Chronic Wasting Disease Diagnostic Discrepancies: The Importance of Testing Both Medial Retropharyngeal Lymph Nodes

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy affecting many cervid species, including white-tailed deer (WTD; Odocoileus virginianus), mule deer (Odocoileus hemionus), elk (Cervus elaphus), moose (Alces alces), and reindeer (Rangifer tarandus; Haley and Hoover 2015; Benestad et al. 2016). The range of CWD in North America is expanding; as of July 2020, CWD had been detected in free-ranging cervids from 24 states (US) and two Canadian provinces and in captive cervid facilities in 17 states and three provinces (Richards and USGS National Wildlife Health Center 2020).

A misfolding (PrP^CWD) of the host normal cellular prion protein (PrP^C) is associated with development of CWD (Colby and Prusiner 2011). Clinical signs are a result of accumulation of misfolded proteins in the central nervous system, and include weight loss, listlessness, ataxia, polyuria and polydipsia, bruxism, and hypersalivation (Williams and Young 1992). Although the zoonotic potential of CWD is considered very low because of differences in microcrystal structures of human and cervid prion proteins, transmission risks remain unclear (Kurt et al. 2015), leading to increased requests for testing hunter-killed animals in states with known CWD-positive animals (Saunders et al. 2012).

With increasing requests for testing by the public and a strong desire by wildlife management agencies to detect the disease as early as possible, a high confidence in test results is needed. A review of diagnostic tests available for CWD can be found in Haley and Richt (2017). Federally recognized tests include immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), and western blot; testing must be performed at approved laboratories, and positive samples must undergo confirmatory testing at the National Veterinary Services Laboratories (US Department of Agriculture 2019).

The current gold standard for confirmatory CWD testing in WTD is IHC of the medial retropharyngeal lymph nodes (MRPLN); however, ELISA is a high-throughput test often used to screen samples prior to IHC (Keane et al. 2008; Haley and Richt 2017). Because of the unknown sensitivity of all testing modalities for CWD detection in the earliest stages of disease, results are currently reported as not detected rather than negative (US Department of Agriculture 2019). In the event of a case being suspect-positive using ELISA but not being detected using IHC, it is current practice to accept the IHC results as final. Hibler et al. (2003) reported the relative sensitivity and specificity of ELISA compared to IHC when IHC results were accepted as truth. Overall agreement between ELISA and IHC was ≥97.6% for MRPLN samples.

Although consistency of results between ELISA and IHC has previously been discussed, consistency of results between contralateral lymph nodes from the same animal has not been explored. In this study, we characterized the prevalence of discrepancies in results of ELISA positive cases with confirmatory IHC testing, and evaluated the prevalence of cases in which contralateral MRPLN from the same ELISA-positive animal yielded inconsistent IHC results.

White-tailed deer (n=101) harvested in Michigan from 2015 to 2019 that were ELISA-positive for CWD at the Michigan State University Veterinary Diagnostic Laboratory (Lansing, Michigan, USA) were included in this study. The ELISAs were performed using a commercially available kit (TeSeE Purification and Detection Kit, Bio-Rad Laboratories, Hercules, California, USA) following the manufacturer's instructions. A homogenate sample from each MRPLN was run in triplicate, and a mean optical density (OD) value was obtained. The cutoff criterion for a nonnegative (initial reactor or suspect) sample was an OD >0.035 plus the mean of the negative controls. The IHC for the prion protein was completed for both MRPLN (n=202) collected from ELISA-positive deer using a commercially available kit for the Ventana Discovery platform (Anti-Prion (99) Research Kit, Roche Diagnostics, Indianapolis, Indiana, USA). The number of IHC-positive follicles in each MRPLN were counted and normalized by the area (cm²) of the lymph node sections to account for different sizes among samples. In addition, intensity of the labeling was ranked on a scale of 1 (lightest) to 3 (darkest). Statistical analyses were performed using R 3.2.3 (R Foundation for Statistical Computing, Vienna, Austria).

Of the 101 deer, 86 were hunter-harvested, 6 were harvested under a disease control permit, 4 were targeted for showing clinical signs related to CWD, 3 were harvested under a crop damage permit, 1 was roadkill, and 1 was culled by US Department of Agriculture wildlife services. Fifty-five were male and 46 were female, and ages based on tooth wear ranged from <1 yr old to 9 yr old.

The OD values from ELISA were not significantly related to age (linear regression; P=0.420) or sex (Student's t-test; P=0.957). The mean number of positive follicles in IHC-positive MRPLN was 48/cm² (range 1–200). Of the 101 ELISA-positive cases, both MRPLN were IHC positive for 88.1% (89/101) of cases. The four deer targeted for showing clinical signs of CWD all had high mean OD values (4.08, 3.49, 3.14, and 4.08) and all were IHC positive in both MRPLN. There were inconsistent results between ELISA and IHC in 11.9% (12/101) of cases. Prion protein was not detected by IHC in either MRPLN in 5.9% (6/101) of ELISA-positive cases. These cases included three females and three males, and all were 4 yr old or less (Table 1). The mean OD value in these cases was 0.79 (range 0.07–3.31). In addition, only one MRPLN was IHC positive for 5.9% (6/101) of cases, whereas prion protein was not detected in the contralateral MRPLN. These cases included three females and three males, and all were 4 yr old or less (Table 2). The mean OD value in these cases was 2.86 (range 0.41–4.11).

In 54/101 cases, we knew which IHC-labeled lymph node was used for the ELISA (n=50 IHC positive and four in which IHC not detected). There was a significant but weak positive relationship between the number of IHC positive follicles and ELISA OD values (linear regression; R²=0.08, P=0.039; Fig. 1). The mean OD value in IHC-positive samples was higher than IHC–not detected samples (Table 3); however, this was not statistically significant (Welch's t-test; P=0.260). In the 50 IHC-positive cases, OD values were not significantly related to labeling intensity (analysis of variance; P=0.531).

We demonstrate that in 5.9% of ELISA-positive MRPLN cases, the diagnosis could not be confirmed by IHC. Cases with inconsistent results were evenly distributed among males and females, and all were aged less than 4 yr old. Inconsistencies in ELISA and IHC results are most likely due to sampling methods and protein distribution within the examined MRPLN (Hibler et al. 2003). Sampling for ELISA involves taking 180–220 mg of tissue that is preferentially collected from the lymph node cortex to improve accuracy, whereas IHC examines cross sections of MRPLN that are 5–7 mm in thickness and include both cortex and medulla. Thus, spatial distribution of prion protein within the MRPLN may play a role in whether protein is detected. This would especially explain disparate results for cases with very low positive OD values, in which there may only be a single positive follicle that may not have been included in sections prepared for IHC examination. However, for one case in the current study, the MRPLN had a high mean OD value (3.31), but repeated IHC testing of numerous sections was unable to detect prion protein. This may also be explained by differences in methodology for ELISA and IHC. For example, tissue digestion and antigen retrieval differ among the two tests. It is also possible that the antiPrP antibody used in the ELISA has greater affinity for PrP^CWD than the monoclonal antibody used for IHC (Hibler et al. 2003). Alternatively, different abnormal forms of prion protein (i.e., strains) have been reported in different cervid species (Pirisinu et al. 2018) and the antibodies may not equally detect such distinct prion protein types. Future studies could utilize multiple antibodies to further analyze these points.

The mean OD values from IHC-positive cases was higher than from IHC–not detected cases; however, this was not statistically significant (P=0.260). There were only four IHC–not detected cases available for analysis, so it is possible that results were not significant because of a small sample size. In addition, the four cases had highly variable mean OD values (0.17, 0.33, 3.72, and 4.06).

No previous studies have compared ELISA and IHC results using both MRPLN collected from the same animal. A key finding in our study was the disparate IHC results between contralateral MRPLN from ELISA-positive WTD. In our study, six ELISA-positive cases (5.9%) had one MRPLN that was IHC positive, and no prion protein was detected by IHC in the contralateral node. Based on our results, a diagnosis of CWD may be missed in 5.9% of cases if only one MRPLN was examined. In addition to spatial distribution biases discussed earlier, prion spread may play a role in these cases. Especially in cases of early infection, it is possible that only one lymph node could be positive, and that the other has not yet accumulated prion protein. It is currently still common practice to submit or test only one of the MRPLN from WTD for CWD testing. Our findings demonstrate the importance of submitting and testing both MRPLN to improve the sensitivity of CWD testing. In addition, it is prudent to take multiple sections for IHC, especially in cases that are ELISA suspect but have no positive labeling on initial IHC. Further sections for IHC in other areas of the node may reveal positive labeling in these cases.

We thank Tom Cooley, Katie Farinosi, and Caitlin Ott-Conn, who assisted with processing samples for analysis. We thank the immunodiagnostics laboratory at the Michigan State University Veterinary Diagnostic Laboratory and Nicole Grosjean for providing us the ELISA results.