Development of a sensitive real-time quaking-induced conversion (RT-QuIC) assay for application in prion-infected blood.

Efforts to prevent human-to-human transmission of variant Creutzfeldt-Jakob disease (vCJD) by contaminated blood would be aided by the development of a sensitive diagnostic test that could be routinely used to screen blood donations. As blood samples from vCJD patients are extremely rare, here we describe the optimisation of real-time quaking-induced conversion (RT-QuIC) for detection of PrPSc (misfolded prion protein, a marker of prion infection) in blood samples from an established large animal model of vCJD, sheep experimentally infected with bovine spongiform encephalopathy (BSE). Comparative endpoint titration experiments with RT-QuIC, miniaturized bead protein misfolding cyclic amplification (mb-PMCA) and intracerebral inoculation of a transgenic mouse line expressing sheep PrP (tgOvARQ), demonstrated highly sensitive detection of PrPSc by RT-QuIC in a reference sheep brain homogenate. Upon addition of a capture step with iron oxide beads, the RT-QuIC assay was able to detect PrPSc in whole blood samples from BSE-infected sheep up to two years before disease onset. Both RT-QuIC and mb-PMCA also demonstrated sensitive detection of PrPSc in a reference vCJD-infected human brain homogenate, suggesting that either assay may be suitable for application to human blood samples. Our results support the further development and evaluation of RT-QuIC as a diagnostic or screening test for vCJD.

Subclinical infection occurs frequently following low dose exposure to prions by blood transfusion.

Infectious prion diseases have very long incubation periods, and the role that subclinical infections play in transmission, persistence and re-emergence of these diseases is unclear. In this study, we used a well-established model of vCJD (sheep experimentally infected with bovine spongiform encephalopathy, BSE) to determine the prevalence of subclinical infection following exposure by blood transfusion from infected donors. Many recipient sheep survived for years post-transfusion with no clinical signs and no disease-associated PrP (PrPSc) found in post mortem tissue samples by conventional tests. Using a sensitive protein misfolding cyclic amplification assay (PMCA), we found that the majority of these sheep had detectable PrPSc in lymph node samples, at levels approximately 105-106 times lower than in equivalent samples from clinically positive sheep. Further testing revealed the presence of PrPSc in other tissues, including brain, but not in blood samples. The results demonstrate that subclinical infection is a frequent outcome of low dose prion infection by a clinically relevant route for humans (blood transfusion). The long term persistence of low levels of infection has important implications for prion disease control and the risks of re-emergent infections in both humans and animals.

BSE can propagate in sheep co-infected or pre-infected with scrapie.

To understand the possible role of mixed-prion infections in disease presentation, the current study reports the co-infection of sheep with bovine spongiform encephalopathy (BSE) and scrapie. The bovine BSE agent was inoculated subcutaneously into sheep with ARQ/ARQ or VRQ/ARQ PRNP genotypes either at the same time as subcutaneous challenge with scrapie, or three months later. In addition, VRQ/VRQ sheep naturally infected with scrapie after being born into a scrapie-affected flock were challenged subcutaneously with BSE at eight or twenty one months-of-age. Sheep were analysed by incubation period/attack rate, and western blot of brain tissue determined the presence of BSE or scrapie-like PrPSc. Serial protein misfolding cyclic amplification (sPMCA) that can detect very low levels of BSE in the presence of an excess of scrapie agent was also applied to brain and lymphoreticular tissue. For VRQ/ARQ sheep challenged with mixed infections, scrapie-like incubation periods were produced, and no BSE agent was detected. However, whilst ARQ/ARQ sheep developed disease with BSE-like incubation periods, some animals had a dominant scrapie western blot phenotype in brain, but BSE was detected in these sheep by sPMCA. In addition, VRQ/VRQ animals challenged with BSE after natural exposure to scrapie had scrapie-like incubation periods and dominant scrapie PrPSc in brain, but one sheep had BSE detectable by sPMCA in the brain. Overall, the study demonstrates for the first time that for scrapie/BSE mixed infections, VRQ/ARQ sheep with experimental scrapie did not propagate BSE but VRQ/VRQ sheep with natural scrapie could propagate low levels of BSE, and whilst BSE readily propagated in ARQ/ARQ sheep it was not always the dominant PrPSc strain in brain tissue. Indeed, for several animals, a dominant scrapie biochemical phenotype in brain did not preclude the presence of BSE prion.

Preclinical transmission of prions by blood transfusion is influenced by donor genotype and route of infection.

Variant Creutzfeldt-Jakob disease (vCJD) is a human prion disease resulting from zoonotic transmission of bovine spongiform encephalopathy (BSE). Documented cases of vCJD transmission by blood transfusion necessitate on-going risk reduction measures to protect blood supplies, such as leucodepletion (removal of white blood cells, WBCs). This study set out to determine the risks of prion transmission by transfusion of labile blood components (red blood cells, platelets, plasma) commonly used in human medicine, and the effectiveness of leucodepletion in preventing infection, using BSE-infected sheep as a model. All components were capable of transmitting prion disease when donors were in the preclinical phase of infection, with the highest rates of infection in recipients of whole blood and buffy coat, and the lowest in recipients of plasma. Leucodepletion of components (<106 WBCs/unit) resulted in significantly lower transmission rates, but did not completely prevent transmission by any component. Donor PRNP genotype at codon 141, which is associated with variation in incubation period, also had a significant effect on transfusion transmission rates. A sensitive protein misfolding cyclic amplification (PMCA) assay, applied to longitudinal series of blood samples, identified infected sheep from 4 months post infection. However, in donor sheep (orally infected), the onset of detection of PrPSc in blood was much more variable, and generally later, compared to recipients (intravenous infection). This shows that the route and method of infection may profoundly affect the period during which an individual is infectious, and the test sensitivity required for reliable preclinical diagnosis, both of which have important implications for disease control. Our results emphasize that blood transfusion can be a highly efficient route of transmission for prion diseases. Given current uncertainties over the prevalence of asymptomatic vCJD carriers, this argues for the maintenance and improvement of current measures to reduce the risk of transmission by blood products.

Neuropathology of Aging in Cats and its Similarities to Human Alzheimer's Disease.

Elderly cats develop age-related behavioral and neuropathological changes that ultimately lead to cognitive dysfunction syndrome (CDS). These neuropathologies share similarities to those seen in the brains of humans with Alzheimer's disease (AD), including the extracellular accumulation of ß-amyloid (Aß) and intraneuronal deposits of hyperphosphorylated tau, which are considered to be the two major hallmarks of AD. The present study assessed the presence and distribution of Aß and tau hyperphosphorylation within the cat brain (n = 55 cats), and how the distribution of these proteins changes with age and the presence of CDS. For this, immunohistochemistry was performed on seven brain regions from cats of various ages, with and without CDS (n = 10 with CDS). Cats accumulate both intracytoplasmic and extracellular deposits of Aß, as well as intranuclear and intracytoplasmic hyperphosphorylated tau deposits. Large extracellular aggregates of Aß were found in elderly cats, mainly in the cortical brain areas, with occasional hippocampal aggregates. This may suggest that these aggregates start in cortical areas and later progress to the hippocampus. While Aß senile plaques in people with AD have a dense core, extracellular Aß deposits in cats exhibited a diffuse pattern, similar to the early stages of plaque pathogenesis. Intraneuronal Aß deposits were also observed, occurring predominantly in cortical brain regions of younger cats, while older cats had few to no intraneuronal Aß deposits, especially when extracellular aggregates were abundant. Intracytoplasmic hyperphosphorylated tau was found within neurons in the brains of elderly cats, particularly in those with CDS. Due to their ultrastructural features, these deposits are considered to be pre-tangles, which are an early stage of the neurofibrillary tangles seen in AD. The largest numbers of pre-tangles are found mainly in the cerebral cortex of elderly cats, whereas lower numbers were found in other regions (i.e., entorhinal cortex and hippocampus). For the first time, intranuclear tau was found in both phosphorylated and non-phosphorylated states within neurons in the cat brain. The highest numbers of intranuclear deposits were found in the cortex of younger cats, and this tended to decrease with age. In contrast, elderly cats with pre-tangles had only occasional or no nuclear labelling.

A prion reduction filter does not completely remove endogenous prion infectivity from sheep blood.

BACKGROUND: Variant Creutzfeldt-Jakob disease (vCJD) is a transmissible spongiform encephalopathy affecting humans, acquired initially through infection with bovine spongiform encephalopathy (BSE). A small number of vCJD cases have been acquired through the transfusion of blood from asymptomatic donors who subsequently developed vCJD. Filter devices that selectively bind the infectious agent associated with prion disease have been developed for removal of infection from blood. This study independently assessed one such filter, the P-CAPT filter, for efficacy in removing infectivity associated with the BSE agent in sheep blood. The sheep BSE model has previously been used to evaluate the distribution of infectivity in clinically relevant blood components. This is the first study to assess the ability of the P-CAPT filter to remove endogenous infectivity associated with blood components prepared from a large animal model. STUDY DESIGN AND METHODS: Paired units of leukoreduced red blood cells (LR-RBCs) were prepared from donors at the clinical stage of infection and confirmed as having BSE. One cohort of recipients was transfused with LR-RBCs alone, whereas a parallel cohort received LR and P-CAPT-filtered RBCs (LR-RBCs-P-CAPT). RESULTS: Of 14 recipients, two have been confirmed as having BSE. These sheep had received LR-RBCs and LR-RBCs-P-CAPT from the same donor. CONCLUSIONS: The results indicate that, after leukoreduction and P-CAPT filtration, there can still be sufficient residual infectivity in sheep RBCs to transmit infection when transfused into a susceptible recipient.

Prion protein-specific antibodies that detect multiple TSE agents with high sensitivity.

This paper describes the generation, characterisation and potential applications of a panel of novel anti-prion protein monoclonal antibodies (mAbs). The mAbs were generated by immunising PRNP null mice, using a variety of regimes, with a truncated form of recombinant ovine prion protein spanning residues 94-233. Epitopes of specific antibodies were mapped using solid-phase Pepscan analysis and clustered to four distinct regions within the PrP molecule. We have demonstrated the utility of these antibodies by use of Western blotting and immunohistochemistry in tissues from a range of different species affected by transmissible spongiform encephalopathy (TSE). In comparative tests against extensively-used and widely-published, commercially available antibodies, similar or improved results can be obtained using these new mAbs, specifically in terms of sensitivity of detection. Since many of these antibodies recognise native PrPC, they could also be applied to a broad range of immunoassays such as flow cytometry, DELFIA analysis or immunoprecipitation. We are using these reagents to increase our understanding of TSE pathogenesis and for use in potential diagnostic screening assays.

Significant differences in incubation times in sheep infected with bovine spongiform encephalopathy result from variation at codon 141 in the PRNP gene.

The susceptibility of sheep to prion infection is linked to variation in the PRNP gene, which encodes the prion protein. Common polymorphisms occur at codons 136, 154 and 171. Sheep which are homozygous for the A(136)R(154)Q(171) allele are the most susceptible to bovine spongiform encephalopathy (BSE). The effect of other polymorphisms on BSE susceptibility is unknown. We orally infected ARQ/ARQ Cheviot sheep with equal amounts of BSE brain homogenate and a range of incubation periods was observed. When we segregated sheep according to the amino acid (L or F) encoded at codon 141 of the PRNP gene, the shortest incubation period was observed in LL(141) sheep, whilst incubation periods in FF(141) and LF(141) sheep were significantly longer. No statistically significant differences existed in the expression of total prion protein or the disease-associated isoform in BSE-infected sheep within each genotype subgroup. This suggested that the amino acid encoded at codon 141 probably affects incubation times through direct effects on protein misfolding rates.

All clinically-relevant blood components transmit prion disease following a single blood transfusion: a sheep model of vCJD.

Variant CJD (vCJD) is an incurable, infectious human disease, likely arising from the consumption of BSE-contaminated meat products. Whilst the epidemic appears to be waning, there is much concern that vCJD infection may be perpetuated in humans by the transfusion of contaminated blood products. Since 2004, several cases of transfusion-associated vCJD transmission have been reported and linked to blood collected from pre-clinically affected donors. Using an animal model in which the disease manifested resembles that of humans affected with vCJD, we examined which blood components used in human medicine are likely to pose the greatest risk of transmitting vCJD via transfusion. We collected two full units of blood from BSE-infected donor animals during the pre-clinical phase of infection. Using methods employed by transfusion services we prepared red cell concentrates, plasma and platelets units (including leucoreduced equivalents). Following transfusion, we showed that all components contain sufficient levels of infectivity to cause disease following only a single transfusion and also that leucoreduction did not prevent disease transmission. These data suggest that all blood components are vectors for prion disease transmission, and highlight the importance of multiple control measures to minimise the risk of human to human transmission of vCJD by blood transfusion.

Infection of cattle with Theileria parva induces an early CD8 T cell response lacking appropriate effector function.

Theileria parva causes an acute lympho-proliferative disease in cattle, which can result in death of susceptible animals within 2-3 weeks of infection. Analyses of the cellular response in the lymph node draining the site of infection demonstrated an early T cell response, with the appearance of large numbers of uninfected lymphoblasts between 6 and 9 days p.i., coinciding with initial detection of parasitised cells. There was a marked increase in the representation of CD8(+) T cells and the emergence of a sizable sub-population of CD2(-) CD8(+) alpha/beta T cells during this period. Analysis of T cell receptor beta chain variable (TCR BV) gene expression did not reveal any evidence for the involvement of a superantigen in stimulating the response. Responding lymph node cells were found to produce increased quantities of IFNgamma and IL-10, and both the CD2(+) CD8(+) and CD2(-) CD8(+) populations expressed IFNgamma transcripts. Purified CD2(+) CD8(+) cells proliferated when stimulated in vitro with autologous parasitised cells or non-specific mitogens, whereas CD2(-) CD8(+) cells were refractory to these stimuli. In contrast to the parasite-specific cytotoxic activity associated with T cell responses in immune cattle, the responses to primary infection exhibited variable levels of non-specific cytotoxic activity. Stimulation of purified CD2(+) CD8(+) T cells in vitro with autologous parasitised cells also failed to reveal evidence of specific cytotoxic activity. These findings indicate that primary infection with T. parva induces an aberrant T cell response that lacks appropriate effector activity.

Comparative analysis of normal prion protein expression on human, rodent, and ruminant blood cells by using a panel of prion antibodies.

BACKGROUND: It is not known whether variant CJD can be transmitted within the human population by blood transfusion. The expression of normal cellular prion protein (PrPC) by different blood cell types may permit selective uptake and dissemination of infectivity. STUDY DESIGN AND METHODS: The normal distribution of PrPC on the major blood cell types of species known to be susceptible to natural or experimental transmissible spongiform encephalopathies was studied. Blood from healthy humans, mice, hamsters, cattle, and sheep was examined by flow cytometry by using a large panel of antibodies with different prion protein (PrP) epitope specificities to maximize the detection of PrP variants across species and cell type. RESULTS: PrP was detected on all major human blood cells types except eosinophils, but was not detected as ubiquitously or uniformly on major blood cell types of different animal species. CONCLUSION: Different animal species have unique patterns of expression of PrPC on blood cell types, with none equivalent to the human pattern. This needs to be considered when extrapolating from animal models of blood-borne transmissible spongiform encephalopathy infectivity, particularly in regard to the risk assessment of potential variant CJD spread within the human population. The relationship between PrP distribution and infectivity distribution in blood needs further investigation.