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.     

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.     

Detection of IgM antibodies against a recombinant nucleocapsid protein of canine distemper virus in dog sera using a dot-blot assay

A dot-blot assay for the detection of IgM antibodies (ABs) against canine distemper virus (CDV) in canine serum is described. The diagnostic potential of this technique was evaluated by analysing sera from three test groups: (i) specific pathogen-free (SPF) beagle dogs experimentally infected with virulent CDV; (ii) SPF dogs immunized with a combined vaccine containing CDV, and (iii) SPF dogs immunized with a CDV-free vaccine. As antigen for the dot-blot assay we used the recombinant nucleocapsid protein (N protein) of the virulent A75/17 CDV strain. All 12 dogs of group 1, infected with virulent CDV, showed detectable CDV-specific IgM levels in their serum. All dogs of group 2 were also positive for anti-CDV IgM after the first immunization with the CDV-containing vaccine. The four dogs immunized with a CDV-free vaccine (group iii) remained negative throughout the course of the experiment. From these results, we conclude that the IgM detection test, which requires only a single serum sample, is a useful method for diagnosing current or recent CDV infection in CDV-infected or CDV-immunized dogs under experimental conditions.     

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.     

Immunophenotypical characterization of monocytes in canine distemper virus infection

Canine distemper virus (CDV) infection induces multifocal demyelination in the central nervous system (CNS). It is thought that the resident macrophages of the CNS, the microglia, as well as invading monocytes associated with the inflammatory reaction may play a central role in the demyelinating process. To evaluate changes in peripheral monocytes in CDV infection their immunophenotype was characterized by flow cytometry during the course of an experimental CDV infection in dogs. The highest number of CDV-infected monocytes was found in dogs developing demyelinating lesions. In CD18, CD45, CD44, and CD14 neither up- nor down-regulation was observed. Marked up-regulation occurred in a number of surface molecules including CD1c, B7-1 and B7-2, MHC I, and CD11b. Peak expression was found at 4-5 weeks post-infection (PI), regardless of clinical outcome. All these molecules play an important role in the host's immune response, notably antigen presentation and cell adhesion. These results demonstrate that CDV infection in vivo may enhance several macrophage functions. This could lead to more effective clearance of the virus but may also increase demyelination through a bystander effect in animals that accumulated significant amounts of CDV in the CNS.     

Cerebellum progesterone concentration decreased in canine distemper virus infection

Progesterone has neuroprotective effects including augmentation of myelination in the central and peripheral nervous system. This study was designed to determine if demyelinating lesions in the cerebellum resulting from canine distemper virus (CDV) infection are associated with progesterone levels. Progesterone was measured using radioimmunoassay in samples of the cerebellum, corpus callosum, medulla oblongata, parietal, frontal, temporal, and occipital cortices as well as cerebrospinal fluid (CSF) and plasma collected from ten CDV infected and six non-infected dogs. The cerebellum progesterone level was significantly different between CDV infected (0.66+/-0.09 ng/g) and control dogs (1.14+/-0.09 ng/g) (p<0.001); however, no difference was observed for the other CNS regions, plasma and CSF (p>0.05). The cerebellum progesterone level was also significantly different between acute (0.71+/-0.0 5 ng/g) and chronic cases (0.61+/-0.09 ng/g) (p<0.05). The CDV infected cerebella were also categorized histopathologically according to the severity of demyelinating lesions as mild (n=5), moderate (n=2), or severe (n=3) among which the cerebellum progesterone level was significantly different (p<0.05). Progesterone concentration was 0.71+/-0.05 ng/g in mild, 0.65+/-0.10 ng/g in moderate, and 0.56+/-0.07 ng/g in severe cases. In conclusion, progesterone concentration decreases in the cerebellum in CDV infection and the severity of demyelinating lesions is the greatest in cerebella with the lowest progesterone concentrations. The results suggest that local impairment of progesterone metabolism may be associated with the initiation and progression of cerebellar lesions in CDV infection.     

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.     

Partial protection and intrathecal invasion of CD8(+) T cells in acute canine distemper virus infection

Initial non-inflammatory demyelination in canine distemper virus infection (CDV) develops against a background of severe immunosuppression and is therefore, thought to be virus-induced. However, recently we found a marked invasion of T cells throughout the central nervous system (CNS) in dogs with acute distemper despite drastic damage to the immune system. In the present study, this apparent paradox was further investigated by immunophenotyping of lymphocytes, following experimental CDV challenge in vaccinated and non-vaccinated dogs. In contrast to CDV infected, unprotected dogs, vaccinated dogs did not become immunosuppressed and exhibited a strong antiviral immune response following challenge with virulent CDV. In unprotected dogs rapid and drastic lymphopenia was initially due to depletion of T cells. In peripheral blood, CD4(+) T cells were more sensitive and depleted earlier and for a longer time than CD8(+) cells which recovered soon. In the cerebrospinal fluid (CSF) we could observe an increase in the T cell to B cell and CD8(+) to CD4(+) ratios. Thus, partial protection of the CD8(+) cell population could explain why part of the immune function in acute distemper is preserved. As found earlier, T cells invaded the CNS parenchyma in these dogs but also in the protected challenged dogs, which did not develop any CNS disease at all. Since markers of T cell activation were upregulated in both groups of animals, this phenomenon could in part be related to non-specific penetration of activated T cells through the blood brain barrier. However, in diseased animals much larger numbers of T cells were found in the CNS than in the protected dogs, suggesting that massive invasion of T cells in the brain requires CDV expression in the CNS.     

Effects of canine distemper virus on natural killer cell activity in dogs

An in vitro 51Cr-release assay was developed to detect the cytotoxicity of natural killer cells (NK) of canine peripheral blood mononuclear leukocytes to canine distemper virus (CDV) target cell membrane-bound antigens. Leukocytes from 23 young (greater than or equal to 1 week of age), CDV-naive gnotobiotic dogs could discriminate between noninfected control and CDV-infected Vero target cells. However, the amount of preinfection NK activity did not positively correlate with the ultimate outcome of the disease process when these same dogs were given virulent R252-CDV. Evaluation of preinfection and postinfection CDV-specific NK activity indicated that infection-associated increases in cytolysis of CDV-infected or noninfected Vero targets did not occur. In vitro infection of peripheral blood leukocytes with CDV did not change the kinetics or magnitude of NK-mediated cytolysis of homologous virus-infected or other NK-susceptible target cells.     

Canine distemper virus-induced thrombocytopenia

Effects of canine distemper virus (CDV) infection on circulating platelet values were studied in gnotobiotic dogs inoculated with R252-CDV. Thrombocytopenia (less than 200,000 platelets/microliter) was present on postinoculation day (PID) 5 and persisted through PID 15. Peak thrombocytopenia occurred on PID 10 (less than 85,000 platelets/microliter). Thrombocytopenia was accompanied by lymphopenia, neutropenia, and monocytopenia. Platelet membrane-bound CDV antigen and IgG were present from PID 7 onward; neither the third component of complement nor IgM was detected on platelets from CDV-inoculated dogs. The mean number of megakaryocytes per unit of bone marrow surface area was unchanged. Megakaryocyte infection was present in dogs euthanatized on PID 4 (0.33%), increased slightly in dogs euthanatized on PID 8 (3%), and increased sharply in dogs euthanatized on PID 9 and 10 to 17.8% and 8.3%, respectively. Phagocytosis of platelets by stellate reticuloendothelial (Kupffer's) cells in the liver was prominent in dogs euthanatized from PID 5 onward. Seemingly, CDV-induced thrombocytopenia was mediated by virus-antibody immune complexes on platelet membranes. Decreased platelet production after PID 8 resulting from direct viral infection of megakaryocytes was a likely contributing factor and occurred against a background of profound virus-induced dysfunctions of all hematopoietic cellular elements.     

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.     

Canine microglial cells: stereotypy in immunophenotype and specificity in function?

Microglial cells represent the endogenous immune system of the central nervous system (CNS). Upon pathological insults they reveal their immunological potential aimed at regaining homeostasis. These reactions have long been believed to follow a uniform and unspecific pattern which is irrespective to the underlying disease entity. Evidence is growing that this view seriously underrates microglial competence as the defenders of the CNS. In the present study, microglial cells of 47 dogs were examined ex vivo by means of flow cytometry. Ex vivo examination included immunophenotypic characterization using eight different surface markers and functional studies such as phagocytosis assay and the reactive oxygen species (ROS) generation test. The dogs were classified according to their histopathological diagnoses in disease categories (controls, canine distemper virus (CDV) induced demyelination, other diseases of the CNS) and results of microglial reaction profiles were compared. Immunophenotypic characterization generally revealed relative high conformity in the microglial disease response among the different groups, however the functional response was shown to be more specific. Dogs with intracranial inflammation and dogs with demyelination showed an enhanced phagocytosis, whereas a significant up-regulation of ROS generation was found in dogs with demyelination due to CDV infection. This strongly suggests a specific response of microglia to infection with CDV in the settings of our study and underlines the pivotal role of microglial ROS generation in the pathogenesis of demyelinating diseases, such as canine distemper.     

犬瘟热病毒自然感染犬淋巴组织中T、B细胞变化的研究

为了调查患犬瘟热病犬淋巴组织中T、B细胞变化的特点及淋巴细胞减少的发病机制,试验通过免疫组织化学的方法观察了T细胞(用CD3和CD45RO检测T细胞)、B细胞(用IgG、IgM抗血清检测B细胞)和犬瘟热病毒(抗犬瘟热病毒抗体)在病犬淋巴组织中的分布。结果表明:在淋巴组织中的淋巴细胞、淋巴小结中树突状细胞和巨噬细胞中均检出了抗病毒阳性反应细胞。在骨髓组织的前髓细胞中也发现抗病毒阳性反应细胞和嗜酸性胞浆内及核内包涵体的存在。与对照组相比,CD3和CD45RO阳性细胞主要存在于T细胞的分布域;但CD3和CD45RO阳性T细胞的数量较少。位于淋巴组织中的巨噬细胞有的被CD45RO染成阳性。在B细胞分布的区域中,IgG、IgM阳性细胞的数量明显减少;一些位于淋巴组织的浆细胞也被IgG或IgM染成阳性。在淋巴组织中淋巴细胞减少的顺序为:IgG阳性细胞减少最明显,其次为IgM和CD45RO阳性细胞,再次为CD3阳性细胞。依据试验结果,作者认为病犬淋巴组织中淋巴细胞减少主要是由B细胞缺乏所引起的;淋巴细胞的增殖能力减弱是引起淋巴组织中淋巴细胞减少的重要原因。     

Canine distemper virus-induced depletion of uninfected lymphocytes is associated with apoptosis

Canine distemper virus (CDV), a negative stranded RNA morbillivirus, causes a multisystemic disease in dogs, which is associated with a severe immune suppression. The aim of the study was to examine the influence of early CDV infection on leukocyte depletion, lymphopenia and virus-induced cell death in dogs infected with a virulent CDV strain. From 10 infected dogs, peripheral blood leukocytes were harvested periodically, phenotyped and analyzed for CDV antigen content and apoptosis using Annexin V-FITC and propidium iodide labeling. CDV infection induced a severe CD3+ T cell and CD21+ B cell depletion in all animals at 3 days post-infection (d.p.i.). For dogs with severe distemper, developing virus persistence in the lymphoid tissue and central nervous system, this lymphopenia lasted until the end of the experiment. Increased levels of lymphocyte apoptosis were found at 3 d.p.i., and monocyte apoptosis at 6 d.p.i. This was more prominent in the group of animals with severe distemper. At 3 d.p.i. no leukocyte infection was detectable indicating that the early lymphocyte depletion and apoptosis was not a direct consequence of virus infection. Taken together, our results demonstrate that CDV-induced lymphopenia is an early event and that the degree of lymphocyte depletion correlates with the severity of disease and virus persistence in the lymphoid tissue and central nervous system.     

Detection of canine distemper virus antigen in canine serum and its application to diagnosis

Canine distemper virus (CDV) antigen was detected in the serum of dogs by an ELISA and the results of this assay were compared with an anti-CDV immunoglobulin M (IgM) antibody test. In paired sera from 26 naturally infected dogs, the antigen-positive rate was 26.9 per cent at the first examination and 11.5 per cent at the second examination two to three weeks later. The antigen was detected in three of the 10 dogs which were negative for anti-CDV IgM antibody at the first examination. It could also be detected in the serum of between eight and two of 40 specific pathogen-free dogs vaccinated against CDV, for up to four weeks after they were vaccinated.     

Presence of anti-platelet IgM and IgG antibodies in dogs naturally infected by Leishmania infantum

Thirty-three dogs, naturally infected by Leishmania infantum, were enrolled in the study and were classified as oligo-symptomatic (n. 15) and symptomatic or markedly symptomatic (n. 18). A control group was 10 healthy dogs. A haematological profile was obtained and the dogs serum was employed to assess the presence of platelet binding IgM and IgG antibodies (PBIgM, PBIgG) using flow cytometry. FITC labelled goat anti-dog IgM or IgG were used to detect PBIgM and PBIgG. Samples with a mean fluorescence intensity (MFI) that was 100 channels higher on a log scale for more than 30% of the platelets than seen in negative control platelets from a healthy dog were considered positive for the presence of anti-platelet antibodies (PBIg). Twenty-one (63.3%) dogs revealed the presence of PBIg. Six of them were oligo-symptomatic while 15 showed moderate or severe clinical signs of illness. All the dogs with PBIg showed the presence of PBIgM, with nine animals showing both PBIgM and PBIgG. Nine of 18 symptomatic or markedly symptomatic dogs showed thrombocytopenia, while normal platelet counts were observed in all oligo-symptomatic animals. Eight of 9 thrombocytopenic animals showed the presence of PBIgM, while six of them showed PBIgG. One thrombocytopenic dog was negative for PBIg. This study is the first report documenting the presence of PBIg in natural canine leishmaniasis implying a pathogenic association between thrombocytopenia and the presence of antibody against platelet membrane.     

犬实验感染CDV的病理学研究

对实验感染犬瘟热病毒的病犬进行了系统的病理学观察,并用酶标ＳＰＡ法对病犬脏器组织中ＣＤＶ抗原进行了定位检查。结果表明,淋巴系统各器官组织是ＣＤＶ急性感染早期首先侵犯的靶器官。脏器组织的病理改变与ＣＤＶ抗原检出呈正相关。脏器组织中包涵体的检出与形态结构具有一定的特征性和示病意义,但采用免疫组化方法检查ＣＤＶ抗原,更具优越性。作者认为,ＣＤＶ９３０３９株和ＣＤＶ９３０４１株是致病力很强的泛嗜性ＣＤＶ。     

Production of antibodies to canine distemper virus in chicken eggs for immunohistochemistry

The present study describes the application of egg yolk antibodies in immunohistochemistry. In order to obtain specific antibodies against canine distemper virus (CDV), chickens were immunized with attenuated virus. Distinct antibody titres in serum and yolk could be detected by means of a modified plaque/focus immunoassay (ELISA) two weeks after a second immunization. The lower concentrations in corresponding yolk globulin preparations are attributed to the loss of antibodies caused by the isolation procedure (dextran and ammonium sulfate precipitation). After verification of the antibody specificity by indirect immunofluorescence technique high titred globulin fractions were employed in immunohistochemistry using the Avidin-Biotin-Complex method. A specific and distinct immunostaining in formalin fixed and paraffin embedded brain sections of CDV-infected dogs was obtained. The advantages of egg yolk antibodies for immunological purposes are discussed in detail.     

Antibodies to Ehrlichia canis, Ehrlichia platys, and Spotted Fever Group Rickettsiae in Louisiana Dogs

Antibodies to Ehrlichia canis, Ehrlichia platys, and spotted fever group (SFG) rickettsiae were detected by indirect immunofluorescence in sera from 27 ill individually owned thrombocytopenic dogs (platelet concentrations less than 200,000 platelets/microliters) and 59 healthy kenneled dogs located in southern Louisiana. Platelet concentrations less than 100,000 platelets/microliters were detected in 63% of ill thrombocytopenic dogs and 6.8% of healthy kennel dogs. One ill thrombocytopenic dog had intracytoplasmic E platys morulae detected within platelets. The prevalence of increased serum antibody titers to E canis and E platys was 25.9% and 40.7% for the ill thrombocytopenic dogs and 20.3% and 54.2% for the healthy kennel dogs, respectively. All dogs with seropositivity to E canis had increased antibody titers of greater than or equal to 1:100 to E platys. Simultaneous examination of increased serum antibody titers (greater than or equal to 1:64) to four SFG rickettsiae indicate that Rickettsia rhipicephali and Rickettsia montana accounted for the majority of the antibodies detected in these dogs. Of 86 dogs tested, 44.2% were seronegative to E canis, E platys, and SFG rickettsiae.     

自发性急性犬瘟热的原发性脱髓性脑病

为了进一步观察犬瘟热病毒引起的原发性脑损伤和包涵体形成的特点，调查脑组织的损伤与神经症状的关系，对10只急性犬瘟热病犬的脑组织进行了详细的病理学研究。为了仔细地观察病变，本试验按照解剖学关系将脑组织分成3个大部分和11个切面，即大脑(4个切面)，脑干(5个切面)和小脑(2个切面)。组织切片经HE、LFB和免疫组织化学染色后进行检查，结果表明：在大脑，脱髓呈弥漫性发生，程度较轻；脑干的周围或靠近第三脑室的白质脱髓较重；小脑在轻度或中度脱髓的基础上常出现严重的多发性脱髓灶。脱髓部呈空泡或海绵状，有少量胶质细胞存在，但无炎性反应。脱髓性病损是非时称性发生，对神经束没有特殊的亲和力。在脑室的室管膜细胞内发现有较多的嗜酸性胞浆或核内包涵体。用抗犬瘟热病毒抗体染色，带有包涵体的室管膜细胞呈现强阳性反应。部分锥体细胞，神经核细胞和漓氏细胞变性、溶解或胞浆深染。胞核浓缩。这种变化以小锥体神经细胞表现得最为明显。根据此研究结果，作者认为由犬瘟热病毒引起的原发性脑组织损伤是一种脱髓性脑病，而不是脑炎变化；位于室管膜细胞内的包涵体对于脑组织犬瘟热的确诊具有重要的作用；由于犬瘟热病毒引起神经细胞的损伤是非特异性的，对脑组织的侵害是非对称性的。对神经束的作用无特殊的亲和力，所以患犬瘟热的犬在临床上可出现不同的神经症状。