Chronic wasting disease (CWD) prion detection in environmental and biological samples from a taxidermy site and nursing facility, and instruments used in surveillance activities

Abstract

Chronic wasting disease (CWD) is a transmissible prionopathy affecting free-ranging and captive cervids. CWD is thought to spread through both direct and indirect transmission mechanisms. Along this line, human activities have not been thoroughly explored for their potential to spread this disease. One area of concern involves taxidermy procedures and surveillance activities as handled animals or carcasses are of unknown CWD statuses. Worrisomely, taxidermy facilities can act as foci of prion infectivity if appropriate biosecurity practices are not implemented. In this study, we evaluated the presence of infectious prions in a taxidermy facility that was possibly exposed to CWD prions. To determine this, we collected biological and environmental specimens from this site and screened them using the protein misfolding cyclic amplification (PMCA) technique. Additionally, we swabbed different surfaces possibly exposed to CWD-infected animals or carcasses. We report the presence of prions in i) waters used to digest tissues from deer carcasses, ii) soils that were in contact with the previously mentioned waters, iii) dermestid beetles used to clean skulls, iv) other insects found in the beetle shed, and iv) dumpsters where animal carcasses were disposed. Additionally, we report that surgical materials used in surveillance practices may also hold CWD prions, even after being washed with aqueous solutions. All these results suggest that CWD prions may be disseminated due to human practices and that protocols should be established to decontaminate potentially contaminated materials.

INTRODUCTION

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy that affects multiple cervid species, including mule deer, white-tailed deer, elk, reindeer, and moose, among others(Escobar et al., 2020). CWD is widely distributed in the United States and Canada in captive and free-ranging cervids. CWD is characterized by long incubation periods, followed by a relatively short clinical phase(Rivera et al., 2019; Williams, 2005). CWD is fatal in all cases when animals are clinical, and to date, there is no feasible treatment for curing either CWD or any other prion disease(Haley & Hoover, 2015). Unfortunately, CWD is rapidly expanding in both number of animals affected and geographical areas(Bartz et al., 2024; National Wildlife Health Center, 2025). Considering this, efficient containment and diagnostic testing strategies are urgently needed.

CWD can spread within animal populations with relative ease(Otero et al., 2021). The propagation of this disease is thought to be more efficient in captive animals considering the higher animal densities found in these environments(Mathiason et al., 2009; Rivera et al., 2019; Shannon L. Bartelt-Hunt & Jason C. Bartz, 2017; Zabel & Ortega, 2017). Nevertheless, CWD transmission is still highly efficient in free-ranging cervid populations. CWD transmission is thought to occur mostly through direct animal contacts, or indirectly through the exposure of naïve animals to contaminated environments(Denkers et al., 2013; Escobar et al., 2020; Haley et al., 2011; Mathiason et al., 2009; Miller Michael W. et al., 2004; Soto et al., 2024). Environmental components, including inert materials and living organisms, are thought to play relevant roles in the spread of CWD(Escobar et al., 2020; Jacobson, Lee, Mckenzie, et al., 2010; Pritzkow et al., 2018). Different studies have shown that infectious prions can enter the environment through decaying carcasses from diseased animals(Soto et al., 2023), gestational tissues released during parturition(Bravo-Risi et al., 2021), saliva(Haley et al., 2011), and excreta(Bravo-Risi et al., 2023). Once released, prions can bind to soil and other natural and manmade components(Johnson et al., 2006; Smith et al., 2011) and become available to susceptible animals. Other organisms, including plants, predators, scavengers, parasites, insects, and annelids may also participate in these processes by interacting with contaminated environmental components or animal tissues(Carlson et al., 2023; Fischer et al., 2013; Inzalaco et al., 2023; Pritzkow et al., 2015; Soto et al., 2024). Overall, multiple components and processes are involved in CWD prion transmission and dissemination. However, most of them are understudied and/or assayed in laboratory-controlled conditions. As consequence, the specific contribution of each process/element in CWD environmental contamination is contentious.

An essential factor to consider in the spreading of CWD prions includes human activities(Mori et al., 2024). Along this line, regulatory agencies already implemented or are currently considering regulations in several of these activities including containment, animal processing, movement of animals, disposal of carcasses, and breeding practices, among others(Mateus-Pinilla et al., 2013; Mysterud & Edmunds, 2019). Although most of these practices have a potential for CWD transmission, their overall importance in CWD epidemiology is understudied. One example of this involves taxidermy procedures. Deer are regarded as one of the most popular game animals, generating a high economic output and involving various activities including deer breeding, taxidermy, meat processing, and others(Arnot et al., 2009; Bishop, 2004) (https://nri.tamu.edu/media/3968/economic-values-of-white-tailed-deer-in-texas-2022-survey-part-i.pdf). Hunters active in areas with reported CWD cases are requested to follow state, wildlife and public health recommendations(Carlson et al., 2018). In general, all agencies strongly suggest CWD animal testing before processing. Many hunters process game at private facilities and, because of no statutory authority or lack of awareness, CWD-relevant guidelines or rules may not exist for meat processing and taxidermy facilities despite their capacity to transmit CWD. Specifically, the movement of carcasses or carcasses parts from areas with a high CWD incidence to areas with little or no incidence may facilitate CWD introduction and spread. In addition, it should be considered that inappropriate disposal of carcasses and animal products may also contribute to the dissemination of this disease(Angers et al., 2009; Benestad et al., 2016). Considering the above-mentioned scenarios, proper surveillance of CWD and management of activities involving potentially infected animals and animal-derived products might help to contain it. If left unchecked, there could be profound negative impacts on human health and wildlife conservation.

In this study, we evaluated the presence of CWD prions in a taxidermy facility where deer heads are processed to prepare skull mounts. Analyses were made using the protein misfolding cyclic amplification (PMCA) technique in various biological and environmental samples. In addition, we also tested the persistence of CWD prions in surgical materials used in surveillance procedures. Our results show, for the first time, CWD-prion detection in a potentially exposed taxidermy facility. In addition, we also tested the persistence of CWD prions in surgical materials used in surveillance procedures. This information has important implications on several fronts of CWD research including environmental spreading, human-related practices, and exposure of humans to infectious animal prions.

MATERIALS AND METHODS

Samples:

The samples were collected from a taxidermy and free-ranging deer rehabilitation facility located on the same property in Val Verde County, Texas. Texas Parks and Wildlife Department (TPWD) staff were initially notified of CWD clinical suspect on this property; this animal was ultimately euthanized and confirmed positive for CWD by the National Veterinary Services Laboratory on January 5^th, 2020. Samples were collected from different areas on this property where deer heads were processed to make skull mounts in close proximity to rehabilitated deer. Water samples were taken from buckets used to soak deer heads along with soil, plant leaves, and grass from other facility areas. In addition, insects such as dermestid beetles and flies were collected from different areas suggesting direct, indirect, or no contact with deer materials. Sheep and deer fecal samples (collected from living animals present at the facility at the moment of collection) and pieces of deer head tissues found in instruments involved in taxidermy practices were also collected. In addition, swabs from different surfaces were obtained. These included: i) a trailer bed where animals’ heads or whole carcasses were transported, ii) hooks holding deer heads, iii) waterer containers, and iv) the facility's primary dumpster. A summary of the samples collected from this facility and tested in this study are summarized in Table 1. In addition, surgical materials used in CWD surveillance practices were tested by swabbing in three different scenarios: i) after being used, and after serial cleaning procedures with ii) phosphate buffer saline (PBS) and iii) a 50% bleach solution. The CWD prion content in the PBS used for these washes was also collected for testing. Sample preparation before PMCA was conducted as described below.

Table 1.

Summary of samples collected in a taxidermy and rehabilitation facility suspected to be exposed to CWD-infected materials.

Sample	Area	Sampling	PMCA Sample
Truck bed	De-meat	Swab	Swab piece
Animal brain piece	De-meat	Tissue	Tissue piece
De-meat area	De-meat	Swab	Swab piece
Hook	De-meat	Swab	Swab piece
Soils	Maceration	20% w/v homogenate	10 μL
Waters	Maceration	200 μL	Sarkosyl/ultracentrifugation pellet
Dermestid beetles	Maceration	20% w/v homogenate	Sarkosyl/ultracentrifugation pellet
Nasal bot	Maceration	20% w/v homogenate, 200 μL	Sarkosyl/ultracentrifugation pellet
Black fly	Beetle shed	20% w/v homogenate, 200 μL	Sarkosyl/ultracentrifugation pellet
Dermestid beetles	Beetle shed	20% w/v homogenate, 200 μL	Sarkosyl/ultracentrifugation pellet
Dumpster	Facility Entrance	Swab	Swab piece
Waterer	Random	Swab	Swab piece
Soil	Random	20% w/v homogenate	10 μL
Grass	Random	20% w/v homogenate	10 μL
Deer saliva	Random	Swab	Swab piece
Deer feces	Random	20% w/v homogenate	10 μL
Sheep feces	Random	20% w/v homogenate	10 μL
Steel toe boots	Random	Swab	Swab piece

Sample homogenization.

Soils, dermestid beetles, flies, sheep and deer feces, plants, grass, and animals’ tissues were prepared at a concentration of 20% weight/volume (w/v) in PBS (Hyclone PBS, GE Healthcare Life Sciences), supplemented with a protease inhibitor cocktail (without EDTA, Roche). These samples were homogenized in a Precellys^® 24 homogenizer, using Precellys^® hard tissue homogenizing CK-28-R 2 mL tubes. The homogenization process was carried out using program # 5, repeating it three times for all samples.

Swabbing of materials present in a taxidermy facility.

Cotton swabs (Swab/Tube Applicators Fisherbrand^™) were submerged in PBS (Hyclone PBS, GE Healthcare Life Sciences) and then used to swab the above-mentioned surfaces. The procedure was conducted by swabbing each surveyed surface five times at random areas within the surface. All swabbing was performed in duplicate. After swabbing, cotton sticks were individually stored in 15 mL screw-capped tubes, labeled, and transferred to UTHealth-Houston facilities at 4 °C. Then, cotton sticks were stored at −20 °C until used.

Swabbing of surgical materials used for CWD surveillance.

Surgical scissors and forceps used in a CWD surveillance activity by Texas Parks and Wildlife Department (TPWD) were included in this study (Medina county, Texas). After completing the procedure, each set of surgical materials, used in a single animal, was stored in sealed plastic bags and transferred to UTHealth-Houston facilities. From these materials, a single set of surgical tools was exposed to a CWD positive animal. The CWD contaminated scissor and forcep, still containing blood and tissues pieces, was swabbed as described above. Then, the scissor was extensively washed in PBS (Hyclone PBS, GE Healthcare Life Sciences), air dried and swabbed again. The PBS used in the washing process was also collected for further analyses. Finally, the contaminated surgical materials were incubated overnight with a 50% bleach solution, rinsed with PBS, air dried, and swabbed again. The same procedure was repeated with surgical materials used in CWD negative animals.

PrP^Sc enrichment using sarkosyl-based precipitation.

Sample homogenates (200 μL) were incubated with 1 volume of 20% sarkosyl for 1 hour at room temperature. Then, samples were centrifuged at 100,000 x g for 1 hour at 4 °C. The supernatants were gently discarded, and the pellets were washed in 2 volumes (400 μL) of PBS (Hyclone PBS, GE Healthcare Life Sciences), supplemented with a protease inhibitor cocktail (without EDTA, Roche). The samples were centrifuged again at 100,000 x g for 30 min at 4 °C. The final pellets were directly resuspended in PMCA substrate.

PrP^Sc enrichment using sarkosyl-based precipitation.

Sample homogenates (200 μL) were incubated with 1 volume of 20% sarkosyl for 1 hour at room temperature. Then, samples were centrifuged at 100,000 x g for 1 hour at 4 °C. The supernatants were gently discarded, and the pellets were washed in 2 volumes (400 μL) of PBS (Hyclone PBS, GE Healthcare Life Sciences), supplemented with a protease inhibitor cocktail (without EDTA, Roche). The samples were centrifuged again at 100,000 x g for 30 min at 4 °C. The final pellets were directly resuspended in PMCA substrate.

Artificial contamination with CWD prions of different materials and surfaces.

CWD-free soil, grass, feces, nasal bots dermestid beetles and black flies were homogenized at 20% w/v in PBS. An aliquot of 100 μL of each homogenate was mixed with 10 μL of the brain extract of a terminally ill, CWD infected white-tailed deer at a 10⁻³ dilution (same stock as positive control mentioned below). CWD-free water (100 μL) was also mixed with the same quantity of prions mentioned above. Ten μL of these samples were tested in PMCA as explained below. Polyurethane and stainless steel surfaces were contaminated with 100 μL of the CWD brain extract (diluted to 10⁻³). Once dried, these surfaces were swabbed and tested by PMCA as described above. Negative controls included equivalent samples and surfaces exposed to brain extracts from CWD-free transgenic mice expressing the deer prion protein (Browning et al., 2004).

Substrate preparation for PMCA.

Mice (tg1536^+/+ mice(Browning et al., 2004)) were euthanized using CO₂ inhalation, following previously approved protocols. Transgenic mice brains were collected after cardiac perfusion with cold perfusion buffer (PBS (Hyclone PBS, GE Healthcare Life Sciences) supplemented with 5 mM EDTA (0.5M, pH 8.0, Molecular Grade, Promega^™)). Then, brains were collected, snap-frozen in liquid nitrogen, and stored at −80 °C until use. The brains were homogenized at a concentration of 10% w/v in conversion buffer (phosphate-buffered saline PBS (Hyclone PBS, GE Healthcare Life Sciences), supplemented with 1% Triton X-100 (Sigma-Aldrich), 150 mM NaCl (Sigma-Aldrich) and a protease inhibitor cocktail (cOmplete, Roche)). Homogenates were centrifuged at 805 x g at 4 °C for 1 minute. The supernatants were collected, vortexed, aliquoted, and stored at −80 °C until used. Before the PMCA reaction was started, the substrate was supplemented with 5 mM EDTA (0.5M, pH 8.0, Molecular Grade, Promega^™) and 0.025% v/v digitonin (5% Digitonin, Invitrogen).

PMCA procedure.

For the homogenized specimens, either 10 μL of the sample, or the direct pellet obtained after the sarkosyl-based concentration, were individually mixed with 90 μL of PMCA substrate. For swabs, ~0.0025 grams of each swab were cut with a disposable blade and directly mixed with 90 μL of PMCA substrate. In addition, ~0.003 grams of deer tissue collected from this facility were inserted into the 90 μL of PMCA substrate. The PMCA procedure was performed as previously described(Morales et al., 2012). Specifically, substrate-sample mixtures were submitted to 144 cycles of incubation and sonication (each PMCA cycle consisting of 29 min and 40 s of incubation and 20 s of sonication). The resulting samples were subjected to two or four additional rounds of PMCA (96 cycles each) by mixing 10 μL of the PMCA products of each round with new aliquots of PMCA substrate (90 μL). PMCA products were treated with proteinase K (PK, Sigma-Aldrich) and examined by western blotting as described below. As controls, each PMCA reaction set (testing approximately ten samples) included serial dilutions of a CWD brain of known PMCA activity and at least four unseeded reactions (NC). These controls are interpreted and used as follows: a) Results from a PMCA set are discarded if any negative control tests positive. b) PMCA data is usually evaluated after three rounds. A maximum amplification up to a dilution of 10⁻¹¹ is expected in this case. Any amplification beyond that dilution may indicate cross contamination. In this scenario, the whole PMCA set is discarded regardless of negative results in the negative control samples. c) Some samples require five PMCA rounds as, due to their inhibitory nature, may need additional cycles of incubation and sonication to display signals. In those cases, amplifications ranging from 10⁻¹¹ to 10⁻¹³ dilutions are expected in the positive control samples.

Proteinase K (PK) treatment.

To assess the potential presence of disease-associated prion proteins (PrP^Sc), 20 μL of PMCA products were treated with 100 μg/mL of proteinase K (PK, Sigma-Aldrich) at 37 °C and 450 rpm shaking (using an Eppendorf thermomixer) for 90 min. PK reactions were stopped by adding 10 μL of LDS sample buffer (Invitrogen^™ NuPAGE^™ LDS Sample Buffer (4X)) and exposure to 90 °C for 10 min.

Electrophoresis and western blot.

Electrophoresis was performed in NuPAGE 4-12% or 12%, Bis-Tris gels (Invitrogen) with MOPS buffer (NuPAGE^™ MOPS SDS Running Buffer (20X)) at 80 V for 20 min., and then 140 V for 1 h and 40 min. Fractionated proteins were transferred to nitrocellulose membranes (GE Healthcare Amersham) at 100 V for 60 min. and 4 °C using transfer buffer 1x (25 mM Tris, 192 mM glycine, 10% methanol). Unspecific antibody binding to the membranes was blocked by incubating them in a 10% w/v non-fat milk solution. Blocked membranes were probed with primary monoclonal 6H4 (Prionics) or 8H4 (Abcam) antibodies diluted 12,500 times in washing buffer for one hour at room temperature. Later, membranes were washed to remove un-specifically bound primary antibody particles and incubated with a polyclonal Anti-Mouse IgG (whole molecule)–Peroxidase antibody produced in sheep (Sigma-Aldrich) diluted 1:3,000 (prepared in PBST) for 1 h at room temperature. The membrane was washed three times for 10 min. with PBST. Finally, membranes were developed using ECL (GE Healthcare Amersham) following the manufacturer’s recommendation, and signals were visualized in a ChemiDoc Imaging System (BIO-RAD).

RESULTS

Screening of CWD prions in environmental and processing elements from a taxidermy facility.

The European mount technique is a widely utilized taxidermy practice aiming to produce a skull of an animal that is free of tissue. The first critical step in making a European mount is removing the flesh from the animal’s head. This is achieved by removing the majority of the external flesh, eyeballs, ears, and the brain. Then, the de-meated skull is subjected to a maceration process using bacteria. The purpose of this step is to soften and degrade any remaining tissue on the skull. This maceration process involves leaving the head of the animal soaking for several days or weeks in a container with water containing bacteria that will expedite the process. This practice is usually performed in the open air. An ideal temperature of 35 °C must be reached for the bacteria to stay alive and reproduce. Water is periodically added as water evaporates from the container; however, it is not completely replaced so as to maintain the bacteria populations needed for the maceration process. Once the maceration process is complete, the water used in this process is typically poured in the soils adjacent to the processing area.

Following maceration, the skull is removed from the water container and residual tissue may be air compressed from the sinuses or skull cavity. Afterwards, the skull is rinsed under clean running water, and allowed to dry. Usually, the latter step is done inside a drying room, free of light, to prevent the bones from cracking. Subsequently, to deep clean the bones, dermestid beetles are used. These dermestid beetles are predatory insects that eat dead and decaying meat, preferably dried(Kadej et al., 2023; Matuszewski & Madra-Bielewicz, 2022).

A strong colony of more than 1,000 beetles can clear a skull in about 5 days (https://bonesandbugs.com). Considering the above-mentioned handling, multiple elements and methods used in these practices may contribute to CWD prion spillovers if contaminated carcasses are improperly handled or disposed. Currently, limited regulations for mitigating CWD transmission related to taxidermy practices are in place. For this purpose, we collected and screened multiple biological and inert environmental samples from a taxidermy and deer rehabilitation facility that was suspected to handle CWD-infected cervid carcasses.

We spatially divided the areas of this facility, depending on the procedures carried out. Specifically, these included the de-meat area (where the most significant amount of tissue is extracted) (Figure 1), the maceration area (where the skulls are soaked in the bacteria-enriched water) (Figure 2), and the beetle shed area (where is the skulls are dried and exposed to dermestid beetles) (Figure 3). In addition, different samples were taken from other locations of this facility, including the dumpster were most of the taxidermy-derived waste is deposited. The different samples (n = 41) were collected either directly or by swabbing (Table 1). The sample number consider replicas from some samples.

Figure 1. Sampling of the taxidermy de-meating area.

A) Schematic representation of samples collected in the de-meating area. B) Evaluation of PMCA products of the samples depicted in (A) via western blotting. Numbers at the right of this panel represent molecular weight markers. All samples were treated with PK with the exception of “PrP^C” that is used as a control of electrophoretic mobility and antibody reactivity. C) Summary of the PMCA results obtained for these samples. Numbers in (A), (B) and (C) represent the same samples.

Figure 2. Environmental specimens collected from a taxidermy maceration area.

A) Schematic representation of samples collected in the maceration area. B) Evaluation of PMCA products of the samples depicted in (A) via western blotting. Numbers at the right of this panel represent molecular weight markers. All samples were treated with PK with the exception of “PrP^C” that is used as a control of electrophoretic mobility and antibody reactivity. C) Summary of the PMCA results obtained for these samples. Numbers in (A), (B) and (C) represent the same samples. Red fonts represent PMCA positive samples.

Figure 3. Insects collected in a taxidermy beetle shed (drying area for skulls).

A) Schematic representation of the insects collected in the beetle shed. B) Evaluation of PMCA products of the samples depicted in (A) via western blotting. Numbers at the right of this panel represent molecular weight markers. All samples were treated with PK with the exception of “PrP^C” that is used as a control of electrophoretic mobility and antibody reactivity. C) Summary of the PMCA results obtained for these samples. Numbers in (A), (B) and (C) represent the same samples. Red fonts represent PMCA positive samples.

In the de-meat area (Figure 1A), we took swab samples from the truck where dead animals, and heads are transported and partially processed. We also swabbed hooks where the heads are hanged during the process. We also collected pieces of tissue in this area (on the ground) and took them as additional specimens. We additionally swabbed other surfaces in this area that were in contact with animal tissues. All the samples tested in the de-meat area were negative for prion detection using PMCA (Figure 1B and C), suggesting either a low or complete absence of prion load in this area, an inaccurate sampling, or the inactivation of prions due to periodic decontamination procedures.

The maceration area (Figure 2A) was outdoors, and included approximately 40 containers with submerged animal heads. We collected water samples from some of these containers. In addition, since this place was outdoors, we took soil samples adjacent to the water containers (the water from these containers was periodically poured in these soils), pieces of plants (leaves and roots) from this place, and some insects around the site, such as nasal bots and beetles. The PMCA analysis of these samples showed presence of CWD prions in some of the water specimens collected from the skull soaking containers (Figure 2B and C). In addition, some of the soil samples collected from this area were also positive by PMCA. All other specimens, including plants, were negative.

As mentioned, additional samples were collected from the place where the skulls were dried (beetle shed, Figure 3A). These samples included dead flies found in the floor of the room, and dermestid beetles from the colony used to clean the animals’ carcasses. The dermestid beetles were housed in a closed, unplugged chest freezer of approximately 120 cm long x 60 cm wide x 60 cm high. Interestingly, all these insects provided strong PMCA signals, suggesting a relevant load of CWD prions on them (Figure 3B and C). Unfortunately, we were unable to test the skulls currently present in this area as this could interfere with the skull mount process.

Finally, a plethora of other potentially relevant samples were collected from this facility (Figure 4A). Feces and saliva were taken from a sheep and a white-tailed deer that roamed the site at the time of sampling. In addition, soil samples and plants from areas not directly exposed to deer materials, and fecal pellets found at random places within the facility, were taken. We also swabbed the outsole of the boots used by the owner, as well as a waterer and a feeder used by free-ranging animals visiting the property. The facility’s primary dumpster was also swabbed. This dumpster was used to collect all types of waste in this facility, including deer tissues, carcasses, and other elements derived from the taxidermy procedures. From all these samples, only the dumpster swabs provided positive detection using the PMCA technique (Figure 4B and C). These data suggest that although CWD prions are present in this facility, they are confined to the areas in direct contact with the animal carcasses.

Figure 4. Additional samples collected in different places across the taxidermy facility.

A) Schematic representation of the additional samples collected in this specific facility. B) Evaluation of PMCA products of the samples depicted in (A) via western blotting. Numbers at the right of this panel represent molecular weight markers. All samples were treated with PK with the exception of “PrP^C” that is used as a control of electrophoretic mobility and antibody reactivity. C) Summary of the PMCA results obtained for these samples. Numbers in (A), (B) and (C) represent the same samples. Red fonts represent PMCA positive samples.

Table 1 includes the type of sample, the area where the sample was taken, and the sampling type and processing. In summary, PMCA seeding activity was detected in 5 out of 41 specimens using the PMCA technique (soils and waters in the maceration area, dermestid beetles and flies in the beetle shed, and the main facility dumpster). These data were collected after 5 PMCA rounds. For these samples, all PMCA rounds were later visualized to assess whether signals appeared earlier, as a proxy of a higher prion load. Two of these samples provided prion seeding activity in PMCA round 3 (dermestid beetles and dumpster swab, Supplementary Figure 1).

All the PMCA reactions described in this experiment were controlled by appropriate positive and negative controls as described in Materials and Methods and Supplementary Figures 2 and 3. The first set of controls included unseeded reactions (negative controls) and reactions spiked with CWD prions of known PMCA activity (positive) in the different endpoints (3 or 5 PMCA rounds) used in this study. All the controls used in this study behaved as expected, and matched the results presented in our previous publications (Bravo-Risi et al., 2023; Kramm et al., 2017, 2019, 2020; Soto et al., 2023, 2024, 2025). Additional controls included the artificial contamination of different CWD-free materials that are commonly found in places such as those sampled for this study. Specifically, water, soil, grass, feces nasal bots, dermestid beetles and black flies were artificially infected with CWD prions to assess whether each matrix interfere in the PMCA assay. Surfaces such as polyurethane and stainless steel were also contaminated. As controls, equivalent materials were exposed to a brain extract that devoid of CWD prions (Supplementary Figure 3). Our results show that while PMCA was able to detect prion contamination in all the CWD-exposed samples, none of the contaminated materials displayed positive signals (Figure 5). In summary, the multiple controls included in this study provide rigor and specificity for the results described for the taxidermy-derived specimens.

Figure 5. Detection of CWD prions in artificially contaminated materials.

A) Schematic representation of the CWD contamination strategy in different materials. As described, while some samples were homogenized before contamination, others were swabbed after prion exposure. All homogenates and swabs were tested in PMCA. Negative controls included materials exposed to a CWD-free brain extract. B) PMCA products of the samples tested in (A) were visualized using western blotting. Numbers at the right of the panels represent molecular weight markers. All samples were treated with PK with the exception of “PrP^C” that is used as a control of electrophoretic mobility and antibody reactivity. R1 and R2 represent two different replicates.

Overall, the data presented so far demonstrate the potential use of prion amplification methods in environmental screening. In addition, our results strongly suggest a role of taxidermy practices in the dissemination of CWD prions.

Screening of surgical tools used in CWD surveillance activities.

To further evaluate the role of human activities in CWD dissemination, we tested surgical tools that were used in surveillance practices in Medina County, Texas. We tested a total of 9 sets of surgical tools, including forceps and scissors, by swabbing them as shown in Figure 6A. Each surgical tool set was used in a single animal (white-tailed deer) to avoid potential cross-contaminations. In a first instance, all surgical tools, still containing blood and tissue debris were swabbed and blindly analyzed by PMCA as described above. Only a single set of surgical tools provided positive results in a third PMCA round (Figure 6B). It was later confirmed that these surgical materials were the only ones used on a CWD positive animal as evaluated by the gold-standard immunohistochemical (IHC) method. All other tools were used in CWD non-detect deer as evaluated by IHC. In order to mimic potential practices used in the field, the surgical tools were washed with an aqueous buffer (PBS) in order to remove all blood and tissue debris, air-dried, and swabbed again (Figure 6A). Surprisingly, PMCA detection was achieved in a second PMCA round, demonstrating a more efficient detection compared with the previous situation. We believe that tissues and blood present in the first swabbing partially interfered with the first PMCA analysis, explaining these differences in prion detection. Nevertheless, this data supports, in a practical scenario (field sample processing), that rinsing surgical tools with aqueous solutions is not enough to remove prion infectivity from surgical instruments. Worrisomely, we observed that the washing solution used in this experiment also contained PMCA activity (Figure 6B), demonstrating that the residual waters used in washing contaminated materials may also participate in the spreading of prions. Finally, treating these materials with a sodium hypochlorite (bleach) solution, eliminated prion seeding activity. As expected, all treatments subjected to surgical materials in contact with CWD non-detect animals were negative in the PMCA testing (Figure 6B).

Figure 6. Analysis of the persistence of CWD prions in surgical materials used in surveillance practices.

A) Schematic representation of the swabbing and washing procedures used to screen for prions in surgical materials. B) Evaluation of PMCA products of the samples depicted in (A) via western blotting. Numbers at the right of this panel represent molecular weight markers. “R01” to “R05” represent the products of the increasing PMCA rounds. “+” and “−” depict surgical materials used in CWD-positive and CWD non-detect animals, respectively. All samples were treated with PK with the exception of “PrP^C” that is used as a control of electrophoretic mobility and antibody reactivity.

DISCUSSION

CWD continues expanding in distribution and prevalence in North America. Hence, an essential factor in limiting the spread of this disease is to monitor and contain infectivity foci. This has proven to be difficult considering the limited diagnostic tools available to effectively identify the CWD infectious agent (prions). Identifying and mitigating the negative role that anthropogenic activities have in promoting CWD transmission is an essential step in developing a prevention strategy. The disease management protocol for white-tailed deer in many states generally involves the reduction of the localized population through culling, prohibition of feeding deer, and restrictions on the translocation of carcasses, among others(Mori et al., 2024).

It has been suggested that CWD has “colonized” multiple geographical areas through practices such as the transport of live deer or deer carcasses(Angers et al., 2009; Benestad et al., 2016; Fischer et al., 2013). One of these practices may include taxidermy.

In taxidermy, animal carcasses are handled, commonly, in outdoor settings. This involves the potential deposition of CWD infected materials in the environment, and the contact of naïve animals to infectious prions. In this study, we sampled different elements from a taxidermy and deer rehabilitation facility suspected to handle CWD-infected deer carcasses. The PMCA technique was used for this purpose considering its high sensitivity and specificity, and its proven efficiency in detection prions in environmental fomites such as plants(Carlson et al., 2023; Pritzkow et al., 2015), insects(Inzalaco et al., 2023; Soto et al., 2024), soils(Jacobson, Lee, Somerville, et al., 2010; Johnson et al., 2006; Smith et al., 2011), and swabbed surfaces(Soto et al., 2023). Prion seeding activity was detected in key elements of the taxidermy facility, including waters used to macerate skulls, soils where the maceration waters were poured, insects in contact with the deer skulls, and the main facility dumpster. The elements named above posit relevant issues in terms of environmental contamination.

As mentioned, the water-filled containers used for maceration were located outdoors, and free-ranging and rehabilitated deer had free access to this area. In fact, deer roaming this property were observed to submerge their heads in these containers. This may explain the relatively recent identification of CWD-infected animals in this area, specifically in spots close to this property. The potential role of these containers in CWD dissemination is strong, as infectious prions were identified in the water and surrounding soil. The latter is further supported in the fact that soil specimens collected far from the maceration area were negative for PMCA seeding activity. Dermestid beetles are also a concern, as when these insects expand to levels that are not practical to be maintained, they are commonly released or disposed of into the environment. The fact that flies collected in the beetle shed were also positive for CWD prions is worrisome, as this potential vector could transmit its infectious cargo at distances far from the original focus of infectivity. Our results also show negative prion detection in multiple areas of the facility, suggesting that prion dissemination was not ubiquitous but rather concentrated to areas with direct and/or repeated exposure to infectious materials. Importantly, we believe that the current data supports the accuracy of our screening method as prions were identified only in samples that have a direct, or extended contact with infected tissues.

Additionally, prions were detected in the primary facility dumpster. As expected, large quantities of potentially infected tissues or taxidermy-derived products were disposed in this container. If not properly contained or disposed of, infected materials may be carried and deposited to other areas, potentially creating new hotspots of CWD infectivity.

The main limitation of this study includes the sampling of a single taxidermy and deer rehabilitation site. Although this is an obvious limitation that does not allow us to generalize to other similar sites, it provides us with a good idea on how these practices can facilitate CWD transmission. While intuitive, regulating agencies should more effectively address proper disposal of possibly CWD infected materials in addition to reducing the risk of CWD exposure to, and movement of, rehabilitated CWD-susceptible species. Considering the data presented in this article, future research is warranted. More specifically, the overall infectivity of PMCA positive elements and their relevance to CWD epidemiology is still contentious. Current experiments in our laboratory are trying to evaluate the infectivity titers present in these samples and whether they are able to transmit disease to naïve deer. Finally, the PMCA methods used in this experiment were not optimized for each component. Previous reports suggest that different biological or inert samples require pre-treatments for optimal detection(Yuan et al., 2022). Here, we used untreated samples or simple concentration methods prior to PMCA detection. Future refinements of prion amplification assays may provide more sensitive and sample-specific platforms to detect CWD prions. Along the same line, the use of non-optimized PMCA protocols may suggest relevant levels of prion infectivity in these samples.

In the second part of this study, we were able to identify prions on surgical instruments used in field surveillance. We show that a simple rinsing with aqueous solution is not enough to remove prions from the surgical instruments. Moreover, prions appear to be disseminated to the environment through the disposal waters used to clean these instruments. These data concur with extensive experimental evidence demonstrating the efficient and recalcitrant binding of CWD prions to different surfaces(Pritzkow et al., 2018; Yuan et al., 2022). However, in this study we performed these analyses in field samples processing naturally infected, free-ranging animals. We also show that bleach treatments are sufficient to remove prion seeding activity from these materials.

CONCLUSION

In summary, the information provided in this report demonstrate how anthropogenic activities, specifically taxidermy practices, animal processing, and rehabilitation of CWD susceptible species, may facilitate CWD transmission through the environmental dissemination of CWD prions. This study, along with future research efforts characterizing the overall level of infectivity, provides relevant information on managing CWD and to control its rapid geographic expansion.

Supplementary Material

Supplementary Figure 1

Supplementary Figure 1. Samples providing positive PMCA signals in a third PMCA round. Representative western blot of PMCA products from tested samples in a third PMCA round. Red fonts and squares represent PMCA positive samples. All samples were treated with PK, with the exception of PrP^C that was used as a control of electrophoretic mobility and antibody specificity. Numbers at the right represent molecular weight markers.

Supplementary Figure 2

Supplementary Figure 2. Description of the control PMCA reactions. Positive PMCA controls, consisting of a 10% w/v brain extract from an experimentally infected, clinical white-tailed deer, was serially diluted (10⁻⁴ – 10⁻¹³) in PMCA substrate and submitted to incubation and sonication cycles (in parallel with the taxidermy site specimens). Negative controls (NC) represent unseeded reactions. This figure depicts representative results of these controls after 3 (A) or 5 ((B) and (C)) PMCA rounds. All samples were treated with PK, with the exception of PrP^C that was used as a control of electrophoretic mobility and antibody specificity. Numbers at the right represent molecular weight markers. MW: molecular weight marker.

Supplementary Figure 3

Supplementary Figure 3. CWD-free materials experimentally exposed to CWD prions. A) Photographs depicting the different materials that were exposed to brain extracts containing CWD prions or lacking them. B) Resulting homogenates from some of the materials shown in (A) (upper panels) or swabbed surfaces (lower panels). C) Summary table displaying the sample quantities tested in PMCA for each sample.

ABBREVIATIONS

CWD

chronic wasting disease

PBS

phosphate buffer saline

PK

proteinase K

PMCA

protein misfolding cyclic amplification

PrP^C

cellular prion protein

PrP^Sc

disease-associated prion protein

TPWD

Texas Parks and Wildlife Department