Use of different RT-QuIC substrates for detecting CWD prions in the brain of Norwegian cervids

Chronic wasting disease (CWD) is a highly contagious prion disease affecting captive and free-ranging cervid populations. CWD has been detected in United States, Canada, South Korea and, most recently, in Europe (Norway, Finland and Sweden). Animals with CWD release infectious prions in the environment through saliva, urine and feces sustaining disease spreading between cervids but also potentially to other non-cervids ruminants (e.g. sheep, goats and cattle). In the light of these considerations and due to CWD unknown zoonotic potential, it is of utmost importance to follow specific surveillance programs useful to minimize disease spreading and transmission. The European community has already in place specific surveillance measures, but the traditional diagnostic tests performed on nervous or lymphoid tissues lack sensitivity. We have optimized a Real-Time Quaking-Induced Conversion (RT-QuIC) assay for detecting CWD prions with high sensitivity and specificity to try to overcome this problem. In this work, we show that bank vole prion protein (PrP) is an excellent substrate for RT-QuIC reactions, enabling the detection of trace-amounts of CWD prions, regardless of prion strain and cervid species. Beside supporting the traditional diagnostic tests, this technology could be exploited for detecting prions in peripheral tissues from live animals, possibly even at preclinical stages of the disease

Prion Protein Polymorphisms Affect Chronic Wasting Disease Progression

Analysis of the PRNP gene in cervids naturally infected with chronic wasting disease (CWD) suggested that PRNP polymorphisms affect the susceptibility of deer to infection. To test this effect, we orally inoculated 12 white-tailed deer with CWD agent. Three different PRNP alleles, wild-type (wt; glutamine at amino acid 95 and glycine at 96), Q95H (glutamine to histidine at amino acid position 95) and G96S (glycine to serine at position 96) were represented in the study cohort with 5 wt/wt, 3 wt/G96S, and 1 each wt/Q95H and Q95H/G96S. Two animals were lost to follow-up due to intercurrent disease. The inoculum was prepared from Wisconsin hunter-harvested homozygous wt/wt animals. All infected deer presented with clinical signs of CWD; the orally infected wt/wt had an average survival period of 693 days post inoculation (dpi) and G96S/wt deer had an average survival period of 956 dpi. The Q95H/wt and Q95H/G96S deer succumbed to CWD at 1,508 and 1,596 dpi respectively. These data show that polymorphisms in the PRNP gene affect CWD incubation period. Deer heterozygous for the PRNP alleles had extended incubation periods with the Q95H allele having the greatest effect

Gene by environment QTL mapping through multiple trait analyses in blood pressure salt-sensitivity: identification of a novel QTL in rat chromosome 5

BACKGROUND: The genetic mechanisms underlying interindividual blood pressure variation reflect the complex interplay of both genetic and environmental variables. The current standard statistical methods for detecting genes involved in the regulation mechanisms of complex traits are based on univariate analysis. Few studies have focused on the search for and understanding of quantitative trait loci responsible for gene × environmental interactions or multiple trait analysis. Composite interval mapping has been extended to multiple traits and may be an interesting approach to such a problem. METHODS: We used multiple-trait analysis for quantitative trait locus mapping of loci having different effects on systolic blood pressure with NaCl exposure. Animals studied were 188 rats, the progenies of an F2 rat intercross between the hypertensive and normotensive strain, genotyped in 179 polymorphic markers across the rat genome. To accommodate the correlational structure from measurements taken in the same animals, we applied univariate and multivariate strategies for analyzing the data. RESULTS: We detected a new quantitative train locus on a region close to marker R589 in chromosome 5 of the rat genome, not previously identified through serial analysis of individual traits. In addition, we were able to justify analytically the parametric restrictions in terms of regression coefficients responsible for the gain in precision with the adopted analytical approach. CONCLUSION: Future work should focus on fine mapping and the identification of the causative variant responsible for this quantitative trait locus signal. The multivariable strategy might be valuable in the study of genetic determinants of interindividual variation of antihypertensive drug effectiveness

Quantitative trait locus analysis of hybrid pedigrees: variance-components model, inbreeding parameter, and power

<p>Abstract</p> <p>Background</p> <p>For the last years reliable mapping of quantitative trait loci (QTLs) has become feasible through linkage analysis based on the variance-components method. There are now many approaches to the QTL analysis of various types of crosses within one population (breed) as well as crosses between divergent populations (breeds). However, to analyse a complex pedigree with dominance and inbreeding, when the pedigree's founders have an inter-population (hybrid) origin, it is necessary to develop a high-powered method taking into account these features of the pedigree.</p> <p>Results</p> <p>We offer a universal approach to QTL analysis of complex pedigrees descended from crosses between outbred parental lines with different QTL allele frequencies. This approach improves the established variance-components method due to the consideration of the genetic effect conditioned by inter-population origin and inbreeding of individuals. To estimate model parameters, namely additive and dominant effects, and the allelic frequencies of the QTL analysed, and also to define the QTL positions on a chromosome with respect to genotyped markers, we used the maximum-likelihood method. To detect linkage between the QTL and the markers we propose statistics with a non-central χ²-distribution that provides the possibility to deduce analytical expressions for the power of the method and therefore, to estimate the pedigree's size required for 80% power. The method works for arbitrarily structured pedigrees with dominance and inbreeding.</p> <p>Conclusion</p> <p>Our method uses the phenotypic values and the marker information for each individual of the pedigree under observation as initial data and can be valuable for fine mapping purposes. The power of the method is increased if the QTL effects conditioned by inter-population origin and inbreeding are enhanced. Several improvements can be developed to take into account fixed factors affecting trait formation, such as age and sex.</p

Detection of Prion Protein in Urine-Derived Injectable Fertility Products by a Targeted Proteomic Approach

BACKGROUND: Iatrogenic transmission of human prion disease can occur through medical or surgical procedures, including injection of hormones such as gonadotropins extracted from cadaver pituitaries. Annually, more than 300,000 women in the United States and Canada are prescribed urine-derived gonadotropins for infertility. Although menopausal urine donors are screened for symptomatic neurological disease, incubation of Creutzfeldt-Jakob disease (CJD) is impossible to exclude by non-invasive testing. Risk of carrier status of variant CJD (vCJD), a disease associated with decades-long peripheral incubation, is estimated to be on the order of 100 per million population in the United Kingdom. Studies showing infectious prions in the urine of experimental animals with and without renal disease suggest that prions could be present in asymptomatic urine donors. Several human fertility products are derived from donated urine; recently prion protein has been detected in preparations of human menopausal gonadotropin (hMG). METHODOLOGY/PRINCIPAL FINDINGS: Using a classical proteomic approach, 33 and 34 non-gonadotropin proteins were identified in urinary human chorionic gonadotropin (u-hCG) and highly-purified urinary human menopausal gonadotropin (hMG-HP) products, respectively. Prion protein was identified as a major contaminant in u-hCG preparations for the first time. An advanced prion protein targeted proteomic approach was subsequently used to conduct a survey of gonadotropin products; this approach detected human prion protein peptides in urine-derived injectable fertility products containing hCG, hMG and hMG-HP, but not in recombinant products. CONCLUSIONS/SIGNIFICANCE: The presence of protease-sensitive prion protein in urinary-derived injectable fertility products containing hCG, hMG, and hMG-HP suggests that prions may co-purify in these products. Intramuscular injection is a relatively efficient route of transmission of human prion disease, and young women exposed to prions can be expected to survive an incubation period associated with a minimal inoculum. The risks of urine-derived fertility products could now outweigh their benefits, particularly considering the availability of recombinant products

Cigarette smoking, health status, socio-economic status and access to health care in diabetes mellitus: a cross-sectional survey

BACKGROUND: In diabetes mellitus, cigarette smoking is associated with increased risk of cardiovascular mortality and microvascular complications. We evaluated cigarette smoking in people with diabetes mellitus in a socio-economically deprived area. METHODS: We carried out a cross-sectional survey of people registered with diabetes mellitus at 29 general practices in inner London. Responses were analysed for 1,899 (64%) respondents out of 2,983 eligible. RESULTS: There were 1,899 respondents of whom 968 (51%) had never smoked, 296 (16%) were current smokers and 582 (31%) were ex-smokers. Smoking was more frequent in white Europeans (men 22%, women 20%), than in African Caribbeans (men 15%, women 10%) or Africans (men 8%, women 2%). Smoking prevalence decreased with age. Smokers were more likely to be living in rented accommodation (odds ratio, OR 2.02, 95% confidence interval 1.48 to 2.74). After adjusting for confounding, current smokers had lower SF-36 scores than subjects who had never smoked (mean difference in physical functioning score -5.6, 95% confidence interval -10.0 to -1.2; general health -6.1, -9.7 to -2.5). Current smokers were less likely to have attended a hospital diabetic clinic in the last year (OR 0.59, 0.44 to 0.79), and their hypertension was less likely to be treated (OR 0.47, 0.30 to 0.74). CONCLUSIONS: Compared with non-smokers, smokers had lower socio-economic status and worse health status, but were less likely to be referred to hospital or treated for their hypertension. People with diabetes who smoke can be regarded as a vulnerable group who need more intensive support and treatment

Modeling Routes of Chronic Wasting Disease Transmission: Environmental Prion Persistence Promotes Deer Population Decline and Extinction

Chronic wasting disease (CWD) is a fatal disease of deer, elk, and moose transmitted through direct, animal-to-animal contact, and indirectly, via environmental contamination. Considerable attention has been paid to modeling direct transmission, but despite the fact that CWD prions can remain infectious in the environment for years, relatively little information exists about the potential effects of indirect transmission on CWD dynamics. In the present study, we use simulation models to demonstrate how indirect transmission and the duration of environmental prion persistence may affect epidemics of CWD and populations of North American deer. Existing data from Colorado, Wyoming, and Wisconsin's CWD epidemics were used to define plausible short-term outcomes and associated parameter spaces. Resulting long-term outcomes range from relatively low disease prevalence and limited host-population decline to host-population collapse and extinction. Our models suggest that disease prevalence and the severity of population decline is driven by the duration that prions remain infectious in the environment. Despite relatively low epidemic growth rates, the basic reproductive number, R0, may be much larger than expected under the direct-transmission paradigm because the infectious period can vastly exceed the host's life span. High prion persistence is expected to lead to an increasing environmental pool of prions during the early phases (i.e. approximately during the first 50 years) of the epidemic. As a consequence, over this period of time, disease dynamics will become more heavily influenced by indirect transmission, which may explain some of the observed regional differences in age and sex-specific disease patterns. This suggests management interventions, such as culling or vaccination, will become increasingly less effective as CWD epidemics progress

Detection of Prion Protein in Urine-Derived Injectable Fertility Products by a Targeted Proteomic Approach

BACKGROUND: Iatrogenic transmission of human prion disease can occur through medical or surgical procedures, including injection of hormones such as gonadotropins extracted from cadaver pituitaries. Annually, more than 300,000 women in the United States and Canada are prescribed urine-derived gonadotropins for infertility. Although menopausal urine donors are screened for symptomatic neurological disease, incubation of Creutzfeldt-Jakob disease (CJD) is impossible to exclude by non-invasive testing. Risk of carrier status of variant CJD (vCJD), a disease associated with decades-long peripheral incubation, is estimated to be on the order of 100 per million population in the United Kingdom. Studies showing infectious prions in the urine of experimental animals with and without renal disease suggest that prions could be present in asymptomatic urine donors. Several human fertility products are derived from donated urine; recently prion protein has been detected in preparations of human menopausal gonadotropin (hMG). METHODOLOGY/PRINCIPAL FINDINGS: Using a classical proteomic approach, 33 and 34 non-gonadotropin proteins were identified in urinary human chorionic gonadotropin (u-hCG) and highly-purified urinary human menopausal gonadotropin (hMG-HP) products, respectively. Prion protein was identified as a major contaminant in u-hCG preparations for the first time. An advanced prion protein targeted proteomic approach was subsequently used to conduct a survey of gonadotropin products; this approach detected human prion protein peptides in urine-derived injectable fertility products containing hCG, hMG and hMG-HP, but not in recombinant products. CONCLUSIONS/SIGNIFICANCE: The presence of protease-sensitive prion protein in urinary-derived injectable fertility products containing hCG, hMG, and hMG-HP suggests that prions may co-purify in these products. Intramuscular injection is a relatively efficient route of transmission of human prion disease, and young women exposed to prions can be expected to survive an incubation period associated with a minimal inoculum. The risks of urine-derived fertility products could now outweigh their benefits, particularly considering the availability of recombinant products

Bayesian shrinkage mapping of quantitative trait loci in variance component models

<p>Abstract</p> <p>Background</p> <p>In this article, I propose a model-selection-free method to map multiple quantitative trait loci (QTL) in variance component model, which is useful in outbred populations. The new method can estimate the variance of zero-effect QTL infinitely to zero, but nearly unbiased for non-zero-effect QTL. It is analogous to Xu's Bayesian shrinkage estimation method, but his method is based on allelic substitution model, while the new method is based on the variance component models.</p> <p>Results</p> <p>Extensive simulation experiments were conducted to investigate the performance of the proposed method. The results showed that the proposed method was efficient in mapping multiple QTL simultaneously, and moreover it was more competitive than the reversible jump MCMC (RJMCMC) method and may even out-perform it.</p> <p>Conclusions</p> <p>The newly developed Bayesian shrinkage method is very efficient and powerful for mapping multiple QTL in outbred populations.</p

Highly Efficient Amplification of Chronic Wasting Disease Agent by Protein Misfolding Cyclic Amplification with Beads (PMCAb)

Protein misfolding cyclic amplification (PMCA) has emerged as an important technique for detecting low levels of pathogenic prion protein in biological samples. The method exploits the ability of the pathogenic prion protein to convert the normal prion protein to a proteinase K-resistant conformation. Inclusion of Teflon® beads in the PMCA reaction (PMCAb) has been previously shown to increase the sensitivity and robustness of detection for the 263 K and SSLOW strains of hamster-adapted prions. Here, we demonstrate that PMCAb with saponin dramatically increases the sensitivity of detection for chronic wasting disease (CWD) agent without compromising the specificity of the assay (i.e., no false positive results). Addition of Teflon® beads increased the robustness of the PMCA reaction, resulting in a decrease in the variability of PMCA results. Three rounds of serial PMCAb allowed detection of CWD agent from a 6.7×10−13 dilution of 10% brain homogenate (1.3 fg of source brain). Titration of the same brain homogenate in transgenic mice expressing cervid prion protein (Tg(CerPrP)1536+/− mice) allowed detection of CWD agent from the 10−6 dilution of 10% brain homogenate. PMCAb is, thus, more sensitive than bioassay in transgenic mice by a factor exceeding 105. Additionally, we are able to amplify CWD agent from brain tissue and lymph nodes of CWD-positive white-tailed deer having Prnp alleles associated with reduced disease susceptibility