Addressing chronic wasting disease in Korean farms: topsoil removal and 2N NaOH treatment before cervid restocking

ABSTRACT

Chronic wasting disease (CWD) is a highly contagious prion disease occurring in free-ranging and farmed cervids. In the Republic of Korea, cases of CWD continue to be detected almost annually, on both new and occasionally previously infected farms. CWD-infected animals contaminate soil and other environmental components by shedding prions through their excreta. Since shed prions remain infectious for years in the environment, they can act as infectivity reservoirs facilitating horizontal transmission of CWD. To prevent the further spread of CWD and allow farms to resume operations, control measures on infected farms, including topsoil removal and thorough environmental treatment with 2N NaOH, have been implemented in the Republic of Korea. Restocking remediated farms with cervids was permitted after confirming the absence of prion seeding activity in soil samples using protein misfolding cyclic amplification (PMCA). A total of 215 samples from 18 remediated farms were collected and analysed using PMCA, with only 3 samples from 3 farms displaying prion seeding activity. While the disease control measures effectively eliminated prion seeding activity in CWD-affected farms, CWD recurred at two of the 18 remediated farms 4 to 5 years after restocking animals. It remains unclear whether the recurrence of CWD at the two farms was due to residual prions in the environment after the control measures, or the introduction of the infected animals from other farms. This uncertainty is heightened by the annual occurrence of CWD at multiple farms and the absence of a traceability system for farmed cervids.

Introduction

Prions are proteinaceous infectious agents composed of ordered multimeric forms of prion protein (PrP^Sc) [1–3]. Prions can infect multiple mammalian species causing fatal neurodegenerative diseases such as Creutzfeldt-Jakob disease in humans, scrapie in sheep and goats. Chronic wasting disease (CWD) is a highly contagious prion disease affecting both farmed and free-ranging cervids [4,5]. Since its first identification in the 1960s in Colorado, USA [6], CWD has been continuously expanding in geographic distribution and prevalence. CWD has now been reported in more than 35 U.S. states and 5 Canadian provinces of North America [7], and also in the Republic of Korea and Scandinavia [8,9]. CWD prions are widely distributed throughout the tissues and excreta of infected animals, including their blood, urine, and faeces, and are shed into the environment contaminating soil and other components [10–14]. Once introduced into the environment, prions bind to soil particles and other environmental matrices, retaining their infectivity for years [15–19]. The interaction between prions and soil can vary depending on prion strains and soil types [15,20]. Notably, clay-rich soils exhibit much higher adsorption capacity for prions compared to sand or sandy soil [19]. Furthermore, prions in the contaminated soil can be internalized by plants, and transported and accumulated in their above-ground tissues [17,21]. The adsorption of prions has also been observed on various environmental surfaces including wood, rocks, plastic, and metals [22–24]. Collectively, by acting as long-lasting environmental reservoirs of CWD infectivity, shed prions can facilitate horizontal transmission of the disease without direct contact between animals [16,18,25].

Prions are notoriously difficult to fully inactivate through routine procedures that are effective against most other pathogens [26–28]. Although numerous decontamination methods involving various chemicals, enzymes, heat, and autoclaving have been developed, primarily for use in hospital settings and research laboratories, they are not practically applicable to the complex environmental components or conditions that may exist on a CWD-infected farm. We previously reported that treating CWD-contaminated farm soil with 2N NaOH under experimental conditions was effective in inactivating CWD prions [29].

In the Republic of Korea, CWD cases have been detected in multiple cervid farms nearly annually over the last several years. Most farm owners have conveyed a strong desire to continue operating as cervid farms after CWD outbreaks, so eliminating CWD prions in the farm environment has become a key focus of the disease control policy in the Republic of Korea. To mitigate the risk of CWD transmission while allowing cervid farms to resume operations, the Korean veterinary authorities implemented strong disease control measures on CWD-affected farms including topsoil removal and thorough environmental treatment with 2N NaOH. Restocking cervids on the remediated farms was permitted only after confirming the absence of prion seeding activity in soil samples using the protein misfolding cyclic amplification (PMCA) technique. PMCA is a highly sensitive prion amplification technique, and prion seeding activity measured by PMCA was shown to correlate closely with prion infectivity [30]. For use as PMCA seeds, CWD prions bound to soil particles need to be extracted from soil; however, recovery of soil-bound prions has been shown to be difficult and inefficient [20,31,32]. While soil-bound prions do not support PMCA amplification efficiently, they still retain infectivity [31,32]. Using a refined extraction technique for soil-bound prions, we recently reported that prions can be reliably detected by PMCA in soil of CWD-affected farms in South Korea [33]. In this study, we have shown that at least one CWD-positive soil sample was identified at each of the six CWD-affected farms investigated, with detection rates ranging from 10% to 80% [33]. Another study by Kuznetsova et al., which employed a refined extraction method of soil-bound PrP^Sc distinct from ours, also reported successful detection of PrP^Sc exclusively in prairie soils collected from the CWD-endemic region with higher prevalence among the two endemic regions studied [34].

This study summarizes 18 CWD-affected farms in which cervid animals were reintroduced following the implementation of those disease control measures. Our results indicate that the disease control measures are effective in eliminating prion seeding activity on CWD-affected farms. However, given the recurrence of CWD at 2 out of 18 remediated farms, additional actions such as traceability and biosecurity measures will need to be considered towards the prevention and eradication of CWD in the Republic of Korea.

Results and discussion

To date, CWD control measures have been applied to 18 CWD-affected farms before the reintroduction of cervids (Table 1). CWD prevalence rates among those farms varied widely, ranging from less than 1% (Farms I and J) to more than 50% (Farms K and R). The number of CWD-positive animals also varied widely across the farms. Only one animal tested positive for CWD on three farms (Farms E, G and I), and two animals were CWD-positive on four farms (Farms J, M, P and Q). In comparison, more than 10 CWD-positive animals were detected on seven farms, with the highest number of CWD-positive animals identified on a single farm being 54 (Farm N; prevalence: 19.6%). Among the nine farms on which three cervid species were raised, CWD-positive animals were identified in only a single species on three of the farms (elk, Farm M or red deer, Farms G and I), in two species on one farm (Farm D), and in all three species on the remaining five farms. Since prions in CWD-infected animals are shed daily through excreta even during the preclinical stage, they can accumulate in the environment over time within each affected farm [11,13,18,35]. Accordingly, farms with differing CWD prevalence may also vary considerably in the amount and distribution of environmental CWD prions. Supporting this possibility, a recent study by Kuznetsova et al. detected CWD prions only in soils from the region with higher prevalence, among the two endemic regions examined, with none detected in the lower-prevalence region [34]. Although 2N NaOH treatment is effective in inactivating CWD prions, a high environmental prion burden may increase the likelihood that some prions might persist in areas inaccessible to disinfectants, even after repeated decontamination. How environmental prion burden influences the risk of prion persistence after decontamination and reinfection following animal reintroduction remains largely unexplored and requires further investigation.

Table 1.

Summary of CWD-infected farms on which cervids were reintroduced following depopulation and implementation of control measures to inactivate CWD prions from the environment.

Farms	Year of CWD occurrence	CWD testing1)				Year approved for animal re-introduction2)	CWD recurrence
		Total	Elk	Sika Deer	Red deer		Yes/No	Year	CWD testing1)
A	2016	17/83	2/48	9/17	6/18	2017	Yes	2022	13/61
B	2016	7/40	-	4/30	3/10	2017	No	-	-
C	2016	10/36	2/9	4/21	4/6	2017	No	-	-
D	2016	3/56	0/45	1/2	2/9	2017	No	-	-
E	2016	1/10	1/3	0/7	-	2018	No	-	-
F3)	2016	0/10	-	0/9	0/1	2018	No	-	-
G	2018	1/83	0/17	0/25	1/41	2019	No	-	-
H	2018	4/22	1/10	1/5	2/7	2019	No	-	-
I	2018	1/161	0/111	0/20	1/30	2020	No	-	-
J	2019	2/221	0/175	-	2/46	2020	Yes	2024	25/258
K	2019	27/53	-	-	27/53	2020	No	-	-
L	2019	14/61	1/27	-	13/34	2020	No	-	-
M	2020	2/118	2/62	0/13	0/43	2021	No	-	-
N	2020	54/275	28/139	25/123	1/13	2021	No	-	-
O	2021	11/104	8/90	1/4	2/10	2022	No	-	-
P	2022	2/15	2/15	-	-	2023	No	-	-
Q	2022	2/75	2/75	-	-	2023	No	-	-
R	2022	16/30	10/18	6/12	-	2023	No	-	-

1. Number of animals positive for CWD/Number of animals tested2. Cervids were restocked on these farms after disease control measures were implemented and veterinary approval was granted. There are currently no specific regulations regarding the animals introduced after this approval.3. While CWD was not detected in all 10 animals tested, one of the soil samples collected from this farm tested positive for CWD by PMCA analysis before implementing the disease control measures. An epidemiological investigation into this farm revealed that several animals had died showing clinical signs suggestive of CWD, but they had not been submitted for CWD testing.

Following the implementation of CWD control measures, including topsoil removal and thorough environmental treatment with 2N NaOH, prion seeding activity on these premises were assessed using PMCA. To this end, two sets of soil samples collected 1–2 months apart (with only one set of soil samples during the period of 2016 – 2018) were analysed using PMCA. Specifically, we performed 10 repeated PrP^Sc extractions from each soil sample, and a sample was considered CWD-positive if a PrP^Sc signal was detected in any of the 10 extracts following three rounds of PMCA. As recently demonstrated [33], PMCA combined with this refined extraction technique successfully detected PrP^Sc in soil samples that had been incubated for 2 to 3 years following exposure to 0.001% CWD brain homogenate (Figure 1). PMCA seeding activity detected in soil samples from unmanaged CWD-affected farms (i.e., before implementation of CWD control measures) was no longer detectable in corresponding soil samples collected after the control measures were implemented (Figure 2). Out of 215 soil samples collected from the 18 farms after implementing the CWD control measures, only 3 samples from 3 farms (Farms I, N and O) tested positive for PrP^Sc signals in PMCA resulting in a positivity rate of 1.4% (Table 2). This rate was remarkably low compared to the previously reported PMCA positivity rate of 34.8%, which was observed in soil samples collected from unmanaged CWD-affected farms and analysed with the identical PMCA methodology (Table 2). Collectively, our findings strongly support the effectiveness of the implemented control measures in significantly reducing or inactivating CWD prions. The three farms on which PMCA seeding activity was detected underwent three additional rounds of 2N NaOH treatment at intervals of 2–3 weeks, followed by further PMCA analysis of two sets of soil samples collected 1–2 months apart. Restocking cervid animals on these remediated farms was permitted after confirming the absence of prion seeding activity through PMCA analysis.

Figure 1.

Detection of prions in soil experimentally exposed to CWD-positive brain homogenates. Soil collected from a CWD-free elk farm was exposed to a 0.001% CWD brain homogenate, and then incubated at 22°C for either 2 or 3 years. Each soil sample underwent 10 repeated PrP^Sc extractions as described in the methods section. The presence of PrP^Sc in the 10 extracts obtained from soil incubated for 2 years (A) or 3 years (B) was assessed after 3 rounds of PMCA. The third round PMCA products were digested with proteinase K then analysed by Western blotting with anti-PrP rabbit serum raised against bovine PrP 106–122 peptide. Molecular weight markers are shown on the left.

Figure 2.

Elimination of prion seeding activity in the soil of CWD-affected farms following implementation of CWD control measures. The CWD control measures, including topsoil removal and repeated environmental treatment with 2N NaOH, were implemented on farms where CWD-positive animals had been detected. Soil samples were collected from the feeding trough, barn, pen and path before (A) and after (B) implementing the CWD control measures. Each soil sample underwent 10 repeated PrP^Sc extractions as described in the methods section. The presence of PrP^Sc in these extracts was assessed after 3 rounds of PMCA. The third round PMCA products were digested with proteinase K and then analysed by Western blotting with anti-PrP rabbit serum raised against bovine PrP 106–122 peptide. Molecular weight markers are shown on the left.

Table 2.

Effective inactivation of prion seeding activity in farms affected by CWD following disease control measures including topsoil removal and environmental treatment with 2N NaOH.

Classification	Farm1)	Soil sample2)	Remarks
CWD-affected farms without disease control measures	8/8 (100%)	16/46 (34.8%)	Reference [33]
CWD-affected farms following disease control measures	3/18 (16.7%)	3/215 (1.4%)

1. Number of farms where one or more soil samples were PMCA-positive/Number of farms where soil samples were collected for PMCA analysis2. Number of soil samples positive in PMCA/Number of total soil samples examined by PMCA.

Among the 18 remediated farms in which cervid animals were restocked, CWD recurred at two farms (Farms A and J, Table 1). On Farm A, CWD was identified five years after the reintroduction of animals in 2017. In the 2022 recurrence, while all 27 sika deer on this farm tested negative for CWD, 13 out of 34 elk were found to be CWD-positive. The overall positivity rate for cervids on Farm A was 21.3%. On Farm J, CWD was identified four years after the reintroduction of animals in 2020. Twenty five out of 258 elk tested positive for CWD on this farm (prevalence: 9.7%). No other cervid species was raised on this farm at the time of recurrence of CWD.

In this study, we reported on the CWD recurrence status at the 18 remediated farms in which cervids were reintroduced after implementing CWD control measures which included topsoil removal and environmental treatment with 2N NaOH. Although these measures were shown to effectively eliminate detectable CWD prion seeding activity in the soil of CWD-affected farms, we observed CWD recurrence at two of the 18 remediated farms 4 to 5 years after restocking with cervids. The occurrence of CWD-positive animals at the two remediated farms could be due to residual prions in the environment, despite the implementation of disease control measures [17,23,36]. Supporting this possibility, controlled studies on scrapie-affected farms have documented recurrence of the disease even after multiple rounds of thorough sodium hypochlorite treatment [37–39], highlighting the difficulty of eradicating scrapie prions that are known to persist in the environment for many years [40,41]. On these farms, environmental dust contaminated with scrapie prions has been implicated as the source of re-infection [38,42].

Notably, the relative prevalence of CWD prior to depopulation observed in our study did not predict the likelihood of disease recurrence. For example, CWD recurred at Farm J with an initial prevalence below 1%, while no reoccurrence has been observed at Farms K and R with prevalence above 50%. Considering the unrestricted movement of animals between cervid farms in South Korea (also discussed below), this discrepancy might be explained by the recent introduction of subclinically infected cervids into those high prevalence farms, limiting the time for substantial environmental prion accumulation. Conversely, the low prevalence observed at farms such as Farm J may reflect underreporting or failure to submit suspected cases for diagnostic testing, as described in our recent publication [33].

An alternative explanation for CWD recurrence at two of the 18 remediated farms is that, following remediation, cervids already infected with CWD in its early stages had been inadvertently introduced to these two farms and remained undetected for some time. This alternative possibility may be particularly relevant to the two farms in which CWD recurred, given the annual detection of CWD cases at multiple farms and the unrestricted movement of animals between cervid farms in the absence of an animal tracking system for farmed cervids in the Republic of Korea.

In conclusion, the control measures including topsoil removal and environmental treatment with 2N NaOH, and subsequent confirmation of the absence of PMCA seeding activity, may represent promising preventive strategies prior to restocking animals into CWD-affected farms. However, given the limited duration of post-restocking surveillance in some farms and the known potential for delayed recurrence of CWD, the long-term effectiveness of these measures remains to be confirmed through continued surveillance. To more effectively prevent and eradicate the spread of CWD in the Republic of Korea, it may also be necessary to implement additional measures such as a traceability system for farmed cervids along with the more stringent enforcement of existing disease control measures.

Materials and methods

Ethical statement

This study utilized tissue samples collected from elk and deer that were either culled or found deceased during surveillance conducted by the Animal and Plant Quarantine Agency (APQA). The animals were not specifically sacrificed for this study. In accordance with the Korean Act on the Prevention of Contagious Animal Diseases, chronic wasting disease (CWD) is designated as a Type 2 infectious disease, and routine disease control measures, including surveillance and diagnostics, are conducted under legal provisions. Thus, no approval from the APQA Institutional Animal Care and Use Committee (IACUC) was required for actions associated with CWD control measures. The enforcement of CWD control measures was performed after obtaining verbal consent from farm owners. All procedures involving laboratory mice for PMCA study were approved by the Institutional Animal Care and Use Committee (IACUC) of the Animal and Plant Quarantine Agency (Permit No: APQA-2018–861).

CWD diagnosis

The diagnosis of CWD in cervid animals was made by the WOAH Reference Laboratory for CWD in the Animal and Plant Quarantine Agency (APQA) in the Republic of Korea. CWD testing of cervid animals was conducted on obex and retropharyngeal lymph node (RPLN) tissues using the IDEXX HerdChek BSE-Scrapie Antigen Test. The diagnosis of CWD was confirmed by TeSeE Western Blot (Bio-Rad) assay.

CWD control measures

When farm owners found cervids dead or sick showing clinical signs consistent with CWD (e.g. loss of body condition, lack of coordination, excessive salivation, excessive drinking and urination) on their farms and reported them to local veterinary authorities, personnel in local veterinary authorities collected tissue samples (obex and retropharyngeal lymph nodes) and sent them to APQA for CWD testing. Once CWD was confirmed, all remaining cervid animals on the affected farm were immediately culled and CWD testing was conducted as described above. Additionally, all animals on other farms which had either received cervids from, or sent cervids to, this CWD-positive farm within the last 5 years were also culled and tested for CWD. Following herd depopulation on positive farms, the upper layer of soil was removed to a depth of 30 cm in areas surrounding barns, feeding troughs and manure storage sites, and to a depth of 5 cm in other areas. All burnable materials (such as bedding and remaining feed) within the farms affected by CWD were incinerated. The scraped topsoil, incineration ash and animal carcasses were then buried on-site at each farm after thorough application of 2N NaOH, and the burial sites were subsequently separated from other areas of the farm by a metal fence. After these measures, the soil and other environmental components of each farm were thoroughly treated with 2N NaOH at least three times, with each treatment occurring at intervals of 2–3 weeks. Subsequently, soil samples were collected and analysed as described below.

Experimental contamination of soil

Experimental contamination of farm soil was conducted as described previously [29,33]. Briefly, soil collected from a CWD-free farm in the Republic of Korea was treated with a CWD-positive elk brain homogenate. This CWD-free farm soil, characterized by a silt loam texture, a pH of 5.5, and a cation exchange capacity (CEC) of 20.2 cmol/kg, is considered representative of typical soil properties found in Korean farmland [29,43]. After saturating 28 g of soil with 5 mL of distilled water in a 50 mL plastic conical screw cap tube and incubating it for 16 hours at room temperature, 3 mL of 0.001% CWD brain homogenate was added weekly for 4 months. Given that the CWD brain homogenate used for soil contamination had an infectivity titre of 10^5.6 LD₅₀ per gram of brain tissue [33], the addition of 3 mL of 0.001% homogenate weekly for 4 months (48 mL total) would be expected to result in an estimated accumulation of 192 LD₅₀ of infectivity in the 28 g of soil. The tubes containing CWD-contaminated soil were maintained at 22°C with 50 ~ 55% humidity.

Soil collection in cervid farms

After implementing the control measures on CWD-affected farms including topsoil removal and thorough treatment with 2N NaOH, soil samples were collected for protein misfolding cyclic amplification (PMCA) analysis if the farm owners intended to restock animals. Soil collection was conducted by the personnel of local veterinary authorities, as described previously [33]. Briefly, soil was collected from four specific locations at each farm: the feeding trough, barn, pen, and path. At each location, soil was collected from at least five spots, placed into a zipper bag (27 cm ×30 cm), and sent to the Animal and Plant Quarantine Agency for PMCA analysis.

Soil extraction and PMCA analysis

PMCA analysis using 10 sequential extracts per soil sample was conducted as described previously [33]. In brief, 0.5 g of soil was transferred into a 15 mL tube containing 3 mL of PBS, vortexed at maximum speed for 20 seconds, and centrifuged at 1,000 × g for 5 minutes. A 500 μL supernatant was collected as the first extract. An equal volume (500 μL) of fresh PBS was then added to the remaining suspension in the 15 mL tube. The vortexing and centrifugation steps were repeated to obtain the second extract (500 μL). This process was repeated 8 additional times to obtain the third to tenth extracts. All 10 sequential extracts from each soil sample were used as PMCA seeds. For use as PMCA substrate, brains were collected from transgenic mice expressing elk prion protein that were kindly provided by Dr. R. Rubinstein (Institute for Basic Research in Developmental Disabilities, USA) [44]. The brains were then prepared as 5% (w/v) homogenates in PMCA conversion buffer and then clarified at 2,000 × g for 1 min. Each PMCA reaction mixture was prepared as 80 μL of substrate, 20 μL of seed and two Teflon beads, and then incubated in a water bath set at 37°C for 16 hours. Subsequently, 56 cycles of 30 s sonication and 9 min 30 s incubation were conducted using a microsonicator (Misonix model 4000) at 75% amplitude, with the reaction tubes immersed in the 37°C water of sonication bath. After transferring 70 μL of the first-round PMCA products into new tubes for future analysis, 70 μL of fresh substrate was added to the original tubes and subjected to a second round of PMCA cycles. This procedure was repeated one more time (third PMCA round). The PMCA products were digested with 200 µg/ml proteinase K (PK) at 37°C for 1 hour, and subsequently analysed by Western blotting using rabbit anti-PrP serum raised against a synthetic peptide corresponding to bovine PrP residues 106–122. Each soil sample was classified as CWD-positive if a PrP^Sc signal was detected in any of the 10 extracts after three rounds of PMCA. The molecular weights of PK-resistant core fragments of PrP^Sc were determined by comparison with Amersham ECL Rainbow molecular weight markers (RPN800E, GE Healthcare).

Funding Statement

This research was supported by the Animal and Plant Quarantine Agency, Ministry for Agriculture, Food and Rural Affairs [B-1543085-24–25–01].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data supporting the findings of this study are available from the corresponding author, Hyun Joo Sohn upon reasonable request.