Differential infection efficiencies of peripheral lung and tracheal tissues in sheep infected with Visna/maedi virus via the respiratory tract (original) (raw)
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Journal of Virological Methods, 2006
In doubtful cases, the histopathological diagnosis of lesions induced by Maedi Visna virus (MVV), a chronic multisystemic lentiviral disease of sheep, needs to be confirmed by the demonstration of MVV in the tissues. The influence of fixatives and the duration of fixation on the detection of MVV by immunohistochemistry (IHC) and PCR in paraffin-embedded tissues was assessed in lung samples with lesions in different degree, from five sheep serologically positive. Samples were fixed in 10% neutral buffered formalin (NBF), Bouin's solution (BS) and a zinc salts-based fixative (ZSF), for different periods of time between 24 h and 30 days. The three fixatives preserved the morphology of the tissues, although in ZSF-fixed samples an increase in the number of desquamated cells was seen in the alveoli. Tissues showed a similar degree of immunolabelling, irrespective of the duration of fixation using ZSF and NBF fixatives. MVV nucleic acids could be detected in samples fixed up to 14 days in NBF and 30 days in ZSF. However, in BS fixed tissues, immunostaining was weak and non-specific signals were observed after 4 days of fixation. Amplification of proviral DNA could not be obtained by PCR in these samples. IHC detected viral antigens in all sheep whereas one sheep with mild lesions was always negative by PCR.
Veterinary Microbiology, 2004
To verify colostral transmission and to study early viral entry in lambs, colostrum samples, and small intestine and mesenteric lymph nodes of lambs born from experimentally infected ewes were examined by histopathology, immunohistochemistry (IHC) and in situ hybridisation (ISH) studies. In particular, newborn lambs were naturally fed maternal colostrum and humanely killed at 10, 24, 48, 72, 96 h and 7 and 10 days after birth; two caesarian-derived lambs served as uninfected controls. No lesions suggestive of MVV infection were found, but marked immunoreactions for MVV capsid antigen (CA, p28) were detected in lambs fed maternal colostrum and in macrophages cultured from colostrum. IHC results in lambs suggest an initial viral absorption by intestinal epithelial cells at the tip of the villi, passage to mononuclear cells in the lamina propria and involvement of ileum Peyers' patches and mesenteric lymph nodes, with different staining patterns depending on infection times. ISH on intestinal sections of the 72 h lamb revealed the presence of proviral DNA in epithelial cells at the tip of the villi, suggesting a role for these cells in early MVV replication. The results contribute to knowledge about the pathogenesis of ovine lentivirus infection suggesting that the small intestine and mesenteric nodes are the sites of entry and propagation of MVV in lambs fed colostrums from infected ewes. #
Clinical & Experimental Immunology, 2008
In order lo investigate the conlribution of lymphocytes to interslilial lung disease in animals with visna-maedi infection, we studied in parallel bronchoalveolar cells and lung tissue from slaughterhouse animals (« = 29) and from colostrum-deprived lambs transtrachcLdly inoculated with field isolates of visna-maedi virus (n = 9) or saline (/i -6). Lymphocyte subpopulations were identitied in bronchoalveolar lavage by immutiolluorcscenceand flow cytometry analysis and in lung tissue using indirect immunohistochemistry. In infected animals a lymphoeytic alveolitis containing CV)4 and CDS lymphtKytes was observed. Peribronchovascular lymphoid nodules comprise mostly CIM lymphocytes. Alveolar lymphocytes of both subsets displayed increased expression of MHC class II antigens in animals with naturally occurring maedi but not in experitnentally infected ones. A sequential process of lymphocyte attraction and activation is likely to occur in vivo as part of the alveolitis.
Clinical & Experimental Immunology, 2008
We have analysed the phenotype of lymphocytes in lung and regional lymph node of symptomatic and asymptomatic sheep infected with the ovine lentivirus, maedi visna virus (MVV). Compared to equilavent tissues from age-matched, non-infected controls, MVV-infected sheep show increased numbers of lymphocytes in the lung, both in the bronchus-associated lymphoid tissue (BALT) and in the alveolar septae. Both CD8+ and CD4+ T lymphocyte numbers in alveolar septae were increased, particularly in animals with clinical respiratory disease. The ratio of CD8 + to CD4+ lymphocytes was similar to that in normal lung. In both MVV-infected and uninfected animals a high proportion of pulmonary lymphocytes, particularly in the alveolar septae, did not express the CD5 antigen, suggesting that they were activated. The number of activated cells was higher in infected sheep. Variable numbers of alveolar macrophages containing MVV-core protein were present in alveolar lumina, the majority of positive cells showing morphological evidence of activation. In regional lymphoid tissue there were increased numbers of CD8+ and yb expressing T cells in lymphoid follicles and germinal centres of infected animals. The specificity of these cells is unknown and we could find no evidence for the presence ofcells productively infected with the virus in these structures. This study shows that activated T lymphocytes, particularly of the CD8 subset, play a major part in the pathogenesis of MVV-induced pulmonary and regional lymph node lesions.
Intratracheal inoculation as an efficient route of experimental infection with maedi–visna virus
Research in Veterinary Science, 2003
Maedi-visna virus (MVV) spreads horizontally via the respiratory route. In order to establish an experimental mucosal infection route, we compared intranasal and intratracheal inoculation using the infectious MVV molecular clone KV1772-kv72/67. For intranasal infection 0.5 Â 10 3-0.5 Â 10 7 TCID 50 of virus was sprayed into the nostrils of the sheep. For the intratracheal infection 10 0-10 6 TCID 50 of virus was injected into the trachea. Successful infection was indicated by development of MVV specific antibodies and virus isolation over a period of 6 months. In the intranasal infection, only the sheep receiving the highest dose i.e., 0.5 Â 10 7 TCID 50 , became infected, suggesting that intranasal application was not an efficient mode of infection. In the intratracheal infection, the sheep infectious dose 50% was 10 1 TCID 50 and virus could be isolated from the central nervous system 4 months post infection with 10 4 TCID 50. Therefore it is concluded that intratracheal infection is a very efficient route for experimental inoculation with MVV.
Maedi-Visna virus (MVV) is a non-oncogenic ovine lentivirus whose main targets are the lung, mammary gland, central nervous system and joints. Cells of the monocyte-macrophage lineage are the major viral target in vivo; other cell types are infected as well, as indicated by several studies, largely based on the examination of animals infected experimentally or on the in vitro infection of cultured cells. Aim of this study was to investigate the cell types harbouring the viral genome in lungs and mammary glands of animals infected naturally by using in situ PCR-associated immunohistochemistry. Several types of cells were infected: in the lung type I and II pneumocytes, interstitial and alveolar macrophages, endothelial cells and fibroblast-like cells. Epithelial cells, macrophages, endothelial cells and fibroblast-like cells were infected also in the mammary gland. These results indicate that the in situ PCR, a powerful technique which combines the high sensitivity of the conventional PCR with the ability to localise the cellular targets within a tissue, can be improved further by its association with the immunohistochemistry. This can be especially advantageous when the presence and localisation of the target sequence are investigated in the context of a tissue with its complex cellular organisation. #