4-month-old, intact female, Rottweiler mix dog (Canis familiars).The puppy had a recent history of increased respiratory rate and effort progressing to dyspnea. A bronchointerstitial pattern on chest radiographs was noted. There was a lack of response to antifungals and antibiotics. The dog had a one week history of prednisone use for suspected protein-losing enteropathy. The dog had a longer history of pyoderma, chronic diarrhea, ascites, and microhepatica. The puppy had been administered two DHPP vaccines sometime prior to 2 months of age. The puppy died at home two days after being discharged.
The puppy was moderately thin with muscle wasting. There was periocular dermatitis, inflammation centered on the mucocutaneous junction, and ventral abdominal pyoderma. The lungs were mottled pale pink to dark pink with multifocal to coalescing, white to beige, flat to slightly raised, semi-firm areas in a branching pattern. The lesions were most prominent in the hilar and mid lobe regions intermixed with slightly sunken, non-crepitant, dark pink to red irregular areas of parenchyma (atelectasis). There were multifocal, fairly well-demarcated, dark red, irregular foci, 1-3 mm in diameter, throughout the lung parenchyma (hemorrhage). On cut section the lungs were mottled dark red to red to pink to beige and semi-firm. The liver was friable and mottled pale pink to dark pink. A delicate fibrin membrane was adherent to the liver capsule. The intestines were moderately dilated with watery to viscous contents. The lymphatics overlying the pancreas and associated with the duodenum and proximal jejunum were prominent.
Lung Multi-focally affecting ~50% of the section there are geographic areas of alveoli filled with neutrophils and alveolar macrophages admixed with accumulations of fibrin, hemorrhage, and siderophages centered around bronchi and bronchioles. The bronchi and bronchioles are lined by mildly to moderately hyperplastic respiratory epithelium and are filled with degenerate and non-degenerate neutrophils and sloughed epithelial cells. Occasionally the bronchial and bronchiolar walls are disrupted by inflammatory infiltrates as previously described and necrotic debris. Within these bronchi and bronchioles, a small to moderate number of scattered epithelial cells, sloughed epithelial cells, bronchial gland epithelium, and rarely pulmonary macrophages have swollen, enlarged nuclei that contain a large, 5-15um, ovoid to round, smudgy, basophilic to amphophilic intranuclear inclusion that typically completely fills the nucleus but occasionally has a thin clear rim (halo) between the inclusion and marginated chromatin. There are moderate to large numbers of lymphocytes and plasma cells, neutrophils and macrophages within the bronchial submucosa and surrounding bronchial glands with mild edema. There are infrequent thrombi within adjacent vessels. There is moderate perivascular edema around large caliber vessels within these areas. The alveolar septa multifocally are mildly expanded by neutrophils, macrophages, lymphocytes, infrequent fibrin, and rarely lined by few plump type II pneumocytes. Within affected alveoli, there are scattered pneumocytes, which are often sloughed, as well as alveolar macrophages that contain intranuclear inclusion bodies as described in the bronchial epithelium.
Pancreas The exocrine pancreatic cells in randomly distributed foci are pale with loss of zymogen granules, occasionally vacuolated (degeneration), or faded and shrunken. Nuclei of these cells are frequently absent and the cells are admixed with amorphous cellular debris (necrosis). There are low numbers of neutrophils, macrophages, and lymphocytes and plasma cells within these areas and in the interstitium. Moderate numbers of acinar cells in these areas have an enlarged nucleus with marginated chromatin and an eosinophilic to basophilic inclusion similar to that described in the bronchial epithelial cells in the lung. Multifocally large areas of the surrounding adipose tissue are hypereosinophilic with absent nuclei and concentric flocculent to amorphous eosinophilic material within the adipocytes or between them (fat necrosis). There are small numbers of macrophages, neutrophils, and lymphocytes along the periphery of these areas.
Mucocutaneous junction (not included): Multifocally there is mild parakeratotic hyperkeratosis, acanthosis, infrequent spongiosis, and occasional areas of ulceration. There are multifocal neutrophilic intracorneal pustules and superficial serocellular crusts composed of serum proteins and degenerate and non-degenerate neutrophils. There are multifocal areas in some sections of glabrous skin, with moderate numbers of lymphocytes and plasma cells, macrophages and neutrophils surrounding the labial glands. The labial glands are often absent, degenerative, or replaced by necrotic debris and sloughed cells with few intraluminal neutrophils and debris. Rarely within these glands the nuclei are enlarged with marginated chromatin and contain a large ovoid basophilic inclusion body.
1. Lung: Necrosuppurative and lymphoplasmacytic bronchitis/bronchiolitis and bronchointerstitial pneumonia, multifocal to coalescing, moderate to marked, subacute with mild bronchial epithelial hyperplasia and intraepithelial intranuclear inclusion bodies (consistent with canine adenovirus type 2).
2. Pancreas: Necrotizing and lymphohistiocytic pancreatitis, multifocal, moderate, subacute with intraepithelial intranuclear inclusion bodies (consistent with canine adenovirus type 2).
Lung (2 specimens) - No growth and no significant growth, respectively
Liver - No growth
Spleen- Staphylococcus intermedius group (mixed culture, 1+)
Canine adenovirus type 2 lung (respiratory epithelium, pneumocytes, alveolar macrophages), exocrine pancreas, and labial glands in the oral mucocutaneous junction - positive
Canine adenovirus type 2 small intestine - inconclusive
Canine distemper virus lung - negative
Canine parvovirus small intestine - negative
Virus isolation lung - Canine adenovirus isolated
Negative stain lung - Adenovirus
Canine Respiratory Panel (PCR) Lung - Canine distemper virus - positive
Canine influenza virus - negative
Canine parainfluenza virus - negative
Canine respiratory coronavirus - negative
Canine Adenovirus 2 - positive
Canine Bordetella bronchiseptica - negative
Canine adenovirus (CAV) is a non-enveloped icosahedral double stranded DNA virus found worldwide that belongs to the genus Mastadenovirus and family Adenoviridae. In addition to affecting domestic dogs, other carnivores such as foxes, coyotes, otters, and wolves are susceptible to CAV infection and antibodies have been detected in fishers, polar and black bears, sea lions, and walruses.(4,8) There are two serotypes of adenovirus, canine adenovirus type 1 (CAV1) and canine adenovirus type 2 (CAV2), that are genetically and antigenically similar yet divergent enough to allow differentiation with various diagnostic methods. The similarities allow vaccination with a parenteral modified live attenuated CAV2 in dogs to provide cross-protection against CAV1 while avoiding the well-known side effects of using a modified live CAV1 vaccine.(8,13) In countries with established systematic vaccination in the domestic dog population, circulation of the virus is limited and thus prevalence of disease is low. In unvaccinated and wild canid populations, seroprevalence is variable but significant (between 30-70% worldwide) indicating exposure to this virus is common.(5,6,8,15) CAV1 is a systemic infection that targets hepatocytes and endothelial cells and mostly affects young dogs with mortality rates between 10-30%. In contrast, CAV2 is typically limited in extent and virulence resulting in a respiratory infection that is often asymptomatic in otherwise healthy dogs. This virus is one of the organisms involved in canine infectious respiratory disease (CIRDC) complex (also called canine infectious tracheobronchitis or kennel cough) that typically occurs after exposure to other dogs (boarding, shelter, dog parks, etc.). CAV2 replicates most readily in non-ciliated bronchiolar and bronchial epithelium as well as the epithelial cells of the tonsillar crypts, pharynx, nasal mucosa, mucus cells of the trachea and bronchi, and occasionally type 2 pneumocytes.(13) In addition, replication can occur to a limited extent in the intestinal epithelial cells and CAV2 has been previously associated outbreaks of diarrhea in one group of dogs.(6)
The extensive pulmonary infection with CAV2 resulting in dyspnea and respiratory failure in this puppy in the absence of a secondary bacterial infection is unusual. Additionally the extent of infection in this puppy including the oral labial glands at the mucocutaneous junction and exocrine pancreatic acinar cells is rare. The pattern of tissue involvement is reminiscent of that described in some immunodeficient Arabian foals with equine adenoviral infection(10) and, to the authors knowledge, involvement of the pancreas with CAV2 infections in the dog has not been previously described in the literature. However, less typical locations to identify adenoviral inclusions have been noted previously in the splenic macrophages and hepatocytes in a bulldog puppy with systemic CAV2 infection.(1) In a separate report, detection of CAV2 via in situ-hybridization in the liver and spleen and via polymerase chain reaction in the brain was noted in a puppy without inclusion bodies present in any of these tissues.(3) The exact cause of the histologic pattern in this puppy is unclear and although mutations have been noted to occur in CAV3, this virus is generally considered genetically stable(8) while the significance of mutations in terms of virulence or cell tropism is unknown.
Clinical and severe pulmonary infection with CAV2 is typically attributed to an immunocompromised status or complication with other viruses or bacteria.(1) Co-infections with CIRDC are common of which infections with multiple respiratory viruses appears to be more common than previously believed.(12,14) In this puppy, both canine distemper virus (CDV) and adenovirus were detected by PCR of lung tissue but interestingly there was no immunoreactivity noted in the lung on CDV IHC and a lack of viral inclusions consistent with CDV. Although PCR and IHC testing of CDV has not been directly compared, the higher sensitivity of PCR over IHC has been noted in another veterinary infectious disease(2) and inclusion bodies were only found in 25% of CDV cases confirmed by other methods in another report7 which may explain the findings in this case. The contribution of CDV in this case is unknown and assuming that the lack of histologic or immunohistochemical detection suggests a low viral load, one of two following scenarios is likely: 1. Early infection prior to significant viral replication and cytopathic effects, or 2. Late infection after significant but incomplete clearance of the virus. With either of these scenarios, the degree of virus-induced immunosuppression can be questioned as the former is before the lymphoid destruction that induces immunosuppression and the latter would imply enough recovery of the immune system to allow significant clearance of the virus. Other reasons for possible immunosuppression also existed in this puppy including recent steroid usage and others conditions including marked lymphangiectasia with hypoproteinemia and evidence of an intrahepatic portosystemic shunt. The occurrence of clinical infection in this vaccinated puppy is likely attributable to interference of maternal antibodies which typically persist until 12-14 weeks of age (2-4 weeks beyond this puppies last vaccine) rather than emergence of a variant strain.(8,13)
1. Lung: Pneumonia, bronchointerstitial, necrotizing, multifocal to coalescing, severe with numerous intraepithelial intranuclear viral inclusion bodies, Rottweiler mix, canine.
2. Pancreas: Pancreatitis, necrotizing, multifocal to coalescing, moderate with numerous intraepithelial intranuclear viral inclusion bodies.
3. Adipose tissue, peripancreatic and mesenteric: Fat necrosis, multifocal to coalescing, with saponification.
In the lung, conference participants readily identified intranuclear inclusion bodies within intact and sloughed epithelial cells, both bronchiolar and type II pneumocytes. Along with expansion of alveolar septa with inflammatory cells and foci of alveolar septal necrosis, and megakaryocytes are also occasionally found in alveolar capillaries. Multifocally, alveoli contain a macrophage-rich exudate and there is type II pneumocyte hyperplasia. Participants also described segmental necrosis of bronchiolar epithelium, epithelial attenuation, and sloughing of epithelial cells into the lumina. Changes identified by participants in the pancreas mirrored those of the contributor and include degeneration and necrosis of pancreatic acinar epithelium; loss of zymogen granules; expansion of the interlobular areas with fibrin and edema; and presence of intranuclear inclusion bodies in the acinar epithelial cells. Other changes include necrosis and mineralization (saponification) of the peripancreatic fat.
Canine adenovirus type 2 is most often associated with mild respiratory disease, although more severe fatal disease has been documented.(1) This dogs history included vaccination only up to 8 weeks, as well as administration of corticosteroids, both of which were suggested as playing a role in the pathogenesis in this case. The differential diagnosis for viral pneumonia in the dog discussed by participants included: canine morbillivirus, canine influenza virus, canine coronavirus and canine parainfluenza virus. In this case, the intranuclear viral inclusion bodies within epithelial cells are considered distinctive for canine adenovirus type 2, making other viral etiologies less likely. Some participants speculated that a secondary bacterial infection also may have contributed to the pulmonary changes in this dog.
Viruses are well-known to play a role in the pathogenesis of bacterial pneumonias by damaging the respiratory epithelium, inhibiting bacterial clearance and facilitating bacterial adhesion. Other participants commented that a contributing bacterial etiology likely would have resulted in a more neutrophil-rich inflammatory component in the lung. Bacterial agents of canine infectious respiratory disease include Bordetella bronchiseptica, Mycoplasma spp., and Streptococcus equi sp. zooepidemicus.(14)
The pancreatic involvement in this case is uncommon in typical infections with canine adenovirus. Pancreatitis due to adenovirus infection is well documented in nonhuman primates; adenoviral infection in a number of non-human primate species can result in pancreatic necrosis and fibrosis with chronic-active inflammation. Pancreatic involvement typically occurs in more clinically severe cases, typically with underlying immunosuppression; other lesions also include hepatitis and nephritis. Adenovirus infection in immunocompetent nonhuman primate hosts usually presents as self-limiting respiratory and gastrointestinal infections.(9) Fowl adenovirus, a common disease in chickens, results in necrotizing pancreatitis, gizzard erosion, and inclusion body hepatitis.(11)
We appreciate the excellent supporting materials with the submission, including laboratory data, gross, immunohistochemistry, and electron microscopy images. These additional materials greatly enhances the teaching value of this case.
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