Detection of Prions in Blood of Cervids at the Asymptomatic Stage of Chronic Wasting Disease

Abstract

Chronic wasting disease (CWD) is a rapidly spreading prion disorder affecting captive and free-ranging cervids. The zoonotic potential of CWD is unknown, as well as the mechanism for its highly efficient transmission. A top priority to minimize further spreading of this disease and its potential impact on environmental prion contamination is the development of a non-invasive, sensitive, and specific test for ante-mortem detection of infected animals. Here, we optimized the protein misfolding cyclic amplification (PMCA) assay for highly efficient detection of CWD prions in blood samples. Studies were done using a blind panel of 98 field-collected samples of whole blood from codon 96 glycine/glycine, captive white-tailed deer that were analyzed for prion infection post-mortem by immunohistochemistry (IHC). The results showed a sensitivity of 100% in animals with very poor body condition that were IHC-positive in both brain and lymph nodes, 96% in asymptomatic deer IHC-positive in brain and lymph nodes and 53% in animals at early stages of infection that were IHC-positive only in lymph nodes. The overall mean diagnostic sensitivity was 79.3% with 100% specificity. These findings show that PMCA might be useful as a blood test for routine, live animal diagnosis of CWD.

Figure 1

PMCA of CWD prions. (A) To optimize CWD PrP^Sc amplification by PMCA and determine the limit of detection, brain extracts from CWD sick animals was serially diluted (10⁻⁴ to 10⁻¹³) in buffer and subjected to various consecutive rounds of 144 PMCA cycles. As negative controls, 5 tubes (C1 to C5) in which PMCA was done without CWD brain homogenate were used to control for possible cross-contamination. After each PMCA round, an aliquot of 10 µL was taken to analyze for PrP^Sc signal by western blot using the 6H4 anti-PrP antibody. All samples, except the normal brain homogenate (NBH), were treated with 10 µg/mL of PK for 1 h at 37 °C, before western blotting to differentiate PrP^Sc from PrP^C. (B) Whole blood from a healthy deer was spiked with CWD brain homogenate at distinct final dilutions (10⁻⁶ to 10⁻¹¹). The same dilutions were spiked in buffer (PBS) as control (right panel). After processing by high-speed centrifugation in the presence of sarkosyl (as described in Methods), samples were subjected to three consecutive rounds of PMCA. The PrP^Sc signal was assessed by Western blot analysis after PK digestion. NBH refers to the transgenic normal (healthy) brain homogenate, used as migration control marker. Dashed lines in some of the blots indicate splicing, done to remove unrelated lanes. Numbers in the right indicate the position of molecular weight markers.

Figure 2

PrP^Sc detection in blood of animals showing clinical symptoms of CWD. Representative samples of whole blood (80 µL) from three white-tailed deer positive for PrP^Sc in obex and MRPLN as examined by IHC and presenting with poor body condition (S1, S2, S3), were analyzed by PMCA. As controls, blood from two deer from a CWD-free control herd (C1 and C2) were analyzed in parallel. Samples were subjected to three serial rounds of PMCA and the results of rounds 2 and 3 are shown. The PRC1 anti-PrP antibody was used for western blotting immunodetection. All samples were treated with PK, except the transgenic mice normal brain homogenate (NBH), used as migration control. Numbers in the right indicate the position of molecular weight markers.

Figure 3

Blind study of PrP^Sc detection in blood of CWD-infected, but asymptomatic animals. Representative samples of whole blood (200 µL) from asymptomatic white-tailed deer, were collected from either CWD-free or CWD-infected herds. Samples analyzed included 49 animals that were positive for PrP^Sc-staining in both the MRPLN and the obex via IHC (B + LN + ), 34 animals that were positive for PrP^Sc-staining only in the MRPLN via IHC (B-LN + ) and 10 that were negative in both brain and MRLPN. The figure shows representative samples from 12 B + LN + , 5 B-LN + and 3 B-LN- (Neg). The entire set of samples was analyzed independently by two different investigators in duplicate. Top and bottom panels show the results from the two different investigators which developed their western blots using two distinct anti-PrP antibodies (PRC1 and 6H4). Samples were subjected to three serial rounds of PMCA and the results obtained in the third round are shown. All samples were treated with PK, except the transgenic mice normal brain homogenate (NBH), used as migration control. Numbers in the right indicate the position of molecular weight markers.

Figure 4

Detection of PrP^Sc in small volume of blood and removal of pre-cleaning step. To estimate the minimum amount of blood needed for detection, different volumes (20, 10, 5, 1 and 0.1 µL) of whole blood from five representative samples that were positive in the blinded study (3 B + LN + and 2 B-LN + ), were directly added to a 10% brain homogenate from cervid transgenic mice. As controls we used two samples from CWD negative animals. Samples were subjected to three sequential rounds of PMCA and PrP^Sc detected by Western blot using the PRC1 antibody. The figure shows the results of the third round of PMCA. As before, all samples were treated with PK, except the transgenic mice normal brain homogenate (NBH), used as migration control. Numbers in the right indicate the position of molecular weight markers.