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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 serially diluted cervid and hamster brain samples.Brain samples were serially diluted 10-3 to 10-6 or 10-3 to 10-10 for cervids (A) and hamsters (B), respectively, and analyzed in RT-QuIC for 60 hours. A ThT fluorescence level above threshold determined positivity. Both cervid and hamster brains from positively inoculated animals demonstrated positivity in all dilutions, while all brain dilutions from naïve animals remained negative.
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pone-0080203-g008: RT-QuIC analysis of serially diluted cervid and hamster brain samples.Brain samples were serially diluted 10-3 to 10-6 or 10-3 to 10-10 for cervids (A) and hamsters (B), respectively, and analyzed in RT-QuIC for 60 hours. A ThT fluorescence level above threshold determined positivity. Both cervid and hamster brains from positively inoculated animals demonstrated positivity in all dilutions, while all brain dilutions from naïve animals remained negative.

Mentions: Using bioassay in cervidized transgenic mice and the Reed-Muench method, the LD50 titer for 1 ml of 10% CWD-positive brain homogenate was determined to be 104.664, or 4.62x104 units/ml (calculated from values in Table 3). End point dilution analysis revealed a failure to cause disease in dilutions greater than 10-5. Serial dilutions of 10% homogenate CWD-positive brain homogenates in RT-QuIC demonstrated consistent positivity to a dilution of 10-6, with 50% converting activity detected in the 10-7 dilution (Figure 8A). SD50 titer for the RT-QuIC assay was calculated for 1 ml of CWD-positive brain and was determined to be 109.544, or 3.5x109 units/ml. These results indicate that the sensitivity of RT-QuIC for CWD detection is greater than animal bioassay.


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 serially diluted cervid and hamster brain samples.Brain samples were serially diluted 10-3 to 10-6 or 10-3 to 10-10 for cervids (A) and hamsters (B), respectively, and analyzed in RT-QuIC for 60 hours. A ThT fluorescence level above threshold determined positivity. Both cervid and hamster brains from positively inoculated animals demonstrated positivity in all dilutions, while all brain dilutions from naïve animals remained negative.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0080203-g008: RT-QuIC analysis of serially diluted cervid and hamster brain samples.Brain samples were serially diluted 10-3 to 10-6 or 10-3 to 10-10 for cervids (A) and hamsters (B), respectively, and analyzed in RT-QuIC for 60 hours. A ThT fluorescence level above threshold determined positivity. Both cervid and hamster brains from positively inoculated animals demonstrated positivity in all dilutions, while all brain dilutions from naïve animals remained negative.
Mentions: Using bioassay in cervidized transgenic mice and the Reed-Muench method, the LD50 titer for 1 ml of 10% CWD-positive brain homogenate was determined to be 104.664, or 4.62x104 units/ml (calculated from values in Table 3). End point dilution analysis revealed a failure to cause disease in dilutions greater than 10-5. Serial dilutions of 10% homogenate CWD-positive brain homogenates in RT-QuIC demonstrated consistent positivity to a dilution of 10-6, with 50% converting activity detected in the 10-7 dilution (Figure 8A). SD50 titer for the RT-QuIC assay was calculated for 1 ml of CWD-positive brain and was determined to be 109.544, or 3.5x109 units/ml. These results indicate that the sensitivity of RT-QuIC for CWD detection is greater than animal bioassay.

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