Reliability of a diagnosis of BSE made on the basis of clinical signs.
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Animals↗
Biomedical subjects
M Vandevelde
Publications and source records attributed to M Vandevelde.
Modelling the expected numbers of preclinical and clinical cases of bovine spongiform encephalopathy in Switzerland.
The objective of this study was to model the expected numbers of cattle incubating bovine spongiform encephalopathy (BSE) and the numbers of clinical cases of BSE in the Swiss cattle population between 1984 and 2005. The results were compared with the observed number of clinical BSE cases and with the results of a culling and testing scheme on herdmates of cattle with BSE. The age distribution of the Swiss cattle population, the age-at-death distribution of the first 235 BSE cases and exposure information were used to calculate the expected number of infected cattle in each birth cohort and the resulting numbers of clinical cases and survivors incubating the disease for each year. The model which did not assume any under-reporting of cases fitted the observed epidemic curve of clinical cases reasonably well, and predicted that the Swiss BSE epidemic would come to an end between 2003 and 2005. The age of survivors incubating BSE is increasing. The higher than expected incidence of subclinical cases observed in animals from the culling scheme is most probably the result of the heterogeneous distribution of infected animals and affected herds in the population. The results of the model need to be taken into account when designing surveillance and testing schemes for BSE.
Age Distribution↗
Early T cell response in the central nervous system in canine distemper virus infection.
The initial demyelinating lesions in canine distemper virus (CDV) infection develop during a period of severe immunosuppression in the absence of inflammation. In vitro and in vivo studies suggest that early demyelination is due to directly virus-induced oligodendroglial changes. In the present spatiotemporal study in experimentally CDV-infected dogs we observed diffuse up-regulation of T cells throughout the central nervous system (CNS) and T cell invasion in early demyelinating lesions. Invasion of T cells in the CNS occurred despite severe immunosuppression and without any perivascular cuffing. However, the major fraction of invading T cells correlated with sites of viral replication and coincided with the demonstration of an early immune response against the nucleocapsid protein of CDV. Activation of microglial cells was thought to have elicited the migration of T cells to the CNS by secretion of chemokines: marked IL-8 activity was found in the CSF of dogs with acute lesions. In areas of early demyelination, large numbers of CD3+ cells accumulated in the tissue in the absence of any morphological sign of inflammation. Whether the T cells at lesion sites contribute to the development of acute demyelination remains uncertain at this stage. Antiviral cytotoxicity was not apparent since viral clearance in demyelinating lesions is only effective when B cells and concurring antiviral antibody production appeared in the subacute and chronic inflammatory stage of the disease. CD3+ cells appear to persist for several weeks after infection since they were also found in recovered dogs that did not develop demyelination. Accumulation of immune cells, including a significant proportion of resting T cells (CD45RA+) in the CNS in the early stages of the disease may facilitate the later development of the intrathecal immune response and associated immunopathological complications.
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Oligodendroglial degeneration in distemper: apoptosis or necrosis?
Canine distemper virus (CDV) causes a multifocal demyelinating disease in dogs. It was previously shown that the initial demyelinating lesions are directly virus induced since a correlation between the occurrence of demyelination and CDV replication in white matter cells was observed. During the course of infection oligodendrocytes undergo distinct morphological alterations, partly due to a restricted CDV infection of these cells, and eventually disappear from the lesions. This phenomenon has been described in vivo as well as in vitro. However, the reason for the morphological alterations and the following oligodendroglial depletion remained unclear. Since virus infection can induce cell death, it was investigated whether apoptosis or necrosis plays a role in the pathogenesis of demyelination in canine distemper. In brain tissue sections from dogs with acute distemper apoptotic cells were not detected within the demyelinating lesions using morphological and biochemical cell death criteria. In chronic distemper, apoptotic cells - presumably inflammatory cells - were seen within the perivascular cuffs. These in vivo findings were correlated to the in vitro situation using CDV-infected primary dog brain cell cultures as well as Vero cells. Infection with culture-adapted CDV lead to massive necrosis but not to apoptosis. After infection with virulent CDV neither apoptosis nor necrosis was a predominant feature in either culture system. These findings suggest that virus-induced demyelination in canine distemper is not the direct consequence of apoptosis or necrosis. It is speculated that another mechanism must be responsible for the observed morphological alterations of oligodendrocytes, ultimately leading to demyelination.
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Validation of a western immunoblotting procedure for bovine PrP(Sc) detection and its use as a rapid surveillance method for the diagnosis of bovine spongiform encephalopathy (BSE).
In this report we document the results of several independent studies testing the sensitivity, specificity and reliability of the Prionics Western blotting (PWB) procedure to detect bovine and ovine disease-specific, protease-resistant prion protein (PrP(Sc)). Validation of the technique was obtained by blind analysis of samples from cattle affected with bovine spongiform encephalopathy (BSE), clinically normal animals or cattle with neurological diseases unrelated to BSE. Overall, very high sensitivity, specificity and reliability was observed. It became clear that sampling of the correct brain region and the method used for protein extraction are important factors for correct diagnosis. Furthermore, we tested the usefulness of the PWB technique as an instrument for surveillance purposes. We analyzed animals from a culling scheme as well as older animals from abattoirs to determine the number of subclinical BSE cases detectable by histopathological examination, immunohistochemistry for PrP(Sc) and PWB. In both studies, BSE-affected animals with no overt clinical symptoms were detected. These results demonstrate the usefulness of the PWB procedure in surveillance systems serving as a rapid diagnostic tool to identify animals subclinically infected with BSE.
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Azodicarbonamide inhibits T-cell responses in vitro and in vivo.
Azodicarbonamide was recently identified as a new anti-HIV agent that targets the zinc finger domains of the HIV-1 NCp7 nucleocapsid protein. Here, we demonstrate that azodicarbonamide inhibits in a dose-dependent manner the responses of purified human CD4+ T lymphocytes stimulated either by monoclonal antibodies against CD3 and CD28 or by allogeneic dendritic cells. These suppressive effects involve a direct action on the calcium mobilization machinery, as azodicarbonamide strongly inhibited the calcium influx induced either by antibodies against CD3 and CD28 or the chemokine RANTES, as well as by thapsigargin, an activator of depletion-activated calcium channels. In vivo, administration of azodicarbonamide into mice blunted their response to polyclonal T-cell activation induced by the injection of monoclonal antibody against CD3 and resulted in delayed rejection of skin allografts. In addition to its anti-HIV properties, azodicarbonamide is a new immunosuppressive agent that might have therapeutic applications in T cell-mediated inflammatory disorders.
Animals↗
Lymphocyte subset distribution in steroid responsive meningitis-arteriitis in comparison to different canine encephalitides.
Steroid responsive meningitis-arteriitis (SRMA) is a well-known disease in dogs, but the aetiology and pathogenesis are not yet understood. In the peripheral blood an overrepresentation of B cells was found. In the present study we therefore evaluated the distribution of lymphocyte subsets in SRMA in paraffin-embedded tissue sections directly at the lesion sites and compared the results to different canine encephalitides. An intriguing finding was that the B cell/T cell distribution varied depending on the aetiology of the disease: in viral encephalitides, T cells were the predominant cell population in perivascular cuffs, whereas in protozoal and bacterial diseases B cells prevailed. In SRMA an overrepresentation of B cells occurred in meningeal lesions, as already found in the peripheral blood. The distribution of lymphocyte subsets was similar to bacterial and protozoal diseases and was not a unique phenomenon for this specific inflammatory lesion in the canine central nervous system (CNS). Multiple mechanisms seem to be responsible for recruitment and activation of different leukocyte subsets after alteration of the CNS tissue by an environmental factor. A specific finding in SRMA was that the distribution of T and B cells depended also on the lesion site. In contrast to meningeal lesions, in inflamed arteries T cells were the only lymphocyte population found. In these vessels, diffuse infiltration with immunoglobulins was revealed. Inactivated or resting lymphocytes and large granular lymphocytes occurred in each of the diseases examined. These similarities between SRMA and infectious CNS diseases of the dog support earlier suggestions that the disease is somehow triggered by a hitherto unknown environmental factor which leads to the dysregulation of the immune system.
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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.
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Nuclear DNA fragmentation and immune reactivity in bovine spongiform encephalopathy.
To investigate whether apoptosis contributes to neuronal degeneration in bovine spongiform encephalopathy (BSE), morphological changes consistent with apoptosis were sought and in-situ end labelling (ISEL) was applied, in a series of 20 BSE cases and 10 age-matched normal control cattle. Apoptotic changes were not found in neurons but were occasionally seen in glial cells. Relatively few ISEL-positive neurons were found, but many labelled nuclei were seen in glial cells in certain areas. None of the labelled cells showed morphological features of apoptosis. ISEL(+)cells occurred in areas of spongiform change and other areas of grey matter lacking spongiform change. Some association was found between degree of cellular DNA fragmentation and accumulation of abnormal prion protein (PrP(Sc)). Interestingly, small or moderate numbers of T lymphocytes, not present in the normal central nervous system (CNS), were detected in the CNS parenchyma in most BSE cases. There was a pronounced astrogliosis, but markers of macrophage or microglial activation were only slightly increased. The results indicate that nuclear DNA vulnerability is enhanced in certain neuroanatomical areas in BSE, but evidence that apoptosis plays a role in neuronal loss in BSE was very limited. 1999 Harcourt Publishers Ltd.
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Detection of bovine spongiform encephalopathy-specific PrP(Sc) by treatment with heat and guanidine thiocyanate.
The conversion of a ubiquitous cellular protein (PrP(C)), an isoform of the prion protein (PrP), to the pathology-associated isoform PrP(Sc) is one of the hallmarks of transmissible spongiform encephalopathies such as bovine spongiform encephalopathy (BSE). Accumulation of PrP(Sc) has been used to diagnose BSE. Here we describe a quantitative enzyme-linked immunosorbent assay (ELISA) that involves antibodies against epitopes within the protease-resistant core of the PrP molecule to measure the amount of PrP in brain tissues from animals with BSE and normal controls. In native tissue preparations, little difference was found between the two groups. However, following treatment of the tissue with heat and guanidine thiocyanate (Gh treatment), the ELISA discriminated BSE-specific PrP(Sc) from PrP(C) in bovine brain homogenates. PrP(Sc) was identified by Western blot, centrifugation, and protease digestion experiments. It was thought that folding or complexing of PrP(Sc) is most probably reversed by the Gh treatment, making hidden antigenic sites accessible. The digestion experiments also showed that protease-resistant PrP in BSE is more difficult to detect than that in hamster scrapie. While the concentration of PrP(C) in cattle is similar to that in hamsters, PrP(Sc) sparse in comparison. The detection of PrP(Sc) by a simple physicochemical treatment without the need for protease digestion, as described in this study, could be applied to develop a diagnostic assay to screen large numbers of samples.
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Neuropathological and aetiological studies of sporadic non-suppurative meningoencephalomyelitis of cattle.
Sporadically occurring non-suppurative encephalitis appears to be a frequent condition of Swiss cattle. Fifty-one such cases diagnosed over a period of 10 years were examined retrospectively to investigate whether they constituted one or more distinct diseases, and to search for aetiological agents. Three cases were characterised by periventricular granulomatous encephalitis, and most probably represented a different disease, but the remaining 48 cases had disseminated non-suppurative encephalitis with widespread neuronal changes. Neuronal degeneration was very marked in the hippocampus of 10 cases and in the cerebellar Purkinje cells of 11. It was thought that the latter cases represented morphological variations of the same disease rather than a different disease because of their overlapping morphological features. The 48 cases had the following features in common: the disease had primarily neurological signs affecting mostly adult cattle, it was a sporadic condition, and there was a clear tendency for it to have a subacute to chronic course. Polymerase chain reaction (PCR) amplification for chlamydial DNA was negative except in one of 32 specimens, and immunohistochemistry did not demonstrate the presence of chlamydial antigens either in the one PCR-positive case or in the other cases examined. Immunohistochemistry for rabies virus, Borna disease virus, and central European tickborne encephalitis virus was negative. In four cases, immunolabelled cells were found in the lesions with antibodies against paramyxovirus antigens.
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Chemotactic activity and IL-8 levels in the cerebrospinal fluid in canine steroid responsive meningitis-arteriitis.
Steroid responsive meningitis-arteriitis (SRMA) is a systemic immune disorder, characterized by inflammatory-stenosing lesions of the meningeal arteries and meningitis. The predilection of the disease for the central nervous system (CNS) remains unexplained. In this study, chemotactic activity and chemotactic factors were measured in the cerebrospinal fluid (CSF) of dogs with SRMA. CSF of dogs with SRMA exerted a marked chemotactic activity for leukocytes. Neutrophils were attracted to a similar degree as by CSF from animals with bacterial encephalitis. Chemotactic activity was also noted for mononuclear cells, however, by far weaker than in CSF from animals with viral encephalitis. While the inflammatory process could be suppressed with glucocorticoid treatment, the chemotactic activity of CSF persisted. We could identify IL-8-like activities using a desensitization assay in the CSF of animals with SRMA and also found increased IgA levels. Increased chemotactic activity for polymorphonuclear leukocytes correlated positively with the levels of IL-8-like activity in CSF. Our observations clearly suggest that in SRMA chemotactic factors are generated in the CNS. These include IL-8, but probably also others. The intensity of this production appears to correlate with IgA levels in the CSF suggesting either a causal link or reflecting the severity of the inflammation.
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Oligodendroglial pathology in canine distemper.
Canine distemper virus (CDV) causes a multifocal demyelinating disease in dogs. The mechanism of acute demyelination in distemper is still poorly understood. The initial demyelinating lesion in distemper is directly virus induced, since there is a clear correlation between the occurrence of demyelination and CDV replication in the cells of the white matter. Yet, there is little evidence for oligodendroglial infection. Changes of these cells have been reported in vitro and in vivo. The in vitro studies showed that--in contrast to other cells such as astrocytes and macrophages--oligodendrocytes hardly express CDV protein. However, we could show that these cells underwent a restricted infection with transcription of CDV RNA and that this phenomenon correlated with down-regulation of myelin gene transcription. The extension of these in vitro findings in vivo was obscured by the lack of reliable oligodendrocyte labelling techniques in canine brain tissue sections. In this study we combined immunohistochemistry with in situ hybridization to examine oligodendrocytes in demyelinating lesions and to investigate the question of oligodendrocyte infection in vivo. We could demonstrate that CDV infection leads to massive down-regulation of myelin gene expression in demyelinating lesions and that this effect correlates in part with a restricted infection of oligodendrocytes.
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Lymphocyte subsets and CD45RA positive T-cells in normal canine cerebrospinal fluid.
In order to evaluate changes in lymphocyte subpopulations of cerebrospinal fluid (CSF) cells in neurological diseases, normal control data have to be established. In this study we evaluated CSF samples from 65 dogs of both sexes and various breeds with an age range between 5 months and 6 years, and 20 one-year-old healthy inbred Beagles. For comparison, blood samples from 10 healthy dogs were examined. 14 different antibodies against leukocyte surface markers were used. The subpopulations were evaluated using flow cytometry (FACS) and immunocytochemistry. It could be shown that lymphocyte populations in CSF differ from peripheral blood in a few subsets. A relatively high degree of individual variation was found, not only in dogs of different breeds and ages, but also in the inbred Beagle population. These large individual variations suggest that repeated paired CSF-blood samples during the course of neurological disease should be examined within the same individual to obtain meaningful results. CD3+ and CD4+ T-cells were significantly lower in normal CSF. Of great interest is the fact, that T-cells, characterized by double staining CD3/CD45RA are present in variable numbers in normal CSF. In other species they are known to be naive or resting T-cells. CD4/CD45RA positive cells seem to be an important subpopulation of these CD45RA positive T-cells. Furthermore, by far more CD11b positive lymphocytes were observed in the CSF than in the peripheral blood and these are not large granular lymphocytes. The present study shows that systematic FACS analysis of CSF is feasible in larger animals such as dogs.
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Mivazerol prevents the tachycardia caused by emergence from halothane anesthesia partly through activation of spinal alpha 2-adrenoceptors.
BACKGROUND: Mivazerol (MIV) is an alpha 2-adrenoceptor agonist designed to prevent adverse cardiac outcome in perioperative patients. The present study was undertaken to determine whether the hyperdynamic state observed at emergence from halothane (HAL) anesthesia in rats could be modulated by MIV and to explore the mode of action of MIV under such conditions. METHODS: Male Sprague Dawley rats were anesthetized with 1% HAL and assisted for respiration (N2O-O2: 70-30%). MIV 2.2-15.3 micrograms.kg-1.h-1 i.v. was infused 30 min before withdrawal of anesthesia and compared for heart rate (HR) and systolic arterial blood pressure (SAP) to control animals treated with saline. In some experiments, animals were pretreated with intrathecal pertussis toxin (T2 level, 0.5 microgram, 7 d), or i.v. rauwolscine (0.34 mg/kg, 5 min) or were bilaterally stellectomized (30 min) prior to withdrawal of HAL. RESULTS: Increases in HR (65 bpm, +20%) and in SAP (25 mmHg, +26%) were observed immediately upon discontinuation of HAL and remained constant for at least 30 min. The increase in HR was abolished by removal of the stellate ganglia. MIV dose-dependently inhibited the increase in HR from 4.8 micrograms.kg-1.h-1 (68% reduction, P < 0.05) without affecting HR or SAP during anesthesia. Inhibition of HR increase was of 98% at 15.3 micrograms.kg-1.h-1. This effect was abolished by rauwolscine, and partially (50%) inhibited by pertussis toxin pre-treatment. CONCLUSION: These results demonstrate that withdrawal of HAL anesthesia in the rat produces a sustained increase in HR due to activation of the sympathetic system and that MIV inhibits this tachycardia via activation of alpha 2-adrenoceptors located at least in part in the spinal cord.
Adrenergic alpha-Agonists↗
[Transmissible spongiform encephalopathies in animals].
Transmissible spongiform encephalopathies in animals are known for centuries. In particular scrapie in sheep and goats occurs worldwide; it spreads as a natural disease and is genetically controlled. Chronic wasting disease (CWD) in the United States (Wyoming and Colorado) also spreads as natural disease among free ranging and captive elk and mule deer. In contrast, transmissible mink encephalopathy (TME) of mink in fur producing farms is caused by contaminated feed; the source of this food contamination is still controversial. The only occurrence of a TSE in an avian species was reported from a flock of ostriches in a German zoo. The origin of the outbreak of bovine spongiform encephalopathy (BSE) in Great Britain could be traced back to feeding concentrates containing animal proteins contaminated with the agent of sheep scrapie. BSE was introduced into other countries, e.g. Switzerland and France, through the import of contaminated feeds from Great Britain. In addition, sporadic cases of TSE occurred in 10 other species, in particular domestic cats and zoo animals, e.g. antelopes and large cats. The diagnosis is based on the neuropathological examination of the brain as well as the demonstration of the disease specific protease resistant prion protein (PrPres). The first measures against the disease aim at eliminating the risk factors. The most important is the prohibition of feeding animal protein concentrates to ruminants. Thanks to this measure the incidence of BSE diminished remarkably. To protect consumers of beef products, in countries with BSE the potentially infectious organs of all cattle are confiscated at slaughter. Yet, in Great Britain this measure was introduced only at a point of time when BSE had already spread all over the country. Therefore, there is a strong probability of an exposition of consumers of beef with the BSE agent, which might have caused the new variant of Creutzfeldt-Jakob disease (nvCJD).
Animal Feed↗
[182 offspring of cows with bovine spongiform encephalopathy (BSE) in Switzerland. 1. Clinical findings].
This study involved 182 calves, heifers and cows that were the first generation progeny of cows with bovine spongiform encephalopathy (BSE). All animals underwent clinical and neurological examinations. Blood, milk, urine, ruminal fluid and cerebrospinal fluid samples were examined. Each cow underwent a specific examination for BSE which included assessment of behaviour, sensitivity and locomotion. This examination was performed twice, and there was very good agreement between the results. The most common symptom was nervousness (14 animals). In the first examination, 68 animals were mildly to moderately sensitive to touching of the head, 44 animals were hypersensitive to a halogen light and 35 animals were hypersensitive to a camera flash. Twenty-four animals reacted by kicking when the hindlimbs were touched with a broom. None of the animals had disturbances in locomotion. Based on the interpretation of all findings and the comparison of the results of the first and second examinations, 173 animals were diagnosed as free of BSE. In 9 animals, the disease could not be ruled out; however, it could not be confirmed in any of them. Histological and immunohistochemical examinations of the brain of all animals were negative for BSE (see Part 2 of the study for details). The results of this study indicate that abnormal clinical findings may occur in clinically healthy cattle. However, none of the progeny of cows with BSE exhibited clinical signs typical of BSE, such as disturbances in behaviour; sensitivity and locomotion.
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[182 offspring of cows with bovine spongiform encephalopathy (BSE) in Switzerland. 2. Epidemiology and pathological findings].
In order to detect lesions of a spongiform encephalopathy and/or accumulation of the protease resistant prion protein (PrPres), 182 offspring of cows affected with BSE were examined neuropathological and immunohistochemically. Neither spongiform encephalopathy nor PrPres accumulation were found. In seven animals other neuropathological lesions were seen, significant ones in three. Because of the small risk of exposure to contaminated feed in these animals, nearly all of which were born after the introduction of the protein feed ban for ruminants, the occurrence of spongiform encephalopathy in this series of BSE offspring would be suggestive of maternal transmission. However, the value of the study in this respect is quite limited. Only half of the animals were old enough to develop clinical and pathological evidence of the disease. If a maternal effect on the risk for the offspring is only to be expected during the last 6 months of the incubation of the dam as suggested by British investigations, only few animals in this study would fulfil the requirement of having been born during this critical period. Since it cannot be entirely excluded that the BSE agent transiently invades extraneural tissues in the early stages of infection, the above mentioned restriction to the final 6 months of the incubation time of the dam would not necessarily be applicable to all situations. We concluded that this study supports previous observations according to which maternal transmission of BSE is at best a rare event.
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