Infectious prions in brains and muscles of domestic pigs experimentally challenged with the BSE, scrapie, and CWD agents

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.IMPORTANCEPrions (PrP^Sc) 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.

Keywords: PMCA; bovine spongiform encephalopathy; chronic wasting disease; muscles; pigs; prion; scrapie; zoonotic infections.

Fig 1

Experimental strategy. Brain (n = 3/inoculum) and skeletal muscle (n_total = 21) specimens from domestic pigs experimentally inoculated with C-type BSE (yellow), scrapie (green), and CWD (pink) prions were interrogated for their prion content using western blotting and PMCA. The routes of inoculations in pigs were intracerebral (I.C), intravenous (I.V), and intraperitoneal (I.P) for the BSE prions, while scrapie and CWD prions were administered by the I.C. route only. The specific skeletal muscles assessed were semitendinosus (pig-adapted BSE), masseter, diaphragm, and psoas major (pig-adapted scrapie and CWD). The PMCA protocol used in this study was optimized to replicate pig PrP^Sc, as described (64). Finally, the zoonotic potential of the pig-adapted prions was evaluated by PMCA reactions using the 129M and 129V human PrP^C variants as substrate. This figure was generated using Biorender.com.

Fig 2

Levels of PK-resistant PrP^Sc in the brains of domestic pigs experimentally challenged with classical BSE, scrapie, and CWD prions. Representative Western blot of PK-resistant PrP levels from samples 310 (pig-BSE), 371 (pig-scrapie), and 423 (pig-CWD). Brain homogenates (BH) were treated with PK (50 µg/mL), then diluted in PBS, and evaluated by Western blot. The specimens from pigs exposed to BSE were performed in duplicates, and those inoculated with scrapie and CWD were performed in triplicates. Numbers at the top of the panels represent the dilution of BH used. The name at the bottom of each panel depicts the prion strain used as inoculum for the domestic pigs. The number at the right represents molecular weight markers (in KDa).

Fig 3

Identification of seeding-competent pig-derived BSE, scrapie, and CWD prions in brain tissues by PMCA. Representative western blots depicting the prion seeding activity (third PMCA round) in brain tissues (samples 310 [pig-BSE], 371 [pig-scrapie], and 399 [pig-CWD]). The number at the top of the panels represents the final concentration of the brain homogenates submitted to the PMCA substrate (ranging from 10⁻² to 10⁻⁶ g/mL). Each sample was analyzed in triplicate. The pig-adapted scrapie and CWD prions were detected by incubating the membranes with the mAb 8H4 at a dilution of 1:5,000. The numbers at the right represent molecular weight markers (in KDa). The MW represents the EZ-Run molecular weight ladder (Fisher Scientific, Waltham, MA, USA). “PrP^C” denotes brain extracts from Tg002 mice (expressing the pig version of PrP^C) not treated with PK and used as an electrophoretic mobility and antibody reactivity control. The dashed lines represent sites where the membrane was cropped/edited. All images come from the same membrane.

Fig 4

Individual results of the seeding activities from pig-derived BSE, scrapie, and CWD prions from domestic pigs’ brains. This figure shows the raw data of PMCA analyses in each brain specimen. Three replicates were tested in each case, at different dilutions. Other information, relevant for this Figure, is presented as follows: ^ǂ Ryder, S. J., Hawkins, S. A. C., Dawson, M., & Wells, G. A. H. (20). The neuropathology of experimental bovine spongiform encephalopathy in the pig. Journal of Comparative Pathology, 122(2–3), 131–143. https://doi.org/10.1053/jcpa.1999.0349. ^¥ Greenlee, J. J., Kunkle, R. A., Smith, J. D., & Greenlee, M. H. W. (56). Scrapie in Swine: a Diagnostic Challenge. Food Safety, 4(4), 110–114. https://doi.org/10.14252/foodsafetyfscj.2016019. ^§ Moore, S. J., West Greenlee, M. H., Kondru, N., Manne, S., Smith, J. D., Kunkle, R. A., Kanthasamy, A., & Greenlee, J. J. (61). Experimental transmission of the chronic wasting disease agent to swine after oral or intracranial inoculation. Journal of Virology, JVI.00926-17. https://doi.org/10.1128/JVI.00926-17. ^£ Observed in some replicates. ^Φ Samples 399, 412, and 423 in this study are from animals 26, 28, and 27 from the reference (61) study, respectively.

Fig 5

Individual results of the in vitro seeding activity of skeletal muscle specimens from domestic pigs experimentally challenged with classical BSE prions. This figure shows the raw data of PMCA analyses in each muscle specimen for pigs infected with BSE prions. Two replicates were tested in each case, at different dilutions.

Fig 6

Individual results of the in vitro seeding activity of skeletal muscle specimens from domestic pigs experimentally challenged with scrapie and CWD prions. This figure shows the raw data of PMCA analyses in each muscle specimen for pigs infected with either scrapie or CWD prions. Three replicates were tested in each case, at different dilutions. Other information, relevant for this figure, is presented as follows: ^Φ Samples 399, 412, and 423 in this study are from animals 26, 28, and 27 from the reference (61) study, respectively.

Fig 7

In vitro conversion efficiency of pig PrP^C templated by pig-derived BSE, scrapie, and CWD prions in skeletal muscles. Representative Western blots depict PrP^Sc detected in muscle specimens. The tissues were serially diluted (10⁻¹ to 10⁻⁵ g/mL) and submitted to three PMCA rounds. (A) Semitendinosus muscle homogenates from a pig inoculated with C-type BSE interrogated in duplicate. (B) Masseter, diaphragm, and psoas major homogenate tissues from domestic pigs experimentally exposed to scrapie and CWD prions were analyzed in triplicate. The numbers at the top of the panels represent the final dilutions of the samples in the pig PMCA substrate. The numbers at the right represent molecular weight markers (in KDa). The MW represents the EZ-Run molecular weight ladder (Fisher Scientific, Waltham, MA, US). “PrP^C” denotes brain extracts from Tg002 mice (expressing the pig version of PrP^C) not treated with PK and used as an electrophoretic mobility and antibody reactivity control. The dashed lines represent sites where the membrane was cropped/edited.

Fig 8

In vitro misfolding of human prion protein (129M) seeded with abnormal prion proteins derived from domestic pigs experimentally inoculated with the classical BSE, scrapie, and CWD agents (Site 1). (A) Humanized PMCA reactions using brain homogenates from swine inoculated with C-type BSE, scrapie (scr), and CWD as inocula. (B) Humanized PMCA reactions using muscle homogenates from swine inoculated with C-type BSE, scrapie (scr), and CWD as inocula. (C) PMCA controls for these experiments included reactions seeded with C-type BSE, classical scrapie, and CWD (Elk) of known seeding activity. PBS was also used as an additional technical control (37). F (frozen) represents aliquots of each reaction collected before amplification and stored at −80 °C for comparison. S (sonicated) denotes samples harvested post-amplification. PMCA products were detected using the mAb 3F4, which recognizes the amino acid sequence between 109 and 112 of the human prion protein and does not cross-react with porcine PrP. The contribution of the inocula to the 3F4-visualized signals was assessed by re-incubating these membranes with the mAb 6H4 (detecting both human and porcine prion sequences). M denotes the molecular weight marker (in KDa). (*) Asterisks indicate undigested PrP material.

Fig 9

In vitro misfolding of human prion protein (129M) seeded with abnormal prion proteins derived from domestic pigs experimentally inoculated with the BSE, scrapie, and CWD agents (Site 2). (A) Humanized PMCA (129M) reactions seeded with brain homogenates from three pigs (IDs 42, 66, and 310) inoculated with C-type BSE. (B) Humanized PMCA (129M) reactions seeded with muscle homogenate from three pigs (IDs 42, 66, and 310) inoculated with C-type BSE. (C) Technical controls consisted of seeded reactions with serial dilutions (10⁻⁹ and 10⁻¹⁰) from the brain homogenate of a vCJD individual. Additionally, unseeded (UN) PMCA reactions were included. PMCA products were detected using the mAb 3F4 (1:5,000) after PK digestion. Numbers at the right of each panel represent molecular weight markers (in KDa). “PrP^C” denotes brain extracts from Tg-HuMM mice (expressing the 129M version of human PrP) not treated with PK and used as an electrophoretic mobility and antibody reactivity controls. The dashed line represents cropped/edited membranes.