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1 Infectious canine hepatitis in red foxes (Vulpes vulpes) in wildlife rescue centres in the United 2 Kingdom 3 4 D. Walker, E. Abbondati, A. L. Cox, G. B. B. Mitchell, R. Pizzi, C. P. Sharp, A. W. Philbey 5 6 David Walker, BSc(Hon), BVM&S, MSc, MRCVS, 7 Royal (Dick) School of Veterinary Studies and the Roslin Institute, University of Edinburgh, Easter Bush, Edinburgh EH25 9RG, United Kingdom 8 Erika Abbondati, DVM, MRCVS, 9 Royal (Dick) School of Veterinary Studies and the Roslin Institute, University of Edinburgh, Easter Bush, Edinburgh EH25 9RG, United Kingdom 10 Alistair L. Cox, BVMS, MSc, FRCPath, MRCVS, 11 Veterinary Services, SAC Consulting, Scotland’s Rural College (SRUC), Edinburgh EH26 0QE, United Kingdom 12 George B. B. Mitchell, BVMS, PhD, 13 Veterinary Services, SAC Consulting, Scotland’s Rural College (SRUC), Auchincruive, Ayr KA6 5AE, United Kingdom 14 Romain Pizzi, BVSc, MSc, DZooMed, DipECZM, MANZCVS, FRES, FRGS, FRSB, MRCVS, 15 Scottish Society for the Prevention of Cruelty to Animals, National Wildlife Rescue Centre, Fishcross, Clackmannanshire FK10 3AN, United 16 Kingdom, and Royal Zoological Society of Scotland, Corstorphine Road, Edinburgh, EH12 6TS, United Kingdom 17 Colin P. Sharp, BSc(Hons), PhD, 18 The Roslin Institute, University of Edinburgh, Easter Bush, Edinburgh EH25 9RG, United Kingdom 19 Adrian W. Philbey, BVSc(Hon), PhD, FRCPath, MANZCVSc(Path), FHEA, MRCVS, 20 Royal (Dick) School of Veterinary Studies and the Roslin Institute, University of Edinburgh, Easter Bush, Edinburgh EH25 9RG, United Kingdom 21 22 23 E-mail for correspondence: [email protected] 2 24 25 Summary Outbreaks of infectious canine hepatitis (ICH) are described in red foxes (Vulpes vulpes) at 26 two wildlife rescue centres in the United Kingdom. Disease occurred in 2 to 4 month old juvenile 27 foxes, which were held in small enclosures in groups of three to eight animals. The foxes died or 28 were euthanased after a short clinical course, sometimes including neurological signs and jaundice, 29 with a high case fatality rate. Four red foxes submitted for postmortem examination had enlarged, 30 congested livers, with rounded borders and mild accentuation of the lobular pattern. On histological 31 examination, there was random, multifocal to massive hepatic necrosis, along with multifocal 32 vasculitis in the central nervous system (CNS) and mild, multifocal glomerulonephritis. Intranuclear 33 inclusion bodies, typical of canine adenovirus type 1 (CAV-1) infection, were present in 34 hepatocytes, vascular endothelial cells in the CNS, renal glomeruli and renal tubular epithelial cells. 35 CAV-1 was detected in tissues from affected foxes by the polymerase chain reaction and 36 sequencing. Congregation of juvenile foxes in wildlife rescue centres is likely to be a risk factor for 37 transmission of CAV-1. Preventative measures in wildlife centres should be implemented to prevent 38 the spread of the virus among conspecifics and to other susceptible species. 39 3 40 41 Introduction Early laboratory studies showed that red foxes (Vulpes vulpes) are susceptible to 42 experimental infection with canine adenovirus type 1 (CAV-1), the cause of infectious canine 43 hepatitis (ICH) (Green and others 1930). However, the pathology and clinical signs of ICH in 44 naturally infected, free-ranging canids are not well characterised. Red foxes in the United Kingdom 45 (Thompson and others 2010) and Italy (Balboni and others 2013) appear to be a wildlife reservoir of 46 CAV-1. Serological exposure to untyped CAV in several free-ranging carnivore species has been 47 reported worldwide (Amundson and Yuill 1981; Thompson and others 2010; Truyen and others 48 1998), suggesting that CAV-1 may be sustained in wild canid populations. However, there are only 49 a few reports of the occurrence of spontaneous ICH in free-ranging foxes (Woods 2001), limited to 50 descriptions of isolated cases in a grey fox (Urocyon cinereoargenteus) (Gerhold and others 2007) 51 and three red foxes (Thompson and others 2010). 52 53 It has been hypothesised that sporadic transmission of CAV-1 from infected red foxes to susceptible 54 (unvaccinated) domestic dogs may occur through contact with infected excretions, such as urine and 55 faeces (Thompson and others 2010). Widespread vaccination of dogs in the United Kingdom, in 56 combination with responsible dog ownership and effective control of stray dogs, is likely to have 57 reduced the incidence of transmission of CAV-1 amongst dogs to a low, and probably 58 unsustainable, level. However, the possibility of infection through direct or indirect contact with 59 susceptible wildlife, or unvaccinated dogs, may be a mechanism allowing the occasional 60 presentation of ICH in dogs to veterinary surgeons in general practice. Furthermore, concern has 61 been expressed about the emergence of infectious diseases, such as canine distemper (Walker and 62 others 2014), rabies and Echinococcus multilocularis (Bourne and others 2015), in the United 63 Kingdom as a consequence of both legal and illicit animal movements. Therefore, veterinary 64 surgeons should be aware of the clinical signs and diagnoses of such diseases, and also be conscious 4 65 that wildlife, such as foxes, which occasionally are presented for veterinary intervention, are also 66 susceptible to many diseases of the domestic dog. 67 68 In the Mediterranean, ICH has been reported as an ‘old’ disease which is re-emerging (Decaro and 69 others 2007). Other cases of ICH in dogs and foxes have been reported in the past decade in Europe 70 (Gleich and others 2009, Müller and others 2010, Thompson and others 2010), Asia (Wen and 71 others 2009, Cheema and others 2012), North America (Wong and others 2012, Headley and others 72 2013) and South America (Inkelmann and others 2007, Oliveira and others 2011). However, this 73 published evidence suggests that the frequency of occurrence of ICH has not necessarily changed 74 substantially in the last 25 years. Despite this, in conjunction with evidence of serological exposure 75 in wildlife, such reports support the hypothesis that there is on-going exposure and transmission of 76 CAV-1 to susceptible species. There is no current evidence to suggest that the incidence of ICH in 77 dogs is higher in areas with a greater wildlife disease burden. However, in principal, there is 78 potential for disease ‘spill over’ events from red foxes to unvaccinated dogs. 79 80 In this paper, the clinical and pathological findings in juvenile red foxes that died during outbreaks 81 of ICH at two wildlife rescue centres in Scotland are described. To the authors’ knowledge, this is 82 the first detailed report of ICH in multiple red foxes affected during disease outbreaks in wildlife 83 rescue centres, whereas previously ICH was reported in free-ranging red foxes in the United 84 Kingdom (Thompson and others 2010). Furthermore, the present study confirms the presence of 85 CAV-1 in the United Kingdom by the polymerase chain reaction (PCR) and sequencing. These 86 findings provide further evidence that CAV-1 is present in red foxes in the United Kingdom, that it 87 sporadically causes fatal disease in this species, and that it may also infect conspecifics. 88 89 Materials and methods 5 90 History and pathological examinations 91 Foxes 1, 2 and 3 were juvenile red foxes from different litters that had been orphaned and 92 were admitted to the Scottish Society for the Prevention of Cruelty to Animals (SSPCA) National 93 Wildlife Rescue Centre, Fishcross, Scotland, in May 2013 to be reared with the aim to release them 94 back to the wild. In early June 2013, fox 1 died and fox 2 was euthanased due to collapse and 95 seizures. Subsequently, fox 3, from the same group of animals, died suddenly overnight. In the 96 same month, a further three associated foxes were found dead and a further three foxes were 97 euthanased after exhibiting seizures. 98 99 Fox 4 died at Hessilhead Wildlife Rescue Centre, Beith, Scotland in June 2011. It was one 100 of four red foxes that had died at the centre within 2 weeks. The cub was enclosed in outdoor pens 101 with five others, one of which exhibited convulsions prior to death. Fox 4 appeared to be normal 102 when inspected by the staff at the centre on the day prior to death. The only other notable finding in 103 the history of this group of animals was evidence of diarrhoea on the floor of the enclosure. 104 105 Post-mortem examination and histopathology 106 Foxes 1, 2 and 3 were submitted to the Royal (Dick) School of Veterinary Studies, 107 Edinburgh, Scotland, for post-mortem examination in June 2013. Samples of liver (foxes 2 and 3), 108 brain (fox 2) and lung (foxes 1 and 3) were collected into viral transport medium (10% phosphate 109 buffered glycerol saline) and stored at -20 °C. Fox 4 was submitted to Scotland’s Rural College 110 (SRUC), Auchincruive, Scotland, for post-mortem examination. A range of tissues, including brain, 111 spleen, heart, mesenteric lymph node, liver, kidney and small intestine, were fixed in formalin, 112 embedded in paraffin wax and stained with haematoxylin and eosin (H&E) for histopathological 113 evaluation. 114 6 115 Polymerase chain reaction and sequencing 116 DNA was extracted from tissue samples from foxes 1, 2 and 3 using the AllPrep DNA/RNA 117 Mini kit (Qiagen, Hilden, Germany). DNA was extracted from ~20 µm thick sections of formalin- 118 fixed paraffin-embedded (FFPE) tissues from fox 4 using the QIAamp FFPE DNA Tissue kit 119 (Qiagen). 120 121 The PCR for adenoviral DNA polymerase sequences was performed using a nested protocol 122 adapted from Wellehan and others (2004). The PCR reaction mixture contained 2 µL DNA, 36.5 µL 123 H2O, 10 µL 5x GoTaq reaction buffer (Promega, Madison, WI, USA), 0.5 µL ‘polFouter’ forward 124 primer (100 µM), 0.5 µL ‘polRouter’ reverse primer (100 µM), 0.3 µL deoxynucleotide 125 triphosphates (10 mM) and 0.2 µL (1 U) GoTaq G2 DNA polymerase (Promega). The samples 126 underwent two rounds of conventional nested PCR (94 °C for 5 minutes, 45 cycles of 94 °C for 30 127 seconds, 46 °C for 1 minute and 72 °C for 1 minute, followed by a final extension at 72 °C for 5 128 minutes); then, 2 µL PCR product from the first round was used as a template for the second round, 129 under the same conditions, using the internal primers ‘polFinner’ and ‘polRinner’, described by 130 Wellehan and others (2004). The expected amplicon size for CAV-1 is 321 base pairs (bp). The 131 resulting amplicons were visualised using the G:BOX gel imaging system (Syngene, Cambridge, 132 UK) following separation by gel electrophoresis in agarose containing SYBR Safe DNA Gel Stain 133 (Invitrogen, Paisley, UK). Sanger sequencing was performed using the internal primers (Edinburgh 134 Genomics, University of Edinburgh, United Kingdom). 135 136 Results 137 Gross pathology 138 Fox 1 7 139 Fox 1 was a juvenile male red fox with a moderate degree of autolysis. The liver was 140 moderately enlarged, friable and brown, with mild accentuation of the lobular pattern. There was 141 mild congestion of the meninges. 142 143 Fox 2 144 In fox 2, a male juvenile red fox, the liver was mildly enlarged and dark red, with 145 multifocal, irregular areas of brown discolouration. The intestinal mucosa had multifocal patches of 146 red discolouration. There was mild, diffuse, congestion of the meninges. 147 148 Fox 3 149 Fox 3 was a juvenile red fox of unknown sex in poor body condition and with jaundice. The 150 liver was mildly enlarged and dark brown, with poorly defined pale patches. The spleen and 151 mesenteric lymph nodes were mildly enlarged. The lumen of the stomach, small intestines and 152 proximal large intestines contained dark red, haemorrhagic fluid. 153 154 Fox 4 155 156 Fox 4 was a female juvenile red fox weighing 2 kg. The liver and kidneys were moderately congested. Moderate splenomegaly was evident. 157 158 Histopathology 159 Fox 1 160 Histopathology of the liver of fox 1 revealed moderate to severe, random, multifocal to 161 massive necrosis of hepatocytes. Amphophilic intranuclear inclusion bodies, including many 162 classical Cowdry type A inclusion bodies, were present within numerous hepatocytes. There was 163 dissociation and vacuolation of hepatocytes. Blood vessels and hepatic sinusoids contained fibrin 8 164 thrombi. In the lungs, there were mild interstitial infiltrates of lymphocytes and macrophages in 165 alveolar walls, along with mild interstitial oedema and formation of fibrin thrombi within blood 166 vessels. Small numbers of intra-alveolar macrophages were present. In random, small blood vessels 167 within the cerebral cortex, there was segmental swelling of vascular endothelial cells, mild 168 perivascular oedema, occasional individual cell degeneration and rare intranuclear inclusion bodies. 169 170 Fox 2 171 The liver of fox 2 exhibited moderate to severe, random, multifocal to massive necrosis of 172 hepatocytes, along with dissociation and vacuolation of hepatocytes, and expansion of the spaces of 173 Disse. Numerous intranuclear inclusion bodies were present in hepatocytes (Figure 1A). In the 174 kidney, there were occasional intranuclear inclusion bodies in probable mesangial cells in glomeruli 175 and in epithelial cells lining the proximal convoluted tubules. Occasional degeneration of individual 176 cells, probably mesangial cells, was evident in glomeruli. There was mild vacuolation of epithelial 177 cells, mainly in the proximal convoluted tubules, along with small amounts of proteinaceous and 178 sometimes cellular material in tubule lumina. There were no significant findings in the brain. 179 180 Fox 3 181 In the liver of fox 3, there was severe, massive, hepatic necrosis, with dissociation, 182 vacuolation and fragmentation of hepatocytes, and numerous intranuclear inclusion bodies. There 183 was a mild increase in the number of neutrophils within hepatic sinusoids. In the lung, there were 184 increased numbers of macrophages and lymphocytes in alveolar walls, along with individual cell 185 degeneration, probably of leucocytes. The brain was not examined. 186 187 Fox 4 9 188 The liver of fox 4 exhibited moderate, random, multifocal hepatic necrosis, with occasional 189 intranuclear inclusions. In the kidney, rare intranuclear inclusion bodies were present in probable 190 mesangial cells in glomeruli (Figure 1B) and there was occasional individual degeneration of 191 epithelial cells lining proximal convoluted tubules. There was moderate lymphocytolysis within 192 lymphoid follicles in the white pulp of the spleen and in the cortex of the mesenteric lymph node. 193 Moderate numbers of intranuclear inclusion bodies were present in endothelial cells of blood 194 vessels apparently scattered at random throughout the brain (Figure 1C), often associated with 195 perivascular haemorrhage and occasionally with mild, focal degeneration and necrosis of neurones 196 (Figure 1D). 197 198 Polymerase chain reaction and sequencing 199 Adenoviral sequences were detected by PCR in frozen tissues processed from foxes 1 200 (lung), 2 (brain and liver) and 3 (lung and liver), as well as in FFPE brain and pooled spleen, 201 mesenteric lymph node and cardiac muscle from fox 4. The primers amplified a 272 bp segment of 202 the adenoviral DNA polymerase gene in foxes 1, 2 and 3 when the primer sequences are excluded 203 (Wellehan and others 2004). Shorter fragments were sequenced from the FFPE samples (fox 4). The 204 sequences from all foxes had a 100% match to CAV-1 (GenBank AC_000003.1), excluding the 205 possibility of infection with CAV-2 (GenBank AC_000020.1). 206 207 Discussion 208 In this study, four cases of spontaneous ICH were confirmed by clinical and pathological 209 examination, supported by PCR and confirmation of CAV-1 by sequencing, in juvenile red foxes 210 that died at two wildlife rescue centres in Scotland. Several other foxes died within a period of 211 weeks during the outbreaks of ICH at both wildlife rescue centres. The clinical histories suggested 212 that the disease was peracute to acute and appears to have followed a similar course to the classical 10 213 “fulminating form” of ICH (Parry 1950). In ICH, clinical signs develop after an incubation period 214 of 2-6 days (Cabasso 1962). 215 216 The gross and histopathological findings in affected red foxes in the present study were consistent 217 with ICH. The most frequent gross changes were enlargement and altered colour of the liver. 218 Jaundice was prominent in one fox. Oedema of the wall of the gall bladder may be observed in dogs 219 with ICH (Decaro and others 2012), but was not evident in the red foxes in the present study. 220 Similarly, there was no evidence of corneal opacity (“blue eye”) in the red foxes included in this 221 investigation. On histological examination of the liver, intranuclear inclusion bodies were evident in 222 hepatocytes, along with hepatocyte necrosis. Inclusion bodies were also seen within glomeruli and 223 occasionally in renal tubular epithelial cells in the kidneys. A notable feature in the present cases 224 was the presence of vasculitis and intranuclear inclusion bodies in vascular endothelial cells in the 225 brain. Central nervous system lesions were not described in a previous study of ICH in free-ranging 226 red foxes, since brain was not available from these cases (Thompson and others 2010). 227 228 Wildlife rescue centres in the United Kingdom receive numerous juvenile foxes in spring and early 229 summer, usually as orphans due to misadventure or disease in the vixens. Congregation of juvenile 230 foxes in pen groups is likely to provide an opportunity for transmission of infectious disease. The 231 juvenile foxes in the current study had a rapid onset and short duration of disease, with a high case 232 fatality rate. Juvenile red foxes are likely to be susceptible to infection and at risk of severe ICH if 233 they have not received maternal antibodies against CAV-1 or if maternal immunity has waned. It is 234 also possible that other diseases, such as parasitism, emaciation or captivity-induced stress, may 235 have affected the susceptibility of fox cubs to ICH. 236 11 237 Further investigation into the origin of the outbreak at one of the wildlife centres revealed that the 238 usual quarantine procedures for new admissions had not been followed. In particular, a newly 239 introduced fox cub was allowed to have direct contact with a group of juvenile foxes that had been 240 at the centre for several weeks. Congregation of susceptible juvenile foxes with conspecifics in 241 captivity is likely to be a risk factor for the spread of CAV-1 and the occurrence of outbreaks of 242 ICH. Transmission of CAV-1 occurs through direct exposure to infected animals, or indirect contact 243 via contaminated urine, faeces or ocular or nasal secretions (Decaro and others 2012; Woods 2001). 244 The duration of persistence of CAV-1 in the environment in such excretions is uncertain, although 245 the virus has been shown to persist and remain infective for several months at temperatures below 246 4°C (Decaro and others 2012). The course of the disease outbreak at the second wildlife centre and 247 the underlying factors are unknown. 248 249 It is clear that ICH is frequently manifested as a fatal disease in both red foxes and dogs. The 250 majority of the literature reporting disease due to ICH in red foxes is based on experimental 251 infections (Green and others 1930) or as reports of isolated cases of naturally acquired disease 252 (Thompson and others 2010). However, there is serological evidence of untyped CAV antibodies in 253 free-ranging red foxes in Europe (Truyen and others 1998; Thompson and others 2010), as well as 254 other parts of the world, suggesting that a proportion of free-ranging red foxes are exposed to the 255 virus and will survive without fatal disease. 256 257 Infectious diseases, such as ICH, should be considered when undertaking veterinary assessment of 258 sick red foxes submitted to wildlife rescue centres. It is also important to note that CAV-1 will be 259 excreted by red foxes with ICH, as well as by animals incubating the disease and possibly by 260 clinically healthy foxes. Moreover, since domestic dogs are susceptible to ICH, the possibility of 261 transmission of CAV-1 from red foxes to dogs (and vice versa), either directly or through fomites, 12 262 should be considered. There is some evidence that CAV-1 can be shed in the urine of apparently 263 recovered dogs for up to 9 months following experimental infection with CAV-1 (Baker and others 264 1954). 265 266 Continued vaccination of domestic dogs against CAV-1 (with CAV-2-based vaccines) is 267 recommended, whereas vaccination of foxes in the wild is unlikely to be practically or 268 economically feasible. Reasonable precautions should be taken in wildlife rescue centres and 269 veterinary hospitals to reduce the risk and to prevent the spread of CAV-1 amongst susceptible 270 species, including red foxes, dogs, other canids, as well as some mustelids. A suitable level of 271 infectious disease control should be implemented in well managed animal rescue centres and 272 veterinary hospitals. 273 274 In one of the wildlife rescue centres in the current investigation, it is likely that a fox with 275 inapparent infection had been introduced and presumably was the source of CAV-1 for other foxes 276 in the same or nearby pens. Therefore, quarantine and close observation of wildlife entering rescue 277 centres and veterinary hospitals is recommended. Considering that the incubation period for ICH 278 may be up to 1 week following exposure to CAV-1 (Cabasso 1962), this length of quarantine 279 should be considered. 280 281 Vaccination of foxes on entry to wildlife rehabilitation centres could be considered, but may only be 282 effective if a quarantine period of 1 to 2 weeks is also applied, to allow sufficient time for 283 vaccinated animals to produce protective neutralising antibodies. Foxes can be vaccinated with live 284 attenuated vaccines, licensed for use in dogs, which contain the cross-protective virus CAV-2; most 285 vaccines available routinely will also contain canine parvovirus, canine distemper virus and canine 286 parainfluenza virus. The number of vaccinations will depend on the age of the animal being 13 287 vaccinated. Long-term ‘residents’ of wildlife rehabilitation centres should be routinely vaccinated, 288 at the manufacturer’s published intervals, to provide immunity to these individuals if the quarantine 289 of new arrivals is not possible. 290 291 In conclusion, multiple cases of spontaneous ICH were identified in juvenile red foxes at two 292 wildlife rescue centres in Scotland. CAV-1 appears to have spread amongst susceptible animals, 293 either directly or indirectly, resulting in a high case fatality rate after a short clinical course. Disease 294 management protocols should be adopted in wildlife rescue centres and veterinary hospitals which 295 regularly receive red foxes, in order to prevent the spread of CAV-1 among susceptible animals. 296 Infectious diseases, such as ICH, which are encountered infrequently in domestic dogs in general 297 practice, should be considered in foxes exhibiting indicative clinical signs. 298 299 Acknowledgements 300 The authors wish to thank staff at SSPCA Fishcross, SRUC Auchincruive, and the 301 histopathology section of the Royal (Dick) School of Veterinary Studies, University of Edinburgh. 302 303 References 304 AMUNDSON, T.E. & YUILL, T.M. (1981) Prevalence of selected pathogenic microbial agents in 305 the red fox (Vulpes fulva) and gray fox (Urocyon cinereoargenteus) of southwestern 306 Wisconsin. Journal of Wildlife Diseases 17, 17-22 307 BALBONI, A., VERIN, R., MORANDI, F., POLI, A., PROSPERI, S. & BATTILANI, M. 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Photomicrographs of histological sections from foxes with infectious canine hepatitis. (A) 374 Liver from fox 2, showing intranuclear inclusion bodies (arrows) in hepatocytes. (B) Kidney from 375 fox 4, showing an intranuclear inclusion body (arrow) in a glomerulus. (C) Brain from fox 4, 376 showing an intranuclear inclusion body (arrow) in a vascular endothelial cell. (D) Brain from fox 4, 377 showing perivascular oedema and haemorrhage, along with focal degeneration and necrosis of 378 neurones (arrows). Haematoxylin and eosin staining. Scale bars = 50 μm.