Opportunistic viral surveillance confirms the ongoing disease threat grey squirrels pose to sympatric red squirrel populations in the UK.

BACKGROUND: Red Squirrels United was a UK landscape-scale grey squirrel management programme undertaken between 2016 and 2020. METHODS: A total of 11034 grey squirrels were removed by culling, with 1506 necropsied and 1405 suitable for adenovirus (AdV) or squirrelpox virus (SQPV) quantitative PCR (qPCR) analysis. Spleen, lip or hair were extracted, and DNA was isolated, with samples tested in duplicate by qPCR. RESULTS: Of 1378 tissue samples, 43% were positive for AdV and 10% for SQPV. Of 1031 hair samples, 11% were positive for AdV and 10% for SQPV. Overall, 762 of 1405 (54%) animals were positive for one or both viruses. LIMITATIONS: Ad hoc sampling was undertaken from limited geographical areas but provided the only dataset from that period, instead of extrapolating from historical data. CONCLUSIONS: The grey squirrel is an asymptomatic reservoir host for AdV and SQPV. Interspecific infection transmission potential is demonstrated. Grey squirrel management by culling is essential for mainland red squirrel viability until other suitable management tools are available.

A novel dicistrovirus in a captive red squirrel (Sciurus vulgaris).

Dicistroviruses are single-stranded RNA viruses in the family Dicistroviridae. The viruses have mainly been detected in arthropods and are the cause of several devastating diseases in many of these species such as honeybees. Increasingly, dicistroviruses have also been detected in both mammalian and avian species in faeces, blood and liver, but with unconfirmed pathology. Here, we report a novel dicistrovirus detected in the intestinal content of a captive red squirrel with enteritis along with the disease history, pathology and genomic characterisation of the virus. Virus particle morphology resembled those of picornaviruses with a diameter of 28-32 nm but failed to be detected using a mammalian/avian pan viral microarray. Next-generation sequencing confirmed a dicistrovirus having a typical dicistrovirus genome organization, but with the polyprotein 1 being shorter by about 100 amino acids, compared to that of other dicistroviruses. Phylogenetic analysis of ORF1 and ORF2 sequences clustered the virus with two yet unassigned dicistroviruses detected in Gorilla gorilla and a freshwater arthropod and likely to be designated to a new genus. Our data further highlights the ever-growing diversity of dicistroviruses, but the clinical significance of the virus in mammalian species and particularly red squirrels has yet to be established.

Mortalities, amyloidosis and other diseases in free-living red squirrels (Sciurus vulgaris) on Jersey, Channel Islands.

Between 2007 and 2014, 337 free-living red squirrels (Sciurus vulgaris) on Jersey, Channel Islands, were examined post mortem as part of a mortality and disease surveillance scheme. Road traffic accidents (RTAs) were attributable for 50.7 per cent (171/337) of the casualties, 34.4 per cent (116/337) succumbed to diseases including fatal exudative dermatitis (FED), 7.1 per cent (24/337) to predation, 6.5 per cent (22/337) to other trauma and 1.2 per cent (4/337) to suspected poisoning. Cat predation accounted for 5 per cent (17/337) of mortalities. Pathologies were diverse and individual animals were often identified with more than one disease process. Squirrelpox virus (SQPV) particles were not detected in selected cases examined by transmission electron microscopy (TEM). Amyloid was identified in 19.3 per cent (65/337) of squirrels, often in conjunction with inflammatory lesions like hepatic capillariasis. A consistent cause of amyloid accumulation was not identified, although there was a significant association of amyloidosis with hepatic capillariasis and FED. In addition to RTAs, amyloidosis and FED have been identified as important causes of squirrel morbidity and mortality on Jersey, while the underlying aetiology and predisposing factors for these two disease complexes are presently unclear. Disease, fragmented woodlands, an increasingly suburban habitat, along with various anthropogenic factors, may jeopardise the long-term viability of this island red squirrel population.

Enhancement of wildlife disease surveillance using multiplex quantitative PCR: development of qPCR assays for major pathogens in UK squirrel populations.

Rapid development in polymerase chain reaction (PCR) technology has revolutionised the speed and accuracy of many diagnostic assays. However, comparatively few wildlife epidemiological studies use quantitative PCR (qPCR) for pathogen detection, even fewer employ an internal control, to ensure confidence in negative results, and PCR's ability to multiplex and therefore detect several targets in a single reaction is underutilised. Here, we describe the development of two multiplex qPCR assays for the red and grey squirrel that detect the pathogens squirrelpox virus (SQPV) and adenovirus in squirrels (SADV), both of which cause mortality in the red squirrel. Both assays use a section of the squirrel phosphoglycerate kinase gene as an endogenous internal control that identifies and compensates for both, inadequate sampling or PCR inhibition. Tests on infected squirrel tissue demonstrate that simple swab samples (particularly from distal antebrachial skin) are sufficient to detect and identify the relative quantity of SQPV DNA in both squirrel species, while rectal swabs and blood cell pellets can be used to reliably indicate SADV infection. These assays are sensitive and specific with an endogenous internal control providing confidence in negative results and allowing comparison across laboratories. Using such assays should prove advantageous in wildlife studies with limited resources while allowing the maximum data yield.

Ljungan virus and an adenovirus in Italian squirrel populations.

We report Ljungan virus infection in Eurasian red squirrels (Sciurus vulgaris) for the first time, and extend the known distribution of adenoviruses in both native red squirrels and alien gray squirrels (Sciurus carolinensis) to southern Europe.

Causes of mortality and pathological lesions observed post-mortem in red squirrels (Sciurus vulgaris) in Great Britain.

BACKGROUND: The red squirrel population in Great Britain has declined dramatically in recent decades, principally due to squirrelpox. Concern exists that red squirrels may become extinct nationally and, as there has been limited research in to diseases other than squirrelpox, this study aimed to identify additional causes of mortality. RESULTS: Post-mortem examinations on 163 red squirrels found dead on Isle of Wight (IoW) England, in Scotland and at other locations in Great Britain showed that 41.7% (n = 68) were killed by road traffic and 9.2% (n = 15) by predators, principally domestic cats and dogs. The overall male/female ratio was 1.08/1. Fleas were recorded on 34.9% of IoW squirrels and on 43.8% of Scottish squirrels but sucking lice and ixodid ticks were only seen on Scottish squirrels. Bacterial infections were significant, particularly in association with respiratory disease (n = 16); two squirrels died of Bordetella bronchiseptica bronchopneumonia. Cases of fatal exudative dermatitis (n = 5) associated with a lukM-positive clone of Staphylococcus aureus occurred only on the IoW. Toxoplasmosis (n = 12) was also confined to IoW where it was responsible for almost one tenth (9.5%) of all deaths. Hepatozoonosis was common, especially in IoW squirrels, but was not considered a primary cause of mortality. Hepatic capillariasis affected four IoW squirrels and one from Scotland. Fungal infections included oral candidiasis, adiaspiromycosis and pulmonary phaeohyphomycosis. Neoplastic conditions diagnosed were: pulmonary carcinoma, gastric spindle cell tumour, renal papillary adenoma and trichoepithelioma. Epidermal hyperplasia of unknown aetiology was seen in squirrels showing crusty lesions of the ear pinnae on IoW (n = 3) and Brownsea Island (n = 1), associated in two cases with cutaneous wart-like growths. Miscellaneous diagnoses included chylothorax, electrocution, intussusception, suspected cholecalciferol rodenticide poisoning and foetal death and mummification. No cases of squirrelpox were diagnosed. CONCLUSIONS: Red squirrels in Britain suffer premature or unnatural mortality due to a number of conditions in addition to squirrelpox, many of which result, directly or indirectly, from human activities: road traffic trauma, pet predation, toxoplasmosis, trap injuries, rodenticide poisoning and electrocution accounted for 61% of all recorded mortality in this study. Red squirrels are also affected by several diseases of unknown aetiology which merit further research.

Characterization of a novel poxvirus in a North American red squirrel (Tamiasciurus hudsonicus).

In 2009, a novel poxvirus was identified in a North American red squirrel (Tamiasciurus hudsonicus) from Yukon, Canada. Initial molecular analyses indicated that this virus was likely to be distinct from all other known mammalian poxviruses, including those previously associated with disease in tree squirrels--squirrel fibroma virus in North America and squirrelpox virus in the UK (UK SQPV). We characterize the Canadian squirrelpox virus (Canadian SQPV) using DNA sequence analysis and negative-contrast transmission electron microscopy (TEM). Sequence analysis revealed that the Canadian SQPV is distinct from all known mammalian poxviruses but most closely related to the parapoxviruses, followed by UK SQPV. In contrast, TEM showed that the ultrastructure of Canadian SQPV is distinct from that of the parapoxviruses and UK SQPV but indistinguishable from that of other chordopoxviruses (a morphological group that includes the orthopoxviruses and leporipoxviruses). Overall, our analyses suggest a potential evolutionary relationship between UK SQPV and Canadian SQPV and supports our assertion that the Canadian virus represents a newly identified poxvirus in North American tree squirrels.

Evaluation of a preclinical blood test for scrapie in sheep using immunocapillary electrophoresis.

An analytical method is described for detection of endogenous disease-associated prion protein in the buffy coat fraction from the blood of sheep infected with scrapie. The method has been improved and evaluated for its performance in the preclinical diagnosis of ovine transmissible spongiform encephalopathies. The test system uses a protocol for sample preparation that includes extraction and concentration and a test method that uses a liquid-phase competitive immunoassay for prion protein. Antibodies directed to a peptide sequence at the C-terminus of the prion protein (PrP) and a fluorescein-labeled peptide conjugate are used in the assay. Free zone capillary electrophoresis with laser-induced fluorescence for detection is used to separate the antibody-bound fluorescently labeled peptide and free labeled peptide. In this assay, the PrP competes with the fluorescently labeled peptide for limited antibody binding sites, which results in a reduction of the peak representing the immunocomplex of the antibody bound to the fluorescently labeled peptide. When blood samples from scrapie-infected sheep aged 7-12 months and of the scrapie-susceptible PrP genotypes VRQ/VRQ and VRQ/ARQ were analyzed, the abnormal PrP was found in blood samples. These results correlated with the post-mortem diagnosis of scrapie. The sheep were preclinical and appeared normal at the time of testing but later died with clinical disease approximately 12 months after testing. In older animals, and those with clinical signs, a smaller percentage of animals tested positive. This study has demonstrated that this technology can be used as a sensitive, rapid preclinical test to detect the disease-associated PrP in the blood of scrapie-infected sheep. Improvements in the extraction protocol and capillary electrophoresis conditions will enhance the robustness of this test.