Phenotyping Core, Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA

4. Shin Kong Wu-Ho-Su Memorial Hospital, Taipei Taiwan111.

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ABSTRACT

Canine distemper virus (CDV) causes a highly contagious disease, which has been reported in Taiwan for many years; however phylogenetic analysis of field CDV, histopathological lesions, and co-infection are poorly understood. The goals of this study were to characterize the pathology of CDV in dogs in Taiwan, and to assess the frequency of CNS demyelination in cases of CDV infection confirmed by immunohistochemistry (IHC) and/or reverse transcription polymerase chain reaction (RT-PCR). Fifty two (IHC or RT-PCR positive) affected dogs were obtained from either animal clinics or dog shelters from 2000 to 2009. Postmortem and laboratory examination included gross findings, histopathology, Luxol-fast blue cresyl echt violet (LFB-CEV) histochemistry, non-biotin HRP anti-CDV IHC, and phosphoprotein gene RT-PCR. Thirty two (32) dogs were submitted from clinic.

Twenty (20) dogs were submitted from shelter. Clinic cases had histories of treatment and or vaccination. Twenty four clinic cases (75%) were puppies less than 6 months old. Seventeen shelter cases (85%) were identified as ‘adults’ greater than 6 months old. There were 27 males and 25 females. Eleven dog breeds were represented, but most dogs (35/52, 67%) were mixed-breed. Totally, 79% (41/52) had lymphoid depletion, 71% (37/52) had interstitial pneumonia, 65% (34/52) had CNS demyelination, and 32% (17/52) had catarrhal enteritis. Younger clinic group

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pneumonia (26/32, 81%), and CNS demyelination (26/32, 81%), also showed statistically significant difference compared to shelter group. Enteritis was identified in about one third of the animals in both groups. The distribution of inclusion bodies also showed significant difference in urinary bladder, lymphoid tissues, lung, and alimentary tract between the two groups. A variety of 29 co-infections and other associated lesions were identified. However, no significant difference in the frequency of occurrence between the two groups, the exception was interstitial nephritis. In conclusion, lymphoid depletion, pneumonia, and CNS demyelination were the most common CDV-infected principal lesions, and high occurrence of inclusion bodies were found in spleen, lymph node, and mucosa epithelium of urinary bladder, pelvis, bronchioles, and stomach.

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INTRODUCTION

Canine distemper virus (CDV), which belongs to morbillivirus genus, family Paramyxoviridae, produces systemic or central nervous system(CNS) infections of dogs and related species, and often associated with high mortality in dogs in Taiwan.¹⁴ Expected pathology findings in CDV infected dogs include lymphocyte depletion in lymphoid tissues, interstitial pneumonia, degenerative changes in epithelia of respiratory organs, urinary bladder and gastrointestinal tract, foot pad hyperkeratosis,¹² and intranuclear or intracytoplasmic eosinophilic inclusion bodies (INIB/ICIB) in epithelial cells of the urinary bladder or gastrointestinal tract.^1,4 Characteristic central nervous system CNS changes include polioencephalomacia,¹⁵ white matter demyelination, astrogliosis, eosinophilic INIB/ICIB in astrocytes and neuron, gemistocytes and occasional multinucleated syncytial giant cell 3,19,24,25 and old dog encephalitis.²⁶

We have noticed that in Taiwan, there have been changes in the suite of histopathological lesions seen in the prevalent infection of CDV since 2000;²⁹ CNS signs are now marked and gastrointestinal involvement is rare.¹⁴ Two distinct disease group of CDV infection, “enteritis and non-enteritis”, have also been noted in Japan.

The non-enteritis type of CD exhibited reduced epitheliotropism, might be the

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seemed lower than what we were experiencing in Taiwan eg. syncytial giant cells in 9

% of infected brains,¹⁹ and eosinophilic intranuclear or cytoplasmic inclusion bodies in only 17-72 %.8, 9, 23, 29

In addition to the lesions of CDV itself, infected dogs may have a wide variety of concurrent infections, including canine adenovirus type 2 (CAV-2),^5,6 coccidiosis,⁹ colibacillosis,²⁸ cryptosporidiosis,⁷ Parainfluenza viruses,⁶ Mycoplasma Cynos,⁵ toxoplasmosis,¹⁷ Tyzzer's Disease,⁹ documented in individual case reports. Based on the previous findings and lack of a large-scale of case analysis, it is interesting to know concurrent infections with CDV and characteristics of CDV-associated lesions in different environment for appropriate management. Thus, we conducted a retrospective study, for the 10 years from 2000 to 2009, to compare CDV histopathological lesions and complications in two groups of dogs in Taiwan, 32 that had been treated in clinics and 20 dogs from shelters.

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MATERIALS AND METHODS

ANIMALS

This retrospective study was based on necropsy cases obtained between March 2000 and December 2009. The RT-PCR associated with the immunohistochemical labeling-confirmed CDV infection in 52 dogs, and the distribution of these cases over the 10-year-study-period was as follows: 2000 (26), 2001 (2), 2002 (4), 2003 (6), 2004 (2), 2005 (8) and 2009 (4). There were 27 males and 25 females. Most of the dogs were crossed-breed (35); the remainder were distributed among 11 breeds:

Beagle (3), Shih tzu (3), Maltese (2), Labrador retriever (2), Chin (1), Chow chow (1), Dachshund (1), Lhasa apso (1), Miniature pinscher (1), Pomeranian (1), and Shiba (1).

Shelter submitted dogs, were submitted to our laboratory as ‘puppies’ considered to be less than 6 months old, or as ‘adults’, considered to be more than 6-month-old.

Thus, for the purposes of comparison here age groups were classified as less than 6 months and more than 6 months. The dogs had died or were humanely euthanized by 80 mg/kg pentobarbital IV.

IMMUNOHISTOCHEMISTRY (IHC)

CDV immunohistochemistry was reported previously,¹⁴ and was performed on

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examined by IHC included spleen, cerebrum, cerebellum, brain stem, urinary bladder, and lung for each dog. Briefly, the Super Sensitive ^TM Non-Biotin HRP Detection System (BioGenex Laboratories, San Ramon, CA, USA) was used. The primary antibody was mouse anti-CDV (MCA 1893, Clone DV2-12; Serotec, Kidlington, Oxford, UK).Substitution of TBS or negative mouse serum for the primary antibody on sections of CDV-infected cerebrum served as a non-specific negative control.

Cerebral sections from dogs with no evidence of distemper infection served as a specific negative control. Positive control sections from a dog with numerous inclusion bodies were included in each IHC staining.