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Infectious canine hepatitis in red foxes (Vulpes vulpes) in wildlife rescue centres in the United
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Kingdom
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D. Walker, E. Abbondati, A. L. Cox, G. B. B. Mitchell, R. Pizzi, C. P. Sharp, A. W. Philbey
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David Walker, BSc(Hon), BVM&S, MSc, MRCVS,
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Royal (Dick) School of Veterinary Studies and the Roslin Institute, University of Edinburgh, Easter Bush, Edinburgh EH25 9RG, United Kingdom
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Erika Abbondati, DVM, MRCVS,
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Royal (Dick) School of Veterinary Studies and the Roslin Institute, University of Edinburgh, Easter Bush, Edinburgh EH25 9RG, United Kingdom
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Alistair L. Cox, BVMS, MSc, FRCPath, MRCVS,
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Veterinary Services, SAC Consulting, Scotland’s Rural College (SRUC), Edinburgh EH26 0QE, United Kingdom
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George B. B. Mitchell, BVMS, PhD,
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Veterinary Services, SAC Consulting, Scotland’s Rural College (SRUC), Auchincruive, Ayr KA6 5AE, United Kingdom
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Romain Pizzi, BVSc, MSc, DZooMed, DipECZM, MANZCVS, FRES, FRGS, FRSB, MRCVS,
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Scottish Society for the Prevention of Cruelty to Animals, National Wildlife Rescue Centre, Fishcross, Clackmannanshire FK10 3AN, United
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Kingdom, and Royal Zoological Society of Scotland, Corstorphine Road, Edinburgh, EH12 6TS, United Kingdom
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Colin P. Sharp, BSc(Hons), PhD,
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The Roslin Institute, University of Edinburgh, Easter Bush, Edinburgh EH25 9RG, United Kingdom
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Adrian W. Philbey, BVSc(Hon), PhD, FRCPath, MANZCVSc(Path), FHEA, MRCVS,
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Royal (Dick) School of Veterinary Studies and the Roslin Institute, University of Edinburgh, Easter Bush, Edinburgh EH25 9RG, United Kingdom
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E-mail for correspondence: [email protected]
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Summary
Outbreaks of infectious canine hepatitis (ICH) are described in red foxes (Vulpes vulpes) at
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two wildlife rescue centres in the United Kingdom. Disease occurred in 2 to 4 month old juvenile
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foxes, which were held in small enclosures in groups of three to eight animals. The foxes died or
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were euthanased after a short clinical course, sometimes including neurological signs and jaundice,
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with a high case fatality rate. Four red foxes submitted for postmortem examination had enlarged,
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congested livers, with rounded borders and mild accentuation of the lobular pattern. On histological
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examination, there was random, multifocal to massive hepatic necrosis, along with multifocal
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vasculitis in the central nervous system (CNS) and mild, multifocal glomerulonephritis. Intranuclear
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inclusion bodies, typical of canine adenovirus type 1 (CAV-1) infection, were present in
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hepatocytes, vascular endothelial cells in the CNS, renal glomeruli and renal tubular epithelial cells.
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CAV-1 was detected in tissues from affected foxes by the polymerase chain reaction and
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sequencing. Congregation of juvenile foxes in wildlife rescue centres is likely to be a risk factor for
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transmission of CAV-1. Preventative measures in wildlife centres should be implemented to prevent
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the spread of the virus among conspecifics and to other susceptible species.
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Introduction
Early laboratory studies showed that red foxes (Vulpes vulpes) are susceptible to
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experimental infection with canine adenovirus type 1 (CAV-1), the cause of infectious canine
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hepatitis (ICH) (Green and others 1930). However, the pathology and clinical signs of ICH in
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naturally infected, free-ranging canids are not well characterised. Red foxes in the United Kingdom
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(Thompson and others 2010) and Italy (Balboni and others 2013) appear to be a wildlife reservoir of
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CAV-1. Serological exposure to untyped CAV in several free-ranging carnivore species has been
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reported worldwide (Amundson and Yuill 1981; Thompson and others 2010; Truyen and others
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1998), suggesting that CAV-1 may be sustained in wild canid populations. However, there are only
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a few reports of the occurrence of spontaneous ICH in free-ranging foxes (Woods 2001), limited to
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descriptions of isolated cases in a grey fox (Urocyon cinereoargenteus) (Gerhold and others 2007)
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and three red foxes (Thompson and others 2010).
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It has been hypothesised that sporadic transmission of CAV-1 from infected red foxes to susceptible
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(unvaccinated) domestic dogs may occur through contact with infected excretions, such as urine and
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faeces (Thompson and others 2010). Widespread vaccination of dogs in the United Kingdom, in
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combination with responsible dog ownership and effective control of stray dogs, is likely to have
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reduced the incidence of transmission of CAV-1 amongst dogs to a low, and probably
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unsustainable, level. However, the possibility of infection through direct or indirect contact with
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susceptible wildlife, or unvaccinated dogs, may be a mechanism allowing the occasional
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presentation of ICH in dogs to veterinary surgeons in general practice. Furthermore, concern has
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been expressed about the emergence of infectious diseases, such as canine distemper (Walker and
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others 2014), rabies and Echinococcus multilocularis (Bourne and others 2015), in the United
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Kingdom as a consequence of both legal and illicit animal movements. Therefore, veterinary
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surgeons should be aware of the clinical signs and diagnoses of such diseases, and also be conscious
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that wildlife, such as foxes, which occasionally are presented for veterinary intervention, are also
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susceptible to many diseases of the domestic dog.
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In the Mediterranean, ICH has been reported as an ‘old’ disease which is re-emerging (Decaro and
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others 2007). Other cases of ICH in dogs and foxes have been reported in the past decade in Europe
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(Gleich and others 2009, Müller and others 2010, Thompson and others 2010), Asia (Wen and
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others 2009, Cheema and others 2012), North America (Wong and others 2012, Headley and others
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2013) and South America (Inkelmann and others 2007, Oliveira and others 2011). However, this
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published evidence suggests that the frequency of occurrence of ICH has not necessarily changed
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substantially in the last 25 years. Despite this, in conjunction with evidence of serological exposure
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in wildlife, such reports support the hypothesis that there is on-going exposure and transmission of
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CAV-1 to susceptible species. There is no current evidence to suggest that the incidence of ICH in
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dogs is higher in areas with a greater wildlife disease burden. However, in principal, there is
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potential for disease ‘spill over’ events from red foxes to unvaccinated dogs.
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In this paper, the clinical and pathological findings in juvenile red foxes that died during outbreaks
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of ICH at two wildlife rescue centres in Scotland are described. To the authors’ knowledge, this is
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the first detailed report of ICH in multiple red foxes affected during disease outbreaks in wildlife
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rescue centres, whereas previously ICH was reported in free-ranging red foxes in the United
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Kingdom (Thompson and others 2010). Furthermore, the present study confirms the presence of
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CAV-1 in the United Kingdom by the polymerase chain reaction (PCR) and sequencing. These
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findings provide further evidence that CAV-1 is present in red foxes in the United Kingdom, that it
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sporadically causes fatal disease in this species, and that it may also infect conspecifics.
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Materials and methods
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History and pathological examinations
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Foxes 1, 2 and 3 were juvenile red foxes from different litters that had been orphaned and
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were admitted to the Scottish Society for the Prevention of Cruelty to Animals (SSPCA) National
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Wildlife Rescue Centre, Fishcross, Scotland, in May 2013 to be reared with the aim to release them
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back to the wild. In early June 2013, fox 1 died and fox 2 was euthanased due to collapse and
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seizures. Subsequently, fox 3, from the same group of animals, died suddenly overnight. In the
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same month, a further three associated foxes were found dead and a further three foxes were
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euthanased after exhibiting seizures.
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Fox 4 died at Hessilhead Wildlife Rescue Centre, Beith, Scotland in June 2011. It was one
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of four red foxes that had died at the centre within 2 weeks. The cub was enclosed in outdoor pens
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with five others, one of which exhibited convulsions prior to death. Fox 4 appeared to be normal
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when inspected by the staff at the centre on the day prior to death. The only other notable finding in
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the history of this group of animals was evidence of diarrhoea on the floor of the enclosure.
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Post-mortem examination and histopathology
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Foxes 1, 2 and 3 were submitted to the Royal (Dick) School of Veterinary Studies,
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Edinburgh, Scotland, for post-mortem examination in June 2013. Samples of liver (foxes 2 and 3),
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brain (fox 2) and lung (foxes 1 and 3) were collected into viral transport medium (10% phosphate
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buffered glycerol saline) and stored at -20 °C. Fox 4 was submitted to Scotland’s Rural College
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(SRUC), Auchincruive, Scotland, for post-mortem examination. A range of tissues, including brain,
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spleen, heart, mesenteric lymph node, liver, kidney and small intestine, were fixed in formalin,
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embedded in paraffin wax and stained with haematoxylin and eosin (H&E) for histopathological
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evaluation.
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Polymerase chain reaction and sequencing
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DNA was extracted from tissue samples from foxes 1, 2 and 3 using the AllPrep DNA/RNA
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Mini kit (Qiagen, Hilden, Germany). DNA was extracted from ~20 µm thick sections of formalin-
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fixed paraffin-embedded (FFPE) tissues from fox 4 using the QIAamp FFPE DNA Tissue kit
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(Qiagen).
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The PCR for adenoviral DNA polymerase sequences was performed using a nested protocol
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adapted from Wellehan and others (2004). The PCR reaction mixture contained 2 µL DNA, 36.5 µL
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H2O, 10 µL 5x GoTaq reaction buffer (Promega, Madison, WI, USA), 0.5 µL ‘polFouter’ forward
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primer (100 µM), 0.5 µL ‘polRouter’ reverse primer (100 µM), 0.3 µL deoxynucleotide
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triphosphates (10 mM) and 0.2 µL (1 U) GoTaq G2 DNA polymerase (Promega). The samples
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underwent two rounds of conventional nested PCR (94 °C for 5 minutes, 45 cycles of 94 °C for 30
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seconds, 46 °C for 1 minute and 72 °C for 1 minute, followed by a final extension at 72 °C for 5
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minutes); then, 2 µL PCR product from the first round was used as a template for the second round,
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under the same conditions, using the internal primers ‘polFinner’ and ‘polRinner’, described by
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Wellehan and others (2004). The expected amplicon size for CAV-1 is 321 base pairs (bp). The
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resulting amplicons were visualised using the G:BOX gel imaging system (Syngene, Cambridge,
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UK) following separation by gel electrophoresis in agarose containing SYBR Safe DNA Gel Stain
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(Invitrogen, Paisley, UK). Sanger sequencing was performed using the internal primers (Edinburgh
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Genomics, University of Edinburgh, United Kingdom).
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Results
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Gross pathology
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Fox 1
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Fox 1 was a juvenile male red fox with a moderate degree of autolysis. The liver was
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moderately enlarged, friable and brown, with mild accentuation of the lobular pattern. There was
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mild congestion of the meninges.
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Fox 2
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In fox 2, a male juvenile red fox, the liver was mildly enlarged and dark red, with
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multifocal, irregular areas of brown discolouration. The intestinal mucosa had multifocal patches of
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red discolouration. There was mild, diffuse, congestion of the meninges.
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Fox 3
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Fox 3 was a juvenile red fox of unknown sex in poor body condition and with jaundice. The
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liver was mildly enlarged and dark brown, with poorly defined pale patches. The spleen and
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mesenteric lymph nodes were mildly enlarged. The lumen of the stomach, small intestines and
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proximal large intestines contained dark red, haemorrhagic fluid.
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Fox 4
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Fox 4 was a female juvenile red fox weighing 2 kg. The liver and kidneys were moderately
congested. Moderate splenomegaly was evident.
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Histopathology
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Fox 1
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Histopathology of the liver of fox 1 revealed moderate to severe, random, multifocal to
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massive necrosis of hepatocytes. Amphophilic intranuclear inclusion bodies, including many
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classical Cowdry type A inclusion bodies, were present within numerous hepatocytes. There was
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dissociation and vacuolation of hepatocytes. Blood vessels and hepatic sinusoids contained fibrin
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thrombi. In the lungs, there were mild interstitial infiltrates of lymphocytes and macrophages in
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alveolar walls, along with mild interstitial oedema and formation of fibrin thrombi within blood
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vessels. Small numbers of intra-alveolar macrophages were present. In random, small blood vessels
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within the cerebral cortex, there was segmental swelling of vascular endothelial cells, mild
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perivascular oedema, occasional individual cell degeneration and rare intranuclear inclusion bodies.
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Fox 2
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The liver of fox 2 exhibited moderate to severe, random, multifocal to massive necrosis of
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hepatocytes, along with dissociation and vacuolation of hepatocytes, and expansion of the spaces of
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Disse. Numerous intranuclear inclusion bodies were present in hepatocytes (Figure 1A). In the
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kidney, there were occasional intranuclear inclusion bodies in probable mesangial cells in glomeruli
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and in epithelial cells lining the proximal convoluted tubules. Occasional degeneration of individual
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cells, probably mesangial cells, was evident in glomeruli. There was mild vacuolation of epithelial
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cells, mainly in the proximal convoluted tubules, along with small amounts of proteinaceous and
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sometimes cellular material in tubule lumina. There were no significant findings in the brain.
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Fox 3
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In the liver of fox 3, there was severe, massive, hepatic necrosis, with dissociation,
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vacuolation and fragmentation of hepatocytes, and numerous intranuclear inclusion bodies. There
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was a mild increase in the number of neutrophils within hepatic sinusoids. In the lung, there were
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increased numbers of macrophages and lymphocytes in alveolar walls, along with individual cell
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degeneration, probably of leucocytes. The brain was not examined.
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Fox 4
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The liver of fox 4 exhibited moderate, random, multifocal hepatic necrosis, with occasional
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intranuclear inclusions. In the kidney, rare intranuclear inclusion bodies were present in probable
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mesangial cells in glomeruli (Figure 1B) and there was occasional individual degeneration of
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epithelial cells lining proximal convoluted tubules. There was moderate lymphocytolysis within
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lymphoid follicles in the white pulp of the spleen and in the cortex of the mesenteric lymph node.
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Moderate numbers of intranuclear inclusion bodies were present in endothelial cells of blood
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vessels apparently scattered at random throughout the brain (Figure 1C), often associated with
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perivascular haemorrhage and occasionally with mild, focal degeneration and necrosis of neurones
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(Figure 1D).
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Polymerase chain reaction and sequencing
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Adenoviral sequences were detected by PCR in frozen tissues processed from foxes 1
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(lung), 2 (brain and liver) and 3 (lung and liver), as well as in FFPE brain and pooled spleen,
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mesenteric lymph node and cardiac muscle from fox 4. The primers amplified a 272 bp segment of
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the adenoviral DNA polymerase gene in foxes 1, 2 and 3 when the primer sequences are excluded
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(Wellehan and others 2004). Shorter fragments were sequenced from the FFPE samples (fox 4). The
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sequences from all foxes had a 100% match to CAV-1 (GenBank AC_000003.1), excluding the
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possibility of infection with CAV-2 (GenBank AC_000020.1).
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Discussion
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In this study, four cases of spontaneous ICH were confirmed by clinical and pathological
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examination, supported by PCR and confirmation of CAV-1 by sequencing, in juvenile red foxes
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that died at two wildlife rescue centres in Scotland. Several other foxes died within a period of
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weeks during the outbreaks of ICH at both wildlife rescue centres. The clinical histories suggested
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that the disease was peracute to acute and appears to have followed a similar course to the classical
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“fulminating form” of ICH (Parry 1950). In ICH, clinical signs develop after an incubation period
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of 2-6 days (Cabasso 1962).
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The gross and histopathological findings in affected red foxes in the present study were consistent
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with ICH. The most frequent gross changes were enlargement and altered colour of the liver.
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Jaundice was prominent in one fox. Oedema of the wall of the gall bladder may be observed in dogs
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with ICH (Decaro and others 2012), but was not evident in the red foxes in the present study.
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Similarly, there was no evidence of corneal opacity (“blue eye”) in the red foxes included in this
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investigation. On histological examination of the liver, intranuclear inclusion bodies were evident in
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hepatocytes, along with hepatocyte necrosis. Inclusion bodies were also seen within glomeruli and
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occasionally in renal tubular epithelial cells in the kidneys. A notable feature in the present cases
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was the presence of vasculitis and intranuclear inclusion bodies in vascular endothelial cells in the
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brain. Central nervous system lesions were not described in a previous study of ICH in free-ranging
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red foxes, since brain was not available from these cases (Thompson and others 2010).
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Wildlife rescue centres in the United Kingdom receive numerous juvenile foxes in spring and early
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summer, usually as orphans due to misadventure or disease in the vixens. Congregation of juvenile
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foxes in pen groups is likely to provide an opportunity for transmission of infectious disease. The
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juvenile foxes in the current study had a rapid onset and short duration of disease, with a high case
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fatality rate. Juvenile red foxes are likely to be susceptible to infection and at risk of severe ICH if
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they have not received maternal antibodies against CAV-1 or if maternal immunity has waned. It is
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also possible that other diseases, such as parasitism, emaciation or captivity-induced stress, may
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have affected the susceptibility of fox cubs to ICH.
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Further investigation into the origin of the outbreak at one of the wildlife centres revealed that the
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usual quarantine procedures for new admissions had not been followed. In particular, a newly
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introduced fox cub was allowed to have direct contact with a group of juvenile foxes that had been
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at the centre for several weeks. Congregation of susceptible juvenile foxes with conspecifics in
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captivity is likely to be a risk factor for the spread of CAV-1 and the occurrence of outbreaks of
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ICH. Transmission of CAV-1 occurs through direct exposure to infected animals, or indirect contact
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via contaminated urine, faeces or ocular or nasal secretions (Decaro and others 2012; Woods 2001).
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The duration of persistence of CAV-1 in the environment in such excretions is uncertain, although
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the virus has been shown to persist and remain infective for several months at temperatures below
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4°C (Decaro and others 2012). The course of the disease outbreak at the second wildlife centre and
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the underlying factors are unknown.
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It is clear that ICH is frequently manifested as a fatal disease in both red foxes and dogs. The
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majority of the literature reporting disease due to ICH in red foxes is based on experimental
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infections (Green and others 1930) or as reports of isolated cases of naturally acquired disease
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(Thompson and others 2010). However, there is serological evidence of untyped CAV antibodies in
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free-ranging red foxes in Europe (Truyen and others 1998; Thompson and others 2010), as well as
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other parts of the world, suggesting that a proportion of free-ranging red foxes are exposed to the
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virus and will survive without fatal disease.
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Infectious diseases, such as ICH, should be considered when undertaking veterinary assessment of
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sick red foxes submitted to wildlife rescue centres. It is also important to note that CAV-1 will be
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excreted by red foxes with ICH, as well as by animals incubating the disease and possibly by
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clinically healthy foxes. Moreover, since domestic dogs are susceptible to ICH, the possibility of
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transmission of CAV-1 from red foxes to dogs (and vice versa), either directly or through fomites,
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should be considered. There is some evidence that CAV-1 can be shed in the urine of apparently
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recovered dogs for up to 9 months following experimental infection with CAV-1 (Baker and others
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1954).
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Continued vaccination of domestic dogs against CAV-1 (with CAV-2-based vaccines) is
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recommended, whereas vaccination of foxes in the wild is unlikely to be practically or
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economically feasible. Reasonable precautions should be taken in wildlife rescue centres and
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veterinary hospitals to reduce the risk and to prevent the spread of CAV-1 amongst susceptible
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species, including red foxes, dogs, other canids, as well as some mustelids. A suitable level of
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infectious disease control should be implemented in well managed animal rescue centres and
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veterinary hospitals.
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In one of the wildlife rescue centres in the current investigation, it is likely that a fox with
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inapparent infection had been introduced and presumably was the source of CAV-1 for other foxes
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in the same or nearby pens. Therefore, quarantine and close observation of wildlife entering rescue
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centres and veterinary hospitals is recommended. Considering that the incubation period for ICH
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may be up to 1 week following exposure to CAV-1 (Cabasso 1962), this length of quarantine
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should be considered.
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Vaccination of foxes on entry to wildlife rehabilitation centres could be considered, but may only be
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effective if a quarantine period of 1 to 2 weeks is also applied, to allow sufficient time for
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vaccinated animals to produce protective neutralising antibodies. Foxes can be vaccinated with live
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attenuated vaccines, licensed for use in dogs, which contain the cross-protective virus CAV-2; most
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vaccines available routinely will also contain canine parvovirus, canine distemper virus and canine
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parainfluenza virus. The number of vaccinations will depend on the age of the animal being
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vaccinated. Long-term ‘residents’ of wildlife rehabilitation centres should be routinely vaccinated,
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at the manufacturer’s published intervals, to provide immunity to these individuals if the quarantine
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of new arrivals is not possible.
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In conclusion, multiple cases of spontaneous ICH were identified in juvenile red foxes at two
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wildlife rescue centres in Scotland. CAV-1 appears to have spread amongst susceptible animals,
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either directly or indirectly, resulting in a high case fatality rate after a short clinical course. Disease
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management protocols should be adopted in wildlife rescue centres and veterinary hospitals which
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regularly receive red foxes, in order to prevent the spread of CAV-1 among susceptible animals.
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Infectious diseases, such as ICH, which are encountered infrequently in domestic dogs in general
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practice, should be considered in foxes exhibiting indicative clinical signs.
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Acknowledgements
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The authors wish to thank staff at SSPCA Fishcross, SRUC Auchincruive, and the
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histopathology section of the Royal (Dick) School of Veterinary Studies, University of Edinburgh.
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Figure legend
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Figure 1. Photomicrographs of histological sections from foxes with infectious canine hepatitis. (A)
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Liver from fox 2, showing intranuclear inclusion bodies (arrows) in hepatocytes. (B) Kidney from
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fox 4, showing an intranuclear inclusion body (arrow) in a glomerulus. (C) Brain from fox 4,
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showing an intranuclear inclusion body (arrow) in a vascular endothelial cell. (D) Brain from fox 4,
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showing perivascular oedema and haemorrhage, along with focal degeneration and necrosis of
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neurones (arrows). Haematoxylin and eosin staining. Scale bars = 50 μm.