Editor: Karina Acevedo-Whitehouse
The emergence of squirrelpox in Ireland
Article first published online: 13 JUL 2012
© 2012 The Authors. Animal Conservation © 2012 The Zoological Society of London
Volume 16, Issue 1, pages 51–59, February 2013
How to Cite
McInnes, C. J., Coulter, L., Dagleish, M. P., Deane, D., Gilray, J., Percival, A., Willoughby, K., Scantlebury, M., Marks, N., Graham, D., Everest, D. J., McGoldrick, M., Rochford, J., McKay, F. and Sainsbury, A. W. (2013), The emergence of squirrelpox in Ireland. Animal Conservation, 16: 51–59. doi: 10.1111/j.1469-1795.2012.00570.x
- Issue published online: 28 JAN 2013
- Article first published online: 13 JUL 2012
- Manuscript Accepted: 7 JUN 2012
- Manuscript Received: 18 JAN 2012
- SQPV ;
- grey squirrels;
- red squirrels;
The native red squirrel Sciurus vulgaris population in Britain has been on the decline for many years. A poxvirus associated with the introduced American grey squirrel S. carolinensis has been recognized as having a major role in the reduction of red squirrel numbers by causing a deleterious disease, known as squirrelpox, from which they seldom recover. In Ireland, red squirrel numbers have also been reducing while the grey squirrel population, first introduced in 1911, has been expanding. Until now, no poxvirus-associated disease had been found in Irish red squirrels and therefore, the role of squirrelpox in the displacement of red squirrels by grey squirrels in Ireland has been questioned. Here we report, for the first time, confirmed squirrelpox disease in two populations of red squirrels in Northern Ireland. In addition, we present serological evidence of the extent of poxvirus infection in the grey squirrels from both the Republic of Ireland and Northern Ireland, including an apparent increase in the seroprevalence of antibodies against the virus in grey squirrels over the period of the study, and discuss the implications of our findings for the conservation tactics employed to protect red squirrels.
The red squirrel Sciurus vulgaris has been in decline in mainland UK for at least the last century (Middleton, 1930; Shorten, 1957; Harris et al., 1995). The reasons for this decline could be manyfold, but there is now general agreement that the North American Eastern Grey squirrel S. carolinensis, introduced to Britain in the latter part of the 19th century, has had a major negative influence on the native red squirrel population. Research aimed at explaining this influence initially concentrated on habitat loss or fragmentation and how this affected the interactions between the two species, their respective behaviour patterns and food preferences or utilization (Moller, 1983; Kenward & Holm, 1989, 1993; Gurnell & Pepper, 1993). However, it was not until outbreaks of disease in red squirrels were taken into account that a convincing explanation was provided for the pattern and rapidity of red squirrel decline observed in England (Rushton et al., 2000, 2006; Tompkins, White & Boots, 2003).
Throughout the 20th century there had been reports of disease being responsible for the local disappearance or extinction of red squirrels (Middleton, 1930; Edwards, 1962; Vizoso, 1968; Reynolds, 1985), but no common agent and no specific link to grey squirrels was found until relatively recently. Sainsbury et al. (2000) suggested that a poxvirus linked to deaths of red squirrels was also highly prevalent, but asymptomatic, in some grey squirrel populations in the UK. It was later confirmed that the virus, now known as squirrelpox virus (SQPV; McInnes et al., 2006), was capable of killing red squirrels while leaving grey squirrels apparently unaffected (Tompkins et al., 2002; Thomas et al., 2003). Serological studies suggested that red squirrel deaths as a result of SQPV infection were not recorded in parts of the country where there were no grey squirrels or where the grey squirrels were seronegative for the presence of the virus (Sainsbury et al., 2000). Modelling work then showed that in parts of the country where SQPV was present in the grey squirrel population, the grey squirrels could replace the red squirrels up to 25 times faster than in parts of the country where the virus was not present (Rushton et al., 2006). This suggested that no matter what other influences were at work, SQPV was likely to have been a major factor in the decline of red squirrels in parts of the UK where the virus was found. Recently, epidemics of disease caused by SQPV have swept through populations of red squirrels, most notably in Formby on Merseyside, the English Lake District, north-east England and the Borders region of Scotland, although the affected populations have not, yet at least, been totally eliminated by disease (McInnes et al., 2009; Atkin et al., 2010; Bruemmer et al., 2010). Grey squirrels are now heavily controlled in these regions in an attempt to reduce the threat to the remaining red squirrels from squirrelpox disease.
The North American Eastern Grey squirrel is not native to Europe, but has survived and multiplied, after being introduced, in Britain, Ireland and Italy. The founder population of grey squirrels in Ireland is thought to be derived from approximately 12 squirrels introduced to Castle Forbes, County Longford in 1911 (Watt, 1923), although other reports suggest eight squirrels were released at Castle Forbes in 1913 (Middleton, 1930) and an undetermined number at Ballymahon, County Longford in 1929 (Lever, 1977). From the initial introduction at Castle Forbes, it is reported that the grey squirrels expanded at a mean rate of 1.94 km per year such that by the mid 1990s, they were found in 22 of the 32 counties of Ireland (Teangana et al., 2000), and ten years later in 26 of the 32 counties (Carey et al., 2007). Much as in Britain, as the grey squirrels expanded in number, the native red squirrels were reported to be decreasing in number and are now considered to be absent from the counties with the highest densities of grey squirrels. As a result, it has recently been suggested that red squirrel protection areas be set up, concentrating mainly on those counties still regarded to be free of grey squirrels or where grey squirrels are yet to be fully established (Carey et al., 2007). In addition, given the dramatic effect that SQPV has had on the red squirrel population in mainland UK, recent attention has been focussed on whether or not it is also present in Ireland and to what extent it has affected the red squirrel population. Here we present the results of serological monitoring of grey squirrels from Ireland over a 12-year period outlining our current understanding of the geographical extent to which grey squirrels have been exposed to the virus and report for the first time squirrelpox disease in red squirrels in Northern Ireland (NI).
SQPV antibody enzyme-linked immunosorbent assay (ELISA)
Blood samples in this study were analysed retrospectively from animals culled across Ireland. Hence, they represent a convenience sample of grey squirrels, from across their range, rather than a structured study of the same woodlands over a period of time. The fact that some woodlands were sampled more than once has, however, allowed limited longitudinal comparisons to be made. Samples were collected from 740 grey squirrels culled in the Republic of Ireland (ROI) in 2005 and in NI between 1997 and 1999, 2004 and 2005, and 2007 and 2009 (Table 1). None of the animals showed obvious clinical signs of being infected with SQPV. Blood was collected post mortem, usually from the chest cavity. Sera were obtained by centrifugation of the blood samples at 2000 g for 5 min and examined for the presence of antibodies against SQPV as described elsewhere (Sainsbury et al., 2000; McInnes et al., 2009). Briefly, 96-well plates were coated with SQPV- and control negative-antigen before incubating with serum samples collected from the squirrels. The plates were washed to remove non-specifically bound protein and anti-SQPV immunoglobulin G was detected by incubating with horseradish peroxidase-conjugated protein G. Colour was developed with 3,3’,5,5’ tetramethylbenzidine peroxidase substrate (SureBlue, Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD, USA) for 5 min before the reaction was stopped with the addition of 0.18 M H2SO4; optical densities at 450 nm were obtained using a Dynex MRX platereader (Dynex Technologies, Chantilly, VA, USA). To discriminate between positive and negative results an optical density of 0.2 above the background was used.
|County||Woodlanda||Squirrels known to be present 2007b||SQPV + ve/number of grey squirrel blood samples tested|
|Monaghan, ROI||Castle Leslie||−||+||–||33/55||–||–|
|Kildare, ROI||Clongowes Wood||−||+||–||13/51||–||–|
|Tipperary, ROI||Knox's Wood||−||+||–||0/23||–||–|
Post mortem examination
Carcasses of 14 red squirrels with suspected squirrelpox disease were submitted, by the Northern Ireland Forest Service, to the Moredun Research Institute for post mortem examination by a veterinary pathologist. Two of the squirrels had been found dead within Tollymore Forest Park, exhibiting signs of infection with SQPV. The remainder had been euthanized on welfare grounds as they were suspected of suffering from squirrelpox. Each animal was weighed and then examined for external and internal lesions, and samples were taken for further analysis. Blood, or fluid from internal cavities, was collected for assessment of seroconversion to SQPV by ELISA (see earlier). Eyelids and brains were frozen until required for PCR and transmission electron microscopy (TEM) analysis (see later). Fleas were collected, if present, as they are known to be a vector in other poxvirus diseases. For histological examination, a range of tissues samples were fixed in 10% formal saline, processed by routine methods, embedded in paraffin wax and 5-μm sections were mounted on glass microscope slides and stained with haematoxylin and eosin.
SQPV DNA was detected by PCR as previously described (McInnes et al., 2009) using the primers SQ026F 5′ATGTCAGTCACGATAAGATT3′ and SQ026R 5′TCATGTCAGTCGGGTGATGA3′ that amplify a 258-bp fragment of the SQPV genome corresponding to a gene that has been found, so far, only in SQPV, the Parapoxvirinae and Molluscum contagiosum virus. The primers are specific for SQPV and no cross-reactivity has been found with other poxvirus DNA or squirrel genomic DNA. In this analysis, DNA was isolated from tissue using the QIAGEN DNeasy® Kit (Qiagen GmbH, Hilden, Germany) following the manufacturer's instructions. Two microlitres of the resulting DNA was incubated with 1 μM of each PCR primer, 200 μM deoxynucleotide triphosphates, 1.5 mM MgCl2, 10 mM Tris-HCL pH 8.3 and 50 mM KCl and 1 unit Taq DNA polymerase (Roche Applied Science, Mannheim, Germany). DNA was denatured by incubating at 94°C for 4 min and amplification was achieved using 30 cycles of 94°C for 1 min, 55°C for 30 s and 72°C for 1 min. This was followed by a final incubation at 72°C for 5 min. Amplification products were electrophoresed in a 1% agarose gel, stained with GelRedTM (Biotium, Inc., Hayward, CA, USA) and visualized by ultraviolet transillumination.
Negative Contrast Stain TEM
The methodology used for these analyses have been fully described previously in Everest et al., (2010).
Serum samples collected between 1997 and 2009 from 740 grey squirrels culled in different counties (Fig. 1) of both the ROI (n = 191) and NI (n = 549) were examined for antibodies against SQPV. The results are presented in Table 1.
Approximately one-third (244/740) of all the animals sampled had antibodies against SQPV. There was a greater proportion of seropositive grey squirrels in 2009 (67%; 35/52) than in 1997–1999 (17%; 31/184), and the trend over all years between 1997 and 2009 was one of an increasing proportion of seropositive grey squirrels. The data may be biased because sampling in 1997 and 2004/2005 was widespread and designed to determine the geographical extent to which the grey squirrels in Ireland had been exposed to SQPV. In contrast, sampling in 2007/2009 tended to concentrate on those woodlands known to contain seropositive grey squirrels. Nevertheless, the general trend of increasing seroprevalence was also found in the three woodlands that were tested on at least three occasions over the period of the study. For example, none of the 21 animals tested from Derrynoyd in 1999 had antibodies against the virus, 20% (1/5) of those tested in 2004/2005 had antibodies and 80% (8/10) of those tested in 2009 were seropositive. Similarly, none (0/13) from Gosford were seropositive in 1999, 29% (6/21) were seropositve in 2004/2005, 57% (4/7) in 2007/2008 and 71% (5/7) in 2009. In Loughgall, 14% (1/7) were seropositive in 1999, 35% in 2004/2005, 40% in (2007/2008) and 66% (8/12) were positive in 2009. There were only four locations, in Antrim, Londonderry and Fermanagh in NI and Tipperary in the ROI, where no seropositive grey squirrels were found; all other woodlands from which grey squirrels were sampled had at least one seropositive grey squirrel. One of these four locations, Learmount in Londonderry, is still regarded as primarily holding red squirrels with only the occasional grey squirrel being found, while the other two in NI are reported to hold both red and grey squirrels. Knox's Wood in Tipperary was regarded at the time of sampling in 2005 as being at the southern extreme of the grey squirrel range in the ROI.
In March 2011, post mortem examinations were performed on the carcasses of two red squirrels that had been found during the preceding week within Tollymore Forest Park, County Down. The squirrels were suspected to be suffering from squirrelpox disease. One had gross lesions of severe dermatitis with scab formation on the eyelids, with similar, but less severe lesions on the footpads and chin very similar to those described in other cases of squirrelpox (Fig. 2 and b). The other had alopoecia and very mild lesions on the left eyelid and chin, which were less suggestive of squirrelpox. Histological examination of the skin lesions from squirrel 1 (Table 2, Fig. 2c) revealed ballooning degeneration in the stratum spinosum at the periphery of severe necro-suppurative dermatitis resulting in full-thickness erosion (ulceration) of the epidermis. A mixed inflammatory cell infiltration was present along with mineralization of necrotic tissue; all consistent with SQPV infection. For squirrel 2, a severe suppurative dermatitis with loss of epithelium was present on the eyelids (Fig. 2d). PCR analysis of the DNA isolated from the eyelid lesions from both squirrels found SQPV DNA to be present. Further PCR analysis of the face and footpad lesions from squirrel 1, the chin lesion from squirrel 2 and fleas taken from both squirrels revealed that all were positive for SQPV DNA (results not shown). Over the next 2 months, a further 12 red squirrel carcasses were examined post mortem and tissues taken for both histological and PCR analysis. Nine of these were from Tollymore Forrest Park and three were from woodlands in the Glens of Antrim, approximately 70 miles north of Tollymore. Of the 14 squirrels examined in total all but one, from the Glens of Antrim, were found to have been infected by SQPV by PCR analysis. Each displayed varying extant signs of SQPV infection, with at least one of the SQPV-positive squirrels exhibiting no visible external lesions. Histological examination, however, suggested SQPV infection was likely even in those animals with few or no gross lesions. The results of the analyses are summarized in Table 2.
|ID||Date||Location||Sex||Gross lesions||Histological lesions||EM||PCR|
|1||23-03-11||Tollymore||F||Lesions on eyelids, mouth and footpads||Consistent with SQPV infection||+ve||+ve||nd|
|2||23-03-11||Tollymore||F||Mild lesions on left eye and chin||Not inconsistent with SQPV infection||+ve||+ve||nd|
|3||29-03-11||Tollymore||M||Scabs on eyelids of both eyes, lower jaw, both axillae and ventral to larynx||Consistent with SQPV infection||+ve||+ve||nd|
|4||12-04-11||Tollymore||F||Alopoecia of skin around both eyes, severe dermatitis around ears, scabs on nose and digit 1 of both fore-paws and large scab in R axilla||Consistent with SQPV infection||+ve||+ve||−ve|
|5||12-04-11||Tollymore||F||Eyes, ears and chin covered in scabs plus dorsal aspect of R carpus, ventral base of tail and back||Consistent with SQPV infection||+ve||+ve||−ve|
|6||12-04-11||Tollymore||M||Eyelids a little swollen||Consistent with SQPV infection||+ve||+ve||−ve|
|7||12-04-11||Tollymore||M||Slight crusting R lower eyelid, left OK||Consistent with SQPV infection||+ve||+ve||−ve|
|8||19-04-11||Glenariff||F||Tiny bit of crusting on lateral aspects of fore-paws and no hair on distal half of tail||No signs consistent with SQPV infection||nd||−ve||nd|
|9||05-05-11||Tollymore||M||Scabs on eyelids L > R, plus on upper and lower muzzle||Consistent with SQPV infection||+ve||+ve||+vea|
|10||05-05-11||Tollymore||F||Scabs on eyelids of both eyes R > L plus some on chin||Consistent with SQPV infection||+ve||+ve||+vea|
|11||05-05-11||Tollymore||F||No skin lesions||Consistent with SQPV infection||−ve||+vea||−ve|
|12||05-05-11||Tollymore||M||Alopoecia of eyelids||Consistent with SQPV infection||+ve||+ve||−ve|
|13||24-05-11||Glenarm||M||Scabs on mouth, eyes and nose||Not inconsistent with SQPV infection||+ve||+ve||nd|
|14||02–06-11||Glenarm||M||Eyelids swollen and lesions present||Bacterial and fungal dermatitis||+ve||+ve||nd|
Scab or skin lesion material from 13 of the 14 squirrels was examined by negative contrast TEM, to confirm the presence of SQPV particles, determining them by size, shape and surface morphology. Of these, 12 animals had virus particles that were typically ovoid in shape and of the appropriate size (c. 160–190 nm × 200–250 nm), and having a surface morphology displaying a polypeptide spiral wound longitudinally around the particle, typical of SQPV. These results are summarized in Table 2.
Although many factors such as competition for habitat and food resources and lack of juvenile recruitment (Wauters, Lurz & Gurnell, 2000) may account for some of the red squirrel losses in Britain, it has been accepted that disease caused by SQPV is an important driver for the ecological replacement of red squirrels by grey squirrels (Tompkins et al., 2003; Rushton et al., 2006). Until the results from this study were gathered, little was known about the presence of SQPV in Ireland and no cases of squirrelpox disease in the red squirrels had been confirmed. Indeed, it had been mooted that red squirrels in Ireland may be immune to SQPV disease. In the most recent Irish Squirrel Survey, conducted in 2004–2005, there is a report of two red squirrels from Shankill near Dublin suffering from a ‘myxomatosis-like’ disease, but both carcasses were destroyed before they could be tested for SQPV (Carey et al., 2007). Here we report the first confirmed cases of squirrelpox affecting red squirrels from two separate locations in Ireland. The first red squirrel examined for disease was found dead within Tollymore Forest Park on 16 March 2011 and had external signs typical of those reported previously for squirrelpox (McInnes et al., 2009). There were visible scabs on the face and feet and a purulent conjunctivitis. Histological examination of the lesions was consistent with a poxvirus infection and PCR analysis of the eyelids found the presence of SQPV DNA. Electron microscopy confirmed the presence of SQPV particles (results not shown).
Over the following 10 weeks, another 10 animals from Tollymore Forest Park and two of the three animals from the Glens of Antrim were all confirmed as suffering from squirrelpox. The extant signs of disease varied in each animal, although most commonly signs of infection were seen in the peri-orbital area. However, at least two animals (11 and 12, Table 2) exhibited little or no signs of disease. No lesions were seen on squirrel 11 and squirrel 12 had a slight alopecia of the eyelids. Strictly managed supplementary feeding of the squirrels within Tollymore Forest Park was a common practice until the outbreak of the disease. This meant that most of the animals were observed frequently, quite often on a daily basis. Few red squirrels seem capable of surviving an infection with SQPV (Sainsbury et al., 2008) and so most of the animals sent for post mortem examination in this study had been euthanized on welfare grounds, not only because of the presence of visible external lesions, but because their behaviour had suggested to observers that they were suffering from disease. Most commonly, they were reported to drink large quantities of water and to exhibit a degree of lethargy and imbalance (Janette Adams, Tollymore Red Squirrel Group, pers. comm.). This is the first time it has been suggested that behavioural changes are noticeable in SQPV-affected animals before obvious external signs of disease are present.
Despite several reports in the literature describing squirrelpox disease in red squirrels, it is still not known why infected red squirrels die (Scott, Keymer & Labram, 1981; Sainsbury & Ward, 1996; Sainsbury et al., 2008; Duff et al., 2010; LaRose et al., 2010). There appears to be no major abnormalities or disease of the internal organs, and in many cases, including those reported herein, the dead squirrels have full stomachs and do not appear to be dehydrated. The report here of an unsteady gait and the suggestion that SQPV DNA had been found in the brains of infected animals elsewhere (Atkin et al., 2010) prompted us to look in detail at the brains of the animals in this study. Although SQPV DNA was detected at comparatively low levels by PCR in two of the squirrel brains, histological examination of these suggested no abnormalities. Extreme care was taken to use separate instruments for each tissue taken during the post mortem process. However, we believe that, given the presence of virus-laden scabs on the eyelids, muzzle and chin in these cases, it is likely the brain samples taken for PCR analysis could have been contaminated with virus during the dissection of the carcasses rather than there being an actual infection of the brain. As in previous reports, we were unable to determine what the primary cause of death was likely to be for SQPV-infected animals.
Disease modelling, matched by empirical observations in the north of England, suggested that red and grey squirrels overlapped spatially for c. 3–4 years before red squirrel numbers started to decline as a result of squirrelpox disease (Rushton et al., 2006). In Scotland, the first cases of disease in red squirrels were not discovered until c. 2 years after grey squirrels, seropositive for antibodies against the virus, were first detected (McInnes et al., 2009). Despite these observations, Tompkins et al. (2003) argued that red squirrels would immediately succumb to the disease on encountering grey squirrels infected with the virus. These theories and observations are not mutually exclusive as the two species of squirrels have been known to inhabit different habitat blocks within the same woodland thereby reducing encounter rates between them and potentially delaying exposure of red squirrels to the virus, this last point presumably being related to the prevalence of the virus within the grey squirrel population. Sainsbury et al. (2000) suggested that the more established grey squirrel populations had higher seroprevalence of antibodies against SQPV, indicating circulation of the virus within that population, and therefore may represent a higher threat to resident red squirrel populations. This appears to have been the case in the three woodlands (Loughgall, Gosford and Derrynoyd) that were tested over the 12 years of this study, with an increasing prevalence of virus in the grey squirrel population being detected over the period. Each of these woodlands are considered to have lost their populations of red squirrels, although it has to be conceded that no pox-diseased red squirrels were ever found within these woods and consequently, we can only speculate at the role of squirrelpox in the local extinction of the red squirrels. The serology results from these particular woodlands, however, also illustrate that the virus can survive in the grey squirrel population, and apparently increase in prevalence, in the absence of red squirrels, a scenario also reported in Hampshire and Staffordshire on mainland Britain (Sainsbury et al., 2000).
Disease in Tollymore Forest Park was detected 7 years after seropositive grey squirrels were first identified there. Whether or not disease was present, but undetected before 2011, we do not know. Tollymore was the first woodland to be given Forest Park status in Ireland in 1955 and in recent years, as a result of a dramatic decline in red squirrel numbers in the late 1990s, it has been proactively managed for the benefit of red squirrels. Since 2004, supplementary feeding has been provided for the red squirrels and a control programme for grey squirrels, which had first appeared in the mid 1990s, has been practised. A survey carried out in 2004 had estimated there to be 11 red squirrels and 24 grey squirrels in the Park, but after 4 years of active management, this had changed to 103 red squirrels and just three grey squirrels. The model developed by Rushton et al. (2006) suggested that culling 60% or more of the grey squirrels in Cumbria would have stopped, or at least would have slowed significantly, the replacement of red squirrels by the greys. However, the authors conceded that if the replacement was driven by disease, and in particular by squirrelpox, this may not have been the case. Although we do not know the current size of the grey squirrel population within Tollymore, between 2004 and 2008, rangers had reduced the grey population by almost 90%. Despite this, squirrelpox still emerged in the red squirrel population, appearing to confirm that a large population of SQPV-positive grey squirrels is not required for the transmission of the virus to red squirrels. This would also strengthen the argument presented by Tompkins et al. (2002), that once the virus enters into a population of red squirrels, transmission among the reds becomes relatively more important in the epidemiology of disease than transmission from the greys. This appears a reasonable assumption because infected red squirrels suffer an exudative dermatitis resulting in scabs heavily laden with virus, that pose a direct threat to other in-contact squirrels as well as a source of virus for contaminating the environment, whereas there is seldom, if ever, signs of disease in infected grey squirrels. This may also help to explain why Reynolds suggested that squirrelpox appeared to have occurred in some localities in Norfolk in the absence of grey squirrels (Reynolds, 1985).
A similar situation to that found in Tollymore has occurred in Formby in northern England and Drumlanrig Castle Estate in the south of Scotland. Both locations have been actively managed for red squirrels with culling of grey squirrels and supplementary feeding of red squirrels being practised. It could be argued the management practices have been so successful that red squirrel numbers have grown to a level greater than could be sustained naturally by the woodlands (Kenward et al., 1998; Wauters et al., 2000). The red squirrels in these localities have suffered greatly from squirrelpox in the last few years in the presence of relatively few grey squirrels. However, increased numbers of red squirrels may, ironically, increase the encounter rate with the remnant grey squirrel population carrying the virus, thus increasing the threat of disease transmission. Increased encounter rates among the red squirrels themselves would also be expected and thus if the virus had already passed into the red squirrels a rapid decline or even local extinction of the population, as predicted by Tompkins et al. (2002, 2003) could result. It may however simply be that disease is more apparent in these populations because the red squirrels, normally regarded as a cryptic species, are considerably more visible than normal because of the increase in red squirrel numbers. Whatever the reason, the results from Tollymore re-enforce the suggestion that conservation measures for red squirrels need to include tactics for reducing the contact between individuals of the two squirrel species to minimize the likelihood of SQPV transmission. However, it needs to be borne in mind that conservation tactics that include supplementary feeding to bolster red squirrel populations, even in the face of concerted grey squirrel culling, may in fact be counter-productive.
Thanks should go to the many agencies and volunteer groups who have helped collect samples and information for this study. In particular, we would like to thank the Northern Ireland Forest Service and the Tollymore Red Squirrel Group. Thanks also go to the various landowners who helped with this study.
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