Two types of adenoviruses CAV-1 and CAV-2 are described in dogs. CAV-1 causes a severe generalized disease called ICH, whereas CAV-2 causes a mild respiratory disease
(Zee et al., 2004). ICH is transmitted via ingestion of faeces, urine, blood, saliva and nasal discharge of infected dogs
(Willis, 2000). Again, animals that survive the infection reportedly carry and shed the virus through nasal discharge and urine, for at least 6 to 9 months and 11 months, respectively
(Piacesi et al., 2010). After being contracted naso-orally, the virus initially replicates in the tonsillar crypts and Peyer patches, followed by viremia and disseminated infection. CAV-1 is known to have tropism for vascular endothelial cells and hepatocytes
(Zee and Maclachlan, 2004), renal parenchyma, spleen and lungs become infected as well.
Haemogram
The haemogram showed lowered white blood cell (WBC) count as leukopenia, neutropenia and lymphopenia. WBC count ranging from 7.1 x 103/µl to 4.2 x 103/µl showed leucopenia (shift to left). Marked thrombocytopenia (11,000 to 78,000/µl) was evident that could be attributed to platelet clumping as observed during the blood smear examination, but increased consumption due to disseminated intravascular coagulation (DIC) could not be ruled out. There was a moderate left shift (neutrophil count 68% consistent with inflammation and moderate lymphopenia 26% that is attributed to stress and/or exogenous glucocorticoids (Table 1). Blood samples collected during clinical sickness showed a severe anaemia with haemoglobin level ranging from 4.1 g% to 7.5 g% (average 5.8 g%). Red blood cell (RBC) count was 3.0-6.5 x 106/µl. Leukopenia and lymphopenia recorded in the study are important paraclinical observations in ICH affected dogs that were also documented earlier
(Mosallanejad et al., 2010). The finding of anaemia in the pups might also be attributed to severe flea infestation concomitant with acute blood loss due to CAV-1 infection.
Serum biochemical analysis
Serum biochemical analysis showed moderate hyperbilirubinemia (6 ìmol/L against normal range up to 1 to 4 μmol/L); marked increase in alkaline phosphatase (ALP) at 201 to 283 U/L (normal range: 9-90 U/L) and aspartate transaminase (AST) at 42 to 71 U/L (normal range 13-15 U/L). It is noteworthy to mention that all the reference range of values have been considered from adult canine (Table 2). Moderate hyperbilirubinemia, marked increase in ALP and AST are consistent with hepatocellular injury and cholestasis. Activity levels of ALT and AST could be evaluated due to marked haemolysis that might have also contributed to increased bilirubin level. The increased levels of these enzymes are in agreement with those reported previously
(Mosallanejad et al., 2010) during CAV-1 infection in dog.
Macroscopical findings at necropsy
In our case study, the pups were less than one year of age and external examination of the carcasses revealed mild emaciation and pale mucous membranes. The four dhole pups died with a high case fatality rate after a short clinical course of exhibiting fever, excessive salivation, lymphadenopathy, abdominal pain, loss of appetite, lethargy, severe icterus, neurological signs, paddling of legs, nystagmus, hind limb ataxia, seizures, paralysis, disorientation, anterior uveitis, petechiation. They also showed signs associated with inflammation, liver disease (jaundice) and dysregulation in coagulation, but lacked the classic ocular signs.
The clinical symptomatology described here agreed with those described by earlier workers (
Zee and Maclachlan, 2004;
Decaro et al., 2008; Piacesi et al., 2010). Similar signs have also been described by earlier workers
(Mosallanejad et al., 2010) in a 3-month-old male German shepherd dog. Neonates are usually protected against CAV infection by maternal antibody
(Willis, 2000), but in the present study as the adult female dhole was not vaccinated, the pups suffered from acute infection. The source of infection might have been from stray dogs those were traced living around the enclosure. There were many reports of adenoviral infections in dogs in and around the region and these stray dogs were not vaccinated against ICH.
Macroscopical findings during necropsy include severe icterus of visible mucous membranes and the subcutaneous tissue. There was generalized icterus, marked haemorrhage in the oral mucosa, subcutaneous tissues in the sub-mandibular region extending from tip of the lower jaw up to the thoracic inlet. Yellow gelatinous material was present in the sub-mandibular area with oedema in 3 out of 4 pups (Fig 2). Liver showed the petechial to ecchymotic haemorrhages, yellowish discoluration with mottled appearance, diffusely enlarged with round borders having a zonal pattern with alternating red and yellow areas due to the haemorrhages and icteric changes on the surface of the liver (Fig 3). Lungs were severely congested with patchy areas of consolidation. (Fig 4). Kidneys were normal in size but yellowish in colour. The pelvis region was more yellowish might be due to soft tissue and urine accumulation (Fig 5). Among lymphoid organs, spleen showed slight enlargement in three pups with black areas on the surface indicating the sub-capsular haemorrhages (Fig 6). Other organs such as lymph nodes, stomach, intestine and hearts were also congested.
Histopathological observations
Light microscopic examination of HandE-stained sections of liver showed marked multifocal hepatocellular necrosis, severe congestion in sinusoids and around the central vein. The hepatocytes in the necrotic zone showed marked changes such as more eosinophilic cytoplasm with karyorrhexis and karyolysis of nuclei. (Fig 7, 8, 9). Hepatocytes and Kupffer cells contained round to ovoid, magenta coloured basophilic intranuclear inclusion bodies measuring 4-8 µm in diameter with marginated chromatin (Fig 10).
The most significant lesion reported in literature and observed in this study was necrohemorrhagic hepatitis
(Chouinard et al., 1998) along with hepatomegaly has been compatible with CAV-1 infection. Gross necropsy findings of all the dead animals are suggestive of ICH
(Greene, 2006). The basophilic intranuclear inclusion bodies observed in the study have earlier been reported within the hepatocytes and also in CNS vascular endothelium
(Caudell et al., 2005). However, both eosinophilic and basophilic (amphophilic) inclusions in peracute ICH were earlier documented in young Alsatian and Labrador male dogs
(Cheema et al., 2012). Based on all these histopathologic findings, the diagnosis was indicative of ICH.
Although viral detection by classical or molecular methods demonstrated the presence of CAV-1 in tissues, the observation of nuclear inclusion bodies in the liver through histopathological examination
(Duarte et al., 2014), which was satisfied in the study. Significant histopathological alterations were observed within most tissues collected during necropsy. Finally, the systemic ICH with involvement of CAV-1 was presumptively diagnosed by the characteristic clinical manifestation of necrotizing hepatitis associated with intranuclear inclusion bodies predominantly within Kupffer cells and hepatocytes
(Brown et al., 2007; Greene, 2012). Similar histopathological findings along with intranuclear inclusion bodies in hepatocytes were also reported in three cases of ICH in free-ranging red foxes (
Vulpes vulpes) in England satisfying the isolation of CAV-1
(Thompson et al., 2010). Liver and kidney have significantly been classified as the ‘sentinel’ organs for CAV-1 infection. Liver is main organ affect during the acute stage of ICH in dogs (Decaro
et al., 2012), while the kidney is involved in the ‘chronic’ and the virus shed in the urine
(Baker et al., 1954). Focal mesangial sclerosing glomerulonephritis and interstitial nephritis were reported in acute spontaneous cases of ICH
(Wright, 2008). Example of CAV-1 infection involving brain lesions (encephalopathy) has also been documented earlier in Labrador retriever pups
(Caudell et al., 2005). The precise cause of death in ICH is uncertain though liver is the primary site of viral injury. However, severe organ damage or the development of DIC might also play role.
Bacteriological culture
Bacteriological culture was found to be negative for significant bacterial pathogens such as
Salmonella,
Campylobacter, enterotoxigenic
Escherichia coli and
Clostridium perfringens in all four cases.
PCR findings
Additional degree of confidence towards confirmatory diagnosis was obtained from the conventional PCR assay that successfully amplified the 508 bp fragment of the E region of CAV-1 (Fig 11) in hepatic tissue homogenates of the suspected pups submitted for necropsy.
Previously, CAV-1 causing ICH was detected using PCR
(Kiss et al., 1996). CAV-1 was reported in a 10-month-old female fennec fox (
Vulpes Zerda) further confirmed by virus isolation and nucleotide sequencing
(Choi et al., 2014). ICH was also reported in juvenile red foxes in wildlife rescue centres in the United Kingdom (UK), where CAV-1 was detected in tissues from affected foxes by PCR and sequencing
(Walker et al., 2016). Nucleic acid-based technique has proved to be most useful in detecting ICH in many other reported instances. Four outbreaks of ICH occurring in Italy between 2001 and 2006 confirmed by PCR have been reported
(Decaro et al., 2007). For the first time,
Pizzurro et al., (2017) reported the isolation and whole genome sequence of a CAV-1 isolate from the liver of a free-ranging wolf (
Canis lupus) in Southern Italy.
Duarte et al., (2014) described fatal CAV-1 acute infection in an unvaccinated 56-day-old Yorkshire terrier pup in Portugal, where this is considered a rare infection.
Knowles et al., (2018) have recently detected ICH in a free-ranging brown bear (
Ursus arctos horribilis) cub from Alaska, USA with demonstration of both intranuclear inclusion bodies in hepatocytes and immunohistochemical reactivity to adenoviral antigens along with sequencing of the hexon protein gene that showed 100% identity to CAV-1.
The infection of the dhole pups in our study could likely be due to a combination of factors. Firstly, the parent dholes were not vaccinated due to practical difficulties. Secondly, the pups could not be vaccinated at 4 and 6 weeks of age, because of the constraint in their handling in free range condition without any restraining cages in the enclosure. Thirdly, there are no guidelines for vaccination schedule and dosage for dholes in India. Since dhole is a canid, zoo vets feel that the vaccination schedule might be similar to dogs and follow the dog vaccination schedule and dog vaccines, since no separate vaccines are available for the said species. Fourthly, it is known that CAV-1 is endemic in the domestic and stray dogs in the nearby places of the Biological Park at Bengaluru. Being housed indoors, the pups could have acquired the infection via contaminated fomites from the stray dogs roaming around their enclosure, for which the entire litter was exposed to infection leading to fatal ICH within a short period of time. The incidence of CAV-1 infection in dogs appears to have decreased worldwide since the 1950’s due to vaccination of dogs with CAV-2, which is antigenically similar to CAV-1
(Philippa, 2010). Modified live vaccines (MLVs) induce specific antibodies persisting for as long as 14 years conferring full protection against ICH. Recently, a canine hepatitis DNA vaccine has also been developed
(Liu et al., 2008).
