Relationship between resistance factors and treatment efficacy when bromadiolone was used against anticoagulant-resistant Norway rats (Rattus norvegicus Berk.) in Wales

We investigated the relationship between the severity and incidence of resistance among Norway rats (Rattus norvegicus) on a farm in Wales and the subsequent outcome of a practical rodent control operation. Bromadiolone resistance factors were estimated for rats trapped on the farm using the blood clotting response test, and were found to be 2 to 3 for male rats and approximately 6 for females. The incidence of resistance in the rat population was high. Infestation size was estimated by census baiting and tracking, and was found to be substantial, with a maximum of 6.5 kg of bait being eaten on a single night. A proprietary rodenticide (Deadline (TM)), containing 0.005% bromadiolone, was used to control the infestation. The duration of baiting was 35 days and, according to the two methods of assessment used, treatment success was in the region of 87 and 93%. No evidence was observed of a significant impact of resistance on the rat control operation, and the remaining rats of this very heavy infestation would probably have been controlled if baiting had continued for longer.

A standardised BCR resistance test for all anticoagulant rodenticides

This paper presents a reappraisal of the blood clotting response (BCR) tests for anticoagulant rodenticides, and proposes a standardised methodology for identifying and quantifying physiological resistance in populations of rodent species. The standardisation is based on the International Normalised Ratio, which is standardised against a WHO international reference preparation of thromboplastin, and allows comparison of data obtained using different thromboplastin reagents. ne methodology is statistically sound, being based on the 50% response, and has been validated against the Norway rat (Rattus norvegicus) and the house mouse (Mus domesticus). Susceptibility baseline data are presented for warfarin, diphacinone, chlorophacinone and coumatetralyl against the Norway rat, and for bromadiolone, difenacoum, difethialone, flocoumafen and brodifacoum against the Norway rat and the house mouse. A 'test dose' of twice the ED50 can be used for initial identification of resistance, and will provide a similar level of information to previously published methods. Higher multiples of the ED50 can be used to assess the resistance factor, and to predict the likely impact on field control.

Accumulation of anticoagulant rodenticides in a non-target insectivore, the European hedgehog (Erinaceus europaeus)

Studies on exposure of non-targets to anticoagulant rodenticides have largely focussed on predatory birds and mammals; insectivores have rarely been studied. We investigated the exposure of 120 European hedgehogs (Erinaceus europaeus) from throughout Britain to first- and second-generation anticoagulant rodenticides (FGARs and SGARs) using high performance liquid chromatography coupled with fluorescence detection (HPLC) and liquid-chromatography mass spectrometry (LCMS). The proportion of hedgehogs with liver SGAR concentrations detected by HPLC was 3-13% per compound, 23% overall. LCMS identified much higher prevalence for difenacoum and bromadiolone, mainly because of greater ability to detect low level contamination. The overall proportion of hedgehogs with LCMS-detected residues was 57.5% (SGARs alone) and 66.7% (FGARs and SGARs combined); 27 (22.5%) hedgehogs contained >1 rodenticide. Exposure of insectivores and predators to anticoagulant rodenticides appears to be similar. The greater sensitivity of LCMS suggests that hitherto exposure of non-targets is likely to have been under-estimated using HPLC techniques.

Anticoagulant resistance in Norway rats (Rattus norvegicus Berk.) in Kent - a VKORC1 single nucleotide polymorphism, tyrosine139phenylalanine, new to the UK

A sample of 10 Norway rats (Rattus norvegicus) was taken for DNA resistance testing from an agricultural site in Kent where applications of the anticoagulant rodenticide bromadiolone had been unsuccessful. All animals tested were homozygous for the single nucleotide VKORC1 polymorphism tyrosine139phenylalanine, or Y139F. This is a common resistance mutation found extensively in France and Belgium but not previously in the UK. Y139F confers a significant level of resistance to first-generation anticoagulants, such as chlorophacinone, and to the second-generation compound bromadiolone. Another compound widely used in the UK, difenacoum, is also thought to be partially resisted by rats which carry Y139F. A silent VKORC1 mutation was also found in all rats tested. The presence of a third important VKORC1 mutation which confers resistance to anticoagulant rodenticides in widespread use in the UK, the others being Y139C and L120Q, further threatens the ability of pest control practitioners to deliver effective rodent control.

Development of rodenticides and the impact of resistance on anticoagulant rodenticides

Cost effective methods are now available to identify physiological resistance in wild populations of Norway rat and House mice that are proving difficult to control. The new molecular methodology is a significant development for resistance management.

Control of a Population of Norway Rats Resistant to Anticoagulant Rodenticides

For the first time, it has been unequivocally shown that multiple-feed second-generation anticoagulant rodenticides were ineffective against a population of rats in N.W. Berkshire, UK because of an unusually high prevalence and high degree of resistance. Use of the non-anticoagulant rodenticide calciferol led to a substantial reduction in the population, although primary poisoning of small birds appeared to be greater than with anticoagulant baits. There was strong evidence that many of the surviving rats had developed an aversion towards calciferol-treated bait. A reduction in the degree of anticoagulant resistance in the population was evident after a period of 17 months without anticoagulant use. The long-term strategy to manage the resistant population should integrate non-anticoagulant and anticoagulant rodenticide use to take advantage of possible pleiotropic costs of resistance.

Resistance to the first and second generation anticoagulant rodenticides: a new perspective

Warfarin resistance was first discovered among Norway rat (Rattus norvegicus) populations in Scotland in 1958 and further reports of resistance, both in this species and in others, soon followed from other parts of Europe and the United States. Researchers quickly defined the practical impact of these resistance phenomena and developed robust methods by which to monitor their spread. These tasks were relatively simple because of the high degree of immunity to warfarin conferred by the resistance genes. Later, the second generation anticoagulants were introduced to control rodents resistant to the warfarin-like compounds, but resistance to difenacoum, bromadiolone and brodifacoum is now reported in certain localities in Europe and elsewhere. However, the adoption of test methods designed initially for use with the first generation compounds to identify resistance to compounds of the second generation has led to some practical difficulties in conducting tests and in establishing meaningful resistance baselines. In particular, the results of certain test methodologies are difficult to interpret in terms of the likely impact on practical control treatments of the resistance phenomena they seek to identify. This paper defines rodenticide resistance in the context of both first and second generation anticoagulants. It examines the advantages and disadvantages of existing laboratory and field methods used in the detection of rodent populations resistant to anticoagulants and proposes some improvements in the application of these techniques and in the interpretation of their results.

A reappraisal of blood clotting response tests for anticoagulant resistance and a proposal for a standardised BCR test methodology

Blood clotting response (BCR) resistance tests are available for a number of anticoagulant rodenticides. However, during the development of these tests many of the test parameters have been changed, making meaningful comparisons between results difficult. It was recognised that a standard methodology was urgently required for future BCR resistance tests and, accordingly, this document presents a reappraisal of published tests, and proposes a standard protocol for future use (see Appendix). The protocol can be used to provide information on the incidence and degree of resistance in a particular rodent population; to provide a simple comparison of resistance factors between active ingredients, thus giving clear information about cross-resistance for any given strain; and to provide comparisons of susceptibility or resistance between different populations. The methodology has a sound statistical basis in being based on the ED50 response, and requires many fewer animals than the resistance tests in current use. Most importantly, tests can be used to give a clear indication of the likely practical impact of the resistance on field efficacy. The present study was commissioned and funded by the Rodenticide Resistance Action Committee (RRAC) of CropLife International.

Resistance as a factor in environmental exposure of anticoagulant rodenticides: a modelling approach

Anticoagulant rodenticide (AR) resistance in Norway rat populations has been a problem for fifty years, however its impact on non-target species, particularly predatory and scavenging animals has received little attention. Field trials were conducted on farms in Germany and England where resistance to anticoagulant rodenticides had been confirmed. Resistance is conferred by different mutations of the VKORC1 gene in each of these regions: tyrosine139cysteine in Germany and leucine120glutamine in England. A modelling approach was used to study the transference of the anticoagulants into the environment during treatments for Norway rat control. Baiting with brodifacoum resulted in lower levels of AR entering the food chain via the rats and lower numbers of live rats carrying residues during and after the trials due to its lower application rate and efficacy against resistant rats. Bromadiolone and difenacoum resulted in markedly higher levels of AR uptake into the rat population and larger numbers of live rats carrying residues during the trials and for long periods after the baiting period. Neither bromadiolone nor difenacoum provided full control on any of the treated farms. In resistant areas where ineffective compounds are used there is the potential for higher levels of AR exposure to non-target animals, particularly predators of rats and scavengers of rat carcasses. Thus, resistance influences the total amount of AR available to non-targets and should be considered when dealing with rat infestations, as resistance-breakers may present a lower risk to wildlife.

Brodifacoum is effective against Norway Rats (Rattus norvegicus) in a tyrosine139cysteine focus of anticoagulant resistance, Westphalia, Germany

BACKGROUND: The single nucleotide polymorphism (SNP), and consequent amino acid exchange from tyrosine to cysteine at location 139 of the vkorc1 gene (i.e. tyrosine139cysteine or Y139C), is the most widespread anticoagulant resistance mutation in Norway rats (Rattus norvegicus Berk.) in Europe. Field trials were conducted to determine incidence of the Y139C SNP at two rat infested farms in Westphalia, Germany, and to estimate the practical efficacy against them of applications, using a pulsed baiting treatment regime, of a proprietary bait (KleratTM) containing 50 ppm brodifacoum. RESULTS: DNA analysis for the Y139C mutation showed that resistant rats were prevalent at the two farms, with an incidence of 80.0% and 78.6% respectively. Applications of brodifacoum bait achieved results of 99.2% and 100.0% control at the two farms, when measured by census baiting, although the treatment was somewhat prolonged at one site due to the abundance of attractive alternative food. CONCLUSION: The study showed that 50 ppm brodifacoum bait is fully effective against the Y139C SNP at the Münsterland focus and is likely to be so elsewhere in Europe where this mutation is found. The pulsed baiting regime reduced to relatively low levels the quantity of bait required to control these two substantial resistant Norway rat infestations. Previous studies had shown much larger quantities of bromadiolone and difenacoum baits used in ineffective treatments against Y139C resistant rats in the Münsterland. These results should be considered when making decisions about the use of anticoagulant against resistant Norway rats and their potential environmental impacts.

Resistance to the anticoagulant rodenticides – the deployment of the new molecular methodology to identify mutations of the VKORC1 ‘resistance gene’, and understanding their potential impact on treatment outcome

Anticoagulants rodenticides have already known for over half a century, as effective and safe method of rodent control. However, discovered in 1958 anticoagulant resistance has given us a very important problem for their future long-term use. Laboratory tests provide the main method for identification the different types of anticoagulant resistances, quantify the magnitude of their effect and help us to choose the best pest control strategy. The main important tests are lethal feeding period (LFP) and blood clotting response (BCR) tests. These tests can now be used to quantify the likely effect of the resistance on treatment outcome by providing an estimate of the ‘resistance factor’. In 2004 the gene responsible for anticoagulant resistance (VKORC1) was identified and sequenced. As a result, a new molecular resistance testing methodology has been developed, and a number of resistance mutations, particularly in Norway rats and house mice. Three mutations of the VKORC1 gene in Norway rats have been identified to date that confer a degree of resistance to bromadiolone and difenacoum, sufficient to affect treatment outcome in the field.