# Zoonotic Potential of Chronic Wasting Disease After Adaptation in Intermediate Species

Tomás Barrio, Sylvie Benestad, Jean-Yves Douet, Alvina Huor, Séverine Lugan, Naïma Aron, Hervé Cassard, Juan Carlos Espinosa, Alicia Otero, Rosa Bolea, et al.

► To cite this version:

Tomás Barrio, Sylvie Benestad, Jean-Yves Douet, Alvina Huor, Séverine Lugan, et al. Zoonotic Potential of Chronic Wasting Disease After Adaptation in Intermediate Species. Emerging Infectious Diseases, 2024, 30 (12), pp.2579 - 2581. 10.3201/eid3012.240536. hal-05051471

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## 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

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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-and-availability/fda-warns-consumers-not-purchase-or-use-ezricare-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 multidrug-resistant 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

# 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 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.

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 free-ranging 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 (PrP<sup>C</sup>) sequences from several species (Figure, panel A).

<footnote>Address for correspondence: Emma R. Price, New Jersey
Department of Health, 135 E State St, Trenton, NJ 08625, USA;
email: emma.price@doh.nj.gov</footnote>
**Figure.** Western blot analysis in a study of zoonotic potential of chronic wasting disease after adaptation in intermediate species. Results show PK-resistant PrP (PrP<sup>res</sup>) banding patterns of a moose CWD isolate from Europe after transmission to transgenic PrP models. A) Original 16–60-P153 CWD isolate compared with reference Dawson and sheep-passaged BSE. B) Transmission of the original moose CWD isolate to mice ovine PrP<sup>C</sup> genotype VRQ (Tg338), resulting in positive transmission with either 19 or 21 kDa PrP<sup>res</sup> on first passage and 21 kDa PrP<sup>res</sup> 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 PrP<sup>res</sup> banding patterns in TgMet and TgVal inoculated with the Tg338-adapted moose CWD isolate or with M1<sup>CJD</sup> and V2<sup>CJD</sup> 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 PrP<sup>C</sup>-expressing mice; BSE, bovine spongiform encephalopathy; CJD, Creutzfeldt-Jakob disease; CWD, chronic wasting disease; PK, proteinase K; PrP, prion protein; PrP<sup>C</sup>, normal prion protein; PrP<sup>res</sup>, PK-resistant prion protein. TgMet, Tg340 mice expressing methionine; TgVal, Tg361 mice expressing valine.

We anesthetized and inoculated 6-to-10-week-
old 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 clini-
cal signs or after the preestablished endpoint of 700
days postinfection (dpi). We conducted a systematic
proteinase K-resistant prion protein (PrP<sup>res</sup>) detec-
tion 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
PrP<sup>C</sup>. We did not observe PrP<sup>res</sup> in brain tissue or
disease occurrence in bovine PrP<sup>C</sup>-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 PrP<sup>res</sup> accu-
mulation in their brain tissue (Figure, panel B). Of
note, the 2 animals showed different PrP<sup>res</sup> banding
patterns, with the nonglycosylated band migrat-
ing to 19 kDa in the first mouse and to 21 kDa in
the second. Both PrP<sup>res</sup>-containing brains transmit-
ted disease with 100% efficacy to second-passage
Tg338 mice, which contained 21-kDa PrP<sup>res</sup> 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 ad-
aptation of the moose CWD prion to the ovine-PrP<sup>C</sup>
expressing model and suggest moose CWD prions
in Europe may have an intrinsic capability to prop-
agate in ovine species with the VRQ genotype.

We next determined whether adaptation of
this moose CWD agent to Tg338 altered its capac-
ity to cross species barriers. For that purpose, we
inoculated Tg338-adapted moose CWD prions
(passaged twice in Tg338) to the same panel of PrP<sup>C</sup>-

A

<figure><img src="image_2.png" /></figure>

B

<figure><img src="image_3.png" /></figure>

C

<figure><img src="image_4.png" /></figure>

D

<figure><img src="image_5.png" /></figure>
<table><caption>**Table.** Transmission of a moose CWD isolate in a study of zoonotic potential of chronic wasting disease after adaptation in intermediate species*</caption><thead><tr><th rowspan="2">Model<br>characteristics</th><th colspan="3">TgMet</th><th colspan="3">TgVal</th><th colspan="3">Tg338</th><th colspan="3">BoTg110</th></tr><tr><th>No./<br>no.†</th><th>Mean dpi<br>(SD)</th><th>PrP<sup>res</sup><br>band<br>type</th><th>No./<br>no.†</th><th>Mean dpi<br>(SD)</th><th>PrP<sup>res</sup><br>band<br>type</th><th>No./<br>no.†</th><th>Mean dpi<br>(SD)</th><th>PrP<sup>res</sup><br>band<br>type</th><th>No./<br>no.†</th><th>Mean dpi<br>(SD)</th><th>PrP<sup>res</sup><br>band<br>type</th></tr></thead><tbody><tr><td colspan="14"><strong>Prion strains</strong></td></tr><tr><td colspan="14"><strong>M1<sup>CJD</sup> (sCJD MM1)</strong></td></tr><tr><td>1st passage</td><td>6/6</td><td>219 (17)</td><td>21 kDa</td><td>6/6</td><td>327 (19)‡</td><td>21 kDa</td><td>ND</td><td></td><td></td><td>ND</td><td></td><td></td></tr><tr><td>2nd passage</td><td>6/6</td><td>239 (8)‡</td><td>21 kDa</td><td>6/6</td><td>286 (16)‡</td><td>21 kDa</td><td>ND</td><td></td><td></td><td>ND</td><td></td><td></td></tr><tr><td colspan="14"><strong>V2<sup>CJD</sup> (sCJD VV2)</strong></td></tr><tr><td>1st passage</td><td>6/6</td><td>618 (81)‡</td><td>21 kDa</td><td>6/6</td><td>168 (12)‡</td><td>19 kDa</td><td>ND</td><td></td><td></td><td>ND</td><td></td><td></td></tr><tr><td>2nd passage</td><td>6/6</td><td>509 (41)‡</td><td>21 kDa</td><td>6/6</td><td>169 (12)‡</td><td>19 kDa</td><td>ND</td><td></td><td></td><td>ND</td><td></td><td></td></tr><tr><td colspan="14"><strong>Classical BSE</strong></td></tr><tr><td>1st passage</td><td>1/12</td><td>739‡</td><td>BSE§</td><td>0/12</td><td>≥750‡</td><td>NA</td><td>6/6</td><td>≥750¶¶</td><td>BSE§</td><td>6/6</td><td>295 (12)#</td><td>BSE§</td></tr><tr><td>2nd passage</td><td>9/12</td><td>613 (43)‡</td><td>BSE§</td><td>0/12</td><td>≥750‡</td><td>NA</td><td>6/6</td><td>682 (52)¶¶</td><td>BSE§</td><td>6/6</td><td>265 (35)#</td><td>BSE§</td></tr><tr><td colspan="14"><strong>Sheep-adapted BSE</strong></td></tr><tr><td>1st passage</td><td>6/6</td><td>690 (83)#</td><td>BSE§</td><td>ND</td><td></td><td></td><td>6/6</td><td>≥750¶¶</td><td>BSE§</td><td>6/6</td><td>254 (19)¶¶</td><td>BSE§</td></tr><tr><td>2nd passage</td><td>5/5</td><td>564 (39)#</td><td>BSE§</td><td>ND</td><td></td><td></td><td>6/6</td><td>653 (32)¶¶</td><td>BSE§</td><td>6/6</td><td>234 (12)¶¶</td><td>BSE§</td></tr><tr><td colspan="14"><strong>Tg338-adapted BSE</strong></td></tr><tr><td>1st passage</td><td>6/6</td><td>596 (92)</td><td>BSE§</td><td>0/6</td><td>≥700</td><td>BSE§</td><td>5/5</td><td>224 (37)</td><td>BSE§</td><td>6/6</td><td>222 (22)</td><td>BSE§</td></tr><tr><td>2nd passage</td><td></td><td></td><td></td><td>ND</td><td></td><td></td><td>ND</td><td></td><td></td><td>ND</td><td></td><td></td></tr><tr><td colspan="14"><strong>Moose CWD (16–60-P153)</strong></td></tr><tr><td>1st passage</td><td>0/12</td><td>≥700</td><td>NA</td><td>0/1<br>2</td><td>≥700</td><td>NA</td><td>2/12</td><td>612, 717</td><td>19<br>kDa,<br>21 kDa</td><td>0/12</td><td>≥700</td><td>NA</td></tr><tr><td>2nd passage</td><td>0/6<br>ND</td><td>≥700</td><td>NA</td><td>ND</td><td></td><td></td><td>5/5<br>6/6</td><td>167 (4)**<br>244 (33)††</td><td>21 kDa<br>21 kDa</td><td>ND<br>ND</td><td></td><td></td></tr><tr><td colspan="14"><strong>Tg338-adapted moose CWD‡‡</strong></td></tr><tr><td>1st passage</td><td>1/8</td><td>561</td><td>19+21<br>kDa</td><td>5/6</td><td>483 (35)</td><td>21 kDa</td><td>7/7</td><td>95 (5)</td><td>21 kDa</td><td>5/5</td><td>431 (32)</td><td>20 kDa</td></tr><tr><td>2nd passage</td><td>ND</td><td></td><td></td><td>4/4</td><td>311 (12)</td><td>21 kDa</td><td>ND</td><td></td><td></td><td>ND</td><td></td><td></td></tr></tbody></table>

Inoculation of the Tg338-adapted 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). PrP<sup>res</sup> 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 PrP<sup>res</sup> (Figure, panel C). On second passage, transmission was 100% and we observed a shorter incubation period (311 ± 12 dpi).

The incubation periods and PrP<sup>res</sup> 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 PrP<sup>C</sup>-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

<footnote>*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; PrP<sup>res</sup>, PK-resistant prion protein; VRQ, valine136-arginine154-glutamine171 ovine PrP<sup>C</sup> variant.</footnote>

<footnote>†No. affected mice/total no. inoculated.</footnote>

<footnote>‡Transmissions reported in (5)</footnote>

<footnote>§Transmissions reported in (6)</footnote>

<footnote>¶Transmissions reported in (7)</footnote>

<footnote>#Transmissions reported in (8)</footnote>

<footnote>**Transmission was performed from the brain of the first-passage mouse that showed a 19 kDa banding pattern.</footnote>

<footnote>††Transmission was performed from the brain of the first-passage mouse that showed a 21 kDa banding pattern.</footnote>

<footnote>‡‡The Tg338-adapted isolate corresponds to the brain of a second-passage Tg338 mouse that was culled at 170 dpi after infection with first-passage 19K brain.</footnote>
the role of this polymorphism in susceptibility
to prions (10).

In summary, our results demonstrate the po-
tential 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 condi-
tions. In addition, the dramatic changes of the zoo-
notic capacity of the CWD isolate we documented
from Europe clearly demonstrate the risk adapta-
tion 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 ani-
mal and public health.

Experiments were performed in compliance with institu-
tional, 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.

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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: tomas.barrio@envt.fr