Adaptation of a real-time PCR method for the detection and quantification of pathogenic leptospires in environmental water.

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 10(7) bacteria/mL, and therefore, the method represents a tool that could be integrated into future public health surveillance programs for recreational freshwater areas.

Are water vole resistant to anticoagulant rodenticides following field treatments?

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-Comté (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.

Assessment of ruminal degradation, oral bioavailability, and toxic effects of anticoagulant rodenticides in sheep.

OBJECTIVE: To assess the rate and extent of ruminal degradation of warfarin, chlorophacinone, and bromadiolone in vitro and determine the oral availability and clinical and hemostatic effects of each anticoagulant rodenticide in adult sheep. ANIMALS: 3 Texel sheep. PROCEDURE: Samples of ruminal fluid were incubated with each of the anticoagulants to assess the kinetics of ruminal degradation over 24 hours. To determine the plasma kinetics of the anticoagulants, each sheep received each of the anticoagulants IV or via a rumenimplanted cannula at 2-month intervals (3 rodenticide exposures/sheep). At intervals during a 240- to 360- hour period after treatment, prothrombin time (PT) was measured, plasma anticoagulant concentration was assessed, and clinical signs of rodenticide poisoning were monitored. In plasma and rumen extracts, anticoagulant concentrations were determined via high-performance liquid chromatography. RESULTS: In the rumen extracts, anticoagulants were slightly degraded (< 15%) over 24 hours. In vivo, oral availability of warfarin, chlorophacinone, and bromadiolone was estimated at 79%, 92%, and 88%, respectively. Although maximum PT was 80 seconds after chlorophacinone and bromadiolone treatments, no clinical signs of toxicosis were detected; PT returned to baseline values within 2 weeks. CONCLUSIONS AND CLINICAL RELEVANCE: In sheep, warfarin, chlorophacinone, and bromadiolone were not degraded in the rumen but their bioavailabilities were high after oral administration; the kinetics of these compounds in sheep and other mammals are quite similar. These data suggest that the lack of susceptibility of ruminants to these anticoagulant rodenticides cannot be explained by either ruminal degradation or the specific toxicokinetics of these anticoagulants.

Evidence of secondary poisoning of free-ranging riparian mustelids by anticoagulant rodenticides in France: implications for conservation of European mink (Mustela lutreola).

Because of the rapid decline of the endangered European mink (Mustela lutreola) populations in France, a national conservation program has been put into action, including research to understand the causes of decline. As part of this research, concentrations of eight anticoagulant rodenticides were examined in livers from 122 carcasses of four species of free-ranging mustelids collected between 1990 and 2002 in southwestern France. Bromadiolone residue was found in all species and 9% of the sample (one of 31 European mink, three of 47 American mink [Mustela vison], five of 33 polecats [Mustela putorius], and two of 11 European otters [Lutra lutra]). Liver concentrations ranged from 0.6 mug/g to 9.0 mug/g. Chlorophacinone residue was found in two species and 4% of the sample (in four of the American mink and in one of the otters), with liver concentrations ranging from 3.4 mug/g to 8.5 mug/g. Two polecats and one American mink had lesions and liver residues indicating bromadiolone was directly responsible for their death. However, most of our study animals survived secondary poisoning until they were caught; this study certainly underestimates the extent of fatal exposure of mustelids to rodenticides. Moreover, anticoagulant poisoning could increase their vulnerability to other causes of death. The current status of the endangered European mink population is such that any additional risk factor for mortality is important, and it is thus urgent to monitor and reduce the extensive use of bromadiolone and chlorophacinone against field rodents in France.