Stefan Endepols

Adaptive Introgression of Anticoagulant Rodent Poison Resistance by Hybridization between Old World Mice

Polymorphisms in the vitamin K 2,3-epoxide reductase subcomponent 1 (vkorc1) of house mice (Mus musculus domesticus) can cause resistance to anticoagulant rodenticides such as warfarin [1-3]. Here we show that resistant house mice can also originate from selection on vkorc1 polymorphisms acquired from the Algerian mouse (M. spretus) through introgressive hybridization. We report on a polymorphic introgressed genomic region in European M. m. domesticus that stems from M. spretus, spans >10 Mb on chromosome 7, and includes the molecular target of anticoagulants vkorc1 [1-4]. We show that in the laboratory, the homozygous complete vkorc1 allele of M. spretus confers resistance when introgressed into M. m. domesticus. Consistent with selection on the introgressed allele after the introduction of rodenticides in the 1950s, we found signatures of selection in patterns of variation in M. m. domesticus. Furthermore, we detected adaptive protein evolution of vkorc1 in M. spretus (Ka/Ks = 1.54-1.93) resulting in radical amino acid substitutions that apparently cause anticoagulant tolerance in M. spretus as a pleiotropic effect. Thus, positive selection produced an adaptive, divergent, and pleiotropic vkorc1 allele in the donor species, M. spretus, which crossed a species barrier and produced an adaptive polymorphic trait in the recipient species, M. m. domesticus.

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.

A scheme for the placement of rodenticide baits for rat eradication on confinement livestock farms.

We investigated whether the allocation of rodenticide baiting points to specific structural elements would result in complete rat eradication on livestock farms, as opposed to assigning the baiting points only to places where there were obvious signs of rat activity. The goal was to establish an effective rodent-control program that is easy for untrained persons to conduct.Rat-control strategies were examined on 25 farms in Velen (Muensterland), Germany, where an average of 20% of trapped rats were resistant for bromadiolone according to a blood-clotting response (BCR) test. All farms were investigated for signs of rat activity prior to and after the control measure. Differences in the percentage level of farmer compliance in setting up the baiting points as prescribed were analysed for each type of baiting point and in total, and were compared between the group of farms which achieved complete rat eradication and those which did not. Farms achieving complete eradication had an average of 81% compliance with prescribed control plans, whereas a significantly lower compliance level of only 51% was recorded on farms that did not achieve eradication. A >/=75% level of implementation of the control plan always resulted in complete control success. The new method of bait-point allocation was incorporated into a self-explanatory computer program, which was verified to be effective during a rat-control campaign in the restricted area after an outbreak of classical swine fever near Soltau in northern Germany, in July 2001. This program, which is available on the Internet, enables the creation of individualised rat-control plans, including complete documentation of the control measure.

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.

Vitamin K requirement and reproduction in bromadiolone-resistant Norway rats

BACKGROUND Nucleotide polymorphisms in the VKORC1 gene can be linked to anticoagulant rodenticide resistance in Norway rats (Rattus norvegicus Berkenhout). This provides a fitness advantage to rats exposed to anticoagulant actives, but may also cause fitness costs. The vitamin K requirement and reproductive parameters of bromadiolone-resistant rats (Westphalian resistant strain; VKOR variant Tyr139Cys) and bromadiolone-susceptible Norway rats were compared. RESULTS At vitamin K deficiency, blood clotting times increased in all homozygous resistant males within 8 days and in 80% of homozygous resistant females within 15 days. There was little effect on blood clotting in heterozygous males and no effect in heterozygous females and VKOR wild-type individuals. Litter size was about 20% higher in sensitive pairs compared with resistant pairs. Testes growth, male gonad weight, sperm motility and testis cell concentration were unaffected by the mutation. CONCLUSIONS The VKOR variant Tyr139Cys causes considerable physiological cost in Norway rats in terms of vitamin K requirement and reproduction. This may affect the distribution and spread of resistant individuals in the wild. Decreased litter size of resistant parents seems to be due to lowered female reproductive performance, as there was no significant effect of the mutation on any aspects of male reproduction considered, but this requires further study.

Rodenticides - indispensable for safe food production

Stefan Endepols, Bayer CropScience-Environmental Science and Chairman of the Rodenticide Resistance Action Committee (RRAC), describes the work of the RRAC in establishing rodenticide resistance management strategies.

Vkorc1 variation in house mice during warfarin and difenacoum field trials

BACKGROUND Field studies guided by genetic monitoring of Vkorc1 need to be done to implicate mutations conclusively with rodent control problems due to the presence of animals resistant to anticoagulant rodenticides. Rodent control success in relation to Vkorc1 genotypes in house mice (Mus musculus domesticus) was studied on two farms (I and II) in Germany. Tests were carried out to determine whether certain resistance profiles and Vkorc1 genotypes displayed dynamics over the course of sequential treatments with warfarin and difenacoum that were consistent with single nucleotide polymorphisms (SNPs) in Vkorc1 as indicators of resistance. RESULTS On farms I and II, respectively, three (A to C) and two (A and B) types of control problem with anticoagulants (i.e. proxies for resistance) were encountered in spatially segregated subunits: A = none; B = control problems with warfarin but not with difenacoum; C = control problems with both anticoagulants. Unexpectedly, resistance was encountered in a population where only Vkorc1 wild-type mice were detected. In addition, the Arg58Gly Vkorc1 variant was found not to correlate with observed control failures. CONCLUSION Control problems were encountered that cannot be explained by Vkorc1 coding or intronic SNPs, and therefore are likely due to non-coding Vkorc1 SNPs or due to other genetic or non-genetic factors.

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.

The potential of coumatetralyl enhanced by cholecalciferol in the control of anticoagulant resistant Norway rats (Rattus norvegicus).

BACKGROUND We evaluated the potential of cholecalciferol as an enhancer of the first-generation anticoagulant coumatetralyl in the Westphalia anticoagulant-resistant strain of the Norway rat (Rattus norvegicus Berkenhout), characterised by the Tyr139Cys polymorphism on the VKOR enzyme. Because today only the most potent, but also most persistent anticoagulant rodenticides of the second generation remain available to control this strain, new rodenticide solutions are required. RESULTS Feeding trials in the laboratory confirmed a significant level of efficacy, which was corroborated by field trials in the Münsterland resistance area. After frequency and level of resistance were assessed by blood clotting response tests, field trials were conducted with bait containing coumatetralyl at 375 mg kg-1 and cholecalciferol at 50 mg kg-1 or 100 mg kg-1 . Control success was 94% when a large rat infestation comprising 42% resistant animals was treated. Another field trial applying the combination to a rat population that had survived a preceding treatment with bromadiolone resulted in a 99.5% control success according to the first census day, but with some increase in rat activity during subsequent census days. CONCLUSION The combination of coumatetralyl and cholecalciferol is a promising alternative approach to the most potent second-generation anticoagulants in resistance management, particularly in respect of environmental risks, such as secondary poisoning.