BSE case associated with prion protein gene mutation

Bovine spongiform encephalopathy (BSE) is a transmissible spongiform encephalopathy (TSE) of cattle and was first detected in 1986 in the United Kingdom. It is the most likely cause of variant Creutzfeldt-Jakob disease (CJD) in humans. The origin of BSE remains an enigma. Here we report an H-type BSE case associated with the novel mutation E211K within the prion protein gene (Prnp). Sequence analysis revealed that the animal with H-type BSE was heterozygous at Prnp nucleotides 631 through 633. An identical pathogenic mutation at the homologous codon position (E200K) in the human Prnp has been described as the most common cause of genetic CJD. This finding represents the first report of a confirmed case of BSE with a potential pathogenic mutation within the bovine Prnp gene. A recent epidemiological study revealed that the K211 allele was not detected in 6062 cattle from commercial beef processing plants and 42 cattle breeds, indicating an extremely low prevalence of the E211K variant (less than 1 in 2000) in cattle.     

Biological and biochemical characterization of M2B cells: Classical BSE prion is conserved in transgenic mice overexpressing bovine prion protein gene

M2B cells with persistent classical bovine spongiform encephalopathy (C-BSE) have been established previously. In this study, we performed strain characterization of the M2B cell line in bovine PrP^C overexpressing mice (Tg 1896). Mice intracranially inoculated with M2B cells and C-BSE survived for 451 ± 7 and 465 ± 31 d post inoculation, respectively. Although biochemical properties, including deglycosylation and conformational stability, differed between M2B cells and C-BSE, inoculation with M2B cell lysate and C-BSE resulted in comparable phenotypes. Comparable vacuolation scores and PrP^Sc depositions were observed in the brain of Tg 1896 inoculated with both M2B cell lysate and C-BSE. Our results show that biochemical and biological characteristics of M2B cells and C-BSE are classifiable in the same strain.     

Effect of bovine PRNP gene polymorphisms on BSE susceptibility in cattle

Prion protein gene (PRNP) variants determine the susceptibility of humans, sheep and mice to prion diseases, whereas polymorphisms in the open reading frame (ORF) of bovine PRNP seem to be unrelated to the incidence of bovine spongiform encephalopathy (BSE). According to the latest reports, the genetic susceptibility of cattle to BSE is associated with polymorphisms ofthe regulatory region of the PRNP gene and the level ofits expression. This review provides information on the bovine PRNP gene, its polymorphism, and recently identified genetic markers for BSE, and attempts to explain the mechanism behind the genetic resistance or susceptibility of cattle to this disease.     

Evaluation of the human transmission risk of an atypical bovine spongiform encephalopathy prion strain

Bovine spongiform encephalopathy (BSE), the prion disease in cattle, was widely believed to be caused by only one strain, BSE-C. BSE-C causes the fatal prion disease named new variant Creutzfeldt-Jacob disease in humans. Two atypical BSE strains, bovine amyloidotic spongiform encephalopathy (BASE, also named BSE-L) and BSE-H, have been discovered in several countries since 2004; their transmissibility and phenotypes in humans are unknown. We investigated the infectivity and human phenotype of BASE strains by inoculating transgenic (Tg) mice expressing the human prion protein with brain homogenates from two BASE strain-infected cattle. Sixty percent of the inoculated Tg mice became infected after 20 to 22 months of incubation, a transmission rate higher than those reported for BSE-C. A quarter of BASE strain-infected Tg mice, but none of the Tg mice infected with prions causing a sporadic human prion disease, showed the presence of pathogenic prion protein isoforms in the spleen, indicating that the BASE prion is intrinsically lymphotropic. The pathological prion protein isoforms in BASE strain-infected humanized Tg mouse brains are different from those from the original cattle BASE or sporadic human prion disease. Minimal brain spongiosis and long incubation times are observed for the BASE strain-infected Tg mice. These results suggest that in humans, the BASE strain is a more virulent BSE strain and likely lymphotropic.     

Evaluation of rapid post-mortem test kits for bovine spongiform encephalopathy (BSE) screening in Japan: Their analytical sensitivity to atypical BSE prions

A classical type of bovine spongiform encephalopathy (C-BSE), recognized in 1987, had a large impact on public health due to its zoonotic link to variant Creutzfeldt-Jakob disease by the human consumption of dietary products contaminated with the C-BSE prion. Thus, a number of countries implemented BSE surveillance using rapid post-mortem test kits that were approved for detection of the C-BSE prion in the cattle brain. However, as atypical BSE (L- and H-BSE) cases emerged in subsequent years, the efficacy of the kits for the detection of atypical BSE prions became a matter of concern. In response to this, laboratories in the European Union and Canada evaluated the kits used in their countries. Here, we carried out an evaluation study of NippiBL®, a kit currently used for BSE screening in Japan. By applying the kit to cattle brains of field cases of C-BSE and L-BSE, and an experimental case of H-BSE, we showed its comparable sensitivities to C, L-, and H-BSE prions, and satisfactory performance required by the European Food Safety Authority. In addition to NippiBL®, two kits (TeSeE® and FRELISA®) formerly used in Japan were effective for detection of the L-BSE prion, although the two kits were unable to be tested for the H-BSE prion due to the discontinuation of domestic sales during this study. These results indicate that BSE screening in Japan is as effective as those in other countries, and it is unlikely that cases of atypical BSE have been overlooked.     

Establishment of bovine prion peptide-based monoclonal antibodies for identifying bovine prion

To obtain high titer monoclonal antibodies (McAbs) which can react with mammalian prion protein (PrP), Balb/C mice were immunized with bovine (Bo) PrP peptide (BoPrP 209—228 aa) coupled to keyhole limpet hemocyanin (KLH). The hybridoma cell lines secreting monoclonal antibodies against the pep-tide were established by cell fusion and cloning. The obtained McAbs were applied to detect recombi-nant human, bovine and hamster PrP, cellular prion protein (PrP^c) in normal bovine brain and patho-genic scrapie prion protein (PrP^Sc) accumulated in the medulla oblongata of bovine spongiform en-cephalopathy(BSE)specimen with Western blot and immunohistochemical detection, respectively. The current procedure might offer a simple, feasible method to raise high titer antibodies for studying bio-logical features of PrP in mammals, as well as detection of transmissible spongiform encephalopathy (TSE) and diagnosis of BSE, in particular.     

Thermodynamic characterization for the denatured state of bovine prion protein and the BSE Associated variant E211K

ABSTRACT

Propagation of transmissible spongiform encephalopathies involves the conversion of cellular prion protein, PrP^C, into a misfolded oligomeric form, PrP^Sc. The most common hereditary prion disease is a genetic form of Creutzfeldt-Jakob disease in humans, in which a mutation in the prion gene results in a glutamic acid to lysine substitution at position 200 (E200K) in PrP. In cattle, the analogous amino acid substitution is found at residue 211 (E211K) and has been associated with a case of bovine spongiform encephalopathy. Here, we have compared the secondary structure of E211K to that of wild type using circular dichroism and completed a thermodynamic analysis of the folding of recombinant wild type and E211K variants of the bovine prion protein. The secondary structure of the E211K variant was essentially indistinguishable from that of wild type. The thermodynamic stability of E211K substitution showed a slight destabilization relative to the wild type consistent with results reported for recombinant human prion protein and its mutant E200K. In addition, the E211K variant exhibits a similarly compact denatured state to that of wild type based upon similar m-value and change in heat capacity of unfolding for the proteins. Together these results indicate that residual structure in the denatured state of bPrP is present in both the wild type protein and BSE associated variant E211K. Given this observation, as well as folding similarities reported for other disease associated variants of PrP it is worth consideration that functional aspects of PrP conformation may play a role in the misfolding process.     

Association and haplotype analysis of purH gene with inosine monophosphate content in chickens

The current study was designed to investigate the effects of the purH gene on chicken muscle inosine monophosphate (IMP) content. Muscle IMP content was measured in five chicken breeds. Single nucleotide polymorphisms (SNPs) were detected by PCR-SSCP and DNA sequencing. Two SNPs were detected, A/T substitution at position 8023 in exon 9, and T/C substitution at position 17446 in exon 16. The results indicated that only T17446C polymorphism was associated with IMP content. The haplotype effect was higher than the single genotype effect. We tentatively conclude that purH gene is a candidate locus or linked to a major gene that affects muscle IMP content. Haplotypes are superior to single genotypes as potential molecular markers for meat quality traits in chicken.     

Association between mitochondrial DNA haplotype compatibility and increased efficiency of bovine intersubspecies cloning

Reconstructed embryos derived from intersubspecies somatic cell nuclear transfer(SCNT) have poorer developmental potential than those from intrasubspecies SCNT.Based on our previous study that Holstein dairy bovine(HD) mitochondrial DNA(mtDNA) haplotype compatibility between donor karyoplast and recipient cytoplast is crucial for SCNT embryo development,we performed intersubspecies SCNT using HD as donor karyoplast and Luxi yellow heifer(LY) as recipient cytoplast according to mtDNA haplotypes determined...     

Association between mitochondrial DNA haplotype compatibility and increased efficiency of bovine intersubspecies cloning

Reconstructed embryos derived from intersubspecies somatic cell nuclear transfer (SCNT) have poorer developmental potential than those from intrasubspecies SCNT. Based on our previous study that Holstein dairy bovine (HD) mitochondrial DNA (mtDNA) haplotype compatibility between donor karyoplast and recipient cytoplast is crucial for SCNT embryo development, we performed intersubspecies SCNT using HD as donor karyoplast and Luxi yellow heifer (LY) as recipient cytoplast according to mtDNA haplotypes determined by polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) analysis. The results demonstrated that intersubspecies mtDNA homotype SCNT embryos had higher pre- and post-implantation developmental competence than intrasubspecies mtDNA heterotype embryos as well as improved blastocyst reprogramming status, including normal H3K9 dimethylation pattern and promoter hypomethylation of pluripotent genes such as Oct4 and Sox2, suggesting that intersubspecies SCNT using LY oocytes maintains HD cloning efficiency and may reprogram HD nuclei to develop into a normal cloned animal ultimately. Our results indicated that karyoplast-cytoplast interactions and mtDNA haplotype compatibility may affect bovine intersubspecies SCNT efficiency. This study on bovine intersubspecies SCNT is valuable for understanding the mechanisms of mtDNA haplotype compatibility between karyoplast and cytoplast impacting the bovine SCNT efficiency, and provides an alternative and economic resource for HD cloning.     

Isolation and molecular characterization of rasfadin, a novel gene in the vicinity of the bovine prion gene

A novel gene, rasfadin (RASSF2) was identified close to the bovine prion gene, and its genomic structure was derived with a combination of exon trapping and RACE. The gene covers at least 28 kb and maps to the same chromosomal region as the prion gene in cattle, sheep, and human. The RASSF2 ORF is composed of 987 base pairs divided into nine exons and shows a high nucleotide (88%) and amino acid similarity (95%) with a previously described human cDNA, KIAA0168. The bovine 3′UTR region is significantly shorter than the human counterpart, but shares with it two highly conserved nucleotide blocks. The expression of the gene was investigated in brain, liver, and spleen. Alternative splicing yields a shorter product in the liver composed of only four exons. Computer analysis showed a highly significant similarity of the rasfadin protein with the Ras association (RalGDS/AF-6) domain family 2 and with the afadin family, respectively, for the longer brain/spleen and the shorter liver variants. Received: 26 June 2000 / Accepted: 8 September 2000     

Characterization of a 320-kb region containing the HEXA gene on bovine chromosome 10 and analysis of its association with BSE susceptibility

Bovine spongiform encephalopathy (BSE) belongs to a group of neurodegenerative diseases known as transmissible prion diseases. Recently, variants in the promoter region of the prion protein ( PRNP ) gene have been shown to have a considerable effect on the susceptibility to BSE. However, a previous genome scan revealed other putative BSE-susceptibility loci. Here, we analysed such a region on BTA10, which contains the functional candidate gene HEXA . Three hundred and twenty kilobases that, besides HEXA , also contain ARIH1 , BRUNOL6 and PARP6 were characterized and screened for polymorphisms. Genotyping of 38 SNPs in Holstein–Friesian animals from the UK (350 diseased and 270 controls) revealed two intronic SNPs that were associated with BSE incidence, with experiment-wise P -values of 3.5 × 10⁻³ and 7.7 × 10⁻³ respectively. Both SNPs were in strong linkage disequilibrium and the rare alleles had a protective effect. These alleles were contained in a haplotype dubbed 'UK-protective' that was significantly overrepresented in the controls with a permuted P -value of 2 × 10⁻³. An association study in German Holstein animals (73 diseased and 627 controls) revealed an opposite effect of the 'UK-protective' haplotype in this population, i.e. it was overrepresented in the diseased animals, although not significant after correction for multiple testing. These findings indicate a causal variant for BSE susceptibility on BTA10 in linkage disequilibrium with the markers studied. Candidate gene analyses of the surrounding region and additional association studies will help to clarify the origin of the protective effects and to identify causal variants for BSE susceptibility on BTA10.     

Association of a lipoprotein-like particle with bovine fetuin

Fetuin is a major protein of fetal bovine serum that exhibits heterogeneity and has been found to be associated with some biological active growth factors. Preliminary studies indicated that commercial fetuin preparations contain lipids. We investigated in detail the nature of lipids associated with fetuin by using ultracentrifugation and agarose gel chromatography followed by lipid analysis. Fetuin was associated with a variety of lipids, predominantly cholesterol, cholesteryl ester with smaller amounts of phospholipids, triglycerides, and free fatty acids. Adjustment of fetuin preparation for various densities followed by ultracentrifugation resulted in a fraction with a density 1.063-1.21 g/ml (1-2% of total protein) that contained the bulk of the lipids. This fraction eluted as a single peak upon high pressure liquid chromatography and agarose gel chromatography. Delipidation of the lipoprotein-like particle followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a major band in the range of fetuin itself. These studies suggest that a fraction of fetuin (or isoform) binds lipids forming a particle with floating characteristics similar to high density lipoproteins.     

Prion protein gene sequence of Canada's first non-imported case of bovine spongiform encephalopathy (BSE).

In May 2003, Canada became the 22nd country outside of the United Kingdom to report a case of bovine spongiform encephalopathy (BSE) in an animal not known to be imported from a country with cattle previously affected by this fatal, transmissible prion disease. Despite extensive testing of thousands of other animals that may have been exposed to contaminated feed at the same time as the affected animal, no evidence has been found for other infections. This finding leaves room for conjectures that the single confirmed case arose spontaneously, perhaps (by analogy with human Creutzfeldt-Jakob disease) as a result of a somatic protein misfolding event or a novel germline mutation. Here we present DNA sequence data from the affected animal's prion protein coding sequence that argue definitively against the latter hypothesis.