Experimental re-infected cats do not transmit SARS-CoV-2

Abstract

SARS-CoV-2 is the causative agent of COVID-19 and responsible for the current global pandemic. We and others have previously demonstrated that cats are susceptible to SARS-CoV-2 infection and can efficiently transmit the virus to naïve cats. Here, we address whether cats previously exposed to SARS-CoV-2 can be re-infected with SARS-CoV-2. In two independent studies, SARS-CoV-2-infected cats were re-challenged with SARS-CoV-2 at 21 days post primary challenge (DPC) and necropsies performed at 4, 7 and 14 days post-secondary challenge (DP2C). Sentinels were co-mingled with the re-challenged cats at 1 DP2C. Clinical signs were recorded, and nasal, oropharyngeal, and rectal swabs, blood, and serum were collected and tissues examined for histologic lesions. Viral RNA was transiently shed via the nasal, oropharyngeal and rectal cavities of the re-challenged cats. Viral RNA was detected in various tissues of re-challenged cats euthanized at 4 DP2C, mainly in the upper respiratory tract and lymphoid tissues, but less frequently and at lower levels in the lower respiratory tract when compared to primary SARS-CoV-2 challenged cats at 4 DPC. Viral RNA and antigen detected in the respiratory tract of the primary SARS-CoV-2 infected cats at early DPCs were absent in the re-challenged cats. Naïve sentinels co-housed with the re-challenged cats did not shed virus or seroconvert. Together, our results indicate that cats previously infected with SARS-CoV-2 can be experimentally re-infected with SARS-CoV-2; however, the levels of virus shed was insufficient for transmission to co-housed naïve sentinels. We conclude that SARS-CoV-2 infection in cats induces immune responses that provide partial, non-sterilizing immune protection against re-infection.

Keywords: COVID-19; SARS-CoV-2; cats; re-infection; transmission.

Figure 1.

Re-infection study design. (a) In each of 2 studies, 6 cats were inoculated with SARS-CoV-2 and 2 sentinel contact cats were introduced at 1 day post primary challenge (DPC). Necropsy was performed on principal infected cats at 4, 7 and 21 DPC. (b) At 21 DPC, cats were re-challenged with SARS-CoV-2 at the same dose as used for primary challenge, and 2 sentinels introduced at day 1 post-second challenge (DP2C). Necropsy of principal re-infected cats were performed at 4, 7 and 14 DP2C, and of the sentinels at 14 PD2C.

Figure 2.

Viral shedding from SARS-CoV-2 infected and re-infected cats. RT-qPCR was performed on nasal (a), oropharyngeal (b) and rectal (c) swabs collected from cats at the indicated days following primary challenge (DPC) and re-challenge (DP2C) from the second re-infection study. Results represent all cats that were swabbed and tested at 0, 1, 3, 5, 7, 10, 14, 18 and 21 DPC, and at 0, 1, 3, 5, 7, 8, 11 and 14 DP2C. Results at 4 DPC and 4 DP2C are representative of only the 2 necropsied cats that were swabbed and tested on these days. (d) Nasal, oropharyngeal and rectal swabs collected from the 3 re-challenged cats from the first re-infection study. Mean and standard deviation of viral RNA copy number (CN) per mL based on the nucleocapsid gene are shown. Asterisks (*) indicate samples with 1 out of 2 of the RT-qPCR reactions below the limit of detection (LOD), indicated by the dotted line.

Figure 3.

SARS-CoV-2 RNA detected in various tissues from infected and re-infected cats. RT-qPCR was used to detect the presence of SARS-CoV-2 in various tissues of cats euthanized at the indicated days after primary challenge (DPC) and re-challenge (DP2C). Viral RNA copy number (CN) per mL (a–c) or mg (d–h) based on the nucleocapsid gene are plotted for individual animals. Coloured symbols corresponding to cat ID numbers, day of necropsy and study are indicated in the figure key. Asterisks (*) indicate samples with 1 out of 2 of the RT-qPCR reactions below the limit of detection (LOD) which is, indicated by the dotted line.

Figure 4.

(a) Histologic lesions in the respiratory tract of primary infected cats. Mock-infected (A–C), 4 (D–F), and 21 days post-challenge (DPC; G–I) with SARS-CoV-2. At 4 DPC, intense neutrophilic rhinitis with luminal exudate (D, arrowhead and arrow, respectively) and lymphohistiocytic and neutrophilic tracheobronchoadenitis with necrosis and obliteration of seromucinous glands (E and F, arrows) were characteristic. At 21 DPC, histologic changes were limited to lymphoid aggregates in the lamina propria of the nasal turbinates (G), trachea (H) and bronchi (I). H&E, 200× total magnification. Bar = 100 μm. (b). SARS-CoV-2 distribution as determined by in situ hybridization in the respiratory tract of primary infected cats. Mock-infected (A–C), 4 (D–F), and 21 DPC (G–I) with SARS-CoV-2. At 4DPC, SARS-CoV-2 RNA was detected and localized within the nasal respiratory epithelium, olfactory neuroepithelium (D) and tracheobronchial glands in trachea and bronchi (E and F). At 21 DPC, no viral RNA was detected in the respiratory tract (G–I). Fast Red, 200× total magnification. Bar = 100 μm. (c). SARS-CoV-2 distribution as determined by immunohistochemistry in the respiratory tract of primary infected cats. Mock-infected (A–C), 4 (D–F), and 21 DPC (G–I) with SARS-CoV-2. At 4DPC, SARS-CoV-2 antigen was present in the nasal respiratory epithelium, olfactory neuroepithelium (D) and tracheobronchial glands in trachea and bronchi (E and F), co-localizing with viral RNA. At 21 DPC, no viral antigen was detected in the respiratory tract (G–I). Fast Red, 200× total magnification. Bar = 100 μm.

Figure 5.

Virus neutralizing antibody responses in primary infected and re-challenged cats. Statistical analysis of virus neutralizing antibody titers were determined by multiple t-test using the Holm-Sidak method, with alpha=5.000%. Only data for a single re-infected cat was available at 14 DP2C therefore significance could not be calculated. Statistical significance is indicated by: *<0.05; **<0.005; ***<0.0005; DPC = days post primary challenge; DP2C = days post second challenge

Figure 6.

(a) Histologic lesions in the respiratory tract of re-challenged cats. 4 (A–C), 7 (D–F), and 14 days post second challenge (DP2C; G–I) with SARS-CoV-2. At all time points, variable lymphoid aggregates expanded the lamina propria of the nasal turbinates (A, D, G), trachea (B, E, H) and bronchi (C, F, I). No disruption of tracheobronchial glands was noted. H&E, 200× total magnification. Bar = 100 μm. (b) SARS-CoV-2 distribution as determined by in situ hybridization in the respiratory tract of re-challenged cats. 4 (A–C), 7 (D–F), and 14 DP2C (G–I) with SARS-CoV-2. No viral RNA was detected in the nasal cavity (A, D, G), trachea (B, E, H) or bronchi (C, F, I) at any time point. Fast Red, 200× total magnification. Bar = 100 μm. (c) SARS-CoV-2 distribution as determined by immunohistochemistry in the respiratory tract of re-challenged cats. 4 (A–C), 7 (D–F), and 14 DP2C (G–I) with SARS-CoV-2. No viral antigen was detected in the nasal cavity (A, D, G), trachea (B, E, H) or bronchi (C, F, I) at any time point. Fast Red, 200× total magnification. Bar = 100 μm.