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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.

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.

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.

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.

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.

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.

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.

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.

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.