Zoonotic Potential of Chronic Wasting Disease after Adaptation in Intermediate Species

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 30, No.12, December 2024 2691 RESEARCH LETTERS About the Author Dr. Price is a veterinary epidemiologist at the Centers for Disease Control and Prevention assigned to New Jersey Department of Health, Trenton, New Jersey, USA. Her research interests focus on healthcare-associated infections and antimicrobial stewardship strategies in companion animals. References 1. Centers for Disease Control and Prevention. 2019 Antibiotic resistance threats report [cited 2023 Dec 6]. https://www.cdc.gov/antimicrobial-resistance/ data-research/threats 2. Hyun JE, Chung TH, Hwang CY. Identification of VIM-2 metallo-β-lactamase-producing Pseudomonas aeruginosa isolated from dogs with pyoderma and otitis in Korea. Vet Dermatol. 2018;29:186–e68. https://doi.org/10.1111/vde.12534 3. Fernandes MR, Sellera FP, Moura Q, Carvalho MPN, Rosato PN, Cerdeira L, et al. Zooanthroponotic transmission of drug-resistant Pseudomonas aeruginosa, Brazil. Emerg Infect Dis. 2018;24:1160–2. https://doi.org/10.3201/eid2406.180335 4. Wang Y, Wang X, Schwarz S, Zhang R, Lei L, Liu X, et al. IMP-45-producing multidrug-resistant Pseudomonas aeruginosa of canine origin. J Antimicrob Chemother. 2014;69:2579–81. https://doi.org/10.1093/jac/dku133 5. US Food and Drug Administration. FDA warns consumers not to purchase or use EzriCare Artificial Tears due to potential contamination [updated 8/25/2023] [cited 2023 Dec 6]. https://www.fda.gov/drugs/drug-safety-andavailability/fda-warns-consumers-not-purchase-or-useezricare-artificial-tears-due-potential-contamination 6. Grossman MK, Rankin DA, Maloney M, Stanton RA, Gable P, Stevens VA, et al.; Multistate Pseudomonas Outbreak Investigation Group. Extensively drug-resistant Pseudomonas aeruginosa outbreak associated with artificial tears. Clin Infect Dis. 2024;79:6–14. https://doi.org/10.1093/ cid/ciae052 7. Livermore DM, Andrews JM, Hawkey PM, Ho PL, Keness Y, Doi Y, et al. Are susceptibility tests enough, or should laboratories still seek ESBLs and carbapenemases directly? J Antimicrob Chemother. 2012;67:1569–77. https://doi.org/10.1093/jac/dks088 8. Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki EP, Zaslavsky L, et al. NCBI prokaryotic genome annotation pipeline. Nucleic Acids Res. 2016;44:6614–24. https://doi.org/10.1093/nar/gkw569 9. Dazio V, Nigg A, Schmidt JS, Brilhante M, Mauri N, Kuster SP, et al. Acquisition and carriage of multidrugresistant organisms in dogs and cats presented to small animal practices and clinics in Switzerland. J Vet Intern Med. 2021;35:970–9. https://doi.org/10.1111/jvim.16038 10. van den Bunt G, Fluit AC, Spaninks MP, Timmerman AJ, Geurts Y, Kant A, et al. Faecal carriage, risk factors, acquisition and persistence of ESBL-producing Enterobacteriaceae in dogs and cats and co-carriage with humans belonging to the same household. J Antimicrob Chemother. 2020;75:342–50. https://doi.org/10.1093/jac/ dkz462 Address for correspondence: Emma R. Price, New Jersey Department of Health, 135 E State St, Trenton, NJ 08625, USA; email: [email protected] Zoonotic Potential of Chronic Wasting Disease after Adaptation in Intermediate Species Tomás Barrio, Sylvie L. Benestad, Jean-Yves Douet, Alvina Huor, Séverine Lugan, Naïma Aron, Hervé Cassard, Juan Carlos Espinosa, Alicia Otero, Rosa Bolea, Juan María Torres, Olivier Andréoletti Author affiliations: Unité Mixte de Recherche de l’Institut National de Recherche pour l’Agriculture, l’Alimentation, et l’Environnement 1225 Interactions Hôtes-Agents Pathogènes, École Nationale Vétérinaire de Toulouse, Toulouse, France (T. Barrio, J.-Y. Douet, A. Huor, S. Lugan, N. Aron, H. Cassard, O. Andréoletti); Norwegian Veterinary Institute, Ås, Norway (S.L. Benestad); Consejo Superior de Investigaciones Científicas, Madrid, Spain (J.C. Espinosa, J.M. Torres); Universidad de Zaragoza, Zaragoza, Spain (A. Otero, R. Bolea) DOI: https://doi.org/10.3201/eid3012.240536 Chronic wasting disease (CWD) is a highly contagious prion disease affecting members of the Cervidae family. CWD is widely spread across North America, where it endangers the survival of freeranging cervid populations. In Europe, CWD was reported in a reindeer (Rangifer tarandus tarandus) from Norway in 2016 (1). Since 2016, several cases have been reported in Norway, Sweden, and Finland in multiple species, including reindeer, red deer (Cervus elaphus), and moose (Alces alces) (2). Whereas CWD strains circulating in North America exhibit some uniformity (3), the cases found in Europe are more variable. Transmission into rodent models has revealed multiple CWD strains that are apparently different than strains in North America, and moose cases in Norway have demonstrated biochemical patterns distinct from previous cases in Europe (4). We characterized the interspecies transmission potential of 1 moose CWD isolate from Norway (Norwegian Veterinary Institute identification no. 16–60-P153) (4) by intracerebral injection of mouse models expressing the normal prion protein (PrPC) sequences from several species (Figure, panel A). Chronic wasting disease (CWD) is an emerging disease in Europe. We report an increase in interspecies transmission capacity and zoonotic potential of a moose CWD isolate from Europe after passage in an ovine prion protein–expressing host. Those results indicated some CWD prions could acquire enhanced zoonotic properties following adaptation in an intermediate species. 2692 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 30, No.12, December 2024 RESEARCH LETTERS We anesthetized and inoculated 6-to-10-weekold mice with 2 mg of equivalent tissue (20 µL of 10% brain homogenate) in the right parietal lobe. We monitored the inoculated animals daily and humanely euthanized animals at the onset of clinical signs or after the preestablished endpoint of 700 days postinfection (dpi). We conducted a systematic proteinase K–resistant prion protein (PrPres) detection by using Western blot. Inoculation of the original CWD isolate did not cause the propagation of detectable prions in Tg340 mice expressing methionine (TgMet) or Tg361 mice expressing valine (TgVal) at position 129 of human PrPC. We did not observe PrPres in brain tissue or disease occurrence in bovine PrPC-expressing mice (BoTg110) after intracerebral inoculation of the CWD isolate (Table; Figure, panel B). We inoculated the CWD isolate in Tg338 mice, which overexpress ovine PrP ≈8 times. At 612 and 717 dpi (Table), 2 of 12 animals showed clinical signs of prion disease, and we detected PrPres accumulation in their brain tissue (Figure, panel B). Of note, the 2 animals showed different PrPres banding patterns, with the nonglycosylated band migrating to 19 kDa in the first mouse and to 21 kDa in the second. Both PrPres-containing brains transmitted disease with 100% efficacy to second-passage Tg338 mice, which contained 21-kDa PrPres in their brains (Figure, panel B). A third passage resulted in the incubation period shortening (95 ± 5 dpi). Our observations are consistent with a progressive adaptation of the moose CWD prion to the ovine-PrPC expressing model and suggest moose CWD prions in Europe may have an intrinsic capability to propagate in ovine species with the VRQ genotype. We next determined whether adaptation of this moose CWD agent to Tg338 altered its capacity to cross species barriers. For that purpose, we inoculated Tg338-adapted moose CWD prions (passaged twice in Tg338) to the same panel of PrPCFigure. Western blot analysis in a study of zoonotic potential of chronic wasting disease after adaptation in intermediate species. Results show PKresistant PrP (PrPres) banding patterns of a moose CWD isolate from Europe after transmission to transgenic PrP models. A) Original 16–60P153 CWD isolate compared with reference Dawson and sheep-passaged BSE. B) Transmission of the original moose CWD isolate to mice ovine PrPC genotype VRQ (Tg338), resulting in positive transmission with either 19 or 21 kDa PrPres on first passage and 21 kDa PrPres on second passage; PrP Sha31 antibodies (right panel) and PrP 12B2 antibodies (left panel). C) Transmission of Tg338-adapted (second passage) moose CWD isolate to Tg338, BoTg110, TgMet, or TgVal. D) Comparison of PrPres banding patterns in TgMet and TgVal inoculated with the Tg338-adapted moose CWD isolate or with M1CJD and V2CJD reference sCJD strains. Immunodetection was performed by using either the Sha31 antibody to detect the amino acid sequence YEDRYYRE (145–152), or the 12B2 antibody to detect the amino acid sequence WGQGG (89–93). Dawson (a reference 21-kDa scrapie strain) is included on all panels except D for molecular weight reference. 1P, 1st passage; 2P, 2nd passage; BoTg110, bovine PrPC-expressing mice; BSE, bovine spongiform encephalopathy; CJD, Creutzfeldt-Jakob disease; CWD, chronic wasting disease; PK, proteinase K; PrP, prion protein; PrPC, normal prion protein; PrPres, PK-resistant prion protein. TgMet, Tg340 mice expressing methionine; TgVal, Tg361 mice expressing valine. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 30, No.12, December 2024 2693 RESEARCH LETTERS expressing mice models. Inoculation of the Tg338adapted isolate to BoTg110 resulted in 100% disease transmission that showed a banding pattern and intermediate molecular weight from 19–21 kDa (Figure, panel C; Appendix Figure, https://wwwnc.cdc. gov/EID/article/30/12/24-0536-App1.pdf) and an incubation period of 431 ± 32 dpi (Table), which suggested the lack of a major transmission barrier. In addition, 1 of 8 inoculated TgMet mice showed clinical signs at 561 dpi (Table). PrPres in the brain of that mouse was revealed by a mixed 19 + 21–kDa banding pattern (Figure, panel C). A second passage in TgMet is underway. Inoculation of TgVal resulted in efficient transmission (5/6 animals); the mean incubation period was 483 ± 35 dpi (Table) and accumulation was 21– kDa PrPres (Figure, panel C). On second passage, transmission was 100% and we observed a shorter incubation period (311 ± 12 dpi). The incubation periods and PrPres biochemical profiles of the CWD prions that propagated in the TgMet and TgVal mice greatly differed from those observed in mice inoculated with the most prevalent human prion strains or with classic bovine spongiform encephalopathy (BSE), sheep-adapted BSE, or Tg338-adapted c-BSE (Table; Figure, panel D). Those results might suggest this CWD-derived prion strain differs from other strains documented in those models. Further investigation is necessary. The evolution of moose CWD zoonotic potential after its passage in an ovine PrPC-expressing host is reminiscent of the well-documented altered capacities of the c-BSE agent to cross the human species barrier after adaptation in sheep and goats (9). The codon 129-dependent response to infection of humanized mice with Tg338-adapted CWD is also compatible with studies demonstrating Table. Transmission of a moose CWD isolate in a study of zoonotic potential of chronic wasting disease after adaptation in intermediate species* Model characteristics TgMet TgVal Tg338 BoTg110 No./ no.† Mean dpi (SD) PrPres band type No./ no.† Mean dpi (SD) PrPres band type No./ no.† Mean dpi (SD) PrPres band type No./ no.† Mean dpi (SD) PrPres band type Prion strains M1CJD (sCJD MM1) 1st passage 6/6 219 (17) 21 kDa 6/6 327 (19)‡ 21 kDa ND ND 2nd passage 6/6 239 (8)‡ 21 kDa 6/6 286 (16)‡ 21 kDa ND ND V2CJD (sCJD VV2) 1st passage 6/6 618 (81)‡ 21 kDa 6/6 168 (12)‡ 19 kDa ND ND 2nd passage 6/6 509 (41)‡ 21 kDa 6/6 169 (12)‡ 19 kDa ND ND Classical BSE 1st passage 1/12 739‡ BSE§ 0/12 >750‡ NA 6/6 >750¶ BSE§ 6/6 295 (12)# BSE§ 2nd passage 9/12 613 (43)‡ BSE§ 0/12 >750‡ NA 6/6 682 (52)¶ BSE§ 6/6 265 (35)# BSE§ Sheep-adapted BSE 1st passage 6/6 690 (83)# BSE§ ND 6/6 >750¶ BSE§ 6/6 254 (19)¶ BSE§ 2nd passage 5/5 564 (39)# BSE§ ND 6/6 653 (32)¶ BSE§ 6/6 234 (12)¶ BSE§ Tg338-adapted BSE 1st passage 6/6 596 (92) BSE§ 0/6 >700 BSE§ 5/5 224 (37) BSE§ 6/6 222 (22) BSE§ 2nd passage ND ND ND Moose CWD (16–60-P153) 1st passage 0/12 >700 NA 0/1 2 >700 NA 2/12 612, 717 19 kDa, 21 kDa 0/12 >700 NA 2nd passage 0/6 >700 NA ND 5/5 167 (4)** 21 kDa ND ND ND 6/6 244 (33)†† 21 kDa ND Tg338-adapted moose CWD‡‡ 1st passage 1/8 561 19+21 kDa 5/6 483 (35) 21 kDa 7/7 95 (5) 21 kDa 5/5 431 (32) 20 kDa 2nd passage ND 4/4 311 (12) 21 kDa ND ND *Results show moose isolate (16–60-P153) from Norway and reference prion strains (human sCJD strains M1CJD and V2CJD, cattle strain c-BSE) in transgenic mouse models expressing human PrPC 129M (TgMet) and 129V (TgVal), ovine VRQ PrPC (Tg338) and bovine PrPC (BoTg110). BSE, bovine spongiform encephalopathy; dpi, days post-inoculation; NA, not available; ND, not done; PrP, prion protein; PrPres, PK-resistant prion protein; VRQ, valine136-arginine154-glutamine171 ovine PrPC variant. †No. affected mice/total no. inoculated. ‡Transmissions reported in (5) §Transmissions reported in (6) ¶Transmissions reported in (7) #Transmissions reported in (8) **Transmission was performed from the brain of the first-passage mouse that showed a 19 kDa banding pattern. ††Transmission was performed from the brain of the first-passage mouse that showed a 21 kDa banding pattern. ‡‡The Tg338-adapted isolate corresponds to the brain of a second-passage Tg338 mouse that was culled at 170 dpi after infection with firstpassage 19K brain. 2694 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 30, No.12, December 2024 RESEARCH LETTERS the role of this polymorphism in susceptibility to prions (10). In summary, our results demonstrate the potential capacity of some CWD agents to transmit to sheep or other farmed animals. Our results highlight the need to experimentally assess and monitor this transmission risk under natural exposure conditions. In addition, the dramatic changes of the zoonotic capacity of the CWD isolate we documented from Europe clearly demonstrate the risk adaptation and propagation of cervid prions into farmed animals represents. Although additional studies are needed to characterize these emerging agents, our findings have major potential implications for animal and public health. Experiments were performed in compliance with institutional, national, and European guidelines and approved by the local Ecole Nationale Vétérinaire de Toulouse ethics committee. This work was funded by the International Coordination of Research on Infectious Animal Diseases, a European Research Area Networks co-funded by the European Union’s Horizon 2020 research and innovation program through project Tackling Chronic Wasting Disease in Europe (grant no. 862605), the European Union’s “HORIZON EUROPE” program through project WiLiMan-ID (grant no. 101083833), Agence National de la Recherche through project European Union-Chronic Wasting Disease (grant no. ANR-20-CE35-0015), and the European Regional Development Fund via the Interreg V-A Spain-France-Andorra program through projects REDPRION (grant no. EFA 148/16) and NEURO-COOP (grant no. EFA 031/01). About the Author Dr. Barrio is a research scientist with Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement. His research interests include animal and human prion diseases and other neurodegenerative disorders linked to misfolded proteins. References 1. 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Sheep and goat BSE propagate more efficiently than cattle BSE in human PrP transgenic mice. PLoS Pathog. 2011;7:e1001319. https://doi.org/ 10.1371/journal.ppat.1001319 10. Deslys JP, Marcé D, Dormont D. Similar genetic susceptibility in iatrogenic and sporadic Creutzfeldt-Jakob disease. J Gen Virol. 1994;75:23–7. https://doi.org/ 10.1099/0022-1317-75-1-23 Address for correspondence: Tomás Barrio, Unité Mixte de Recherche de l’Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement 1225 Interactions Hôtes-Agents Pathogènes, , 23 chemin des Capelles 31076 Toulouse, France; email: [email protected]