Distribution of Borrelia burgdorferi sensu lato in Ixodes ricinus Populations Across Central Britain

Abstract

Lyme borreliosis is rapidly emerging in the United Kingdom, with over 1000 cases per annum now reported. Lyme borreliosis is caused by the Borrelia burgdorferi sensu lato (s.l.) group of spirochetes, which are transmitted by ixodid ticks. In the United Kingdom, Ixodes ricinus is recognized as the principal vector of the spirochetes, and this tick species is widely distributed across the country. However, as yet, it is unclear whether the distribution of B. burgdorferi essentially mirrors that of its vector, or if there are marked differences between the 2. The aim of this survey was to investigate the prevalence of B. burgdorferi in I. ricinus populations across northern England, north Wales, and the Scottish Border region. We surveyed for questing I. ricinus nymphs and adults at 17 sites, encountering ticks at 12. At 8 sites, large numbers (>160) ticks were collected, and at 3 of these sites B. burgdorferi infection prevalence was significantly higher (>7.5%) than the other 5 (<1.0%). Habitat type was associated with B. burgdorferi prevalence, with ticks from deciduous and mixed woodland being significantly more likely to be infected than those from other habitat types. Identification of infecting Borrelia species indicated that Borrelia valaisiana was the most common and widespread species encountered. B. garinii was common at sites where infection prevalence was high, and B. afzelii was also occasionally encountered at these sites. The survey revealed a surprisingly uneven distribution of B. burgdorferi s.l. across the region, thereby indicating that the presence of ticks does not necessarily mean the presence of the pathogen. Indeed, the spirochete appears to be absent or very rare at some sites where ticks are abundant.

Introduction

T he hard tick, Ixodes ricinus, is a vector for many of the arthropod-borne diseases of humans and livestock in temperate zones of the Northern Hemisphere. Lyme borreliosis, the most common tick-borne disease in Europe and North America, is caused by spirochetes belonging to the Borrelia burgdorferi sensu lato (s.l.) complex. Within this complex, numerous species have been delineated and, of these, Borrelia afzelii, Borrelia bissettii, Borrelia burgdorferi sensu strictu (s.s.). Borrelia garinii, Borrelia lusitaniae, and Borrelia valaisiana have been most frequently encountered in Europe. Many surveys of B. burgdorferi s.l. in European I. ricinus populations have now been reported (e.g., Kirstein et al. 1997, Rauter and Hartung 2005, Halos et al. 2010, Kjelland et al. 2010, Nazzi et al. 2010, Reye et al. 2010), and these have demonstrated not only that the overall prevalence of infection can significantly differ between survey sites, but that the relative contribution each complex member makes to the local I. ricinus–associated B. burgdorferi community varies markedly between sites. The reasons for this variation are not fully understood, but are undoubtedly, in part, a reflection of the local vertebrate host fauna. This fauna influences not only I. ricinus population dynamics but is also a determinant of the B. burgdorferi s.l. complex composition, because different members of this complex appear to have adapted to exploit different vertebrates as reservoir hosts; B. garinii and B. valaisiana are associated with birds whereas B. afzelii is associated with small mammals (Kurtenbach et al. 2002, Hanincová et al. 2003a,b). Conversely, B. burgdorferi s.s., in North America at least is far more of a generalist, exploiting a wide range of hosts (Ginsberg et al. 2005, Hanincová et al. 2006).

Lyme borreliosis is an emerging disease in the United Kingdom. Over the past decade, the number of cases being diagnosed each year has increased 10-fold, such that in 2010, over 1000 serologically confirmed infections were reported (Health Protection Agency 2010, Health Protection Scotland 2010). In addition, it has recently been estimated that as many as 2000 cases per year may go unconfirmed (British Infection Association 2011). Why Lyme borreliosis should have emerged so dramatically in the United Kingdom over the past decade or so is unclear. Improvements in medical awareness, diagnostics, and reporting are likely contributory factors, but they may also reflect an increased natural presence of B. burgdorferi. Although some efforts have been made to establish the national distribution of I. ricinus, and how this is changing (Kirby et al. 2004, Pietzsch et al. 2005, Scharlemann et al. 2008, Gray et al. 2009, Smith et al. 2011), there have been, to date, only a handful of surveys that have attempted to quantify the presence of B. burgdorferi in UK I. ricinus populations (Livesley et al. 1994, Kirstein et al. 1997, Kurtenbach et al. 1998, Davidson et al. 1999, Ling et al. 2000, Vollmer et al. 2011), hence knowledge of the distribution of the pathogen is primarily derived from Lyme borreliosis patient histories (Smith et al. 2000; Health Protection Agency 2010). These data suggest that there are apparent “Lyme borreliosis hot spots” in the United Kingdom (Smith et al. 2000), but, because the natural presence of B. burgdorferi within hot spots is apparently no greater than elsewhere, the higher contact rates between infected ticks and humans result from a higher frequency of human visits to these sites (Dobson et al. 2011). One other possible determinant of “hot spots” may be the composition of the “guild” of Borrelia species present. Some species are far more frequent zoonotic agents than others; thus, the medical significance of B. afzelii, B. burgdorferi s.s., and B garinii is well established, whereas the importance of B. valaisiana, B. lusitaniae, and B. bissettii remains uncertain (Ruzic-Sabljic et al. 2008).

We investigated the distribution of borreliae-infected ticks across central Britain. We chose this region because previous studies have focused on southern England or the Scottish Highlands and, anecdotal evidence suggests that central Britain contains areas where ticks are known to be abundant, but from which few cases of Lyme borreliosis have been reported.

Materials and Methods

Sample collection

Potential survey sites were first identified using satellite imagery (Google Earth) to locate forest/woodland areas across central Britain. The suitability of these sites, largely in terms of the presence of ticks and/or deer, and accessibility, was assessed through contact with, primarily, Forestry Commission rangers. Because surveying all sites deemed suitable was beyond our capacity, we selected 17 that were geographically dispersed across the study area (Fig. 1). Survey of these sites was carried out on warm (14–27°C), rain-free days between May and July, 2010, and most sites were visited only once. Questing I. ricinus ticks were collected by blanket dragging, with nymphs and adults being removed into 70% ethanol. Each drag was, typically, 10–15 meters long, although we did not attempt to standardize dragging because we were not attempting to quantify tick populations. Dragging was repeated until we estimated that at least 200 nymphs had been collected. At some sites, no or very low numbers of ticks were encountered. These sites were surveyed for a minimum of 90 min by at least 3 people, and we estimate that an area of ground in excess of 1000 square meters was dragged at these sites.

FIG. 1.

Map of central Britain showing locations of sites used in this study. Bb, Borrelia burgdorferi; CB, Coed y Brenin; CR, Crowden; DF, Dalby Forest; GI, Gilsland; GF, Gisburn Forest; GR, Graig-fechan; HW, Hampsfell Woods; HF, Hamsterley Forest; HA, Harwood; HI, Hiraethog; KF, Kielder Forest; LV, Lake Vyrnwy; LD, Loch Doon; MA, Mabie Forest; MC, Macclesfield Forest; MF, Mell Fell; NF, Naddle Forest.

DNA extraction, B. burgdorferi detection, and Borrelia species delineation

DNA extraction was performed in a dedicated laboratory located apart from those in which PCR products were handled. Each I. ricinus adult or nymph was placed in an individual 1.7-mL Eppendorf tube, and a crude DNA extract was prepared from it using alkaline lysis as previously described (Guy and Stanek 1991). One “blank” (no tick) sample was co-processed with every 5 ticks. Extracts were individually tested for the presence of Borrelia species by real-time PCR amplification of a fragment of the 23S ribosomal RNA encoding gene, as previously described (Courtney et al. 2004). For nymphal ticks only, reverse line blotting (RLB) (Alekseev et al. 2001; Poupon et al. 2006) was used to determine from which species within the B. burgdorferi s.l. complex all real-time PCR products were obtained. In addition, the identity of the Borrelia species in a random subset of extracts from nymphal ticks was also tested by comparative analysis of 5S–23S rDNA intergenic spacer region sequences, following amplification of this region as previously described (Rijpkema et al. 1995).

Statistical analysis

To investigate the potential effects of habitat type (as a proxy for host community structure) and tick developmental stage (because adult ticks have been previously been reported to be infected more commonly than nymphs; e.g., Rauter and Hartung 2005) on the probability of a tick being infected with B. burgdorferi s.l., we used generalized linear mixed models (GLMMs) with a binomial error term and a logit link. Maximal models had individual tick infection status as the response variable, with tick developmental stage and habitat type as fixed effects. Site was included as a random effect to account for possible pseudoreplication associated with information for each habitat being based on a number of different sites. Model selection was based on backward stepwise model selection with only variables significant at the p<0.05 level being retained in the final model. Analyses were undertaken using R 2.14 (http://www.r-project.org/).

Results

I. ricinus nymphs and adults were found at 12 of the 17 sites visited (Table 1). The 5 sites where ticks were not found were geographically dispersed (Fig. 1) and were of differing habitat categories. Four sites yielded only between 8 and 71 ticks, but at the remaining 8 sites at least 173 ticks were collected.

Table 1.

Location of Sites Used in this Study and Details of I. ricinus Collections and B. burgdorferi s.l. Infections in Collected Ticks

			I. ricinus collected		Infected ticks
Site	Ordnance survey grid ref.	Habitat ^a	Adults	Nymphs	Adults	Nymphs (% prevalence: exact binomial 95% confidence intervals)
Coed y Brenin, Gwynedd	SH7227	Mixed woodland	2	40	0	0 (0: 0–8.81)
Crowden, Derbyshire	SK0799	Moorland	0	0
Dalby Forest, North Yorkshire	SE8587	Mixed woodland	20	237	2	19 (8.02: 4.89–12.24)
Gilsland, Northumbria	NY6768	Conifer plantation/grassland	16	55	0	0 (0: 0–6.49)
Gisburn Forest, Lancashire	SD7457	Conifer plantation/grassland	14	253	2	2 (0.79: 0.09–2.83)
Graig-fechan, Clwyd	SJ1554	Moorland	22	212	0	2 (0.94: 0.11–3.37)
Hampsfell Woods, Cumbria	SD4078	Deciduous woodland	29	245	6	19 (7.75: 4.73–11.85)
Hamsterley Forest, County Durham	NZ0931	Mixed woodland	0	0
Harwood, Northumberland	NY9994	Conifer plantation/grassland	6	167	0	0 (0: 0–2.18)
Hiraethog, Clwyd	SJ0351	Mixed woodland	0	0
Kielder Forest, Northumbria	NY6096	Conifer plantation/grassland	100	185	0	1 (0.54: 0–2.97)
Lake Vyrnwy, Powys	SH9524	Moorland	0	0
Loch Doon, South Ayrshire	NX4794	Conifer plantation/grassland	0	8	nt^b	nt
Mabie Forest, Dumfriesshire	NX9371	Deciduous woodland	18	232	0	19 (8.19: 5.0–12.49)
Macclesfield Forest, Cheshire	SJ9670	Deciduous woodland	0	0
Mell Fell, Cumbria	NY4024	Deciduous woodland	2	28	0	1 (3.57: 0.09–18.35)
Naddle Forest, Cumbria	NY4914	Moorland	72	249	0	0 (0: 0–1.47)

Habitat definitions: Deciduous woodland, >80% of trees deciduous; conifer plantation, >80% trees conifers; mixed woodland, woodland in which conifers/deciduous each contribute 20–80% of trees; moorland, generally treeless, heather, bracken, vegetation typically <50 cm high; grassland, generally treeless, juncus grass, hair grass, vegetation typically >50 cm high.

Not tested.

All ticks, except those collected at Loch Doon, were tested for the presence of B. burgdorferi DNA. Ticks from Loch Doon were not tested because too few were collected to yield useful data. Of the 2204 ticks (nymphs and adults) tested, 73 yielded an amplicon, giving an overall prevalence of infection of 3.31% (95% confidence interval [CI] 2.60–4.15). Of the 1903 nymphs tested, 63 (3.31%, 95% CI 2.55–4.22) yielded an amplicon. No evidence of B. burgdorferi infection was found at four sites (Coed y Brenin, Gilsland, Harwood, and Naddle Forest). The prevalence of infection in nymphs at the other sites varied between 0.54% (95% CI 0–2.97) and 8.19% (95% CI 5.0–12.49) (Table 1).

GLMM analyses indicated that habitat type, but not tick developmental stage, significantly influenced the probability of a tick being infected with B. burgdorferi (Table 2). Relative to those collected from deciduous forest, ticks from moorland were significantly less likely to be infected with B. burgdorferi (odds ratio [OR]=0.048, 95% CI 0.012–0.2). That is, the odds of a tick being infected in deciduous woodland are approximately 20.64 (i.e., 1/0.048) times greater than they are in moorland. Similarly, ticks from conifer plantation/grassland habitats were also significantly less likely to be infected than those from deciduous woodland (OR=0.07, 95% CI 0.028–0.18). No significant difference was found between ticks collected from deciduous and mixed woodland (OR for mixed woodland=−0.16, 95% CI 0.30–1.23).

Table 2.

Parameter Estimates (Logit Scale) and Standard Errors for the Model of Infection with B. burgdorferi in Ticks (Deciduous Woodland Is the Reference Habitat Type)

Parameter	Coefficient (standard error)	z value	Probability>z
Intercept	−2.43 (0.16)	−15.59	<0.001
Mixed woodland	−0.16 (0.28)	−0.57	0.57
Moorland	−3.03 (0.73)	−4.17	<0.001
Plantation/grassland	−2.64 (0.48)	−5.56	<0.001

The identities of Borrelia species present in 72 of the 73 nymphs that were found to contain B. burgdorferi s.l. DNA were successfully determined using RLB and comparative sequence analysis. Seventy-one of the 73 nymphs were tested using RLB (for 2 nymphs, insufficient DNA was available), with infecting Borrelia species being identified in 70 (DNA from 1 nymph reacted only with the B. burgdorferi s.l. probe). Sequence data for a single Borrelia species was obtained from 12 of the 15 nymphs tested (for 3, chromatographic data were of insufficient quality to be trustworthy). For 10 nymphs, the identities of infecting Borrelia species were determined by both comparative sequence analysis and RLB; 9 produced identical results, whereas for the 10^th nymph, comparative sequence analysis indicated a B. garinii infection and RLB indicated a mixed infection of B. garinii and B. valaisiana. In total, 41 nymphs (58%) contained B. valaisiana DNA and 24 nymphs (33%) contained B. garinii DNA. Two nymphs (3%) contained B. afzelii DNA. The remaining 5 nymphs contained DNA from both B. valaisiana and B. garinii (Fig. 2).

FIG. 2.

Identity of Borrelia species contributing to spirochete communities in infected I. ricnus ticks collected from seven sites during this study. DF, Dalby Forest; GF, Gisburn Forest; GR, Graig-fechan; HW, Hampsfell Woods; KF, Kielder Forest; MA, Mabie Forest; MF, Mell Fell. For 1 tick at MA, the RLB indicated the presence of a B. burgdorferi s.l. member but was unable to delineate which species within the complex to which this organism belonged. In several ticks, mixed infections of B. garinii and B. valasisiana were detected.

B. valaisiana was encountered at all 7 of the sites where B. burgdorferi s.l.–infected nymphs were found. B. garinii and mixed infections were found at the 3 deciduous forest sites with the highest overall prevalence of B. burgdorferi s.l. infection. B. afzelii was also found at 2 of these sites (Fig. 2).

Discussion

The generally accepted view of B. burgdorferi distribution in Europe is that the bacteria are present more or less wherever there are I. ricinus ticks (Dobson et al. 2011). The evidence to support this assumption is that only very rarely have surveys of I. ricinus failed to yield any evidence of infection; indeed, in a recent systematic review of the prevalence of B. burgdorferi in I. ricinus in Europe, only 1 of 110 studies from 24 countries reported such a failure (Rauter and Hartung 2005). This study was carried out in coastal deciduous woodland in northern Italy and reported the quantification of I. ricinus seasonal dynamics but repeated, unsuccessful attempts to detect infection by and/or exposure to B. burgdorferi in (>250) questing ticks and vertebrates frequenting the woodland (Mannelli et al. 1999). No other study included in the systematic review reported a B. burgdorferi infection prevalence of less than 1.5%, and only 5 reported a prevalence of less than 3% (Rauter and Hartung 2005). Thus, our finding that B. burgdorferi infections appear to be absent from, or present at extremely low prevalences at, numerous sites in central Britain is noteworthy. Five of the sites we surveyed appeared to support a high density of ticks in which B. burgdorferi s.l. infection prevalence ranged between 0% and 1.5%. These sites were geographically distinct, lying in north Wales, Lancashire, eastern Cumbria, and Northumberland, and were interspersed with sites that apparently supported high tick densities in which B. burgdorferi infection prevalence was significantly higher and akin to prevalence values commonly reported at sites in southern England (Vollmer et al. 2011).

Our findings point to a patchy distribution for B. burgdorferi in central British I. ricinus populations and are thus inconsistent with the impression created by previously reported studies carried out elsewhere in the United Kingdom, in which B. burgdorferi is almost always found (Livesley et al. 1994, Kurtenbach et al. 1998, Davidson et al. 1999, Ling et al. 2000). However, there is at least 1 exception; Vollmer and colleagues (2011) reported an absence of B. burgdorferi infections in 82 nymphs surveyed at Rhossili Down in South Wales. Furthermore, the same authors reported infection in only 1 of 71 nymphs collected in Richmond Park, Surrey (Vollmer et al. 2011), although others have reported higher infection prevalence values in this park (Dobson et al. 2011). Very recent work from elsewhere in Europe has also indicated that the distribution of B. burgdorferi in I. ricinus populations may be more patchy than previously recognized. Gassner and colleagues (2011) surveyed I. ricinus populations at 24 sites across The Netherlands, but found B. burgdorferi infections at only 16. The authors suggested that an absence of evidence for B. burgdorferi at these sites may have been due to too few ticks being tested, but did not specify how many ticks had been examined from these sites. However, the study did present data indicating an absence of B. burgdorferi infections at sites supporting up to 50 I. ricinus nymphs per 100 square meters (Gassner et al. 2011).

Given the cross-sectional nature of our study, we cannot rule out that the observed absence of ticks and/or B. burgdorferi (at some of our tick-infested sites) was temporary. Previously reported longitudinal studies have observed annual fluctuations in the prevalence of B. burgdorferi infections in I. ricinus populations at specific sites (Mejlon and Jaenson 1993, Wielinga et al. 2006), and these fluctuations were often dramatic enough that interannual B. burgdorferi prevalence values were significantly different from one another, even when relatively few ticks were sampled. Although we can find no record of an apparent temporary absence of infections in I. ricinus populations that otherwise maintained B. burgdorferi, a 4-year longitudinal study of Ixodes pacificus populations in the western United States was unable to detect B. burgdorferi at some sites during the first year of survey, but subsequently consistently detected the spirochete at those sites (Eisen et al. 2004). Clearly, repeated sampling at our sites would address this shortfall, although sporadic attempts to detect B. burgdorferi in I. ricinus ticks collected in Kielder Forest over the past 8 years have never succeeded (K.B., unpublished observations). Furthermore, discussion with a local general practice suggested that, although practitioners had attended Lyme disease patients, they had no record of locally acquired cases (R.B., personal communication).

We can only speculate why we observed such patchiness, but our data suggest that habitat type influences the prevalence of B. burgdorferi in questing ticks. Infection prevalence in ticks collected from deciduous and mixed woodland sites was significantly higher than at other sites where conifer plantation, grassland, or moorland was the dominant habitat. The effect of habitat on prevalence of B. burgdorferi has previously been reported in Europe and the United States (Wielinga et al. 2006, Brownstein et al. 2005, Halos et al. 2010). This is likely to be a reflection of the different host community structure found in different habitats. Interestingly, 1 of the sites where we did not detect any B. burgdorferi was Naddle Forest in eastern Cumbria, a location where B. burgdorferi has previously been encountered (Ogden et al. 1997). This report detailed the maintenance of B. burgdorferi at the site via sheep primarily by co-feeding adult and nymphal ticks. Although our sampling at this site included 72 adults, in which no infections were diagnosed, these were collected on a specific area of moorland from which sheep had been excluded for several years. Thus, the absence of B. burgdorferi infections in our sample is not inconsistent with the enzootic cycle proposed by Ogden and colleagues (1997). Indeed, such cycles may exist at other sites where we tested few adults and encountered low prevalence or absence of B. burgdorferi infections in questing nymphs.

Despite the unexpected patchiness in distribution of B. burgdorferi infections across our study area, our work confirms the presence of the pathogen in several previously unexplored tick populations, thereby adding to the expanding knowledge of B. burgdorferi distribution in the United Kingdom. However, as pointed out previously (Dobson et al. 2011), the public health risk posed by the presence of B. burgdorferi in these populations is tempered by the extent of their contact with humans. All 3 sites where we recorded a high prevalence of B. burgdorferi in questing ticks were close to towns (Dumfries, Grange-over-Sands, and Pickering) and were well visited; indeed Mabie Forest and Dalby Forest are specifically promoted as tourist/recreation sites, with well over 500,000 visitors per annum between them (http://www.forestry.gov.uk/), equating to a density of visits to Dalby Forest of approximately 12,000 visits/km² per year.

Our observation of B. afzelii at 2 sites in northern England suggests a widespread presence of this species in the country. Previously, the species has only been observed at sites in south and southwestern England (Couper et al. 2010, Vollmer et al. 2010). However, as also observed by Vollmer and colleagues (2011), the species represented only a minor component of the Borrelia community at the 2 sites where we found it. The apparent scarcity of B. afzelii in southern Britain is in marked contrast to the apparent abundance of the species in the Scottish highlands (Ling et al. 2000). Furthermore, we did not encounter B. burgdorferi s.s., another species that appears to be rare in the United Kingdom outside the Scottish Highlands (Kurtenbach et al. 1998, Ling et al. 2000, Couper et al. 2010, Vollmer et al. 2011). In keeping with earlier work (Kurtenbach et al. 1998, Ling et al. 2000, Couper et al. 2010, Vollmer et al. 2011), we observed that B. valaisiana and B. garinii were the species that contributed most to Borrelia communities at our study sites. B. valaisiana was the most widespread, being present at all sites where we encountered Borrelia-infected ticks. That only B. valaisiana was identified at sites where Borrelia infections were extremely rare may simply reflect the widespread dominance of this species across southern Britain, however, but also likely reflects the dispersal efficiency of this and other avian-adapted species (Vollmer et al. 2011), and thus their enhanced potential as invaders of remote tick communities in which borreliae may or may not be established. This process has been elegantly demonstrated as B. burgdorferi emerges in southern Canada (Ogden et al. 2010), but is clearly ongoing throughout the distribution of the spirochete.

One of our most unexpected observations was the apparent absence of I. ricinus from some of our survey sites, despite them possessing a flora and fauna at least superficially akin to those of sites where I. ricinus was present in abundance. The patchiness of I. ricinus distribution on a relatively local scale has been described previously (Milne 1944) and correlated to drainage and vegetation, but this study was restricted to Northumbrian farmland used for livestock grazing rather than woodland. Three of the sites in our study matched the “rough grazing” habitat that Milne (1944) associated with the presence of I. ricinus; we encountered ticks at 2 (Graigfechan and Naddle Forest) but not at the 3^rd (Crowden). The absence of ticks from Hamsterley Forest in County Durham is particularly bemusing given the high density of roe deer in the vicinity (Andrew Rothwell, Forestry Commission Wildlife Ranger, personal communication). A passive surveillance programme for UK ticks, managed by the Health Protection Agency, has been in place since 2005, and collation of the data this program has assimilated with historical records has provided the most complete picture of I. ricinus distribution in the United Kingdom to date (Jameson and Medlock 2011). These data do not currently include any ticks from the vicinity of Hamsterley Forest or, indeed, anywhere in the Pennine Hills in between the Yorkshire Dales and the Tyne valley, perhaps suggesting I. ricinus is absent from a large part of this region. However, by its very nature, this is a survey of where ticks are, rather than where they are not. Active survey would clearly help clarify I. ricinus distribution in the region.

In conclusion, we demonstrated similar patterns of Borrelia infection in tick populations in parts of northern England, north Wales, and southern Scotland to those found in the south of England and Northern Ireland, but differing from those found in the north of Scotland. In addition we identified sites at which (1) ticks were unexpectedly absent, or (2) B. burgdorferi appeared not to be established, despite an abundant tick population. Further studies to determine why such differences exist are warranted, as they could help to predict Lyme borreliosis risk as well as explore the complex ecology of this pathogen.

Footnotes

Acknowledgments

We would like to thank members of the Forestry Commission GB and the Field Studies Council for their help in identifying and providing access to sampling sites. We also acknowledge funding from the Henry Lester Trust (to F.Z.) and from The Wellcome Trust (to M.R.) and the Veterinary Training and Research Initiative (VTRI) by the Higher Education Funding Council for England and the United Kingdom's Department for the Environment, Food and Rural Affairs (to J.B.).

Author Disclosure Statement

No competing financial interests exist.

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