In utero transmission and tissue distribution of chronic wasting disease-associated prions in free-ranging Rocky Mountain elk

Abstract

The presence of disease-associated prions in tissues and bodily fluids of chronic wasting disease (CWD)-infected cervids has received much investigation, yet little is known about mother-to-offspring transmission of CWD. Our previous work demonstrated that mother-to-offspring transmission is efficient in an experimental setting. To address the question of relevance in a naturally exposed free-ranging population, we assessed maternal and fetal tissues derived from 19 elk dam-calf pairs collected from free-ranging Rocky Mountain elk from north-central Colorado, a known CWD endemic region. Conventional immunohistochemistry identified three of 19 CWD-positive dams, whereas a more sensitive assay [serial protein misfolding cyclic amplification (sPMCA)] detected CWD prion seeding activity (PrPCWD) in 15 of 19 dams. PrPCWD distribution in tissues was widespread, and included the central nervous system (CNS), lymphoreticular system, and reproductive, secretory, excretory and adipose tissues. Interestingly, five of 15 sPMCA-positive dams showed no evidence of PrPCWD in either CNS or lymphoreticular system, sites typically assessed in diagnosing CWD. Analysis of fetal tissues harvested from the 15 sPMCA-positive dams revealed PrPCWD in 80 % of fetuses (12 of 15), regardless of gestational stage. These findings demonstrated that PrPCWD is more abundant in peripheral tissues of CWD-exposed elk than current diagnostic methods suggest, and that transmission of prions from mother to offspring may contribute to the efficient transmission of CWD in naturally exposed cervid populations.

Fig. 1.

PrP^CWD detection in dilutions of CWD-positive elk brain following sPMCA. Representative Western blots for detection of PrP^CWD in CWD-positive elk brain homogenate (E2) following sPMCA (dilutions 10^− 2, 10^− 6 and 10^− 12, rounds 1, 3, 5 and 7 shown). PrP^CWD was not detected in a 10 % homogenate of E2 prior to sPMCA or after one round of sPMCA. After three and five rounds of E2 sPMCA, PrP^CWD was detected at 10^− 6. PrP^CWD was detected at 10^− 12 following seven rounds of sPMCA. sPMCA controls (R7– and R7+) showed complete proteinase K (PK) digestion of negative elk brain homogenate (R7–) and proteinase K-resistant PrP^CWD in E2 CWD-positive elk brain homogenate (R7+) (10 % homogenates, undiluted, round 7). Western blot controls (no sPMCA) showed complete proteinase K digestion of PrP in negative white-tailed deer brain homogenate and proteinase K-resistant PrP^CWD in CWD-positive white-tailed deer brain homogenate (10 % homogenates, undiluted, no sPMCA).

Fig. 2.

Infectivity titres of serially diluted E2 CWD brain isolate. Groups of 10 mice were inoculated intracranially with the E2 isolate of CWD prions diluted into normal brain homogenate at the indicated serial dilutions. End-point prion titres were determined as described previously (Reed & Muench, 1938). Error bars are smaller than the indicated points for mean infectivity on the graph. Dashed line indicates extrapolated values that lie outside of the dynamic range of the bioassay. Dotted lines converging from y = 100 and x = 10^− 11.5 represent the extrapolated dilution of E2 brain homogenate (10^− 11.5) containing 1 LD₅₀ U (100) (g tissue)^− 1. Dotted lines converging from x = 10^− 12 and y = 10^− 0.5 represent the extrapolated infectivity titre [0.32 LD₅₀ U (g tissue)^− 1] of a 10^− 12 dilution of E2 brain homogenate that is the PMCA seeding dose for seven rounds of PMCA.

Fig. 3.

PrP^CWD detection in elk fetal and dam tissues following seven versus five rounds (R#) of sPMCA. Representative Western blots for detection of PrP^CWD in elk fetal umbilicus (Sample 1), fetal intestine (Sample 4) and dam uterus (Samples 15 and 22) following seven rounds of sPMCA. PrP^CWD was not detected in these tissues after five rounds of sPMCA. sPMCA controls (–C and +C) showed complete proteinase K (PK) digestion of negative elk brain homogenate (–C) and proteinase K-resistant PrP^CWD in E2 CWD-positive elk brain homogenate (+C) (10 % homogenates, undiluted, round 7). Western blot controls (no sPMCA) showed complete proteinase K digestion of PrP in a negative white-tailed deer brain homogenate and PrP^CWD signal post-proteinase K digestion in a CWD-positive white-tailed deer brain homogenate (10 % homogenate, undiluted, no sPMCA).

Fig. 4.

(a) Category classification and numbers of tissues found PrP^CWD-positive by sPMCA in elk dams. Numbers represent the numbers of dams found positive in that tissue or category of tissues. (b) PrP^CWD distribution in sPMCA-positive adult elks. Total dams found positive by sPMCA in either category (n = 15) of total elk dams studied from the endemic area (n = 19). Total dams with at least one C tissue positive, n = 7; total dams with at least one L tissue positive, n = 7; total dams with at least one R tissue positive, n = 13; and total dams with at least one S tissue positive, n = 13.

Fig. 5.

PrP^CWD detection in elk dam tissues following seven rounds of sPMCA. Representative Western blots for detection of PrP^CWD in dam tissues. sPMCA controls showed the lack of amplification for a CWD-negative sample and the successful amplification of a known CWD-positive white-tailed deer. Western blot controls (no sPMCA) showed complete proteinase K (PK) digestion of PrP in a negative white-tailed deer brain homogenate and PrP^CWD signal post-proteinase K digestion in a CWD-positive white-tailed deer brain homogenate (10 % homogenate, undiluted, no sPMCA). Asterisks denote sPMCA + CWD amplification. Tissue codes: C, control. (+, positive; –, negative); AD, adipose tissue; BL, urinary bladder; BR, brain; CT, cotyledon; K, kidney; LNG, lung; LVR, liver; MG, mammary gland; NE, nasal epithelium; OB, obex; OV, ovary; PA, pancreas; PL, placenta; RLN, retropharyngeal lymph node; SG, salivary gland; SP, spleen; TO, tongue; UR, ureter; UT, uterus.

Fig. 6.

PrP^CWD detection in elk fetal tissues following seven rounds of sPMCA. Representative Western blots for detection of PrP^CWD in fetal tissues. sPMCA controls showed the lack of amplification for a CWD-negative sample and the successful amplification of a known CWD-positive white-tailed deer. Western blot controls (no sPMCA) showed complete proteinase K (PK) digestion of PrP in a negative white-tailed deer brain homogenate and PrP^CWD signal post-proteinase K digestion in a CWD-positive white-tailed deer brain homogenate (10 % homogenate, undiluted, no sPMCA). Tissue codes: C, control (+, positive; –, negative); BR, brain; CL, colon; IT, intestine; LNG, lung; LVR, liver; PLN, popliteal lymph node; RLN, retropharyngeal lymph node; RT, rectum; SP, spleen; TY, thymus; UM, umbilicus; VG, vagus nerve. Asterisks denote sPMCA+ CWD amplification.

Fig. 6.

PrP^CWD detection in elk fetal tissues following seven rounds of sPMCA. Representative Western blots for detection of PrP^CWD in fetal tissues. sPMCA controls showed the lack of amplification for a CWD-negative sample and the successful amplification of a known CWD-positive white-tailed deer. Western blot controls (no sPMCA) showed complete proteinase K (PK) digestion of PrP in a negative white-tailed deer brain homogenate and PrP^CWD signal post-proteinase K digestion in a CWD-positive white-tailed deer brain homogenate (10 % homogenate, undiluted, no sPMCA). Tissue codes: C, control (+, positive; –, negative); BR, brain; CL, colon; IT, intestine; LNG, lung; LVR, liver; PLN, popliteal lymph node; RLN, retropharyngeal lymph node; RT, rectum; SP, spleen; TY, thymus; UM, umbilicus; VG, vagus nerve. Asterisks denote sPMCA+ CWD amplification.