Non-cytocidal infection of keratinocytes by canine distemper virus in the so-called hard pad disease of canine distemper

A late, but not uncommon sequel to canine distemper virus (CDV) infection of dogs is thickening of footpads and nasal planum, the so-called hard pad disease, originally described as vacuolar degeneration of epidermal keratinocytes with inclusion body formation and massive hyperkeratosis. However, in a recent study of footpads of naturally CDV-infected dogs only hyperkeratosis was observed without any of the other changes. Instead, acanthosis was frequently noticed. CDV nucleoprotein was present in the suprabasal keratinocytes and eccrine epithelial glands only. No CDV nucleoprotein was present in basal keratinocytes. This observation in combination with lack of obvious cytocidal changes strongly suggested the possibility of a restricted viral infection with presence of viral mRNA but without protein expression. Therefore, the presence of CDV nucleoprotein mRNA was investigated using in situ hybridization and compared to the localization of the nucleoprotein in footpads of clinically healthy and distemper dogs. Viral nucleoprotein and nucleoprotein mRNA in nearly all cases co-localized to the same compartments and basal keratinocytes did not contain nucleoprotein mRNA. These findings dispute the idea of a restricted viral infection of footpad keratinocytes in dogs with natural CDV infection. Instead, a migration of the virus to the epidermal surface along with the proliferating and differentiating epithelium is the most likely explanation for the lack of virus antigen in basal keratinocytes.     

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.     

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.     

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.     

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 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.     

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.     

Serum antibody response to canine parvovirus, canine adenovirus-1, and canine distemper virus in dogs with known status of immunization: study of dogs in Sweden

Serum antibody titers to canine parvovirus (CPV), canine adenovirus-1 (CAV-1), and canine distemper virus (CDV) were measured in dogs with known immunization status. The dogs represented 3 groups: nonvaccinated dogs less than 12 months old; vaccinated dogs less than 12 months old; and adult dogs greater than 12 months old. For practical reasons, the population from which the specimens were obtained could be considered as free from natural infection with CAV-1 and CDV. In nonvaccinated dogs less than 12 months old, antibodies against all 3 viruses were measured at the time the dogs were given their first vaccination. Altogether, 50.7% of the dogs had titer greater than or equal to 1:10 to CPV, and 26.1 and 46.2% had titer greater than or equal to 1:8 to CAV-1 and CDV, respectively. The concentration of maternal antibody seemed to be of major importance for failure of immunization with use of inactivated CPV vaccine, but not with CAV-1 and CDV vaccination. In dogs less than 12 months old and vaccinated against CPV infection with inactivated virus, only 11.5% had titer greater than or equal to 1:80. In dogs vaccinated against infectious canine hepatitis and canine distemper, 63.2 and 78.3%, respectively, had titer greater than or equal to 1:16. In adult dogs greater than 2 months old and vaccinated against CPV infection, less than 50% had titer greater than or equal to 1:80, regardless of time after vaccination. There was no significant difference in titer between vaccinated and nonvaccinated dogs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Three-year duration of immunity in dogs following vaccination against canine adenovirus type-1, canine parvovirus, and canine distemper virus

A challenge-of-immunity study was conducted to demonstrate immunity in dogs 3 years after their second vaccination with a new multivalent, modified-live vaccine containing canine adenovirus type 2 (CAV-2), canine parvovirus (CPV), and canine distemper virus (CDV). Twenty-three seronegative pups were vaccinated at 7 and 11 weeks of age. Eighteen seronegative pups, randomized into groups of six dogs, served as challenge controls. Dogs were kept in strict isolation for 3 years following the vaccination and then challenged sequentially with virulent canine adenovirus type 1 (CAV-1), CPV, and CDV. For each viral challenge, a separate group of six control dogs was also challenged. Clinical signs of CAV-1, CPV, and CDV infections were prevented in 100% of vaccinated dogs, demonstrating that the multivalent, modified-live test vaccine provided protection against virulent CAV-1, CPV, and CDV challenge in dogs 7 weeks of age or older for a minimum of 3 years following second vaccination.     

同时检测犬瘟热病毒和犬细小病毒双重PCR方法的建立

为建立可以同时检测犬瘟热病毒(CDV)和犬细小病毒(CPV)的双重PCR方法,本研究根据GenBank登录的CDV N蛋白序列和CPV NS基因保守序列,设计合成2对特异性引物。通过优化反应条件,对CDV阳性病毒株反转录后的cDNA模板和CPV的DNA模板进行双重PCR扩增,同时得到2条与试验设计相符的669 bp(CDV)和392 bp(CPV)特异性条带,建立了同时检测CDV和CPV的双重PCR方法。实验结果表明:在同一PCR反应体系中可以同时检测这2种病毒,而对犬腺病毒Ⅰ型、犬腺病毒Ⅱ型、狂犬病毒检测均为阴性;CDV和CPV的最低检出限分别为101.8TCID50和101.4TCID50。采用该方法对在黑龙江省不同地区所采集的30份犬病料样品进行检测,CDV阳性率为30%;CPV阳性率为23.33%,表明建立的PCR方法可以用于临床诊断。     

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.     

Immunohistochemical distribution of alpha B-crystallin in the cerebellum of dogs infected with canine distemper virus

The cerebella of 12 dogs infected with canine distemper virus (CDV) and those of three normal dogs were examined. The avidin-biotin-peroxidase complex technique was used to detect alphaB-crystallin (alphaB-c) immunoreactivity and immunolocalisation of the CDV antigen. CDV antigens, immunopositive astrocytes, oligodendrocytes and granular neurons were seen in both the white and grey matter of the infected dogs. In the controls, alphaB-c immunopositive glial cells were seen in the white matter and around the Purkinje cells. In dogs with distemper, alphaB-c immunoreactivity was not observed in some of the glial cells around the Purkinje cells. A significant negative correlation of P < 0.01 level was found between areas of severe demyelination and the number of alphaB-c immunopositive cells in dogs infected with CDV. Such correlation was not observed between mild and moderate demyelinating areas and alphaB-c immunostaining. The alphaB-crystallin/ total number of cells ratio was found to be significant in severely affected demyelinating areas (P < 0.05). These data indicate that there was a relationship between the degrees of CDV associated with demyelination and the level of alphaB-c expression in the glial cells.     

Antibody titers for canine parvovirus type-2, canine distemper virus, and canine adenovirus type-1 in adult household dogs

Serum antibody titers for canine parvovirus type-2 (CPV-2), canine distemper virus (CDV) and canine adenovirus type-1 (CAV-1) were investigated in 1031 healthy adult household dogs (2 to 18 years old) given an annual inoculation in the previous 11 to 13 months. The number of dogs retaining significant titers of antibodies against CPV-2, CDV, and CAV-1 were 888 (86%), 744 (72%), and 732 (71%), respectively. There were no differences between males and females in antibody titers against the 3 viruses. Antibody titer for CPV-2 was significantly higher in younger dogs than in older dogs, CDV antibody was significantly higher in older dogs than in younger dogs, and CAV titer was not associated with age.     

New members to Arctic-like lineage of canine distemper virus from Turkey

Canine distemper virus (CDV) causes a multisystemic fatal disease, briefly named as distemper, in domestic and wild animals. Molecular characterization studies serve to identify local strains, accordingly, helps to determine the scope of vaccination in prevention of distemper. We aimed with this study to update the molecular status of CDV in domestic dogs in Turkey.Sequence analysis of the H gene revealed that novel Turkish sequences formed a separated clade in Arctic-like lineage. Italian clade which mainly included strains originated from wild canid or non-canid localized nearly to novel Turkish clade. Codons 530th and 549th determining the affinity of domestic or wild animals to distemper were Asparagine and Tyrosine, respectively.This report presented the presence of CDV strains belonging to Arctic-like lineage for the first time in domestic dogs in Turkey. The findings pave the way for the reassessment of the circulation and geographical shifting of Arctic-like lineages of CDV.     

表达犬瘟热病毒H基因重组新城疫病毒的构建及其免疫原性研究

为研制安全、有效的新型犬瘟热疫苗,本研究利用新城疫病毒(NDV) LaSota弱毒疫苗株反向遗传操作系统,构建出表达犬瘟热病毒(CDV)弱毒疫苗株Rockborn-20/8血凝素(H)蛋白的重组病毒rLa-CDV-H,并对其生物学特性进行鉴定,评估其作为犬瘟热活载体疫苗的安全性和有效性.通过免疫荧光和western blot试验证明了CDV H蛋白的正确表达;重组病毒株保持了LaSota亲本株的低致病性和高滴度鸡胚生长特性;重组病毒rLa-CDV-H接种12周龄比格犬后,可以诱导显著的CDV中和抗体反应.本研究表明重组病毒rLa-CDV-H具有作为犬瘟热重组病毒活载体疫苗的潜力.     

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.     

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.     

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 investigation of cerebellum in dogs infected with canine distemper virus

The cerebella of 21 dogs with canine distemper virus (CDV) infection and four normal dogs were examined histopathologically and immunohistochemically. Cerebella of CDV-infected dogs showed nonsuppurative demyelinating encephalomyelitis, classified as acute, subacute or chronic. Immunolocalisation of CDV antigen also confirmed the infection. Tissues were examined for co-localisation of the CDV antigen with either an astrocyte-specific marker, glial fibrillary acidic protein (GFAP), or an oligodendrocyte-specific marker, galactocerebroside (GalC). Immunoreactive cells were counted in demyelinating areas of the white matter. The number of astrocytes (GFAP positive) was significantly (p < 0.05) higher in CDV-infected dogs compared to controls. In contrast, the number of oligodendrocytes (GalC positive) was significantly (p < 0.001) lower in CDV-infected dogs and was much lower in chronic cases (p < 0.05). Approximately 41% of astrocytes and 17% of oligodendrocytes were immunoreactive for CDV. The ratio of CDV-infected oligodendrocytes and astrocytes remained almost constant during the progression of the disease (P > 0.05). In conclusion, CDV infects both astrocytes and oligodendrocytes. The gradual loss of oligodendrocytes is most likely responsible for the progressive demyelination in CDV infection. Astrocytosis in CDV infection should be further investigated if it occurs to stimulate oligodendrocytes for myelin production to compensate for the loss or to induce oligodendrocyte degeneration.     

Comparative analyses of canine distemper viral isolates from clinical cases of canine distemper in vaccinated dogs

Sequence and phylogenetic analyses of three isolates of canine distemper virus (CDV) isolated from three dogs with a vaccination history were compared with the same analyses of vaccine virus isolated from a vaccine used for dogs. The three dogs showed clinical signs of a recent major type of CD in Japan, including oculonasal discharge and diarrhea, and pathological findings including non-suppurative encephalitis, pneumonia, mild gastroenteritis and lymphoid depletion. Inclusion bodies were in the stomach without inflammation and encephalitis was without clinical signs. One of the highest titers of CDV in different organs of the three dogs was commonly systemic lymphatic organs, including the spleen, lymph nodes and tonsils. New isolates of CDV joined to the clades of the Asia 1 group that is far from the vaccine group. These results surely indicate that wild strains of CDV from dogs with a vaccination history were not reversed vaccine virus, and that the dogs showed characteristics of recent CD in Japan.