Immunohistochemical characterization of inflammatory cells in brains of dogs with granulomatous meningoencephalitis.

The inflammatory cells of eleven dogs with canine granulomatous meningoencephalitis were characterized immunohistochemically. Macrophages were identified by antibodies directed against lysozyme and the DH82 antigen (expressed by cells of a malignant histiocytosis). T cells were demonstrated by CD3, CD43, and CD45R antigen, and B cells by immunoglobulin G and immunoglobulin M expression. Furthermore, staining for the major histocompatibility complex (MHC) class II antigen was evaluated. Diseased animals ranged from 1 to 9 years of age. Small and medium-sized breeds were affected predominantly. Lesions were widespread and localized mainly in the brain stem, less frequently in the cerebrum or cerebellum. Alterations were represented by perivascular cuffs, parenchymal granulomas, and leptomeningeal infiltrates. Lymphocytes and macrophages comprised the dominant cell populations; their percentage varied substantially between different animals and between sections from the same individual. Immunohistochemically, the bulk of lymphocytes were CD3 antigen-positive T cells, while only a few cells were CD43 and CD45R antigen-positive or were classified as B cells. The majority of macrophages expressed both lysozyme and DH82 antigen; however, some were positive for only one antigen. MHC class II antigen-expression, observed only within and in close proximity to the lesions, was found on all inflammatory cells, pericytes/endothelial cells, and microglia. Results were negative for canine distemper virus antigen and nucleoprotein mRNA, rabies virus antigen, fungi, bacteria, and protozoal agents. This immunomorphologic study reveals that inflammatory lesions in canine granulomatous meningoencephalitis consist of a heterogeneous population of MHC class II antigen-positive macrophages and predominantly CD3 antigen-positive lymphocytes. The data suggest a T cell-mediated delayed-type hypersensitivity of an organ-specific autoimmune disease as a possible pathogenic mechanism for this unique canine brain lesion.

In vivo and in vitro detection of canine distemper virus nucleoprotein gene with digoxigenin-labelled RNA, double-stranded DNA probes and oligonucleotides by in situ hybridization.

A single-stranded RNA, two double-stranded (ds) DNA probes and 10 oligonucleotides labelled with digoxigenin were comparatively evaluated for their usefulness to detect canine distemper virus (CDV) nucleoprotein RNA in in vitro infected Vero cells and in tissues of dogs with spontaneous CDV infection by in situ hybridization (ISH). In addition, results were compared to CDV nucleoprotein antigen distribution as demonstrated by immunohistochemistry. The RNA probe was derived from the virulent A75/17 strain, the DNA and oligonucleotide probes from the avirulent Onderstepoort strain of CDV. The two DNA probes were 287 and 126 base pairs long. For ISH, various factors including fixatives, proteolytic digestion, probe concentration, hybridization conditions and detection systems were compared. All probes were suitable for demonstration of CDV RNA in in vitro infected cells, regardless of the CDV strain employed. In vivo CDV nucleic acid was detected by RNA and the dsDNA probes. However, the probes varied substantially with respect to sensitivity and specificity. The CDV RNA probe was far superior in sensitivity when compared to the DNA probes. Furthermore, the shorter DNA probe displayed a higher sensitivity, indicating that length of the probe is an important parameter when selecting probes. Oligonucleotides displayed only rarely a positive signal and caused frequently hybridization signals in the nucleus, which where considered not specific for CDV. Summarized, the present study reveals that RNA probes are currently the most sensitive tool for detection of CDV RNA in tissues.

Abundant expression of viral nucleoprotein mRNA and restricted translation of the corresponding viral protein in inclusion body polioencephalitis of canine distemper.

Brain and other tissues of three dogs aged 4-21 months with inclusion body polioencephalitis caused by canine distemper virus (CDV) were examined for CDV nucleoprotein (N) antigen and mRNA distribution. Two animals (nos 3 and 1) had suddenly shown central nervous system (CNS) signs 4 days and 5 months, respectively, after vaccination with a modified live CDV vaccine; animal no. 2 had shown similar signs 4 weeks after vaccination with an unknown product. Lesions in the CNS, which were restricted to the grey matter, occurred most frequently in the diencephalon, mesencephalon, medulla oblongata and, in one animal, in the cerebral cortex. Changes were characterized by mild to moderate perivascular lymphohistiocytic cuffs, loss of neurons, neuronal necrosis, glial nodules, and oedema. Intranuclear and cytoplasmic inclusion bodies, especially prominent in neurons, were observed. By in-situ hybridization, CDV N mRNA expression was confirmed with a non-radioactively labelled N-specific mRNA probe. The corresponding RNA translation product was detected immunohistochemically with a proteinspecific monoclonal antibody. Viral antigen and mRNA were observed in the same cell types and brain compartments. However, the number of cells expressing N mRNA exceeded the number of cells containing viral antigen greatly in two animals and slightly in one. Some areas with abundant viral mRNA expression were almost completely devoid of viral antigen. mRNA and the corresponding translation product were demonstrated in neurons and less frequently in astrocytes, but not in perivascular inflammatory cells. It would appear that distemper inclusion-body polioencephalitis may be due to a non-productive CDV infection of neurons, characterized by abundant expression of CDV N mRNA and reduced translation of the corresponding viral protein. These findings suggest that in distemper the pathogenesis of grey-matter lesions differs substantially from that of white-matter lesions, which constitute the most common manifestation of distemper encephalitis.

Metaphyseal bone lesions in young dogs with systemic canine distemper virus infection.

Bone lesions, restricted to the metaphyses of long bones, were observed in young dogs with systemic distemper following experimental and spontaneous infection. Canine distemper virus (CDV) antigen was found immunocytochemically in hematopoietic marrow cells, osteoclasts, osteoblasts and rarely in osteocytes. In experimentally infected dogs, viral antigen was demonstrated in the metaphysis between 5 and 36 days after infection. Associated lesions, characterized by necrosis of osteoclasts, persistence of primary spongiosa and atrophy and necrosis of osteoblasts and marrow cells, were mild and most prominent between 8 and 32 days postinfection. Metaphyseal osteosclerosis (MO) of the long bones, varying from mild to severe, was observed macroscopically in 8 (19%) out of 42 dogs with spontaneous distemper. Affected animals were between 3 and 6 months of age and belonged mainly to the large breeds. In these animals, MO was characterized histologically by persistence of primary spongiosa, loss of bone marrow cells and necrosis of osteoclasts and bone marrow cells varying from mild to severe. Summarized, CDV-associated bone lesions were only transient and there were no indications of viral persistence in bones of dogs experimentally infected with CDV. Although no clinical signs related to the bones were observed, the present study reveals that infection of metaphyseal bone cells is common in young dogs with systemic distemper and occurrence of viral antigen in these cells results in defects in bone modelling.

[Detection of distemper virus N protein RNA in the brain of dogs with spontaneous distemper encephalitis using a digoxigenin-labeled, double-stranded DNA probe for in situ hybridization]. / Nachweis von Staupevirus-N-Protein-RNS im Gehirn von Hunden mit spontaner Staupeenzephalitis mittels einer Digoxigenin-markierten, doppelsträngigen DNS-Sonde in der in situ Hybridisierung.

A digoxigenin-labelled dsDNA-probe of 287 basepairs length complementary to the nucleoprotein-gene of canine distemper virus (CDV) was generated by the polymerase-chain-reaction. The dsDNA-probe hybridized specifically with base sequences of 8 different CDV strains, whereas no hybridization was observed with a porpoise and a canine parainfluenza virus and only a weak signal was obtained with measles virus. In formalin-fixed, paraffin-embedded brain sections of 35 immunohistologically CDV antigen positive dogs with spontaneous distemper encephalitis CDV-RNA could be detected in 25 cases by in situ hybridization. The reason for the lack of RNA detection in some immunohistologically positive dogs may be due to the low stability of DNA-RNA-hybrids. Degradation of RNA by RNAses or diffusion out of autolysed cells can not be excluded.