Blood Clotting Response Research Articles

Anticoagulant resistance: a relevant issue in sewer rat (Rattus norvegicus) control?

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.

A standardised BCR resistance test for all anticoagulant rodenticides*

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.

Relationship between resistance factors and treatment efficacy when bromadiolone was used against anticoagulant-resistant Norway rats (Rattus norvegicus Berk.) in Wales*

We investigated the relationship between the severity and incidence of resistance among Norway rats (Rattus norvegicus) on a farm in Wales and the subsequent outcome of a practical rodent control operation. Bromadiolone resistance factors were estimated for rats trapped on the farm using the blood clotting response test, and were found to be 2 to 3 for male rats and approximately 6 for females. The incidence of resistance in the rat population was high. Infestation size was estimated by census baiting and tracking, and was found to be substantial, with a maximum of 6.5 kg of bait being eaten on a single night. A proprietary rodenticide (Deadline™), containing 0.005% bromadiolone, was used to control the infestation. The duration of baiting was 35 days and, according to the two methods of assessment used, treatment success was in the region of 87 and 93%. No evidence was observed of a significant impact of resistance on the rat control operation, and the remaining rats of this very heavy infestation would probably have been controlled if baiting had continued for longer.

Susceptibility to the anticoagulants bromadiolone and coumatetralyl in wild Norway rats ( Rattus norvegicus ) from the UK and Germany*

A new blood clotting response test was used to determine the susceptibility, to coumatetralyl and bromadiolone, of laboratory strains of Norway rat from Germany and the UK (Hampshire), and wild rats trapped on farms in Wales (UK) and Westphalia (Germany). Resistance factors were calculated in relation to the CD strain of Norway rat. An outbred strain of wild rats, raised from rats trapped in Germany, was found to be more susceptible to coumatetralyl by a factor of 0.5 – 0.6 compared to the CD strain. Homozygous and heterozygous animals of a strain of resistant rats from Westphalia were cross-resistant to coumatetralyl and bromadiolone, with a higher resistance factor for bromadiolone than that found in both UK strains. Our results show that the degree of altered susceptibility and resistance varies between strains of wild rat and between resistance foci. Some wild rat strains may be more susceptible than laboratory rat strains. Even in a well-established resistance area, it may be difficult to find infestations with resistance high enough to suspect control problems with bromadiolone, even after decades of use of this compound.

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

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

Blood-clotting response tests for resistance to diphacinone and chlorophacinone in the Norway Rat ( Rattus norvegicus Berk.)

Resistance baselines were obtained for the first generation anticoagulant rodenticides chlorophacinone and diphacinone using laboratory, caesarian-derived Norway rats ( Rattus norvegicus) as the susceptible strain and the blood clotting response test method. The ED 99 estimates for a quantal response were: chlorophacinone, males 0.86 mg kg −1, females 1.03 mg kg −1; diphacinone, males 1.26 mg kg −1, females 1.60 mg kg −1. The dose-response data also showed that chlorophacinone was significantly ( p<0.0001) more potent than diphacinone for both male and female rats, and that male rats were more susceptible than females to both compounds ( p<0.002). The ED 99 doses were then given to groups of five male and five female rats of the Welsh and Hampshire warfarin-resistant strains. Twenty-four hours later, prothrombin times were slightly elevated in both strains but all the animals were classified as resistant to the two compounds, indicating cross-resistance from warfarin to diphacinone and chlorophacinone. When rats of the two resistant strains were fed for six consecutive days on baits containing either diphacinone or chlorophacinone, many animals survived, indicating that their resistance might enable them to survive treatments with these compounds in the field.

Genomic assignment of the warfarin resistance locus, Rw, in the rat.

The locus responsible for resistance to the anticoagulants warfarin and bromadiolone (locus symbol Rw) was integrated into the rat (Rattus norvegicus) microsatellite genome map. Seventh-generation offspring of a segregating strain of rats heterozygous resistant to both compounds were tested with a blood-clotting-response (BCR) test. No recombination between resistance to warfarin and bromadiolone was observed, indicating a common genetic basis. No recombinants were found between Rw and D1Arb18 (Myl2) located at the MIT-microsatellite map position 95.90 (SHRSP x BN F2-cross) or 82.24 (FHH x ACI F2-cross). Resistance segregated in a ratio expected for single, dominant gene responses. An equal number of females and males were resistant, but females retained higher percentage blood coagulation activities (PCA) after anticoagulant administration. Partial synteny between rat, mouse, and human suggests that Myl2 may serve as anchor to map the Rw homologs in mouse and human.

Endothelial Protein C Receptor

Endothelial Protein C Receptor

A review of secondary‐poisoning studies with rodenticides

Rodenticides can become a danger for predators and scavengers if they prey on dead or moribund rodents. One possibility for assessing the hazard potential is to conduct trials in which poisoned rodents are fed to predators. Over the years, numerous such studies have been carried out with a wide range of carnivorous mammals and birds. Since an internationally recognized standard method only recently came into existence, the studies differ in details of trial procedure. The most significant end point is the mortality rate; in addition, blood clotting response often serves as a biomarker. This review includes studies on brodifacoum, bromadiolone, chlorophacinone, coumatetralyl, difenacoum, flocoumafen, warfarin and zinc phosphide which have been published in the open literature or which have been submitted by industry in the authorization procedure for plant protection products.

Resistance to anticoagulant rodenticides in Germany and future strategies to controlRattus norvegicus

Widespread anticoagulant resistance was discovered in populations of the brown rat (Rattus norvegicus Berk.) in an area of roughly 8000 km2 in north-west Germany. Resistance testing was performed by feeding tests and/or by measuring blood clotting response after intraperitoneal injection of a sublethal testing solution. A hierarchical resistance system was found with warfarin resistance at the base followed by bromadiolone/coumatetralyl resistance to difenacoum resistance at the top. Warfarin resistance was spread over the whole area with reduced incidence towards the edges. Difenacoum resistance represented the highest level found so far and was restricted to the inner zone of the resistance area where it occurred with a frequency of 6% of all individuals tested. Breeding experiments with bromadiolone-resistant rats showed that expression of the bromadiolone resistance gene differed in the two sexes, suggesting additional sex-linked modifying effects to the resistance gene. Control strategy within the resistance area with respect to prevention of further selection for resistance is discussed.

Activation waves in a model of platelet aggregation: existence of solutions and stability of travelling fronts.

Platelets cohere to one another to form platelet aggregates as part of the blood's clotting response. The ability of a platelet to participate in this process depends on its prior 'activation' by chemicals released into the blood plasma by other activated platelets. We study the piecewise-linear system of reaction-diffusion equations which, in one spatial dimension, describe the chemically-mediated spread of platelet activation. We establish the existence of classical solutions to this system of equations, and show that these solutions do not blow up in finite time. We also explicitly construct travelling front solutions and discuss their stability. Finally, we present numerical evidence which suggests that for a broad range of initial data with the correct limiting values at +/- infinity, the solution to the initial value problem rapidly evolves into the travelling front solution provided the front is linearly stable.

The development of a blood clotting response test for discriminating between difenacoum-resistant and susceptible Norway rats ( Rattus norvegicus, berk.)

1. A new test for identifying levels of difenacoum resistance in the Norway rat is described, based upon the differential physiological response to difenacoum administration. 2. This test is based on changes in blood clotting activity over 4 days, following administration of the rodenticide difenacoum in conjunction with menadione (vitamin K 3). 3. The anticoagulant effect is reduced only in rats that are resistant or tolerant to difenacoum. 4. This test procedure is quicker than traditional feeding tests, and identifies the degree of resistance in both laboratory and wild rats that have difenacoum resistance genes.