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In vitro detection of prionemia in TSE-infected cervids and hamsters.

Elder AM, Henderson DM, Nalls AV, Wilham JM, Caughey BW, Hoover EA, Kincaid AE, Bartz JC, Mathiason CK - PLoS ONE (2013)

Bottom Line: Thus, it is important to monitor cervid and human blood products to ensure herd health and human safety.Current methods for detecting blood-associated prions rely primarily upon bioassay in laboratory animals.Our results indicate that RT-QuIC methodology as modified can provide consistent and reliable detection of blood-borne prions in preclinical and symptomatic stages of two animal TSEs, offering promise for prionemia detection in other species, including humans.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, United States of America.

ABSTRACT
Blood-borne transmission of infectious prions during the symptomatic and asymptomatic stages of disease occurs for both human and animal transmissible spongiform encephalopathies (TSEs). The geographical distribution of the cervid TSE, chronic wasting disease (CWD), continues to spread across North America and the prospective number of individuals harboring an asymptomatic infection of human variant Creutzfeldt-Jakob Disease (vCJD) in the United Kingdom has been projected to be ~1 in 3000 residents. Thus, it is important to monitor cervid and human blood products to ensure herd health and human safety. Current methods for detecting blood-associated prions rely primarily upon bioassay in laboratory animals. While bioassay provides high sensitivity and specificity, it requires many months, animals, and it is costly. Here we report modification of the real time quaking-induced conversion (RT-QuIC) assay to detect blood-borne prions in whole blood from prion-infected preclinical white-tailed deer, muntjac deer, and Syrian hamsters, attaining sensitivity of >90% while maintaining 100% specificity. Our results indicate that RT-QuIC methodology as modified can provide consistent and reliable detection of blood-borne prions in preclinical and symptomatic stages of two animal TSEs, offering promise for prionemia detection in other species, including humans.

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Related in: MedlinePlus

RT-QuIC analysis of fresh versus frozen whole blood.Blood was collected from a CWD-infected and CWD-naïve white-tailed deer and aliquots were analyzed immediately (fresh) or frozen (-80C). Serial blood sample dilutions (neat to 10-6) were assayed by RT-QuIC in duplicate for 60 hours and ThT fluorescence level above threshold determined positivity. Detection of PrPC-converting activity for each replicate is shown for blood analyzed fresh (A) and frozen (B).
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pone-0080203-g002: RT-QuIC analysis of fresh versus frozen whole blood.Blood was collected from a CWD-infected and CWD-naïve white-tailed deer and aliquots were analyzed immediately (fresh) or frozen (-80C). Serial blood sample dilutions (neat to 10-6) were assayed by RT-QuIC in duplicate for 60 hours and ThT fluorescence level above threshold determined positivity. Detection of PrPC-converting activity for each replicate is shown for blood analyzed fresh (A) and frozen (B).

Mentions: In order to determine if historical blood samples were adequately preserved to initiate PrPC-converting activity in RT-QuIC, whole blood was collected from contemporary naïve and CWD-infected white-tailed deer and compared as fresh versus frozen samples. Samples were processed in various dilutions ranging from undiluted to 10-6 to determine the optimal dilution for PrPD detection using frozen whole blood in the RT-QuIC assay. While PrPC-converting activity was detected in fresh whole blood, blood that had been processed through the freeze-thaw procedure yielded higher and more consistent detection of PrPC-converting activity (2/2 replicates in the 10-3, 10-4 and 10-6 dilutions; 1/2 replicates in the 10-5 dilution) (Figure 2). PrPC-converting activity was not observed in wells containing only substrate or naïve cervid blood. To determine if the results observed in the anticoagulant study were due solely to the use of fresh blood, the experiments were repeated on frozen blood collected in all three anticoagulants. Results revealed identical outcomes for both CPDA and EDTA blood while showing an increased sensitivity in heparin, as described above (data not shown). All subsequent RT-QuIC analyses included heparin-preserved whole blood that had undergone four freeze-thaw cycles.


In vitro detection of prionemia in TSE-infected cervids and hamsters.

Elder AM, Henderson DM, Nalls AV, Wilham JM, Caughey BW, Hoover EA, Kincaid AE, Bartz JC, Mathiason CK - PLoS ONE (2013)

RT-QuIC analysis of fresh versus frozen whole blood.Blood was collected from a CWD-infected and CWD-naïve white-tailed deer and aliquots were analyzed immediately (fresh) or frozen (-80C). Serial blood sample dilutions (neat to 10-6) were assayed by RT-QuIC in duplicate for 60 hours and ThT fluorescence level above threshold determined positivity. Detection of PrPC-converting activity for each replicate is shown for blood analyzed fresh (A) and frozen (B).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815098&req=5

pone-0080203-g002: RT-QuIC analysis of fresh versus frozen whole blood.Blood was collected from a CWD-infected and CWD-naïve white-tailed deer and aliquots were analyzed immediately (fresh) or frozen (-80C). Serial blood sample dilutions (neat to 10-6) were assayed by RT-QuIC in duplicate for 60 hours and ThT fluorescence level above threshold determined positivity. Detection of PrPC-converting activity for each replicate is shown for blood analyzed fresh (A) and frozen (B).
Mentions: In order to determine if historical blood samples were adequately preserved to initiate PrPC-converting activity in RT-QuIC, whole blood was collected from contemporary naïve and CWD-infected white-tailed deer and compared as fresh versus frozen samples. Samples were processed in various dilutions ranging from undiluted to 10-6 to determine the optimal dilution for PrPD detection using frozen whole blood in the RT-QuIC assay. While PrPC-converting activity was detected in fresh whole blood, blood that had been processed through the freeze-thaw procedure yielded higher and more consistent detection of PrPC-converting activity (2/2 replicates in the 10-3, 10-4 and 10-6 dilutions; 1/2 replicates in the 10-5 dilution) (Figure 2). PrPC-converting activity was not observed in wells containing only substrate or naïve cervid blood. To determine if the results observed in the anticoagulant study were due solely to the use of fresh blood, the experiments were repeated on frozen blood collected in all three anticoagulants. Results revealed identical outcomes for both CPDA and EDTA blood while showing an increased sensitivity in heparin, as described above (data not shown). All subsequent RT-QuIC analyses included heparin-preserved whole blood that had undergone four freeze-thaw cycles.

Bottom Line: Thus, it is important to monitor cervid and human blood products to ensure herd health and human safety.Current methods for detecting blood-associated prions rely primarily upon bioassay in laboratory animals.Our results indicate that RT-QuIC methodology as modified can provide consistent and reliable detection of blood-borne prions in preclinical and symptomatic stages of two animal TSEs, offering promise for prionemia detection in other species, including humans.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, United States of America.

ABSTRACT
Blood-borne transmission of infectious prions during the symptomatic and asymptomatic stages of disease occurs for both human and animal transmissible spongiform encephalopathies (TSEs). The geographical distribution of the cervid TSE, chronic wasting disease (CWD), continues to spread across North America and the prospective number of individuals harboring an asymptomatic infection of human variant Creutzfeldt-Jakob Disease (vCJD) in the United Kingdom has been projected to be ~1 in 3000 residents. Thus, it is important to monitor cervid and human blood products to ensure herd health and human safety. Current methods for detecting blood-associated prions rely primarily upon bioassay in laboratory animals. While bioassay provides high sensitivity and specificity, it requires many months, animals, and it is costly. Here we report modification of the real time quaking-induced conversion (RT-QuIC) assay to detect blood-borne prions in whole blood from prion-infected preclinical white-tailed deer, muntjac deer, and Syrian hamsters, attaining sensitivity of >90% while maintaining 100% specificity. Our results indicate that RT-QuIC methodology as modified can provide consistent and reliable detection of blood-borne prions in preclinical and symptomatic stages of two animal TSEs, offering promise for prionemia detection in other species, including humans.

Show MeSH
Related in: MedlinePlus