Alan Buckle

Anticoagulant resistance in the United Kingdom and a new guideline for the management of resistant infestations of Norway rats (Rattus norvegicus Berk.).

Anticoagulant resistance was first discovered in UK Norway rats (Rattus norvegicus Berk.) in 1958 and has been present ever since. The possible detrimental impact of resistance on effective rodent control was quickly recognised, and, for almost three decades, extensive research was conducted on the geographical distribution and severity of anticoagulant resistance in UK rats. Various schemes for the eradication of resistant rats were also implemented. At first, surveys showed resistance only to the first-generation anticoagulants, such as warfarin, chlorophacinone and coumatetralyl, but, later, resistance to the more potent second-generation anticoagulants, such as difenacoum and bromadiolone, was also discovered. Unlike some European countries, where only one or two resistance mutations occur, virtually all known rat resistance mutations occur in the United Kingdom, and five (Leu128Gln, Tyr139Ser, Tyr139Cys, Tyr139Phe and Leu120Gln) are known to have significant impacts on anticoagulant efficacy. Little is currently known of the geographical extent of anticoagulant resistance among Norway rats in the United Kingdom because no comprehensive survey has been conducted recently. At an operational level, anticoagulants generally retain their utility for Norway rat control, but it is impossible to control resistant rats in some areas because of restrictions on the use of the more potent resistance-breaking compounds. This paper reviews the development of resistance in Norway rats in the United Kingdom, outlines the present situation for resistance management and introduces a new resistance management guideline from the UK Rodenticide Resistance Action Group.

A standardised BCR resistance test for all anticoagulant rodenticides

Abstract 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. The 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.

Blood-clotting response tests for resistance to diphacinone and chlorophacinone in the Norway Rat (Rattus norvegicus Berk.)

Resistance baselines were obtained for the first generation anticoagulant rodenticides chlorophacinone and diphacinone using laboratory, caesarian-derived Norway rats (Rattus norvegicus) as the susceptible strain and the blood clotting response test method. The ED99 estimates for a quantal response were: chlorophacinone, males 0.86 mg kg−1, females 1.03 mg kg−1; diphacinone, males 1.26 mg kg−1, females 1.60 mg kg−1. The dose-response data also showed that chlorophacinone was significantly (p<0.0001) more potent than diphacinone for both male and female rats, and that male rats were more susceptible than females to both compounds (p<0.002). The ED99 doses were then given to groups of five male and five female rats of the Welsh and Hampshire warfarin-resistant strains. Twenty-four hours later, prothrombin times were slightly elevated in both strains but all the animals were classified as resistant to the two compounds, indicating cross-resistance from warfarin to diphacinone and chlorophacinone. When rats of the two resistant strains were fed for six consecutive days on baits containing either diphacinone or chlorophacinone, many animals survived, indicating that their resistance might enable them to survive treatments with these compounds in the field.

Susceptibility to the anticoagulants bromadiolone and coumatetralyl in wild Norway rats (Rattus norvegicus) from the UK and Germany

Abstract A new blood clotting response test was used to determine the susceptibility, to coumatetralyl and bromadiolone, of laboratory strains of Norway rat from Germany and the UK (Hampshire), and wild rats trapped on farms in Wales (UK) and Westphalia (Germany). Resistance factors were calculated in relation to the CD strain of Norway rat. An outbred strain of wild rats, raised from rats trapped in Germany, was found to be more susceptible to coumatetralyl by a factor of 0.5 – 0.6 compared to the CD strain. Homozygous and heterozygous animals of a strain of resistant rats from Westphalia were cross-resistant to coumatetralyl and bromadiolone, with a higher resistance factor for bromadiolone than that found in both UK strains. Our results show that the degree of altered susceptibility and resistance varies between strains of wild rat and between resistance foci. Some wild rat strains may be more susceptible than laboratory rat strains. Even in a well-established resistance area, it may be difficult to find infestations with resistance high enough to suspect control problems with bromadiolone, even after decades of use of this compound.

Resistance testing and the effectiveness of difenacoum against Norway rats (Rattus norvegicus) in a tyrosine139cysteine focus of anticoagulant resistance, Westphalia, Germany

BACKGROUND Anticoagulant resistance in Norway rats at foci in Belgium, Denmark, France, Germany, the Netherlands and the United Kingdom is genetically characterised by the same 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). The purpose of this study was to assess the degree of resistance among rats at two infested farm sites in the Y139C focus in Westphalia, Germany, using blood clotting response (BCR) tests, and to determine the practical efficacy of applications of a commercial 50 ppm difenacoum bait (Neokil™) against them. RESULTS BCR tests showed that the difenacoum resistance factor (RF) among the Y139C rats was about 2.5. DNA analysis for the Y139C mutation revealed that it was present among rats at the two sites with a prevalence of 75 and 93%. Applications of difenacoum bait at the two sites achieved 86.8 and 59.9% control. The different outcomes did not appear to be due to differences either in the degree and prevalence of resistance or in the quantities of poisoned bait consumed. CONCLUSION The study showed that, although the RF for difenacoum among rats carrying the Y139C SNP was apparently low, an acceptable level of control of resistant Norway rat infestations was not achieved using difenacoum. Continued use of anticoagulants against rats that are resistant to them will exacerbate resistance problems in terms of both increased severity and prevalence. These conclusions are likely to apply elsewhere in Europe where the Y139C SNP occurs.

Effects of tamper-resistant bait boxes on bait uptake by Norway rats (Rattus norvegicus Berk.)

We compared the quantity of wheat bait consumed by Norway rats (Rattus norvegicus) from: (i) wooden bait trays, made as safe as possible from non-target animals using materials available at trial sites, and (ii) three different, proprietary tamper-resistant rat bait boxes. A balanced Latin square experimental design was used to overcome operational biases that occur when baits of different types are applied simultaneously at the same sites. The consumption of bait from the four different types of bait placement differed significantly and accounted for more than 76% of the total variation. The amount of bait eaten by rats from the bait trays was approximately eight times greater than the quantity eaten from the tamper-resistant bait boxes. The three bait box designs appeared to deter bait consumption by rats to a similar extent. Tamper-resistant bait boxes are essential tools in the application of rodenticides in many circumstances but their use should not be mandatory when it is possible to make baits safe from non-target animals by other means.

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

BACKGROUND The tyrosine to cysteine amino acid substitution at location 139 of the vkorc1 protein (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 the incidence of the Y139C mutation 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 (Klerat™) containing 0.005% 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, possibly owing to the abundance of attractive alternative food. CONCLUSION The study showed that 0.005% brodifacoum bait is fully effective against Norway rats possessing the Y139C mutation 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 largely ineffective treatments against Y139C resistant rats in the Münsterland. These results should be considered when making decisions about the use of anticoagulants against resistant Norway rats and their potential environmental impacts.

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.

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

Abstract 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™), 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.

Use of Anticoagulant Rodenticides in Different Applications Around the World

Anticoagulant rodenticides (ARs) are used worldwide to manage adverse effects of rodents in plant production, public health and conservation. Mainly commensal pest rodent species, Rattus rattus, Rattus norvegicus and Mus musculus, but also field rodents such as Bandicota bangalensis, Mastomys spp., Microtus spp., Rattus argentiventer and R. tanezumi, are targets of AR applications. Pest rodents cause immense monetary losses, health risks, food security issues, and conservation problems to society in many parts of the world. Losses are rarely quantified, but the few examples available suggest that the worldwide cost of pest rodent activity reaches several billion US