Detection of feline infectious peritonitis virus-like antigen in ferrets.
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Animals↗
Biomedical subjects
M Reinacher
Publications and source records attributed to M Reinacher.
Clear cell trichoblastomas in two dogs.
Two cases of clear cell trichoblastomas were diagnosed in young dogs. The tumour had ribbons of basaloid cells as seen in ribbon trichoblastomas, as well as differentiation to external root sheath of the hair follicle and few cells with sebaceous differentiation. This is the first report of clear cell trichoblastoma in dogs.
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Age-related dynamics of constitutive cytokine transcription levels of feline monocytes.
Monocytes/macrophages are central mediators of inflammation and immunity and therefore of major interest in the study of immunosenescence. In healthy adult cats, monocytes have been shown to constitutively transcribe pro- and anti-inflammatory cytokines. However, in order to characterize the effect of age, feline monocyte functions were examined for changes in cytokine transcription levels in early stages of immunosenescence. For this purpose, isolated, short-term cultured monocytes from barrier-maintained adult cats of different ages (15 mo to 10 yr) were examined for transcription of IL-1 beta, IL-6, IL-10, IL-12 p40 and TNF-alpha by real-time PCR. Transcription levels of cytokines varied and were generally highest for IL-1 beta. For IL-1 beta, IL-6 and IL-12 p40, both young and old cats exhibited highest levels. The age association was significant. TNF-alpha appeared to be transcribed at similar levels over the examination period, whereas IL-10 tended to decline with age but without any statistical significant differences. The observed age association of the constitutive transcription of some cytokines indicates a drop in monocyte activities from youth to middle age, which is then followed by a (progressive) increase with increasing age. This provides evidence that monocytes are in part responsible for the pro-inflammatory status observed with ageing.
Aging↗
Morphologic features and development of granulomatous vasculitis in feline infectious peritonitis.
Feline infectious peritonitis (FIP) is a fatal, coronavirus (CoV)-induced systemic disease in cats, characterized by granulomas in organs and granulomatous vasculitis. This study describes the morphologic features of granulomatous vasculitis in FIP as well as its development in the course of monocyte-associated feline CoV (FCoV) viremia in five naturally infected Domestic Shorthair cats with FIP. Monocyte-associated FCoV viremia was demonstrated by immunohistology, RNA in situ hybridization, and electron micropscopy. Granulomatous phlebitis at different stages of development was observed. Vasculitic processes ranged from attachment and emigration of FCoV-infected monocytes to vascular/perivascular granulomatous infiltrates with destruction of the vascular basal lamina. Monocytes as well as perivascular macrophages were activated because they were strongly positive for CD18 and expressed cytokines (tumor necrosis factor-alpha and interleukin-1beta) and matrix metalloproteinase-9. In addition, general activation of endothelial cells, represented by major histocompatibility complex II upregulation, was observed in all cases. These results confirm FIP as a monocyte-triggered systemic disease and demonstrate the central role of activated monocytes in FIP vasculitis.
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High viral loads despite absence of clinical and pathological findings in cats experimentally infected with feline coronavirus (FCoV) type I and in naturally FCoV-infected cats.
Specified pathogen-free cats were naturally infected with FCoV or experimentally infected with FCoV type I. Seroconversion was determined and the course of infection was monitored by measuring the FCoV loads in faeces, whole blood, plasma and/or monocytes. Tissue samples collected at necropsy were examined for viral load and histopathological changes. Experimentally infected animals started shedding virus as soon as 2 days after infection. They generally displayed the highest viral loads in colon, ileum and mesenteric lymph nodes. Seroconversion occurred 3-4 weeks post infection. Naturally infected cats were positive for FCoV antibodies and monocyte-associated FCoV viraemia prior to death. At necropsy, most animals tested positive for viral shedding and FCoV RNA was found in spleen, mesenteric lymph nodes and bone marrow. Both experimentally and naturally infected cats remained clinically healthy. Pathological findings were restricted to generalized lymphatic hyperplasia. These findings demonstrate the presence of systemic FCoV infection with high viral loads in the absence of clinical and pathological signs.
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Reduced constitutive cytokine transcription in isolated monocytes of clinically healthy cats, infected with an FIV strain of low pathogenicity.
Twenty-five barrier-maintained cats had been experimentally infected for 9.5 months with an FIV strain of low pathogenicity, FIV Zurich 2. Animals were clinically healthy and did not exhibit any haematological changes. FIV proviral DNA was demonstrated in peripheral blood lymphocytes of all cats and in monocytes of most animals, identifying FIV Zurich 2 as a both lympho- and monocytotropic strain. Monocytes were isolated from FIV-infected cats as well as from age-matched uninfected control cats, short-term cultured and examined for cytokine (IL-1beta, IL-6, IL-10, IL-12 p40 and TNF-alpha) transcription by real-time PCR. Constitutive transcription of cytokines in monocytes from FIV-infected cats was restricted to IL-1beta and, in the majority of samples, TNF-alpha. For all cytokines, transcription levels were significantly lower in FIV-infected cats than in control cats. Transcription was often least intense in those samples where FIV infection of the monocyte fraction was not demonstrated. Results show that infection of cats with an FIV strain of low pathogenicity was associated with depression of constitutive cytokine transcription in monocytes even if clinical and haematological changes were not observed.
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Canine osteosarcoma of the penile bone.
A 13-year-old mixed breed dog was presented with difficulties to urinate, blood in the urine and a nodule at the root of the penis. The penis was completely resected, and an osteosarcoma of the penile bone was diagnosed. Radiographically, there was no evidence to suggest tumours in other bones or distant metastases. The dog was euthanized as a consequence of tumour recurrence after 2 months. This is believed to be the first reported case of a primary osteosarcoma of the penile bone.
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Cytokine mRNA levels in isolated feline monocytes.
Real-time PCR systems were developed to quantitate cytokine expression in short-time cultivated feline monocytes. Feline-specific interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor-alpha (TNF-alpha) primers as well as TaqMan probes were designed and were adapted to a quantitative PCR system which had been previously established for feline IL-10 and IL-12 p40. Quantitative analysis of cytokine messenger RNA (mRNA) transcription based on the comparison of the cytokine with the housekeeping gene feline glyceraldehyde-3-phosphate dehydrogenase (GAPDH), providing universally expressed mRNA. GAPDH mRNA was readily detectable in cDNA prepared from short-time cultivated peripheral blood monocytes. Cytokine mRNA was demonstrated in all samples at variable amounts. IL-1beta and TNF-alpha mRNA was constitutively expressed whereas IL-6, IL-10 and IL-12 p40 mRNA was generally expressed at a lower level and was occasionally not detected. There was a great variability of cytokine production between individual cats and at different time points in the same cat.
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Comparative examination of cats with feline leukemia virus-associated enteritis and other relevant forms of feline enteritis.
Cats with feline leukemia virus (FeLV)-associated enteritis (FAE), enteritis of other known viral etiology (parvovirus [PV], enteric coronavirus [CoV]), and enteritis of unknown etiology with histologic features similar to those of FAE and PV enteritis (EUE) and FeLV-negative and FeLV-positive cats without enterocyte alterations were examined. Amount and types of infiltrating leukocytes in the jejunum and activity and cellular constituents of mesenteric lymph nodes, spleen, and bone marrow were determined. PV and CoV infections were confirmed by immunohistologic demonstration of PV and CoV antigen, ultrastructural demonstration of viral particles in the intestinal content, and in situ hybridization for PV genome. FeLV infection was detected by immunohistology for gp70, p27, and p15E. Latent FeLV infection was excluded by polymerase chain reaction methods for exogenous FeLV DNA. Enterocyte lesions involved the crypts in cats with PV enteritis, FAE, and EUE and the villous tips in cats with CoV enteritis. Inflammatory infiltration was generally dominated by mononuclear cells and was moderate in the unaltered intestine and in cats with PV enteritis and marked in cats with FAE, CoV enteritis, and EUE. In cats with EUE, myeloid/histiocyte antigen-positive macrophages were relatively numerous, suggesting recruitment of peripheral blood monocytes. Lymphoid tissues were depleted in cats with PV enteritis and with EUE but were normal or hyperplastic in cats with FAE. Bone marrow activity was decreased in cats with PV enteritis; in cats with FAE or EUE and in FeLV-positive cats without enterocyte alterations, activity was slightly increased. In cats with FAE and PV enteritis, a T-cell-dominated response prevailed. EUE showed some parallels to human inflammatory bowel disease, indicating a potential harmful effect of infiltrating macrophages on the intestinal epithelium.
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Expression of viral proteins in feline leukemia virus-associated enteritis.
Fourteen cases of feline leukemia virus (FeLV)-associated enteritis were immunohistologically examined for the expression of FeLV proteins gp70, p27, and p15E in the jejunum, mesenteric lymph nodes, spleen, and bone marrow. Results were compared with those of FeLV-infected cats without intestinal alterations. Other viral infections and specific bacterial, fungal, and parasitic infections were excluded by standard microbiologic methods, histopathology, immunohistology, and in situ hybridization. In FeLV-associated enteritis, FeLV gp70 and p15E were strongly expressed in intestinal crypt epithelial cells. In contrast, FeLV-positive cats without intestinal alterations showed only faint staining for gp70 and p15E and comparatively strong p27 expression in these cells. Findings suggest a direct relation between FeLV infection and alterations in intestinal crypt epithelial cells that may be attributed to the envelope proteins gp70 and p15E and/or their precursor protein. Distinct similarities to the intestinal changes in the experimentally induced FeLV-feline AIDS syndrome are obvious, suggesting that naturally occurring feline AIDS variants may be responsible for FeLV-associated enteritis.
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Histopathological alterations of lymphatic tissues in cats without feline infectious peritonitis after long-term exposure to FIP virus.
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Animals↗
Feline infectious peritonitis presenting as a tumour in the abdominal cavity.
This paper describes six cases of feline infectious peritonitis (FIP) in which an abdominal tumour had been suspected clinically. Pathological changes were mainly restricted to the massive enlargement of a mesenteric lymph node due to necrogranulomatous lymphadenitis. FIP was diagnosed on the basis of the immunohistological demonstration of coronavirus antigen in intact macrophages within the necrogranulomatous lesions. In the affected lymph node lymphoid tissue was either almost completely effaced or restricted to follicles composed mainly of variable numbers of blasts. From one to many plasma cells positive for coronavirus-specific antibodies were present in the marginal sinuses or capsules. In addition, necrogranulomas were present in the gut-associated lymphoid tissue of the caecum of one cat, and adjacent to the affected lymph node of another.
Abdominal Neoplasms↗
Cellular composition, coronavirus antigen expression and production of specific antibodies in lesions in feline infectious peritonitis.
Twenty-three cats with spontaneous feline infectious peritonitis (FIP) were examined by light microscopy including immunohistology and histochemistry in order to determine the cellular composition and the expression of viral antigen in lesions in FIP. Furthermore, the presence of plasma-cells producing coronavirus-specific antibodies was evaluated in situ. Macrophages and neutrophils were demonstrated by an antibody against calprotectin (leukocyte protein L1, myeloid/histiocyte antigen), neutrophils were recognized due to their chloroacetate esterase activity, and B- and T-lymphocytes were identified by antibodies against the CD3 antigen and the CD45R antigen, respectively. Expression of viral antigen was immunohistologically demonstrated by a monoclonal antibody (mAb) against coronavirus while coronavirus-specific antibodies in situ were identified by the application of feline coronavirus prior to the coronavirus antibody. Lesions were classified as diffuse alterations at serosal surfaces, granulomas with areas of necrosis, granulomas without extended necrosis, focal and perivascular lymphoplasmocytic infiltrates, and granulomatous-necrotizing vasculitis. Diffuse alterations on serosal surfaces were represented either by activated mesothelial cells with single coronavirus antigen-bearing macrophages or by layers of precipitated exudate containing single to numerous granulomas with areas of necrosis. In liver and spleen, the exudate was often underlaid by a small band of subcapsular B-cells with an occasional plasma-cell producing coronavirus-specific antibodies. In other locations, a variably broad band of B-cells and plasma-cells, often infiltrating between underlying muscle fibers, separated the exudate from the unaltered tissue. Some of these plasma-cells were positive for coronavirus-specific antibodies. In granulomas with areas of necrosis, the central necrosis was surrounded by macrophages usually expressing considerable amounts of viral antigen. Few B-cells and plasma-cells were found in the periphery. In granulomas without extended necrosis, the number of macrophages were lower. Only few macrophages expressing low amounts of viral antigen were present. B-cells and plasma-cells formed a broad rim. Few plasma-cells stained positive for coronavirus-specific antibodies. In both types of granulomas, few neutrophils were found between macrophages. Few T-cells were seen scattered throughout the lesions. Focal and perivascular lymphoplasmocytic infiltrates were mainlyseen in omentum and leptomeninx. B-cells were the predominant cells; some plasma-cells were positive for coronavirus-specific antibodies. Viral antigen was not readily detected in these alterations. Granulomatous-necrotizing vasculitis was occasionally found in kidneys and leptomeninx. It was dominated by macrophages which often stained strongly positive for coronavirus antigen. Different types of alteration were often seen in the same animal and even the same tissue. There was no obvious correlation between the cat's age, gross pathological changes, and the histological types of alteration. Single plasma-cells positive for coronavirus-specific antibodies were found around blood vessels distant from inflammatory alterations, within the lung parenchyma, as infiltrating cells in the mucosa of the small intestine, and in spleen and mesenteric lymph node. Results show that alterations in FIP are heterogeneous concerning cellular composition and expression of viral antigen. The dominance of B-cells in part of the lesions together with the presence of plasma-cells positive for coronavirus-specific antibodies indicate that these cells may play a role in the maintenance of inflammatory processes in FIP.
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Fatal enteritis associated with coronavirus infection in cats.
This report describes five cases of naturally occurring feline coronavirus enteritis. The affected animals, aged 2 months to 7 years, had a clinical history of intestinal symptoms, including diarrhoea or vomiting, or both. They exhibited variable histological changes in the epithelium of the small intestine, ranging from degeneration of single cells and detachment of groups of cells from the villous tips to regenerative processes of the crypt epithelia. Post-mortem diagnosis was based on the immunohistochemical demonstration of coronavirus antigen within intestinal epithelial cells and on the electron microscopical demonstration of coronavirus particles in the faeces. In addition, one animal was immunohistochemically positive for antigens of feline leukaemia virus (FeLV) and exhibited intestinal changes consistent with FeLV-associated enteritis. Two cats were tested serologically for feline immunodeficiency antibodies, with negative results. The findings indicate that natural coronavirus infection is a potential cause of severe enteritis in juvenile and adult cats.
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Feline immunodeficiency virus (FIV) infection in cats at necropsy: a serological study.
Sera collected post mortem during a 6-month period from cats were tested for feline immunodeficiency virus (FIV)-specific antibodies by (1) an enzyme-linked immunosorbent assay (ELISA), (2) an indirect peroxidase-based immunocytological test (IP), (3) a Western immunoblotting (WB) method with FIV-infected cell lysates, and (4) a WB method with purified viral antigen. All four methods were capable of detecting FIV-specific antibodies in haemolysed sera. However, the ELISA showed the lowest "positive predictive value" (PVpos = 22%) followed by the IP (PVpos 50-60%). Serum was FIV antibody-positive in 6% (15/255) of all cats examined. The mean age of seropositive cats was 9 years (4 years among seronegative cases) and the male-to-female ratio in such cats was 1.8 to 1 (overall ratio 0.8 to 1). Forty per cent of the seropositive cats were in the final phase of acquired immune deficiency syndrome. Feline leukaemia virus (FeLV) predominated among viral co-infections. It was concluded that (1) a combination of the IP and WB reliably detected FIV-specific antibodies in sera collected post mortem, and (2) at post-mortem examination, cats from high-risk groups (male, > 5 years old, hypercellular bone marrow) were frequently infected with FIV.
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Recombinant FeLV vaccine: long-term protection and effect on course and outcome of FIV infection.
The efficacy and the long-term protection of a recombinant feline leukemia virus (FeLV) vaccine were determined in 30 specified pathogen free cats for over 3 years. At the same time, in order to specify the effects of feline immunodeficiency virus (FIV) on the immune system, one half of the cats (n = 15) were previously infected with the Swiss isolate FIV Zurich 2. The second half of the animals (n = 15) served as non-infected controls. Eighteen (nine FIV-negative, nine FIV-positive) vaccinated and 12 (six FIV-negative, six FIV-positive) non-vaccinated cats were intraperitoneally challenged with FeLV A. Seventeen of 18 vaccinated cats were protected against persistent viremia, while ten of 12 non-vaccinated controls became infected. An increase of antibodies against FeLV SU was found in all protected cats after the challenge exposure. No difference in vaccine efficacy was found between FIV-negative and FIV-positive animals. The whole group of cats was observed for over 3 years. There were no further vaccinations during this period. CD4+ and CD8+ cell subsets, clinical outcome and time of survival of the cats were recorded. FIV-negative and FIV-positive animals were kept in two different rooms. However, FeLV-negative and FeLV viremic cats were housed together in both rooms in order to imitate a natural FeLV exposure situation. Anti-recombinant FeLV SU antibodies were measured by enzyme-linked immunosorbent assay. Although a continuous decline of antibodies was found in FeLV vaccinated cats, they remained protected against constant FeLV challenge for over 3 years. FIV infection had a stronger effect on the depression of the CD4+:CD8+ ratio than FeLV infection. Within the group of FIV-positive cats, the FeLV-vaccinated animals had significantly better survival rates as well as better clinical and laboratory parameters. FIV- and FeLV-coinfected cats showed the lowest CD4+:CD8+ ratio, mainly caused by decreased CD4+ lymphocyte counts. CD8+ lymphocytes with strong fluorescence (CD8(high)) disappeared and cells with weak fluorescence (CD8(low)) appeared instead. Prevention of coinfection by immunizing FIV-positive cats against FeLV infection improved the clinical outcome and prolonged the cat's life expectancy.
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Immunohistological demonstration of feline infectious peritonitis virus antigen in paraffin-embedded tissues using feline ascites or murine monoclonal antibodies.
Feline infectious peritonitis (FIP) virus antigen was demonstrated after methanol, ethanol or formalin fixation in paraffin-embedded tissues by means of monoclonal and polyclonal antibodies. The monoclonal antibody was induced by immunization with transmissible gastroenteritis virus. Polyclonal antibodies were obtained by purification on protein A-Sepharose of ascites fluid from a cat with FIP. Almost all cats diagnosed as suffering from FIP by postmortem and histological examination exhibited FIP virus (FIPV) antigen in macrophages in granulomas whereas FIPV antigen was only once demonstrable in another location.
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[The significance of FeLV infection for diseases in necropsied cats].
Persistent FeLV infection was demonstrated in more than 3000 necropsied cats by an immunohistological method. The findings were associated with the diagnoses established by means of post-mortem examination, histopathological, bacteriological, virological, and parasitological investigations. Statistically significant differences between FeLV-positive and FeLV-negative cats in the relative risk for certain lesions could be demonstrated for the first time. As a consequence, the importance of FeLV infection for the development of certain lesions in cats is to be seen under new aspects for some diagnoses. The relative risk for development of anemia, e.g., is only slightly increased in FeLV-positive animals whereas the relative risk for focal myocardial necrosis and coccidiosis is elevated tremendously in these cats. The relative risk for some lesions and diagnoses is much higher in FeLV-negative cats than in FeLV-positive animals. This is true, e.g., for myocardiopathy and hemorrhagic cystitis.
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