Canine distemper virus infection: proliferation of canine footpad keratinocytes

The proliferation of footpad keratinocytes of canine distemper virus (CDV)-infected dogs was investigated. Footpads of 19 dogs inoculated experimentally with a virulent distemper strain (A75/17) and of two noninoculated control dogs were collected at necropsy. Dogs were divided into four groups according to results of the postmortem examination: dogs with severe distemper (group 1), dogs with mild distemper (group 2), inoculated dogs without distemper (group 3) and noninoculated dogs (group 4). There was no distinct difference of epidermal thickness among the four groups. Infection of the footpad epidermis with CDV was demonstrated using immunohistochemistry for viral nucleoprotein and in situ hybridization for nucleoprotein messenger ribonucleic acid (mRNA). Only group 1 dogs had viral antigen and mRNA in the footpad epidermis with the same distribution. Footpad epidermis of group 1 dogs had more mitotic figures in the basal layer, and significantly more basal keratinocytes were positive for the proliferation markers Ki-67 and proliferating cell nuclear antigen. Double-staining for Ki-67 and viral nucleoprotein identified rare double-labeled basal keratinocytes. These findings suggest that the presence of CDV particles in the footpad epidermis is associated with keratinocyte proliferation.     

Canine distemper virus associated proliferation of canine footpad keratinocytes in vitro

Infection of canine footpads with canine distemper virus (CDV) can result in so-called hard pad disease characterized by footpad epidermal proliferation and hyperkeratosis. Cultured canine footpad keratinocytes (CFK) were inoculated with a virulent canine distemper virus strain (A75/17-CDV) to study the effects of CDV-infection on keratinocyte proliferation. Infection was analyzed by immunohistochemistry and in situ hybridization for CDV nucleoprotein (N-protein) antigen and mRNA. CDV caused a persistent, non-cytocidal infection with spread from single cells to infection of the confluent cell layer 7 days post infection (p.i.). Absolute cell numbers were significantly higher in infected cultures compared to control cultures from day 4 until day 6 p.i. Infected cultures contained significantly more total DNA on day 5 p.i. compared to controls. Immunohistochemical investigation of proliferation markers Ki67 and BrdU demonstrated a nearly two-fold increase in numbers of positive cells on day 5 p.i. compared to controls. These findings demonstrate that canine distemper virus infection of canine footpad keratinocytes in vitro was associated with proliferation.     

Canine distemper virus infection of canine footpad epidermis

Infection of the footpad epidermis can occur in natural canine distemper virus (CDV) infection of dogs. Footpads from 19 dogs experimentally inoculated with virulent distemper strain A75/17 and from two nonexposed dogs were examined histopathologically and assessed for the presence of viral antigen and nucleoprotein mRNA, as well as number of inflammatory and apoptotic cells. Dogs were divided into four groups based on inoculation status and postmortem examination: inoculated dogs with severe distemper (group 1, n = 7); inoculated dogs with mild distemper (group 2, n = 4); inoculated dogs without distemper (group 3, n = 8); and noninoculated dogs (group 4, n = 2). Footpads from dogs of all groups had a comparably thick epidermis. Eosinophilic viral inclusions and syncytial cells were present in footpad epidermis of one dog of group 1. Footpads of group 1 dogs contained viral antigen and mRNA in the epidermis with strongest staining in a subcorneal location. Additionally, in these dogs footpad dermal structures including eccrine glands and vascular walls were positive for virus particles. No CDV antigen or mRNA was present in the footpad epidermis and dermis of any other dog. Group 1 dogs had more CD3-positive cells and apoptotic cells within the basal layer of the epidermis when compared to the other groups. These findings demonstrate that in experimental infection CDV antigen and mRNA were colocalized in all layers of the infected canine footpad epidermis. The scarcity of overt pathological reactions with absence of keratinocyte degeneration indicates a noncytocidal persisting infection of footpad keratinocytes by CDV.     

Histopathology and immunohistochemistry of canine distemper virus-induced footpad hyperkeratosis (hard Pad disease) in dogs with natural canine distemper

Hard pad disease represents an uncommon manifestation of canine distemper virus (CDV) infection with a still uncertain pathogenesis. To study the pathogenesis of this uncommon, virally induced cutaneous lesion, the footpads of 19 dogs with naturally occurring distemper were investigated for histologic changes and distribution pattern of CDV antigen. All dogs displayed clinical signs of distemper, which had lasted from 10 to 75 days. Overt digital hyperkeratosis was observed in 12 animals (group A), whereas the footpads of the remaining seven dogs appeared normal macroscopically (group B). Orthokeratotic hyperkeratosis (12/12; 100%), irregular acanthosis (11/12; 92%), thickened rete ridges (10/12; 83%), and mild mononuclear perivascular (10/ 12; 83%) and periadnexal (7/12; 58%) dermatitis were the most common findings in dogs with hard pad disease. Surprisingly, orthokeratotic hyperkeratosis (5/7; 71%), irregular acanthosis (5/7; 71%), and thickened rete ridges (4/7; 57%) were also seen in the dogs without clinical evidence of digital hyperkeratosis. CDV-specific inclusion bodies and ballooning degeneration were not observed in the footpad epidermis of the 19 dogs. Immunohis-tochemistry revealed that CDV antigen was most frequently found in the stratum spinosum and granulosum and in the epithelial cells of the eccrine sweat glands and only rarely in the basal layer. Fibroblasts, pericytes, endothelial cells, and hair follicles were also positive in some animals. Despite the obvious difference regarding the macroscopic picture, the microscopic changes were less prominent between the animal groups. The selective infection of keratinocytes in the stratum spinosum might be the key event for the development of hard pad disease in the dog.     

Up-regulation of cytokeratin expression in canine distemper virus-infected canine footpad epidermis

Cytokeratin expression was assessed in footpad epidermis from dogs using immunohistochemistry. Four groups of dogs were studied: dogs with experimentally induced distemper and with canine distemper virus (CDV) in footpad epidermis (group 1, n = 7); dogs with experimentally induced distemper and without CDV in footpad epidermis (group 2, n = 4); inoculated dogs without distemper and without CDV in the footpad epidermis (group 3, n = 8), and noninoculated dogs without distemper (group 4, n = 2). No increase in thickness of the footpad epidermis was present in any of these groups. Sections of metacarpal or metatarsal pads were stained for cytokeratin (CK)14 (proliferation-associated), CK10 (correlated with early differentiation), and for involucrin (associated with terminal differentiation). CK14 was present in basal keratinocytes of all groups, but staining intensity decreased towards the corneal layer in groups 2-4, but not in group 1. CK10 was present in the spinous and granular layer of all groups, but staining of the granular layer was much stronger in group 1. Involucrin was present in the granular layer of footpads of group 1 and only in the upper part of this layer in groups 2-4. The results demonstrate increased staining intensity and/or wider distribution within the footpad epidermis in group 1 dogs when compared to the other groups. This was interpreted as up-regulation in expression of these proteins. These findings suggest that presence of CDV antigen and mRNA in footpad epidermis was associated with an increase in expression of CK14, CK10 and involucrin. The potential role of this up-regulation in cytokeratin expression in the development of CDV-induced digital hyperkeratosis remains speculative at the moment and requires further studies.     

Immunohistochemical demonstration of the putative canine distemper virus receptor CD150 in dogs with and without distemper

Signaling lymphocyte activation molecule (SLAM) or CD150 can function as a receptor for the canine distemper virus (CDV) in vitro. The expression of SLAM was studied using immunohistochemistry in order to evaluate the presence and distribution of the receptor in dogs in vivo. Additionally, receptor expression was assessed after experimental infection of dogs with CDV. In 7 control dogs without distemper virus, the receptor was found in various tissues, mostly on cells morphologically identified as lymphocytes and macrophages. In 7 dogs with early distemper lesions characterized by presence of the virus, higher numbers of SLAM-expressing cells were found in multiple tissues recognized as targets of CDV compared with those in control dogs. These findings suggest that SLAM, a putative distemper receptor, is expressed in dogs in vivo. Additionally, virus infection is associated with up-regulation of SLAM, potentially causing an amplification of virus in the host.     

Identification of a genetic variant of canine distemper virus from clinical cases in two vaccinated dogs in Mexico

Canine distemper virus (CDV) is a highly contagious viral pathogen of worldwide distribution that can cause lethal disease in dogs and other mammals. Genetic diversity is found among reference strains and isolates of CDV, mainly in the haemagglutinin protein (H), fusion protein (F) and nucleoprotein (N), and this may be associated with the increasing incidence of distemper in dogs. CDV was identified by RT-PCR in serum samples taken from two clinically diseased, previously vaccinated Mexican dogs. Subsequently, in both samples, a fragment of the CDV N gene was sequenced revealing a 100% identity between nucleotide sequences. However, the sequence obtained was different to that found in virus strains used in vaccines and in isolates reported elsewhere, but was closely related to A75/17, 1127/Gi95, and 2495/Gi95 sequences from USA and Germany, and clustered with 1127/Gi95 and 2495/Gi95 strains. The results suggest that a novel CDV lineage may be present in Mexico.     

Experimental and naturally occurring transplacental transmission of canine distemper virus

In the present study, 2 different effects of experimentally induced infection with virulent canine distemper virus (CDV) on pregnant CDV-susceptible dogs were studied. In 1 bitch, abortion occurred 7 days after viral inoculation and there was no evidence of fetal infection. Another bitch had subclinical infection and delivered 3 CDV-infected pups. Sequential clinical, immunologic, and virologic studies of a litter of gnotobiotic pups (3rd bitch) that were congenitally infected with CDV demonstrated the heightened susceptibility to CDV in the neonatal period. The data presented add canine distemper to the list of transplacental infectious diseases in the canine species.     

Apoptosis in the cerebellum of dogs with distemper

Canine distemper virus (CDV) may induce multifocal demyelination in the central nervous system of infected dogs. The pathogenesis of this process is not clear. The present work identifies the presence of apoptotic cells in white and grey matter of dogs'cerebellum, naturally infected with CDV. Fifteen dogs with clinical signs of canine distemper that tested positive for CDV nucleoprotein were used. Brain specimens were processed and embedded in paraffin. Sections 5 microm thick were stained with hematoxylin-eosin and Shorr. Other sections were submitted to TUNEL reaction and to immunohistochemistry for CDV nucleoprotein detection. Acute and chronic demyelinated plaques were observed in the white matter, while apoptosis occurred particularly in the granular layer of grey matter. Apoptosis seems to play an important role in the pathogenesis of canine distemper demyelination.     

Restricted virus protein translation in canine distemper virus inclusion body polioencephalitis.

In this study, inclusion body polioencephalitis, an uncommon form of canine distemper virus (CDV)-induced encephalitis, was investigated for viral protein and mRNA expression by immunohistochemistry (IH) and in situ hybridization and, in addition, infiltrating cells were characterized by IH. Lesions were predominantly found in the grey matter of the brain stem and the immune response, dominated by T cells, was associated with a strong MHC II upregulation. Abundant expression of all viral protein mRNAs and reduced or lacking protein translation, especially of the matrix protein were the most important findings, indicating that restricted virus infection in the grey matter might represent a mechanism for viral persistence in distemper polioencephalitis.     

Anosmia associated with canine distemper

The sense of smell in dogs infected with canine distemper virus (CDV) was examined by use of EEG olfactometry, behavioral olfactometry, and electro-olfactography. Infection with CDV was confirmed by a direct immunofluorescence technique in 8 active cases and was suggested by clinical history compatible with canine distemper 10 to 26 weeks earlier in 6 cases. Pathologic alterations of the olfactory mucosa in 3 clinically affected dogs was examined by light microscopy. Infection with CDV was found to be associated with anosmia and lack of recorded responses on electro-olfactogram in 8 of 8 dogs with clinical signs of acute distemper from naturally acquired infections. Anosmia was found in 5 of 6 dogs that had recovered from acute distemper 10 to 26 weeks earlier. The sixth dog had hyposmia, with abnormalities on the electro-olfactogram. Histologic examination was not performed on the 6 dogs that had recovered. Histologic lesions observed at necropsy in 3 dogs that had had clinical signs of acute distemper were those of subacute purulent rhinitis and atrophy of the olfactory epithelium. Altered olfactory function could be explained by mucopurulent exudate blocking odors from olfactory receptors in the acutely affected dogs, but alteration of olfactory function in the dogs that had recovered without clinical evidence of rhinitis could not be explained.     

Natural infection with canine distemper virus in a Japanese monkey (Macaca fuscata)

A case of encephalitis in a Japanese monkey (Macaca fuscata) was examined histopathologically and serologically. The animal had brain lesions consisting of perivascular cuffs, malacia, inclusion bodies and giant cells. Monoclonal antibody to the nucleoprotein of canine distemper virus (CDV) stained the inclusions, and the distribution of the virus antigen was closely associated with that of the histological lesions. Serologically, all the 22 monkeys in the same group as the diseased monkey had relatively high titers of neutralizing antibody to CDV, but not to measles virus (MV). The pattern of the antibody titers to CDV and MV closely resembled that of cynomolgus monkeys experimentally inoculated with CDV, but differed from that of monkeys inoculated with MV. These findings suggest that an epidemic of CDV occurred in these Japanese monkeys, associated with one case of fatal viral encephalitis. This is believed to be the first report of a natural infection by CDV in non-human primates.     

Immunohistochemical demonstration of canine distemper virus antigen as an aid in the diagnosis of canine distemper encephalomyelitis

Brain tissue from 33 dogs with non-suppurative encephalitis was examined for evidence of canine distemper virus (CDV) encephalitis. Sections were examined for lesions, inclusion bodies, syncytial cells and CDV antigen using a double bridge unlabelled antibody enzyme technique. Histopathological lesions considered to be typical of granulomatous meningoencephalomyelitis were found in seven dogs. They all lacked inclusion bodies, syncytial cells and CDV antigen. The remaining 26 dogs all had histopathological lesions typical of CDV encephalitis. Inclusion bodies were found in 24 dogs, four of which also had syncytial cells and CDV antigen was detected immunocytochemically in 25. One dog had no inclusion bodies or syncytial cells and was immunohistochemically negative. Syncytial cells have been found to be of limited diagnostic value for the diagnosis of CDV encephalitis. While inclusion bodies proved to be a good diagnostic criterion for the confirmation of CDV infection, the immunohistochemical demonstration of CDV antigen proved to be superior. CDV antigen was more prevalent than inclusion bodies in tissue sections and much more easily detectable.     

Immunohistochemical detection of canine distemper virus in haired skin, nasal mucosa, and footpad epithelium: a method for antemortem diagnosis of infection.

A reliable antemortem diagnostic method is needed for determining infection with canine distemper virus (CDV). The utility of immunohistochemical detection of CDV antigen was examined was examined for samples of nasal and footpad epithelium and haired skin in dogs with and without detectable CDV antigen in the lung and/or brain. Tissues from 57 dogs at risk of CDV infection were tested. Viral antigen was found in the lung and/or brain of 28 dogs. Among these dogs, viral antigen was demonstrated in the epithelial cells of the nasal mucosa in 24 of 27 dogs, in the footpad epithelium in 24 of 26 dogs, and in the haired skin of the dorsal neck in 26 of 27 dogs. Among the 29 dogs without CDV antigen in either the lung or brain, 1 dog had positive staining for viral antigen in the skin and nasal mucosa. Biopsies of haired skin of the dorsal neck, which is relatively simple to sample, can be used for immunohistochemical testing for acute and subacute infection with CDV.     

Canine distemper virus-associated hypocalcemia.

A retrospective study was done to correlate serum calcium concentrations and parathyroid gland ultrastructure to clinical, immunologic, and pathologic changes experimentally induced in gnotobiotic dogs by canine distemper virus (CDV). Dogs infected with CDV had significantly reduced serum calcium concentrations associated with ultrastructural evidence of parathyroid gland inactivity, degeneration, and viral inclusions. Although CDV-infected dogs exhibited neurologic signs, minimal lesions were present in the central nervous system. It is suggested that viral-induced parathyroid dysfunction may contribute to neutrologic disturbance of CDV infection.     

Myocarditis caused by naturally acquired canine distemper virus infection in 4 dogs

Canine distemper virus (CDV) has long been recognized as a cause of myocarditis; however, cases of myocarditis caused by naturally acquired CDV infection have been reported only rarely in dogs. We describe here our retrospective study of naturally acquired systemic CDV infection in 4 dogs, 4–7 wk old, that had myocarditis, with myocardial necrosis and fibrosis. One of the 4 dogs had intracytoplasmic eosinophilic inclusion bodies in cardiomyocytes. Other lesions included bronchointerstitial pneumonia (4 of 4), necrotizing hepatitis (2 of 4), splenic lymphoid necrosis (2 of 4), encephalitis (1 of 3; brain was not submitted in 1 case), and necrotizing gastroenteritis (1 of 4). The presence of CDV in the heart was confirmed by immunohistochemistry in all 4 dogs.     

Lymphoid apoptosis in acute canine distemper

The relationship between the canine distemper virus (CDV) infection and apoptosis in the canine lymphoid tissues was investigated using immunostaining for single stranded DNA (ssDNA), TdT-mediated dUTP-biotin nick end-labeling (TUNEL) method, and electron microscopy. Twenty-six lymphoid tissues from 8 spontaneously CDV-infected dogs and 1 non-infected dog were used, and lesions were classified into 4 groups according to frequency of the CDV-antigen. Histologically, the degree of lymphoid depletion tended to depend on amount of CDV antigen. The numbers of ssDNA- and TUNEL-labeling cells were significantly high in the lymphoid tissues with abundant viral antigen. However, ssDNA- and TUNEL-positive lymphocytes were also frequently found even in the lymphoid tissues where there was only a small amount of CDV-antigen in sinus histiocytes. The incidence and distribution of apoptotic cells in the CDV-antigens-negative lymphoid tissues from infected dogs were equal to those from a non-infected dog. Double labeling immunostaining using a ssDNA and a CDV nucleocapsid protein (CDV-NP) antibody revealed that there were ssDNA positive but CDV-NP negative cells besides those stained doubly positive. Ultrastructurally, lymphocytes in the CDV-infected lymphoid tissues frequently had characteristic morphological features of apoptosis such as apoptotic bodies. All these results suggest that CDV leads to lymphocytic apoptosis directly or indirectly, resulting in severe lymphoid depletion and immunosuppression in acute or subacute phase of CDV infection.     

Identification of a new genotype of canine distemper virus circulating in America

Canine Distemper is a highly contagious viral systemic disease that affects a wide variety of terrestrial carnivores. Canine Distemper virus (CDV) appears genetically heterogeneous, markedly in the hemagglutinin protein (H), showing geographic patterns of diversification that are useful to monitor CDV molecular epidemiology. In Mexico the activity of canine distemper remains high in dogs, likely because vaccine prophylaxis coverage in canine population is under the levels required to control effectively the disease. By phylogenetic analysis based on the nucleoprotein (N) and on the H genes, Mexican CDV strains collected between 2007 and 2010 were distinguished into several genovariants, all which constituted a unique group, clearly distinct from field and vaccine strains circulating worldwide, but resembling a CDV strain, 19876, identified in Missouri, USA, 2004, that was genetically unrelated to other North-American CDV strains. Gathering information on the genetic heterogeneity of CDV on a global scale appears pivotal in order to investigate the origin and modalities of introduction of unusual/novel CDV strains, as well as to understand if vaccine breakthroughs or disease epidemics may be somewhat related to genetic/antigenic or biological differences between field and vaccine strains.