Canine Distemper Encephalitis Research Articles

Beneficial and detrimental impact of transplanted canine adipose-derived stem cells in a virus-induced demyelinating mouse model

In recent years stem cell therapies have been broadly applied in various disease models specifically immune mediated and degenerative diseases. Whether adipose-derived stem cells might represent a useful therapeutic option in virus-triggered central nervous system diseases has not been investigated so far. Theiler’s murine encephalomyelitis (TME) and canine distemper encephalitis are established, virus-mediated animal models sharing many similarities with multiple sclerosis (MS). Canine adipose-derived stem cells (ASC) were selected since dogs might serve as an important translational model for further therapeutic applications. The aim of the present study was to investigate whether canine ASC influence clinical signs, axonal damage, demyelination and inflammation during TME. ASC were transplanted intravenously (iv) or intra-cerebroventricularly (icv) at 7 (early) or 42 (late) days post infection (dpi) in TME virus (TMEV) infected mice.TMEV/ASC iv animals transplanted at 7dpi displayed a transient clinical deterioration in rotarod performance compared to TMEV/control animals. Worsening of clinical signs was associated with significantly increased numbers of microglia/macrophages and demyelination in the spinal cord. In contrast, late transplantation had no influence on clinical findings of TMEV-infected animals. However, late TMEV/ASC iv transplanted animals showed reduced axonal damage compared to TMEV/control animals. Screening of spinal cord and peripheral organs for transplanted ASC revealed no positive cells. Surprisingly, iv transplanted animals showed pulmonary follicular aggregates consisting of T- and B-lymphocytes. Thus, our data suggest that following intravenous application, the lung as priming organ for lymphocytes seems to play a pivotal role in the pathogenesis of TME. Consequences of T-lymphocyte priming in the lung depend on the disease phase and may be responsible for disease modifying effects of ASC.

犬ジステンパー脳炎の診断におけるリファレンス抗体測定の有用性

犬ジステンパー脳炎（CDE）を血清学的に診断する際の脳脊髄液への血液成分混入の指標（リファレンス抗体）として犬パルボウイルス抗体と犬アデノウイルス抗体の測定の有用性を検討した．両抗体の血清中力価/脳脊髄液中力価（S/C）値の比は検討した30頭すべて測定誤差の許容範囲内（2：1，1：1 又は1：2）であり，どちらの抗体を指標としても同様な成績が得られることが示された．CDEの診断基準である犬ジステンパーウイルス（CDV）抗体のS/C値＜256を示した神経症例29頭中1頭だけがCDV抗体のS/C値がリファレンス抗体のそれに比べて低く（1：8），CDEと診断されたが，残りの28頭の両抗体のS/C値の比は誤差範囲内で診断は成立しなかった．このことはCDEの診断に際しCDV抗体だけでは誤診を招く危険性があることを示すものである．

Emergence of canine distemper in Bavarian wildlife associated with a specific amino acid exchange in the haemagglutinin protein

A widespread, severe outbreak of canine distemper encephalitis was observed in wildlife in Southern Bavaria in the spring and summer of 2008. The haemagglutinin (HA) genes of six representative canine distemper virus (CDV) samples originating from five red foxes and one badger during this outbreak had a Y549H amino acid substitution in the HA protein compared to sequences from two captive domesticated ferrets which succumbed to CDV in the same area 2 years earlier. As this specific substitution at the receptor-binding site has been hypothesised to contribute to the emergence of CDV and its spread to novel hosts, the outbreak in wildlife in Southern Bavaria might, directly or indirectly, be associated with a Y549H amino acid exchange.

Acute canine distemper encephalitis is associated with rapid neuronal loss and local immune activation

For most virus infections of the central nervous system (CNS), immune-mediated damage, the route of inoculation and death of infected cells all contribute to the pathology observed. To investigate the role of these factors in early canine distemper neuropathogenesis, we infected ferrets either intranasally or intraperitoneally with the neurovirulent canine distemper virus strain Snyder Hill. Regardless of the route of inoculation, the virus primarily targeted the olfactory bulb, brainstem, hippocampus and cerebellum, whereas only occasional foci were detected in the cortex. The infection led to widespread neuronal loss, which correlated with the clinical signs observed. Increased numbers of activated microglia, reactive gliosis and different pro-inflammatory cytokines were detected in the infected areas, suggesting that the presence and ultimate death of infected cells at early times after infection trigger strong local immune activation, despite the observed systemic immunosuppression.

Simultaneous canine distemper encephalitis and canine parvovirus infection with distemper-associated cardiac necrosis in a pup

Simultaneous infection of canine distemper virus and canine parvovirus associated with distemper myocardial degeneration and necrosis is described in a pup. The dog demonstrated myoclonus, nystagmus, enamel hypoplasia, abdominal pustules, and bilateral corneal ulceration clinically. Demyelinating encephalitis, myocardial degeneration and necrosis with mineralization, and necrosis, hemorrhage and fusion of intestinal villi were observed. The lesions observed in this dog are characteristic of a dual infection of canine distemper virus and canine parvovirus.

Glial Fibrillary Acidic Protein (GFAP)-immunoreactive Astrocytes in Dogs Infected with Canine Distemper Virus

An experiment based on astrocyte immunoreactivity to glial fibrillary acidic protein (GFAP) was designed to determine whether the astrocyte response in canine distemper encephalitis (CDE) was associated with the age of the animal, type of lesion and the cerebellar region affected. Four histopathological types of CDE lesion were examined, namely acute (11 dogs), acute with necrosis (four dogs), subacute (22 dogs) and chronic (six dogs). The animals were divided into three age groups, namely, 0–2 years (27 dogs), 2·1–4 years (12 dogs), and 4·1–12 years (four dogs). Three different cerebellar regions were evaluated. Cerebellar sections from three healthy dogs were used for control purposes. The highest number of astrocytes occurred in the cerebellar white matter and in dogs with acute distemper encephalopathy. In animals with subacute distemper encephalitis, the numbers of astrocytes appeared to increase with age, but the opposite effect occurred in dogs with acute or chronic encephalitis; age appeared not to influence the astrocyte numbers in dogs suffering from acute encephalitis with necrosis.

Disappearance of beta2-adrenergic receptors on astrocytes in canine distemper encephalitis: possible implications for the pathogenesis of multiple sclerosis.

It has been reported that astrocytes in the white matter of patients with multiple sclerosis (MS) lack beta2-adrenergic receptors. This abnormality might explain why astrocytes in active MS plaques aberrantly express major histocompatibility (MHC) class II molecules, which play an important role in the immunological cascade leading to myelin destruction. Canine distemper (CD) virus primarily infects astrocytes and causes a demyelinating disease in dogs that closely resembles MS. In control dogs, including three dogs with another inflammatory disease, beta2-adrenergic receptor immunoreactivity was observed on both neurons and astrocytes. In dogs with CD encephalitis, beta2-adrenergic receptors were present on neurons, but were absent on astrocytes in acute lesions, demyelinated lesions, and normal-appearing white matter. Similar to MS, several astrocytes in demyelinated lesions expressed MHC class II. These findings suggest that MS and the demyelinating stages of CD encephalitis have a common pathogenetic factor, and that the loss of astrocytic beta2-adrenergic receptors in MS might be induced by a viral infection of astrocytes.

Up-regulation of the hyaluronate receptor CD44 in canine distemper demyelinated plaques.

CD44 antigen (CD44), the principle cell surface receptor for hyaluronate, is up-regulated in the human demyelinating disease multiple sclerosis on fibrous astrocytes. As astrocytes are the main target cell of canine distemper virus (CDV), the consequences of a CDV infection on the CD44 expression and distribution in brains with spontaneous demyelinating canine distemper encephalitis (CDE) were of interest. Thirteen acute, 35 subacute, and 11 chronic plaques of nine dogs with immunohistologically confirmed CDE and brains of control dogs were included in the study. For light microscopy, 5-micron-thick serial sections were stained with H&E and incubated with monoclonal antibodies (mAbs) against CD44 and canine distemper virus nucleoprotein and polyclonal antibodies (pAbs) against glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP). For immunoelectron microscopy, 90-nm-thick sections were double stained with anti-GFAP and anti-CD44 mAbs to specify CD44-expressing structures. In controls, CD44 was diffusely distributed in the white matter and single meningeal cells exhibited a marginal expression of the antigen. In acute and more prominently in subacute demyelinating encephalitis, there was a plaque-associated up-regulation of CD44 which paralleled GFAP. In chronic demyelinating lesions, a reduction of CD44 associated with a loss of GFAP-positive astrocytes was noted. Additionally, in chronic plaques, CD44 was expressed on the cell membrane of perivascular mononuclear cells. Immunoelectron microscopically, in controls, CD44 was rarely demonstrated on astrocytic cell processes. In contrast, in brains with CDE CD44 was found on the cell membrane of broadened astrocytic cell processes. In summary, CD44 is up-regulated on astrocytes in the early phase of CDE and seems to represent a marker for the activation of immune cells in the late phase of the infection.

Phenotypical characterization of T and B cell areas in lymphoid tissues of dogs with spontaneous distemper

CD3, CD4, CD5, and CD8 antigen expression of T cells and IgG expression of B cells and canine distemper virus (CDV) antigen distribution were immunohistochemically examined in lymphoid tissues (lymph node, spleen, thymus, and tonsil) of control dogs and animals with spontaneous canine distemper. In addition, CNS tissue of all animals was studied for neuropathological changes and CDV antigen distribution. Based on the degree of depletion distemper dogs were classified into two groups. Group I represented animals with moderate to marked lymphoid depletion, while group II dogs displayed mild or no depletion. CDV antigen was mainly found in lymphocytes and macrophages of group I dogs, whereas CDV expression was most prominent in dendritic cells of group II animals. In group I dogs, a marked loss of CD3, CD4, CD5, CD8, and IgG expression was noticed, hereby loss of CD4+ cells was more prominent than depletion of CD8+ cells. In the lymphoid tissues of group II animals, a significant increase in the number of T and B cells was observed compared to group I dogs. The number of CD3+, CD4+, and CD8+ cells in group II dogs was similar to the findings in controls, however, CD5 and IgG expression was mildly reduced in T and B cell areas, respectively. Additionally, in groups I and II dogs, CD3+ and CD5− T cells were detected in T cell areas. Whether this cell population represents a cell type with autoimmune reactive potential remains to be determined. Surprisingly in group II animals, viral antigen was found predominantly in dendritic cells indicating a change in the cell tropism of CDV during chronic infection and a possible mechanism of viral persistence. The two patterns of lymphoid depletions correlated to two different types of canine distemper encephalitis (CDE). Group I dogs displayed acute non-inflammatory CDE, whereas group II dogs suffered from chronic inflammatory demyelinating CDE, indicating a pathogenic relationship between lymphocytic depletion and inflammatory brain lesions in distemper.

Avaliação física, citológica, conteúdo de proteínas e determinação qualitativa de globulinas do liquor de cães normais e de cães com encefalite por cinomose

0 presente experimento teve como objetivos estabelecer padrões de referência para constituintes do líquido cefalorraquidiano de cães normais e de cães jovens portadores de encefalite por cinomose. Foram utilizados 40 animais, sendo 20 cães normais (de seis a 48 meses de idade) e 20 cães com cinomose (até 18 meses de idade). Os exames físico, citológico e bioquímico do líquor dos cães normais permitiram o estabelecimento de valores para densidade: 1007± 1,83, pH: 8,30+0,34. contagem global de hemácias: 6,65 + 5,27/pl, contagem global de leucócitos: 2.85+ 1,63/pl e dosagem de proteínas: 38.70+ 15,49 mg/dl. Os exames físico, citológico e bioquímico do líquor de cães com cinomose permitiram o estabelecimento de valores para densidade: 1010+1,50, pH: 8,35 + 0,46, contagem global de hemácias: 8,55 + 6,48/pl, contagem global de leucócitos: 99,05 + 106,93/pl e dosagem de proteínas: 219,03 + 81,08 mg/dl. O líquor apresentou-se límpido e incolor em todas as amostrasdos cães normais e na maioria das amostras de cães com cinomose. Algumas amostras dos cães com cinomose encontravam-se discretamente turvas e/ou xantocrômicas. O Teste de Pandy foi negativo em todas as amostras de cães normais e positivo, variando de duas a três cruzes, nos animais com cinomose. A contagem diferencial de células do líquor de cães com cinomose revelou uma predominância (67% a 97%) de linfócitos em todas as amostras, presença de 2% a 24% de monócitos e 2% a20% de plasmócitos. Em algumas amostras observaram-se macrófagos, neutrófilos segmentados e células ependimárias. Verificou-se uma correlação positiva entre o número de plasmócitos, os níveis protéicos totais e a taxa de globulinas do líquor de cães com cinomose.

Up-regulation of major histocompatibility complex class II antigen expression in the central nervous system of dogs with spontaneous canine distemper virus encephalitis.

Major histocompatibility complex class II (MHC II) and canine distemper virus (CDV) antigen expression were compared by immunohistochemistry in the cerebellar white matter of ten dogs with naturally occurring canine distemper encephalitis. In addition, infiltrating mononuclear cells were characterized by employing poly- and monoclonal antibodies directed against human CD3, canine MHC II, CD5, B cell antigen and CDV-specific nucleoprotein. Positive antigen-antibody reaction was visualized by the avidin-biotin-peroxidase complex method on frozen sections. Histologically, neuropathological changes were categorized into acute, subacute, and chronic. In control brains, MHC II expression was weak and predominantly detected on resident microglia of the white matter and on endothelial, perivascular and intravascular cells. In CDV antigen-positive brains, MHC II was mainly found on microglia and to a lesser extent on endothelial, meningeal, choroid plexus epithelial, ependymal and intravascular cells. In addition, virtually all of the perivascular cells expressed MHC II antigen. CDV antigen was demonstrated most frequently in astrocytes. Of the perivascular lymphocytes, the majority were CD3-positive cells, followed by B cells. Only a small proportion of perivascular cells expressed the CD5 antigen. In addition, B cells and CD3 and CD5 antigen-positive cells were found occasionally in subacute and frequently in chronic demyelinating plaques. In acute encephalitis, CDV antigen exhibited a multifocal or diffuse distribution, and MHC II was moderately up-regulated throughout the white matter and accentuated in CDV antigen-positive plaques. In subacute encephalitis, moderate multifocal CDV antigen and moderate to strong diffuse MHC II-specific staining, especially prominent in CDV antigen-positive lesions, were observed. In chronic encephalitis, CDV antigen expression was restricted to single astrocytes at the edge of the lesions or was absent, while MHC II expression, especially prominent on microglia, was strongly up-regulated throughout the white matter, most pronounced in demyelinated plaques. In summary, in acute and subacute lesions without perivascular cuffs, MHC II expression correlated with the presence of CDV antigen. In contrast, in chronic lesions, MHC II expression on microglial cells was the most prominent despite a few CDV antigen-positive astrocytes, indicating that nonviral antigens may play an important role as triggering molecules for the process of demyelination.

Neural cells from dogs with spontaneous distemper encephalitis express class II major histocompatibility complex molecules

Expression of class II major histocompatibility complex (MHC) molecules by non-immune cells (e.g., parenchymal cells) leads to the presentation of self-antigens, and may have a role in the pathogenesis of many diseases mediated by autoimmunity. Such diseases, characterized by demyelination of the central nervous system and expression of class II MHC molecules on neural cells, include multiple sclerosis, experimental allergic encephalitis and Theiler's murine encephalomyelitis virus infection. Canine distemper encephalitis probably does not have an autoimmune character, but it shares many similarities with the aforementioned diseases. For this reason, the expression of class II MHC molecules in the brains of dogs with canine distemper encephalitis was investigated immunohistochemically. The results presented here demonstrate that canine microglia and astrocytes "upregulate" class II MHC expression in cases of encephalitis associated with chronic canine distemper.

Diagnosis of inflammatory and infectious diseases of the central nervous system in dogs: a retrospective study.

The medical records of 220 dogs with inflammatory/infectious diseases of the central nervous system (CNS) were retrospectively examined. The aims of the study were to determine if clinical and clinicopathologic data (not including biopsy or necropsy examination) could distinguish inflammatory CNS diseases from diseases of other types, and to search for criteria allowing differentiation of specific inflammatory diseases. The signalment, historical findings, extraneural and neurological signs, and the lesion site contributed marginally to a specific diagnosis. Multifocal signs were only noticed in one third of the dogs with inflammatory/infectious diseases. Particular neurological abnormalities were more frequent in certain diseases than in others (eg, myoclonus was frequent in dogs with distemper, but it was also found in those with other meningoencephalomyelitides). Hematologic findings contributed to the diagnosis in certain conditions (eg, canine distemper encephalitis, protozoal encephalomyelitis, steroid-responsive meningitis-arteritis). Cerebrospinal fluid examinations, including immunoglobulin G index and cytology were useful to separate meningoencephalomyelitides from the other CNS diseases and to distinguish certain conditions from others. In most cases a specific diagnosis depended on a combination of clinical signs and ancillary diagnostic aids. Still, a specific diagnosis remained very difficult, if not impossible, in at least one third of the dogs.

Restricted expression of viral surface proteins in canine distemper encephalitis.

Sixteen dogs with naturally occurring acute and chronic canine distemper virus (CDV) encephalitis were examined immunohistochemically for the presence of the five major CDV-specific proteins in the central nervous system. Monoclonal antibodies (mAbs) directed against two, three, four and five epitopes of the nucleo- (N), phospho- (P), fusion (F), and hemagglutinin (H) protein, respectively, and a polyclonal monospecific antibody recognizing the matrix (M) protein were used. Both core proteins and their epitopes, three F protein epitopes and the M protein were demonstrated in all animals examined. A fourth F protein epitope was found only in 13 animals. The H-2 and H-3 epitope of the H protein were detected in 15, the H-1 and H-5 epitope in 14, and the H-4 epitope in 3 animals. All viral proteins were observed in the same types of brain cells including neurons and astrocytes. The N and P protein were demonstrated in nucleus, cytoplasm and cell processes, whereas M, H and F protein were observed in the cytoplasm only and rarely in cell processes. In addition, the M protein was detected occasionally in the nucleus of neurons and reactive astrocytes. Intralesional distribution of CDV-specific proteins varied between core and surface proteins. In acute and subacute lesions without associated inflammation, expression of the M, H and F protein was only slightly diminished compared to the N and P protein. However, plaques with severe inflammation were either devoid of viral antigen or exhibited N- and P protein-specific immunoreactivity exclusively at the periphery, whereas expression of surface proteins was severely reduced or absent. These results are suggestive of restricted synthesis of CDV envelope proteins in acute, and more prominent in chronic, distemper encephalitis.

Canine distemper virus-immune complexes induce bystander degeneration of oligodendrocytes.

Demyelination in chronic canine distemper encephalitis may be the result of a bystander effect in which the antiviral immune response is involved. In the present report we demonstrate that canine distemper virus-antiviral antibody immune complexes induce oligodendroglial degeneration in mixed brain cell cultures, particularly at the level of the cell processes. The involvement of macrophages as effector cells in this process was confirmed by depletion of these cells from the cultures which prevented the immune complex-mediated oligodendroglial degeneration. Canine distemper virus-immune complex-induced oligodendroglial pathology is thought to be mediated by toxic factors released from stimulated macrophages, this bystander effect demonstrated here in vitro may be relevant to the mechanisms of demyelination in vivo, in which virus persistence plays an important role.

Selective Degeneration of Oligodendrocytes Mediated by Reactive Oxygen Species

The mechanism underlying demyelination in inflammatory canine distemper encephalitis is uncertain. Macrophages and their secretory products are thought to play an important effector role in this lesion. Recently, we have shown that anti-canine distemper virus antibodies, known to occur in chronic inflammatory lesions, stimulate macrophages leading to the secretion of reactive oxygen species (ROS). To investigate whether ROS could be involved in demyelination, dog glial cell cultures were exposed to xanthine/xanthine oxidase (X/XO), a system capable of generating O2-. This treatment resulted in a specific time-dependent degeneration and loss of oligodendrocytes, the myelin producing cells of the central nervous system. Initial degeneration was not associated with a decrease in viability of oligodendrocytes as judged by trypan blue and propidium iodide exclusion. Astrocytes and brain macrophages were not affected morphologically by this treatment. Further, an evaluation of the effect of several ROS scavengers, transition metal chelators and inhibitors of poly (ADP-ribose) polymerase suggests that a metal dependent formation of .OH or a similar highly oxidizing species could be responsible for the observed selective damage to oligodendrocytes.

Antibody-induced generation of reactive oxygen radicals by brain macrophages in canine distemper encephalitis: a mechanism for bystander demyelination.

The mechanism of inflammatory demyelination in canine distemper encephalitis (CDE) is uncertain but macrophages are thought to play an important effector role in this lesion. Serum and cerebrospinal fluid (CSF), containing anti-canine distemper virus and anti-myelin antibodies from dogs with CDE were tested for their ability to generate reactive oxygen species (ROS) in macrophages in primary dog brain cell cultures using a chemiluminescence (CL) assay. The majority of serum samples and several CSF samples from animals with inflammatory demyelination elicited a CL signal in infected dog brain cell cultures. In contrast, none of these samples induced a positive response in uninfected cultures which contained large numbers of myelin antigen-presenting cells, although defined anti-myelin antibodies lead to a marked secretion of ROS in this system. It was concluded that antiviral antibody-induced secretion of ROS, known to be highly toxic for brain tissue, may play an important role in white matter damage in inflammatory lesions supporting a previous hypothesis of bystander demyelination in CDE. No evidence was found for a similar antibody-dependent cellular cytotoxicity-like mechanism mediated by anti-myelin antibodies in CDE, which does not support the concept of autoimmunity in this disease.

Demyelinating canine distemper encephalomyelitis: measurement of myelin basic protein in cerebrospinal fluid

Beagle dogs were experimentally infected with the Cornell A75-17 strain of canine distemper virus. At three time points post-infection (PI), immunoreactive myelin basic protein (MBP) was measured in cerebrospinal fluid (CSF). Levels were correlated with neuropathological findings, interferon in CSF and virus isolation from the brain. CSF from animals inoculated with Cornell A75-17 strain often showed detectable immunoreactive MBO late in the disease course. As anticipated from earlier morphological studies, CSF drawn around day 20 PI lacked MBP while subsequent samples were positive. Dogs with severe demyelination had elevated values of immunoreactive MBP while dogs with only mild inflammation had little or none. Release of MBP or MBP peptides into CSF of dogs with canine distemper may be a valuable laboratory test in studies of the natural history of this disease and in assessing the response to treatment. Whether an immune response to MBP plays an immunopathogenic role in the chronic, demyelinating phase of canine distemper encephalitis remains to be determined.

Studies on the intrathecal humoral immune response in canine distemper encephalitis

Albumin and IgG were quantitated in paired cerebrospinal fluid (CSF) and serum samples from dogs with demyelinating canine distemper virus (CDV) infection by means of rocket immunoelectrophoresis. The IgG index as indicator for intrathecal immunoglobulin synthesis was normal in animals with non-inflammatory demyelinating lesions and elevated in dogs with inflammatory myelin lesions. Specific antibodies against CDV and myelin were quantitated in CSF and serum from 8 dogs with an elevated IgG index. Eight of these dogs had significant amounts of antimyelin antibody and 4 dogs had neutralizing anti-CDV antibody in the CSF. Whereas the pathogenetic significance of antimyelin antibodies remains uncertain, the intrathecal antiviral immune response provides a plausible explanation for immunopathologic destruction of myelin in distemper.

Immunological and pathological findings in demyelinating encephalitis associated with canine distemper virus infection.

Nine dogs with canine distemper encephalitis (CDE) were examined with immunological techniques including demonstration of antibodies against canine distemper virus (CDV) in the serum and against myelin basic protein (MBP) in serum and in CSF. Mitogen stimulation tests of lymphocytes were also done. The brains were examined pathologically and immunoglobulin and C3 were demonstrated in lesions by means of immunohistological techniques. Six dogs with acute CDE had none or low antibody levels against CDV or MBP, and there was no immunoglobulin in demyelinating lesions. Some of these dogs had depressed lymphocyte mitogen responses. Two dogs with chronic CDE showed recovery of lymphocyte mitogen responses. One of these had a significant antibody response against CDV and MBP in the serum. Both dogs with chronic CDE had very high antibody titers against MBP in the CSF and demyelinating lesions contained immunoglobulin. These results suggest that acute demyelination in CDE is probably due to some direct viral activity and that the progression of demyelination in chronic CDE is associated with a local immune response.