Incidental finding of a human-like tusavirus in a lamb with lip lesions and fatal pneumonia.

Tusaviruses in the genus Protoparvovirus of family Parvoviridae were first identified in a diarrhoeic Tunisian child in 2014. Thereafter, high prevalence of a genetically similar virus was demonstrated in faeces from caprine and ovine species in Hungary. Here, we describe an investigation into the cause of scabby lip lesions in a 6 month-old lamb, submitted from a farm experiencing weight loss and scouring in lambs in England. Transmission electron microscopy visualised small circular particles of 18 and 22 nm in diameter in lip lesions identified as tusavirus and flumine parvovirus by Next Generation Sequencing. Liver, kidney, lung, small intestine content and faeces were also strongly positive for the tusavirus DNA as well as 10 % of faecal samples of the flock collected 2 months after the initial lip sampling. NS1 and VP1 amino acid sequences of this tusavirus displayed 99.5 and 92.89 % identity to those of a human tusavirus, respectively. These amino acid identities were at 95.5 and 89.68 % when compared to those of a goat tusavirus. Phylogenetic analysis of the NS1 and VP1 also grouped the virus in the genus Protoparvovirus and close to tusaviruses detected in human, ovine and caprine species. Wider surveillance of the virus indicated a broader geographical distribution for the virus in England. Histology of the lip tissue revealed localised areas of epidermal hyperplasia and hyperkeratosis affecting haired skin, with mild leucocyte infiltration of the subjacent dermis, but no changes to implicate virus involvement. Flumine parvovirus was concluded to be an environment contaminant. Broader studies in prevalence of these virus in UK sheep flocks and human population, animal models and experimental infections could provide insights into the pathogenesis of these novel viruses and their zoonotic potential.

Investigation of nasal epithelial cells as a surrogate for bronchial epithelial cells in the research of equine asthma.

Equine asthma, previously known as Recurrent Airway Obstruction (RAO) or Inflammatory Airway Disease (IAD), is an often-debilitating condition that may severely affect both performance and quality of life. Research is hindered by the low sample numbers of subjects recruited to studies, a consequence in part of the invasive nature of the sampling methods of bronchial brushing and biopsy. We present an alternative method of sampling equine airway epithelial cells, the 'nasal brush method' (NBM). Obtained by light brushing of the ventral meatus whilst the horse is under standing sedation, these cells express the same markers of differentiation as their deeper counterparts. Grown as 3-D spheroids or as air-liquid interface cultures, nasal epithelial cells are responsive to the inflammatory cytokine interleukin-13. This may be attenuated by modulation of the Notch signalling pathway using the gamma-secretase inhibitor Semagecestat; a previously unreported finding that cements the link between equine and human asthma research and strengthens the case for a One Health approach in researching asthma pathophysiology and therapeutic intervention.

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.

Galleria mellonella as an infection model for the virulent Mycobacterium tuberculosis H37Rv.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is a leading cause of infectious disease mortality. Animal infection models have contributed substantially to our understanding of TB, yet their biological and non-biological limitations are a research bottleneck. There is a need for more ethically acceptable, economical, and reproducible TB infection models capable of mimicking key aspects of disease. Here, we demonstrate and present a basic description of how Galleria mellonella (the greater wax moth, Gm) larvae can be used as a low cost, rapid, and ethically more acceptable model for TB research. This is the first study to infect Gm with the fully virulent MTB H37Rv, the most widely used strain in research. Infection of Gm with MTB resulted in a symptomatic lethal infection, the virulence of which differed from both attenuated Mycobacterium bovis BCG and auxotrophic MTB strains. The Gm-MTB model can also be used for anti-TB drug screening, although CFU enumeration from Gm is necessary for confirmation of mycobacterial load reducing activity of the tested compound. Furthermore, comparative virulence of MTB isogenic mutants can be determined in Gm. However, comparison of mutant phenotypes in Gm against conventional models must consider the limitations of innate immunity. Our findings indicate that Gm will be a practical, valuable, and advantageous additional model to be used alongside existing models to advance tuberculosis research.

An Opportunistic Assessment of the Impact of Squirrelpox Disease Outbreaks upon a Red Squirrel Population Sympatric with Grey Squirrels in Wales.

Native red squirrels (Sciurus vulgaris) persisted in the coastal mainland woodlands of northern Gwynedd whilst sympatric with an invasive grey squirrel (Sciurus carolinensis) population suppressed by culling. Squirrelpox disease in the red squirrel population was recorded in 2017 and 2020/21. An autumn 2020 outbreak was associated with only 17.4% of animals caught and marked in the preceding June known to be present in March 2021. Despite an opportunistic data collection lacking the rigour of empirical experimental design, we observed low local survival rates similar to previously published accounts reported during major squirrelpox outbreaks. The use of a conservation dog to detect red squirrel carcasses resulted in positive detection and confirmation of a temporal and spatial expansion of one disease outbreak. The study is the first in Wales to use conservation dogs and the findings reinforce the vital strategic importance of geographical isolation reducing sympatry of red with grey squirrels in European regions where the introduced congener is a source of the squirrelpox infection.

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.

Novel Arterivirus Associated with Outbreak of Fatal Encephalitis in European Hedgehogs, England, 2019.

In the fall of 2019, a fatal encephalitis outbreak led to the deaths of >200 European hedgehogs (Erinaceus europaeus) in England. We used next-generation sequencing to identify a novel arterivirus with a genome coding sequence of only 43% similarity to existing GenBank arterivirus sequences.

A novel biosafety level 2 compliant tuberculosis infection model using a ΔleuDΔpanCD double auxotroph of Mycobacterium tuberculosis H37Rv and Galleria mellonella.

Mammalian infection models have contributed significantly to our understanding of the host-mycobacterial interaction, revealing potential mechanisms and targets for novel antimycobacterial therapeutics. However, the use of conventional mammalian models such as mice, are typically expensive, high maintenance, require specialized animal housing, and are ethically regulated. Furthermore, research using Mycobacterium tuberculosis (MTB), is inherently difficult as work needs to be carried out at biosafety level 3 (BSL3). The insect larvae of Galleria mellonella (greater wax moth), have become increasingly popular as an infection model, and we previously demonstrated its potential as a mycobacterial infection model using Mycobacterium bovis BCG. Here we present a novel BSL2 complaint MTB infection model using G. mellonella in combination with a bioluminescent ΔleuDΔpanCD double auxotrophic mutant of MTB H37Rv (SAMTB lux) which offers safety and practical advantages over working with wild type MTB. Our results show a SAMTB lux dose dependent survival of G. mellonella larvae and demonstrate proliferation and persistence of SAMTB lux bioluminescence over a 1 week infection time course. Histopathological analysis of G. mellonella, highlight the formation of early granuloma-like structures which matured over time. We additionally demonstrate the drug efficacy of first (isoniazid, rifampicin, and ethambutol) and second line (moxifloxacin) antimycobacterial drugs. Our findings demonstrate the broad potential of this insect model to study MTB infection under BSL2 conditions. We anticipate that the successful adaptation and implementation of this model will remove the inherent limitations of MTB research at BSL3 and increase tuberculosis research output.

Novel enteric viruses in fatal enteritis of grey squirrels.

Astro- and kobuviruses infect both humans and animals. Here, we report on the disease history, detection and genomic characterization of novel astro- and kobuviruses from fatal diarrhoea of two juvenile grey squirrels. The virus particles had enterovirus-like morphology and a diameter of 28-32 nm. Next-generation sequencing confirmed astro- and kobuviruses and sequence analysis revealed typical astrovirus and picornavirus genome organizations. The astrovirus ORF2 sequence clustered with a clade of unassigned astroviruses, with marmot and rodent mamastroviruses as closest relatives. For the kobuvirus, divergences greater than 49.4 % for P1 and 43.5 % in the non-structural proteins indicated a novel species. However, phylogenetic analysis of the 3D polymerase showed that it clustered with that of the newly classified ludopivirus A1, suggesting a previous recombination event in the evolution of the kobuvirus. Our data provide further insights into the diversity of astro- and kobuviruses and broaden the spectrum of viruses infecting grey squirrels.

Prevalence in Britain of abnormal prion protein in human appendices before and after exposure to the cattle BSE epizootic.

Widespread dietary exposure of the population of Britain to bovine spongiform encephalopathy (BSE) prions in the 1980s and 1990s led to the emergence of variant Creutzfeldt-Jakob Disease (vCJD) in humans. Two previous appendectomy sample surveys (Appendix-1 and -2) estimated the prevalence of abnormal prion protein (PrP) in the British population exposed to BSE to be 237 per million and 493 per million, respectively. The Appendix-3 survey was recommended to measure the prevalence of abnormal PrP in population groups thought to have been unexposed to BSE. Immunohistochemistry for abnormal PrP was performed on 29,516 samples from appendices removed between 1962 and 1979 from persons born between 1891 through 1965, and from those born after 1996 that had been operated on from 2000 through 2014. Seven appendices were positive for abnormal PrP, of which two were from the pre-BSE-exposure era and five from the post BSE-exposure period. None of the seven positive samples were from appendices removed before 1977, or in patients born after 2000 and none came from individuals diagnosed with vCJD. There was no statistical difference in the prevalence of abnormal PrP across birth and exposure cohorts. Two interpretations are possible. Either there is a low background prevalence of abnormal PrP in human lymphoid tissues that may not progress to vCJD. Alternatively, all positive specimens are attributable to BSE exposure, a finding that would necessitate human exposure having begun in the late 1970s and continuing through the late 1990s.

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.

Identification of Novel Cetacean Poxviruses in Cetaceans Stranded in South West England.

Poxvirus infections in marine mammals have been mainly reported through their clinical lesions and electron microscopy (EM). Poxvirus particles in association with such lesions have been demonstrated by EM and were previously classified as two new viruses, cetacean poxvirus 1 (CePV-1) and cetacean poxvirus 2 (CePV-2). In this study, epidermal pox lesions in cetaceans stranded in South West England (Cornwall) between 2008 and 2012 were investigated by electron microscopy and molecular analysis. PCR and sequencing of a highly conserved region within the viral DNA polymerase gene ruled out both parapox- and orthopoxviruses. Moreover, phylogenetic analysis of the PCR product clustered the sequences with those previously described as cetacean poxviruses. However, taking the close genetic distance of this gene fragment across the family of poxviridae into account, it is reasonable to postulate further, novel cetacean poxvirus species. The nucleotide similarity within each cluster (tentative species) detected ranged from 98.6% to 100%, whilst the similarity between the clusters was no more than 95%. The detection of several species of poxvirus in different cetacean species confirms the likelihood of a heterogeneous cetacean poxvirus genus, comparable to the heterogeneity observed in other poxvirus genera.

Mortality associated with avian reovirus infection in a free-living magpie (Pica pica) in Great Britain.

BACKGROUND: Avian reoviruses (ARVs) cause a range of disease presentations in domestic, captive and free-living bird species. ARVs have been reported as a cause of significant disease and mortality in free-living corvid species in North America and continental Europe. Until this report, there have been no confirmed cases of ARV-associated disease in British wild birds. CASE PRESENTATION: Sporadic individual magpie (Pica pica) mortality was detected at a single site in Buckinghamshire, England, April-September 2013. An adult female magpie was found moribund and subsequently died. Post-mortem examination identified hepatomegaly and splenomegaly as the most severe macroscopic abnormalities. Histopathological examination revealed extensive hepatic and splenic necrosis. Transmission electron microscopy (TEM) identified virions of a size (circa 78 nm diameter) and morphology consistent with ARV in both the liver and the small intestinal (SI) contents. Nucleic acid extracted from pooled liver and spleen was positive on both a pan-reovirus nested PCR targeting the RNA-dependent RNA polymerase gene and a PCR using primers specific to the ARV sigma C protein gene. Virus isolated from the liver and the SI contents was characterised by a syncytial-type cytopathic effect, a reovirus-like appearance on TEM and sequence identical to that from PCR of tissues. In situ hybridisation confirmed co-localisation of ARV with lesions in the liver and spleen, implicating ARV as the causative agent. Splenic lymphoid atrophy and necrotic stomatitis associated with Aspergillus fumigatus infection were consistent with generalised immunosuppression and resultant opportunistic infection. CONCLUSIONS: The pathology and comprehensive virus investigations in this case indicate ARV as the primary pathogen in this magpie, with concurrent secondary infection subsequent to immunosuppression, as has been observed with reoviral infections in other bird species. ARV should be considered as a differential diagnosis for magpie, and potentially other corvid, disease and mortality incidents. This is the first demonstration of ARV-associated mortality in a wild bird in Britain. The prevalence and significance of ARV infection in British wild birds, and its implications for poultry and captive bird health, are currently unknown.