Detection of chronic wasting disease in the lymph nodes of free-ranging cervids by real-time quaking-induced conversion

Abstract

Chronic wasting disease (CWD), a transmissible spongiform encephalopathy of deer, elk, and moose, is the only prion disease affecting free-ranging animals. Since the disease was first identified in northern Colorado and southern Wyoming in 1967, new epidemic foci of the disease have been identified in 20 additional states, as well as two Canadian provinces and the Republic of South Korea. Identification of CWD-affected animals currently requires postmortem analysis of brain or lymphoid tissues using immunohistochemistry (IHC) or an enzyme-linked immunosorbent assay (ELISA), with no practical way to evaluate potential strain types or to investigate the epidemiology of existing or novel foci of disease. Using a standardized real-time (RT)-quaking-induced conversion (QuIC) assay, a seeded amplification assay employing recombinant prion protein as a conversion substrate and thioflavin T (ThT) as an amyloid-binding fluorophore, we analyzed, in a blinded manner, 1,243 retropharyngeal lymph node samples from white-tailed deer, mule deer, and moose, collected in the field from areas with current or historic CWD endemicity. RT-QuIC results were then compared with those obtained by conventional IHC and ELISA, and amplification metrics using ThT and thioflavin S were examined in relation to the clinical history of the sampled deer. The results indicate that RT-QuIC is useful for both identifying CWD-infected animals and facilitating epidemiological studies in areas in which CWD is endemic or not endemic.

FIG 1

Summary of retropharyngeal lymph node samples evaluated and positive sample locations. Samples included 100 white-tailed deer lymph nodes from New York State, 695 white-tailed deer lymph nodes from Illinois, 280 white-tailed and mule deer lymph nodes from Nebraska, 126 mule deer lymph nodes from Texas, and 42 moose lymph nodes from Colorado. Of 1,243 samples evaluated, 11 RT-QuIC assay-positive deer were identified in Illinois, 10 in Nebraska, and one in Texas.

FIG 2

RT-QuIC results from CWD-positive deer. Samples that were positive in the initial screening were reanalyzed in triplicate in three separate experiments, using either thioflavin T (ThT) or thioflavin S (ThS). Positive controls (CBP6) and multiple negative controls (CWD-negative lymph nodes and untreated Syrian hamster [SH] recombinant PrP) were included on each experimental plate. The threshold for amplification (orange dotted line) was determined by averaging the relative fluorescence units (RFUs) of negative-control samples over the course of the experiment and adding five standard deviations. Seeded amplification is demonstrated by increases in ThT and ThS fluorescence over time in the positive-control sample as well as each of three positive lymph nodes from study deer; negative-control samples do not show seeded amplification.

FIG 3

Variable importance weights of location, species, sex, age, and amino acid predictors of QuIC metrics. Where there was clearly a best predictor variable, variable importance weights were >0.4 (vertical dashed line). Of the correlates analyzed, likely predictors of age included ThT slope and ThS amplitude, while ThS score was correlated with sex and ThS slope seemed to be a good predictor of amino acid 96 (aa96) identity (for full model comparison tables, see Table S1 in the supplemental material).

FIG 4

Distribution of QuIC metric data associated with the best predictor variable in each case (see Fig. 3). Coefficients of variation are given in each panel. These results show a positive correlation between ThS slope and cervid PrP amino acid 96 identity (for full model comparison table, see Table S1 in the supplemental material). The dashed line represents the trend line determined from the data.