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

Pelz, H.-J., Rost, S., Müller, E., Esther, A., Ulrich, R.G., Müller, C.R

VKORC1 sequence variants associated with resistance to anticoagulant rodenticides in Irish populations of Rattus norvegicus and Mus musculus domesticus

While resistance to anticoagulant rodenticides is known to occur in many European populations of Norway rat and house mouse, to-date no data is available on the occurrence in Ireland of such resistance. No genetic evidence for the occurrence of resistance was found in 65 Norway rat samples analysed, indicative of an absence, or low prevalence, of resistance in rats in at least the Eastern region of the island of Ireland. The presence of two of the most commonly found amino acid substitutions Leu128Ser and Tyr139Cys associated with house mouse resistance to anticoagulant rodenticides was confirmed. The occurrence of two such mutations is indicative of the occurrence of resistance to anticoagulant rodenticides in house mice in the Eastern region of the island of Ireland

Mus musculus populations in Western Australia lack VKORC1 mutations conferring resistance to first generation anticoagulant rodenticides: Implications for conservation and biosecurity

Background Humans routinely attempt to manage pest rodent populations with anticoagulant rodenticides (ARs). We require information on resistance to ARs within rodent populations to have effective eradication programs that minimise exposure in non-target species. Mutations to the VKORC1 gene have been shown to confer resistance in rodents with high proportions of resistance in mice found in all European populations tested. We screened mutations in Mus musculus within Western Australia, by sampling populations from the capital city (Perth) and a remote island (Browse Island). These are the first Australian mouse populations screened for resistance using this method. Additionally, the mitochondrial D-loop of house mice was sequenced to explore population genetic structure, identify the origin of Western Australian mice, and to elucidate whether resistance was linked to certain haplotypes. Results No resistance-related VKORC1 mutations were detected in either house mouse population. A genetic introgression in the intronic sequence of the VKORC1 gene of Browse Island house mouse was detected which is thought to have originated through hybridisation with the Algerian mouse (Mus spretus). Analysis of the mitochondrial D-loop reported two haplotypes in the house mouse population of Perth, and two haplotypes in the population of Browse Island. Conclusions Both house mouse populations exhibited no genetic resistance to ARs, in spite of free use of ARs in Western Australia. Therefore weaker anticoagulant rodenticides can be employed in pest control and eradication attempts, which will result in reduced negative impacts on non-target species. Biosecurity measures must be in place to avoid introduction of resistant house mice, and new house mouse subspecies to Western Australia

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

Pelz, H.-J., Rost, S., Müller, E., Esther, A., Ulrich, R.G., Müller, C.R

Assessing the sustainability of anticoagulant-based rodent control for wildlife conservation in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Conservation Biology at Massey University, Palmerston North, New Zealand

Figures 1.4, 1.5 & 1.6 were removed for copyright reasons, but may be accessed via their respective sources: Schulman & Furie, 2015 Fig 1; Ishizuka et al., 2007 Fig 1; & Ishizuka et al., 2008 Fig 3.Brodifacoum is used extensively to control invasive rodent pest populations in New Zealand. However, there are major concerns regarding non-target poisoning due to brodifacoum, its high persistence and risk of emergence of resistance in targeted rodents. In the present study, I assessed brodifacoum resistance in ship rats and house mice using blood-clotting response (BCR) tests. Mature ship rats of both sexes were live trapped from Akatarawa forest, an area of no known anticoagulant use history in Wellington. A ranging study was performed whereby healthy ship rats were administered increasing doses of brodifacoum to calculate the effective dose, which in ship rats is considered to be the dose giving a 3.6-fold increase in blood-clotting time (this proportional increase is referred to as the International Normalised Ratio, or INR). An unexpectedly high effective dose of 2.9 and 3.8 mg/kg was calculated for male and female ship rats respectively. The calculated effective dose was used to access brodifacoum susceptibility in ship rats captured from nine areas of known brodifacoum use history in the Wellington region and Palmerston North. A total of 54 ship rats were successfully tested, and there was a significant decrease in INR with increasing number of years of brodifacoum use in an area. Despite this evidence of anticoagulant resistance revealed by BCR tests, no mutations conferring anticoagulant resistance were found in VKORC1 gene sequences in tested ship rats. This suggests that resistance may be caused by other pathways. Similarly, BCR tests were performed in house mice using the effective dose from published literature, i.e. 0.52 mg/kg for males and 0.46 mg/kg for females. Twenty out of 26 house mice assessed were found to be resistant to administered dose of brodifacoum, meaning the INR was >5. However, no relationship was observed between the INR value and the number of years of brodifacoum use in an area. Seven of the tested mice were found to have a non-synonymous mutation, Tyr139Cys in exon 3 of the VKORC1 gene. The house mouse individuals carrying this mutation are known to be fully resistance to all first-generation anticoagulants and a second-generation anticoagulant, bromadiolone, but only minor resistance is known to occur towards more potent second-generation anticoagulants. At present, only technical resistance to brodifacoum has been reported in ship rats and house mice, and brodifacoum may still be used effectively to control these rodent populations. However, continual use of brodifacoum may encourage further resistance. Effective long-term control of anticoagulant-resistant populations can only be achieved by use of alternative non- anticoagulant rodenticides

Novel mutations in the VKORC1 gene of wild rats and mice – a response to 50 years of selection pressure by warfarin?

<p>Abstract</p> <p>Background</p> <p>Coumarin derivatives have been in world-wide use for rodent pest control for more than 50 years. Due to their retarded action as inhibitors of blood coagulation by repression of the vitamin K reductase (VKOR) activity, they are the rodenticides of choice against several species. Resistance to these compounds has been reported for rodent populations from many countries around the world and poses a considerable problem for efficacy of pest control.</p> <p>Results</p> <p>In the present study, we have sequenced the <it>VKORC1 </it>genes of more than 250 rats and mice trapped in anticoagulant-exposed areas from four continents, and identified 18 novel and five published missense mutations, as well as eight neutral sequence variants, in a total of 178 animals. Mutagenesis in <it>VKORC1 </it>cDNA constructs and their recombinant expression revealed that these mutations reduced VKOR activities as compared to the wild-type protein. However, the <it>in vitro </it>enzyme assay used was not suited to convincingly demonstrate the warfarin resistance of all mutant proteins</p> <p>Conclusion</p> <p>Our results corroborate the <it>VKORC1 </it>gene as the main target for spontaneous mutations conferring warfarin resistance. The mechanism(s) of how mutations in the <it>VKORC1 </it>gene mediate insensitivity to coumarins <it>in vivo </it>has still to be elucidated.</p

Efficacy of anticoagulant drugs as rodenticides and genetic variation on Vkorc1 of Mus musculus from Buenos Aires Province (Argentina).

The relationship between anticoagulant rodenticides and the gene for vitamin K epoxide reductase has been extensively studied. The significance of mutations in Vkorc1 on the degree of response to anticoagulants is controversial. The implication of the VKORC1 function in pest control drives the attention to a wide range of factors that may be influencing the resistance to rodenticides, an issue that will be discussed in this article. Anticoagulants are used as rodenticides as well as to improve human health. The interactions between Vkorc1 and Cyp4f18 (cytochrome P450) are complex. It is likely that the coagulation response may be due to a diverse expression of the Vkorc1 gene and its interaction with the expression of Cyp4f18, and to the polymorphisms present in both genes. We analyzed the presence of polymorphisms (especially the presence of mutations that produce substitutions of Tyr139Cys and Leu128Ser) in Vkorc1 of the genome of Mus musculus belonging to local wild populations living in farms. The animals studied were from areas in which rodenticide anticoagulants (ej. bromadiolone) are commonly used as pest control. None of the studied mice showed any signs of anticoagulant resistance-related mutations in the Vkorc1 gene. This study will enable a proper selection of the rodenticide method for pest control in local areas. Key words: Vitamin K epoxide reductase, rodenticides, rodents.Fil: Espinosa, Maria Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Parque Centenario. Instituto de Investigaciones en Biociencias Agricolas y Ambientales; Argentina

VKORC1 mutation in European populations of Rattus norvegicus with first data for Italy and the report of a new amino acid substitution.

In the Norway rat, Rattus norvegicus, anticoagulant rodenticide resistance is mainly associated with mutations in the third exon of the Vitamin K epoxide reductase complex subunit 1 (VKORC1). Identification of the resistant wild populations is very important to improve the control practices and to limit the damages due to inadequate use of the anticoagulant rodenticide. In this study, we determined the distribution of the third exon mutations in poorly investigated areas of Africa, Europe and the Middle East. In particular, we investigated the phenomenon for the first time in the Italian peninsula. We obtained sequences of the third exon for 133 Norway rats from 37 localities in Africa, Europe and the Middle East. For additional analysis, we retrieved information in literature on amino acid substitution in 1136 third exon sequences of Norway rats from Europe, the Far East, North America and South America. However, we found third exon mutations only in Europe and the Far East with the Y139F mutation shared between the two areas. Europe has the higher number of mutant individuals and Y139C mutation prevails. In Italy, we found a single missense mutation (I123S) in a Venetian locality. This homozygote mutation, is not know in literature to be associated with resistance, but it is very similar to a mutation that confers resistance in humans (I123N). This similarity and its high local frequency makes it a good candidate for future testing. Our results provide useful data to better understand the resistance phenomenon and to plan targeted control actions

A genetic investigation of anticoagulant rodenticide resistance in Mus musculus of Western Australia: Implications for conservation and biosecurity

Human-wildlife interactions have developed since the agricultural revolution that occurred 10,000 years ago, and the expansion of commensal species’ geographical distribution led to conflicts that prompted humans to adopt a wide range of control methods for pest species (Horvitz, Wang, Wan, & Nathan, 2017; Riyahi et al., 2013; Saraswat, Sinha, & Radhakrishna, 2015). The order Rodentia is characterised by a high number of successful invaders, which humans have attempted to manage with the use of anticoagulant rodenticides (ARs) since the 1940s (Capizzi, Bertolino, & Mortelliti, 2014; Ruiz-Suárez et al., 2014). The rise and spread of a genetic mutation that infers AR resistance among mice and rats led to the production of stronger second generation compounds, which are characterised by higher toxicity and longer persistence in the liver tissue (Lohr & Davis, 2018; Ruiz-Suárez et al., 2014). These traits have led to AR residues being detected in a variety of non-target organisms from both terrestrial and aquatic environments (Kotthoff et al., 2018; López-Perea, Camarero, Sánchez-Barbudo, & Mateo, 2019; Rattner, Lazarus, Elliott, Shore, & Van Den Brink, 2014a). Additionally, the impact of ARs on non-target species is exacerbated when rodents are resistant to the poison as they become capable of transmitting high doses to their predators. To reduce negative effects on the ecosystem, rodent eradication requires information on the presence of resistance within populations; this has been intensely studied in Europe through laboratory feeding trials, blood clotting response tests and genetic screening of the Vkorc1 gene (Goulois, Lambert, Legros, Benoit, & Lattard, 2016; Grandemange, Lasseur, Longin-Sauvageon, Benoit, & Berny, 2010; Mayumi Ishizuka et al., 2007; Pelz et al., 2012). In Australia, the only available information on resistance of rodent populations comes from a study in 1975 on black rats from Sydney, and another study on Mus musculus of Lord Howe Island, New South Wales (Billing, 2000; Saunders, 1978; Wheeler et al., 2018). Since the house mouse (Mus musculus) exhibits a degree of natural tolerance to rodenticides (Cowan et al., 2017), and its eradication has higher failure rate compared to rats (Howald et al., 2007), it is vital to know whether particular populations possess mutations that may infer resistance, and how common the mutations are within the population. The aim of this study was to produce the first data showing whether Vkorc1 mutations that may provide anticoagulant resistance in house mouse are present in Western Australia by sampling populations from the Perth metropolitan area, which is continuously exposed to ARs, and from Browse Island, which has no history of exposure. Additionally, the mitochondrial D-loop of house mice was sequenced to investigate population genetic structure, identify the origin of Western Australian mice, and to elucidate whether resistance was linked to certain haplotypes. No resistance-related Vkorc1 mutations have been detected in either house mouse populations. A genetic introgression in the intronic sequence of the Vkorc1 gene of Browse Island house mouse was detected and it is thought to have originated through hybridisation with the Algerian mouse (Mus spretus). Analysis of the mitochondrial D-loop reported two haplotypes in the house mouse population of Perth, and two haplotypes in the population of Browse Island. The findings suggest that both house mouse populations exhibit no genetic resistance to ARs, and therefore less strong rodenticides can be employed in pest control and eradication attempts, which will result in a less negative impact on nontarget species. Biosecurity measures must be in place to avoid potentially resistant house mice to enter Western Australia, and to prevent the introduction of new house mouse subspecies on mainland, such as the one found on Browse Island