Alexandra Esther

Spatial and temporal exposure patterns in non-target small mammals during brodifacoum rat control

Worldwide pest rodents on livestock farms are often regulated using anticoagulant rodenticides (ARs). Second generation ARs in particular can cause poisoning in non-target species due to their high toxicity and persistence. However, research on exposure of small mammals is rare. We systematically investigated spatial and temporal exposure patterns of non-target small mammals in a large-scale replicated study. Small mammals were trapped at different distances to bait stations on ten farms before, during and after brodifacoum (BR) bait application, and liver samples of 1178 non-target small mammals were analyzed for residues of eight ARs using liquid chromatography coupled with tandem mass spectrometry. BR residues were present in 23% out of 742 samples collected during and after baiting. We found clear spatial and temporal exposure patterns. High BR residue concentrations mainly occurred within 15m from bait stations. Occurrence and concentrations of residues significantly decreased with increasing distance. This pattern was found in almost all investigated taxa. After baiting, significantly more individuals contained residues than during baiting but concentrations were considerably lower. Residue occurrence and concentrations differed significantly among taxa, with the highest maximal residue concentrations in Apodemus species, which are protected in Germany. Although Sorex species are known to be insectivorous we regularly found residues in this genus. Residues of active agents other than brodifacoum were rare in all samples. The confirmation of substantial primary exposure in non-target small mammals close to the baiting area indicates considerable risk of secondary poisoning of predators, a pathway that was possibly underestimated until now. Our results will help to develop risk mitigation strategies to reduce risk for non-target small mammals, as well as their predators, in relation to biocidal AR usage.

Distribution and frequency of VKORC1 sequence variants conferring resistance to anticoagulants in Mus musculus.

BACKGROUND Emerging resistance to anticoagulant rodenticides may significantly impair house mouse (Mus musculus L.) control. As in humans and rats, sequence variants in the gene vitamin K epoxide reductase complex subunit 1 (VKORC1) of house mice are strongly implicated in the responses of mice to anticoagulants. This study gives a first overview of the distribution and frequency of such potentially resistance-conferring sequence variants in house mice, based on tissue samples from 30 populations in Germany, Switzerland and the Azores. RESULTS Except for one population from south Germany, sequence variants were found in individuals from all locations sampled (29 out of 30 sites surveyed), with less than 10% of the individuals matching the wild-type genotype. The most frequent and widespread amino acid substitutions were Leu128Ser, Tyr139Cys and a group of linked sequence changes (Arg12Trp/Ala26Ser/Ala48Thr/Arg61Leu). Where these substitutions occurred as the sole variant, the proportion of homozygous individuals was 72-83%. CONCLUSIONS An evaluation of published data revealed that the three most frequently found sequence variants are associated with a substantial loss of rodenticide efficacy of first-generation anticoagulants (e.g. warfarin, coumatetralyl), as well as the second-generation compound bromadiolone and most probably also difenacoum. Knowledge of the distribution and frequency of resistance-conferring sequence variants will stimulate their further functional characterisation and facilitate the choice of effective active substances for house mouse control.

Relation between Intensity of Biocide Practice and Residues of Anticoagulant Rodenticides in Red Foxes (Vulpes vulpes)

Anticoagulant rodenticides (ARs) are commonly used to control rodent infestations for biocidal and plant protection purposes. This can lead to AR exposure of non-target small mammals and their predators, which is known from several regions of the world. However, drivers of exposure variation are usually not known. To identify environmental drivers of AR exposure in non-targets we analyzed 331 liver samples of red foxes (Vulpes vulpes) for residues of eight ARs and used local parameters (percentage of urban area and livestock density) to test for associations to residue occurrence. 59.8% of samples collected across Germany contained at least one rodenticide, in 20.2% of cases at levels at which biological effects are suspected. Second generation anticoagulants (mainly brodifacoum and bromadiolone) occurred more often than first generation anticoagulants. Local livestock density and the percentage of urban area were good indicators for AR residue occurrence. There was a positive association between pooled ARs and brodifacoum occurrence with livestock density as well as of pooled ARs, brodifacoum and difenacoum occurrence with the percentage of urban area on administrative district level. Pig holding drove associations of livestock density to AR residue occurrence in foxes. Therefore, risk mitigation strategies should focus on areas of high pig density and on highly urbanized areas to minimize non-target risk.

Distribution of rodenticide resistance and zoonotic pathogens in Norway rats in Lower Saxony and Hamburg, Germany.

BACKGROUND Genetically based resistance to anticoagulants has led to increasing difficulties in the control of rodents over recent decades. The possible impact of rodenticide-resistant rats on the infection risk of humans and livestock by zoonotic pathogens is generally unknown. Hence, in a monitoring programme in the German federal states of Lower Saxony and Hamburg, more than 500 Norway rats were analysed for both Tyr139Cys polymorphisms within the VKORC1 gene and zoonotic agents. RESULTS Evidence of resistance was almost completely restricted to the known resistance area in southern Lower Saxony. Homozygous mutations were only found in urban areas sampled owing to the occurrence of rat control problems and were missing in bycatches of rats by muskrat trappers in rural areas. In more than 25% of the rats, zoonotic bacteria (Leptospira, Salmonella, Yersinia and Coxiella) were detected. There was no obvious correlation between the occurrence of rats carrying zoonotic pathogens and anticoagulant resistance. CONCLUSION Zoonotic agents and genetically based resistance conferred by the Tyr139Cys polymorphism are both unevenly distributed in Lower Saxony. The study provides the basis for further studies focusing on districts with high levels of pathogens and resistance to assess the potential health risk of their combined occurrence.

Identification of weather parameters related to regional population outbreak risk of common voles (Microtus arvalis) in Eastern Germany

Context Common vole (Microtus arvalis) populations can increase to several thousand individuals per hectare during outbreaks. In central Europe such outbreaks usually extend across large areas but there can be significant regional differences in outbreak intensity, general outbreak risk and associated crop damage. Aims We tested whether weather parameters can be used to explain the regional variability in outbreak risk of common voles in an area of Eastern Germany where common vole outbreaks are common. Method Suitable weather parameters were identified by principal component analysis (PCA). Time series of common vole abundance from 50 locations across 36 500 km2 sampled in 1973–97 were related to weather parameters selected by PCA and multiple linear regression. A hierarchical cluster analysis was used on relevant weather parameters to display the temporal and spatial variability in vole abundance. An overlay of risk class transformed abundances allowed for the identification of appropriate threshold values to define vole outbreaks. Key results Weather parameters were closely related to the variation in regional outbreak risk of common voles. Mostly weather parameters in winter and early spring were identified to be highly important. All risk thresholds tested revealed similar patterns for the distribution of risk classes across locations and years. While most years of very low or very high outbreak risk clustered well according to weather parameters, some cases of medium risk classes did not cluster well. Conclusions Weather parameters especially in winter and early spring are related to common vole outbreak risk in the following autumn. This is the case for extremely high and low outbreak risks and is largely independent of the choice of particular threshold values for outbreak risk. Implications Weather parameters could be used to develop automated forecast systems at the spatial resolution of single weather stations. Combined with other parameters that are easily available, such as information on soil characteristics, such forecasts might be as reliable as more complex biological models developed in the past.

Topography and soil properties contribute to regional outbreak risk variability of common voles (Microtus arvalis)

Context Common voles (Microtus arvalis) are the most common vertebrate pests in central European agriculture. During outbreaks common vole populations can increase to an enormous number of individuals; however, this outbreak risk varies regionally. Aims In this study we tested whether topography and soil properties are suited to explain the regional variability in the outbreak risk of the common vole in Eastern Germany. This study provides the first detailed large-scale study of the association of site characteristics and small mammal outbreak risk at a regional scale. Methods Data on common vole outbreaks were recorded by active burrow counts at 82 sampling sites in Eastern Germany from almost four decades. Data on topography and soil properties – i.e. groundwater fluctuation index, soil air capacity, saturated hydraulic conductivity, soil class and elevation – were obtained from soil maps and a digital elevation model in a geographic information system and were related to outbreak risk classes, applying classification and regression trees (CART). Based on these results a map of the outbreak risk was developed for the area. Key results Classification and regression tree analyses revealed that the mean elevation, area-related percentage of Chernozem soils and soil air capacity were the site characteristics best suited to explain local variability in outbreak risk. In the northern German lowland, below an elevation of ~83 m above sea level, the outbreak risk is generally very low. The region of the central upland has an increased risk for outbreaks of common voles. Within the region of the central uplands the risk was again elevated if the area covered by Chernozem soils was higher than 36%, and increased further if the area covered by soils with a moderate soil air capacity was higher than 90%. Conclusions Topography and soil properties, and accordingly the character of a landscape, are static parameters that affect the local risk of common vole outbreak. Further detailed field investigations of soil properties are required to link the variation in regional outbreak risk to site characteristics with relevance to common vole ecology. Implications Areas of varying regional outbreak risk of common voles can be defined according to static site characteristics identified in this study. They can provide a spatial framework to relate dynamic parameters, such as meteorological parameters, as well as biological parameters, such as food availability, to common vole outbreaks. This could be used in the future to develop improved predictive models to forecast common vole outbreaks.

Prey composition modulates exposure risk to anticoagulant rodenticides in a sentinel predator, the barn owl

Worldwide, small rodents are main prey items for many mammalian and avian predators. Some rodent species have pest potential and are managed with anticoagulant rodenticides (ARs). ARs are consumed by target and non-target small mammals and can lead to secondary exposure of predators. The development of appropriate risk mitigation strategies is important and requires detailed knowledge of AR residue pathways. From July 2011 to October 2013 we collected 2397 regurgitated barn owl (Tyto alba) pellets to analyze diet composition of owls on livestock farms in western Germany. 256 of them were fresh pellets that were collected during brodifacoum baiting. Fresh pellets and 742 liver samples of small mammals that were trapped during baiting in the same area were analyzed for residues of ARs. We calculated exposure risk of barn owls to ARs by comparing seasonal diet composition of owls with AR residue patterns in prey species. Risk was highest in autumn, when barn owls increasingly preyed on Apodemus that regularly showed AR residues, sometimes at high concentrations. The major prey species (Microtus spp.) that was consumed most frequently in summer had less potential to contribute to secondary poisoning of owls. There was no effect of AR application on prey composition. We rarely detected ARs in pellets (2 of 256 samples) but 13% of 38 prey individuals in barn owl nests were AR positive and substantiated the expected pathway. AR residues were present in 55% of 11 barn owl carcasses. Fluctuation in non-target small mammal abundance and differences in AR residue exposure patterns in prey species drives exposure risk for barn owls and probably other predators of small mammals. Exposure risk could be minimized through spatial and temporal adaption of AR applications (avoiding long baiting and non-target hot spots at farms) and through selective bait access for target animals.

Assessing the effects of three potential chemical repellents to prevent bird damage to corn seeds and seedlings

BACKGROUND Bird damage to seeds and seedlings of maize (Zea mays) and other crops is widespread, especially in organic farming, because no adequate seed protection is available. In this study, the effect of seed treatments with three substances likely to affect bird feeding behaviour (anthraquinone, pulegone and methyl anthranilate) was tested. Their repellent effect was tested in food and seedling choice (treated versus untreated) experiments with feral pigeons (Columba livia f. domestica) in aviaries. Most efficient variants were additionally tested in the field, where wild birds had unlimited access. RESULTS In aviaries, untreated seeds were clearly preferred over treated seeds by pigeons. The highest feeding deterrence effect occurred with the treatment variants pulegone 1.4 mL kg(-1) and methyl anthranilate 0.085 mL kg(-1). In contrast, there was no repellent effect if seedlings were offered to the pigeons in aviaries. The same applies to the number of maize seeds and seedlings grown from treated and untreated seeds, which were damaged in the field mainly by pheasants (Phasianus colchicus). CONCLUSION The study shows that the chemicals failed to repel feeding by birds in Germany. Further studies should concentrate on alternative, systemic effective substances possibly based on plant secondary metabolites to yield a bird repellent usable in organic farming.

VKOR variant and sex are the main influencing factors on bromadiolone tolerance of the house mouse (Mus musculus L.).

BACKGROUND After reports of management problems in practice, a survey was conducted to determine the presence of bromadiolone-resistant animals in different house mouse (Mus musculus L.) populations in Serbia. A 21 day no-choice feeding test was carried out to examine the resistance of house mice to bromadiolone. Eighty house mice collected from four locations (ten males and ten females per location) were tested for bromadiolone tolerance. Surviving animals and their F1 offspring were screened for mutations. The influence of VKOR variant, zygosity and sex on bromadiolone tolerance was analysed. RESULTS Bait intake and changes in body weight revealed different animal responses regarding susceptibility or resistance. Leu128Ser, Tyr139Cys and a new Ala21Thr polymorphism were detected in wild-born survivors and their F1 generation. However, not every individual with the polymorphisms Leu128Ser and Tyr139Cys survived the feeding test. VKOR variants and sex caused variations in bromadiolone tolerance. CONCLUSION For the first time it was shown that the VKOR variant, along with sex, is responsible for bromadiolone tolerance in house mice. Other factors influencing bromadiolone tolerance, including sex-specific factors, cannot be excluded. The tolerance levels of VKOR variants should be determined in further studies in order to evaluate the effectiveness of bromadiolone in sustainable management.

Development of Resistance to Anticoagulant Rodenticides in Rodents

Control of commensal rodents worldwide relies on the use of rodenticides. In Europe, mostly anticoagulants are available. First generation anticoagulants were developed in the late 1950s’. Their use resulted in the selection of resistant strains of rats and mice. As a consequence, second generation anticoagulants were developed, more potent but also more stable and persistent. These second generation anticoagulants are active after a single feeding and can overcome the resistance of rats and mice. Resistance is a genetic trait related to a single mutation in the Vkorc1 gene. This gene encodes for Vitamin K epoxyde reductase, an enzyme involved in the catalytic cycle of vitamin K. The mutated strains produce an enzyme which is much less susceptible to inhibition by anticoagulants and resistant rats or mice can tolerate much higher amounts of rodenticides than susceptible individuals. Many different mutations have been identified in Europe or worldwide and their relative potency also described. Today, it is possible to determine the potential susceptibility of a rodent strain just by identifying the mutation. There is evidence to support the general idea that mice are less susceptible to anticoagulants than rats, especially in Spain or France where the house mouse crossbred with the Algerian mouse and integrated a mutated portion of the Vkorc1 gene of high potency. Resistance may also be conferred by increased metabolic degradation of rodenticides, but this type of resistance is much less potent and less commonly observed. It has been described in all commensal rodent species. Overall, resistance is present in most areas sampled in Europe and up to 60% of rodents trapped in large surveys can carry a mutation. It is possible to detect 100% mutated individuals in some locations. Resistance monitoring today should be implemented at the reginal/national scale by means of genetic identification of suspected strains, in order to adapt the use of rodenticides locally.