Infectious prions in brains and muscles of domestic pigs experimentally challenged with the BSE, scrapie, and CWD agents
Authors: Francisca Bravo-Risi, Fraser Brydon, Angela Chong, Kane Spicker, Justin J. Greenlee https://orcid.org/0000-0003-2202-3054, Glenn Telling, Claudio Soto https://orcid.org/0000-0002-3412-0524, Sandra Pritzkow, Marcelo A. Barria, Rodrigo Morales https://orcid.org/0000-0001-7766-5770 rodrigo.moralesloyola@uth.tmc.edu
ABSTRACT
Experimental studies suggest that animal species not previously described as naturally infected by prions are susceptible to prion diseases affecting sheep, cattle, and deer. These interspecies transmissions may generate prions with unknown host ranges. Pigs are susceptible to prions from different origins, including deer chronic wasting disease (CWD), sheep scrapie, and bovine spongiform encephalopathy (BSE). Here, we studied prions in brains and muscles from pigs previously infected with these different prion sources. Specifically, we measured the total prion protein (PrP) and PK-resistant PrP by western blot. Seeding activity in these tissues was evaluated using the protein misfolding cyclic amplification (PMCA) technique. We found that BSE-infected pigs contained substantially more seeding competent prions compared with those infected with CWD and scrapie. Moreover, the zoonotic potential of porcine-BSE prions seems to be relevant, as both brains and muscles from BSE-infected pigs induced the misfolding of the human prion protein in vitro. This study helps to understand the potential fate of naturally existing prion strains in a relevant host and calls for caution considering the co-existence between feral swine and other prion-susceptible animal species.
IMPORTANCE
Prions (PrPSc) are proteinaceous, infectious pathogens responsible for prion diseases. Some livestock are highly susceptible to prion diseases. These include cattle (bovine spongiform encephalopathy, BSE), sheep and goat (scrapie), and cervids (chronic wasting disease, CWD). Unfortunately, BSE has been reported to be naturally transmitted to humans and other animal species. Domestic pigs, a relevant livestock animal, have not been reported to be naturally affected by prions; however, they are susceptible to the experimental exposure to BSE, scrapie, and CWD prions. Given the widespread consumption of porcine food products by humans, we aimed to evaluate the levels of pig-derived BSE, scrapie, and CWD prions from experimentally challenged domestic pigs in brain and meat cuts (leg, cheek meat, skirt meat, and tenderloin). We detected pig-adapted prions in the brains and some muscles of these animals. Additionally, we evaluated the in vitro compatibility between pig prions and the human prion protein (as a surrogate of zoonosis). Our results show that only pig-derived BSE prions were able to induce the misfolding of the cellular human prion protein. This data highlights the consequences of prion spillovers to other animal species and their potential availability to humans.
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DISCUSSION
In this study, we evaluated the presence and relative quantities of prions in brains and muscles from pigs infected with three different prion isolates: C-type BSE, scrapie, and CWD. Our findings confirm previous reports by demonstrating the presence of disease-associated prions in the brains of these animals (20, 56, 61). In addition, we evaluated the replication capacity of the prions present in these samples using the PMCA technique. The presence of prions in the brains of these prion-infected pigs appears to be variable depending on the original prion source. Although prion seeding activity was identified in the brains of pigs from all three groups, we found that those from animals infected with BSE contained the most active seeds. It is relevant to mention that most of the pigs infected with either scrapie or CWD did not display neurological signs linked with prion disease, yet prion seeding activity was still detected in their brains. The Moore et al. publication (61) states the presence of sparse PK-resistant PrP accumulation in the brain of CWD-infected pigs, suggesting a low amount of PrPSc in these samples. To improve the detection, the authors treated brain extracts with a sarkosyl/ultracentrifugation protocol and loaded at 100 mg of brain tissue for detection via western blots. Due to the limited amount of sample available for the present study, we did not enrich the sample and loaded 2 mg of brain tissue on western blots. This might have affected the detection of the PrP signals using this method (Fig. 2; Fig. S2). The low amount of PrPSc in these samples was further confirmed by PMCA, as seeding activity was present in just some replicates (Fig. 4), mostly at low dilutions. Nevertheless, the identification of subclinical prion infection in these brains is consistent with previous studies suggesting the same when bioassays in transgenic mice expressing the porcine PrPC were used in the BSE and CWD transmissions (26, 56). The fact that subclinical prion infection was also identified in pigs orally exposed to BSE and CWD (56, 61, 62) suggests that transmissions in natural scenarios are possible and an eventual adaptation of the agent in pigs (e.g., considering the cannibalistic behavior of wild pigs) is plausible.
In addition to the prion content in brains, we evaluated the anatomical distribution of PrPSc in several skeletal muscles with the goal of understanding the tropism of the adapted prions in these tissues. The specific muscles tested included semitendinosus (leg), masseter (cheek meat), diaphragm (skirt meat), and psoas major (tenderloin). These pork cuts are commonly consumed by people and are commercially available. Unfortunately, the muscle tested for the BSE-infected group (semitendinosus) differed from those analyzed in the scrapie and CWD groups (masseter, diaphragm, and psoas) due to the availability of archived materials. Regardless, these samples are useful to understand whether prions in pigs accumulate in muscles that might be available for human consumption. Our results indicate that the muscles collected from the BSE-infected pigs contain higher levels of seeding-competent prions compared with those in other groups. Nevertheless, the same cautions regarding the routes of administration of BSE materials in pigs explained above must be considered when interpreting this data. The comparison between scrapie and CWD inoculated pig groups, that were inoculated with the same quantities of infectious materials and the same route, and where the same tissues were collected, allows us to make more accurate associations. In summary, our findings demonstrate that muscles closer to the animal head (masseter) supported a better seeding activity compared to those located at the thorax (diaphragm) and hind levels (psoas major). One explanation for this could be explained by the centrifugal spreading of prions, which agrees with the findings by Headman and colleagues (58) describing the detection of PrPSc in upper muscles of clinically affected pigs inoculated intracerebrally with sheep-BSE prions. In that study, two swine harbored PrPSc in muscles associated with the eyes (oculomotor muscle), while prions were identified in the semitendinosus muscle of just one animal. Recently, the same group analyzed the peripheral tissues of these pigs by PMCA, detecting seeding activity in the oculomotor muscle in all the animals exposed to the BSE-derived agent in dilutions as low as 10−4 (67). Due to the limited amount of muscle samples from the pigs inoculated with BSE, we were unable to perform a detailed anatomical analysis. However, we showed that the semitendinosus muscle, which is located at the hind limbs, contained relevant levels of seeding competent PrPSc. The relevantly abundant presence of prions in this particular group of pigs could be due to the fact that they were peripherally exposed to the agent, or by the peripheral tropism of BSE prions as it has been observed for the transmission of this particular prion strain to multiple other animal species (62, 68–80). The data describing higher quantities of seeding-competent prions in the brains of BSE-infected pigs needs to be interpreted with caution. As described, BSE-infected pigs received infectious materials at higher doses and through three simultaneous routes of administration compared to the animals exposed to the scrapie and CWD agents (20, 56, 61). If we restrict these comparisons to the latter two groups (scrapie- and CWD-infected pigs) that were exposed to the same prion doses (750 µL of 10% [w/v] pooled sheep and cervid brain homogenate, respectively), and through the same route of inoculation (intracerebral), we found that pig-adapted scrapie prions exhibited a higher amount of seeding-competent PrPSc in both brain and muscle tissues compared with their CWD-exposed counterparts. The differences in seeding capacities and tropisms between these newly generated porcine prions can be explained in the generation of different prion strains in each case. Nevertheless, it is also relevant to consider that these studies used unique scrapie and CWD strains from the many described in experimental and natural settings (31, 33, 40, 41). Along this line, future studies should focus on the differential susceptibilities experienced by pigs when exposed to different variants associated with these animal prionopathies.
Importantly, our studies evaluating zoonotic potentials showed that pig-adapted BSE prions were able to induce the misfolding of the human prion protein in vitro. These results were confirmed in two different laboratories, a fact that provides rigor to this study. Importantly, these analyses also show that muscles from these BSE-infected pigs may present a concern if introduced in the human food chain. Importantly, the zoonotic risk of these materials seems to be restricted to the 129M polymorphic version of the human prion protein, in line with the higher susceptibility of BSE to the population carrying this specific protein. Previous reports have shown that mice expressing the 129M human PrPC variant (HuPrP-tg 650 and 340) exhibited a species barrier to BSE prions that was overcome in a first transmission passage (81). Similar outcomes were observed when pig-adapted BSE prions were inoculated in HuPrP-tg340 mice, although with incomplete attack rates (50%) in a first passage. Serial transmissions of this infectious material in the same mice resulted in complete attack rates and reduction in the incubation periods, suggesting that the infectious material successfully adapted in the new host (60). On the contrary, extensive evidence demonstrates that the species barrier between scrapie and CWD prions and humans is strong, if not absolute (39, 42, 82). However, the existence of different prion strains associated with these animal prionopathies urges for a systematic analysis including the whole pathogenic spectrum associated with them. In our study, scrapie- and CWD-adapted prions were not able to induce the misfolding of the human prion protein. However, considering the data from the porcine-BSE prions, we cannot conclude whether the lower load of prions in the CWD- and scrapie-porcine tissues was responsible for these results.
Some of the limitations of the current study have been already discussed. One of the issues involves the unknown prion infectivity titers of the BSE-, scrapie-, and CWD-bearing tissues used to infect pigs. This importantly limits comparisons between the groups. Prion infectivity titers can affect tissue tropisms, especially in subclinical transmissions (as prions may need additional time to reach target tissues). This could be considered an additional source of variability for the dissimilar presence of prions in muscles for the different animal groups included in this study. Another limitation involves the uneven testing of muscle tissues across the three groups and the lack of analysis of other muscles representing different anatomical distributions within the body. The latter may be relevant for understanding the tropism of prions induced by each injectate. Finally, the scrapie and CWD groups were treated with specific prion strains from many that have been described. This is relevant considering that different strains may adapt with different efficiencies into new hosts and result in infectious particles with unique host ranges. Future studies should focus on the above-mentioned limitations. However, we believe that the current study provides a solid background to justify these analyses.
In summary, our data shows the dynamic of animal prions when exposed to infectious pigs, as well as their distributions and zoonotic potentials. The data presented here may be relevant to understanding the fate of naturally existing prions in a sympatric animal species relevant for human consumption. This acquires importance considering a recent report describing the interaction between CWD and wild pigs in natural settings.
https://journals.asm.org/doi/10.1128/mbio.01800-25
Price of TSE Prion Poker goes up drastically!
Detection of Prions in Wild Pigs (Sus scrofa) from Areas with Reported Chronic Wasting Disease Cases, United States
Volume 31, Number 1—January 2025
Abstract
Using a prion amplification assay, we identified prions in tissues from wild pigs (Sus scrofa) living in areas of the United States with variable chronic wasting disease (CWD) epidemiology. Our findings indicate that scavenging swine could play a role in disseminating CWD and could therefore influence its epidemiology, geographic distribution, and interspecies spread.
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Conclusions
In summary, results from this study showed that wild pigs are exposed to cervid prions, although the pigs seem to display some resistance to infection via natural exposure. Future studies should address the susceptibility of this invasive animal species to the multiple prion strains circulating in the environment. Nonetheless, identification of CWD prions in wild pig tissues indicated the potential for pigs to move prions across the landscape, which may, in turn, influence the epidemiology and geographic spread of CWD.
https://wwwnc.cdc.gov/eid/article/31/1/24-0401_article
Although the current U.S. feed ban is based on keeping tissues from TSE infected cattle from contaminating animal feed, swine rations in the U.S. could contain animal derived components including materials from scrapie infected sheep and goats. These results indicating the susceptibility of pigs to sheep scrapie, coupled with the limitations of the current feed ban, indicates that a revision of the feed ban may be necessary to protect swine production and potentially human health.
2. Determined that pigs naturally exposed to chronic wasting disease (CWD) may act as a reservoir of CWD infectivity. Chronic wasting disease is a naturally occurring, fatal, neurodegenerative disease of cervids. The potential for swine to serve as a host for the agent of CWD disease is unknown. The purpose of this study was to investigate the susceptibility of swine to the CWD agent following experimental oral or intracranial inoculation. Pigs were assigned to 1 of 3 groups: intracranially inoculated; orally inoculated; or non-inoculated. At market weight age, half of the pigs in each group were tested ('market weight' groups). The remaining pigs ('aged' groups) were allowed to incubate for up to 73 months post inoculation (MPI). Tissues collected at necropsy were examined for disease-associated prion protein (PrPSc) by multiple diagnostic methods. Brain samples from selected pigs were bioassayed in mice expressing porcine prion protein. Some pigs from each inoculated group were positive by one or more tests. Bioassay was positive in 4 out of 5 pigs assayed. Although only small amounts of PrPSc were detected using sensitive methods, this study demonstrates that pigs can serve as hosts for CWD. Detection of infectivity in orally inoculated pigs using mouse bioassay raises the possibility that naturally exposed pigs could act as a reservoir of CWD infectivity. Currently, swine rations in the U.S. could contain animal derived components including materials from deer or elk. In addition, feral swine could be exposed to infected carcasses in areas where CWD is present in wildlife populations. The current feed ban in the U.S. is based exclusively on keeping tissues from TSE infected cattle from entering animal feeds. These results indicating the susceptibility of pigs to CWD, coupled with the limitations of the current feed ban, indicates that a revision of the feed ban may be necessary to protect swine production and potentially human health.
https://www.ars.usda.gov/research/publications/publication/?seqNo115=353091
https://www.ars.usda.gov/research/project/?accnNo=432011&fy=2017
https://www.ars.usda.gov/research/publications/publication/?seqNo115=337105
https://www.ars.usda.gov/research/publications/publication/?seqNo115=326166
Docket No. FDA-2003-D-0432 (formerly 03D-0186) Use of Material from Deer and Elk in Animal Feed
PUBLIC SUBMISSION
Comment from Terry Singeltary Sr.
Posted by the Food and Drug Administration on May 17, 2016 Comment
Docket No. FDA-2003-D-0432 (formerly 03D-0186) Use of Material from Deer and Elk in Animal Feed Singeltary Submission
https://www.regulations.gov/comment/FDA-2003-D-0432-0011
https://www.regulations.gov/docket/FDA-2003-D-0432
WEDNESDAY, AUGUST 13, 2025
Revised Bovine Spongiform Encephalopathy BSE TSE Prion Standards
https://bovineprp.blogspot.com/2025/08/revised-bovine-spongiform.html
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