Biochemical typing of pathological prion protein in aging cattle with BSE

BACKGROUND: The broad enforcement of active surveillance for bovine spongiform encephalopathy (BSE) in 2000 led to the discovery of previously unnoticed, atypical BSE phenotypes in aged cattle that differed from classical BSE (C-type) in biochemical properties of the pathological prion protein. Depending on the molecular mass and the degree of glycosylation of its proteinase K resistant core fragment (PrPres), mainly determined in samples derived from the medulla oblongata, these atypical cases are currently classified into low (L)-type or high (H)-type BSE. In the present study we address the question to what extent such atypical BSE cases are part of the BSE epidemic in Switzerland. RESULTS: To this end we analyzed the biochemical PrPres type by Western blot in a total of 33 BSE cases in cattle with a minimum age of eight years, targeting up to ten different brain regions. Our work confirmed H-type BSE in a zebu but classified all other cases as C-type BSE; indicating a very low incidence of H- and L-type BSE in Switzerland. It was documented for the first time that the biochemical PrPres type was consistent across different brain regions of aging animals with C-type and H-type BSE, i.e. independent of the neuroanatomical structure investigated. CONCLUSION: Taken together this study provides further characteristics of the BSE epidemic in Switzerland and generates new baseline data for the definition of C- and H-type BSE phenotypes, thereby underpinning the notion that they indeed represent distinct prion disease entities.

Bovine prion protein gene (PRNP) promoter polymorphisms modulate PRNP expression and may be responsible for differences in bovine spongiform encephalopathy susceptibility

The susceptibility of humans to the variant Creutzfeldt-Jakob disease is greatly influenced by polymorphisms within the human prion protein gene (PRNP). Similar genetic differences exist in sheep, in which PRNP polymorphisms modify the susceptibility to scrapie. However, the known coding polymorphisms within the bovine PRNP gene have little or no effect on bovine spongiform encephalopathy (BSE) susceptibility in cattle. We have recently found a tentative association between PRNP promoter polymorphisms and BSE susceptibility in German cattle (Sander, P., Hamann, H., Pfeiffer, I., Wemheuer, W., Brenig, B., Groschup, M., Ziegler, U., Distl, O., and Leeb, T. (2004) Neurogenetics 5, 19-25). A plausible hypothesis explaining this observation could be that the bovine PRNP promoter polymorphisms cause changes in PRNP expression that might be responsible for differences in BSE incubation time and/or BSE susceptibility. To test this hypothesis, we performed a functional promoter analysis of the different bovine PRNP promoter alleles by reporter gene assays in vitro and by measuring PRNP mRNA levels in calves with different PRNP genotypes in vivo. Two variable sites, a 23-bp insertion/deletion (indel) polymorphism containing a RP58-binding site and a 12-bp indel polymorphism containing an SP1-binding site, were investigated. Band shift assays indicated differences in transcription factor binding to the different alleles at the two polymorphisms. Reporter gene assays demonstrated an interaction between the two postulated transcription factors and lower expression levels of the ins/ins allele compared with the del/del allele. The in vivo data revealed substantial individual variation of PRNP expression in different tissues. In intestinal lymph nodes, expression levels differed between the different PRNP genotypes.

Squirrel Monkeys (Saimiri sciureus) Infected with the Agent of Bovine Spongiform Encephalopathy Develop Tau Pathology

Squirrel monkeys (Saimiri sciureus) were infected experimentally with the agent of classical bovine spongiform encephalopathy (BSE). Two to four years later, six of the monkeys developed alterations in interactive behaviour and cognition and other neurological signs typical of transmissible spongiform encephalopathy (TSE). At necropsy examination, the brains from all of the monkeys showed pathological changes similar to those described in variant Creutzfeldt-Jakob disease (vCJD) of man, except that the squirrel monkey brains contained no PrP-amyloid plaques typical of that disease. Constant neuropathological features included spongiform degeneration, gliosis, deposition of abnormal prion protein (PrP(TSE)) and many deposits of abnormally phosphorylated tau protein (p-Tau) in several areas of the cerebrum and cerebellum. Western blots showed large amounts of proteinase K-resistant prion protein in the central nervous system. The striking absence of PrP plaques (prominent in brains of cynomolgus macaques [Macaca fascicularis] with experimentally-induced BSE and vCJD and in human patients with vCJD) reinforces the conclusion that the host plays a major role in determining the neuropathology of TSEs. Results of this study suggest that p-Tau, found in the brains of all BSE-infected monkeys, might play a role in the pathogenesis of TSEs. Whether p-Tau contributes to development of disease or appears as a secondary change late in the course of illness remains to be determined.

Comparison of brain PrPd distribution in ovine BSE and scrapie

Scrapie and bovine spongiform encephalopathy (BSE) are both prion diseases affecting ruminants, and these diseases do not share the same public health concerns. Surveillance of the BSE agent in small ruminants has been a great challenge, and the recent identification of diverse prion diseases in ruminants has led to the development of new methods for strain typing. In our study, using immunohistochemistry (IHC), we assessed the distribution of PrP(d) in the brains of 2 experimentally BSE-infected sheep with the ARQ/ARQ genotype. Distribution of PrP(d) in the brain, from the spinal cord to the frontal cortex, was remarkably similar in the 2 sheep despite different inoculation routes and incubation periods. Comparatively, overall PrP(d) brain distribution, evaluated by IHC, in 19 scrapie cases with the ARQ/ARQ, ARQ/VRQ, and VRQ/VRQ genotypes, in some cases showed similarities to the experimentally BSE-infected sheep. There was no exclusive neuroanatomical site with a characteristic and specific PrP(d) type of accumulation induced by the BSE agent. However, a detailed analysis of the topography, types, and intensity of PrP(d) deposits in the frontal cortex, striatum, piriform cortex, hippocampus, mesencephalon, and cerebellum allowed the BSE-affected sheep group to be distinguished from the 19 scrapie cases analyzed in our study. These results strengthen and emphasize the potential interest of PrP(d) brain mapping to help in identifying prion strains in small ruminants.

Molecular analysis of carbohydrate N-acetylgalactosamine 4-O sulfotransferase 8 (CHST8) as a candidate gene for bovine spongiform encephalopathy susceptibility

Endogenous prion proteins (PrP) play the central role in the pathogenesis of transmissible spongiform encephalopathies. The carbohydrate N-acetylgalactosamine 4-O sulfotransferase 8 (CHST8) promotes the conversion of the cellular PrP(C) into the pathogenic PrP(d). Six sequence variants within the CHST8 gene were identified by comparative sequencing and genotyped for a sample of 623 animals comprising bovine spongiform encephalopathy (BSE)-affected and healthy control cows representing German Fleckvieh (German Simmental), German Holstein (Holstein-Friesian) and Brown Swiss. Significant differences in the allele, genotype and haplotype frequencies between BSE-affected and healthy cows indicate an association of sequence variant g.37254017G>T with the development of the disease in Brown Swiss cattle.

Sleep-wake disturbances in sporadic Creutzfeldt-Jakob disease

BACKGROUND: The prevalence and characteristics of sleep-wake disturbances in sporadic Creutzfeldt-Jakob disease (sCJD) are poorly understood. METHODS: Seven consecutive patients with definite sCJD underwent a systematic assessment of sleep-wake disturbances, including clinical history, video-polysomnography, and actigraphy. Extent and distribution of neurodegeneration was estimated by brain autopsy in six patients. Western blot analyses enabling classification and quantification of the protease-resistant isoform of the prion protein, PrPSc, in thalamus and occipital cortex was available in four patients. RESULTS: Sleep-wake symptoms were observed in all patients, and were prominent in four of them. All patients had severe sleep EEG abnormalities with loss of sleep spindles, very low sleep efficiency, and virtual absence of REM sleep. The correlation between different methods to assess sleep-wake functions (history, polysomnography, actigraphy, videography) was generally poor. Brain autopsy revealed prominent changes in cortical areas, but only mild changes in the thalamus. No mutation of the PRNP gene was found. CONCLUSIONS: This study demonstrates in sporadic Creutzfeldt-Jakob disease, first, the existence of sleep-wake disturbances similar to those reported in fatal familial insomnia in the absence of prominent and isolated thalamic neuronal loss, and second, the need of a multimodal approach for the unambiguous assessment of sleep-wake functions in these patients.

PRNP promoter polymorphisms are associated with BSE susceptibility in Swiss and German cattle

BACKGROUND: Non-synonymous polymorphisms within the prion protein gene (PRNP) influence the susceptibility and incubation time for transmissible spongiform encephalopathies (TSE) in some species such as sheep and humans. In cattle, none of the known polymorphisms within the PRNP coding region has a major influence on susceptibility to bovine spongiform encephalopathy (BSE). Recently, however, we demonstrated an association between susceptibility to BSE and a 23 bp insertion/deletion (indel) polymorphism and a 12 bp indel polymorphism within the putative PRNP promoter region using 43 German BSE cases and 48 German control cattle. The objective of this study was to extend this work by including a larger number of BSE cases and control cattle of German and Swiss origin. RESULTS: Allele, genotype and haplotype frequencies of the two indel polymorphisms were determined in 449 BSE cattle and 431 unaffected cattle from Switzerland and Germany including all 43 German BSE and 16 German control animals from the original study. When breeds with similar allele and genotype distributions were compared, the 23 bp indel polymorphism again showed a significant association with susceptibility to BSE. However, some additional breed-specific allele and genotype distributions were identified, mainly related to the Brown breeds. CONCLUSION: Our study corroborated earlier findings that polymorphisms in the PRNP promoter region have an influence on susceptibility to BSE. However, breed-specific differences exist that need to be accounted for when analyzing such data.

Neuropathological and molecular comparison between clinical and asymptomatic bovine spongiform encephalopathy cases

Interest in the proper neuropathological and molecular characterization of bovine spongiform encephalopathy (BSE) has increased since asymptomatic and atypical cases were detected in the cattle population by active disease surveillance. In this respect we investigated a total of 95 confirmed BSE cases originating from different active and passive surveillance categories (clinical suspects, emergency-slaughter, fallen stock and routinely slaughter) in Switzerland for their neuropathological and molecular phenotype. We looked for measurable differences between these categories in lesion profile, severity of spongiform change, degree of astrocytosis as well as immunohistochemical and molecular patterns of the disease-associated isoform of the prion protein (PrPd) in the caudal brainstem. Our results indicate significantly higher intensities of spongiform change in clinically affected compared to asymptomatic BSE cases. Similar effects were in trend observed for the intensities of PrPd deposition and astrocytosis, whereas the frequencies of morphological PrPd types and the molecular patterns in Western immunoblot were not different. Importantly, none of the animals included in this study revealed features of atypical BSE. Taken together, this study suggests that both clinically affected as well as asymptomatic Swiss BSE cases in cattle share the neuropathological and molecular phenotype of classical BSE and that asymptomatic classical BSE cases are at a pre-clinical stage of the disease rather than representing a true sub-clinical form of BSE.

Atypical scrapie in a Swiss goat and implications for transmissible spongiform encephalopathy surveillance

Different types of transmissible spongiform encephalopathies (TSEs) affect sheep and goats. In addition to the classical form of scrapie, both species are susceptible to experimental infections with the bovine spongiform encephalopathy (BSE) agent, and in recent years atypical scrapie cases have been reported in sheep from different European countries. Atypical scrapie in sheep is characterized by distinct histopathologic lesions and molecular characteristics of the abnormal scrapie prion protein (PrP(sc)). Characteristics of atypical scrapie have not yet been described in detail in goats. A goat presenting features of atypical scrapie was identified in Switzerland. Although there was no difference between the molecular characteristics of PrP(sc) in this animal and those of atypical scrapie in sheep, differences in the distribution of histopathologic lesions and PrP(sc) deposition were observed. In particular the cerebellar cortex, a major site of PrP(sc) deposition in atypical scrapie in sheep, was found to be virtually unaffected in this goat. In contrast, severe lesions and PrP(sc) deposition were detected in more rostral brain structures, such as thalamus and midbrain. Two TSE screening tests and PrP(sc) immunohistochemistry were either negative or barely positive when applied to cerebellum and obex tissues, the target samples for TSE surveillance in sheep and goats. These findings suggest that such cases may have been missed in the past and could be overlooked in the future if sampling and testing procedures are not adapted. The epidemiological and veterinary public health implications of these atypical cases, however, are not yet known.

6th International Immunoglobulin Symposium: poster presentations

The posters presented at the 6th International Immunoglobulin Symposium covered a wide range of fields and included both basic science and clinical research. From the abstracts accepted for poster presentation, 12 abstracts were selected for oral presentations in three parallel sessions on immunodeficiencies, autoimmunity and basic research. The immunodeficiency presentations dealt with novel, rare class-switch recombination (CSR) deficiencies, attenuation of adverse events following IVIg treatment, association of immunoglobulin (Ig)G trough levels and protection against acute infection in patients with X-linked agammaglobulinaemia (XLA) and common variable immunodeficiency (CVID), and the reduction of class-switched memory B cells in patients with specific antibody deficiency (SAD). The impact of intravenous immunoglobulin on fetal alloimmune thrombocytopenia, pregnancy and postpartum-related relapses in multiple sclerosis and refractory myositis, as well as experiences with subcutaneous immunoglobulin in patients with multi-focal motor neuropathy, were the topics presented in the autoimmunity session. The interaction of dendritic cell (DC)-SIGN and alpha2,6-sialylated IgG Fc and its impact on human DCs, the enrichment of sialylated IgG in plasma-derived IgG, as wells as prion surveillance and monitoring of anti-measles titres in immunoglobulin products, were covered in the basic science session. In summary, the presentations illustrated the breadth of immunoglobulin therapy usage and highlighted the progress that is being made in diverse areas of basic and clinical research, extending our understanding of the mechanisms of immunoglobulin action and contributing to improved patient care.

BETASCAN: probable beta-amyloids identified by pairwise probabilistic analysis

Amyloids and prion proteins are clinically and biologically important beta-structures, whose supersecondary structures are difficult to determine by standard experimental or computational means. In addition, significant conformational heterogeneity is known or suspected to exist in many amyloid fibrils. Recent work has indicated the utility of pairwise probabilistic statistics in beta-structure prediction. We develop here a new strategy for beta-structure prediction, emphasizing the determination of beta-strands and pairs of beta-strands as fundamental units of beta-structure. Our program, BETASCAN, calculates likelihood scores for potential beta-strands and strand-pairs based on correlations observed in parallel beta-sheets. The program then determines the strands and pairs with the greatest local likelihood for all of the sequence's potential beta-structures. BETASCAN suggests multiple alternate folding patterns and assigns relative a priori probabilities based solely on amino acid sequence, probability tables, and pre-chosen parameters. The algorithm compares favorably with the results of previous algorithms (BETAPRO, PASTA, SALSA, TANGO, and Zyggregator) in beta-structure prediction and amyloid propensity prediction. Accurate prediction is demonstrated for experimentally determined amyloid beta-structures, for a set of known beta-aggregates, and for the parallel beta-strands of beta-helices, amyloid-like globular proteins. BETASCAN is able both to detect beta-strands with higher sensitivity and to detect the edges of beta-strands in a richly beta-like sequence. For two proteins (Abeta and Het-s), there exist multiple sets of experimental data implying contradictory structures; BETASCAN is able to detect each competing structure as a potential structure variant. The ability to correlate multiple alternate beta-structures to experiment opens the possibility of computational investigation of prion strains and structural heterogeneity of amyloid. BETASCAN is publicly accessible on the Web at http://betascan.csail.mit.edu.

Continuous monitoring of bovine spongiform encephalopathy rapid test performance by weak positive tissue controls and quality control charts

Bovine spongiform encephalopathy (BSE) rapid tests and routine BSE-testing laboratories underlie strict regulations for approval. Due to the lack of BSE-positive control samples, however, full assay validation at the level of individual test runs and continuous monitoring of test performance on-site is difficult. Most rapid tests use synthetic prion protein peptides, but it is not known to which extend they reflect the assay performance on field samples, and whether they are sufficient to indicate on-site assay quality problems. To address this question we compared the test scores of the provided kit peptide controls to those of standardized weak BSE-positive tissue samples in individual test runs as well as continuously over time by quality control charts in two widely used BSE rapid tests. Our results reveal only a weak correlation between the weak positive tissue control and the peptide control scores. We identified kit-lot related shifts in the assay performances that were not reflected by the peptide control scores. Vice versa, not all shifts indicated by the peptide control scores indeed reflected a shift in the assay performance. In conclusion these data highlight that the use of the kit peptide controls for continuous quality control purposes may result in unjustified rejection or acceptance of test runs. However, standardized weak positive tissue controls in combination with Shewhart-CUSUM control charts appear to be reliable in continuously monitoring assay performance on-site to identify undesired deviations.

A Targeted Survey for Scrapie in Jos Plateau State, Nigeria

Scrapie, a disease of sheep and goats with a progressive course and fatal outcome, has not been identified in Nigeria. Anecdotal scrapie reports by livestock workers abound. Livestock diseases like scrapie form huddles in livestock economics of countries. For 8 months we surveyed for scrapie targeting emergency/casualty slaughter sheep and goats in Jos, Nigeria. We clinically examined 510 sheep and 608 goats of local breeds, aged from 12 months to 5 years. In total 31 (5.10%) goats and no sheep were clinically suspicious for scrapie. Caudal brainstem tissues of suspect animals collected postmortem were analyzed for the disease specific form of the prion protein, PrPSc, using Bio-Rad’s TeSeE ELISA rapid test kit. No sample was positive for scrapie. Fluorescent antibody test for rabies and H&E staining on samples were carried out for differential diagnosis. These showed no pathological lesions indicative for neurological disease. While our findings do not exclude the presence of scrapie in Jos, we demonstrate that targeted sampling of small ruminants for neuroinfectious disease is feasible in developing countries, pointing to the possibility of implementing such a monitoring scheme in Nigeria to prevent economic losses in small ruminant livestock as scrapie caveats from endemic countries have shown.

A pilot study for targeted surveillance of bovine spongiform encephalopathy in Nigeria.

Bovine spongiform encephalopathy (BSE), popularly known as 'mad cow disease', led to an epidemic in Europe that peaked in the mid-1990s. Its impact on developing countries, such as Nigeria, has not been fully established as information on livestock and surveillance has eluded those in charge of this task. The BSE risk to Nigeria's cattle population currently remains undetermined, which has resulted in international trade restrictions on commodities from the cattle population. This is mainly because of a lack of updated BSE risk assessments and disease surveillance data. To evaluate the feasibility of BSE surveillance in Nigeria, we carried out a pilot study targeting cattle that were presented for emergency or casualty slaughter. In total, 1551 cattle of local breeds, aged 24 months and above were clinically examined. Ataxia, recumbency and other neurological signs were topmost on our list of criteria. A total of 96 cattle, which correspond to 6.2%, presented clinical signs that supported a suspect of BSE. The caudal brainstem tissues of these animals were collected post-mortem and analysed for the disease-specific form of the prion protein using a rapid test approved by the International Animal Health Organization (OIE). None of the samples were positive for BSE. Although our findings do not exclude the presence of BSE in Nigeria, they do demonstrate that targeted sampling of clinically suspected cases of BSE is feasible in developing countries. In addition, these findings point to the possibility of implementing clinical monitoring schemes for BSE and potentially other diseases with grave economic and public health consequences.