We screened pig tissues by using the protein misfolding cyclic amplification (PMCA) technique (Appendix). To screen for CWD and porcine-adapted CWD prions in wild pigs, we analyzed the sensitivity and specificity of PMCA to detect prions from different animal species. To evaluate, we first tested the PMCA efficiency of deer CWD prions in both deer and pig substrates. We found the in vitro replication of CWD prions was highly efficient in the deer substrate, and detectable even at high (10⁻¹¹) dilutions in a first PMCA round (Appendix Figure 2). As a counterpart, we used a porcine-adapted PrP^Sc (scrapie isoform of infectious prion proteins) pool generated through >10 PMCA rounds in Tg002 substrate (Appendix Figure 3). That inoculum displayed limited PMCA seeding activity after 1 round, confirming the ability of our in vitro prion replication systems to discriminate between homologous (deer) and heterologous (porcine) PrP^Sc sources (Appendix Figure 2, panel A).

The specificity of our cervid and porcine PMCA systems was further confirmed when using the pig-derived PMCA substrate (Appendix Figure 2, panel B). Specifically, although we detected no amplification in the system when using the CWD seeds, the porcine-adapted PMCA product displayed great amplification. Overall, those results demonstrated that PMCA can be used to identify prions from deer and porcine sources, provide species specificity, and potentially enable tracking of the source of the infectious agent in tissues collected from wild pigs.

Figure 1

Figure 1. Prion detection using cervid-PMCA in wild pig tissues originating from a CWD-endemic region of Arkansas, USA, in a study of prions in wild pigs (Sus scrofa) from areas...

We then investigated the interaction between wild pigs and CWD prions in tissues from animals trapped in areas with different CWD epidemiology. The first cohort of pigs (cohort 1) included animals trapped in Newton and Searcy Counties, Arkansas, USA, where CWD is endemic in free-ranging white-tailed deer (Odocoileus virginianus) and elk (Cervus elaphus canadensis) (https://www.agfc.com/hunting/deer/chronic-wasting-disease/cwd-in-arkansas). The tissues considered for this screening included retropharyngeal lymph nodes (RPLN) and submandibular lymph nodes (SMLN) because those sites have been described to replicate prions shortly after infection in multiple animal species (Appendix). We also tested brainstem samples from the same animals. The first part of the screening involved the use of deer PrP substrate to assess for the potential exposure of wild pigs to CWD prions. We analyzed all samples in duplicate. We were able to detect CWD seeding activity in several wild pig lymphoid tissues and a higher proportion in the SMLN (37.26%) compared with RPLN (16.67%) (Figure 1). As expected, brains displayed a lower detection; just 14.90% of the specimens provided positive signals (Figure 1). We considered a tissue as positive for CWD prions if we noted positive signals in >1 of the 2 replicates. Along that line, a fraction (12.75%) of PMCA-positive SMLN tissues provided positive PMCA results in both replicates, suggesting a higher concentration of CWD prions in SMLN than RPLN or brains (≈3%) (Figure 1). Of note, the CWD infectivity titers in wild pig tissues appeared to be limited because just a fraction of Tg1536 mice (expressing the deer prion protein) inoculated with selected specimens displayed subclinical prion infection (Appendix Table 1, Figure 4).

Figure 2

Figure 2. Prion detection using porcine-PMCA in wild pig tissues originating from a CWD-endemic region of Arkansas, USA, in a study of prions in wild pigs (Sus scrofa) from areas...

Screening of the same tissues using porcine-PMCA substrate provided a considerably lower number of positive results (Figure 2). Although 5.88% of wild pigs were porcine-PMCA positive at RPLNs, only 1.96% were positive at SMLNs. Of note, none of the tissues that tested PMCA-positive using the porcine substrate provided positive signals in both replicates, suggesting low quantities of seeding-competent porcine prions (Figure 2). None of the brains from cohort 1 provided PMCA seeding activities when the porcine substrate was used, thus confirming those results (Figure 2). Moreover, bioassays in Tg002 mice expressing the porcine prion protein resulted in no transmission (Appendix Table 2).

Figure 3

Figure 3. Prion detection using cervid-PMCA in wild pig tissues originating from a CWD-endemic region of Texas, USA, in a study of prions in wild pigs (Sus scrofa) from areas...

Figure 4

Figure 4. Prion detection using porcine-PMCA in wild pig tissues originating from a CWD-endemic region of Texas, USA, in a study of prions in wild pigs (Sus scrofa) from...

Next, we conducted a screening on a second cohort of animals (cohort 2) collected in Texas, USA. Pigs from cohort 2 were retrieved from counties where either no or low-prevalence CWD had been reported in wild deer. The cervid-adapted PMCA analysis revealed that 15.79% of animals tested positive for CWD prions in RPLNs (Figure 3). Brains (13.16%) and SMLN (8.10%) were also positive, albeit in a lower percentage than for RPLN (Figure 3). Regardless, the overall proportion of PMCA-positive tissues was considerably lower compared with those found for cohort 1, in line with the low prevalence of CWD in free-ranging cervids in the Texas study region. In agreement with the presumably lower exposure of CWD prions for pigs from cohort 2, none of the tissues provided PMCA-positive signals when evaluated in the porcine PMCA system (Figure 4).

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.