Emerging Microbes & Infections 2021, VOL. 11 https://doi.org/10.1080/22221751.2021.2011625 LETTER Experimental and field investigations of exposure, replication and transmission of SARS-CoV-2 in pigs in the Netherlands Reina S. Sikkema a*, Tijs Tobias b*, Nadia Oreshkova c, Erwin de Bruin a, Nisreen Okba a, Felicity Chandlera, Marcel M. Hulst c, Jordi Rodon d, Manon Houben e, Kees van Maanene, Hans Bultmane, Marina Meester b, Nora M. Gerhards c, Martijn Bouwknegt f, Bert Urlings f, Bart Haagmans a, Jan Kluytmans g, Corine H. GeurtsvanKessel a, Wim H.M. van der Poel c, Marion P.G. Koopmans a† and Arjan Stegeman b† a Department Viroscience, Erasmus Medical Centre, Rotterdam, The Netherlands; bDepartment Population Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands; cWageningen Bioveterinary Research, Lelystad, The Netherlands; dIRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Bellaterra (Cerdanyola del Vallès), Spain; eRoyal GD Animal Health, Deventer, The Netherlands; fVion Food Group, Boxtel, The Netherlands; gJulius Center for Health Sciences and Primary Care, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands ABSTRACT In order to assess the risk of SARS-CoV-2 infection, transmission and reservoir development in swine, we combined results of an experimental and two observational studies. First, intranasal and intratracheal challenge of eight pigs did not result in infection, based on clinical signs and PCR on swab and lung tissue samples. Two serum samples returned a low positive result in virus neutralization, in line with findings in other infection experiments in pigs. Next, a retrospective observational study was performed in the Netherlands in the spring of 2020. Serum samples (N =417) obtained at slaughter from 17 farms located in a region with a high human case incidence in the first wave of the pandemic. Samples were tested with protein micro array, plaque reduction neutralization test and receptor-binding- domain ELISA. None of the serum samples was positive in all three assays, although six samples from one farm returned a low positive result in PRNT (titers 40–80). Therefore we conclude that serological evidence for large scale transmission was not observed. Finally, an outbreak of respiratory disease in pigs on one farm, coinciding with recent exposure to SARS-CoV-2 infected animal caretakers, was investigated. Tonsil swabs and paired serum samples were tested. No evidence for infection with SARS-CoV-2 was found. In conclusion, Although in both the experimental and the observational study few samples returned low antibody titer results in PRNT infection with SARS-CoV-2 was not confirmed. It was concluded that sporadic infections in the field cannot be excluded, but large-scale SARS-CoV-2 transmission among pigs is unlikely. ARTICLE HISTORY Received 21 October 2021; Revised 20 November 2021; Accepted 23 November 2021 KEYWORDS Swine; Coronavirus; SARS-CoV-2; antibody; One Health Knowledge on host species susceptibility and trans- studies for transmission under field conditions can mission of emerging pathogens is essential to assess be limited. Therefore, additional observational studies the risk for reservoir development. SARS-CoV-2 can need to be considered [4,8]. In this letter, we report the infect multiple animal species, which has become results of an experimental challenge study combined clear from both experimental [1,2] and observational with a repeated cross-sectional serosurvey of pigs in studies [3]. Previous experimental infection studies the Netherlands in 2020 and an outbreak investigation in pigs (Appendix 1-1) reported no evidence for of pigs with respiratory signs linked to two human SARS-CoV-2 replication [1,2,4–7], with the exception SARS-CoV-2 cases. The aim was to investigate suscep- of one study that reported the isolation of SARS-CoV- tibility, replication, and transmissibility of SARS-CoV- 2 from a single lymph node 13 days post-infection 2 in pigs. (DPI) [4]. Neutralizing antibodies have been occasion- First, eight pigs were challenged with 106,2 TCID50 ally detected in pigs [4–6], possibly indicating infec- SARS-CoV-2 (SARS-CoV-2/human/NL/Lelystad/ tion. The validity of experimental transmission 2020 (wild-type D614G), Genbank accession number CONTACT Tijs Tobias T.J.Tobias@uu.nl Department Population Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 7, 3584CL Utrecht, the Netherlands *Shared first authors. † Shared last authors. Supplemental data for this article can be accessed https://doi.org/10.1080/22221751.2021.2011625. © 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group, on behalf of Shanghai Shangyixun Cultural Communication Co., Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 92 R. S. SIKKEMA ET AL. Figure 1. Study design and results for the SARS-CoV-2 pig challenge study. Challenge and sampling timeline (A), effect of SARS- CoV-2 challenge on pig body weight (B), and pig body temperature (C). Neutralizing antibodies (D) and (subgenomic) PCR results (E, F, G, H, I). MZ144583 [9]) both intranasally and intratracheally Netherlands both with a high incidence of human (Appendix 1-2, Figure 1A). Animals were sacrificed SARS-CoV-2 cases during the early phase of the epi- on DPI3 (n = 2), DPI6 (n = 2), DPI10 (n = 2) and demic (cumulative infection rate 318 cases/100,000 DPI21 (n = 2). Clinical signs were not observed during inhabitants on March 31st, 2020) (Appendix 1-4) as the 21-day study period. Low levels of viral RNA were well as a high incidence of SARS-CoV-2 affected found in nasal swabs on DPI1 (n = 2; 2.4 and 5.3 Log10 mink farms [3]. Twenty-one pig farms were selected TCID50/ml PCR equivalents), and in oropharyngeal (of which 17 participated), based on region and avail- swabs on DPI1 (n = 8; range 2.8–4.1 Log10 TCID50/ ability to collect blood samples during exsanguination ml PCR equivalents) and DPI2 (n = 2; 2.9 Log10 at slaughter. Based on the assumption that potential TCID50/ml PCR equivalents). Rectal swabs all tested transmission in a group of pigs would result in a negative. Low amount of RNA was found in lung final size of >50% infected pigs (expected value when samples (n = 2 on DPI3, n = 1 on DPI6 and n = 2 R0 = 2 [11]), we aimed to obtain six blood samples on DPI10), but no evidence for virus replication was of slaughter pigs at two timepoints per farm. Totally, found given the negative subgenomic RNA PCR 417 serum samples were collected from pigs of 17 results [10] (Figure 1I). Neutralizing antibodies were farms between March and July 2020 (Appendix 1-4). detected in two out of four pigs on DPI10 (Figure A testing algorithm was designed and validated 1D). One of the two animals still had antibodies on (Appendix 1-3), using a protein microarray (PMA) DPI14, while the other was euthanized on DPI10. [12] with the SARS-CoV-2 S ectodomain antigen for Second, a retrospective observational study was screening, followed by a plaque reduction neutraliz- conducted in pigs reared in a region in the ation test (PRNT) and an receptor-binding domain EMERGING MICROBES & INFECTIONS 93 (RBD)-ELISA to confirm positive results ([13], available, whereas with the emergence of new SARS- Appendices 1-3 and 1-4). A total of 29 sera, that CoV-2 variants, also host range may change, as was showed reactivity in the PMA, were included in seen in susceptibility of mice for some novel variants PRNT. In six out of 29 samples, derived from one of concern [15]. Therefore, it is important to regularly farm at one timepoint, low titers were found in the monitor possible animal reservoirs. A One Health PRNT (titer 40-80). None of these 29 samples tested approach and vigilance of researchers, veterinarians above cut-off in the RBD-ELISA. Other serum samples and farmers are needed to detect and recognize future of pigs from the same farm tested negative (n = 35) in host species jumps. PMA and PRNT, including serum samples obtained 2 months prior (n = 12) and 3 months after (n = 18) the timepoint with PRNT positive serum samples. Acknowledgements Finally, in February 2021 in another region in the The work described in this manuscript was funded by the Netherlands, two pig caretakers were confirmed Ministry of Agriculture, Nature and Food Safety of the SARS-CoV-2 positive. They had close contact with Netherlands and by the Coalition for Epidemic Prepared- ness Innovations (CEPI). Positive control pig sera were the pigs in the days before onset of symptoms, obtained from the CBIG Consortium (constituted by which coincided with an episode of nonspecific respir- IRTA-CReSA, BSC, & IrsiCaixa, ES) and supported by Gri- atory clinical signs in weaned pigs, rearing gilts and fols (ES). Hans Nauwynck (U-Ghent, BE) is acknowledged sows. Clinical signs were characterized by cough and for providing PHEV serum. hyperthermia. Tonsil swabs and paired serum samples (27 paired and 3 single sera) were collected from 30 pigs (18 exhibited respiratory signs), to detect a mini- Disclosure statement mal prevalence of ∼10% with 95% confidence. All ton- No potential conflict of interest was reported by the author(s). sil swabs tested negative for SARS-CoV-2 by E gene PCR [3,14]. In 5 out of 57 sera SARS-CoV-2 spike- binding binding antibodies were detected with PMA. ORCID These sera were negative in the PRNT and RBD- Reina S. Sikkema http://orcid.org/0000-0001-7331-6274 ELISA. Tijs Tobias http://orcid.org/0000-0002-0747-9531 Nadia Oreshkova http://orcid.org/0000-0001-8830-7055 In conclusion, some pig sera of one farm had low Erwin de Bruin http://orcid.org/0000-0003-0826-5102 neutralizing antibody titers in a virus neutralization Nisreen Okba http://orcid.org/0000-0002-2394-1079 assay, known to be very specific for human sera [13]. Marcel M. Hulst http://orcid.org/0000-0001-8020-8853 Low antibody titers were also seen in experimentally Jordi Rodon http://orcid.org/0000-0002-1032-9091 infected pigs (Appendix 1-2 and [5]). In the field, we Manon Houben http://orcid.org/0000-0001-6852-7632 neither found serological evidence for large-scale Marina Meester http://orcid.org/0000-0001-8004-8546 Nora M. Gerhards http://orcid.org/0000-0001-7367-9008 transmission among pigs from farms in a high-risk Martijn Bouwknegt http://orcid.org/0000-0002-3446- region, nor for human–pig transmission on a farm 5338 with a known outbreak among animal caretakers. Fur- Bert Urlings http://orcid.org/0000-0002-9299-4211 thermore, no experimental evidence for viral replica- Bart Haagmans http://orcid.org/0000-0001-6221-2015 tion in pigs was found, which is in line with the Jan Kluytmans http://orcid.org/0000-0003-2665-6844 Corine H. Geurtsvankessel http://orcid.org/0000-0002- literature [1,2,4–7]. Therefore, we conclude that 7678-314X sporadic infections in the field cannot be excluded, Wim H.M. van der Poel http://orcid.org/0000-0002-7498- but large-scale SARS-CoV-2 transmission among 8002 pigs in the field is unlikely. Neutralizing antibodies Marion P.G. Koopmans http://orcid.org/0000-0002-5204- seen after experimental infection may be induced by 2312 the inoculum and cross-neutralization by porcine cor- Arjan Stegeman http://orcid.org/0000-0003-4361-3846 onavirus infections needs to be investigated in more detail in future studies. 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