Ticks harbor and excrete chronic wasting disease prions

Abstract

Chronic wasting disease (CWD) is a fatal neurodegenerative disease caused by infectious prions (PrP^CWD) affecting cervids. Circulating PrP^CWD in blood may pose a risk for indirect transmission by way of hematophagous ectoparasites acting as mechanical vectors. Cervids can carry high tick infestations and exhibit allogrooming, a common tick defense strategy between conspecifics. Ingestion of ticks during allogrooming may expose naïve animals to CWD, if ticks harbor PrP^CWD. This study investigates whether ticks can harbor transmission-relevant quantities of PrP^CWD by combining experimental tick feeding trials and evaluation of ticks from free-ranging white-tailed deer (Odocoileus virginianus). Using the real-time quaking-induced conversion (RT-QuIC) assay, we show that black-legged ticks (Ixodes scapularis) fed PrP^CWD-spiked blood using artificial membranes ingest and excrete PrP^CWD. Combining results of RT-QuIC and protein misfolding cyclic amplification, we detected seeding activity from 6 of 15 (40%) pooled tick samples collected from wild CWD-infected white-tailed deer. Seeding activities in ticks were analogous to 10-1000 ng of CWD-positive retropharyngeal lymph node collected from deer upon which they were feeding. Estimates revealed a median infectious dose range of 0.3-42.4 per tick, suggesting that ticks can take up transmission-relevant amounts of PrP^CWD and may pose a CWD risk to cervids.

Figure 1

Recovery of chronic wasting disease (CWD) prions (PrP^CWD) from spiking and membrane feeding experiments for analysis using the real-time quaking-induced conversion (RT-QuIC) assay. Comparison of amyloid formation rates (AFR) by RT-QuIC of (a–c) defibrinated bovine whole blood spiked with a 10^–3 dilution of the same 10% brain homogenate as used for the brain dilution series, and artificial membrane-fed tick homogenates spiked in the same manner as the blood (“b” and “c” depict AFRs of all 8 technical replicates for spiked whole blood or tick homogenates averaged in “a”) or (d) tenfold dilutions of 10% brain homogenate (BH) (from the obex region) from a CWD-positive white-tailed deer. Membrane feeding units were constructed using (e) cured silicon membranes adhered to the base of (f) assembled feeding chambers. (g) Depiction of the assembled feeding unit with feeding chambers being held upright by the chamber supports. (h-j) Comparison of AFRs by RT-QuIC of homogenates from membrane-fed PrP^CWD exposed or negative control ticks and tick frass (“i “and “j” depict AFRs for all 24 technical replicates (from 3 biological replicates run on 3 separate plates) for frass or membrane-fed tick homogenates averaged in “h”). Negative controls (NC) in each AFR plot are representative for the same sample type.

Figure 2

Presence of chronic wasting disease (CWD) prions (PrP^CWD) in tissues and Ixodes scapularis from hunter-harvested white-tailed deer (WTD) assessed by two protein amplification assays. Comparisons of real-time quaking-induced conversation (RT-QuIC) amyloid formation rates (AFR) for (a) deer retropharyngeal lymph node (RPLN), ear tissue (pinna), and pooled tick samples from 15 CWD-positive (ID 1–15) and two of the 15 CWD-negative (ID 16,17) WTD (all other negative sample results are shown in Supplementary Table S2, Supplementary Figure S1 and S2) and (b) tenfold dilutions of ear tissue homogenates (ID 1,4,11). Samples are grouped by WTD IDs on the x-axis. Data points represent mean AFR ± standard deviation of 8 technical replicates. Negative controls (NC) represent the same sample types. (c–f) Scatterplots with fitted linear regression line with 95% confidence intervals comparing AFR relationships between (c) RPLN to ear samples, (d) tick to ear samples, (e) RPLN to tick samples, or (f) pooled tick samples with pooled tick sample weight in milligrams (mg). Each point represents the mean AFR of 8 technical replicates from WTD ID 1–15. (g–i) Western blot analysis of PMCA products to assess the presence of prion seeding activity for; (g) 14 pooled tick samples (WTD IDs 2–15) stored in RT-QuIC sample buffer evaluated in “a”; (h) 10% tick homogenates (ID 17–19 are NCs); (i) serial dilutions of a CWD-positive (CWD +) brain homogenate (BH) (PMCA positive control), and unseeded (UN) or cellular prion (PrP^C) (PMCA NCs). (Original uncropped blots/gels are presented in Supplementary Figure S3) Samples analyzed in this figure were tested in duplicate and represent a third PMCA round. Numbers at the right of each panel represent molecular weight markers in kilodaltons (kDa). Red font indicates samples determined to contain PrP^CWD by either RT-QuIC or PMCA.

Figure 3

Comparison of chronic wasting disease (CWD) prion (PrP^CWD) loads by Ixodes scapularis following infected blood meal with retropharyngeal lymph node (RPLN) from free-ranging white-tailed deer (WTD). Comparisons of relative PrP^CWD loads present in three separate pooled tick homogenates (ID numbers 1, 4, 11) from Fig. 2a, which tested CWD-positive by our parameters, against a tenfold dilution series of RPLN from corresponding WTD. Data points for 8 technical replicates are depicted for each sample ± standard deviation.