Progression of chronic wasting disease in white-tailed deer analyzed by serial biopsy RT-QuIC and immunohistochemistry

Abstract

Chronic wasting disease (CWD) continues to spread or be recognized in the United States, Canada, and Europe. CWD is diagnosed by demonstration of the causative misfolded prion protein (PrP CWD) in either brain or lymphoid tissue using immunodetection methods, with immunohistochemistry (IHC) recognized as the gold standard. In recent years, in vitro amplification assays have been developed that can detect CWD prion seeding activity in tissues, excreta, and body fluids of affected cervids. These methods potentially offer earlier and more facile detection of CWD, both pre-and post-mortem. Here we provide a longitudinal profile of CWD infection progression, as assessed by both real-time quaking-induced conversion (RT-QuIC) and IHC on serial biopsies of mucosal lymphoid tissues of white-tailed deer orally exposed to low doses of CWD prions. We report that detection of CWD infection by RT-QuIC preceded that by IHC in both tonsil and recto-anal lymphoid tissue (RAMALT) in 14 of 19 deer (74%). Of the 322 biopsy samples collected in post-exposure longitudinal monitoring, positive RT-QuIC results were obtained for 146 samples, 91 of which (62%) were concurrently also IHC-positive. The lower frequency of IHC positivity was manifest most in the earlier post-exposure periods and in biopsies in which lymphoid follicles were not detected. For all deer in which RT-QuIC seeding activity was detected in a tonsil or RAMALT biopsy, PrP CWD was subsequently or concurrently detected by IHC. Overall, this study (a) provides a longitudinal profile of CWD infection in deer after low yet infectious oral prion exposure; (b) illustrates the value of RT-QuIC for sensitive detection of CWD; and (c) demonstrates an ultimate high degree of correlation between RT-QuIC and IHC positivity as CWD infection progresses.

Very low oral exposure to prions of brain or saliva origin can transmit chronic wasting disease

“…We correlated the codon 96 PRNP genotype to each deer’s first positive IHC result within the separate cohorts. In cohorts 1 & 2, three of four deer (75%) with PRNP 96GG displayed a positive result prior to 96GS deer [ 44 ]. Cohort 3 were all 96GG and all were negative.…”

“…The codon 96 PRNP genotypes of the deer in above groups were assessed (cohorts 4 and 6). Four of six deer (67%) of PRNP 96GG became CWD+ vs. one of the two 96GS deer, and all four 96GG deer developed first indication of CWD infection before 96GS deer in their respective cohorts [ 44 ]. Cohort 5 (96GG (n = 3); 96GS (n = 1)) were all negative.…”

“…Cohorts 8, 9, 11, & 12 biopsies were analyzed for PrP CWD detection by IHC only. Biopsies from Cohort 10 (10 mg) were analyzed by RT-QuIC for seeding activity and IHC for PrP CWD deposition [ 44 ].…”

“…For each RT-QuIC reaction, we adapted the original protocol [ 35 ] to our current substrate mix [ 40 , 42 , 44 ], composed of 0.10 mg/ml rPrP, 10 μM thioflavin T (ThT), 320 mM NaCl, 1mM EDTA and 1X PBS, to which 96 μl were added to each well of a 96-well plate (Greiner Bio-One black, optical-bottom, VWR). Two [ 2 ] μl of each sample prepared with 0.1% sodium dodecyl sulfate (SDS) was then added to each well.…”

Association of chronic wasting disease susceptibility with prion protein variation in white-tailed deer (Odocoileus virginianus)

“…When PrP variant C and PrP variant F were compared, the deer that carried PrP variant F were proportionately less common among positive than among negative deer than were deer carrying PrP variant C. The incubation period of CWD in naturally infected animals is unknown, but in captive elk most natural cases occur in animals 3 to 8 years old [ 52 ], and it has been estimated that the majority of Cervid species probably develop the disease within the first 3 years of infection [ 6 ]. However, under inoculation experiments in white-tailed deer, prion shedding as early as 3 months after CWD exposure was detected by a real-time quaking-induced conversion method [ 20 , 53 ]. Following oral inoculation, the average survival period of WTD that had glutamine at codon 95 and glycine at 96 was found to be 693 days while deer with p.[(Gly96=)];[(Gly96Ser)] survived 956 days, and the deer with p.[(Gln95=)];[(Gln95His)] started to show the disease symptoms much later and survived a much longer period (1508 days) after inoculation [ 54 ], suggesting that PrP variant F with p.(Gln95His) may slow disease progression more than PrP variant C, which has p.(Gly96Ser).…”