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Anticoagulant resistance in Norway rats (Rattus norvegicus Berk.) in Kent – a VKORC1 single nucleotide polymorphism, tyrosine139phenylalanine, new to the UK
Abstract
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.
... VKORC1 mutations in Norway rats (NR) and House mouse (HM) in UK. From: Pelz et al. 2005;Rost et al. 2009;Prescott et al. 2010;Pelz and Prescott, 2015;Clarke and Prescott, 2015 unpublished ...
... A recent practical failure of a bromadiolone treatment resulted in the DNA sequencing of tissue samples from rats at the site. The Y139F mutation, never before found in the UK, was identified (Prescott et al., 2010). This SNP is found in several European countries including France, Belgium and The Netherlands (Buckle, 2013;Pelz and Prescott, 2015). ...
... DNA sequencing has been used for surveys of resistance in several EU countries including Germany (Pelz, 2007;Pelz et al., 2011), The Netherlands (van der Lee et al., 2011) and Belgium © 2018 Campaign for Responsible Rodenticide Use UK (Baert et al., 2011). In the UK, the technique was used for the first time to identify the resistance SNP present in the Kent resistance focus (Prescott et al., 2010). ...
SUMMARY
1. The status of anticoagulant resistance in the UK is unique in several ways. Most importantly, more than fifty years of continuous research into this phenomenon, both in Norway rats and house mice, has provided an extensive platform of knowledge upon which to base practical advice on anticoagulant use and recommendations on resistance management. Regrettably, an axiom of resistance management, not to use active substances in areas where they are resisted, has been difficult to apply in practice because of a long-standing regulatory policy, virtually unknown elsewhere, wherein the most potent anticoagulant rodenticides were precluded from use in the management of resistant Norway rats because of perceived risks to the environment. This policy has now been superseded and all five second-generation anticoagulant rodenticides (SGARs) can now be used against Norway rat infestations outdoors but undoubtedly its legacy is still felt. Finally, and again uniquely, the UK is home to more anticoagulant resistance mutations in Norway rats than any other country world-wide, with five having practical impacts.
2. Developments in the last decade have revolutionised the study of anticoagulant resistance, in terms of our understanding of its genetic basis, physiological mechanisms and geographical distribution. New resistance tests based on DNA extraction and sequencing, permit rapid, cheap, accurate and humane resistance monitoring. These tests, however, still rely on older techniques, involving laboratory studies using either live rodents or blood samples taken from them and field efficacy testing, to understand practical impacts of resistance mutations on the outcome of anticoagulant applications. Fortunately, these two information threads come together in the UK.
3. Among UK Norway rats we have identified a total of nine genetical mutations in areas of the genome that are known to be important for the action of anticoagulants. Among these, three (L120Q, Y139C, Y139F) confer resistance to the first-generations anticoagulants (FGARs) and to one or more of the second-generation anticoagulants (SGARs). Among the remainder, two (Y139S, L128Q) confer significant levels of resistance to FGARs, one (R33P) has been found to confer resistance to warfarin in the laboratory, two (F63C, Y39N) impair protein function and one (A26T) is thought to have no practical consequences. Both mutations found in UK house mice (L128S, Y139C) confer resistance to FGARs and to one or more SGARs.
4. This report presents additional results of anticoagulant resistance monitoring conducted at the Vertebrate Pests Unit, the University of Reading, for both Norway rats and house mice since the publication of the report of 2017 (Prescott et al., 2017). A total of 37 tissue samples of Norway rat were sequenced during the period March to August 2018. Among these 24 (64.9%) were either homozygous or heterozygous for one or more resistance mutations and 13 (35.1%) were homozygous susceptible. Tissue samples from nine house mice were obtained and eight of these carried one or more resistance mutations. Although the numbers sampled are small, they serve to support the long-held assumption that resistance is now so prevalent in UK house mouse infestations that FGARs should not be used against them.
5. These data confirm the extent of L120Q resistance in Norway rats, the most severe form of resistance in this species, across the whole of central southern England. The ubiquity of Y139F resistance among rats in Kent and East Sussex is also apparent. Of concern are further isolated records of these mutations, far from their core areas, suggesting either transportation of resistant rodents or the de novo development of new foci. Y139C, another relatively severe form of resistance, is also widely dispersed. Several new foci of resistance are reported including, for the first time, both Y139F and Y139C embedded within the resistance focus in Wales that was previously exclusively Y139S, and the continuing spread of both L120Q and Y139C in many foci virtually nationwide. It seems unlikely that these foci have sprung up since the last report. It is more likely that these resistant populations were fostered during the period when only bromadiolone and difenacoum were available for use against rat infestations.
6.
7. Much of the UK remains untested because our laboratory has been unable to obtain biological material from many areas. It is particularly apparent that we have no information for large areas of the Midland counties. A special effort is needed to rectify this unsatisfactory situation. It is unsafe to assume, however, that the absence of a sample showing resistance from any particular area indicates that resistance is absent. Furthermore, the scarcity of wild-type (i.e. fully susceptible) Norway rats, particularly in central-southern and south-east England, suggests that it is reasonable to assume that almost any rodent infestation in those areas will contain rats carrying one or other of the severe L120Q or Y139F mutations. A sample of house mice from south-east England has been tested and results are given in this report for the first time. It is perhaps not surprising that, although the sample is small, both known house mouse resistance mutations (L128S, Y139C) were found at high frequency, with some individuals worryingly possessing both mutations.
8. Recommendations in this report about the use of anticoagulant rodenticides against resistant rodent infestations are reproduced from recently revised resistance management guidelines published by the UK Rodenticide Resistance Action Group (RRAG).
... The detection of resistance using the in vitro methodology based on DNA sequencing of the VKORC1 gene has simplified the resistance monitoring process. This technique is the most cost-effective, and has identified a number of mutations in resistant populations of Norway rats and House mice, that were the subject of research publications as far back as the 1960's (Pelz et al., 2005), (Grandemange et al., 2009b), (Prescott et al., 2010). In many cases, the potential impact of a VKORC1 mutation can be inferred from previous research (Buckle 2012), while for new mutations, or mutations that have been less well studied, other in vitro or in vivo tests are available to provide this information (Grandemange et al., 2009a), (Hodroge et al., 2011), RRAG, RRAC). ...
... The detection of resistance using the in vitro methodology based on DNA sequencing of the VKORC1 gene has simplified the resistance monitoring process. This technique is the most cost-effective, and has identified a number of mutations in resistant populations of Norway rats and House mice, that were the subject of research publications as far back as the 1960's (Pelz et al., 2005;Grandemange et al., 2009b;Prescott et al., 2010). For mutations already described and when the resistance potential has already been evaluated (see Buckle, 2012), in vitro DNA sequencing should be recommended as the first step. ...
... Since the early 60s, resistance to anticoagulant rodenticides has been described across Europe and appears to be quite common in Norway rats (Pelz et al., 2005;, Grandemange et al., 2009b;Baert et al., 2012;Prescott et al., 2010) and in House mice ((Pelz et al., 2005); (Song et al., 2011)). However, to date our understanding of the geographical distribution of the different types resistance in these species is lacking in many MSs. ...
The Biocidal Products Directive (98/8 CE) has been implemented to evaluate the safety and efficacy of products used in households and industry and other buildings to control pests. Rodenticides were the first products to be evaluated and active substances were reviewed, evaluated and eventually registered. In a second step, commercial products were evaluated individually under a mutual recognition process. Marketing authorisations are valid for 5 years and in 2014 the first revision process should start. In view of the various potential toxic risks associated with anticoagulant rodenticides (non-target poisoning in domestic and wild animals primarily), as well as evidence of resistance spreading, the EU Commission requested an expert report on potential risk mitigation measures that could apply to all member states and harmonize the authorization process as well as preserving safe and effective rodenticides The Expert Report is due in September and the proposed harmonized risk mitigation measures as well as general resistance management strategies will be presented in this communication. A survey was also conducted among stakeholders (PCOs' and rodenticide manufacturing companies) to have a more quantitative evidence of the reality of resistance and non-target poisoning issues. Recommendations will also be presented qith respect to monitoring tools to evaluate short term or long term efficacy and toxicity of anticoagulant rodenticides.
... Fortunately, much of this work had already been done in the United Kingdom, as described above. 10 Rost et al., 14 Prescott et al. 27 A list of DNA resistance mutations found in UK Norway rats, and some locations of their occurrence, is given in Table 1. Thus, it is now known that the original Scottish focus harboured rats carrying the Leu128Gln (or L128Q) mutation, the extensive focus on the Anglo-Welsh border was Tyr139Ser (or Y139S) and rats in Hampshire and Berkshire carry the Leu120Gln (or L120Q) mutation. ...
... Knowledge of resistance in the United Kingdom continues to increase, and a new focus, or more likely an old one that had remained undetected for many years, was recently identified in Kent with a DNA mutation (Tyr139Phe or Y139F) not previously found in the United Kingdom. 27 Also, rats carrying L120Q have been found in south-west England, the West Midlands and in south-west Scotland (Clarke D, private communication). In all, nine resistance mutations have been identified in Norway rats in the United Kingdom, more than in any other country. ...
... The Y139F mutation, never before found in the United Kingdom, was identified. 27 There is little wileyonlinelibrary.com/journal/ps published evidence about the practical effectiveness of anticoagulants against Y139F. ...
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. Copyright © 2012 Society of Chemical Industry.
... VKORC1 mutations in Norway rats (NR) and House mouse (HM) in UK. From: Pelz et al., 2005;Rost et al., 2009;Prescott et al., 2010;Pelz and Prescott, 2015;Clarke and Prescott, 2015 unpublished report. Major resistance mutations with known practical consequences shown in bold. ...
... However, that was the last work that was done on this resistance until, almost 30 years later, researchers at the University of Reading received a report of the failure of bromadiolone to control an infestation of Norway rats at a poultry farm in Kent. Analysis of the DNA of rats from the farm confirmed the presence of the Y139F mutation (Prescott et al., 2010). ...
1. During the period 2009 and 2022, in which these DNA resistance surveys have been conducted, first at the University of Reading and now at the Animal and Plant Health Agency, a total of 489 Norway rat and 129 house mouse tissue samples have been examined and DNA has been extracted from them and sequenced. Among these samples we found that 77.9% of rats and 94.6% of mice carried one or more single nucleotide polymorphisms (SNPs) which are known significantly to affect the efficacy of anticoagulant rodenticides. These results may not reflect the true frequency of resistance in the two species, however, because samples are generally sent by those experiencing difficulties in obtaining control of rodent infestations with anticoagulants.
2. Norway rats in the UK carry five different resistance mutations that are known to have adverse consequences for the effectiveness of anticoagulants (Y128Q, Y139S, L120Q, Y139F and Y139C), and house mice carry three such mutations, (L128S, Y139C and the ‘spretus introgression’). The latter having been found for the first time in 2022 in three mice from Hertfordshire.
3. Large numbers of samples permit the geographical distribution of resistance in Norway rats in the UK to be determined. L128Q is largely restricted to Scotland and the north of England. Y139S is found mainly in Wales, on the Anglo-Welsh border and in an expanding focus in North Yorkshire. L120Q is very widespread across central southern England. Y139F is found mainly in Kent, East Sussex and Greater London. Y139C is ubiquitous, with no distinctive geographical central focus.
4. However, and particularly with regard to the three most severe Norway rat mutations, namely L120Q, Y139F and Y139C, outlying resistant foci occur with increasing frequency almost anywhere in England, either disseminated by natural rodent movement or by human transportation systems. Although, there remains evidence of an area of remnant susceptibility in some counties of the Midlands and on the English north-east coast, but these areas are now increasingly infiltrated by resistance.
5. Fewer house mouse samples are obtained but these show that anticoagulant resistance is also widespread in this species. In this respect, it is the position of the Rodenticide Resistance Action Group (RRAG) that all UK house mouse infestations should be assumed to carry resistance and treatments should be conducted against them accordingly.
6. As foci of resistance in both rats and mice spread and overlap there is increasing occurrence of ‘hybrid resistance’, in which individuals carry more than one different resistance SNP. We know little of the consequences of hybrid resistance on rodenticide efficacy but evidence is emerging from studies of house mice in France that hybrid resistance may render rodents less susceptible to anticoagulants than those that carry only one SNP.
7. The maps of Norway rat and house mouse resistance foci presented in this report permit reasonably fine-grained advice to be given to rodenticide users about which interventions to use and which to avoid, following recommendations of the RRAG. Implementation of that advice would: 1) facilitate faster and more effective rodent control for the better protection of human and animal health, 2) prevent the increasing severity and spread of anticoagulant resistance, and of great important to the objectives of the Campaign for Responsible Rodenticide Use (CRRU) and rodenticide stewardship, 3) reduce unnecessary and ineffective emissions of anticoagulants into wildlife and the wider environment.
8. The information presented here should be the subject of a concerted effort of dissemination in an attempt to prevent the purchase of certain rodenticides in areas where there is compelling evidence that their use would be ineffective.
... Extensive studies on the distribution and prevalence of Vkorc1 mutations have been carried out mainly in the European regions 16,[26][27][28][29][30] . The growth and geographical expansion of mutant rat populations may compel an increased reliance on physical population control methods, thus increasing the cost burden to health and pest control authorities. ...
... The other four mutations at codons 103, 107, 108 and 137 were silent. Allelic frequencies for both species of rats in exon 3 are summarised in Table 1 27.7% (13 out of 47, 3 homozygous and 10 heterozygous) of the samples had the silent SNP mutation in codon 108 (Leu108Leu). ...
Anticoagulant rodenticides are commonly used in rodent control because they are economical and have great deployment versatility. However, rodents with Single Nucleotide Polymorphism (SNP) mutations within the Vkorc1 gene are resistant to the effects of anticoagulant rodenticide use and this influences the effectiveness of control strategies that rely on such rodenticides. This study examined the prevalence of rat SNP mutations in Singapore to inform the effectiveness of anticoagulant rodenticide use. A total of 130 rat tail samples, comprising 83 Rattus norvegicus (63.8%) and 47 Rattus rattus complex (36.2%) were conveniently sampled from November 2016 to December 2019 from urban settings and sequenced at exon 3 of Vkorc1. Sequencing analysis revealed 4 synonymous and 1 non-synonymous mutations in Rattus rattus complex samples. A novel synonymous mutation of L108L was identified and not previously reported in other studies. Non-synonymous SNPs were not detected in the notable codons of 120, 128 and 139 in R. norvegicus, where these regions are internationally recognised to be associated with resistance from prior studies. Our findings suggest that the prevalence of anticoagulant rodenticide resistance in Singapore is low. Continued monitoring of rodenticide resistance is important for informing rodent control strategies aimed at reducing rodent-borne disease transmission.
... There is no evidence to date that the silent mutation Il82Il is implicated in anticoagulant resistance, however it has been suggested that the mutation may alter the efficiency of translation of the VKORC1 gene and therefore alter the biological response to anticoagulant exposure 19 . ...
... In Kent the substitution Tyr139Phe was also found in resistant Norway rats at a site treated with bromadiolone bait. All rats tested from the Kent site were found to be homozygous for the mutation 19 . Mutations associated with resistance to anticoagulant rodenticides have not been detected in Norway rats in this study, indicative of an absence, or low prevalence of genetic evidence of resistance to anticoagulant rodenticides in Norway rats in at least the Eastern region of the island of Ireland. ...
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.
... Knowledge of resistance in UK continues to increase and a new focus, or more likely an old one that had remained undetected for many years, was recently identified in Kent with a DNA mutation (Y139F) not previously found in the UK. 27 Also, rats carrying L120Q have been found in the West Midlands and in south-west Scotland (D. Clarke, pers. comm.). ...
... The Y139F mutation, never before found in the UK, was identified. 27 There is little published evidence about the practical effectiveness of anticoagulants against Y139F. However, Grandemange 44 conducted laboratory experiments using BCR testing to determine the effectiveness of chlorophacinone, bromadiolone, difenacoum, and difethialone against Y139F. ...
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 UK and five 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 UK because no comprehensive survey has been conducted recently. At an operational level, anticoagulants generally retain their utility for Norway rat control but it is virtually impossible to control resistant rats in some areas because of restrictions on the use of the more potent resistance-breaking compounds. This paper describes the development of resistance in Norway rats in the UK, outlines the present situation for resistance management and introduces a new resistance management guideline from the UK Rodenticide Resistance Action Group (RRAG, 2010).
... musculus) resistant to anticoagulants of the first and second generation and mutation of Vkorc1 gene can be found in numerous publications including review papers [Pelz et al., 2005[Pelz et al., , 2012Goulois et al., 2017;Mooney et al., 2018;McGee., 2020]. Anticoagulant resistance in house mice and Norway rats has been well studied in Western Europe [Pelz et al., 2005[Pelz et al., , 2012Prescott et al., 2010;Buckle et al., 2013;Meerburg et al., 2014;Haniza et al., 2015;Goulois et al., 2017;Mooney et al., 2018;Buckle et al., 2020]. In Norway rats a number of mutations have been found that provide resistance to anticoagulants of the first and second generations (Leu128Ser, Leu120Gln, Ile82Ile, Il82Il, Trp59Gly, Tyr139Cys, Tyr139Ser, Tyr139Phe, Leu128Gln, Arg35Pro) [Pelz et al., 2005[Pelz et al., , 2012. ...
Genetic resistance to anticoagulants caused by mutations in the Vkorc1 gene of the most invasive rodent species - Norway rats and house mice - has not been studied in Russia. We analyzed the variability of the Vkorc1 gene in house mice and Norway rats in various settlements of Russia, and identified mutations responsible for resistance to rodenticides. Two exons of the Vkorc1 gene were analyzed in 71 Norway rats from four cities (Moscow, Tyumen, Chita, Rostov-on-Don) and 108 house mice from cities and small settlements (Moscow region, Tormosin, Nizhny Tsasuchei). Three Norway rats (15.8% of the studied individuals) in Moscow have a heterozygous state of the Tyr139Ser mutation, which is responsible for resistance. House mice were not found to have mutations in the Vkorc1 gene responsible for resistance to anticoagulants of the first and second generation in the Leu128Ser and Tyr139Cys positions located in the third exon. However, in cities, we identified two heterozygous mutations in the first exon have not be described previously in scientific literature: Lys58Arg and Ser31Trp. In Russia, the genetic resistance to rodenticides in settlements in the populations of house mice and Norway rats is significantly lower than in Western Europe.
... The H68H has already been described in China 50 . Although these point mutations do not alter the amino acid translation, Prescott et al. suggested that it could affect the efficiency of translation of the Vkorc1 gene leading to an alteration of the biological response to anticoagulant exposure 51 . Further studies will be necessary to characterize the consequences of such silent mutations. ...
Anticoagulant rodenticides (AR) remain the most effective chemical substances used to control rodents in order to limit their agricultural and public health damage in both rural and urban environments. The emergence of genetically based resistance to AR worldwide has threatened effective rodent control. This study gives a first overview of the distribution and frequency of single nucleotide polymorphism in the vitamin K epoxide reductase subcomponent 1 (Vkorc1) gene in rodents in Lebanon. In the Mus genus, we detected two missense mutations Leu128Ser and Tyr139Cys, that confer resistance to anticoagulant rodenticides in house mice and a new missense mutation Ala72Val in the Mus macedonicus species, not previously described. In the Rattus genus, we found one missense mutation Leu90Ile in the roof rat and one missense mutation Ser149Ile in the Norway rat. This is the first study to demonstrate potential resistance to AR in Lebanese rodents and therefore it provides data to pest control practitioners to choose the most suitable AR to control rodents in order to keep their efficacy.
... musculus) resistant to anticoagulants of the first and second generation and mutation of Vkorc1 gene can be found in numerous publications including review papers (Pelz et al., 2005(Pelz et al., , 2012Goulois et al., 2017;Mooney et al., 2018;McGee., 2020). Anticoagulant resistance in house mice and Norway rats has been well studied in Western Europe (Pelz et al., 2005(Pelz et al., , 2012Prescott et al., 2010;Buckle et al., 2013;Meerburg et al., 2014;Haniza et al., 2015;Goulois et al., 2017;Mooney et al., 2018;Buckle et al., 2020). In Norway rats a number of mutations have been found that provide resistance to anticoagulants of the first and second generations (Leu128Ser, Leu120Gln, Ile82Ile, Il82Il, Trp59Gly, Tyr139Cys, Tyr139Ser, Tyr139Phe, Leu128Gln, Arg35Pro) (Pelz et al., 2005(Pelz et al., , 2012. ...
Genetic resistance to anticoagulants caused by mutations in the Vkorc1 gene of the most invasive rodent species-Norway rats and house mice, has not been studied in Russia. We analyzed the variability of the Vkorc1 gene in house mice and Norway rats in various settlements of Russia, and identified mutations responsible for resistance to rodenticides. Two exons of the Vkorc1 gene were analyzed in 71 Norway rats from four cities (Moscow, Tyumen, Chita, Rostov-on-Don) and 108 house mice from cities and small settlements (Moscow region, Tormosin, Nizhny Tsasuchei). Three Norway rats (15.8% of the studied individuals) in Moscow have a heterozygous state the Tyr139Ser mutation, which is responsible for resistance. House mice were not found to have mutations in the Vkorc1 gene responsible for resistance to anticoagulants of the first and second generation in the Leu128Ser and Tyr139Cys positions located in the third exon. However, in cities in the first exon, we identified two heterozygous mutations have not be described previously in the scientific literature: Lys58Arg and Ser31Trp. In Russia, the genetic resistance to rodenticides in settlements in the populations of house mice and Norway rats is significantly lower than in Western Europe.
... VKORC1 mutations in Norway rats (NR) and House mouse (HM) in UK. From: Pelz et al., 2005;Rost et al., 2009;Prescott et al., 2010;Pelz and Prescott, 2015;Clarke and Prescott, 2015 unpublished report. Major resistance mutations with known practical consequences shown in bold. ...
SUMMARY
1. New resistance data are presented for tissue samples from Norway rats (Rattus norvegicus) and house mice (Mus musculus) collected in the period September 2019 to February 2020. Coronavirus restrictions at the University of Reading prevented laboratory work after that date. Once again, efforts were made to obtain samples in geographical areas in the UK from which none had been collected in the past.
2. A total of 54 Norway rat tissue samples were analysed, among which 14 were anticoagulant-susceptible and 40 carried one or more of five different resistance mutations (Y139S, Y139C, Y139F, L120Q, L128Q), in either homozygous or heterozygous form. Therefore the prevalence of anticoagulant resistance in this Norway rat sample was 74.1%.
3. For the first time more rats were found to carry the Y139C resistance mutation than the widespread L120Q mutation. This may be because fewer samples were submitted and sequenced from the large and well-known L120Q focus. The observation from previous years was repeated in that resistant rats were again found in places which would not have been expected from prior knowledge of resistance foci. For example, Y139C was found for the first time on the coast of West Sussex. Rats carrying the Y139S mutation (i.e. ‘Welsh’ resistance) were again recorded from North Yorkshire, far outside the original Welsh focus, at a greater frequency than previously, and the focus had apparently spread into County Durham.
4. These ‘break-out’ foci, and the increasing geographical spread of existing foci, have resulted in a phenomenon not previously reported for Norway rats in England, that of ‘hybrid resistance’. This is where a single individual carries more than one resistance mutation. A surprising 20% of resistant rats carried two different mutations in this limited sample. This is the result of previously distinct resistance foci meeting, merging and interbreeding. The impact of this new phenomenon of hybrid resistance on our ability to manage resistant rodents in the future is discussed.
5. Only six house mouse tissue samples were submitted for analysis. Among these five (83.3%) carried one or more resistance mutations. Although the total number of records for house mouse is small, both for the year reported here and for the accumulated total for all years, these continue to show the wide extent of house mouse resistance to anticoagulants across the UK. Therefore, attention is again drawn to the situation in which permanent anticoagulant baiting is the predominant method for the control of the house mouse among professional pest control practitioners. Yet only the widely resisted difenacoum and bromadiolone active substances are permitted for use in permanent baiting.
... The Prescott et al., 2011 study first reported that the Tyr139Phe mutation was present in the UK and suggested that it would be prudent to halt the use of FGARs and bromadiolone in instances where this mutation was identified in R. norvegicus populations. The mutations Leu120Gln, Tyr139Cys and Tyr139Phe were postulated to be the most potent mutations conferring AVK resistance in UK R. norvegicus populations which threatened effective rodent control (Buckle, 2013;Prescott et al., 2011: Jones et al., 2019. Hodroge et al. (2011) demonstrated in vitro that the mutations Leu120Gln, Leu128Gln, Tyr139Cys, Tyr139Phe and Tyr139Ser conferred significant resistance to certain FGARs (warfarin and chlorophacinone) and to a lesser extent the SGAR, bromadiolone. ...
Anti-vitamin K (AVK) compounds are highly potent anticoagulants which are particularly effective for controlling rodent species populations. AVKs have been the most widely used chemical rodenticide option employed since the 1950s for the control of rodents infesting stored commodities and storage facilities, and also in a wide range of other scenarios. However, reports of AVK resistance in wild rodent populations are becoming increasingly common. This could potentially lead to a substantial reduction in AVK efficacy resulting in an impaired ability to manage rodent infestations in the future. The current state of knowledge regarding AVK resistance mechanisms in common pest species is still incomplete. This review draws together reported incidences of AVK resistance in the literature and the underlying mechanisms suspected of conferring resistance for the three main pest rodent species Rattus norvegicus, Rattus rattus and Mus musculus. The purpose of this review is to compare and contrast the underlying resistance mechanisms in these species and demonstrate how this should influence programs for monitoring and avoiding the development of AVK resistance in target rodent species.
... © 2019 Campaign for Responsible Rodenticide Use UK A list of the VKORC1 mutations found in Norway rats and house mice in the UK is shown in Table 1. Pelz et al. 2005;Rost et al. 2009;Prescott et al. 2010;Pelz and Prescott, 2015;Clarke and Prescott, 2015 unpublished ...
SUMMARY
1. New resistance data are presented for tissue samples from Norway rats (Rattus norvegicus) and house mice (Mus musculus) collected in the period September 2018 to September 2019. Particular efforts were made to obtain samples in geographical areas in the UK from which none had been collected in the past.
2. A total of 140 Norway rat tissue samples were analysed, among which 55 were anticoagulant-susceptible and 85 carried one of five different resistance mutations (Y139S, Y139C, Y139F, L120Q, L128Q) in either the homozygous or heterozygous form. Therefore the prevalence of anticoagulant resistance in this Norway rat sample was 60.7%.
3. These new Norway rat resistance records extended the known area of the extensive L120Q resistance across the south of England, provided for the first time information about the prevalence of resistance in rats in Greater Manchester and identified a third new resistance mutation (Y139F) among rats in Greater London. The records also appear better to define the extent of a Y139C focus in the western counties along the entire course of the river Severn and the extent of a focus of the same mutation among the sub-counties of Yorkshire. Also, for the first time, we record the occurrence of the Y139S mutation from sites far removed from its origin on the Anglo-Welsh border.
4. A total of 35 house mouse tissue samples were collected, all showing one or other of the highly prevalent Y139C and L128S mutations. Although the total number of records for house mouse is small, these new data show the wide extent of house mouse resistance to anticoagulants across the UK and bring to 93.2% the prevalence of resistance in that species.
5. Attention is drawn to the situation in which permanent anticoagulant baiting is the predominant method for the control of the house mouse among professional pest control practitioners, house mice are widely resistant to difenacoum and bromadiolone, these two active substances are not recommended for use against house mice but are the only ones permitted for use in permanent baiting.
... Cancers are made up of multiple subclones with distinct mutations that may confer resistance to treatment (18), and high intratumoral heterogeneity provides more opportunities for resistance. Invasive rodents are subject to the same dynamics but evolve resistance less frequently than cancers do (19). This may be due to the low population sizes of rodents, as there are on average 10 3 rats on islands for which exterminations have been attempted (20) compared with the 10 9 cells in a 1-cm 3 neoplasm (21). ...
To cure a patient's cancer is to eradicate invasive cells from the ecosystem of the body. However, the ecological complexity of this challenge is not well understood. Here we show how results from eradications of invasive mammalian species from islands-one of the few contexts in which invasive species have been regularly cleared-inform new research directions for treating cancer. We first summarize the epidemiological characteristics of island invader eradications and cancer treatments by analyzing recent datasets from the Database of Invasive Island Species Eradications and The Cancer Genome Atlas, detailing the superior successes of island eradication projects. Next, we compare how genetic and environmental factors impact success in each system. These comparisons illuminate a number of promising cancer research and treatment directions, such as heterogeneity engineering as motivated by gene drives and adaptive therapy; multi-scale analyses of how population heterogeneity potentiates treatment resistance; and application of ecological data mining techniques to high-throughput cancer data. We anticipate that interdisciplinary comparisons between tumor progression and invasive species would inspire development of novel paradigms to cure cancer.
... An assessment of potential resistance of the rats on the Shiants to bromadiolone rodenticide was carried out by Reading University (Vertebrate Pests Unit), using protocols developed to extract and sequence DNA for the identifi cation of anticoagulant resistance mutations in brown rats (Pelz, et al., 2005;Prescott, et al., 2010). A similar protocol developed specifi cally for black rat rodenticide resistance testing was not available. ...
A successful ground-based eradication of black rats (Rattus rattus) was undertaken on the remote, uninhabited Shiant Isles of north-west Scotland over winter (14 October–28 March) 2015–16. The rat eradication was carried out as part of the Shiants Seabird Recovery Project, which aims to secure long-term breeding habitat for protected seabirds and to attract European storm petrels and Manx shearwaters to nest on the Shiants. Throughout the eradication operation, teams were stationed on two of the three main Shiant islands (Eilean an Tighe, Eilean Mhuire), with access to the third (Garbh Eilean) via a boulder causeway from Eilean an Tighe. Bait (Contrac® blocks containing the anticoagulant bromadiolone 0.005% w/w), was deployed in a grid of 1,183 bait stations covering all areas of the islands and sea stacks. Bait stations were set 50 m apart, with intervals reduced to 25 m in coastal areas of predicted high rat density. Difficult areas were accessed by boat and cliff s of ~120 m in height were accessed by abseiling down ropes made safe using either bolted anchors or ground stakes. The team of staff and volunteers worked through difficult conditions, deploying bait and monitoring intensively for any surviving rats using a combination of tools. The islands were declared rat free in March 2018. This ambitious and challenging project has greatly enhanced UK capacity in rodent eradications for the purposes of conservation.
... bromadiolone, difenacoum, flocoumafen, difethialone, and brodifacoum) were developed in the 1970s/1980s. Nevertheless, both primary and secondary poisoning of non-target species (due to increased persistence of these more effective compounds within the body) were described (Hughes et al. 2013;Langford et al. 2013) as well as possible evidence of rodent resistance (Prescott et al. 2011). Resistance to antivitamin K rodenticides is attributed to single-nucleotide polymorphisms (SNPs) in the vitamin K epoxide reductase complex subunit 1 (Vkorc1) gene (Pelz et al. 2005;Rost et al. 2004). ...
Brown rats are a prolific synanthropic pest species, but attempts to control their populations have had limited success. Rat population dynamics, dispersal patterns, and resistance to rodenticides are important parameters to consider when planning a control programme. We used population genetics and genotyping to investigate how these parameters vary in contrasting landscapes, namely one urban and two rural municipalities from eastern France. A total of 355 wild brown rats from 5 to 6 sites per municipality were genotyped for 13 microsatellite loci and tested for mutations in the Vkorc1 gene which confers resistance to some rodenticides. Results revealed a strong genetic structure of the sampled rat populations at both regional (between municipalities) and local (between sites within municipalities) levels. A pattern of isolation by distance was detected in the urban habitat and in one of the rural municipalities. GeneClass and DAPC analyses identified 25 (7%) and 36 (10%) migrants, respectively. Migrations occurred mostly between sites within each municipality. We deduced that rat dispersal is driven by both natural small-scale movements of individuals and longer-distance (human-assisted) movements. Mutation Y139F on gene Vkorc1 was significantly more prevalent in rural (frequency 0.26–0.96) than in urban sites (0.00–0.11), likely due to differences in selection pressures. Indeed, pest control is irregular and uncoordinated in rural areas, whereas it is better structured and strategically organised in cities. We conclude that simultaneous pest control actions between nearby farms in rural habitats are highly recommended in order to increase rat control success while limiting the spread of resistance to rodenticides.
... 10 In the United Kingdom, where virtually all known resistance mutations are found, resistant R. norvegicus were found in Cambridge/Essex, Nottinghamshire, Kent, Gloucestershire, Norfolk and Lincolnshire, south-west Scotland, Hampshire and Berkshire, the Anglo-Welsh border, central southern Scotland, Yorkshire and Lancashire. 14,15 Resistance against second-generation rodenticides is also present in a number of other European countries, 2 namely across the whole of Denmark (Bornholm, Fünen, Jutland and Zealand), Belgium (Flanders) and large parts of France (e.g. Yonne, Eure and Loire). ...
... We also found that six BCR positive rats had no VKORC1 amino acid substitutions, that is, they were BCR false positives, unless there is an as yet undiscovered, alternative resistance mechanism (Pelz et al., 2005), and a similar result has been reported elsewhere (Heiberg, 2009). The Leu120Gln variant was identified in two BCR positive rats that were initially thought to be false positives, whilst in a further two apparent BCR false positives, a Tyr139Phe amino acid substitution not known from the UK at the time of our study was found, although this VKORC1 variant has since been reported in rats from Kent (Prescott et al., 2010). Several other newly-discovered VKORC1 variants have recently been reported from the UK (Buckle, 2013). ...
The brown rat (Rattus norvegicus) is a relatively recent (<300 years) addition to the British fauna, but by association with negative impacts on public health, animal health and agriculture, it is regarded as one of the most important vertebrate pest species. Anticoagulant rodenticides were introduced for brown rat control in the 1950s and are widely used for rat control in the UK, but long-standing resistance has been linked to control failures in some regions. One thus far ignored aspect of resistance biology is the population structure of the brown rat. This paper investigates the role population structure has on the development of anticoagulant resistance. Using mitochondrial and microsatellite DNA, we examined 186 individuals (from 15 counties in England and one location in Wales near the Wales-England border) to investigate the population structure of rural brown rat populations. We also examined individual rats for variations of the VKORC1 gene previously associated with resistance to anticoagulant rodenticides. We show that the populations were structured to some degree, but that this was only apparent in the microsatellite data and not the mtDNA data. We discuss various reasons why this is the case. We show that the population as a whole appears not to be at equilibrium. The relative lack of diversity in the mtDNA sequences examined can be explained by founder effects and a subsequent spatial expansion of a species introduced to the UK relatively recently. We found there was a geographical distribution of resistance mutations, and relatively low rate of gene flow between populations, which has implications for the development and management of anticoagulant resistance.
... Recentelijk werd deze mutatie ook in Kent, Verenigd Koninkrijk teruggevonden. Dit op een plaats waar ze in de praktijk bromadiolone resistentie hadden vastgesteld(Prescott et al. 2011). Deze resultaten kom overeen met onze bevindingen. ...
... 10 In the United Kingdom, where virtually all known resistance mutations are found, resistant R. norvegicus were found in Cambridge/Essex, Nottinghamshire, Kent, Gloucestershire, Norfolk and Lincolnshire, south-west Scotland, Hampshire and Berkshire, the Anglo-Welsh border, central southern Scotland, Yorkshire and Lancashire. 14,15 Resistance against second-generation rodenticides is also present in a number of other European countries, 2 namely across the whole of Denmark (Bornholm, Fünen, Jutland and Zealand), Belgium (Flanders) and large parts of France (e.g. Yonne, Eure and Loire). ...
Background
Rodenticide resistance to anticoagulants in Rattus norvegicus will lead to increased difficulties in combating these pest animals. Here, we present the results of a survey in the Netherlands where tissue samples and droppings were tested using a newly-developed TaqMan PCR-test for genotypic variation at codon 139 in the Vkorc1 gene associated with anticoagulant rodenticide resistance. Test results are linked to results of a questionnaire that was conducted among pest controllers.ResultsGenetic mutations at codon 139 of the Vkorc1 gene in R. norvegicus can be encountered in many parts of the Netherlands. In 34/61 rat tails a genotype was found that is linked to anticoagulant rodenticide resistance (56%). In droppings, 42/169 samples (25%) showed a resistance mediating genotype. In addition we found indications for a clear genetic sub-structure in the Netherlands. In some regions, only resistance mediating genotypes were found corroborating results from the questionnaire in which pest controllers indicated they suspected resistance to anticoagulant rodenticides.Conclusion
This is the first study that demonstrates the presence of multiple genetic mutations at codon 139 of the Vkorc1 gene in R. norvegicus in the Netherlands. As rodenticides should keep their efficacy because they are a last resort in rodent management, more studies are urgently needed that link specific genetic mutations to the efficacy of active substances.
... this mutation is common in France where it also confers resistance to bromadiolone (GrandeManGe et al., 2009). More recently it was also found in Korea and in the UK (rOst et al., 2009;PrescOtt et al., 2011), the latter in a place where applications of the anticoagulant rodenticide bromadiolone had been unsuccessful. the situation in France as well as in the UK is consistent with our findings. ...
Anticoagulant resistance is known as one of the major factors interfering with rodent control. Within this scope we investigated the distribution of anticoagulant resistance in Flanders, northern Belgium. From 2003 to 2005, we tested 691 rats from different locations with blood clotting response tests for their susceptibility to the anticoagulant compounds warfarin, bromadiolone and difenacoum. 119 of these rats were also screened for a mutation in the VKORC1 gene that is suspected to be responsible for anticoagulant resistance. Warfarin resistant rats were found in the western and eastern parts. The same distribution pattern holds for bromadiolone with the exception of the south eastern area, where this form of resistance was largely absent. We detected difenacoum resistance in only six rats and did not observe any resistant rats in the central part of Flanders. Susceptible rats were found all over Flanders. Genetic analyses showed that anticoagulants resistance in Belgium was related to two different mutations in VKORC1 namely Y139F and L120Q. Our results indicated that rodent control should be regionally tailored to be most effective.
KEY WORDS:
blood clotting response, rodent control, warfarin, bromadiolone, difenacoum, VKORC1
Mutations in Exon 1, 2 and 3 of the vitamin K epoxide reductase complex subunit 1 (Vkorc1) gene are known to lead to anticoagulant rodenticide resistance. In order to investigate their putative resistance in rodenticides, we studied the genetic profile of the Vkorc1 gene in Turkish black rats (Rattus rattus) and brown rats (Rattus norvegicus). In this context, previously recorded Ala21Thr mutation (R. rattus) in Exon 1 region, Ile90Leu mutation (R. rattus, R. norvegicus) in Exon 2 region and Leu120Gln mutation (R. norvegicus) in Exon 3 region were identified as ''missense mutations'' causing amino acid changes. Ala21Thr mutation was first detected in one specimen of Turkish black rat despite the uncertainty of its relevance to resistance. Ile90Leu mutation accepted as neutral variant was detected in most of black rat specimens. Leu120Gln mutation related to anticoagulant rodenticide resistance was found in only one brown rat specimen. Furthermore, Ser74Asn, Gln77Pro (black rat) and Ser79Pro (brown rat) mutations that cause amino acid changes in the Exon 2 region but unclear whether they cause resistance were identified. In addition, ''silent mutations'' which do not cause amino acid changes were also defined; these mutations were Arg12Arg mutation in Exon 1 region, His68His, Ser81Ser, Ile82Ile and Leu94Leu mutations in Exon 2 region and Ile107Ile, Thr137Thr, Ala143Ala and Gln152Gln mutations in Exon 3 region. These silent mutations were found in both species except for Ser81Ser which was determined in only brown rats.
Anticoagulant rodenticides are commonly used in rodent control because they are economical and have great deployment versatility. However, rodents with Single Nucleotide Polymorphism (SNP) mutations within the Vkorc1 gene are resistant to the effects of anticoagulant rodenticide use and this influences the effectiveness of control strategies that rely on such rodenticides. This study examined the prevalence of rat SNP mutations in Singapore to inform the effectiveness of anticoagulant rodenticide use. A total of 130 rat tail samples, comprising 83 Rattus norvegicus (63.8%) and 47 Rattus rattus spp. (36.2%) were conveniently sampled from November 2016 to December 2019 from urban settings and sequenced at exon 3 of Vkorc1 . Sequencing analysis revealed 4 synonymous and 1 non-synonymous mutations in Rattus rattus spp . samples. A novel synonymous mutation of L108L was identified and not previously reported in other studies. Non-synonymous SNPs were not detected in the notable codons of 120, 128 and 139 in Norway rats, where these regions are internationally recognised to be associated with resistance from prior studies. Our findings suggest that the prevalence of anticoagulant rodenticide resistance in Singapore is low. Continued monitoring of rodenticide resistance is important for informing rodent control strategies aimed at reducing rodent-borne disease transmission.
The frequencies of each of the 257 468 complete protein coding sequences (CDSs) have been compiled from the taxonomical divisions
of the GenBank DNA sequence database. The sum of the codons used by 8792 organisms has also been calculated. The data files
can be obtained from the anonymous ftp sites of DDBJ, Kazusa and EBI. A list of the codon usage of genes and the sum of the
codons used by each organism can be obtained through the web site http://www.kazusa.or.jp/codon/ . The present study also
reports recent developments on the WWW site. The new web interface provides data in the CodonFrequency-compatible format as
well as in the traditional table format. The use of the database is facilitated by keyword based search analysis and the availability
of codon usage tables for selected genes from each species. These new tools will provide users with the ability to further
analyze for variations in codon usage among different genomes.
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.
In the present study, we have sequenced the VKORC1 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 VKORC1 cDNA constructs and their recombinant expression revealed that these mutations reduced VKOR activities as compared to the wild-type protein. However, the in vitro enzyme assay used was not suited to convincingly demonstrate the warfarin resistance of all mutant proteins
Our results corroborate the VKORC1 gene as the main target for spontaneous mutations conferring warfarin resistance. The mechanism(s) of how mutations in the VKORC1 gene mediate insensitivity to coumarins in vivo has still to be elucidated.
Coumarin derivatives such as warfarin represent the therapy of choice for the long-term treatment and prevention of thromboembolic events. Coumarins target blood coagulation by inhibiting the vitamin K epoxide reductase multiprotein complex (VKOR). This complex recycles vitamin K 2,3-epoxide to vitamin K hydroquinone, a cofactor that is essential for the post-translational gamma-carboxylation of several blood coagulation factors. Despite extensive efforts, the components of the VKOR complex have not been identified. The complex has been proposed to be involved in two heritable human diseases: combined deficiency of vitamin-K-dependent clotting factors type 2 (VKCFD2; Online Mendelian Inheritance in Man (OMIM) 607473), and resistance to coumarin-type anticoagulant drugs (warfarin resistance, WR; OMIM 122700). Here we identify, by using linkage information from three species, the gene vitamin K epoxide reductase complex subunit 1 (VKORC1), which encodes a small transmembrane protein of the endoplasmic reticulum. VKORC1 contains missense mutations in both human disorders and in a warfarin-resistant rat strain. Overexpression of wild-type VKORC1, but not VKORC1 carrying the VKCFD2 mutation, leads to a marked increase in VKOR activity, which is sensitive to warfarin inhibition.
Anticoagulant compounds, i.e., derivatives of either 4-hydroxycoumarin (e.g., warfarin, bromadiolone) or indane-1,3-dione (e.g., diphacinone, chlorophacinone), have been in worldwide use as rodenticides for >50 years. These compounds inhibit blood coagulation by repression of the vitamin K reductase reaction (VKOR). Anticoagulant-resistant rodent populations have been reported from many countries and pose a considerable problem for pest control. Resistance is transmitted as an autosomal dominant trait although, until recently, the basic genetic mutation was unknown. Here, we report on the identification of eight different mutations in the VKORC1 gene in resistant laboratory strains of brown rats and house mice and in wild-caught brown rats from various locations in Europe with five of these mutations affecting only two amino acids (Tyr139Cys, Tyr139Ser, Tyr139Phe and Leu128Gln, Leu128Ser). By recombinant expression of VKORC1 constructs in HEK293 cells we demonstrate that mutations at Tyr139 confer resistance to warfarin at variable degrees while the other mutations, in addition, dramatically reduce VKOR activity. Our data strongly argue for at least seven independent mutation events in brown rats and two in mice. They suggest that mutations in VKORC1 are the genetic basis of anticoagulant resistance in wild populations of rodents, although the mutations alone do not explain all aspects of resistance that have been reported. We hypothesize that these mutations, apart from generating structural changes in the VKORC1 protein, may induce compensatory mechanisms to maintain blood clotting. Our findings provide the basis for a DNA-based field monitoring of anticoagulant resistance in rodents.
More than 21 million prescriptions for warfarin are written yearly in the U.S. Despite its importance, warfarin's target, vitamin K epoxide reductase (VKOR), has resisted purification since its identification in 1972. Here, we report its purification and reconstitution. HPC4, a calcium-specific antibody that recognizes a 12-aa tag, was used to purify and identify VKOR. Partial reconstitution is achieved on the column by washing with 0.4% dioleoylphosphatidylcholine/0.4% deoxycholate. Activity is completely recovered by dialysis against a buffer containing a reducing agent but lacking dioleoylphosphatidylcholine/deoxycholate. Removal of detergent from the eluted proteins apparently facilitates liposome formation. Purified recombinant VKOR with tag is approximately 21 kDa, as expected; fully active; and > 93% pure. The concentration of warfarin for 50% inhibition is the same for purified protein and microsomes. It has been reported that VKOR is a multisubunit enzyme. Our results, however, suggest that a single peptide can accomplish both the conversion of vitamin K epoxide to vitamin K and vitamin K to reduced vitamin K. This purification will allow further characterization of VKOR in relation to other components of the vitamin K cycle and should facilitate its structural determination.
Natural selection has traditionally been understood as a force responsible for pushing genes to states of higher translational efficiency, whereas lower translational efficiency has been explained by neutral mutation and genetic drift. We looked for evidence of directional selection resulting in increased unpreferred codon usage (and presumably reduced translational efficiency) in three divergent clusters of eukaryotic genomes using a simple optimal-codon-based metric (Kp/Ku).
Here we show that for some genes natural selection is indeed responsible for causing accelerated unpreferred codon substitution, and document the scope of this selection. In Cryptococcus and to a lesser extent Drosophila, we find many genes showing a statistically significant signal of selection for unpreferred codon usage in one or more lineages. We did not find evidence for this type of selection in Saccharomyces. The signal of positive selection observed from unpreferred synonymous codon substitutions is coincident in Cryptococcus and Drosophila with the distribution of upstream open reading frames (uORFs), another genic feature known to reduce translational efficiency. Functional enrichment analysis of genes exhibiting low Kp/Ku ratios reveals that genes in regulatory roles are particularly subject to this type of selection.
Through genome-wide scans, we find recent selection for unpreferred codon usage at approximately 1% of genetic loci in a Cryptococcus and several genes in Drosophila. Unpreferred codons can impede translation efficiency, and we find that genes with translation-impeding uORFs are enriched for this selection signal. We find that regulatory genes are particularly likely to be subject to selection for unpreferred codon usage. Given that expression noise can propagate through regulatory cascades, and that low translational efficiency can reduce expression noise, this finding supports the hypothesis that translational efficiency may be suppressed in some cases to reduce stochastic noise in gene expression.
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.
Control of rodent populations is performed worldwide with coumarin derivatives, such as warfarin. After widespread use, their effect has been diminished by the rapid spread of resistant rodents. Warfarin resistance in Rattus loseas in Jiangmen and Zhanjiang City, Guangdong Province, was investigated by lethal feeding tests. Twenty-three of 30 R. loseas trapped in Jiangmen City were assayed as warfarin-resistant individuals, whereas only 1 of 30 rodents in Zhanjiang was resistant. These results emphasize the need for thorough resistance monitoring as a basis for adequate control measures to prevent the use of ineffective rodenticides in Jiangmen City. The resistance mechanism mainly involves VKORC1, the molecular target for coumarin drugs. VKORC1 mRNA expression in wild-caught resistant animals showed no difference compared with that in susceptible individuals. Mutation screening of VKORC1 was carried out and an Arg58Gly mutation was identified as the prevailing type in R. loseas from Jiangmen City, which may constitute the genetic basis of anticoagulation resistance in R. losea in this resistance region.
Anticoagulant rodenticides are commonly used to control rodent pests all over the world. These pesticides inhibit one enzyme of the vitamin K cycle, Vkorc1, and thus prevent blood clotting and cause death by haemorrhage. Resistance to anticoagulants was first observed in Scotland in 1958, and more potent anticoagulants have been developed to overcome this obstacle. Unfortunately, these chemicals are very toxic and cannot be used everywhere. Some authors have shown that resistance to anticoagulants seems closely linked with single nucleotide polymorphism (SNP) in the Vkorc1 gene.
This study draws a map of SNP and haplotypes found in Vkorc1 in rats from different areas of France. Some of them had never been described before. Moreover, the Y139F mutation, described previously in France and Belgium, is the most frequent in France. This mutation is known to be associated with a strong resistance to anticoagulants, and it was found in 28% of the samples.
This biomolecular approach to studying and detecting resistance is easier to carry out than the phenotypic approach measuring blood coagulation time because it can be conducted on biological samples from dead animals, and it is less dangerous for the operator.
In humans, warfarin is used as an anticoagulant to reduce the risk of thromboembolic clinical events. Warfarin derivatives are also used as rodenticides in pest control. The gene encoding the protein targeted by anticoagulants is the Vitamin K-2,3-epoxide reductase subunit 1 (VKORC1). Since its discovery in 2004, various amino acid and transcription-regulatory altering VKORC1 mutations have been identified in patients who required extreme antivitamin K dosages, or wild populations of rodents that were difficult to control with anticoagulant rodenticides. One unresolved question concerns the dependency of the VKORC1 on the genetic background in humans and rodents that respond weakly or not at all to anticoagulants. Moreover, an important question requiring further analyses concerns the role of the Vkorc1 gene in mediating resistance to more recently developed warfarin derivatives (superwarfarins).
In this study, we bred a quasicongenic rat strain by using a wild-caught anticoagulant resistant rat as a donor to introduce the Y>F amino acid change at position 139 in the Vkorc1 into the genetic background of an anticoagulant susceptible Spraque-Dawley recipient strain.
In this manuscript we report the prothrombin times measured in the F7 generation after exposure to chlorophacinone, bromadiolone, difenacoum and difethialone. We observed that the mutation Y139F mediates resistance in an otherwise susceptible genetic background when exposed to chlorophacinone and bromadiolone. However, the physiological response to the super-warfarins, difenacoum and difethialone, may be strongly dependent on other genes located outside the congenic interval (28.3 cM) bracketing the Vkorc1 in our F7 generation congenic strain.
The majority of rat problems in cities are thought to be related to defective sewers, and the use of anticoagulant rodenticides in such places is often implemented as part of regular urban rodent control. Knowledge pertaining to the resistance status of sewer rat populations is non-existent, which may be leading to control problems in cities. It has become crucial to provide knowledge on the prevalence of resistance and how different control strategies have affected its prevalence among sewer rat populations. The prevalence of resistance was investigated in six sewer locations in Copenhagen and its suburban area by means of the blood clotting response (BCR) test and amplification refractory mutation system polymerase chain reaction (ARMS PCR) analysis, and by additional sequencing of the VKORC1 gene. The sewer locations were chosen to represent three different control strategies: (i) no anticoagulant use for approximately 20 years; (ii) no anticoagulant use for the last 5 years; (iii) continuous use for several decades up to the present.
A low level of anticoagulant resistance was found in the sewers regardless of control strategy. Surprisingly, none of the rats, including the resistant rats, had resistance-related mutations in the VKORC1 gene.
The results of this study suggest that the genetic background of anticoagulant resistance may have to be redefined in respect of resistance-related changes in the VKORC1 gene.
The 2-stage determination is based on changes in blood coaggulation activity brought about both by the administration of warfarin in conjunction with vitamin K1 epoxide and by feeding a vitamin K-free diet for 4 days. When it was applied to laboratory-bred rats of known warfarin-resistance genotype, 35/35 homozygous susceptible, 44/44 homozygous resistant and 131/133 heterozygous rats were correctly classified. This method was equally effective in identifying the genotype of wild rats carrying the warfarin-resistance gene, Rw2. The procedure is rapid and accurate.
The anticoagulant difenacoum was tested at two concentrations, 0·005 and 0·01%, in bait against warfarin-resistant rat infestations in farm buildings. Twelve out of the 14 treatments in which the lower concentration of the anticoagulant was used resulted in complete control. One of the remaining two treatments was probably also completely successful, but in the other a few rats, that were not eating the poisoned baits, were still active after 30 days of baiting. All six treatments done using the stronger concentration of poison were completely effective.
Since it took as long to control infestations with 0·01% as with 0·005% difenacoum in treatments carried out under similar conditions, the lower concentration is recommended for use against warfarin-resistant rats.
The management of warfarin therapy is complicated by a wide variation among patients in drug response. Variants in the gene encoding vitamin K epoxide reductase complex 1 (VKORC1) may affect the response to warfarin.
We conducted a retrospective study of European-American patients receiving long-term warfarin maintenance therapy. Multiple linear-regression analysis was used to determine the effect of VKORC1 haplotypes on the warfarin dose. We determined VKORC1 haplotype frequencies in African-American, European-American, and Asian-American populations and VKORC1 messenger RNA (mRNA) expression in human liver samples.
We identified 10 common noncoding VKORC1 single-nucleotide polymorphisms and inferred five major haplotypes. We identified a low-dose haplotype group (A) and a high-dose haplotype group (B). The mean (+/-SE) maintenance dose of warfarin differed significantly among the three haplotype group combinations, at 2.7+/-0.2 mg per day for A/A, 4.9+/-0.2 mg per day for A/B, and 6.2+/-0.3 mg per day for B/B (P<0.001). VKORC1 haplotype groups A and B explained approximately 25 percent of the variance in dose. Asian Americans had a higher proportion of group A haplotypes and African Americans a higher proportion of group B haplotypes. VKORC1 mRNA levels varied according to the haplotype combination.
VKORC1 haplotypes can be used to stratify patients into low-, intermediate-, and high-dose warfarin groups and may explain differences in dose requirements among patients of different ancestries. The molecular mechanism of this warfarin dose response appears to be regulated at the transcriptional level.
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