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Projet éthique et bien-être animal: contrôle de la douleur liée à l'hyperthermie au cours du processus infectieux. Journées d'animation scientifique de la fédération de recherche en infectiologie (FéRI)

International audience; The anticoagulant rodenticide bromadiolone is widely used as plant protection product to control Water vole outbreaks in European grasslands. Following a treatment, vole population declines rapidly and reaches very low density 3 weeks after bait applications. Then, the plot could be recolonized by voles coming from surrounding plots. When buried in artificial galleries, the persistence of bromadiolone in baits has been showed to be short (DT50 ~ 3 to 6 days). However, baits may be stored in cavities by voles, which increases dramatically the persistence of bromadiolone in the environment (DT50 from 25 to 43 days, Sage et al. 2007, Environ. Pollut. 147). In this study, we aim to document the exposure to bromadiolone of Water voles recolonizing a plot several months after a treatment. To simulate the recolonization, some water voles were trapped alive in non-treated grasslands and marked. Then, they were reintroduced in an experimental plot 1.5, 3 and 6 months after bait application, trapped during the 7 to 10 days following their re-introduction and bromadiolone residues were measured in their tissues. Measured residues were compared to those measured by Sage et al. (2008 STOTEN 407) in voles trapped during the days following bait application. Most of the time, concentrations were lower, but our results show that bromadiolone may persist in vole populations during several months after a treatment and thus, may lead to chronic exposure of wildlife to low doses of rodenticides.

International audience; Rodent predators are largely exposed to anticoagulant rodenticides (ARs) and, to mitigate their exposure, drivers of transfer should be better characterized. The measurement of ARs residues in faeces appears as a potential non-invasive indicator to assess vole predator exposure. However it is unknown whether ARs residues in faeces could be related to treatment patterns. In 2011, fox-like faeces were sampled in 2 contexts of ARs usage, "plant protection product (PPP)/biocide" or "biocide only". PPP treatments using bromadiolone were carried out to control water vole Arvicola terrestris populations. PPP treatments were quantified and located. In each usage, 160 faeces of vole predators were geo-referenced and then, stored at 20°C for further ARs titration (LC-ESI/MS/MS) and species identification (DNA-PCR). DNA was amplified for 37.2% of faeces. Among them, the most frequent species was the red fox (73.9%), then cat (21.8%) and finally dog (4.2%). ARs occurrence did not differ between fox, cat and PCR unidentified faeces (p=0.35). Every positive faeces contained only bromadiolone except one (biocide context) with chlorophacinone. ARs were detected more frequently where PPP treatments occurred (p

International audience; In many countries, the fox (Vulpes vulpes), predator of small mammals, is particularly affected by anticoagulant rodenticides such as bromadiolone due to secondary poisoning. Nevertheless, to date, no method of exposure monitoring is applicable in the field over large areas, and no toxicological data are available concerning sensitivity of foxes to bromadiolone. The aim of this work was to compare excretion kinetics of bromadiolone in fox faeces with clinical and haemostatic effects after repeated exposure to intoxicated voles. A sensitive method for the quantification of bromadiolone excretion in fox faeces and plasma was developed, using liquid chromatography combined with electrospray ionisation mass spectrometry (LC-ESI-MS). The LoD was 0.9 μg/kg and 0.15 μg/L, and the LoQ was 3.0 μg/kg and 0.5 μg/L, in faeces and in plasma, respectively. Four captive foxes were fed for 2 or 5 days with water voles (Arvicola terrestris Sherman) spiked with bromadiolone at concentrations close to those measured in the field. Faeces and blood were collected for bromadiolone titration, and blood-clotting tests were performed to monitor fox health daily during ten days and then every 3-4 days until the end of the experiment (D28). Then, after euthanasia, a complete necropsy was performed, and levels of bromadiolone residues in the liver were determined. Bromadiolone residues were detected in faeces 15 hours after the first exposure. They increased dramatically during the exposure period and then gradually decreased, but they remained detectable at the end of the experiment, i.e., 26 days after the last exposure. Bromadiolone residues in plasma showed a similar pattern but were no longer detectable 7 to 24 days after the last exposure. Two foxes presented very severe external haemorrhages, requiring the administration of the antidote vitamin-K1. Bromadiolone residues in faeces and their relationships with exposure and other directmarkers that were measured is discussed. Liver residues and the toxicity data of our study will help to interpret data from fox carcasses collected by wildlife disease surveillance networks. These findings provide a basis for programs aiming to monitor the exposure of wild fox populations to bromadiolone using non-invasive methods based on standard sampling and analysis of residues in faeces.

Anticoagulants (AVKs) are used worldwide for rodent control, the European regulation authorizing their use as biocides (non-agricultural use at the vicinity of houses) and pesticides (agricultural treatments in fields). AVKs induce adaptations by selection of resistant genotypes in target populations and the poisoning of rodent predators with possible impacts on populations. Here, we present the RODENT program which aims at (1) determining critical parameters of resistance fixation in rodent populations; (2) assessing which factors control AVKs transfer to small mammals in natura and measure impacts on predators (red fox). Several sites are selected in Eastern France according to the dominant AVK use (biocide vs pesticide) and human habitat type (rural vs urban). The prevalence of VKORC1 mutations will be quantified in rats and linked with the characteristics of the studied populations (size, isolation...), human habitats and treatments. In each site, the spatial distribution of AVK accumulation in small mammal community will be described over time. Relationships between resistance and bioaccumulation will be quantified in rats. Concerning predators, non-invasive markers of AVK exposure are under development for the red fox. This will allow determining factors influencing population exposure. Finally, the dynamic of fox populations will be monitored and linked with AVK use, rodent accumulation and exposure at local and regional scales. Based on collaboration with institutions and professionals, this program will contribute to develop new management methods to limit (1) fixation and diffusion of resistance in target rodents and (2) impacts of AVKs on predators. RODENT is funded by the French Agence Nationale de la Recherche from 2009 to 2013.

Pesticides are used for invasive and pest species regulations. Evidence of their negative impact on wildlife has been provided worldwide. In some countries, particularly in Europe, bromadiolone, a Second Generation Anticoagulant Rodenticide (SGAR), is intensively used in the field for controlling the population outbreaks of the Water vole Arvicola terrestris. Among non target species, dozens to hundreds of secondary poisonings of Red foxes Vulpes vulpes have been reported each year. To date, SGAR monitoring has been carried out only based on necropsy and toxicological investigation of carcasses. We have shown that the molecule is measurable in faeces of captive foxes fed with bromadiolone-contaminated voles. The present work aims at (1) assessing the feasibility of bromadiolone analysis in faeces collected in situ, (2) determining if there is a relationship between direct (liver residues) and indirect (faeces bromadiolone residues) markers of exposure. In a 50 km² area, 48% of the faeces collected 15 to 45 days after treatment contained bromadiolone. In an other area of 125 km² treated 1 to 6 month before sampling, 85% of fox livers and 29% of faecal pellets from rectum, contained bromadiolone. We demonstrated an exponential relationship between pellet and liver concentrations. This may suggest a gradient between animals having been exposed long ago and those exposed more recently. Our results indicate that bromadiolone analysis in faeces can be a sensitive and non-invasive tool, and pave the way for further studies aiming at assessing fox population exposure at large spatial scale, before any mortality hazard.

International audience; We document the kinetics of bromadiolone in two rodent populations after a field control of water voles, and their implications for predator exposure. Water voles and common voles were trapped aboveground and underground from 1 to 135 days after bromadiolone treatment in the field. Livers, digestive tracts, and rests of the body were analyzed separately. Our results indicate that 99.6% of the water voles trapped underground and 41% of the common voles trapped aboveground contain bromadiolone residues. Concentrations were maximal between 3.3 and 6.5 days after treatment, according to the tissues examined and the model applied for water voles, and after 1.3 to 3.7 days for common voles. Water voles appeared available almost exclusively for foraging predators. Common voles, found less likely to be poisoned and exhibiting weaker concentrations, were mainly sampled aboveground. The liver, primarily eaten by some predators and scavengers, contains a larger bromadiolone quantity (59% of the total amount found in water voles). The rejection of the digestive tract by those species may lead to a subsequent consumption of voles with higher bromadiolone concentrations (from +3.8 to +5.8% of concentration) and provide a moderate risk increase. After 135 days, eight of the ten water voles and one of the two common voles exhibited detectable residues. Additionally, one specimen presented higher concentrations than the others, and similar to those measured in Voles trapped between the first 15-20 days. This may have consequences on predator intoxications several months after treatment. These results integrate individual differences for the two main rodent species present in treated areas. Implications for predator exposure were investigated at the end of the study and suggest that, if the risk of secondary poisoning is maximal during the first 15-20 days when the rodent densities remain high, exposure conditions are maintained for at least 135 days.

Chaque année, entre 55 000 et 70 000 tonnes de substances actives phytopharmaceutiques, incluant celles utilisables en agriculture biologique et de biocontrôle, sont vendues sur le territoire français métropolitain et d’outre-mer et sont utilisées pour la protection des cultures ou l’entretien des jardins, espaces végétalisés et infrastructures (JEVI). Dans le même temps, le rapport sur l’évaluation mondiale de la biodiversité et des services écosystémiques établi en 2019 par la Plateforme intergouvernementale scientifique et politique sur la biodiversité et les services écosystémiques (IPBES) dresse le bilan alarmant d’une érosion sans précédent de la biodiversité. La pollution chimique générée par les activités humaines, incluant les produits phytopharmaceutiques (PPP), est identifiée parmi les causes de cette érosion. Cette pollution s’ajoute à d’autres pressions, comme les destructions d’habitats causées par l’urbanisation, l’intensification des pratiques agricoles et sylvicoles, et les conséquences du changement climatique. Face à ce constat, la réglementation européenne en matière de mise sur le marché des PPP vise un degré élevé de protection, avec en particulier pour principe d’éviter tout effet inacceptable sur l’environnement. Toutefois, elle ne parvient pas complètement à atteindre cet objectif, en raison notamment d’une prise en compte insuffisante de la diversité des interactions telles qu’elles se produisent dans l’environnement (entre substances, entre organismes, avec une variété de facteurs physico-chimiques, etc.).C’est dans ce contexte que les ministres chargés de l’Environnement, de l’Agriculture et de la Recherche ont sollicité INRAE et l’Ifremer pour réaliser un état des lieux des connaissances scientifiques relatives aux impacts des PPP sur la biodiversité et les services écosystémiques. La précédente expertise scientifique collective (ESCo) portant sur Pesticides, agriculture et environnement avait été réalisée en 2005. Leprésent exercice consiste à en actualiser les résultats, en les élargissant à l’ensemble du continuum terre-mer et en incluant les usages de PPP relevant des zones non agricoles (JEVI). A la différence de celle de 2005, cette ESCo est positionnée en aval de l’utilisation des PPP, pour traiter du devenir et des impacts de ces substances une fois introduites dans l’environnement. Elle ne traite pas des pratiques ou systèmes agricoles permettant de réduire les utilisations de PPP, ni des stratégies préventives de régulation des bioagresseurs. Ces thématiques font l’objet d’autres travaux en cours, notamment une autre ESCo conduite par INRAE sur la gestion des couverts végétaux pour la régulation naturelle des bioagresseurs dont les résultats sont attendus à l’automne 2022. Ces deux exercices s’inscrivent dans le cadre du Plan Ecophyto II+, en complément de l’expertise Pesticides et santé humaine publiée par l’Inserm en 2021.Le périmètre de la présente ESCo couvre les différents milieux (terrestre, atmosphérique, aquatiques continental et marin, à l’exception des eaux souterraines) dans leur continuité, du lieu d’application jusqu’à l’océan, en France métropolitaine et d’outre-mer, à partir de connaissances produites ou transposables dans ce type de contexte (climat, PPP utilisés, biodiversité présente, etc.). Il intègre tous les produits destinés à la protection des cultures ou à l’entretien des JEVI, qu’il s’agisse de PPP conventionnels ou de produits ou agents de biocontrôle, dès lors qu’ils sont susceptibles de se retrouver dans l’environnement du fait d’une utilisation actuelle ou plus ancienne. Le cadre d’analyse mis en place considère la biodiversité dans ses dimensions structurelle et fonctionnelle, et il intègre la question des services écosystémiques. L’attention est ainsi plus particulièrement portée sur des travaux qui documentent la mise en évidence des risques et des effets dans des conditions environnementales réalistes, et à des niveaux d’organisation biologique (ex. individu, population, communauté, écosystème) susceptibles de faciliter le lien à établir avec la biodiversité ainsi qu’avec les fonctions et services écosystémiques.

International audience; Chaque année, entre 55 000 et 70 000 tonnes de substances actives phytopharmaceutiques, incluant celles utilisables en agriculture biologique et de biocontrôle, sont vendues sur le territoire français métropolitain et d’outre-mer et sont utilisées pour la protection des cultures ou l’entretien des jardins, espaces végétalisés et infrastructures (JEVI). Dans le même temps, le rapport sur l’évaluation mondiale de la biodiversité et des services écosystémiques établi en 2019 par la Plateforme intergouvernementale scientifique et politique sur la biodiversité et les services écosystémiques (IPBES) dresse le bilan alarmant d’une érosion sans précédent de la biodiversité. La pollution chimique générée par les activités humaines, incluant les produits phytopharmaceutiques (PPP), est identifiée parmi les causes de cette érosion. Cette pollution s’ajoute à d’autres pressions, comme les destructions d’habitats causées par l’urbanisation, l’intensification des pratiques agricoles et sylvicoles, et les conséquences du changement climatique. Face à ce constat, la réglementation européenne en matière de mise sur le marché des PPP vise un degré élevé de protection, avec en particulier pour principe d’éviter tout effet inacceptable sur l’environnement. Toutefois, elle ne parvient pas complètement à atteindre cet objectif, en raison notamment d’une prise en compte insuffisante de la diversité des interactions telles qu’elles se produisent dans l’environnement (entre substances, entre organismes, avec une variété de facteurs physico-chimiques, etc.).C’est dans ce contexte que les ministres chargés de l’Environnement, de l’Agriculture et de la Recherche ont sollicité INRAE et l’Ifremer pour réaliser un état des lieux des connaissances scientifiques relatives aux impacts des PPP sur la biodiversité et les services écosystémiques. La précédente expertise scientifique collective (ESCo) portant sur Pesticides, agriculture et environnement avait été réalisée en 2005. Leprésent exercice consiste à en actualiser les résultats, en les élargissant à l’ensemble du continuum terre-mer et en incluant les usages de PPP relevant des zones non agricoles (JEVI). A la différence de celle de 2005, cette ESCo est positionnée en aval de l’utilisation des PPP, pour traiter du devenir et des impacts de ces substances une fois introduites dans l’environnement. Elle ne traite pas des pratiques ou systèmes agricoles permettant de réduire les utilisations de PPP, ni des stratégies préventives de régulation des bioagresseurs. Ces thématiques font l’objet d’autres travaux en cours, notamment une autre ESCo conduite par INRAE sur la gestion des couverts végétaux pour la régulation naturelle des bioagresseurs dont les résultats sont attendus à l’automne 2022. Ces deux exercices s’inscrivent dans le cadre du Plan Ecophyto II+, en complément de l’expertise Pesticides et santé humaine publiée par l’Inserm en 2021.Le périmètre de la présente ESCo couvre les différents milieux (terrestre, atmosphérique, aquatiques continental et marin, à l’exception des eaux souterraines) dans leur continuité, du lieu d’application jusqu’à l’océan, en France métropolitaine et d’outre-mer, à partir de connaissances produites ou transposables dans ce type de contexte (climat, PPP utilisés, biodiversité présente, etc.). Il intègre tous les produits destinés à la protection des cultures ou à l’entretien des JEVI, qu’il s’agisse de PPP conventionnels ou de produits ou agents de biocontrôle, dès lors qu’ils sont susceptibles de se retrouver dans l’environnement du fait d’une utilisation actuelle ou plus ancienne. Le cadre d’analyse mis en place considère la biodiversité dans ses dimensions structurelle et fonctionnelle, et il intègre la question des services écosystémiques. L’attention est ainsi plus particulièrement portée sur des travaux qui documentent la mise en évidence des risques et des effets dans des conditions environnementales réalistes, et à des niveaux d’organisation biologique (ex. individu, population, communauté, écosystème) susceptibles de faciliter le lien à établir avec la biodiversité ainsi qu’avec les fonctions et services écosystémiques.; Impacts des produits phytopharmaceutiques sur la biodiversité et les services écosystémiques Synthèse de l'expertise scientifique collective-Mai 2022 Direction de l'expertise scientifique collective, de la prospective et des études (DEPE)

The use of pesticides can affect non-target species by causing population declines through indirect intoxication. Small mustelids (SMs; weasels, Mustela nivalis L.; stoats, Mustela erminea L.) consume water voles (WVs, Arvicola scherman S.) and can be exposed to bromadiolone, an anticoagulant rodenticide used in some countries to reduce WV damage to grasslands. Here, we investigated whether bromadiolone affected SM abundance.We monitored SM abundance using footprint tracking tunnels in spring and autumn at ten sites. Among these sites, four were treated with bromadiolone, while six were not treated. We found reduced SM abundance at these four sites from spring to autumn (treated sites, mean ± SE SM abundance change = -1.68 ± 0.42; untreated sites, 0.29 ± 0.25). Using a linear model, we observed that SM abundance decreased as a function of the quantity of bromadiolone applied during the 3 months before the autumn estimate. We found that WV abundance increased at treated sites (linear model, treated sites, mean ± SE WV abundance change = 1.4 ± 0.4; untreated sites, 0.33 ± 0.25). Thus, at treated sites, SM abundance declined despite increased food availability. By analyzing residues in vole livers and SM scats we showed that SMs may be exposed to bromadiolone at the sites where this compound was used.This study is the first to document the relationship between SM abundance and bromadiolone usage for small mammal control. Declines in SM abundance were observed at treated sites, where bromadiolone residue was found in SM scats. This correlative approach suggests that bromadiolone treatment may lead to seasonal SM declines and associated WV increases. © 2018 Society of Chemical Industry.

International audience

Vitamin K antagonists (VKAs) remain the oral anticoagulant of choice in venous thromboembolic disease. These drugs are characterized by a large inter-individual variability requiring frequent dose tailoring. Genetic polymorphisms for cytochrome CYP2C9 and VKORC1 explain some of the variability, especially in warfarin and acenocoumarol responses. The aim of this study was to assess, in cell models, the role of ABC transporters in the intestinal transfer of the main coumarin derivatives (warfarin, acenocoumarol) and indanedione derivatives (phenindione, fluindione). The results show a basal to apical polarized transport for fluindione, phenindione and acenocoumarol only. Experimental studies using specific inhibitors of transport protein demonstrate the implication of MRPs and BCRP proteins and to a lesser extent P-gp. Warfarin and acenocoumarol seem to be poor inhibitors of MRPs protein, whereas fluindione and phenindione have a slight or no effect. The regulation of the expression of ABC transporters by exposure to VKAs was also investigated in Caco-2 cells. The expression of mRNA P-gp, MRP1, MRP2 and BCRP was weakly or not modified after 24 h of VKAs exposure. In conclusion, the intestinal transfer of indanedione derivatives and acenocoumarol could be influenced by transport proteins of the ABC superfamily. Coumarin derivatives are poor inhibitors of these proteins and AVKs have a slight effect on the mRNA ABC transporter expression level. Copyright © 2016 John WileySons, Ltd.

Anticoagulant rodenticides are commonly used to control rodent pests worldwide. They specifically inhibit the vitamin K epoxide reductase (VKORC1), which is an enzyme encoded by the Vkorc1 gene, involved in the recycling of vitamin K. Therefore, they prevent blood clotting. Numerous mutations of Vkorc1 gene were reported in rodents, and some are involved in the resistant to rodenticides phenotype. Two hundred and sixty‐six mice tails were received from 65 different locations in France. Coding sequences of Vkorc1 gene were sequenced in order to detect mutations. Consequences of the observed mutations were evaluated by the use of recombinant VKORC1. More than 70% of mice presented Vkorc1 mutations. Among these mice, 80% were homozygous. Contrary to brown rats for which only one predominant Vkorc1 genotype was found in France, nine missense single mutations and four double mutations were observed in house mice. The single mutations lead to resistance to first‐generation antivitamin K (AVKs) only and are certainly associated with the use of these first‐generation molecules by nonprofessionals for the control of mice populations. The double mutations, probably obtained by genetic recombination, lead to in vitro resistance to all AVKs. They must be regarded as an adaptive evolution to the current use of second‐generation AVKs. The intensive use of first‐generation anticoagulants probably allowed the selection of a high diversity of mutations, which makes possible the genetic recombination and consequently provokes the emergence of the more resistant mutated Vkorc1 described to date.

Leptospirosis is a zoonosis found worldwide that is caused by a spirochete. The main reservoirs of Leptospira, which presents an asymptomatic infection, are wild rodents, including the brown rat (Rattus norvegicus). Experimental studies of the mechanisms of its renal colonization in rats have previously used an intraperitoneal inoculation route. However, knowledge of rat-rat transmission requires the use of a natural route of inoculation, such as a mucosal or subcutaneous route. We investigated for the first time the effects of subcutaneous and mucosal inoculation routes compared to the reference intraperitoneal route during Leptospira infection in adult rats. Infection characteristics were studied using Leptospira renal isolation, serology, and molecular and histological analyses. Leptospira infection was asymptomatic using each inoculation route, and caused similar antibody production regardless of renal colonization. The observed renal colonization rates were 8 out of 8 rats, 5 out of 8 rats and 1 out of 8 rats for the intraperitoneal, mucosal and subcutaneous inoculation routes, respectively. Thus, among the natural infection routes studied, mucosal inoculation was more efficient for renal colonization associated with urinary excretion than the subcutaneous route and induced a slower-progressing infection than the intraperitoneal route. These results can facilitate understanding of the infection modalities in rats, unlike the epidemiological studies conducted in wild rats. Future studies of other natural inoculation routes in rat models will increase our knowledge of rat-rat disease transmission and allow the investigation of infection kinetics., Author Summary Leptospirosis (infection with pathogenic Leptospira spp.) is a public health concern worldwide. The brown rat (Rattus norvegicus), as the most ubiquitous animal of urban wildlife, is potentially the primary source of Leptospira spp. for humans, dogs and livestock. For understanding the Leptospira maintenance in rat colonies, the experimental studies required the use of natural route of transmission between the rats. We investigated the effects of the mucosal and bite’s transmission (conjunctival-mucosal and subcutaneous routes) compared to the reference route (intraperitoneal) during infection in adult rats. With serology, we showed that the antibody production was independent of the inoculation route. By isolation, molecular and histological analyses, we found that the mucosal route was more efficient at renal colonization and leptospires excretion than the subcutaneous route. These results can be useful in understanding the infection modalities in rat that could prevent the human leptospirosis.

We report the first two cases of pulmonary presence of leptospires in apparently healthy rats captured in a city park in Lyon (France). Only renal carriage ofLeptospirahas been described in the literature. Blood serology was performed in parallel with molecular and histological analyses of the kidney and lung samples. We isolated leptospires from the kidneys of two out of three seropositive wild rats. These results were confirmed by specific detection of pathogenicLeptospiraby real-time PCR. Moreover,LeptospiraDNA was detected in lung tissues. Immunohistochemistry and Warthin-Starry staining revealed that leptospires were present on the surface of the ciliated epithelium of the bronchi. Using PCR of therrs(16S) gene and Multispacer Sequence Typing, DNA extracts of the kidney and lung were identified as belonging toLeptospira interrogansserovar Icterohaemorrhagiae “CHU Réunion.” This first observation of the presenceLeptospirain the lung with simultaneous renal carriage will require further study in future on several target organs to gain a better understanding of theLeptospirainfection in wild rat.

International audience; BACKGROUNDIn spite of intensive use of bromadiolone, rodent control was inefficient on a farm infested by rats in Zaragoza, Spain. While metabolic resistance was previously described in this rodent species, the observation of a target resistance to antivitamin K rodenticides had been poorly documented in Rattus rattus. RESULTSFrom rats trapped on the farm, cytochrome b and Vkorc1 genes were amplified by PCR and sequenced in order to identify species and detect potential Vkorc1 mutations. VKORC1-deduced amino acid sequences were thus expressed in Pichia pastoris, and inhibition constants towards various rodenticides were determined. The ten rats trapped on the farm were all identified as R. rattus. They were found to be homozygous for the g.74A>T nucleotide replacement in exon 1 of the Vkorc1 gene, leading to p.Y25F mutation. This mutation led to increased apparent inhibition constants towards various rodenticides, probably caused by a partial loss of helical structure of TM4. CONCLUSIONThe p.Y25F mutation detected in the Vkorc1 gene in R. rattus trapped on the Spanish farm is associated with the resistance phenotype to bromadiolone that has been observed. It is the first evidence of target resistance to antivitamin K anticoagulants in R. rattus.

The mycotoxin zearalenone (ZEA) is produced by a variety of Fusarium fungi and contaminates numerous cereals, fruits and vegetables. Interacting with the oestrogen receptors, ZEA and reduced metabolites zearalenols (ZOLs) cause hormonal effects in animals, such as abnormalities in the development of the reproductive tract and mammary gland in female offspring. Limited information is available on the pharmacokinetics of ZEA and its metabolites, particularly in pregnant females, foetuses and newborns. Our study was conducted to characterise the tissue distribution and metabolism of ZEA in male and female rats in various physiological states (virgin female, pregnant female) and exposure conditions (subcutaneous versus oral exposure, single versus repeated exposure to 1 mg/kg ZEA). Respective placental and mammary transfer to foetuses and newborns was evaluated. In all states and exposure conditions, α-ZOL and the glucuronides of ZEA and α-ZOL were the predominant metabolites, mostly concentrated in the intestine, the liver and the urine. Toxins were very low or undetectable in most of the tissues 24 h after ZEA exposure, except in foetal livers. Absorption and intestinal glucuronidation of ZEA were higher in males than females. α-ZOL concentration was significantly higher in the intestine and liver of males and pregnant females, compared to virgin females. ZEA and all its metabolites easily crossed the placental barrier and transferred into the milk. ZEA was metabolised in the foetal and neonatal stages, glucuronides being the main form detected in all organs. Metabolite elimination was slower in foetal tissues than in maternal tissues. All toxin concentrations in the foetal and neonatal tissues strongly increased in cases of repeated maternal exposure. A better knowledge of the metabolism and transfer of ZEA in foetuses and newborns will help to evaluate the health risk that such endocrine disruptors represent in these stages.

To elaborate constant rate infusion (CRI) protocols for xylazine (X) and xylazine/butorphanol (XB) which will result in constant sedation and steady xylazine plasma concentrations.Blinded randomized experimental study.Ten adult research horses.Part I: After normal height of head above ground (HHAG = 100%) was determined, a loading dose of xylazine (1 mg kg(-1) ) with butorphanol (XB: 18 μg kg(-1) ) or saline (X: equal volume) was given slowly intravenously (IV). Immediately afterwards, a CRI of butorphanol (XB: 25 μg kg(-1) hour(-1)) or saline (X) was administered for 2 hours. The HHAG was used as a marker of depth of sedation. Sedation was maintained for 2 hours by additional boluses of xylazine (0.3 mg kg(-1)) whenever HHAG50%. The dose of xylazine (mg kg(-1) hour(-1)) required to maintain sedation was calculated for both groups. Part II: After the initial loading dose, the calculated xylazine infusion rates were administered in parallel to butorphanol (XB) or saline (X) and sedation evaluated. Xylazine plasma concentrations were measured by HPLC-MS-MS at time points 0, 5, 30, 45, 60, 90, and 120 minutes. Data were analyzed using paired t-test, Wilcoxon signed rank test and a 2-way anova for repeated measures (p0.05).There was no significant difference in xylazine requirements (X: 0.69, XB: 0.65 mg kg(-1) hour(-1)) between groups. With treatment X, a CRI leading to prolonged sedation was developed. With XB, five horses (part I: two, part II: three) fell down and during part II four horses appeared insufficiently sedated. Xylazine plasma concentrations were constant after 45 minutes in both groups.Xylazine bolus, followed by CRI, provided constant sedation. Additional butorphanol was ineffective in reducing xylazine requirements and increased ataxia and apparent early recovery from sedation in unstimulated horses.Data were obtained on unstimulated healthy horses and extrapolation to clinical conditions requires caution.

National audience; This study deals with the use of a chronic exposure scenario of zebrafish (Danio rerio) in laboratory conditions to evaluate the genotoxic potential of diuron and azoxystrobin, two pesticides intensively used in vineyard agriculture. Adult male zebrafish were exposed during three weeks in the semi static mode in four 20 L aquaria. Treatment allowed to each aquarium was: negative control (untreated), positive control (methyl methane sulphonate 1 µM), diuron 4.3 nM and azoxystrobin 1.2 nM. Once per week, genotoxicity was assessed (6 fish/treatment) by the use of two complementary biomarkers: the primary DNA damages evaluated in somatic (liver) and germ (spermatozoa) cells by the alkaline version of the Comet assay and the micronucleus formation assessed in erythrocytes. Very low basal DNA damages were obtained with both biomarkers in negative control during the three consecutive weeks and a significant genotoxic response was obtained in 1 µM MMS exposed fish, both in liver and germ cells with the Comet assay and in erythrocytes with the micronucleus test, respectively starting after one and three weeks. With this chronic exposure scenario, both diuron and azoxystrobine revealed a genotoxic potential at realistic environmental concentrations and a significant response was obtained in all cell types investigated and with both biomarkers used, mainly after 7 or 14 days, thus stressing the interest of long-term exposure scenario. Further studies will be undertaken in order to evaluate whether DNA damage observed in spermatozoa of fish exposed to environmental concentration of pesticides could lead to subsequent reproductive disorders.

Background Urban leptospirosis has increasingly been reported in both developing and developed countries. The control of the disease is limited because our understanding of basic aspects of the epidemiology, including the transmission routes of leptospires among rat populations, remains incomplete. Through the ability to distinguish among Leptospira strains in rats, multispacer sequence typing (MST) could provide a modern understanding of Leptospira epidemiology; however, to our knowledge, the distribution of Leptospira strains among urban rat colonies has not been investigated using MST. Aims and Methodology The objective of this study was to identify the Leptospira strains present in rats (Rattus norvegicus) in Lyon (France) using MST and to characterize their spatial distribution. Kidneys and urine were collected from rats trapped live in seven locations in the city and in one suburban location. Each location was considered to represent a rat colony. Bacterial cultures and quantitative polymerase chain reaction (qPCR) assays were performed, and the L. interrogans DNA identified was then genotyped using MST. The distributions of Leptospira strains were spatially described. Key Results Among 84 wild rats, MST profiles were obtained in 35 of 37 rats that had a positive result for L. interrogans by bacterial culture and/or qPCR analyses. All of the MST profiles were related to reference strains previously isolated from human patients that belong to the serogroup Icterohaemorrhagiae and the serovars [strain(s)] Copenhageni [Wijinberg or M20] (n = 26), Icterohaemorrhagiae [CHU Reunion] (n = 7), Icterohaemorrhagiae [R1] (n = 1) and Copenhageni [Shibaura 9] (n = 1). Each colony was infected with leptospires having the same MST profile. Major Conclusions This study demonstrated that MST could be used for the purpose of field studies, either on culture isolates or on DNA extracted from kidneys and urine, to distinguish among L. interrogans isolates in rats. MST could thus be used to monitor their distributions in urban rats from the same city, thereby providing new knowledge that could be applied to explore the circulation of L. interrogans infection in rat colonies. Because the strains are related to those previously found in humans, this application of MST could aid in the source tracking of human leptospirosis, and the findings would be relevant for public health purposes according to the One Health principle.

This study attempted to develop a list of priority pathogens. It is part of a European Union (EU) project dedicated to the surveillance of emerging or re-emerging pathogens of wildlife. Partners of the consortium established an initial list of 138 pathogens of concern, which was reduced to a smaller list of 65 pathogens likely to affect ruminants (i.e., the most costly animal group in the EU over the last 15 years). These 65 pathogens underwent a two-step, expert-based risk analysis: 92 experts graded them with respect to their global importance for animal welfare, species conservation, trade/economic impacts and public health. In step 2, the top 15 pathogens from step 1 were assessed by 69 experts considering seven weighted epidemiological criteria (pathogen variability, host specificity, potential for contagion, speed of spread, presence in Europe, difficulty of surveillance in wildlife and persistence in the environment) for which four options were possible. The responses concerned a wide geographic coverage. The resulting top-list pathogens were ranked as follows: 1. Salmonella enterica, 2. Coxiella burnetii, 3. foot-and-mouth disease virus, 4. Mycobacterium bovis, 5. bluetongue virus, and 6. European tick-borne encephalitis virus. The influence of the characteristics of the respondents, the importance of the levels of uncertainty/variability and the implication of the results are discussed. This work highlights the relevance of developing such lists for preparedness.

Rodent control is an important issue in human health and agriculture. Oil palm plantations are rapidly expanding in Indonesia and this is having a major economic and ecological impact. Rodent control in oil palm plantations is based principally on the use of anti-vitamin K (AVK), the main anticoagulant used being coumatetralyl, a first-generation AVK. We conducted a comparative study in two well established oil palm plantations in Indonesia: (1) one without chemical control in Riau and (2) another with intensive coumatetralyl use on Bangka Island. Rat species were identified by the molecular barcoding method. Susceptibility to coumatetralyl was then assessed within the two populations and we screened for mutations in vkorc1, which encodes the molecular target of AVK. Different species were found in the two areas: Rattus tiomanicus in Riau, and a mix of R. tanezumi and a close relative one in Bangka. The rats in Riau were much more susceptible to coumatetralyl than those in Bangka. This study is the first to demonstrate physiological tolerance to AVK in these species. vkorc1 displayed low levels of polymorphism, and no SNP was associated with the high-tolerance phenotypes of R. tanezumi clade, even those exposed to very high concentrations (32 x the effective dose of 0.36 mg kg(-1)). The biochemical basis of this tolerance remains unknown, but may involve the vkorc1 promoter and/or cytochrome P450 metabolism. We discuss our results and the selective role of anticoagulant use in the occurrence of phenotypic tolerance.

Leptospirosis is a major zoonotic disease that affects humans and animals in all continents, in both rural and urban areas. In Europe, metropolitan France is the most affected country, with about 300 human cases declared per year. In France, although leptospirosis is now mostly considered as a recreational disease related to freshwater areas, isolation of pathogenic leptospires from environmental water samples still remains difficult. It thus seemed important to set up an efficient method to detect and quantify these bacteria in this environment. We determined a DNA extraction method suitable for freshwater samples and adapted a real-time quantitative PCR based on the detection of the LipL32 gene using the SYBR green chemistry. The method developed is specific for pathogenic Leptospira. It permits the detection of all the pathogenic strains tested and none of the saprophytic strains. Quantification is possible between 10 and 107 bacteria/mL, and therefore, the method represents a tool that could be integrated into future public health surveillance programs for recreational freshwater areas.

International audience; . Background: The VKORC1 gene codes for the VKORC1 enzyme, which is responsible for the reduction of vitamin K epoxide into vitamin K. VKORC1 enzyme is the target of vitamin K antagonists (VKA). Twenty-eight rare single mutations in the VKORC1 coding sequence have been reported from resistant patients receiving unusually high doses of VKA to achieve therapeutic anticoagulation. Objectives: It has been suggested that these mutations are responsible for the resistant phenotype, while biochemical consequences of these mutations on the VKORC1 enzyme have not yet been evaluated. Therefore, the aim of this study was to investigate the causality of the VKORC1 mutations in the resistance phenotype. Methods: Wild-type VKORC1 and its spontaneous mutants were expressed in Pichia pastoris and susceptibility to VKA was assessed by the in vitro determination of kinetic and inhibition constants. Results and Conclusions: The in vitro analysis revealed that six mutations only (A26P, A41S, V54L, H68Y, I123N and Y139H) were associated with increase in Ki, suggesting their involvement in the resistance phenotype observed in patients. A41S and H68Y led to selective resistance, respectively, to indane-1,3-dione and 4-hydroxycoumarine derivatives. The other mutations did not increase the Ki. Furthermore, 10 mutations (S52L, S52W, W59L, W59R, V66M, V66G, G71A, N77S, N77T and L128R) led to an almost complete loss of activity. These results suggest the existence of other resistance mechanisms.

International audience; In this study, osprey (Pandion haliaetus) tissues collected in France were used for contaminants analyses by a non-invasive approach. 53 pesticides (organochlorine, organophosphate, carbamate, pyrethroids, herbicides, anticoagulant rodenticides), 13 PCBs, and 5 metals (mercury, lead, cadmium, copper and arsenic) were quantified in liver of 14 individual samples. PCBs and mercury were frequent (mean 0.5 mg/kg, range < d.l.-2.6 mg/kg and mean 3.4 mg/kg, range < d.l. -16.3 mg/kg wet weight, respectively). Inter-individual variations in contaminant diversity and amounts were noticed. Immediate conservation of the species in the country does not seem to be threatened by global contamination.

Objective To determine constant rate infusion (CRI) protocols for romifidine (R) and romifidine combined with butorphanol (RB) resulting in constant sedation and romifidine plasma concentrations. Study design Blinded randomized crossover study. Animals Ten adult research horses. Methods Part I: After determining normal height of head above ground (HHAG = 100%), loading doses of romifidine (80 μg kg −1 ) with butorphanol (RB: 18 μg kg −1 ) or saline (R) were given intravenously (IV). Immediately afterwards, a butorphanol (RB: 25 μg kg −1 hour −1 ) or saline (R) CRI was administered for 2 hours. The HHAG was used as marker of sedation depth. Sedation was maintained for 2 hours by additional romifidine (20 μg kg −1 ) whenever HHAG > 50%. The dose rate of romifidine (μg kg −1 hour −1 ) required to maintain sedation was calculated for both treatments. Part II: After loading doses, the romifidine CRIs derived from part I were administered in parallel to butorphanol (RB) or saline (R). Sedation and ataxia were evaluated periodically. Romifidine plasma concentrations were measured by HPLC-MS-MS at 0, 5, 10, 15, 30, 45, 60, 90, 105, and 120 minutes. Data were analyzed using paired t -test, Fisher's exact test, Wilcoxon signed rank test, and two-way anova for repeated measures ( p Results There was no significant difference in romifidine requirements (R: 30; RB: 29 μg kg −1 hour −1 ). CRI protocols leading to constant sedation were developed. Time to first additional romifidine bolus was significantly longer in RB (mean ± SD, R: 38.5 ± 13.6; RB: 50.5 ± 11.7 minutes). Constant plasma concentrations of romifidine were achieved during the second hour of CRI. Ataxia was greater when butorphanol was added. Conclusion Romifidine bolus, followed by CRI, provided constant sedation assessed by HHAG. Butorphanol was ineffective in reducing romifidine requirements in unstimulated horses, but prolonged the sedation caused by the initial romifidine bolus. Clinical relevance Both protocols need to be tested under clinical conditions.

Zearalenone (ZEN) is a non-steroid estrogen mycotoxin produced by numerous strains of Fusarium which commonly contaminate cereals. After oral administration, ZEN is reduced via intestinal and hepatic metabolism to alpha- and beta-zearalenol (alpha ZEL and beta zEL). These reduced metabolites possess estrogenic properties, alpha ZEL showing the highest affinity for ERs. ZEN and reduced metabolites cause hormonal effects in animals, such as abnormalities in the development of the reproductive tract and mammary gland in female offspring, suggesting a fetal exposure to these contaminants. In our previous work, we have suggested the potential impact of ZEN on placental cells considering this organ as a potential target of xenobiotics. In this work, we first compared the in vitro effects of alpha ZEL and beta ZEL on cell differentiation to their parental molecule on human trophoblast (BeWo cells). Secondly, we investigated their molecular mechanisms of action by investigating the expression of main differentiation biomarkers and the implication of nuclear receptor by docking prediction. Conversely to ZEN, reduced metabolites did not induce trophoblast differentiation. They also induced significant changes in ABC transporter expression by potential interaction with nuclear receptors (LXR, PXR, PR) that could modify the transport function of placental cells. Finally, the mechanism of ZEN differentiation induction seemed not to involve nuclear receptor commonly involved in the differentiation process (PPAR gamma). Our results demonstrated that in spite of structure similarities between ZEN, alpha ZEL and beta zEL, toxicological effects and toxicity mechanisms were significantly different for the three molecules. (C) 2012 Elsevier Inc. All rights reserved.

The antivitamines K (AVKs) are molecules which can be inhibiting the activation of PVKDs, preventing or delaying the blood clotting. Today, the treatment by AVKs is the first cause of iatrogenic accidents medicated in humans. In addition, the benefits are significantly higher than the risk so they are used to control the populations of rodent pests. The AVKs are acting as inhibitor for the enzyme VKORC1. The phenomena of hypersensitivity and the resistance to AVKs have been emerged and several spontaneous mutations have been discovered in the gene vkorc1. These mutations were associated with resistance despite the fact that this link has been never been demonstrated. The objective of this work is to confirm or deny these causal links. This study helps to demonstrate the responsibility of the mutations Leu120Gln, Leu128Gln and Tyr139Phe, Ser or Cys in the emergence of the phenotype resistant to AVKs 1ST generation among the wild rat. The catalytic property explains the biological cost associated with the emergence of this resistance in some strains of wild rats. Thus, the mutations at position 139 are responsible for the misappropriation of the catalysis with majority production of hydroxyvitamine K which is directly consumable by the body. In humans, 29 mutations out of 30 have been characterized. While these mutations have been observed in patients resistant to AVKs, the causality of these mutations has been demonstrated only for 6 mutations (Ala26Pro, Val41Ser, Val54Leu, His68Tyr, Ile123Asn, Phe139His) and the other mutations would not be responsible for the phenotype observed, Les anticoagulants antivitamines K (AVKs) sont des molécules qui par inhibition de l’activation des PVKDs, empêchent ou retardent la coagulation sanguine. Aujourd’hui, le traitement par AVKs est la première cause d’accidents iatrogènes médicamenteux chez l’homme. Cependant le bénéfice par rapport au risque étant largement supérieur. Elles sont également utilisées dans le contrôle des populations de rongeurs nuisibles. Les AVKs agissent en inhibant l’enzyme VKORC1. Des phénomènes d’hypersensibilité et de résistance aux AVKs sont apparus et plusieurs mutations spontanées ont été découvertes dans le gène vkorc1. Ces mutations ont alors été associées à la résistance sans que ce lien n’ait jamais été démontré. Le travail, présenté dans ce mémoire, a pour objectif de confirmer ou d’infirmer ces liens de causalité. Cette étude a permis de démontrer la responsabilité des mutations Leu120Gln, Leu128Gln et Tyr139Phe, Ser et Cys dans l’apparition du phénotype résistant aux AVKs de 1ère génération chez le rat sauvage. Les propriétés catalytiques expliquent de plus le coût biologique associé à l’apparition de cette résistance chez certaines lignées de rats sauvages. Ainsi, les mutations en position 139 sont responsables d’un détournement de la catalyse avec production majoritaire d’hydroxyvitamine K directement éliminable par l’organisme. Chez l’homme, 29 mutations sur 30 ont été caractérisées. Alors que ces mutations ont été observées chez des patients résistants aux AVKs, la causalité de ces mutations n’a été démontrée que pour 6 mutations (Ala26Pro, Val41Ser, Val54Leu, His68Tyr, Ile123Asn, Phe139His). Les autres mutations ne seraient pas responsables du phénotype observé

The antivitamines K (AVKs) are molecules which can be inhibiting the activation of PVKDs, preventing or delaying the blood clotting. Today, the treatment by AVKs is the first cause of iatrogenic accidents medicated in humans. In addition, the benefits are significantly higher than the risk so they are used to control the populations of rodent pests. The AVKs are acting as inhibitor for the enzyme VKORC1. The phenomena of hypersensitivity and the resistance to AVKs have been emerged and several spontaneous mutations have been discovered in the gene vkorc1. These mutations were associated with resistance despite the fact that this link has been never been demonstrated. The objective of this work is to confirm or deny these causal links. This study helps to demonstrate the responsibility of the mutations Leu120Gln, Leu128Gln and Tyr139Phe, Ser or Cys in the emergence of the phenotype resistant to AVKs 1ST generation among the wild rat. The catalytic property explains the biological cost associated with the emergence of this resistance in some strains of wild rats. Thus, the mutations at position 139 are responsible for the misappropriation of the catalysis with majority production of hydroxyvitamine K which is directly consumable by the body. In humans, 29 mutations out of 30 have been characterized. While these mutations have been observed in patients resistant to AVKs, the causality of these mutations has been demonstrated only for 6 mutations (Ala26Pro, Val41Ser, Val54Leu, His68Tyr, Ile123Asn, Phe139His) and the other mutations would not be responsible for the phenotype observed, Les anticoagulants antivitamines K (AVKs) sont des molécules qui par inhibition de l’activation des PVKDs, empêchent ou retardent la coagulation sanguine. Aujourd’hui, le traitement par AVKs est la première cause d’accidents iatrogènes médicamenteux chez l’homme. Cependant le bénéfice par rapport au risque étant largement supérieur. Elles sont également utilisées dans le contrôle des populations de rongeurs nuisibles. Les AVKs agissent en inhibant l’enzyme VKORC1. Des phénomènes d’hypersensibilité et de résistance aux AVKs sont apparus et plusieurs mutations spontanées ont été découvertes dans le gène vkorc1. Ces mutations ont alors été associées à la résistance sans que ce lien n’ait jamais été démontré. Le travail, présenté dans ce mémoire, a pour objectif de confirmer ou d’infirmer ces liens de causalité. Cette étude a permis de démontrer la responsabilité des mutations Leu120Gln, Leu128Gln et Tyr139Phe, Ser et Cys dans l’apparition du phénotype résistant aux AVKs de 1ère génération chez le rat sauvage. Les propriétés catalytiques expliquent de plus le coût biologique associé à l’apparition de cette résistance chez certaines lignées de rats sauvages. Ainsi, les mutations en position 139 sont responsables d’un détournement de la catalyse avec production majoritaire d’hydroxyvitamine K directement éliminable par l’organisme. Chez l’homme, 29 mutations sur 30 ont été caractérisées. Alors que ces mutations ont été observées chez des patients résistants aux AVKs, la causalité de ces mutations n’a été démontrée que pour 6 mutations (Ala26Pro, Val41Ser, Val54Leu, His68Tyr, Ile123Asn, Phe139His). Les autres mutations ne seraient pas responsables du phénotype observé

In Africa, metal and pesticide contamination of wetlands is supposed to continue on a permanent basis. However, the ecotoxicological status of these ecosystems remains poorly documented. The aim of the present work is to assess the value of the Nile monitor (Varanus niloticus) as a sentinel species for the environmental contamination of continental wetlands in sub-Saharan Africa. Lead and cadmium on the one hand, and organochlorine and organophosphate pesticides on the other, have been quantified in several tissues by atomic absorption spectrophotometry and gas chromatography, respectively. Samples come from 71 specimens originating from four sites considered unequally contaminated (in Mali and Niger). Although clear differences appear between sites, the environmental contamination turns out to be moderate at the four sites, and does not seem to represent a significant risk neither for the monitors themselves, nor for occasional human consumers. However, the interindividual variability is important. The organotropisms relative to the detected pollutants are consistent with those described in previous studies. Concerning the pesticides, no gender effect has been found, whereas females were more contaminated by metals. The relation between other factors (size, proportion of fat) and tissue concentrations has been considered too. Nile monitors can reveal subtle differences in local pollution and the spatial resolution of this tool seems to be very sharp. Its practical relevance is thus validated. Additionally, an experimental work has been carried out on captive monitors to go into the subject in greater depth; En Afrique, la contamination des zones humides par les métaux et les pesticides va se poursuivre durablement. Pour autant, le statut toxicologique de ces milieux reste trop peu documente. Le but du présent travail est d’estimer la valeur du varan du Nil (Varanus niloticus) en tant qu’espèce sentinelle pour la contamination des zones humides continentales d’Afrique sub-Saharienne. Plomb, cadmium, et pesticides organochlorés et organophosphorés ont été quantifies, par spectrométrie d’absorption atomique et chromatographie gazeuse (respectivement), dans plusieurs tissus provenant de 71 spécimens issus de quatre sites (au Mali et au Niger) juges a priori inégalement contamines. Bien que des différences claires apparaissent, la contamination environnementale s’avère modérée sur les quatre sites, et ne semble pas constituer un risque notable pour les varans ni pour les humains qui s’en nourrissent occasionnellement. Toutefois la variabilité interindividuelle est importante. Les organotropismes des polluants détectés sont cohérents avec ceux préalablement décrits. Si l’on n’a pu mettre en évidence de différence liée au sexe en ce qui concerne les pesticides, les femelles présentaient des charges en métaux supérieures. La relation entre d’autres facteurs (taille, proportion de graisse) et les concentrations tissulaires a également été considérée. Les varans sont susceptibles de révéler des différences subtiles de contamination environnementale entre sites, et la résolution spatiale de l’outil semble très fine. La possibilité pratique d’utiliser cet indicateur se trouve donc validée. Un travail expérimental sur des varans captifs a par ailleurs été mené pour approfondir l’étude

Colocasia esculenta L SCHOTT is a valuable tuber widely consumed in Chad by humans and livestock. There is very little available literature about the cultivation and the post-harvest traditional technologies of the Chadian varieties of taro. A survey was carried out in the Mayo-Kebbi region ( Chad) where taro is a widely cultivated. There are two main varieties of taro in Chad : the " Gouning souol " variety, a local variety characterized by an important level of acridity and the prolonged cooking time (6 to 8 hours). The presence of these properties led to the replacement of the cultivation of this variety by the " Gouning sosso " variety which presents a lower level of acridity and a shorter cooking time (45 to 60 minutes). A traditional process to reduce the acridity and decrease the cooking time consists in dipping the slices of fresh taro in the water, or in a corn solution or in a tamarind infusion for a period of 24 to 48 hours according to the intensity of the acridity. After drying in the sun, the dried chips are ground into flour. This flour is then used to prepare mixtures of porridge, soups and cakes. Analyses were carried out on samples of taro flour produced in accordance with the traditional methods of soaking to determine the effects of this traditional process. Fresh taro chips were dipped over a period of 0, 1, 3, 6, 12 and 24 hours in the water, or in the corn infusion or in the tamarind infusion. Each sample was then dried in the sun for 48 hours and ground into flour. Dry matter, crude protein, ash, crude fiber, minerals and various trace elements were assessed. The in vitro digestibility of taro starch using α-amylase enzyme and the amount of total oxalates in the different taro flour were also determined. The soaking in the tamarind infusion induced a significant (P < 0.001) decrease in protein content (from 3.26 % for the control sample to 2.68 % for the treatment with the tamarind infusion at T24). The other treatments (corn solution and water) reduced also the protein level of taro but not significantly. The crude fibre content was not affected by the tamarind infusion but decreased significantly in the other treatments at the threshold level of 5 %. All methods of soaking led to a significant decrease of the mineral contents, except for an increase in sodium (due to the quality of the water used to prepare solutions) and in phosphorus (probably due to the hydrolysis of phytates by the corn infusion). A significant loss in trace element (Iron) was observed, decreasing from 144 mg / kg of DM (control sample) to 78 mg / kg of DM (after 12 hours of water soaking) corresponding to a loss of 45.83 %. The lowest decrease in iron level was observed after the treatment by the tamarind infusion (31.25 % after 24 hours). All the soaking processes were seen to have improved the digestibility of taro flour. By simply cooking (at a temperature of 95°C for 30 minutes), the in vitro digestibility increased from 39.30 % for the control sample to 78.67 % for the water treated samples of taro. After three hours of soaking, the taro flour treated by the solution of corn becomes significantly more digestible (77.12 %) than by the two other treatments [tamarind (60.86 %), water (60.45 %)] that are statistically similar. (...); Le taro (Colocasia esculenta L SCHOTT) est un tubercule d’une grande importance alimentaire au Tchad pour la consommation humaine et animale. La littérature sur les pratiques culturales et les technologies traditionnelles post-récolte des variétés tchadiennes demeure presque inexistante. Une enquête a donc été menée dans la région du Mayo-Kebbi (Tchad) où le taro est très cultivé. Il résulte qu’il existe principalement deux variétés de taro au Tchad : la variété « Gouning souol» ou variété locale qui est caractérisée par une âcreté importante et un temps de cuisson prolongé (6 à 8 h). Ces propriétés ont entraîné l’abandon de la culture de cette variété au profit de la variété « Gouning sosso » qui présente une âcreté moindre et un temps de cuisson plus court (45 à 60 minutes). Une technique traditionnelle pour réduire l’âcreté et diminuer le temps de cuisson consiste à tremper les tranches de taro frais soit dans l’eau, soit dans une solution de trempage de maïs ou soit dans une infusion de tamarin pendant 24 à 48 h selon l'intensité de l'âcreté. Après séchage au soleil, les cossettes sèches sont transformées en farine. Ces produits de transformation servent à la préparation de boules de pâtes, de bouillie, de soupes ou de beignets. Des analyses ont été effectuées sur une gamme d’échantillon de farine de taro produite conformément à la recette artisanale dans l’optique de comprendre l’effet de cette technologie traditionnelle. Des cossettes fraîches de taro ont été trempées pendant 0, 1 h, 3 h, 6 h, 12 h et 24 h dans de l’eau, ou dans une solution de trempage de maïs ou dans une infusion de tamarin. Chaque échantillon a ensuite été séché au soleil pendant 48 h puis broyé en farine. La matière sèche, les protéines brutes, les cendres, les fibres brutes, les macroéléments minéraux et oligoéléments ont été évalués dans le but de déterminer les effets du trempage traditionnel sur le profil nutritionnel du taro. La digestibilité in vitro de l’amidon sous l’action de l’α-amylase et la teneur en oxalates de différentes farines de taro ont été également déterminées. Il ressort de ces analyses que seul le trempage dans une infusion de tamarin a induit une baisse significative (P < 0,001) de la teneur en protéines brutes (de 3,26 % pour T0 à 2,68 % pour le traitement au tamarin à T24). L’infusion de tamarin n’a pas modifié la teneur en fibres brutes alors que les autres traitements l’ont assez significativement diminuée (P < 0,001). Toutes les méthodes de trempage ont entraîné une diminution significative des teneurs en minéraux, à l’exception d’une augmentation des teneurs en sodium (due à la qualité de l’eau des préparations) et en phosphore (probablement due à l’hydrolyse des phytates du maïs). Une perte significative en fer a été constatée passant de 144 mg/kg de MS (témoin) à 78 mg/kg de MS (échantillon traité à l’eau pendant 12 h), soit une perte de 45,83 %. La plus faible diminution de taux de fer est observée dans le cas de l'échantillon trempé dans l'infusion de tamarin avec une perte maximale de 31,25 % après 24 h. La teneur en zinc ne varie pas. Les procédés de trempage entraînent globalement une amélioration de la digestibilité du taro après une simple cuisson (95°C pendant 30 minutes) qui passe de 39,30 % (échantillon témoin non trempé) à 78,67 % pour le taro trempé dans l’eau. Après 3 h de trempage, la farine de taro traitée par la solution de maïs est significativement plus digestible (77,12 %) que les échantillons résultant de deux autres traitements [tamarin (60,86 %), eau (60,45 %)] qui sont statistiquement similaires. (...)

International audience; Antivitamin K anticoagulants have been commonly used to control rodent pest all over the world for more than 50 years. These compounds target blood coagulation by inhibiting the vitamin K epoxide reductase (VKORC1), which catalyzes the reduction of vitamin K 2,3-epoxide to vitamin K. Resistance to anticoagulants has been reported in wild rat populations from different countries. From these populations, several mutations of the rVkorcl gene have been reported. In this study, rat VKORC1 and its most frequent mutants L120Q-, L128Q-, Y139C-, Y139S- and Y139F-VKORC1 were expressed as membrane-bound proteins in Pichia pastoris and characterized by the determination of kinetic and inhibition parameters. The recombinant rVKORC1 showed similar properties than those of the native proteins expressed in the rat liver microsomes, validating the expression system as a good model to study the consequences of VKORC1 mutations. The determination of the inhibition parameters towards various antivitamin K anticoagulants demonstrated that mutations at Leu-120, Leu-128 and Tyr-139 confer the resistance to the first generation AVKs observed in wild rat populations.

International audience; The anti-vitamin Ks (AVKs) are widely used to control rodent populations. They inhibit Vitamin K regeneration by the Vitamin K Epoxide Reductase (VKOR) and cause a fatal hemorrhagic syndrome. Because of repeated use, some populations of commensal rodents have expressed resistance to these compounds. In Franche- Comte´ (France), the water vole exhibits cyclic population outbreaks. A second generation AVK, bromadiolone, has been used for the last 20 years to control vole populations. The aim of this study is to determine whether these repeated treatments could have led to the development of resistance to AVKs in water vole populations. We conducted enzymatic and genetic studies on water voles trapped in treated and non treated plot. The results indicate that voles from the most heavily treated area exhibit enzymatic changes in VKOR activity hence arguing for resistance to AVKs and that an intronic haplotype on the vkorc1 gene seems to be associated with these enzymatic changes.

National audience

International audience; BACKGROUND: Anticoagulant rodenticides are commonly used to control rodent pests all over the world. These pesticides inhibit one enzyme of the vitamin K cycle, Vkorc1, and thus prevent blood clotting and cause death by haemorrhage. Resistance to anticoagulants was first observed in Scotland in 1958, and more potent anticoagulants have been developed to overcome this obstacle. Unfortunately, these chemicals are very toxic and cannot be used everywhere. Some authors have shown that resistance to anticoagulants seems closely linked with single nucleotide polymorphism (SNP) in the Vkorc1 gene. RESULTS: This study draws a map of SNP and haplotypes found in Vkorc1 in rats from different areas of France. Some of them had never been described before. Moreover, the Y139F mutation, described previously in France and Belgium, is the most frequent in France. This mutation is known to be associated with a strong resistance to anticoagulants, and it was found in 28% of the samples. CONCLUSION: This biomolecular approach to studying and detecting resistance is easier to carry out than the phenotypic approach measuring blood coagulation time because it can be conducted on biological samples from dead animals, and it is less dangerous for the operator.

International audience; Fatty acid composition of the adipose tissue of six carnivorous mammalian species (European otter Lutra lutra, American mink Mustela vison, European Mink Mustela lutreola, European polecat Mustela putorius, stone marten Martes foina and European wild cat Felis silvestris) was studied. These species forage to differing degrees in aquatic and terrestrial food webs. Fatty acid analysis revealed significant differences in polyunsaturated fatty acid composition between species. More specifically, our results underline a gradual significant decrease in the docosahexaenoic acid (DHA)/linoleic acid (LNA) ratio of carnivore species as their dependence on aquatic food webs decreases. In conclusion, the use of the DHA/LNA ratio in long-term studies is proposed as a potential proxy of changes in foraging behaviour of semi-aquatic mammals.

Les endectocides de type avermectines (ivermectine, abamectine, doramectine) ou mylbemycines (moxidectine) constituent un groupe de produits a large spectre insecticide et nématocide, très utilisées dans le monde entier. Etant donné leurs caractéristiques pharmacodynamiques (large spectre d'action) et pharmacocinétiques (faible métabolisation, longue rémanence, excrétion fécale), ces molécules font aujourd'hui l'objet d'une vigilance scientifique particulière. Or, l'autorisation de mise sur le marché de l'ivermectine sous la forme de bolus intestinal à relargage progressif du produit a précédé de peu la promulgation de la directive 93/40/CEE. Ainsi, l'évaluation du risque écotoxicologique associé a été sommaire, alors que des travaux scientifiques antérieurs avaient déjà attiré l'attention sur le risque environnemental de cette molécule.Nos travaux ont montré que l'ivermectine administrée sous forme de bolus intra­ ruminal se retrouve a des concentrations importantes dans les féces de bovins pour une période supérieure à deux mois après le début du traitement. L'éprinomectine distribuée par voie trans-cutanée est excrétée à raison d'un cinquième de la dose par voie fécale, avec des concentrations maximales au cours de la première semaine suivant l'administration.L'exposition d'insectes coprophages à des bouses provenant d'animaux traités a démontré l'effet toxique de l'ivermectine sur le développement larvaire d'Aphodius constans (Coleoptere, Aphodiidae) à de faibles concentrations.L'ivermectine biologiquement active vis-a-vis des nématodes reste presente dans les bouses pendant une période prolongée apres l’administration du bolus. Toutefois, la biodisponiblité pour les nématodes semble faible mais les concentrations existantes encore actives, semblent suffire pour entrainer la sélection de parasites résistants.Les études réalisées sur les bactéries et champignons du sol n'ont pas permis la mise évidence d'effets globaux de l'ivermectine malgré l'utilisation de concentrations de produit tres en excès des concentrations attendues dans le sol. Ces résultats sont probablement en rapport avec la faible biodisponibilité de l'ivermectine résultant de sa très faible hydrosolubilité.Le bivalve d'eau douce Dreissena polymorpha est un mollusque dont l'activité de filtration lui confère un pouvoir de bioaccumulation des composés organiques ou minéraux. En aquarium, ces mollusques ont été exposés a de l'ivermectine incorporée à des microparticules. En fait, la sensibilité des dreissènes a l'ivermectine est très marquée et compatible avec une exposition a des résidus dissous.

Un suivi de la physico-chimie des eaux (formes de l'azote et du phosphore et matières en suspension, MES, essentiellement) est réalisé pour douze sites localisés sur des ruisseaux du bassin Lémanique et deux sites sur des ruisseaux du Massif Armoricain en Basse Normandie présentant des degrés d'anthropisation différents (Projet AQUAE). Pour les premiers, il s'agit d'un prélèvement ponctuel toutes les semaines pendant 2 ans et pour les seconds d'un suivi en continu des MES et d'un prélèvement tous les 15 jours pendant 1,5 an. Les résultats des analyses permettent de classer les sites lémaniques selon un gradient de trophie et d'opposer les sites normands en fonction des flux et concentrations en MES. Parallèlement, ont été réalisés dans les deux régions des suivis de la structure de communautés (invertébrés benthiques, diatomées épilithiques et macrophytes aquatiques) et d'un organisme sentinelle (truite au stade embryolarvaire). Sur les sites lémaniques, les réponses des communautés microbiennes épilithiques et des sédiments ainsi que d'un bivalve, la dreissène, sont analysées. Il s'avère que les indices biocénotiques normalisés (IBGN, IBD et IBMR) ne révèlent des différences de qualité des milieux que si le contraste en matière de trophie ou de MES est suffisamment important. Sur le dispositif de lémanique, le gradient d'état trophique observé pour les différents bassins est suffisamment marqué pour que l'IBD et l'IBMR y soient corrélés. L'IBGN, qui réagit surtout à des variations en O2 dissous, n'est guère pertinent dans le dispositif étudié. Plus généralement, dans les cours d'eau de faibles ordres de drainage, la géomorphologie locale et la structure de l'habitat (plus particulièrement la faible hétérogénéité "naturelle" en substrats) constituent des variables de forçage plus importantes que les perturbations anthropiques sur la réponse de ces biocénoses. La sensibilité de la réponse de ces communautés bio-indicatrices peut être cependant améliorée dans les contextes étudiés en analysant plus finement la liste taxonomique (prise en compte d'un plus grand nombre de taxons et calcul de l'IPS pour les diatomées, part relative des taxons les plus polluo-sensibles chez les invertébrés et des taxons sténoèces chez les macrophytes). Par ailleurs, sur la base des listes faunistiques établies pour le calcul de l'IBGN, on améliore la sensibilité de la réponse de la communauté d'invertébrés aux perturbations étudiées par l'analyse des spectres de répartition de traits biologiques ou par l'établissement de courbes de dominance (abondance et biomasse). Le gradient trophique des sites lémaniques est aussi discriminé par des activités enzymatiques des biofilms microbiens telles le "potentiel dégradatif" (glucosidase, phosphatase, aminopeptidases) mais aussi par des paramètres plus généraux comme la respiration et la dénitrification. Parmi les réponses de la dreissène (Dreissena polymorpha Pallas), le pourcentage de mortalité est un paramètre qui permet de discriminer les sites de trophie différente. Les réponses fonctionnelles comme l’indice de condition ou la respiration, fluctuent régulièrement mais la variabilité intrinsèque de ces facteurs est supérieure à la variabilité mesurée dans le gradient d'exposition. De la même façon, l’induction de protéines comme la métallothionéine n'a pas permis de différencier les sites. Dans les têtes de bassin, zones privilégiées de reproduction de la truite (Salmo trutta), il est étudié la réponse des stades les plus sédentaires (œufs enfouis dans les graviers pendant plusieurs semaines) et les plus sensibles à la pollution. La survie embryo-larvaire présente un bonne sensibilité tant à la trophie du milieu qu’aux MES. Les principaux paramètres physico-chimiques incriminés sont une diminution dans le milieu interstitiel de l'O2 dissous et des teneurs en N-NH4 élevées. Outre un impact sur les survies, les perturbations du milieu engendrent un retard de croissance des embryons à l'émergence. Si la survie embryo-larvaire de la truite s'avère un bon témoin de la qualité des milieux, il est nécessaire dans sa mise en œuvre d'une part de disposer d'œufs de bonne qualité et d'autre part de réaliser un grand nombre de réplicats vue la forte variabilité des réponses intra-site. Ce travail révèle l'utilité d'une approche multi-bioindicateur et l'intérêt des réponses fonctionnelles des communautés et organismes en complément des analyses de biodiversité pour étudier les impacts de perturbations de la qualité des eaux.

International audience; Eleven Microcebus murinus (lemur) primates were intracerebrally or orally infected by bovine spongiform encephalopathy (BSE) or macaque-adapted BSE (MBSE) brain homogenates. In many BSE and MBSE infected lemurs, but not in animals inoculated with normal bovine brain, persistent behavioral changes occurred as early as 3 months, and neurological signs as early as 13 months after infection. Immunohistochemical examination of animals sacrificed during the incubation period revealed an abnormal accumulation of 'prion' protein (PrP) in the intestinal wall, intestinal nervous plexus, mesenteric lymph nodes and spleen, and in the clinical stage, also in the brain. In MBSE-inoculated animals, proteinase K resistance of the PrP (PrPres) was confirmed by Western blot in the spleen and the brain. Obvious signs of neurodegeneration were observed in all infected animals characterized by hyperaggregated and paired-helical filaments-immunoreactive Tau proteins, beta 42-amyloid plaques and astrogliosis. Additionally, PrPres was present in the ganglion cells of the retina in diseased animals after either intracerebrally or oral infection by the BSE or MBSE agent. These results show that the microcebe is susceptible to the BSE infectious agent via intracerebral and oral routes with comparatively short incubation periods compared to simians, and could be a useful animal model to study the pathophysiology of disease transmission in primates.

Poster *INRA UMR ENVL Métabolisme et Toxicologie Comparés des Xénobiotiques 69280 Marcy l'Etoile (FRA) Diffusion du document : INRA UMR ENVL Métabolisme et Toxicologie Comparés des Xénobiotiques 69280 Marcy l'Etoile (FRA); absent

Summary • Variability in the fungal endophytes of 83 natural populations of Lolium perenne (perennial ryegrass) from Europe was assessed. • One plant per population was used for endophyte isolation and mycotoxin analysis. Variability in three isozymes, colony morphology and growth rate on potato dextrose agar (PDA), and synthesis of ergovaline, lolitrem B and peramine was recorded. • Three species were found among 94 strains isolated: Neotyphodium lolii, Neotyphodium sp. (LpTG-2) and Gliocladium-like. The most frequent species was N. lolii, which showed high variability. In 12 populations, a single plant harboured two different endophytes. One-third of the isolates of N. lolii did not produce ergovaline whereas a few isolates did not produce lolitrem B. Ergovaline and lolitrem B-deficient strains, but not the few peramine-deficient isolates, had characteristic morphologies on PDA. No isolate was deficient for both ergovaline and lolitrem B synthesis. • Selection of ergovaline and lolitrem-deficient strains based only on the morphology of the isolates in culture may be possible.

INRA ENVL Métabolisme et Toxicologie Comparés des Xénobiotiques 69260 Marcy l'Etoile (FRA) Diffusion du document : INRA ENVL Métabolisme et Toxicologie Comparés des Xénobiotiques 69260 Marcy l'Etoile (FRA); absent

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