Comparison of the inhibition effect of different anticoagulants on vitamin K epoxide reductase activity from warfarin-susceptible and resistant rat

Anti-vitamin K drugs are widely used as anticoagulant in human thromboembolic diseases. Similar compounds have also been used as rodenticides to control rodent population since 1950s. Massive use of first generation anticoagulants, especially warfarin, has lead to the development of genetic resistances in rodents. Similar resistances have been reported in human. In both cases, polymorphisms in VKORC1 (Vitamin K epoxide reductase subunit 1), the subunit 1 of the VKOR (Vitamin K epoxide reductase) complex, were involved. In rats (Rattus norvegicus), the Y139F mutation confers a high degree of resistance to warfarin. Little is known about the in vitro consequences of Y139F mutation on inhibitory effect of different anticoagulants available. A warfarin-susceptible and a warfarin-resistant Y139F strain of wild rats (Rattus norvegicus) are maintained in enclosures of the Lyon College of Veterinary Medicine (France). Using liver microsomes from susceptible or resistant rats, we studied inhibition parameters by warfarin (K_i = 0.72 ± 0.1 μM; 29 ± 4.1 μM), chlorophacinone (K_i = 0.08 ± 0.01 μM; 1.6 ± 0.1 μM), diphacinone (K_i = 0.07 ± 0.01 μM; 5.0 ± 0.8 μM), coumachlor (K_i = 0.12 ± 0.02 μM; 1.9 ± 0.2 μM), coumatetralyl (K_i = 0.13 ± 0.02 μM; 3.1 ± 0.4 μM), difenacoum (K_i = 0.07 ± 0.01 μM; 0.26 ± 0.02 μM), bromadiolone (K_i = 0.13 ± 0.02 μM; 0.91 ± 0.07 μM), and brodifacoum (K_i = 0.04 ± 0.01 μM; 0.09 ± 0.01 μM) on VKOR activity. Analysis of the results leads us to highlight different anticoagulant structural elements, which influence inhibition parameters in both susceptible and Y139F resistant rats.     

珠江三角洲地区黄毛鼠抗药性的发生趋势及其Vkorc1基因多态性

为阐明珠江三角洲地区黄毛鼠Rattus losea对第1代抗凝血灭鼠剂的抗性发生趋势及其遗传机制,以杀鼠灵为标准药物,采用致死期食毒法对2017—2021年在广东省江门市捕获的165只黄毛鼠进行生理抗性检测,并测定每只试鼠的维生素K环氧化物还原酶复合物亚单位1(vitamin K epoxide reductase complex subunit 1,Vkorc1)的编码基因序列,分析其突变情况。结果显示,江门市黄毛鼠对第1代抗凝血灭鼠剂杀鼠灵的抗性率为27.03%～50.00%,在黄毛鼠Vkorc1基因中检测到6个不同的突变位点,包括2个错义突变位点Arg58Gly及Tyr139Cys和4个沉默突变位点Ala41Ala、Cys96Cys、Arg98Arg及Ala143Ala,突变率分别为87.27%、0.61%、1.21%、0.61%、1.21%和0.61%,其中Ala143Ala是在黄毛鼠中新发现的沉默突变位点。表明珠江三角洲地区黄毛鼠已对第1代抗凝血灭鼠剂产生了群体抗性并呈上升趋势,第58位的精氨酸突变成甘氨酸(Arg58Gly)是黄毛鼠抗性基因Vkorc1的主要突变位点。     

Large-scale identification of rodenticide resistance in Rattus norvegicus and Mus musculus in the Netherlands based on Vkorc1 codon 139 mutations

BACKGROUND

Resistance to rodenticides has been reported globally and poses a considerable problem for efficacy in pest control. The most-documented resistance to rodenticides in commensal rodents is associated with mutations in the Vkorc1 gene, in particular in codon 139. Resistance to anticoagulant rodenticides has been reported in the Netherlands since 1989. A study from 2013 showed that 25% of 169 Norway rats (Rattus norvegicus) had a mutation at codon 139 of the Vkorc1 gene. To gain insight in the current status of rodenticide resistance amongst R. norvegicus and house mice Mus musculus in the Netherlands, we tested these rodents for mutations in codon 139 of the Vkorc1 gene. In addition, we collected data from pest controllers on their use of rodenticides and experience with rodenticide resistance.

RESULTS

A total of 1801 rodent samples were collected throughout the country consisting of 1404 R. norvegicus and 397 M. musculus. In total, 15% of R. norvegicus [95% confidence interval (CI): 13–17%] and 38% of M. musculus (95% CI: 33–43%) carried a genetic mutation at codon 139 of the Vkorc1 gene.

CONCLUSION

This study demonstrates genetic mutations at codon 139 of the Vkorc1 gene in M. musculus in the Netherlands. Resistance to anticoagulant rodenticides is present in R. norvegicus and M. musculus in multiple regions in the Netherlands. The results of this comprehensive study provide a baseline and facilitate trend analyses of Vkorc1 codon 139 mutations and evaluation of integrated pest management (IPM) strategies as these are enrolled in the Netherlands. © 2022 The Dutch Pest and Wildlife. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

Neospora caninum detected in feral rodents

The role of rodents in the epidemiology of neosporosis was investigated by assaying brain tissue of feral mice (Mus musculus) and rats (Rattus norvegicus) for Neospora caninum. Both mouse and rat brain tissue were extracted for total DNA, and subjected to two different N. caninum-specific nested polymerase chain reaction (PCR) assays. A portion of brain tissue from the mice and rats were also assayed for N. caninum in gerbils or gamma-interferon gene knockout (KO) mice. Of the 105 feral mice tested, 10% were positive in the N. caninum-specific PCR assays. Of the 242 rats tested, 30% were positive in both assays. Although mice and rats had N. caninum by PCR testing, clinical signs of N. caninum infection were not observed nor were N. caninum parasites observed in gerbils or KO mice inoculated with the rodent brain tissue.     

A rare autosomal recessive condition,congenital erythropoietic porphyria,found in the canefield rat Rattus sordidus Gould 1858

Congenital erythropoietic porphyria (CEP) is a rare autosomal recessive condition that has been reported in humans and in some animals, in which uroporphyrin 1 is deposited in the bones, teeth and urine, resulting in pink coloration and fluorescence of the tissues and urine under long‐wave ultraviolet (UV) light. We observed red teeth in nine of 450 canefield rats (Rattus sordidus) captured in a small, isolated patch of sugarcane in Tully, north Queensland, Australia. The skeletons of these animals were excised and were found to be bright red under normal day light. Under UV light, the skeleton had a bright red fluorescence. It is plausible that the canefield rat population in this isolated patch of sugarcane is small and inbreeding might have occurred, resulting in incidences of the autosomal recessive genes that cause CEP. The canefield rat can be used as an animal model for research into porphyria.     

余庆县1986～2008年褐家鼠种群动态及繁殖特征分析

通过对贵州省余庆县1986～2008年褐家鼠种群动态和繁殖特征的变化规律进行研究.结果表明,褐家鼠主要分布于住宅区,是余庆县家栖鼠优势种,占总鼠数的85.14%.研究期间住宅区共捕获褐家鼠504只,种群总性比(♀/♂)0.98,平均捕获率1.53%,平均怀孕率20.40%,平均胎仔数7.33只,平均繁殖指数0.74,平均睾丸下降率27.95%.褐家鼠全年均可繁殖,每年在3月、6月、11月出现3个数量高峰期,在2～3月和10～11月出现2个繁殖高峰期.因此,每年3月和10月为最佳防治时期,毒饵站灭鼠技术是目前防治褐家鼠最好的方法之一.本研究同时还比较了国内其他地区褐家鼠种群动态和繁殖特征的变化规律,以期为其种群数量预测预报和防治提供科学依据.     

Reproductive success of bromadiolone-resistant rats in absence of anticoagulant pressure

Resistance to anticoagulant rodenticides in brown rats (Rattus norvegicus Berk.) is associated with pleiotropic effects, notably with an increased dietary vitamin K requirement. Owing to this disadvantage, resistance is believed to be selected against if anticoagulant selection is absent. In small experimental populations of wild brown rats, an investigation was carried out to establish whether tolerance to anticoagulant exposure changed over a period of 2 years. In the same populations, DNA microsatellite markers were used to infer parentage, and this made it possible to estimate reproductive success of sensitive and resistant rats and estimate effective population size, Ne. Even though there was evidence for a selection against resistant rats with high vitamin K requirement, anticoagulant tolerance was not seen to be significantly influenced in the absence of bromadiolone selection. As the population size under investigation was small, random genetic drift may have played a role in this. In the presence of bromadiolone selection, however, the tolerance was significantly increased, suggesting that continuous selection will increase the proportion of highly resistant rats in the population. It was found that, for both males and females, surprisingly few individuals contributed to the next generation with numerous offspring, and most breeders contributed with none or a single offspring. The expected higher reproductive success and consequent increase in proportional numbers of sensitive rats in the absence of anticoagulant selection could not be observed. Among the resistant rats, moderately resistant females were found to be better breeders than highly resistant breeders, but for resistant males the reverse was true. This could be explained by the fact that the increased vitamin K requirement results in sex differential selection; in highly resistant males the selection presumably takes place at the immature stage, whereas in females the vitamin K requirement becomes crucial at the reproductive stage, as vitamin K is not only essential for the blood clotting process but also for bone formation.     

The genetic mechanisms of warfarin resistance in Rattus rattus found in the wild in Japan

Warfarin is commonly used worldwide as a rodenticide. It inhibits blood coagulation by inhibiting vitamin K 2,3-epoxide reductase (VKOR) activity leading to hemorrhage. However, it has been reported that repeated or long-term treatment with warfarin results in resistance emerging in wild rodents. Such resistance may explain why it is difficult to control rodents in many regions in Japan. In this report, we studied mutations in the VKOR gene (including the VKOR complex subunit 1 (VKORC1)), while also analyzing VKOR and clotting factor activity in black rats (Rattus rattus) in order to understand better the mechanism of warfarin resistance in this species.We sequenced the VKORC1 gene from 275 rats living in the wild in Japan. We found several types of novel base substitutions, some of which conferred warfarin resistance.There was no difference in coagulation times between warfarin-sensitive and resistant rats measured under physiological conditions. However, after warfarin administration, no effect was noted in warfarin-resistant rats, although a prolonged coagulation time was noted in warfarin-sensitive rats.We also determined the kinetic differences in hepatic microsomal VKOR-dependent activity between warfarin-resistant and sensitive rats. Warfarin-resistant rats showed 2-3-fold lower V_max/K_m values than did sensitive rats. In addition, we report that resistant rats found in the Tokyo area had a VKOR activity which was poorly inhibited by warfarin.Finally, we conclude that reduced VKOR activity and warfarin resistance in the Japanese black rat might be due to mutations in the VKORC1 gene. However, further study is needed to clarify how such rats can maintain adequate vitamin K-dependent clotting factor levels, while simultaneously exhibiting low VKOR activity and warfarin resistance.     

花生结荚期鼠害的防治指标

研究花生结荚期以黄毛鼠为优势种的鼠害防治指标的结果表明。花生结荚期鼠密度(x)与花生果荚受害率(y)之间呈线性相关(r=0.9619(?));花生结荚期鼠害的动态防治指标可用模型 x=625×CF/YPE+4.2475来估计;根据福建的现状,花生结荚期的经济允许损失率为1.167%,相应的鼠害防治指标为鼠密度11.54%.     

Managing resistance to anticoagulant rodenticides: An appraisal

The action necessary for resistance management is specified and compared with what has actually been done, with reference to executive, extension and research activities and to the role of rodenticides and rodenticide development. Some obstacles to resistance management are discussed. To account for the fact that no plausible programme to manage resistance has been developed over the last 36 years, the hypothesis is examined that resistance management is injurious to the parties concerned and, hence, that resistance is perceived not as a problem but as an exploitable asset. If correct, this hypothesis yields the prediction that the action necessary for resistance management will be taken when such action is judged to be economically efficient. The establishment of the Rodenticide Resistance Action Committee indicates that this time may be approaching.