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3.2.1 DNA samples

In order to identify the spread of the various resistance mutations and delimit the foci wherein they were discovered, sites across the south-east of England were visited in order to obtain DNA samples. Due to ease of access and generally high Norway rat abundance, farms were most often visited. Farms were identified using Ordnance Survey maps (Ordnance Survey Ltd, Southampton, Hampshire, UK), and owners or tenants approached on site by the researchers. Initially sites with no recent history of anticoagulant rodenticide treatment were preferentially selected. This was to prevent inflation of the number of resistant animals discovered, as recent or ongoing treatments would kill off susceptible animals, assuming correct application of rodenticides. This practice was subsequently abandoned when it became clear that stakeholders applied rodenticides with a frequency that made finding rat populations that had undergone no recent chemical control untenable. Alternatively,

stakeholders were contacted or made aware of the study, with the intention that they would supply DNA samples of their own volition. Stakeholders included landowners, gamekeepers, farmers, public health officials and PCOs. PCOs were contacted via industry contacts, and the study was publicised by various print and television media outlets.

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3.2.2 Tissue samples

DNA samples were obtained from tail tips taken from recently killed Norway rats. Rats were caught by researchers in live traps and dispatched using a Home Office schedule 1 method. Tails tip were removed immediately and stored in 70 – 80% IMS and then frozen at -200C until analysis. Stakeholders were requested to follow the same methodology in order to prevent degradation of tissue. Stakeholders were requested not to take tail tips from rats found dead at sites where anticoagulant rodenticides were being used, and not to take tail tips from rats that had been dead for more than 24 hours. However, whether all stakeholders abided by these rules could not be ascertained.

3.2.3 Faecal samples

By 2013 it had become clear that most of the larger pest control companies – the main source of tails sent to the University – were hesitant to get involved with supplying samples, despite adverts in trade publications and repeated appeals from senior figures in the industry. In addition, despite clear instructions being repeatedly provided to suppliers of tissue samples, the number of samples where it was not possible to extract viable DNA due to degradation or contamination indicated that some tail samples were being handled or selected incorrectly. Due to the neophobic nature of Norway rats (Barnett, 1958; Inglis et al., 1996) and the large study area, it was decided that having researchers attempt to make up the bulk of samples via direct captures of live rats would be inefficient. Therefore, from September until December 2013, researchers visited farms in Kent and East Anglia and collected faecal samples for analysis. Suitably fresh samples were identified by sight. Because it was impossible to ascertain which samples came from different individuals, selected droppings were collected together in vials. Once at the laboratory, faecal samples were frozen at -200C until analysis.

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3.2.4 DNA extraction

DNA was extracted from tail tips using the DNeasy Blood and Tissue Kit following a modified version of the manufacturer’s instructions (Qiagen Ltd, Crawley, West Sussex, UK). See Section 2.1.4. See Prescott et al. (2010) for details of samples taken prior to 2012.

DNA was extracted from faecal samples using the QIAamp DNA Stool Mini Kit (Qiagen Ltd, Crawley, West Sussex, UK) following a modified version of the manufacturer’s instructions for human faecal samples. See Section 2.1.4.

3.2.5 DNA amplification

Elutions resulting from DNA extraction of faecal and tail samples were amplified by PCR (see Section 2.1.4). Because the study was exploratory, with the presumed potential for any mutation to be exhibited by rats in any location, it was necessary to detect all the possible single nucleotide polymorphisms of the VKORC1 gene. In order to cover this range of sites of potential SNPs, primers were obtained from Invitrogen (Life Technologies Ltd, Paisley, Renfrewshire, UK) that flanked codons from before and after as many of the known SNP sites as possible in each of the three exons. Primers used from 2012 onward for detecting SNPs in exon 1 were F: GAG GAG CCC TGG ACG TTT and R: AGG AGA AGA CGC GGG AAC; primers used for exon 2 were F: GGT GGA GCA CGT GTT AGG AG and R: GGT CAC CAA GAC ATG AGG TG; primers used for exon 3 were F: TGA GTT CCC TGG TGT CTG TC and R: TTT TAG GGA CCC ACA CAC GA. PCR products were resolved by electrophoresis and the resulting gel examined under UV light. Providing the PCR was shown to be successful, PCR products were purified using the QIAquick PCR

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Purification Kit (Qiagen Ltd, Crawley, West Sussex, UK). Primers used prior to 2012 were based on those used by Rost et al. (2004).

3.2.6 Sequencing and interpretation

PCR products were sent to Source Bioscience for Sanger sequencing (Source BioScience plc, Nottingham, Nottinghamshire, UK). Chromas Lite and Finch TV were used to open the resulting chromatogram files; with the majority of the three exons of the VKORC1 gene displayed onscreen, mutations could be identified simply by visual analysis of all relevant codons and comparison with those found in the genomic DNA of a wild-type Norway rat. In order to measure the size of confirmed mutation foci and identify areas where further samples were required, location data for every individual site where results could be obtained were displayed as point shapefiles on ArcGIS (Esri UK Ltd, Aylesbury, Buckinghamshire, UK).