Short Communication
Combined distemper-adenoviral pneumonia in a dog
Luis E. Rodríguez-Tovar, Rafael Ramírez-Romero, Alicia M. Nevárez., Jxxxx J ZárateRamos and Alfonso López
Departamento de Patología, Facultad de Medicina Veterinaria y Zootecnia, UANL. Avenida
Lázaro Cárdenas 4600 pte. Unidad Universitaria Mederos, Monterrey, N.L. CP 64930,
México (Rodríguez-Tovar, Ramírez Romero, Valdez-Nava, Nevárez-Garza, Zárate-Ramos);
Department of Pathology and Microbiology, Atlantic Veterinary College, UPEI. 550
University Avenue, Charlottetown, PEI, C1A4P3, Canada (López).
Correspondence to
Dr. A. López
lopez@upei.ca
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Summary
A 2 ½-months-old, female, pug with history of anorexia, dyspnea, oculonasal discharge and
seizures was submitted for postmortem examination with the tentative diagnosis of canine
distemper. On necropsy the lungs had moderate crainoventral consolidation.
Microscopically, there was necrotizing bronchointerstitial pneumonia with two distinct types
of inclusion bodies. The fist type consisted of oval acidophilic and intracytoplasmic
inclusions consistent with canine distemper virus (CDV) infection. The second type consisted
of large intranuclear inclusion compatible with canine adenovirus type 2 (CAV-2). Also,
intranuclear inclusion bodies were found in the cerebellar white matter. CDV and CAV-2
antigens tested positive in the lung by immunohistochemistry. Crystalline arrays consistent
with CAV-2 were also observed by EM in the nuclei of the respiratory epithelium. Although
dual CD and CAV-2 infection appear to be rare in dogs, a recent report suggests that
combined infections are more common in some countries than previously suspected and
confirmation should be done by histochemistry.
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An 8-week-old female pug puppy purchased from a local kennel was taken to a local
veterinarian for routine check-up and vaccination. On clinical examination the puppy
appeared healthy and was vaccinated for distemper, ____ [listar agentes y nombre de la
vacuna]. __ wks later a second boost was given to the puppy. ____ days after receiving the
second vaccination the dog became lethargic, anorectic and started to developed dyspnea with
bilateral serous oculonasal discharge. The dog was taken to the local veterinarian and was
treated with antibiotics [cuales, dosis, via de administracion], vitamins [cuales, dosis I vía]
and electrolytes [dosis y via]. The respiratory signs continued in spite of treatment and on
day ___ after initial presentation the puppy developed neurologic signs characterized by
rhythmic contraction of the masticatory muscles and progressive myoclonus. On day ___
the puppy started having recurrent episodes of seizures. Based on the respiratory and
neurological signs canine distemper was suspected. ¿se hizo hematología? On day 25 the
dog died and was submitted for postmortem examination to the Diagnostic Laboratory of the
Faculty of Veterinary Medicine, University of Nuevo León.
At necropsy, the puppy was in fair bodily and showed moderate dehydration.
[¿conjunctivitis o descarga nasal?] Significant postmortem findings were confined to the
respiratory and lymphatic systems. The lungs had cranioventral consolidation affecting __%
of the pulmonary parenchyma. [¿Se notó falta de colapso, impresiones costales o textura
elástica que pudieran haber sugerido neumonía intersticial?]. On cut surface, purulent
exudate was present in the major bronchi of the consolidated lung?. Severe atrophy of the
palatine tonsils was also noted. No other gross lesions were observed and tissue samples from
lung, lymph nodes, tonsils, heart, liver, spleen and brain were fixed in 10% buffered formalin
solution for histopathological examination.
Important microscopic lesions were restricted to the lungs and brain. The lungs
exhibited severe necrotizing bronchiolitis characterized by swelling and exfoliation of
epithelial cells that often resulted in segmental ulceration of the airway mucosa. Mitotic
figures and non-ciliated flat cells consistent with proliferating cells at the early stages of
repair were occasionally observed at the margins of ulcerated basement membranes. The
bronchial and bronchiolar lumens contained many neutrophils admixed with macrophages and
multinucleated syncytial giant cells (Fig 1). Numerous neutrophils were also present in the
mucosa and submucosa of the airways, and the bronchial glands were distended and filled
with neutrophils and cell debris. Some alveoli were packed with neutrophils, foamy
macrophages and multinucleated cells and protein- rich edematous fluid. The most
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remarkable alveolar lesion was a diffuse thickening of the walls because of type II
pneumonocyte hyperplasia and interstitial edema.
Numerous round to oval eosinophilic inclusion bodies were present in the cytoplasm
of bronchial and bronchiolar cells, alveolar macrophages and multinucleated syncytial cells
(Fig. 1). Another striking microscopic finding was the distinct presence of large basophilic
inclusions in the nuclei of bronchial, bronchiolar and alveolar cells (Fig 2). Some of these
basophilic inclusions filled the entire nucleus causing complete margination of the chromatin.
Microscopic changes in the CNS were confined to the cerebellar white matter and
consisted of multifocal neuropil vacuolation, astrocytic swelling and intranuclear acidophilic
inclusions in glial cells. There was no evidence of perivascular infiltration of lymphocytes
(perivascular cuffing) in the brain. Less significant microscopic lesions in other organs
included a moderate lymphoid depletion, extramedullary hemopoieisis in the spleen and
intranuclear and intracytoplasmic acidophilic inclusions in the epithelial cells of the renal
pelvis. Based on the microscopic findings a diagnosis of dual canine distemper-adenovirus
bronchointerstitial pneumonia was made.
Paraffin-embedded lung was submitted to the Prairie Diagnostics in Saskatoon and
tested by the avidin-biotine complex-peroxidase technique for canine distemper virus (CDV)
and canine adenovirus-2 (CAV-2). Results revealed abundant morbillivirus antigen in the
cytoplasm of the bronchial, bronchiolar, alveolar and glandular epitheliums (Fig. 1). Positive
reaction was also observed in alveolar macrophages and multinucleated giant cells. In
addition, CAV-2 antigen was detected in the nuclei of the bronchial epithelium, particularly in
those cells that contained the large basophilic inclusions (Fig. 2).
Thin paraffin sections of lung cut at 5 µm were deparaffinized and processed for
transmission electron microscopy. Paracrystalline arrays of electrondense particles measuring
50 ± 2.0 nm in diameter were found in the nuclei of epithelial cells (Fig. 2). These
electrondense particles were ultrastructurally consistent with CAV-2. Based on
histopathology, histochemistry and electron microscopy the final diagnosis of dual CDV and
CAV-2 infection was made in this puppy.
It was not possible to ascertain the weather infection was related to the vaccination
itself, to natural infection because the puppy was immune-incompetent or had maternal
antiboides, or because the vaccine were ineffective (2). A recent report indicates that canine
distemper in some countries like Japan is occurring with increasingly frequency in vaccinated
dogs (3).
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Bronchointerstitial pneumonias are frequently attributed to viral infections and to a
much lesser extent to chemical and immunemediated injury to the lung (4,5). In the absence
of inclusion bodies it is generally difficult to achieve etiological diagnosis in
bronchointerstitial pneumonias. In this particular case however, the presence of two distinct
types of pulmonary clearly make the tentative diagnosis in this puppy relative simple.
Intracytoplasmic inclusions along with clinical history of respiratory and neurological signs
are highly suggestive of canine distemper, while karyomegaly with large intranuclear
basophilic inclusion bodies are most consistent with CAV-2 infection (6).
Histochemistry proved rather valuable in confirming dual CDV and CAV-2 infection
in this puppy since culture of these virus is difficult (1,6).
The intralesional distribution of
viral antigens in the bronchioles and alveoli was typical of bronchointerstitial pneumonia (4)
At the early stages of infection viral antigens are centered in the areas of bronchiolar necrosis
while in later stages of the disease the viral antigen is also present the alveolar macrophages
as it was the case with this puppy (5,6). Exfoliated cells positively staining for CDV and to a
lesser extent CAV-2 were present in the bronchiolar lumens. The mitotic activity along the
denuded bronchiolar basement membranes and the hyperplasia of alveolar type II
pneumonocytes clearly indicate that at the time of death the lung was undergoing repair (4,5).
These findings were chronologically consistent with 2-3 wks history of respiratory distress
4).
Canine distemper has a worldwide distribution and is particularly prevalent in
geographic areas where vaccination is not routinely done. This disease is generally fatal since
it affects the respiratory tract causing bronchointerstitial pneumonia, rhinosinusitis and
conjunctivitis; the CNS causing non-suppurative encephalitis and demyelination; and the
digestive tact causing gastroenteritis, diarrhea and dehydration (2,7). Although neuropil
vacuolation and intranuclear inclusions in glial cells were present in the brain of this puppy,
there was no histological evidence of perivascular lymphocytes which are typically seen in
the non-suppurative encephalitis of canine distemper (7).
CAV-2 is another highly contagious viral agent incriminated in canine respiratory tract
disease, particularly in young dogs kept in crowded environment such as pet stores, boarding
kennels and veterinary hospitals (kennel cough) (8). Infection with CAV-2 is generally
transient and seldom fatal unless it is complicated with a secondary bacterial
bronchopneumonia (5,8). In fatal cases, there is a severe bronchointerstitial pneumonia
characterized by necrosis of the bronchiolar and alveolar epithelium, and in some cases, large
intranuclear inclusion bodies are seen in bronchiolar and alveolar cells. Like canine
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distemper, etiological diagnosis of CAV-2 infection is achieved by FAT,
immunohistochemistry or viral isolation.
The suppurative changes observed in the lungs of this puppy were consistent with as
secondary bacterial pneumonia. Like many other respiratory viruses, CDV and CAV-2
impair the defense mechanisms and predisposes dogs, particularly young puppies, to
secondary bacterial bronchopneumonia (5,8). Bordetella bronchiseptica is the most common
cause of secondary bacterial pneumonia in dogs. Unfortunately, the lungs of this puppy were
not cultured and therefore the cause of the suppurative bronchopneumonia remained
unknown. It is possible that distemper encephalitis may have predisposed the puppy to
aspiration pneumonia in spite the fact that food particles were not microscopically seen in the
lung. Although frequently reported in dogs with CDV infection, there was no microscopic
evidence of opportunistic infection in this puppy (4,9).
A recent retrospective study of canine viral pneumonias conducted in Mexico revealed
two important facts regarding CVD and CAV-2 infections in dogs. Firstly, single or
combined respiratory infections involving CDV, CAV2 and canine parainfluenza (CPIV)
occur more frequent in the canine population than previously thought (6). Secondly,
histopathology alone is not always reliable as a diagnostic tool since inclusion bodies, the
hallmarks for CDV and CAV-2 infections are not always present in the lung. This is
particularly true during the late stages of the disease where inflammation and repair occurs
and epithelial cells no longer exhibit inclusions. Although dual infections with CDV and
CAV-2 as the one seen in this puppy are relatively rare, similar cases have been previously
reported in Mexico and other countries (10-12). Bronchointerstitial pneumonia along with the
two distinct types of inclusion bodies was almost conclusive of a dual viral infection in the
puppy but routine laboratory confirmation always requires of immunohistochemistry.
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References
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Fig 1. Necrotizing bronchiolitis with large numbers of neutrophilis in the lumen. Note
CAV-2 intranuclear inclusion bodies in the bronchiolar cells (arrow head). Also note
intracytoplasmic eosinophilic inclusion bodies typical of CDV (arrows).
HE bar 100µm.
Inset: Canine distemper virus antigen in epithelial cells cell, Avidin-biotin peroxidase
reaction.
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Fig 2. Necrotizing bronchiolitis with neutrophilic exudation. Note large intranuclear
inclusion bodies in the bronchiolar epithelium (arrows). HE bar 100µm. Left Inset:
CAV-2 antigen positive reaction in bronchiolar cell, Avidin-biotin peroxidase reaction.
Right inset: Paracrystalline arrays of electrondense particles (50 ± 2.0 nm in diameter).
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