SQUIRRELPOX
J. PAUL DUFF
A nimal Health and Veterinary Laboratories Agency Diseases of Wildlife Scheme (AHVLA DoWS), Great Britain Wildlife Disease Surveillance Partnership, Penrith, Cumbria, UK
Squirrelpox disease (SQPD) (synonyms squirrel pox viral disease (SQPVD), squirrel distemper, squirrel myxomatosis, squirrel parapox disease and squirrel pox) is confined to the red squirrel (Sciurus vulgaris) and the introduced grey squirrel ( S.
carolinensis), where they occur together in the UK. A disease of red squirrels, almost certainly SQPD but not confirmable at that time, was first described in England around 1930.The disease has conservation importance, as slowly but efficiently it extirpates (destroys) the red squirrel metapopulation (a series of continuous populations linked over a geographically large area) in mainland Britain. Complete metapopulation destruction caused by infectious disease is unusual even in global terms.
AETIOLOGY
Squirrelpox virus (SQPV) causes SQPD. As a typical poxvirus, SQPV is robust, large (300 ? 80nm), spherical or bullet- shaped, with basketweave- like cross striations that cover the surface of virions and are obvious on electron microscopy. The virus is considered to be resistant to chemicals and some disinfectants and, based on the properties of other poxviruses although not yet confirmed in SQPV, the virus is assumed to survive for some time — perhaps days or weeks — in the environment. The genome has been characterized and classifies virulent SQPV on its own in a separate clade of the poxviruses(11) as a previously unrecognized genus of the Chordopoxvirinae and not a parapoxvirus as was originally thought. Other pox-like viruses are found in North American squirrel species, but these are not closely related to SQPV.
EPIDEMIOLOGY
The red squirrel has a wide Eurasian distribution, but the disease only occurs in populations of red squirrels in contact with introduced, SQPV-infected grey squirrels.
The grey squirrel is the reservoir host or carrier of the virus(12) and is very resistant to disease, with only a single clinical case reported. The combination of both species of squirrel and SQPV together, which leads to the disease, currently occurs only in the UK and Ireland, with the first Irish cases confirmed in 2011 (D. Everest, personal communication). Populations of grey squirrels co- exist with red squirrels in Central Scotland and Italy but are seronegative for SQPV, and consequently neither virus nor disease are present there. Accidental host species have not been identified.As part of the Veterinary Laboratory Agency Diseases of Wildlife Scheme (VLADoWS) national wildlife disease scanning surveillance, 606 sick or dead red squirrels from northern England were examined between 1998 and 2011, from which 164 cases of SQPD were diagnosed(13) (Duff, personal observations). Findings from these cases are used in this description together with findings published elsewhere. Of the 164 cases, 48 were subadults or juveniles, the remainder being adults. There was a sex bias towards males (107 males, 53 females, 4 not recorded) possibly linked to male behaviour questing for territories and mating opportunities, which render them more likely to encounter infective grey squirrels. A small number of red squirrels survive infection in the wild (2 from the 606; about 1.2% of SQPD cases) but the mechanisms involved are not known.
SQPD may occur in any month of the year, this study showed there were peaks in April (27 cases in total) and July (20 cases), with an average of 12 cases in each of the remaining months. The April and July maxima may correspond to the two breeding seasons in the UK. There was an increased frequency during the summer as opposed to the winter (103 cases in 6 months from April versus 61 cases in 6 months from October).
EPIDEMIOLOGICAL ROLE OF AFFECTED SPECIES
SQPD is a well-documented example of pathogen- mediated competition between an invasive species acting as a reservoir for a pathogen (the grey squirrel) and a native species that is very susceptible to the pathogen (the red squirrel)(14).
Red squirrels can also be considered as accidental spillover hosts of a virus carried by an introduced species. Over 60% of sampled grey squirrels in England and Wales had antibody to the virus, supporting the reservoir host theory(12). In the absence of the virus, grey squirrels usually outcompete red squirrels in most habitats; however, the rate of red squirrel replacement by grey squirrels is 17 to 25 times faster in those areas where grey squirrels carry the virus, compared with those areas where they do not(15). Following introduction from North America, the grey squirrel population, once established, then expanded and has now replaced the red squirrel throughout mainland England south of Liverpool. As the grey squirrel extends its distribution northward into Scotland, an epidemic front of SQPD follows in the wake of the line of population expansion, while behind this epidemic front the red squirrel population has effectively disappeared and has been replaced by the grey squirrel.SQPD epidemics appear to follow a repeating pattern; grey squirrels first enter an area and after a delay, local sporadic outbreaks of SQPD occur but fade out when the susceptible animals are killed. The local outbreaks become more frequent and eventually the once- continuous red squirrel population is fragmented. In good-quality red squirrel habitat there may be a ‘sink effect’ where the remaining animals are drawn in from catchment areas and persist for years after the species has disappeared from the rest of the region. Twenty cases of SQPD have now been reported in southern Scotland, and the disease may eventually extend beyond Central Scotland, perhaps coming to a natural barrier in northern Scotland, where only red squirrels survive. SQPV may spare these far- north red squirrel populations if grey squirrels cannot colonize these areas. SQPD exists only in epidemic form and only where red and grey squirrels are in contact, suggesting that red- to-red squirrel transmission does not spatially extend epidemics; that is, the disease has not spread independently of the grey squirrel(16).
However, the increased number of cases during the red squirrel breeding season suggests that red- to- red squirrel transmission may account for some mortality during local outbreaks. Seropositive red squirrels that survive infection are generally in poor condition and appear too scattered temporally and spatially to constitute a viable but possibly immune breeding population.TRANSMISSION
Direct and indirect transmission of SQPV is thought to occur; however, evidence-based information is lacking. Arthropod parasites may possibly transmit the virus mechanically, but a significant arthropod vector has not been found. Both species of squirrel are solitary and the likelihood of direct contact is relatively low. Indirect transmission may involve virus particles left in shared dreys, on branches or at feeding sites as the possible sources of infection between species. It is unlikely that SQPV is picked up from faeces or urine. The relatively short clinical course and the near 99% case-fatality rate suggest that red squirrels are only infective for a period of days. Supplementary feeders used by squirrels, if virus contaminated, are likely to be a source of infectious virus for other squirrels.
PATHOGENESIS, PATHOLOGY AND IMMUNITY
The means of acquisition of infection are not known but may occur when poxvirus penetrates small breaks in the continuity of the skin. Bilateral lesions on the inner aspects of the front feet seem to be associated with the animals rubbing their facial lesions, and these suggest that the virus may invade intact skin. PCR testing has detected low titres of virus in internal organs. From experimental infection studies of red squirrels, disease was produced by a virus titre of 100,000 virus particles per millilitre given in 0.2 ml doses by skin scarification and as subcutaneous injections. SQPV causes exudative erythematous dermatitis and severe dehydration. The infection typically presents as a necrotizing purulent dermatitis, which often extends into the underlying subcutis, producing a deeper-sited cellulitis.
In the VLA study(13), analysis of 157 red squirrels with pox lesions, which were considered well enough preserved for critical evaluation, the head was invariably affected with pox lesions on the eyelids, lips, ear and face. These had often expanded and coalesced to cover much of the face, which became alopecic. All but five (97%) had unilateral or bilateral conjunctivitis with a yellow ocular discharge and eyelid oedema. Lip lesions were seen in 113/157 (72%), usually extending onto the chin or nares. Lesions on the digits and feet were seen in 59/157 (37%). There was variable but usually lesser involvement of skin on neck, proximal limbs, scrotum, anus, dorsum and flanks. Preputial lesions were seen in 34% of males. Permanent skin scarring was not detected, but this was not unexpected as the survival rate from SQPV is very low.The early acute uncomplicated primary histopathological lesions consist of multifocal areas of ballooning degeneration and spongiosis concentrated in the mid to upper epidermal layers. In contrast to pox infections in many other animal species, which are classically associated with strongly staining inclusion bodies, only small numbers of poorly defined weakly eosinophilic intracytoplasmic structures are seen with SQPV. As the disease progresses, there is extensive loss of infected epidermis on the skin surface, often extending into the hair follicles and associated adnexal glands. The marked mixed dermal inflammatory cell infiltrate is predominated by neutrophils and histiocytes closely associated with progressive erosion and ulceration. These ulcers quickly became colonized by opportunistic mixed bacterial species — predominantly staphylococci. A thick adherent layer of serocellular debris, bacteria and plant material often covers well- established lesions. The cellular reaction becomes more mononuclear in character in the subacute stages with prominent plasma cell populations. Deeper extension into the underlying subcutis occurs in many cases with resultant purulent or pyogranulomatous cellulitis tracking along fascial planes.
Dermal and subcutaneous blood vessels within these areas show marked activation of endothelium, neutrophil dia- pedesis and adventitial cuffing. Thrombosis and associated haemorrhage are frequent sequelae related to local bacterial toxin production in situ. Early activation of angioblasts and fibroblasts may be detected if the animal survives long enough for attempts at healing to occur. A similar pattern of reaction is seen in infected non-hairy locations such as conjunctivae. Pox-associated lesions have never been convincingly described within internal organs.Secondary staphylococcal infections of pox lesions and septicaemia are important sequelae, and of the study cases cultured, Staphylococcus aureus was isolated from 48 animals and S. scuiri from 19 cases. In total, 56 animals had staphylococcal infections of internal organs indicative of septicaemia. In a significant number of animals however, mild pox lesions and the absence of cultured bacteria makes it unclear precisely how the disease killed the animal. Affected animals lose weight and are significantly dehydrated. Of the164 study animals, 62 were judged in poor body condition, 86 were in normal condition (condition not recorded in 16).
The humoral antibody response appears protective to a degree, the few surviving animals have had very high enzyme-linked immunosorbent assay (ELISA) titres. How these antibodies neutralize virus is not known. The persistence of protective humoral antibody again is not known, or if it confers immunity to reinfection.
CLINICAL SIGNS AND TREATMENT
I n the VLA survey(13), 70/164 red squirrels were found dead, 6 were found in-extremis and quickly died, 3 were reported as ‘curled in a ball’ lying on the ground for up to 6 hours before dying, 15 were euthanized on capture (or shot to prevent infection of others), and 39 were easily caught and submitted for veterinary treatment, but of these all but 6 eventually died — on average 5 days after submission. The 6 treated animals made a full clinical recovery and were rehabilitated in captivity. Of those listed that were observed alive, the clinical signs most frequently reported were dullness, weakness, lethargy, dehydration, anorexia and inanition. Blindness was reported in 2, however corneal opacity in a further 3 of those found dead, and in addition others in this group, in which the eyelids were matted together by thick dried crusts indicated effective blindness. Rarer signs were gasping and incoordination. Published reports described poor ability to climb, difficulty in moving, dullness and rubbing the facial lesions with their paws. The animals may remain for long periods around feeders and still occasionally eat. Captive animals can be briefly active and aggressive when handled.
Treatment is difficult, as gauntlet handling is essential for medication and therefore euthanasia must be considered at the outset. Rehydration is an immediate priority, and antibiotics effective against staphylococci are required; however, these bacteria can still be readily cultured from lesions well into the course of treatment. Interferon has been used frequently but with equivocal results. The patients do not adjust well to captivity, and animals apparently responding to treatment for several days with resolving lesions may die unexpectedly. It is not known whether recovered animals remain infective; consequently they usually are not released.
DIAGNOSIS
Diagnosis is based on finding the range of typical clinico- pathological findings in cases where both squirrel species co-exist. Typical pox lesions in the areas of skin mentioned are clearly visible and have characteristic pox lesion histopathology. Laboratory confirmation by electron microscopy of scabs and removed skin lesions has proven effective even in autolysed animals. Although electron microscopy is sensitive, PCR for viral nucleic acid is probably a more sensitive detection test. The virus has been cultured on cell lines. ELISA serology is used to detect antibody and has been useful in epidemiological studies, for example to assess whether populations of squirrels are naive for the virus.
MANAGEMENT, CONTROL AND REGULATIONS
The current multi-agency strategy in the UK is to preserve the red squirrel in defined areas of good-quality red squirrel habitat (usually coniferous woodland) and in these to exclude the presence of grey squirrels by trapping. Surveillance for the disease is essential, and the local public in England strongly support surveillance, management and control initiatives. When SQPD occurs in an area, removal of garden feeders is generally advised. Disease modelling has predicted that a grey squirrel population control of >60% effective kill was needed to stop the decline in red squirrel populations1-15). Vaccine has a reasonable chance of conferring immunity. Repeated administration of killed vaccine may be necessary, and it is unlikely that the commercial orf vaccine would cross-protect. Live attenuated vaccine is unlikely to be licensed while live recombinant vaccine incorporating a gene coding for immunogenic portions of the SQPV may have a use, optimally by oral delivery.
There are no EU regulations and no international regulations regarding SQPV.
PUBLIC HEALTH CONCERN
No disease threats to humans or farmed species are apparent in the 80-year history of the disease; however, the precautionary principle should be applied and protective clothing used if animal handling is essential. There is considerable public concern over the disappearance of the once familiar red squirrel from the British countryside. The story of SQPD is a salutary lesson in the dangers of introducing non-native wildlife. Risk assessments on grey squirrels had they been done at introduction were unlikely to have predicted a virus like SQPV in the absence of disease expression.
SIGNIFICANCE AND IMPLICATIONS IN ANIMAL HEALTH
SQPD is a disease of conservation importance related to the threatened destruction of red squirrel populations where they, grey squirrels and SQPV occur together; there are no known threats to other species.
ACKNOWLEDGEMENTS
The help of Robert J. Higgins, AHVLA, in writing the pathology sections, particularly histopathology, is gratefully acknowledged.
MYXOMATOSIS AND OTHER