key: cord-0683421-1ts6llx2 authors: WANG, Huanan; HIRABAYASHI, Miyuki; CHAMBERS, James K.; UCHIDA, Kazuyuki; NAKAYAMA, Hiroyuki title: Immunohistochemical studies on meningoencephalitis in feline infectious peritonitis (FIP) date: 2018-10-17 journal: J Vet Med Sci DOI: 10.1292/jvms.18-0406 sha: 64531e8dca54414b55f67981cd6e67b77fe8c43c doc_id: 683421 cord_uid: 1ts6llx2 The present study describes the association between inflammatory cell types and feline infectious peritonitis virus (FIPV) antigen in the brain of 4 cats diagnosed as feline infectious peritonitis (FIP). Immunohistochemically, FIPV antigens were detected in the inflammatory foci of the leptomeninges, choroid plexus and ventricles in 3 of the 4 cats. In 3 cases, inflammatory foci mainly consisted of CD204- and Iba1-positive macrophages, and the FIPV antigens were found in the macrophages. In the other case which was negative for FIPV antigen, severe inflammation predominantly consisting of CD20-positive B lymphocytes was observed in the leptomeninges and subventricles, accompanied with diffuse proliferation of gemistocytic astrocytes. The difference in histopathology may reflect the inflammatory process or the strain variation of FIP virus. FIP). For case Nos. 3 and 4, the antibody titer of FCoV was elevated (FIP: >100). All tissues were fixed in 10% neutral-buffered formalin, routinely processed and embedded in paraffin wax. Tissue sections of 4 µm-thick were stained with hematoxylin and eosin (HE). Selected brain sections were subjected to immunohistochemical examinations. Normal feline brain tissue was also stained as a control. Deparaffinized and rehydrated sections were treated through the antigen retrieval methods listed in Table 2 . After being immersed in 3% hydrogen peroxide in methanol at room temperature for 5 min, the sections were treated with 8% skim milk at 37°C for 40 min. The sections were then incubated with the primary antibodies listed in Table 2 at 4°C overnight, and then incubated with the Dako EnVision + System horseradish peroxidase (HRP)labelled anti-rabbit (Dako-Japan, Tokyo, Japan) or anti-mouse secondary antibodies (Dako-Japan) at 37°C for 40 min. The antigenantibody reaction was visualized with 0.05% 3.3′-diaminobenzidine and 0.03% hydrogen peroxide in tris-hydrochloric buffer. Counterstaining was performed with hematoxylin. Inflammatory changes were observed in the brain and spinal cord of all the 4 cases. They were located in the leptomeninges (Fig. 1a) , choroid plexus ( Fig. 1c ) and subventricular area (Fig. 1e ) of the lateral, third and fourth ventricles (Table 3) . These inflammatory lesions were characterized by perivascular accumulations of inflammatory cells, and the inflammatory cells consisted of macrophages, lymphocytes, plasma cells and neutrophils. Some macrophages showed an epithelioid cell morphology, forming granulomatous lesions. In all cases there were no notable neuronal degeneration or glial nodules in the brain parenchyma. In the three cases (Case Nos. 1 to 3), viral antigens were detected within the inflammatory foci but not in the brain parenchymal cells including neurons and glial cells (Figs. 1b, 1d and 1f ). On the other hand, in case No. 4, viral antigens were observed neither in the inflammatory foci nor brain parenchyma (Table 3 ). Further differently, in three of the 4 cases (Case Nos. 1 to 3), brain parenchymal lesions were not evident, while in case No. 4, glial fibrillary acidic protein (GFAP)-positive gemistcytic astrocytes proliferated diffusely in the subventricular parenchyma adjacent to severe inflammatory lesions ( Fig. 2a and 2b) . In Case Nos. 1 (Fig. 3a-f ), 2 and 3, viral antigens ( Fig. 3b) were scattered within the perivascular area where a large number of Iba1-and CD204-positive macrophages accumulated ( Fig. 3c and 3d ). Besides, a moderate number of CD20-positive B lymphocytes accumulated around the glia limitans adjacent to the inflammatory foci (Fig. 3e) . A few CD3-positive T lymphocytes were diffusely observed in the foci (Fig. 3f ). Neutrophils were observed in various degrees. In contrast, in case No. 4, Iba1-and CD204-positive macrophages were sparsely scattered in the inflammatory foci (Fig. 4a , 4c and 4d) without a distinct granuloma formation. No FIPV antigens were detected in the foci (Fig. 4b) . A large number of CD20-positive B lymphocytes accumulated mainly in the perivascular areas (Fig. 4e) . The distribution pattern of CD3-positive T lymphocytes was similar to those of the other three cases (Fig. 4f ). Neutrophils were observed in various degrees. In addition to brain lesions, severe inflammatory lesions were observed in the lung (Case Nos. Table 1 ). The type of inflammatory cells in each organ were similar to the brain. Inflammation was also found in adipose tissue and serosa of the thoracic and abdominal organs for Case Nos. 2 and 3, and of the abdominal organs for Case No. 4. In the present study, all the 4 cases examined were diagnosed as FIP based on the clinical and pathologic findings. Viral antigens were detected in macrophages around vessels of the lesions in the leptomeninges and subventricles of Case Nos. 1 to 3. These findings were in conformity with those in a recent report on FIP meningoencephalitis [15] . On the other hand, viral antigen was not detected in the brain of Case No. 4, though the cat was clinically diagnosed as FIP because of neurological symptoms and elevated antibody titer of FCoV. Pathological diagnosis of FIP is mainly performed by immunofluorescence (IF) using fresh tissues or immunohistochemistry on formalin-fixed paraffin sections [7] . Immunohistochemistry was believed to be more specific than IF [15] , while some previous reports also raised questions about its specificity [4] . The antibody used in this study, FIPV3-70, is known to react with the nucleocapsid antigen of FIPV and is considered a valuable diagnostic tool for FIP [8] . However, a previous paper demonstrated antigenic heterogeneity in FIPV nucleocapsid proteins [14] . Thus, it is possible that the difference in FIPV antigen type cause the lack of viral antigen in lesions of Case No. 4. For the constitution of inflammatory cells in the lesion, CD204-and/or Iba1-positive macrophages, CD20-positive B cells, CD3positive T lymphocytes and neutrophils were detected in all 4 cases with various severities. In three cases (Case Nos. 1 to 3), the majority of inflammatory cells was CD204-and/or Iba1-positive macrophages and a moderate number of CD20-positive B lymphocytes accumulated around the glia limitans adjacent to the inflammatory foci. On the contrary, in Case No. 4, the majority of the inflammatory cells was CD20-positive B lymphocytes and moderate to severe diffuse GFAP-positive astrogliosis in the subventricular lesions was also observed adjacent to inflammatory foci. Similar astrocytic response together with microglial reaction have been reported also in previous FIP cases [12] . In a previous study on FIP, it was reported that viral antigens were detected in macrophage dominant lesions, and B lymphocytes and plasma cells which were positive for coronavirus-specific antibodies were observed as bands around or the basal side of the lesion. On the other hand, viral antigens were rarely detected in B lymphocytes and plasma cells dominant lesions. B lymphocytes and plasma cells are considered to induce FIP virus-specific immune responses and be replaced by macrophages [8, 9] . Therefore, in the present study, the lesion of Case No.4 with abundant CD20-positive B lymphocytes and GFAP-positive astrocytes was considered to be a subacute to chronic stage lesion of FIP that lacked active virus replication. Feline infectious peritonitis. 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