Detection by real-time quaking-induced conversion (RT-QuIC), ELISA, and IHC of chronic wasting disease prion in lymph nodes from Pennsylvania white-tailed deer with specific PRNP genotypes

Abstract

Chronic wasting disease (CWD) is a prion-mediated, transmissible disease of cervids, including deer ( Odocoileus spp.), which is characterized by spongiform encephalopathy and death of the prion-infected animals. Official surveillance in the United States using immunohistochemistry (IHC) and ELISA entails the laborious collection of lymphoid and/or brainstem tissue after death. New, highly sensitive prion detection methods, such as real-time quaking-induced conversion (RT-QuIC), have shown promise in detecting abnormal prions from both antemortem and postmortem specimens. We compared RT-QuIC with ELISA and IHC for CWD detection utilizing deer retropharyngeal lymph node (RLN) tissues in a diagnostic laboratory setting. The RLNs were collected postmortem from hunter-harvested animals. RT-QuIC showed 100% sensitivity and specificity for 50 deer RLN (35 positive by both IHC and ELISA, 15 negative) included in our study. All deer were also genotyped for PRNP polymorphism. Most deer were homozygous at codons 95, 96, 116, and 226 (QQ/GG/AA/QQ genotype, with frequency 0.86), which are the codons implicated in disease susceptibility. Heterozygosity was noticed in Pennsylvania deer, albeit at a very low frequency, for codons 95GS (0.06) and 96QH (0.08), but deer with these genotypes were still found to be CWD prion-infected.

Evaluation of Real-Time Quaking-Induced Conversion, ELISA, and Immunohistochemistry for Chronic Wasting Disease Diagnosis

“…We found that our RT-QuIC results were comparable to those of the IHC and ELISA, with only 0.5% (7 samples out of 1,300) of discordant results between the three techniques when testing WTD RLNs ( Table 2 ). In previous studies that compared the three assays, 100% of matching results were observed ( 23 , 30 ). One possible explanation for the observed difference is the fact that the previous studies did not include as many positive WTD RLNs.…”

“…The development of in vitro amplification assays, as protein misfolding cyclic amplification and RT-QuIC have helped to enhance the sensitivity of CWD prion detection in the last few years. The RT-QuIC assay has been reported to amplify PrP CWD seeds present in brain dilutions in the femtogram range ( 44 , 45 ) and has been used to detect PrP CWD in a variety of anatomic sites, body fluids, and excreta ( 13 , 22 – 30 ), but the technique has yet to be approved for official CWD surveillance.…”

“…RT-QuIC assay offers a newer, high-throughput approach that can detect minute amounts of prions in tissues and secretions of CWD-infected animals with the additional advantage of avoiding the proteolytic or acidic pretreatments commonly required for the conventional CWD detection assays (ELISA and IHC). Even though RT-QuIC has been reported to efficiently amplify PrP CWD in a variety of tissues (e.g., RLNs, brain, ear pinna, third eyelid, muscle), body fluids (e.g., nasal swabs, CSF, blood), and excreta (e.g., saliva, urine, feces) of animals at clinical and, sometimes, at preclinical stages of disease, the method is not yet approved for official CWD surveillance programs ( 13 , 22 – 30 ). Nevertheless, sensitive and rapid detection of low levels of prions in early stages of infection would aid substantially in understanding the epidemiology of the disease and enable assessment of mitigation based on management strategies to minimize disease transmission.…”

“…A few studies have investigated the utility of RT-QuIC in comparison to current immunodetection assays on free-ranging cervids ( 23 , 28 , 30 ). In the present study, the consistency of results between RT-QuIC, ELISA and IHC for the detection of CWD was analyzed in 1,300 RLNs of white-tailed deer (WTD, Odocoileus virginianus ) collected between 2015 and 2021 in Michigan.…”

Assessment of Real-Time Quaking-Induced Conversion (RT-QuIC) Assay, Immunohistochemistry and ELISA for Detection of Chronic Wasting Disease under Field Conditions in White-Tailed Deer: A Bayesian Approach

“…We employed weakly informative priors (i.e., distributions with higher uncertainty) for the Se and Sp estimates for ELISA/IHC because of the limited studies of performance available, of which the majority were applied under laboratory conditions or with limited sample size [ 29 , 31 , 32 , 64 ]. We assumed that ELISA and IHC accuracy was similar given the previous review available, indicating that both diagnostic assays performed similarly [ 45 ].…”