Dec;36(6):513-22

Unité de Pharmacologie Cellulaire et Moléculaire & Louvain Drug Research Institute, Université Catholique de Louvain, Brussels, Belgium.

Pseudomonas aeruginosa causes severe nosocomial pneumonia in Intensive Care Unit (ICU) patients, with an increased prevalence of multiresistant strains. We examined the impact of the use of antipseudomonal antibiotic(s) on the susceptibility of P. aeruginosa isolated from ICU patients with clinically suspected hospital-acquired pneumonia collected in five teaching hospitals (110 non-duplicate initial isolates; 62 clonal pairs of initial and last isolates during treatment). Minimum inhibitory concentrations (MICs) were determined for amikacin, ciprofloxacin, meropenem, piperacillin/tazobactam (TZP), cefepime and ceftazidime (used in therapy) as well as five reporter antibiotics (aztreonam, colistin, gentamicin, piperacillin and ticarcillin) using Clinical and Laboratory Standards Institute (CLSI) methodology. Susceptibility was assessed according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) and CLSI breakpoints. Resistance rates prior to treatment exceeded 25% for cefepime, ceftazidime, piperacillin, ticarcillin and aztreonam (EUCAST and CLSI) and for gentamicin, TZP and colistin (EUCAST only). The highest rates of cross-resistance were noted for ceftazidime and cefepime and the lowest rate for amikacin. Mean MIC values were systematically higher in isolates from patients previously exposed (1 month) to the corresponding antibiotic. For clonal pairs, a systematic increase in MIC between initial and last isolates (significant for amikacin, cefepime, meropenem and TZP) was noted. There was a significant correlation between the use of antibiotics (adjusted for respective proportional use of each drug) and loss of susceptibility at the population level when using EUCAST breakpoints. The high level of resistance of P. aeruginosa in ICU patients with nosocomial pneumonia as well as its further increase during treatment severely narrows the already limited therapeutic options. Further observational studies and the development of early diagnosis for resistant isolates are warranted.
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http://dx.doi.org/10.1016/j.ijantimicag.2010.08.005DOI Listing
December 2010

Drug resistance is affected by colocalization of P-glycoproteins in raft-like structures unexpected in eggshells of the nematode Haemonchus contortus.

Biochem Cell Biol 2010 Jun;88(3):459-67

Multiresistances and Antiparasitic drugs team, Institut national de la recherche agronomique, Unité de recherche 1282, Animal Infectiology & Public Health, Nouzilly, F-37380, France.

In nematodes as in other eukaryotes, there is increasing evidence that drug resistance depends on both changes in the drug cellular targets and in nonspecific mechanisms, involving cellular detoxification by efflux pumps. In vertebrates, P-glycoproteins (Pgp) are membrane efflux pumps responsible for the elimination of xenobiotic agents, especially drugs. We previously reported the presence of Pgp pumps in eggshells and cuticles of the nematode Haemonchus contortus. Eggshells and cuticles are different from cell membranes, in particular they include a chitin layer. Nevertheless these structures present some common biological features with cell membranes and play a role in xenobiotic transport. Pgp activity has been shown to depend on the lipid environment and, in particular, on the cholesterol content in both vertebrate and nematode models. In vertebrates, Pgp is in part located in membrane cholesterol-enriched microdomains, the rafts. We describe here, for the first time, lipid microdomains in eggshells that could correspond with raft-like structures (RLSs). Moreover, a large proportion of the Pgp was shown to colocalize with these RLSs. The functional consequences of the colocalization for xenobiotic transport and thus drug resistance in nematodes were analyzed and compared with results obtained in vertebrates. An understanding of such mechanisms is crucial in overcoming the failure of drug treatments due to the development of resistance.
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http://dx.doi.org/10.1139/o09-126DOI Listing
June 2010

Cryptic color change in a crab spider (Misumena vatia): identification and quantification of precursors and ommochrome pigments by HPLC.

J Chem Ecol 2010 Apr 12;36(4):412-23. Epub 2010 Mar 12.

IRBI, Institut de Recherche de la Biologie des Insectes, UMR CNRS 6035, Université François-Rabelais de Tours, Tours, France.

Mimicry is used widely by arthropods to survive in a hostile environment. Often mimicry is associated with the production of chemical compounds such as pigments. In crab spiders, the change of color is based on a complex physiological process that still is not understood. The aim of this study was to identify and quantify the ommochrome pigments and precursors responsible for the color change in the mimetic crab spider Misumena vatia (Thomisidae). A modified high performance reverse phase ion-pair chromatography technique enabled us to separate and quantify the ommochrome pigments, their precursors, and related metabolites in individual spiders. Compounds such as tryptophan, kynurenine, and kynurenic acid occurred only or mainly in white crab spiders. In contrast, compounds such as 3-hydroxy-kynurenine, xanthommatin, and ommatin D occurred only or mainly in yellow crab spiders. Factor analysis ranked the different color forms in accordance with their metabolites. The biochemical results enabled us to associate the different phases of formation of pigment granules with specific metabolites. Yellow crab spiders contain many unknown ommochrome-like compounds not present in white crab spiders. We also found large quantities of decarboxylated xanthommatin, whose role as precursor of new pathways in ommochrome synthesis needs to be assessed. The catabolism of ommochromes, a process occurring when spiders revert from yellow to white, warrants further study.
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http://dx.doi.org/10.1007/s10886-010-9765-7DOI Listing
April 2010

Synthesis and antimicrobial evaluation of amphiphilic neamine derivatives.

J Med Chem 2010 Jan;53(1):119-27

Universite de Grenoble I/CNRS, UMR 5063, Departement de Pharmacochimie Moleculaire, ICMG FR 2607, France.

The aminoglycoside antibiotics bind to the 16S bacterial rRNA and disturb the protein synthesis. One to four hydroxyl functions of the small aminoglycoside neamine were capped with phenyl, naphthyl, pyridyl, or quinolyl rings. The 3',4'- (6), 3',6- (7a), and the 3',4',6- (10a) 2-naphthylmethylene derivatives appeared to be active against sensitive and resistant Staphylococcus aureus strains. 10a also showed marked antibacterial activities against Gram (-) bacteria, including strains expressing enzymes modifying aminoglycosides, efflux pumps, or rRNA methylases. 7a and 10a revealed a weak and aspecific binding to a model bacterial 16S rRNA. Moreover, as compared to neomycin B, 10a showed a lower ability to decrease (3)H leucine incorporation into proteins in Pseudomonas aeruginosa. All together, our results suggest that the 3',4',6-tri-2-naphthylmethylene neamine derivative 10a should act against Gram (-) bacteria through a mechanism different from inhibition of protein synthesis, probably by membrane destabilization.
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http://dx.doi.org/10.1021/jm900615hDOI Listing
January 2010

Relationships between sterol/phospholipid composition and xenobiotic transport in nematodes.

Parasitol Res 2007 Apr 9;100(5):1125-34. Epub 2007 Feb 9.

Multiresistances and Antiparasitic Drugs, INRA: UR1282, Animal Infections and Public Heath, IASP, 37380 Nouzilly, France.

Therapeutic failure limits prophylaxis of nematode diseases and has been mainly attributed to mutations in cellular targets of anthelmintics. Besides these specific mechanisms, alterations of drug transport also occur in parasites resistant to anthelmintics and depend on both the presence of membrane pumps such as P-glycoprotein (Pgp) and on the lipid composition of membranes. We recently showed in the nematode Haemonchus contortus, using eggs as a model, that the total cholesterol (TC) concentration alters the transport of lipophilic molecules due to membrane pumps such as P-glycoprotein and the resistance to anthelmintics. The effect of TC may depend on the presence of other lipids interacting with TC. Therefore, we analysed the lipid composition and its relationship with Pgp and resistance to anthelmintics. Better correlations were found between Pgp and free cholesterol (FC) than with TC. We also showed that the relationships between lipid composition and resistance to anthelmintics or Pgp depended on the equilibrium between FC and phospholipids (PLs), mainly PLs known to be present primarily in either the external leaflets of cell membranes or the internal leaflets. The PLs phosphatidylcholine and phosphatidylethanolamine played the most significant role, but phosphatidic acid also influenced drug resistance.
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http://dx.doi.org/10.1007/s00436-006-0455-xDOI Listing
April 2007

Immunolocalisation of an ABC transporter, P-glycoprotein, in the eggshells and cuticles of free-living and parasitic stages of Haemonchus contortus.

Parasitol Res 2005 Jun 26;96(3):142-8. Epub 2005 Apr 26.

MultiResistances and Antiparasitic Drugs, INRA-Tours: UR086-BioAgressors, Health and Environment, 37380, Nouzilly, France.

Recent data have suggested that P-glycoprotein (Pgp), working as membrane efflux "pumps", plays a major role in the transport of anthelmintic drugs in parasitic nematodes of ruminants. Flow cytometry analyses has shown that active Pgp is probably present in the external layers of Haemonchus contortus eggshells, following staining with the mouse monoclonal anti-human MDR1 antibody UIC2, which binds to Pgp in its active conformation. We evaluated the presence and distribution of this protein in the envelopes (eggshells and cuticles) of H. contortus and compared the various stages (eggs, L1-L2 larvae, L3 larvae, adult male and female worms). Electrophoresis revealed a 170-kDa band, corresponding to the molecular weight of Pgp in all stages. Indirect immunofluorescence staining with UIC2 showed Pgp to be located in the external layer of eggshells or cuticles. Transmission electron microscopy was used to localise Pgp more accurately in the three layers of the eggshells and cuticles. The conformation and biological functions of this protein, which we did not expect to find in such structures, remain to be determined.
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http://dx.doi.org/10.1007/s00436-005-1345-3DOI Listing
June 2005

Increased resistance to anthelmintics of Haemonchus contortus eggs associated with changes in membrane fluidity of eggshells during embryonation.

Parasitol Res 2005 Mar 28;95(4):266-72. Epub 2005 Jan 28.

MultiResistances et Antiparasitic drugs, INRA-Tours: UR086-BioAgressors, Health and Environment, 37380 , Nouzilly, France.

The embryonation of nematode eggs has been shown to increase their resistance to anthelmintics when parasites are submitted to egg hatch assays. Nevertheless, no mechanism has been suggested to explain this phenomenon. Earlier observations by other authors showed that the biochemical composition of eggshells is altered during the embryonation of eggs. The functional consequences of these changes have not been identified. We studied the changes in membrane environment (eggshells) of Haemonchus contortus eggs during the embryonation by fluidity measurements and their effects on nonspecific mechanisms of resistance to anthelmintics. We previously demonstrated that these mechanisms imply P-glycoproteins (Pgp) belonging to the multi-drug resistance (MDR) system and that the Pgp activity is very susceptible to their lipidic environment. The results obtained here show that the embryonation induced a significant and gradual increase in eggshell fluidity which was associated with an increased resistance to anthelmintics. Differences were observed between H. contortus isolates with various levels of resistance which might result from their specific biology and/or membrane composition. The membrane environment changes could act both on the solubilization of anthelmintics into the eggs and on the efflux of these lipophilic molecules by Pgp.
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http://dx.doi.org/10.1007/s00436-004-1288-0DOI Listing
March 2005

Modulation of the multidrug resistance (MDR) system in the nematode Haemonchus contortus by changing cholesterol content: effects on resistance to anthelmintics.

J Antimicrob Chemother 2003 Aug 15;52(2):180-7. Epub 2003 Jul 15.

Equipe Multi-résistances et Antiparasitaires, Santé et Environnement, Nouzilly, France.

Objectives: The efficiency of the anthelmintics used to treat small domestic ruminants infected with nematodes is compromised by the emergence of resistant parasites. Both specific and non-specific mechanisms of resistance exist. The non-specific mechanisms involve multiple resistance phenomena and are dependent on the multidrug resistance (MDR) system, which is also responsible for the development of chemotherapy-resistant tumour cells. We showed previously that the system also exists in nematodes. Membrane 'pumps', known as P-glycoproteins (Pgp), are activated in the MDR system. The nature of the membrane, in particular the lipids, appears to condition the activity of the pumps. Thus, we studied the effects of cholesterol on drug transport activity in the nematode Haemonchus contortus.

Materials And Methods: We used methyl-beta-cyclodextrin to carry out cholesterol depletion and cholesterol loading experiments. The resulting changes in resistance were estimated by measuring changes in drug transport (a) by means of in vitro egg hatch assays in the presence of a benzimidazole anthelmintic, thiabendazole and (b) by measuring the transport of rhodamine 123 (R123), a specific substrate of Pgp. We used biochemical assays to estimate the cholesterol concentration in the parasites.

Results: Changes in the cholesterol content induced changes in anthelmintic resistance; cholesterol depletion gave increased resistance and cholesterol loading gave decreased resistance. These changes also altered the transport of R123.

Conclusion: Cholesterol depletion or cholesterol loading allow modulation of xenobiotic resistance in nematode eggs as they do in tumour cells. The effect appears to be correlated with changes in the function of membrane P-glycoproteins. The lipid environment thus influences the nematode Pgp activity.
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http://dx.doi.org/10.1093/jac/dkg332DOI Listing
August 2003