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© 2019 Campaign for Responsible Rodenticide Use UK
Contents
page
Contents
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Summary
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1. Introduction
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2. Materials and Methods
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2.1 Origins of samples
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2.2 Methods of DNA analysis
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2.3 The Rodenticide Resistance Action Committee (RRAC) interactive
global resistance map
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3. Results
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3.1 Norway rats
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3.2 House mice
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4. Discussion
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5. References
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© 2019 Campaign for Responsible Rodenticide Use UK
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.
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© 2019 Campaign for Responsible Rodenticide Use UK
1. Introduction
Previous reports produced for the Campaign for Responsible Rodenticide Use (CRRU)
(UK) on the status of anticoagulant resistance among Norway rats (Rattus norvegicus) and house
mice (Mus musculus) in the UK have presented background information on resistance mutations,
explained resistance testing methodologies and provided information on the occurrence and
geographical distribution of resistance (see Prescott et al., 2017 and 2018). This previously-
presented information will not be reproduced in this report; rather a summary is provided of new
information that has been obtained since the last report was prepared as the result of genomic
resistance testing conducted at the University of Reading and funded by the Rodenticide
Resistance Action Committee of CropLife International (http://www.rrac.info/).
This report has been prepared for CRRU in response to a requirement of the Health and Safety
Executive (HSE) and the Government Oversight Group (GOG) to provide resistance monitoring
information on an annual basis to support their evaluation of the progress of the UK Rodenticide
Stewardship Regime (HSE, 2019) under the heading “Competent Workforce”.
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© 2019 Campaign for Responsible Rodenticide Use UK
2. Materials and Methods
2.1 Origins of samples
The tissue samples analysed for genetical mutations were either submitted by pest control
technicians or were collected after trapping by staff of the Vertebrate Pests Unit (VPU) at the
University of Reading. Thus, samples were generally received from areas in which technicians
had experienced difficulties in obtaining effective control with anticoagulants, possibly because
of resistance or, in the case of VPU sampling, were taken from the borders of known resistance
areas in an attempt to identify their boundaries.
During 2019 additional effort was expended in obtaining samples from areas of the UK from
which samples had not previously been received. The maps presented in previous reports had
shown that samples have not been obtained, for example, from a large area in the centre of the
country, including many counties of the Midlands. This area is of particular interest because,
from the very few samples that have been received, there appears to be a low incidence of
anticoagulant resistance among Norway rats. Consequently, calls were put out in the magazines
serving the UK professional pest control community asking for samples from these areas (see for
example Jones and Talavera, 2019; https://www.thinkwildlife.org/free-tests-and-new-guide-
tackle-spread-of-resistant-rats/).
2.2 Methods of DNA analysis
As in the previous studies described above, genetical material was obtained from the field
in the form of either tail tip samples or fresh droppings. Where possible, samples were placed in
tubes containing 80% alcohol and then stored at -20°C as quickly as possible. Some unfrozen
samples were shipped to the laboratory using a courier service, surface mail or by hand delivery,
and were frozen on receipt.
Genomic DNA was extracted using the Qiagen DNeasy tissue extraction kit following the
manufacturer’s recommendations (Qiagen Ltd., Crawley, West Sussex, UK). Briefly, a small
quantity of tissue (approximately 3mm x 2mm x 2mm) was shaved from each tail using a sterile
sharp razor blade, and then placed in a 1.5ml microtube. Pre-warmed extraction buffer ATL (180
µl) was added, followed by 20 µl of proteinase K. The mixture was vortexed and incubated at 55
˚C on a rocking platform overnight (approx. 17 h). Genomic DNA was then purified and eluted
from spin-purification columns in 80 µl of elution buffer and the quality and yield were assessed
spectrophotometrically using a nano-drop instrument.
The three exons of the VKORC1 gene, designated 1, 2 and 3, were amplified by PCR following
the methodology of Rost et al. (2004). PCR products were purified using the QIAquick PCR
purification kit (Qiagen Ltd., Crawley, West Sussex, UK). Product samples (3.5µl) were then
sequenced with BigDye version 3.1 terminator chemistry (ABI) on a 9700 ABI thermal cycler,
and the terminated products were resolved on an ABI 3130XL capillary sequencer. The sequence
trace files were visually analysed and any ambiguous bases were edited using the DNASTAR
Lasergene software. The sequence alignments were compiled using ClustalW2.
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© 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.
Table 1. 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.
Species
Mutation
Abbreviations
Where present
NR
Leucine128Glutamine
L128Q†
Central Southern Scotland, Yorkshire,
Lancashire
NR
Tyrosine139Serine
Y139S†
Anglo-Welsh border
NR
Leucine120Glutamine
L120Q†
Hampshire, Berkshire, Essex, Norfolk
and elsewhere
NR
Tyrosine139Cysteine
Y139C†
Gloucestershire, Norfolk, Lincolnshire,
Yorkshire, SW Scotland and elsewhere
NR
Tyrosine139Phenylalanine
Y139F†
Kent, Sussex, Norfolk, Suffolk
NR
Argenine33Proline
R33P‡
Nottinghamshire
NR
Phenylalanin63Cysteine
F63C*
Cambridge/Essex
NR
Tyrosine39Asparagine
Y39N*
Cambridge/Essex
NR
Alanine26Threonine
A26T#
Cambridge/Essex
HM
Tyrosine139Cysteine
Y139C†
Reading
HM
Leucine128Serine
L128S†
Cambridge
† Known either from field experiments and/or field experience to have a significant practical effect on
anticoagulant efficacy
‡ Known from laboratory experiments to confer warfarin resistance
* Shown in laboratory experiments to have a significant impact on protein function
# Unlikely to confer any significant degree of resistance
2.3 The Rodenticide Resistance Action Committee (RRAC) interactive global resistance
map
The results from this study were provided to the funding body, the Brussels-based RRAC
of CropLife International (http://www.rrac.info/). The results are collated with those obtained
from other global studies and presented in an interactive form on the RRAC web-site. The maps
available (see example for the UK at: http://guide.rrac.info/resistance-maps/united-kingdom/) use
Google ‘heatmap’ technology to ascribe different weightings to records depending on the
numbers of positive samples and the frequencies of their closest neighbours. Users of the maps
are able to scroll in to find their own location, that of the nearest confirmed incidence of
anticoagulant resistance, the mutation of that record and to obtain advice about the correct use of
anticoagulants in the area. It is anticipated that this scheme will help pest control practitioners to
make informed choices about which anticoagulant active substance to use and will support a
‘competent workforce’.
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© 2019 Campaign for Responsible Rodenticide Use UK
3. Results
3.1 Norway rats
During the period September 2018 and September 2019 a total of 140 Norway rat tissue
samples was received that were capable of analysis using the gene sequencing technique. Among
these, 85 were found to possess one of the five known Norway rat resistance mutations and 55
were found to be susceptible animals (Table 2). Hence, 60.7% of the samples received possessed
one of the resistance mutations, in either their homozygous or heterozygous form.
Table 2. The numbers of Norway rats tissue samples received and analysed and their status of
resistance or susceptibility. (See Table 1 for further explanations of the different resistance
mutations.)
Resistance Mutation
Homozygous
Heterozygous
Total
L120Q
16
9
25
L128Q
12
15
27
Y139S
3
8
11
Y139F
3
5
8
Y139C
5
9
14
Susceptible
55
0
55
Total
94
46
140
The geographical origins of these new samples are shown in Figure 1. The discovery of several
new resistance foci are revealed when a comparison is made of these finding and those published
in the previous report (Prescott et al., 2018). Of course, it is impossible to determine whether
these are newly-developed resistance foci or have been present for some time but had long been
undetected.
One of the most interesting findings was the discovery of the Y139F mutation in rats taken in
Central London. This mutation had previously been found only in Kent, East Sussex and East
Anglia, with an outlier in north-west Shropshire. Rats from Greater London were previously
found to be susceptible or to possess the L120Q or Y139C mutations.
In the north of England, rats possessing the L128Q mutation were found for the first time on the
north-east coast in the counties of Durham, Northumberland and Tyneside. The first survey of
rats from Greater Manchester revealed a complicated resistance picture. The dominant mutation
was once again, the L128Q genotype. This was found mostly in its homozygous form, which
suggests that the focus had been established for some time. However, some rats from the locality
carried the L120Q and Y139C mutations, both in their heterozygous form. No susceptible rats
were found in the samples received from Greater Manchester, which totalled 20 rat tissue
samples. This presents a complex picture of Norway rat resistance in this very large conurbation
but one in which anticoagulant resistance must be considered with more attention.
Elsewhere, the Y139S mutation that previously had been found almost exclusively on the Anglo-
Welsh border was found in three new locations. A rat from North Yorkshire was found to be
homozygous resistant for Y139S, and heterozygous Y139S rats were also found in Merseyside
and in Essex.
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© 2019 Campaign for Responsible Rodenticide Use UK
Figure 1. Map showing the geographical locations of Norway rat tissue samples submitted to the
Vertebrate Pests Unit in the period September 2018 to September 2019 and their resistance status.
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© 2019 Campaign for Responsible Rodenticide Use UK
Figure 2. Map showing all available data on the occurrence of resistance mutations among
Norway rats in the UK.
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© 2019 Campaign for Responsible Rodenticide Use UK
A new focus of Y139C resistance was demonstrated in North Yorkshire, within the same
sampling area that contained the homozygous Y139S rat that was mentioned previously.
Although this is the first record of Y139C for this county, it had previously been recorded in the
neighbouring counties of West, South and East Yorkshire and probably confirms a large
contiguous focus. The Y139C mutation was also found for the first time in Worcestershire and
this suggests a possible large focus associated with the valley of the River Severn, with Y139C
rats having been found previously in north Somerset, Gloucestershire and Shropshire. Y139C is
an advanced form of resistance and one against which the UK Rodenticide Resistance Action
Group (RRAG) recommends that only brodifacoum, difethialone or flocoumafen should be used
(RRAG, 2018).
Finally, the dimensions of the very large focus of the severe L120Q resistance, which covers
much of the south of England, was extended into a new county, namely Devon. A heterozygous
resistant rat carrying this resistance gene was found in the extreme east of the county near the
town of Holsworthy. Previously, the furthest west this mutation had been found was in central
Somerset near Taunton.
A special effort was made this year to obtain samples from some of the Midland counties that had
previously been unrecorded. This was not especially successful because only four samples were
received from those locations but these efforts will be continued. However, those limited rat
tissue samples received from south-west Staffordshire, the West Midlands, Leicestershire and
Northamptonshire were all found to have come from susceptible Norway rats.
The map shown in Figure 2 gives all accumulated data on the distribution of anticoagulant
resistance for Norway rats in the UK and includes the 2019 data.
3.2 House mice
The results from the analysis of a total of 35 house mouse tissue samples submitted in the
period September 2018 to September 2019 are shown in Table 3. Among 35 samples examined,
none carried the fully susceptible genotype. Table 1 shows that one or other of the two resistance
mutations commonly found among house mice in the UK were present in all animals. Either
Y139C or L128S was found in homozygous form in 21 animals and in heterozygous form in a
further 11 animals, while three individuals carried both mutations each heterozygous.
Table 3. The numbers of house mouse tissue samples received and analysed and their status of
resistance or susceptibility. (See Table 1 for further explanations of the different resistance
mutations.)
Mutation
Homozygous
Heterozygous
Total
L128S
14
6
20
Y138C
7
5
12
L128S and Y139C
0
3*
3
Susceptible
0
0
0
Total samples
21
14
35
*These three animals were heterozygous for each of two the
resistance mutations.
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© 2019 Campaign for Responsible Rodenticide Use UK
The geographical distribution of the 35 samples analysed during September 2018 to September
2019 and reported here is shown in Figure 3. The combined data for all years is shown in Figure
4. Resistance distribution data for house mice recorded in the previous reports (Prescott et al.,
2017 and 2018) were mainly from Greater London and the south-east of England. The samples
now reported were, for the first time, much more widely dispersed and demonstrate conclusively
for the first time the extent of anticoagulant resistance in UK house mice.
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© 2019 Campaign for Responsible Rodenticide Use UK
Figure 3. Map showing the geographical locations of house mouse tissue samples submitted to
the Vertebrate Pests Unit in the period September 2018 to September 2019 and their resistance
status.
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© 2019 Campaign for Responsible Rodenticide Use UK
Figure 4. Map showing all available data on the occurrence of resistance mutations among house
mice in the UK.
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© 2019 Campaign for Responsible Rodenticide Use UK
The L128S mutation appears to be very widely distributed across much of England, from
Tyneside in the north-east to the Channel coast of East and West Sussex, whereas the Y139C
mutation seems to be somewhat more restricted in distribution to the southern and eastern
counties. However, we still lack data for the house mouse and many of the records are for either
single animals or very small samples.
Earlier reports provided information on a total of 53 house mouse samples and these are now
augmented by a further 35 samples. Among the previous 53 a total of 47 (88.7%) carried one or
more resistance mutations. With the addition of the 35 samples reported here, all resistant, the
prevalence of resistance in UK house mice is now 82 resistant samples out of a total of 88, or
93.2%.
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© 2019 Campaign for Responsible Rodenticide Use UK
4. Discussion
This report is the third in a series compiled for CRRU UK by the University of Reading
to document the distribution of resistance to anticoagulants among Norway rats (Rattus
norvegicus) and house mice (Mus musculus) in the UK. When new resistance foci are discovered
it is impossible to know if these have only just developed or if they have in fact existed in the
recorded localities for some time. This is because no consistent and wide-scale inventory of
resistance foci has ever been attempted using the modern DNA sequencing technique (se Pelz and
Prescott, 2015). Instead, samples are received on an ad hoc basis, mainly from technicians in the
professional pest control industry who have experienced difficulty in achieving control using their
customary methods and products. This scheme for the acquisition of samples, of course, causes
bias because, if the cause of the difficulty is indeed resistance, it increases the likelihood that
resistance mutations will be found.
With this important proviso, it is possible to draw some conclusions with a fair degree of
certainty from the data that has been collected over the past three years and presented here.
Firstly, anticoagulant resistance in Norway rats is predominant and widespread across the whole
of southern England, either in the form of the highly resistant L120Q genotype or, further to the
east, as the only slightly less resistant Y139F genotype. With these resistances dominant over
such a large area, and an area in which so much of the country’s commercial and agricultural
activity occurs, it is hardly surprising that these resistance mutations are spreading. This appears
to be happening, firstly, on the boundaries of the main focus, hence the finding of L120Q in the
far west in Devon and, in the far east, on the borders of Surrey and West Sussex with Kent and
East Sussex. A similar ‘spread’ phenomenon may be found in the discovery of rats carrying the
Y139F mutation in central London, which is on the western boundary of the known focus of that
mutation.
However, new L120Q Norway rat resistance foci now appear in places often far removed from
the original heartland’ of Hampshire and Berkshire, such as in East Anglia and West Yorkshire
and it is hard to conceive that the true extent of the focus is actually contiguous with these
outliers. Rather it seems likely that new foci have either developed de novo or resistant rats have
been transported from the main focus and have flourished in new localities. Rats carrying Y139F
are also found far from the original Kent focus in the north of East Anglia and the far west of
Shropshire.
Elsewhere, the data reported here support the likely existence of other large, but previously
unknown, resistance foci. For example, we now have Y139C resistant rat samples from all of the
counties along the Severn Valley, from Somerset in the south to the north western-most edge of
Shropshire and this probably indicates a single focus, rather than several small foci. Similarly,
there appears to be a substantial Yorkshire Y139C focus, since this resistance has been found in
all the Yorkshire sub-counties. Figure 2 appears to show an association between the rivers
Severn, Thames, Humber, Mersey and Dee with this resistance mutation, which is the only one to
be found in both Germany and Denmark (Pelz and Prescott, 2015). But the observations might as
easily be explained by the courses of the motorways M2, M4, M5 and M62.
There remains a central core of the country, including most of the Midland counties, from which
we have very few Norway rat samples but in which we have only detected susceptible animals.
Why the centre of the country should retain anticoagulant susceptibility, while almost completely
surrounded by resistance foci, is a question that may only be answered by more detailed genetical
studies.
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© 2019 Campaign for Responsible Rodenticide Use UK
The purpose of these resistance studies is to provide information that will permit professional pest
control technicians to make informed decisions about their choice of rodenticide active
substances. However its effect in documenting the increasingly extensive occurrence in the UK
of highly anticoagulant-resistant Norway rats is likely to be that there is a shift towards the use of
the three most potent ‘resistance-breaking’ compounds, brodifacoum, difethialone and
flocoumafen. The fact that the latter two active substances remain proprietary to single
commercial entities, while brodifacoum is much more widely available, makes it likely that this
latter active substance that will be the one in predominant use in the UK’s growing Norway rat
resistance foci.
So far this discussion has been restricted to the situation regarding Norway rats. The extent of
resistance in house mouse seems to be even more pervasive. None of the 35 samples collected
during the period of the current study (September 2018 to September 2019) carried any
susceptible genetic material. If these are added to the samples previously reported (Prescott et al.,
2017 and 2018) we arrive at a prevalence of resistance among UK house mice of 93.2%.
This observation draws attention to a regulatory anomaly. The UK Rodenticide Resistance
Action Group published guidance on the use of anticoagulant rodenticides to permit effective
rodent pest management and the prevention of the spread of resistance (RRAG, 2012 and 2018).
In its guidance on the control of house mice with anticoagulants (RRAG, 2012), RRAG
recommends that bromadiolone and difenacoum should not be used against house mice because
of the occurrence of resistance to them. The predominant method for the management of house
mice in all commercial and (especially) in food storage/preparation/sale premises is the
deployment of permanent tamper-resistant mouse bait boxes containing anticoagulant baits. This
use is fundamental to the protection of human health and hygiene. However, we draw attention to
the new rules on permanent baiting, embodied in current product labels, which only permit the
resisted bromadiolone and difenacoum to be used in permanent baiting programmes (CRRU,
2019). This situation requires immediate attention. I t seems particularly unfortunate that we
have just emerged from the virtual prolonged ban on the use of effective resistance-breaking
anticoagulants against Norway rats, which has undoubtedly contributed to the massive spread of
resistant Norway rats in the UK, and we now find ourselves in a similarly contrary regulatory
position with House mice.
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© 2019 Campaign for Responsible Rodenticide Use UK
5. References
Clarke, D. and C. Prescott. (2015). Investigation of the current status of anticoagulant resistance in
UK Norway rats by VKORC1 genotyping. Summary of results – February 2015. University of
Huddersfield, University of Reading. Confidential report. 22 pp.
CRRU UK. (2019). CRRU UK Guidance Permanent Baiting. Revised August 2019. The
Campaign for Responsible Rodenticide Use (CRRU) UK. 11 pp.
HSE (2019). Report on the Rodenticides Stewardship Regime - Assessment of Implementation.
Rodenticides Stewardship Government Oversight Group. Health and Safety Directorate,
Redgrave Court, Merton Road, Bootle, Merseyside. January 2019. 9 pp. Available from:
http://www.hse.gov.uk/biocides/eu-bpr/rodenticides.htm. Date accessed: 30.09.19.
Jones, C. and Talavera, M. (2019). Rodenticide resistance: Will you help find the gaps? PEST
Magazine, Issue August/September 2019, pp 10-11, Available at:
http://www.pestmagazine.co.uk/. Date accessed: 01.10.19.
Pelz, H.-J., S. Rost, M. Hűnerburg, A. Fregin, A-C. Heiberg, K. Baert, A. MacNicoll, C. Prescott,
A-S. Walker, J. Oldenberg and C. Muller. (2005). The genetic basis of resistance to
anticoagulants in rodents. Genetics 170 (4): 1839-1847.
Pelz, H.-J., and C. V. Prescott. (2015). Chapter 9. Resistance to Anticoagulants. Pp 187-208. In:
Rodent Pests and their Control. A. P. Buckle and R. H. Smith (eds.). CAB International,
Wallingford, Oxon, UK. 422 pp.
Prescott, C. V., A. P. Buckle, J. G. Gibbings, E. N. W. Allan and A. M. Stewart. (2010).
Anticoagulant resistance in Norway rats (Rattus norvegicus Berk.) in Kent – a VKORC1 single
nucleotide polymorphism, tyrosine139phenylalanine, new to the UK. International Journal of
Pest Management 57(1): 61-65.
Prescott, C., Baxter, M., Coan, E., Jones, C., Rymer, D. and Buckle, A. (2017). Anticoagulant
Resistance in Rats and Mice in the UK – Current Status in 2017. Report from the Campaign for
Responsible Rodenticide Use (CRRU) UK for the Government Oversight Group. Vertebrate
Pests Unit, University of Reading, UK. 30 pp. Available at:
http://www.thinkwildlife.org/downloads_resources/. Date accessed: 30.09.19.
Prescott, C., Coan, E., Jones, C., Rymer, D. and Buckle, A. (2018). Anticoagulant Resistance in
Rats and Mice in the UK – Current Status in 2018. Report from the Campaign for Responsible
Rodenticide Use (CRRU) UK for the Government Oversight Group. Vertebrate Pests Unit,
University of Reading, UK. 35 pp. Available at:
http://www.thinkwildlife.org/downloads_resources/. Date accessed: 30.09.19.
RRAG. (2012). RRAG House Mouse Resistance Guideline. Rodenticide Resistance Action Group
of the UK. 15 August 2012. 15 pp.
RRAG. (2018). Anticoagulant resistance in the Norway rat and Guidelines for the management of
resistant rat infestations in the UK. Rodenticide Resistance Action Group of the UK. Revised
September 2018. 8 pp.
Rost, S., H.-J. Pelz, S. Menzel, A. MacNicoll, V. León, K-J. Song. T. Jäkel, J. Oldenburg and C.
Műller. (2009). Novel mutations in the VKORC1 gene of wild rats and mice - a response to 50
years of selection pressure by warfarin. BMC Genetics 10(4): 9.
RRAG (2012). RRAG House Mouse Resistance Guideline. Rodenticide Resistance Action Group,
UK. Derby. 11pp.
RRAG (2018). Anticoagulant resistance in the Norway rat and Guidelines for the management of
resistant rat infestations in the UK. Rodenticide Resistance Action Group, UK. Derby. 9pp.
