The stop-feed effect of cholecalciferol (vitamin D3) and the efficacy of brodifacoum combined with cholecalciferol in Y139C-resistant Norway rats (Rattus norvegicus).

Second-generation anticoagulant rodenticides potentially build persistent residues in animals and accordingly pose a risk of secondary poisoning. We examined the effect of a low concentration of cholecalciferol in brodifacoum bait on bait consumption by Norway rats (Rattus norvegicus Berkenhout 1769) and on the control success in a laboratory study and in field trials. Additionally, the efficacy of both baits was determined against resistant Y139C rats. Cholecalciferol caused a strong stop-feed effect after two days in the laboratory study. On two field study sites each, bait containing either 25 mg kg-1 brodifacoum or 25 mg kg-1 brodifacoum and 100 mg kg-1 cholecalciferol was applied to treat infestations of Norway rats. Infestations were assessed pre- and post-treatment. Rats were radio-tagged, and carcasses were searched for during the treatment period. DNA of each rat was genotyped to determine the resistance status conferred by the VKORC1 gene. On all farms, control success exceeded 90%. On farms treated with brodifacoum only, the ratio of total bait consumption to pre-treatment census was significantly higher (6.6 and 4.8 times) than on farms treated with the combination (2.7 and 2.9 times). 78.8% of 183 rats were confirmed Y139C resistant. Bait ingestion was reduced by almost fifty per-cent when cholecalciferol was added to the bait with no impact on control success. All treatments resulted in control levels exceeding 90%, despite a high proportion of anticoagulant-resistant rats. When the use of highly toxic compounds is required in resistance management, addition of cholecalciferol to these baits may reduce the transfer of residues to the environment.

Anticoagulant rodenticide blood-clotting dose-responses and resistance factors for Tyrosine139Cysteine (Y139C) heterozygous- and homozygous-resistant house mice (Mus musculus).

BACKGROUND: The house mouse (Mus musculus) is a globally distributed rodent pest species against which anticoagulant rodenticides are widely used for the protection of human and animal health and the conservation of threatened wildlife. Anticoagulant-resistant house mice have been known for more than half a century. A house mouse strain was developed in the laboratory that was homozygous resistant for the single nucleotide polymorphism (SNP) Tyrosine139Cysteine (Y139C) and, subsequently, heterozygous resistant animals were produced from this strain by crossing with the homozygous susceptible strain. RESULTS: Using blood clotting response tests, resistance factors at the ED50 level in the homozygous resistant strain for the first-generation anticoagulants warfarin, chlorophacinone, diphacinone and coumatetralyl were in the range 31.5 to 628.0 for males (M) and 21.6 to 628.0 for females (F), thus indicating that Y139C house mice are substantially resistant to all these substances. Resistance factors at the ED50 level for the homozygous strain generated against the second-generation compounds were: brodifacoum (M, 1.7; F, 1.9), bromadiolone (M, 16.6; F, 21.0), difenacoum (M, 1.2; F, 2.7), difethialone (M, 1.5; F, 1.5), and flocoumafen (M, 0.9; F, 1.2). Equivalent values for the heterozygous strain were: brodifacoum (M, 1.6; F, 1.4), bromadiolone (M, 5.6; F, 6.5), difenacoum (M, 1.0; F, 1.3), difethialone (M, 1.1; F, 1.1), flocoumafen (M, 0.9; F, 1.1). CONCLUSION: Y139C SNP homozygous resistant mice are more resistant to anticoagulants than heterozygous resistant animals. All first-generation anticoagulants are highly resisted and, among the second-generation compounds, Y139C mice are resistant to bromadiolone and sometimes to difenacoum. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Effects of anticoagulant rodenticide poisoning on spatial behavior of farm dwelling Norway rats.

Commensal rodent species cause damage to crops and stored products, they transmit pathogens to people, livestock and pets and threaten native flora and fauna. To minimize such adverse effects, commensal rodents are predominantly managed with anticoagulant rodenticides (AR) that can be transferred along the food chain. We tested the effect of the uptake of the AR brodifacoum (BR) by Norway rats (Rattus norvegicus) on spatial behavior because this helps to assess the availability of dead rats and residual BR to predators and scavengers. BR was delivered by oral gavage or free-fed bait presented in bait stations. Rats were radio-collared to monitor spatial behavior. BR residues in rat liver tissue were analyzed using liquid chromatography coupled with tandem mass spectrometry. Norway rats that had consumed BR decreased distances moved and had reduced home range size. Treatment effects on spatial behavior seemed to set in rapidly. However, there was no effect on habitat preference. Ninety-two percent of rats that succumbed to BR died in well-hidden locations, where removal by scavenging birds and large mammalian scavengers is unlikely. Rats that ingested bait from bait stations had 65% higher residue concentrations than rats that died from dosing with two-fold LD50. This suggests an overdosing in rats that are managed with 0.0025% BR. None of the 70 BR-loaded rats was caught/removed by wild predators/scavengers before collection of carcasses within 5-29 h. Therefore, and because almost all dead rats died in well-hidden locations, they do not seem to pose a significant risk of AR exposure to large predators/scavengers at livestock farms. Exposure of large predators may originate from AR-poisoned non-target small mammals. The few rats that died in the open are accessible and should be removed in routine searches during and after the application of AR bait to minimize transfer of AR into the wider environment.

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. © 2016 Society of Chemical Industry.

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.

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.

Resistance tests and field trials with bromadiolone for the control of Norway rats (Rattus norvegicus) on farms in Westphalia, Germany.

BACKGROUND: The aim of this study was to determine the incidence and the level of resistance to bromadiolone among rats on farms suspected of being foci of resistance by using the international normalised ratio (INR)-based blood clotting response (BCR) test. Whether the level of reduced susceptibility constitutes 'practical resistance' was subsequently determined in field trials. RESULTS: The 2.5 multiple of the ED(50) baseline was used to test for the incidence of resistance, and higher multiples in the range of the suspected resistance factor were used to investigate the degree of resistance. The ED(50) values of bromadiolone in resistant rats were confirmed in the range 4.70-7.05 mg kg(-1) for males and 4.62-6.61 mg kg(-1) for females. Variations within these ranges appeared between farms. According to the BCR resistance tests, 50-100% of rats were classified as resistant prior to the field trials; 29-100% of rats survived the treatments. CONCLUSION: BCR tests based on the use of the INR and baselines are suitable for determining the incidence and for assessing the level of resistance in populations of Norway rats. The majority of rats of the Westphalian resistant strain, characterised by the Y139C marker in VKOR, are resistant to bromadiolone under practical control conditions.

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.

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.