Leukoencephalopathy associated with parvovirus infection in Cretan hound puppies

Abstract

Leukoencephalopathies in dogs encompass presumably inherited conditions such as leukodystrophies, hypomyelination or spongiform degeneration, but other causes, such as virus infections and toxic or nutritional factors, might also play a contributory role. In this report, we provide evidence of parvovirus infection and replication in the brains of five 6-week-old Cretan hound puppies suffering from a puppy shaker syndrome and leukoencephalopathy. Although these puppies belonged to two different litters, they were closely related, tracing back two generations to the same sire. Histologically, a mild to moderate lymphohistiocytic meningitis, with focal lymphohistiocytic leukoencephalitis in two animals, and a mild to moderate vacuolation with myelin loss, mainly in the white matter of the cerebellum was detected. Vacuolation was also found in the corpus callosum, fimbria hippocampi, mesencephalon, capsula interna, basal ganglia, and hypothalamus. By immunohistology and in situ hybridization, either parvoviral antigen, DNA, mRNA, or replicative intermediate DNA were detected in the cerebellum, hippocampus, periventricular areas, corpus callosum, cerebral cortex, medulla oblongata, and spinal cord. Parvovirus antigen, DNA, and mRNA were present in cells of the outer granular layer of the cerebellum and in periventricular cells, most likely representing spongioblasts, glial cells, neurons, endothelial cells, occasional macrophages, and ependymal cells. Sequencing revealed canine parvovirus type 2 stretches. Thus, an association of parvovirus infection with the leukoencephalopathy seems likely, possibly facilitated by a genetic predisposition due to the mode of inbreeding in this particular dog breed.

FIG. 1.

Histological changes in the brains of the Cretan hound puppies. (A) Vacuolization of the white matter of the cerebellum (arrows). H&E staining was performed. Bar, 50 μm. (B) Luxol fast blue (LFB) staining of vacuolated area of the white matter of the cerebellum. Note the decreased LFB staining in this area (arrows). Bar, 50 μm.

FIG. 2.

Demonstration of parvovirus antigen, DNA, and mRNA or replicative intermediate in the brain of the Cretan hound puppies. (A and B) Immunohistochemical labeling of parvovirus antigen within cells (arrows) in the periventricular area (A) and cerebellum (B). (C to F) ISH using the T3 (C and D) or the T7 (E and F) RNA probe directed against the parvovirus DNA to detect positive cells (arrows) in the periventricular area (C and E) and cerebellum (D and F). Bar, 50 μm.

FIG. 3.

Gel electrophoresis of the real-time PCR. L, 20-bp ladder. Tested sample (lane 1) and positive control (lane 2), both at an 84-bp length, and a negative control (lane 3) of the first PCR are shown. Tested sample (lane 4) and positive control (lane 5) both at a 130-bp length, and negative control (lane 6) of the second PCR are also shown.

FIG. 4.

Melting curve of the real-time PCR. The tested sample (arrow 1) and positive control (arrow 2), both at an 84-bp length, and a negative control (arrow 3) of the first PCR are indicated. The tested sample (arrow 4) and positive control (arrow 5), both at a 130-bp length, and a negative control (arrow 6) of the second PCR are also indicated.