Experimental intravenous, intratracheal, and intranasal inoculation of swine with SARS-CoV-2

Authors: Buckley, Alexandra; Falkenberg, Shollie; MARTINS, MATHIAS - Cornell University; Palmer, Mitchell; Lager, Kelly; DIEL, DIEGO - Cornell University

Submitted to: International Pig Veterinary Society (IPVS)
Publication Type: Proceedings
Publication Acceptance Date: 6/5/2022
Publication Date: 6/23/2022
Citation: Devries, A.C., Falkenberg, S.M., Martins, M., Palmer, M.V., Lager, K.M., Diel, D. 2022. Experimental intravenous, intratracheal, and intranasal inoculation of swine with SARS-CoV-2. Pig Veterinary Society International Congress Proceedings. Pg.6825 Abstract 473030. https://doi.org/10.3390/v13081506.
DOI: https://doi.org/10.3390/v13081506

Interpretive Summary:

Technical Abstract: Introduction Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is novel coronavirus that causes disease (COVID-19) in humans, and it is hypothesized to have a zoonotic origin. Since swine and other livestock species are susceptible to other coronaviruses, research has been initiated to determine the susceptibility of livestock species to SARS-CoV-2 and if livestock can play a role in spread of the virus. Multiple groups have reported that swine are not susceptible to SARS-CoV-2 via the intranasal inoculation route; therefore, two different routes of inoculation (intravenous and intratracheal) were evaluated to determine if swine were susceptible to SARS-CoV-2. Materials and Methods Twenty-four 3-week-old pigs were purchased from a commercial swine herd and transported to the National Animal Disease Center in Ames, IA. Control pigs were housed in ABSL-2, while challenged pigs and their contacts were housed in a BSL-3AG facility. Three challenge routes were performed: intravenous (IV, n=4, 344-347), intratracheal (IT, n=4, 348-351) and intranasal (IN, n=4, 352-355). Sham cell culture lysate challenge was administered to control pigs (n=2/route) with 2 control pigs untreated. Finally, one contact pig was added to each challenge group and the control room on day 2 post inoculation. Virus was isolated from a Malayan tiger that developed respiratory signs after infection with SARS-CoV-2 (TGR/NY/20). IV pigs received 2 mL, while both IT and IN groups received 5 mL of 6.8 x 106 TCID50/ml virus solution. Temperatures were recorded daily using a subcutaneous microchip. Nasal/oral swabs, rectal swabs, and group oral fluids were collected on 0-7, 10, 12, 14, 18 and 21 days post inoculation (dpi). Serum and whole blood were obtained at 0, 3, 7, 14, and 21 dpi. Samples were tested by PCR for the presence of virus and serum was utilized in a serum virus neutralization assay to test for neutralizing antibodies. At 21 dpi animals were euthanized and tissues were collected for PCR testing and histology. Results Immediately after IV challenge, pigs that received either virus or sham cell culture lysate began to vomit, but quickly recovered. Two IT pigs had increased temperatures 2-3 dpi, but only one pig had a temperature recorded greater than 40.5°C on 13 dpi from the IV group. Otherwise, pigs in this study did not demonstrate any clinical signs. The IV group had two pigs (344, 347) PCR positive on 3 dpi in the buffy coat and one pig (345) positive in a nasal/oral swab on 4 dpi. The remaining samples were all negative by PCR. All pigs in the IV group developed virus neutralizing antibody (VN) titers of 1:32 and 64 on 7 dpi but a reduction in those titers was observed on 14 dpi (1:8 to 1:32). By 21 dpi all pigs had titers of 1:8. In the IT group, all pigs were positive in nasal/oral swabs on 1 dpi, suggesting likely detection of the inoculum virus. On 2 dpi, SARS-CoV-2 RNA was detected in only two pigs (350, 351). Beyond 2 dpi, one pig (351) was PCR positive on 7 dpi, with RNA being detected on nasal/oral swabs. In rectal swabs, pig 350 was positive on 1, 2, and 7 dpi. Pig 351 had positive rectal swabs on 1 and 3 dpi. In addition, the IT group had positive oral fluids on 6 dpi. Pig 350 developed a titer of 1:32 on 21, while pig 351 had a titer of 1:16 on 14 dpi and dropped back down to 1:8 on 21 dpi. All animals in the IN group were positive in nasal/oral swabs on 1 dpi, which again could be detection of inoculum given the route of inoculation. Two animals (353, 355) were positive on 2 dpi and one animal (354) on 3 dpi. In rectal swabs, pig 355 had positive swabs on 1 and 2 dpi. Of note, pig 352 had positive rectal swabs on 2, 3, 4, 5, 10, and 12. In addition, this group was PCR positive in oral fluids on 4 dpi. Only pig 352 seroconverted with a titer of 1:32 on 14 dpi and 1:16 on 21 dpi. The contact pigs did not present any PCR p