Gene expression alterations in Rocky Mountain elk infected with chronic wasting disease

Chronic wasting disease (CWD) is an invariably fatal neurologic disease that naturally infects mule deer, white tailed deer and elk. The understanding of CWD neurodegeneration at a molecular level is very limited. In this study, microarray analysis was performed to determine changes in the gene expression profiles in six different tissues including brain, midbrain, thalamus, spleen, RPLN and tonsil of CWD-infected elk in comparison to non-infected healthy elk, using 24,000 bovine specific oligo probes. In total, 329 genes were found to be differentially expressed (> 2.0-fold) between CWD negative and positive brain tissues, with 132 genes upregulated and 197 genes downregulated. There were 249 DE genes in the spleen (168 up- and 81 downregulated), 30 DE genes in the retropharyngeal lymph node (RPLN) (18 up- and 12 downregulated), and 55 DE genes in the tonsil (21 up- and 34 downregulated). Using Gene Ontology (GO), the DE genes were assigned to functional groups associated with cellular process, biological regulation, metabolic process, and regulation of biological process. For all brain tissues, the highest ranking networks for DE genes identified by Ingenuity Pathway Analysis (IPA) were associated with neurological disease, cell morphology, cellular assembly and organization. Quantitative real-time PCR (qRT-PCR) validated the expression of DE genes primarily involved in different regulatory pathways, including neuronal signaling and synapse function, calcium signaling, apoptosis and cell death and immune cell trafficking and inflammatory response. This is the first study to evaluate altered gene expression in multiple organs including brain from orally infected elk and the results will improve our understanding of CWD neurodegeneration at the molecular level.     

Gene expression alterations in Rocky Mountain elk infected with chronic wasting disease

Chronic wasting disease (CWD) is an invariably fatal neurologic disease that naturally infects mule deer, white tailed deer and elk. The understanding of CWD neurodegeneration at a molecular level is very limited. In this study, microarray analysis was performed to determine changes in the gene expression profiles in six different tissues including brain, midbrain, thalamus, spleen, RPLN and tonsil of CWD-infected elk in comparison to non-infected healthy elk, using 24,000 bovine specific oligo probes. In total, 329 genes were found to be differentially expressed (> 2.0-fold) between CWD negative and positive brain tissues, with 132 genes upregulated and 197 genes downregulated. There were 249 DE genes in the spleen (168 up- and 81 downregulated), 30 DE genes in the retropharyngeal lymph node (RPLN) (18 up- and 12 downregulated), and 55 DE genes in the tonsil (21 up- and 34 downregulated). Using Gene Ontology (GO), the DE genes were assigned to functional groups associated with cellular process, biological regulation, metabolic process, and regulation of biological process. For all brain tissues, the highest ranking networks for DE genes identified by Ingenuity Pathway Analysis (IPA) were associated with neurological disease, cell morphology, cellular assembly and organization. Quantitative real-time PCR (qRT-PCR) validated the expression of DE genes primarily involved in different regulatory pathways, including neuronal signaling and synapse function, calcium signaling, apoptosis and cell death and immune cell trafficking and inflammatory response. This is the first study to evaluate altered gene expression in multiple organs including brain from orally infected elk and the results will improve our understanding of CWD neurodegeneration at the molecular level.     

Autoantibody to glial fibrillary acidic protein in the sera of cattle with bovine spongiform encephalopathy

It is desirable to make the diagnosis in live cattle with bovine spongiform encephalopathy (BSE), and thus surrogate markers for the disease have been eagerly sought. Serum proteins from BSE cattle were analyzed by 2‐D Western blotting and TOF‐MS. Autoantibodies against proteins in cytoskeletal fractions prepared from normal bovine brains were found in the sera of BSE cattle. The protein recognized was identified to be glial fibrillary acidic protein (GFAP), which is expressed mainly in astrocytes in the brain. The antigen protein, GFAP, was also found in the sera of BSE cattle. The percentages of both positive sera in the autoantibody and GFAP were 44.0% for the BSE cattle, 0% for the healthy cattle, and 5.0% for the clinically suspected BSE‐negative cattle. A significant relationship between the presence of GFAP and the expression of its autoantibody in the serum was recognized in the BSE cattle. These findings suggest a leakage of GFAP into the peripheral blood during neurodegeneration associated with BSE, accompanied by the autoantibody production, and might be useful in understanding the pathogenesis and in developing a serological diagnosis of BSE in live cattle.     

BSE in France: epidemiological analysis and predictions

Attention throughout Europe continues to focus on bovine spongiform encephalopathy (BSE) with increasing evidence linking it to the new variant of Creutzfeldt-Jakob disease (vCJD) in humans. The age- and cohort-specific incidence of BSE in French cattle was modelled as a function of the survival distribution, the cohort-specific incidence of BSE infection, the underreporting rate of BSE cases, and the age-specific probability, conditional on survival, that an infected animal would experience clinical onset. The results reveal that thousands of French cattle were infected with BSE over the course of the epidemic. However, case incidence is predicted to decline in future years.     

The identification of candidate genes and SNP markers for classical bovine spongiform encephalopathy susceptibility

Classical bovine spongiform encephalopathy is a transmissible prion disease that is fatal to cattle and is a human health risk due to its association with a strain of Creutzfeldt-Jakob disease (vCJD). Mutations to the coding region of the prion gene (PRNP) have been associated with susceptibility to transmissible spongiform encephalopathies in mammals including bovines and humans. Additional loci such as the retinoic acid receptor beta (RARB) and stathmin like 2 (STMN2) have also been associated with disease risk. The objective of this study was to refine previously identified regions associated with BSE susceptibility and to identify positional candidate genes and genetic variation that may be involved with the progression of classical BSE. The samples included 739 samples of either BSE infected animals (522 animals) or non-infected controls (207 animals). These were tested using a custom SNP array designed to narrow previously identified regions of importance in bovine genome. Thirty one single nucleotide polymorphisms were identified at p < 0.05 and a minor allele frequency greater than 5%. The chromosomal regions identified and the positional and functional candidate genes and regulatory elements identified within these regions warrant further research.     

The identification of candidate genes and SNP markers for classical bovine spongiform encephalopathy susceptibility

Classical bovine spongiform encephalopathy is a transmissible prion disease that is fatal to cattle and is a human health risk due to its association with a strain of Creutzfeldt-Jakob disease (vCJD). Mutations to the coding region of the prion gene (PRNP) have been associated with susceptibility to transmissible spongiform encephalopathies in mammals including bovines and humans. Additional loci such as the retinoic acid receptor beta (RARB) and stathmin like 2 (STMN2) have also been associated with disease risk. The objective of this study was to refine previously identified regions associated with BSE susceptibility and to identify positional candidate genes and genetic variation that may be involved with the progression of classical BSE. The samples included 739 samples of either BSE infected animals (522 animals) or non-infected controls (207 animals). These were tested using a custom SNP array designed to narrow previously identified regions of importance in bovine genome. Thirty one single nucleotide polymorphisms were identified at p < 0.05 and a minor allele frequency greater than 5%. The chromosomal regions identified and the positional and functional candidate genes and regulatory elements identified within these regions warrant further research.     

Strain-specific barriers against bovine prions in hamsters

We investigated the susceptibilities of Syrian golden hamsters to transmissible spongiform encephalopathy agents from cattle. We report efficient transmission of the L-type atypical bovine spongiform encephalopathy (BSE) agent into hamsters. Importantly, hamsters were also susceptible to the transmissible mink encephalopathy agent from cattle, which has molecular features similar to those of the L-type BSE agent, as also shown in bovinized transgenic mice. In sharp contrast, hamsters could not be infected with classical or H-type BSE agents from cattle. However, previous adaptation of the classical BSE agent in wild-type mice led to efficient transmission. Thus, this study demonstrates the existence of distinct "strain barriers" upon the transmission of bovine prions in hamsters.     

H-Type Bovine Spongiform Encephalopathy

We previously reported that some cattle affected by bovine spongiform encephalopathy (BSE) showed distinct molecular features of the protease-resistant pion protein (PrPres ) in Western blot, with a 1-2 kDa higher apparent molecular mass of the unglycosylated PrPres associated with labelling by antibodies against the 86-107 region of the bovine PrP protein (H-type BSE). By Western blot analyses of PrPres, we now showed that the essential features initially described in cattle were observed with a panel of different antibodies and were maintained after transmission of the disease in C57Bl/6 mice. In addition, antibodies against the C-terminal region of PrP revealed a second, more C-terminally cleaved, form of PrPres (PrPres #2), which, in unglycosylated form, migrated as a ≈ 14 kDa fragment. Furthermore, a PrPres fragment of ≈ 7kDa, which was not labelled by C-terminus-specific antibodies and was thus presumed to be a product of cleavage at both N- and C-terminal sides of PrP protein, was also detected. Both PrPres #2 and ≈ 7 kDa PrPres were detected in cattle and in C57Bl/6 infected mice. These complex molecular features are reminiscent of findings reported in human prion diseases. This raises questions regarding the respective origins and pathogenic mechanisms in prion diseases of animals and humans.     

Genome-wide scan identifies loci associated with classical BSE occurrence

Classical bovine spongiform encephalopathy (BSE) is an acquired prion disease that is invariably fatal in cattle and has been implicated as a significant human health risk. Sequence variations in the coding region of the prion gene (PRNP) have been associated with acquired transmissible spongiform encephalopathy (TSE) susceptibility in mammals; however, this is not the case in cattle. It has been hypothesized that genes, in addition to the prion gene, contribute to genetic susceptibility of acquired TSEs. Accordingly, genetic studies of classical BSE in cattle identified loci other than PRNP that are associated with disease incidence. The objective of this study was to utilize a genome-wide association study to test for genetic loci associated with classical BSE. The samples include 143 BSE affected (case) and 173 unaffected half sib (control) animals collected in the mid 1990s in Southern England. The data analysis identifies loci on two different chromosomes associated with BSE disease occurrence. Most notable is a single nucleotide polymorphism on chromosome 1 at 29.15 Mb that is associated with BSE disease (p = 3.09E-05). Additionally, a locus on chromosome 14, within a cluster of SNPs showed a trend toward significance (p = 5.24E-05). It is worth noting that in a human vCJD study markers on human chromosome 8, a region with shared synteny to the region identified on cattle chromosome 14, were associated with disease. Further, our candidate genes appear to have plausible biological relevance with the known etiology of TSE disease. One of the candidate genes is hypothetical gene LOC521010, similar to FK506 binding protein 2 located on chromosome 1 at 29.32 Mb. This gene encodes a protein that is a member of the immunophilin protein family and is involved in basic cellular processes including protein folding. The chromosomal regions identified in this study and candidate genes within these regions merit further investigation.     

Comparative molecular analysis of the abnormal prion protein in field scrapie cases and experimental bovine spongiform encephalopathy in sheep by use of Western blotting and immunohistochemical methods

Since the appearance of bovine spongiform encephalopathy (BSE) in cattle and its linkage with the human variant of Creutzfeldt-Jakob disease, the possible spread of this agent to sheep flocks has been of concern as a potential new source of contamination. Molecular analysis of the protease cleavage of the abnormal prion protein (PrP), by Western blotting (PrP(res)) or by immunohistochemical methods (PrP(d)), has shown some potential to distinguish BSE and scrapie in sheep. Using a newly developed enzyme-linked immunosorbent assay, we identified 18 infected sheep in which PrP(res) showed an increased sensitivity to proteinase K digestion. When analyzed by Western blotting, two of them showed a low molecular mass of unglycosylated PrP(res) as found in BSE-infected sheep, in contrast to other naturally infected sheep. A decrease of the labeling by P4 monoclonal antibody, which recognizes an epitope close to the protease cleavage site, was also found by Western blotting in the former two samples, but this was less marked than in BSE-infected sheep. These two samples, and all of the other natural scrapie cases studied, were clearly distinguishable from those from sheep inoculated with the BSE agent from either French or British cattle by immunohistochemical analysis of PrP(d) labeling in the brain and lymphoid tissues. Final characterization of the strain involved in these samples will require analysis of the features of the disease following infection of mice, but our data already emphasize the need to use the different available methods to define the molecular properties of abnormal PrP and its possible similarities with the BSE agent.     

Sheep-passaged bovine spongiform encephalopathy agent exhibits altered pathobiological properties in bovine-PrP transgenic mice

Sheep can be experimentally infected with bovine spongiform encephalopathy (BSE), and the ensuing disease is similar to scrapie in terms of pathogenesis and clinical signs. BSE infection in sheep is an animal and human health concern. In this study, the transmission in BoPrP-Tg110 mice of prions from BSE-infected sheep was examined and compared to the transmission of original cattle BSE in cattle and sheep scrapie prions. Our results indicate no transmission barrier for sheep BSE prions to infect BoPrP-Tg110 mice, but the course of the disease is accelerated compared to the effects of the original BSE isolate. The shortened incubation period of sheep BSE in the model was conserved in subsequent passage in BoPrP-Tg110 mice, indicating that it is not related to infectious titer differences. Biochemical signature, lesion profile, and PrP(Sc) deposition pattern of both cattle and sheep BSE were similar. In contrast, all three sheep scrapie isolates tested showed an evident transmission barrier and further adaptation in subsequent passage. Taken together, those data indicate that BSE agent can be altered by crossing a species barrier, raising concerns about the virulence of this new prion towards other species, including humans. The BoPrP-Tg110 mouse bioassay should be considered as a valuable tool for discriminating scrapie and BSE in sheep.     

H-Type Bovine Spongiform Encephalopathy: Complex Molecular Features and Similarities with Human Prion Diseases

We previously reported that some cattle affected by bovine spongiform encephalopathy (BSE) showed distinct molecular features of the protease-resistant prion protein (PrP^res) in Western blot, with a 1–2 kDa higher apparent molecular mass of the unglycosylated PrP^res associated with labelling by antibodies against the 86–107 region of the bovine PrP protein (H-type BSE). By Western blot analyses of PrP^res, we now showed that the essential features initially described in cattle were observed with a panel of different antibodies and were maintained after transmission of the disease in C57Bl/6 mice. In addition, antibodies against the C-terminal region of PrP revealed a second, more C-terminally cleaved, form of PrP^res (PrP^res #2), which, in unglycosylated form, migrated as a ≈ 14 kDa fragment. Furthermore, a PrP^res fragment of ≈7 kDa, which was not labelled by C-terminus-specific antibodies and was thus presumed to be a product of cleavage at both N- and C-terminal sides of PrP protein, was also detected. Both PrP^res #2 and ≈7 kDa PrP^res were detected in cattle and in C57Bl/6 infected mice. These complex molecular features are reminiscent of findings reported in human prion diseases. This raises questions regarding the respective origins and pathogenic mechanisms in prion diseases of animals and humans.Key Words: prion, BSE, Creutzfeldt-Jakob, Gerstmann-Sträussler-Scheinker, Western blot, amyloid     

Intraspecies transmission of BASE induces clinical dullness and amyotrophic changes

The disease phenotype of bovine spongiform encephalopathy (BSE) and the molecular/ biological properties of its prion strain, including the host range and the characteristics of BSE-related disorders, have been extensively studied since its discovery in 1986. In recent years, systematic testing of the brains of cattle coming to slaughter resulted in the identification of at least two atypical forms of BSE. These emerging disorders are characterized by novel conformers of the bovine pathological prion protein (PrP(TSE)), named high-type (BSE-H) and low-type (BSE-L). We recently reported two Italian atypical cases with a PrP(TSE) type identical to BSE-L, pathologically characterized by PrP amyloid plaques and known as bovine amyloidotic spongiform encephalopathy (BASE). Several lines of evidence suggest that BASE is highly virulent and easily transmissible to a wide host range. Experimental transmission to transgenic mice overexpressing bovine PrP (Tgbov XV) suggested that BASE is caused by a prion strain distinct from the BSE isolate. In the present study, we experimentally infected Friesian and Alpine brown cattle with Italian BSE and BASE isolates via the intracerebral route. BASE-infected cattle developed amyotrophic changes accompanied by mental dullness. The molecular and neuropathological profiles, including PrP deposition pattern, closely matched those observed in the original cases. This study provides clear evidence of BASE as a distinct prion isolate and discloses a novel disease phenotype in cattle.     

Comparison of the local structural stabilities of mammalian prion protein (PrP) by fragment molecular orbital calculations

Bovine spongiform encephalopathy (BSE), a member of the prion diseases, is a fatal neurodegenerative disorder suspected to be caused by a malfunction of prion protein (PrP). Although BSE prions have been reported to be transmitted to a wide range of animal species, dogs and hamsters are known to be BSE-resistant animals. Analysis of canine and hamster PrP could elucidate the molecular mechanisms supporting the species barriers to BSE prion transmission. The structural stability of 6 mammalian PrPs, including human, cattle, mouse, hamster, dog and cat, was analyzed. We then evaluated intramolecular interactions in PrP by fragment molecular orbital (FMO) calculations. Despite similar backbone structures, the PrP side-chain orientations differed among the animal species examined. The pair interaction energies between secondary structural elements in the PrPs varied considerably, indicating that the local structural stabilities of PrP varied among the different animal species. Principal component analysis (PCA) demonstrated that different local structural stability exists in bovine PrP compared with the PrP of other animal species examined. The results of the present study suggest that differences in local structural stabilities between canine and bovine PrP link diversity in susceptibility to BSE prion infection.     

BSE infection of the small short-lived primate Microcebus murinus

Eleven Microcebus murinus (lemur) primates were intracerebrally or orally infected by bovine spongiform encephalopathy (BSE) or macaque-adapted BSE (MBSE) brain homogenates. In many BSE and MBSE infected lemurs, but not in animals inoculated with normal bovine brain, persistent behavioral changes occurred as early as 3 months, and neurological signs as early as 13 months after infection. Immunohistochemical examination of animals sacrificed during the incubation period revealed an abnormal accumulation of 'prion' protein (PrP) in the intestinal wall, intestinal nervous plexus, mesenteric lymph nodes and spleen, and in the clinical stage, also in the brain. In MBSE-inoculated animals, proteinase K resistance of the PrP (PrPres) was confirmed by Western blot in the spleen and the brain. Obvious signs of neurodegeneration were observed in all infected animals characterized by hyperaggregated and paired-helical filaments-immunoreactive Tau proteins, beta 42-amyloid plaques and astrogliosis. Additionally, PrPres was present in the ganglion cells of the retina in diseased animals after either intracerebrally or oral infection by the BSE or MBSE agent. These results show that the microcebe is susceptible to the BSE infectious agent via intracerebral and oral routes with comparatively short incubation periods compared to simians, and could be a useful animal model to study the pathophysiology of disease transmission in primates.     

Intraspecies transmission of L‐type‐like bovine spongiform encephalopathy detected in Japan

It has been assumed that the agent causing BSE in cattle is a uniform strain (classical BSE); however, different neuropathological and molecular phenotypes of BSE (atypical BSE) have been recently reported. We demonstrated the successful transmission of L‐type‐like atypical BSE detected in Japan (BSE/JP24 isolate) to cattle. Based on the incubation period, neuropathological hallmarks, and molecular properties of the abnormal host prion protein, the characteristics of BSE/JP24 prion were apparently distinguishable from the classical BSE prion and closely resemble those of bovine amyloidotic spongiform encephalopathy prion detected in Italy.     

Conversion of the BASE prion strain into the BSE strain: the origin of BSE?

Atypical neuropathological and molecular phenotypes of bovine spongiform encephalopathy (BSE) have recently been identified in different countries. One of these phenotypes, named bovine "amyloidotic" spongiform encephalopathy (BASE), differs from classical BSE for the occurrence of a distinct type of the disease-associated prion protein (PrP), termed PrP(Sc), and the presence of PrP amyloid plaques. Here, we show that the agents responsible for BSE and BASE possess different biological properties upon transmission to transgenic mice expressing bovine PrP and inbred lines of nontransgenic mice. Strikingly, serial passages of the BASE strain to nontransgenic mice induced a neuropathological and molecular disease phenotype indistinguishable from that of BSE-infected mice. The existence of more than one agent associated with prion disease in cattle and the ability of the BASE strain to convert into the BSE strain may have important implications with respect to the origin of BSE and spongiform encephalopathies in other species, including humans.     

Ion channels and membrane potential in stimulus-secretion coupling in adrenal paraneurons

Cultured bovine adrenal medulla cells have been shown to contain several different ion channels (Na+, Ca2+, acetylcholine receptor regulated) whose activation leads to the secretion of catecholamines. The pharmacology of these ion channels and their interactions during secretion have been examined. The mechanisms of agonist-induced calcium influx are of particular interest since this is an early obligatory event during secretion from the adrenal medulla. Data obtained on catecholamine release and 45Ca2+ uptake indicate that both voltage-dependent and voltage-independent calcium influx mechanisms operate in cultured bovine adrenal medulla cells. The significance of these results in understanding the mechanism of action of the physiological stimulus acetylcholine (Ach) will be discussed. The alkaloid channel neurotoxins D-600, batrachotoxin, veratridine, and aconitine were shown to exert a noncompetitive inhibitory effect on Ach-induced ion flux in adrenal medulla cells, presumably through an interaction with the nicotinic receptor regulated channel. Lipid-soluble neurotoxins may interact with multiple ion channels in nerve and muscle membrane.     

Different prion disease phenotypes result from inoculation of cattle with two temporally separated sources of sheep scrapie from Great Britain

Background

Given the theoretical proposal that bovine spongiform encephalopathy (BSE) could have originated from sheep scrapie, this study investigated the pathogenicity for cattle, by intracerebral (i.c.) inoculation, of two pools of scrapie agents sourced in Great Britain before and during the BSE epidemic. Two groups of ten cattle were each inoculated with pools of brain material from sheep scrapie cases collected prior to 1975 and after 1990. Control groups comprised five cattle inoculated with sheep brain free from scrapie, five cattle inoculated with saline, and for comparison with BSE, naturally infected cattle and cattle i.c. inoculated with BSE brainstem homogenate from a parallel study. Phenotypic characterisation of the disease forms transmitted to cattle was conducted by morphological, immunohistochemical, biochemical and biological methods.

Results

Disease occurred in 16 cattle, nine inoculated with the pre-1975 inoculum and seven inoculated with the post-1990 inoculum, with four cattle still alive at 83 months post challenge (as at June 2006). The different inocula produced predominantly two different disease phenotypes as determined by histopathological, immunohistochemical and Western immunoblotting methods and biological characterisation on transmission to mice, neither of which was identical to BSE. Whilst the disease presentation was uniform in all scrapie-affected cattle of the pre-1975 group, the post-1990 inoculum produced a more variable disease, with two animals sharing immunohistochemical and molecular profile characteristics with animals in the pre-1975 group.

Conclusion

The study has demonstrated that cattle inoculated with different pooled scrapie sources can develop different prion disease phenotypes, which were not consistent with the phenotype of BSE of cattle and whose isolates did not have the strain typing characteristics of the BSE agent on transmission to mice.