Neuropathological changes in auditory brainstem nuclei in cattle with experimentally induced bovine spongiform encephalopathy

Bovine spongiform encephalopathy (BSE) is characterized by the appearance of spongy lesions in the brain, particularly in the brainstem nuclei. This study evaluated the degenerative changes observed in the central auditory brainstem of BSE-challenged cattle. The neuropathological changes in the auditory brainstem nuclei were assessed by determining the severity of vacuolation and the presence of disease-associated prion protein (PrP(Sc)). Sixteen female Holstein-Friesian calves, 2-4 months of age, were inoculated intracerebrally with BSE agent. BSE-challenged animals developed the characteristic clinical signs of BSE approximately 18 months post inoculation (mpi) and advanced neurological signs after 22 mpi. Before the appearance of clinical signs (i.e. at 3, 10, 12 and 16 mpi), vacuolar change was absent or mild and PrP(Sc) deposition was minimal in the auditory brainstem nuclei. The two cattle sacrificed at 18 and 19 mpi had no clinical signs and showed mild vacuolar degeneration and moderate amounts of PrP(Sc) accumulation in the auditory brainstem pathway. In the animals challenged with BSE agent that developed clinical sings (i.e. after 20 mpi), spongy changes were more prominent in the nucleus of the inferior colliculus compared with the other nuclei of the auditory brainstem and the medial geniculate body. Neuropathological changes characterized by spongy lesions accompanied by PrP(Sc) accumulation in the auditory brainstem nuclei of BSE-infected cattle may be associated with hyperacusia.     

Comparative ultrastructural neuropathology of naturally occurring bovine spongiform encephalopathy and experimentally induced scrapie and Creutzfeldt-Jakob disease.

We report the ultrastructural neuropathology of bovine spongiform encephalopathy (BSE), a recently described slow virus disease first recognized in Friesian/Holstein cattle, and compare it to that of experimental scrapie and Creutzfeldt-Jakob disease. The spongiform change, which was most pronounced in the central grey matter of the midbrain, consisted of membrane-bound vacuoles within neuronal processes, containing curled membrane fragments, secondary chambers and vesicles. Axons and dendrites accumulated whorls of neurofilaments and other subcellular organelles, such as mitochondria and dense bodies, which were entrapped within the filamentous masses. Other neurites accumulated electron-dense bodies, and still others electron-lucent cisterns and branching tubules. Membrane-bound neuronal inclusions, composed of tubules measuring 10 nm in diameter, were found in axonal terminals. Tubulovesicular structures were loosely packed and were occasionally surrounded by a common membrane, a finding previously described only in natural scrapie in sheep. Except for the intraneuronal inclusions, all of the ultrastructural features of BSE resembled those found in scrapie and Creutzfeldt-Jakob disease.     

Immunohistochemical approach to the pathogenesis of bovine spongiform encephalopathy in its early stages

An immunohistochemical and histochemical study was carried out on the brains of nine cases of BSE-diagnosed cattle as part of the surveillance plan in Catalonia, Spain. The animals had no clinical symptoms reported and were thus at early stages of the disease. The first part of the study consisted of a characterization of PrP(BSE) deposits throughout the encephalon. The behaviour of the different immuno-labelling patterns was analysed and tropism of some patterns towards certain brain areas was described. This tropism is principally directed to the brain stem region; however, an association of the stellate pattern was found with areas where PrP(BSE) is deposited less abundantly, such as the cerebral cortex. Secondly, distinct pathogenesis mechanisms that take place in the early stages of BSE, which would include these cases were investigated. This study describes the glial response to the presence of PrP(BSE) (using antibodies against astrocytic glial fibrillary acidic protein and lectin from Griffonia simplicifolia to identify microglia), the presence of mild oxidative stress phenomena (antibodies against metallothioneins I and II and against nitrated aminoacidic residues: nitrotyrosine), the apparent absence of apoptotic cellular death (cleaved caspase 3) and the preservation of synaptic proteins synaptophysin and small synaptosome-associated 25 kDa protein immuno-labelling. Finally, no alteration of the extra-cellular matrix was detected with the use of Wisteria floribunda agglutinin, a marker for perineuronal nets.     

Pathogenesis of bovine spongiform encephalopathy in sheep

The pathogenesis of bovine spongiform encephalopathy (BSE) in sheep was studied by immunohistochemical detection of scrapie-associated prion protein (PrP^Sc) in the gastrointestinal, lymphoid and neural tissues following oral inoculation with BSE brain homogenate. First accumulation of PrP^Sc was detected after 6 months in the tonsil and the ileal Peyer’s patches. At 9 months postinfection, PrP^Sc accumulation involved all gut-associated lymphoid tissues and lymph nodes as well as the spleen. At this time point, PrP^Sc accumulation in the peripheral neural tissues was first seen in the enteric nervous system of the caudal jejunum and ileum and in the coeliac-mesenteric ganglion. In the central nervous system, PrP^Sc was first detected in the dorsal motor nucleus of the nervus Vagus in the medulla oblongata and in the intermediolateral column in the spinal cord segments T7–L1. At subsequent time points, PrP^Sc was seen to spread within the lymphoid system to also involve all non-gut-associated lymphoid tissues. In the enteric nervous system, further spread of PrP^Sc involved the neural plexi along the entire gastrointestinal tract and in the CNS the complete neuraxis. These findings indicate a spread of the BSE agent in sheep from the enteric nervous system through parasympathetic and sympathetic nerves to the medulla oblongata and the spinal cord.     

Predominant involvement of the cerebellum in guinea pigs infected with bovine spongiform encephalopathy (BSE)

This study reports the experimental transmission of bovine spongiform encephalopathy (BSE) to guinea pigs and describes the cerebellar lesions in these animals. Guinea pigs were inoculated intracerebrally with 10% brain homogenates from BSE-affected cattle. These animals were designated as the first passage. Second and third passages were subsequently performed. All guinea pigs developed infection at each passage. The mean incubation period of the first passage was 370 days post-infection (dpi) and this decreased to 307 dpi and 309 dpi for the second and third passages, respectively. Mild to severe spongiform degeneration and gliosis were observed in the cerebral cortex, thalamus and brainstem. In addition, the affected animals had marked pathological changes in the cerebellum characterized by severe cortical atrophy associated with Bergmann radial gliosis of the molecular layer and reduction in the width of the granular cell layer. Immunohistochemically, intense PrP^Sc deposition and scattered plaque-like deposits were observed in the molecular and granular cell layers. Cerebellar lesions associated with severe atrophy of the cortex have not been reported in animal prion diseases, including in the experimental transmission of PrP^Sc to small rodents. These lesions were similar to the lesions of human kuru or the VV2 variant of sporadic Creutzfeldt–Jakob disease, although typical kuru plaques or florid plaques were not observed in the affected animals.     

Post-mortem immunodiagnosis of scrapie and bovine spongiform encephalopathy

Two polyclonal antisera were raised in rabbits against the scrapie-associated fibril protein (PrP) prepared from sheep and mice which were terminally infected with experimental scrapie. The anti-mouse PrP serum identifies the proteins of scrapie-associated fibrils (SAF) from all the host species studied (mouse, hamster, sheep and goat) and bovine spongiform encephalopathy (BSE) fibrils from cow. The anti-sheep PrP serum displays species restricted immunoreactivity. While it identifies several PrP polypeptides from terminally affected sheep, goat and cow material, only the highest molecular weight band is recognised from hamster and there is no detection of mouse PrP. The use of these antisera in routine laboratory testing at post mortem provides a highly sensitive test for scrapie and BSE and may allow the identification of infected animals prior to the onset of clinical signs.     

Gadofluorine M-enhanced MRI shows involvement of circumventricular organs in neuroinflammation

Background  

Circumventricular organs (CVO) are cerebral areas with incomplete endothelial blood-brain barrier (BBB) and therefore regarded as "gates to the brain". During inflammation, they may exert an active role in determining immune cell recruitment into the brain.     

The neuropathology of experimental bovine spongiform encephalopathy in the pig

In an experimental study of the transmissibility of BSE to the pig, seven of 10 pigs, infected at 1-2 weeks of age by multiple-route parenteral inoculation with a homogenate of bovine brain from natural BSE cases developed lesions typical of spongiform encephalopathy. The lesions consisted principally of severe neuropil vacuolation affecting most areas of the brain, but mainly the forebrain. In addition, some vacuolar change was identified in the rostral colliculi and hypothalamic areas of normal control pigs. PrP accumulations were detected immunocytochemically in the brains of BSE-infected animals. PrP accumulation was sparse in many areas and its density was not obviously related to the degree of vacuolation. The patterns of PrP immunolabelling in control pigs differed strikingly from those in the infected animals.     

Perceived risk of bovine spongiform encephalopathy and dietary behavior

The German BSE crisis in early 2001 can be considered as a natural experiment with strong behavioral consequences. The present study investigated psychological and other factors associated with reduced meat consumption compared to the first months of the previous year. As expected, all types of meat, with the exception of poultry and game, were eaten less often. The effect was strongest in beef, where almost half of the sample reported reduced meat consumption. As predicted by the health belief model, perceived threat was associated with subjective vulnerability. It was not, however, strongly associated with perceived seriousness of BSE, probably due to the ubiquitous public discussion of the topic. Reduced beef consumption has three to four times more frequent in the event of subjective threat.     

Decontamination studies with the agents of bovine spongiform encephalopathy and scrapie

Summary Macerates of bovine brain infected with bovine spongiform encephalopathy (BSE) agent, and rodent brain infected with the 263K or ME7 strains of scrapie agent, were subjected to porous-load autoclaving at temperatures between 134 and 138 °C for 60 min. Bioassay in rodents showed that none of the regimes produced complete inactivation. Homogenates of BSE-infected bovine brain were exposed for 120 min to solutions of sodium hypochlorite or sodium dichloroisocyanurate containing 16,500 ppm available chlorine. There was no detectable survival of infectivity after the hypochlorite treatments but none of the dichloroisocyanurate solutions produced complete inactivation. Homogenates of BSE-infected bovine brain, and rodent brain infected with the 263K and ME7 strains of scrapie agent, were exposed for 120 min to 1M or 2M sodium hydroxide but no procedure produced complete inactivation of all agents tested.     

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.     

Immunohistochemical distinction between preclinical bovine spongiform encephalopathy and scrapie infection in sheep

Sheep are susceptible experimentally to bovine spongiform encephalopathy (BSE), the clinical signs being indistinguishable from those of scrapie. Because of the possibility of natural ovine BSE infection, laboratory tests are needed to distinguish between scrapie and BSE infection. The objectives of this study were to determine whether (1) PrPSc accumulates in biopsy samples of the tonsil or third eyelid, or both, of BSE-infected sheep before the appearance of clinical disease, and (2) such samples from BSE- and scrapie-infected sheep differ in respect of PrPSc accumulations. Homozygous ARQ sheep (n = 10) were dosed orally at 4-5 months of age with a brain homogenate from BSE-infected cattle. Third eyelid and tonsillar biopsy samples were taken at < or = 6 monthly intervals post-infection and examined immunohistochemically for PrPSc. Third eyelid protuberances were difficult to identify, resulting in many unsuitable samples; however, third eyelid samples shown to contain lymphoid follicles were invariably negative for PrPSc. In contrast, tonsillar biopsy samples became positive for PrPSc from 11 to 20 months post-infection. Consistent differences in the morphology of PrPSc granules in tingible body macrophages (TBMs) between BSE- and scrapie-infected sheep were detected with anti-peptide antibodies directed towards amino acids 93-106 of the ovine prion protein: thus, PrPSc appeared as single granules in TBMs of tonsillar sections from BSE-infected sheep, whereas clusters of PrPSc granules were observed within TBMs in the tonsils of scrapie-infected sheep. In contrast, antibodies against epitopes situated N- and C-terminally from the 93-106 region of the ovine prion protein revealed no differences between BSE- and scrapie-infected sheep in terms of PrPSc granules in TBMs.     

Analysis of the risk of transmitting bovine spongiform encephalopathy through bone grafts derived from bovine bone

Bone substitutes of bovine origin are widely used for treatment of bone defects in dental and orthopedic surgery. Due to the occurrence of BSE and the new variant of Creutzfeldt Jakob Disease risks of transmitting diseases through the use of such materials need to be carefully evaluated. Risk analysis can either be based on theoretical assessments or experimental evidence. Here we present a comparative study on two bovine bone substitutes (Bio-Oss and Osteograf/N) which is based on theoretical values. Furthermore, for one of these materials, i.e. Bio-Oss, the prion inactivation capacity of one of the production steps was experimentally evaluated. Theoretical and experimental data indicate that the use of these materials does not carry a risk of transmitting BSE to patients.     

A biochemical theory to explain the cause of bovine spongiform encephalopathy and other encephalopathies

The purpose of this study is to present a hypothesis to explain the aetiology of bovine spongiform encephalopathy (BSE) which is more credible than any at present available, and to increase its credibility by varying the hypothesis to supply explanations for Alzheimer's disease, Parkinson's disease and certain other conditions.The method used has been to utilize material from biochemical textbooks and similar sources.It has been concluded that BSE is caused by the failure to synthesize sufficient cyclic guanosine monophosphate (cGMP), with the result that neurons die because they are no longer able to prevent the entry of toxic quantities of calcium ions into their cytoplasm. Several causes for the failure to synthesize sufficient cGMP have been identified; these involve selenium and folate deficiencies, and problems with the availability of nicotinamide adenosine dinucleotide (NAD). It is proposed that BSE is initiated by a combination of selenium deficiency and the destruction of NAD by a bacterial toxin of the same type as causes cholera, that folate deficiency is the predominant cause of Alzheimer's disease, and that the failure to synthesize sufficient tetrahydrobiopterin and cGMP from guanosine triphosphate results in Parkinson's disease.     

Atypical status of bovine spongiform encephalopathy in Poland: a molecular typing study

Summary The aim of this study was to analyze molecular features of protease-resistant prion protein (PrP^res) in Western blots of BSE cases diagnosed in Poland with respect to a possible atypical status. Confirmed cases were analyzed by Western blotting with several monoclonal antibodies directed at N-terminal and core epitopes of prion protein (PrP). Most cases showed the classical glycoprofile characterized by the dominance of the di- over the monoglycosylated PrP^res band, yielding di-/mono- ratios well above 2 and by reactivity with antibodies having their epitopes in bovine PrP region 110–242 (C-type cases). Surprisingly, seven cases of BSE were atypical. Six were classified as L-type based on a slightly lower molecular mass (M_r) of the non- glycosylated band with respect to C-types and a conspicuously low di-/mono- ratio of glycosylated PrP^res bands approaching unity. One case was classified as H-type because of a higher M_r of PrP^res bands on the blot when compared with C-type cases. A characteristic epitope of H-type PrP^res occurred in the 101–110 region of PrP for which only antibody 12B2 had a sufficient affinity. The occurrence of atypical cases only in animals 9 years of age and older raises questions about the mechanisms of prion diseases and the origin of BSE. Correspondence: Miroslaw Polak, National Veterinary Research Institute, Partyzantow 57, 24-100 Pulawy, Poland     

Antigen retrieval using sodium hydroxide for prion immunohistochemistry in bovine spongiform encephalopathy and scrapie

Formalin-fixed and paraffin wax-embedded (FFPE) tissue sections are usually used for histopathological and immunohistochemical analyses in prion diseases in animals and man. However, formalin fixation cross-links proteins, reducing disease-associated prion protein (PrP^Sc) immunolabelling. To detect PrP^Sc in animals naturally affected with bovine spongiform encephalopathy (BSE) and scrapie, we applied minimal pretreatment with sodium hydroxide (NaOH). This simple pretreatment, combined with enzymatic digestion using proteinase K (PK), was equally effective in the detection of PrP^Sc in FFPE tissue, and superior in terms of speed, compared with the usual autoclaving method. The most effective results, without any section loss, were obtained with 10 μg/ml PK in phosphate buffered saline containing 0.1% Triton-X at room temperature for 10 min and 150 mM NaOH at 60°C for 10 min. By this simple procedure, PrP^Sc was visualized in the brain of animals with BSE and scrapie using a range of anti-PrP primary antibodies.     

Autoantibodies to brain components and antibodies to Acinetobacter calcoaceticus are present in bovine spongiform encephalopathy

Bovine spongiform encephalopathy (BSE) is a neurological disorder, predominantly of British cattle, which belongs to the group of transmissible spongiform encephalopathies together with Creutzfeldt-Jakob disease (CJD), kuru, and scrapie. Autoantibodies to brain neurofilaments have been previously described in patients with CJD and kuru and in sheep affected by scrapie. Spongiform-like changes have also been observed in chronic experimental allergic encephalomyelitis, at least in rabbits and guinea pigs, and in these conditions autoantibodies to myelin occur. We report here that animals with BSE have elevated levels of immunoglobulin A autoantibodies to brain components, i.e., neurofilaments (P < 0.001) and myelin (P < 0.001), as well as to Acinetobacter calcoaceticus (P < 0.001), saprophytic microbes found in soil which have sequences cross-reacting with bovine neurofilaments and myelin, but there were no antibody elevations against Agrobacterium tumefaciens or Escherichia coli. The relevance of such mucosal autoantibodies or antibacterial antibodies to the pathology of BSE and its possible link to prions requires further evaluation.