Acumulación de Pretensiones

Very soon after the initial discovery of anticoagulant resistance, rats were captured in the wild and brought into the laboratory to be subjected to tests intended to elucidate the impact that resistance would have on future attempts at practical control in the field. Breeding experiments utilising the offspring of these wild progenitors revealed that resistance is controlled by a single, dominant autosomal gene mapped to orthologous linkage groups in both Norway rats (Rw; chromosome 1) and house mice (Raw; chromosome 7) (Greaves and Ayres, 1969; Wallace and MacSwiney, 1976). Just as laboratory feeding tests were used to confirm efficacy of potential anticoagulant rodenticides prior to their marketing and

subsequent use in the field (Bentley, Hammond and Taylor, 1955; Bentley and Rowe, 1956; Bentley and Larthe, 1959; Redfern, Gill and Hadler, 1976) so they were used to confirm suspected instances of resistance (Boyle, 1960; Cuthbert, 1963; Lund, 1964) or to confirm their efficacy against various resistant strains (Redfern and Gill, 1978; Gill, 1992). As the scale of resistance in both Norway rats and house mice became clear (Dodsworth, 1961; Bentley, 1968), laboratory tests on these rodents became less reactive and more proactive, focusing on level of tolerance that resistance mutations conferred against the various

anticoagulants. These tests involved offering warfarin baits or warfarin-infused foodstuffs to rats at given rates, and monitoring their survivability in what appeared to be largely ad-hoc experiments. Individuals that died contributed to the generation of lethal feeding periods (LFP01-99) rather than lethal doses (LD01-99), because it was not possible to determine the

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exact volume of warfarin each individual had consumed. As described in Section 2.2.1, although a standard test methodology was established (six days’ feeding on 0.005% warfarin bait) that appeared to identify rats resistant to warfarin (Drummond and Bentley, 1965; Bentley, 1968; Drummond and Wilson, 1968), a method that was quicker and less questionable in its humaneness (because it did not rely on mortality of test animals) was quickly posited (Greaves and Ayres, 1967). BCR resistance tests were expanded to include a variety of anticoagulant active ingredients, establishing discriminating doses for FGARs and SGARs against susceptible rats, and subjecting laboratory-resistant animals of known origin and strain to the same test. Using this information, assuming researchers had identified a particular strain correctly (i.e. by its nominal geographic designation), these resistance tests could be applied to wild rats, and the wild population in question designated as resistant or susceptible based on whether or not individuals were “responders” (i.e. to be significantly affected by the anticoagulant administered). This designation was based upon the percentage coagulation activity (PCA) of test animals. Calibration curves based on dilutions of normal plasma in saline are used to convert measured coagulation times in seconds to PCA in order to ascertain the level of coagulation capability that blood samples have retained. Examples of these tests include chlorophacinone and diphacinone against “Welsh” and “Hampshire” resistant strains (Prescott and Buckle, 2000); warfarin against “Welsh” rats (Greaves and Ayres, 1967; Martin et al., 1979; MacNicoll and Gill, 1993a), “Scottish” and “Hampshire” rats (MacNicoll and Gill, 1993a); and bromadiolone against “Welsh”, “Scottish”,

“Hampshire” and “Berkshire” rats (Gill et al., 1994). Difenacoum tests were carried out against “Welsh”, “Scottish” and “Hampshire” rats (Gill et al., 1993), but because these aimed to replicate the results of LFP tests as well as monitor PCA (that is, cause mortality in

susceptible rats whilst monitoring PCA over several days, rather than classifying rats as responders or non-responders in a one-time test) discriminating doses are much higher than

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expected, and of little use. They also lacked the bonuses of reduced time, cost and

humaneness issues. There was little consistency between these tests. SGAR tests utilised a 96-hour period between dosing and blood sampling, whereas a 24-hour period was

established when testing FGARs. Furthermore, in SGAR tests rats were considered

responders if their PCA was less than 10%; rats in tests involving FGARs were classified as responders if their PCA fell below 17% (see also Baert et al., 2012). The level and standard of efficacy necessary to discriminate between susceptible and resistant rats was not consistent between studies. Only in the tests involving bromadiolone, diphacinone and chlorophacinone was the point of discrimination (ED99 or the upper 95% fiducial limit thereof) established

prior to testing by Probit analysis (EPPO, 1999b). Furthermore, utilising a different method of analysis (e.g. generating a Probit log dose-response rather than a Probit dose-response) will create varying estimates of ED99. The methods available to generate dose response data

are primarily designed to estimate the ED50; as effective dose responses approach 99% (or

1%) the fiducial limits associated with the predicted effective dose grow wider, compared with the size of the predicted effective dose. This effect is compounded when these methods are used in tests against vertebrate animals, numbers of which are reduced for humaneness reasons wherever possible. So even when standardised, tests to determine ED99s are not

reliable. In addition to the inherent problems with generating and utilising ED99s, the

application of these tests was also limited because their main use was simply to ascertain whether rats of a given resistant strain could tolerate a dose of a given anticoagulant that would kill a given number of susceptible animals. These data cannot be compared without making assumptions based on extrapolation and guesswork. The tests were all specific to each strain, and other than the nominal geographic naming tendencies in the UK, there was no guarantee that results from a particular study could be applied to practical pest control in other areas of the UK, or elsewhere. In addition, in the field there are frequently rats

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heterozygous for the resistance mutations in question, which respond differently to

anticoagulant and vitamin K-deficient stimuli, than do susceptible or homozygous-resistant rats (see Section 1.4.2).

With the initial identification of the resistance-conferring mutations (Li et al., 2004; Rost et al., 2004) and their confirmed locations (e.g. Pelz et al., 2005), data from all future resistance tests – and any previous tests wherein viable tissue of the test subjects had been retained, or the strain in question was still extant with no further introductions of foreign animals – could potentially contribute to the sum of all researchers’ knowledge, regardless of the animals’ provenance. Until now, most research has focused on identification and

delimitation of resistance foci. Given the extent of the L120Q focus in the south-east of England (see Section 3.3.1), there is a clear requirement to quantify the level of tolerance (resistance factor: RF) to anticoagulants that the various L120Q strains confer.